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Purinergic Signal. 2010 June; 6(Suppl 1): 1–162.
Published online 2010 June 17. doi:  10.1007/s11302-010-9187-6
PMCID: PMC2908743

Purines 2010: Adenine Nucleosides and Nucleotides in Biomedicine

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Abstracts from Purines 2010 Meeting

Tarragona-Barcelona

30th May–2nd June

Guest Editors

Francisco Ciruela

Artur Llobet

Mireia Martín-Satué

Carles Solsona

Organizing Committee

Ana M. Sebastião, Artur Llobet, Carles Solsona, Enrique Castro, Esmerilda Delicado, Francisco Ciruela, Geoffrey Burnstock, Gloria Rosell, Jesús Pintor, Joaquim A. Ribeiro, Josefa Mallol, Lydia Giménez-Llort, Mairena Martin, Marçal Pastor-Anglada, Mari CarmenMontesinos, Maria Pia Abbracchio, María Teresa Miras-Portugal, Mireia Martín-Satué, Pablo Pelegrin, Paulo Correia-de-Sá, Pedro Rotllán, Pier Andrea Borea, Rafael Franco, Raquel Sen, Rodrigo Cunha, Sergio Castillón.

Scientific Committee

Adriaan P. IJzerman, Alan North, Annmarie Surprenant, Beata Sperlagh, Bertil B. Fredholm, Bruce N. Cronstein, Christa E. Müller, Claire H. Mitchell, David Erlinge, Edward W. Inscho, Eugene M. Silinsky, Felicita Pedata, Francesco Di Virgilio, George R. Dubyak, Gloria Cristalli, Hiroyasu Nakata, Herbert Zimmermann, Igor Feoktistov, Imogen R. Coe, Italo Biaggioni, Ivana Novak, Jashvant D. Unadkat, Jean-Marie Godfraind, Jean Sévigny, Jean-Marie Boeynaems, Jiang F. Chen, Joel Linden, José L. Boyer, Juan Pablo Huidobro-Toro, Karl-Norbert Klotz, Katya Ravid, Kazuhide Inoue, Kenneth A. Jacobson, Luis Sobrevía, Michail V. Sitkovsky, Michael R Blackburn, Patrizia Popoli, Paul A. Insel, Peter Illes, Richard C Boucher, Scott A. Rivkees, Sergi Ferré, Simon C. Robson, Stephen A. Baldwin, Stephen J. Hill, Sylvester Vizi.

Sponsors:

Universitat de Barcelona

Diputació de Tarragona

Patronat Municipal de Turisme de Tarragona

Comissionat per a Universitats i Recerca-Generalitat de Catalunya

Ministerio de Ciencia e Innovación-Gobierno de España

Addittional support from:

Can-Fite BioPharma Ltd.

Tocris Bioscience

Laboratorios Almirall

Deyman

PLENARY LECTURES

PL1

The therapeutic potential of purinergic signalling

Geoffrey Burnstock

Autonomic Neuroscience Centre, University College Medical School, UK

After a brief initial description of some of the important conceptual steps in the establishment of purinergic signalling, the talk will focus on some of the exciting developments relating to purinergic pathophysiology and potential therapeutic applications. There will be discussion of the long-term trophic effects of purines and pyrimidines in blood vessel remodelling in restenosis; plasticity of purinergic cotransmission of diseased urinary bladder and hypertensive rats; sperm motility in IVF; purinergic mechanosensory transduction in visceral pain and the role of spinal microglial purinoceptors in neuropathic pain; the potential for P2X7 receptor antagonists in osteoporosis and kidney failure; the growing literature about the roles of purinergic signalling of disorders of the central nervous system; and the role of ATP in the treatment of cancer. Finally, a novel hypothesis will be presented for the involvement of purinergic signalling in acupuncture.

PL2

Adenosine-dependent stabilization of the circadian rhythm protein Per2 mediates a metabolic switch critical for myocardial adaptation to ischemia

Holger K. Eltzschig

Mucosal Inflammation Program, Department of Medicine, University of Colorado, USA

Studies of metabolic adaptation during environmental stress have broad applications to human disease. Adenosine signaling has been implicated in cardiac adaptation to limited oxygen availability. Serendipitously, a wide search for adenosine-elicited cardio-adaptive responses identified the circadian rhythm protein period2 (Per2). Subsequent pharmacologic or genetic studies confirmed adenosine-dependent stabilization of Per2 during myocardial ischemia. Moreover, we observed elevated PER2 levels in cardiac biopsies from patients with ischemic heart disease. Functional studies of myocardial ischemia in Per2−/− mice revealed larger infarct sizes and abolished cardioprotection by ischemic preconditioning. Metabolic studies during myocardial ischemia uncovered a limited ability of Per2−/− mice to utilize carbohydrates via oxygen-efficient glycolysis. Similarly, cardiac stabilization of Per2 via light-exposure transcriptionally enhanced glycolytic pathways, and provided period-specific cardioprotection from myocardial ischemia. Together, these studies identify Per2 as a key regulator of ischemia tolerance through reprogramming of the cardiac metabolism and provide important clues for adenosine-dependent metabolic adaptation of the heart.

PL3

The P2X7 receptor supports cell growth

Francesco Di Virgilio

Department of Experimental and Diagnostic Medicine, Section of General Pathology, University of Ferrara, Italy

The P2X7 receptor is well known for its cytotoxic activity. However this is the result of activation by pharmacological doses of extracellular ATP, i.e. levels that can reasonably be reached in vivo only under severe pathophysiological conditions. There is now accruing evidence that in the absence of stimulation by exogenously-added ATP the P2X7 receptor may sustain cell growth, and may thus be implicated in normal or tumor cell proliferation. Evidence in favor of this role is manifold: 1) extracellular ATP supports proliferation of T lymphocytes via a P2X7-like receptor; 2) P2X7 transfection confers growth advantage; 3) P2X7 transfection increases mitochondrial metabolism and ATP synthesis; 4) LPS-dependent growth arrest in microglia is mediated via down-regulation of the P2X7 receptor; 5) several malignant tumours express high P2X7 levels; 6) the ATP concentration in tumour interstitium is several fold higher than in normal tissues; 7) P2X7 transfection increases the endoplasmic reticulum calcium level; 8) P2X7 activates the growth-promoting transcription NFATc1. We believe that a better understanding of the role played by P2X7 in growth promotion will provide insight into the mechanism of normal and malignant cell growth and point to innovative anti-tumour therapies.

References

Di Virgilio F, Ferrari D, Adinolfi, E. 2009. P2X7: a growth-promoting receptor. Purinergic Signal 5:251–256.

Adinolfi E, Callegari MG, Cirillo M, Pinton P, Giorgi C, Cavagna D, Rizzuto R, Di Virgilio F. 2009. Expression of the P2X7 receptor increases the Ca2+ contento f the endoplasmic reticulum, activates NFATc1 and protects from apoptosis. J Biol Chem 284:10120–10128.

Adinolfi E, Cirillo M, Woltersdorf R, Falzoni S, Chiozzi P, Pellegatti P, Callegari MG, Sandonà D, Markwardt F, Schmalzing G, Di Virgilio F. 2010. Trophic activity of a naturally-occurring truncated isoform of the P2X7 receptor. FASEB J in press.

PL4

CF101 for the treatment of autoimmune inflammatory diseases: data from phase 2 clinical trials in patients with rheumatoid arthritis, psoriasis and dry eye syndrome

Pnina Fishman, Sara Bar Yehuda, Clinical study groups

Can-Fite BioPharma, Can-Fite BioPharma Israel

Objective: A3 adenosine receptor (A3AR) is over-expressed in inflammatory cells and was suggested as a novel target to combat inflammation. Pharmacology studies show that CF101 (IB-MECA), a highly selective A3AR agonist, acts as an anti-inflammatory agent via a mechanism which entails de-regulation of the NF-κB signaling pathway and inhibition of TNF-α. This study presents a summary of 3 human Phase II clinical trials looking at the safety and efficacy of CF101 in patients with Rheumatoid Arthritis (RA), Psoriasis and Dry Eye syndrome. Methods: The studies were Phase 2, multicenter, randomized, double-masked, parallel-group. Patients were treated orally with either CF101 (0.1, 1, 2 or 4 mg) or matching placebo given twice daily for 12 weeks. Efficacy was tested as follows: RA - % of patients achieving ACR20; Psoriasis - Area and Severity index (PASI) and Physician’s Global Assessment (PGA) scores; Dry Eye Syndrome – corneal fluorescein staining. Safety was evaluated by clinical and laboratory tests. Results: CF101 was very well tolerated and treatment resulted in a statistically significant improvement of the clinical signs and symptoms of each disease. Furthermore, in the RA study, statistically significant correlation between A3AR expression at baseline and patients’ response to CF101 was observed, suggesting the receptor as a biological predictive marker. Conclusions: The anti-inflammatory and the excellent safety profile of CF101supports further clinical development of this drug candidate for the treatment of autoimmune inflammatory disease.

PL5

Nucleoside transporter proteins in the crossroads between purinergic signaling and nucleoside salvage

Marçal Pastor-Anglada

Department of Biochemistry and Molecular Biology, Institute of Biomedicine (IBUB), University of Barcelona and CIBER EHD, Spain

Most mammalian cells express several genes belonging to Solute Carrier gene families SLC28 and SLC29, encoding Concentrative (CNT) and Equilibrative (ENT) Nucleoside Transporter proteins, respectively. Although adenosine is a physiological substrate for most NT proteins, the biological rationale for the co-expression in most cell types of plasma membrane carriers with overlapping substrate selectivity is still poorly understood. In this contribution we will compare the NT protein expression pattern and the selective regulation of particular NTs in a variety of cell types to get further insight into the relative role of salvage processes compared to other physiological functions of NT proteins. Epithelial cell types such as enterocytes, renal tubular cells, epithelial cells from the syncytiotrophoblast and hepatocytes will be compared with other cell models also studied in our group, including neurons and immune system cells. Overall, results support the view that vectorial flux of nucleosides for salvage purposes is highly variable in epithelial barriers, whereas CNT-type transporters, particularly CNT2, a high affinity adenosine carrier, is under purinergic control and has tight links with intracellular pathways regulating energy metabolism in various cell types. Moreover CNT-type proteins also mediate the uptake of nucleoside-derived drugs which actually exert their pharmacological action mostly by interfering salvage processes. Understanding CNT regulatory mechanisms may also prove to be suitable for improvement of nucleoside-based therapies.

PL6

P2X7and epithelial cancers

George Gorodeski

Case Western Reserve, University Cleveland University of Bonn, USA

Recent expression and structure-function data in epithelia revealed a novel role for the P2X7 system.

  1. The main biological role of the P2X7 is mediation of apoptosis.
  2. Initiation of apoptosis involves formation of P2X7 pores, which allow uncontrolled influx of Ca2+.
  3. Formation of P2X7 pores (and hence apoptosis) is normally controlled by low affinity of the P2X7-R to extracellular ATP (the natural ligand of the P2X7-R), and by the truncated P2X7-j form, which is constitutively expressed in epithelial cells, and can hetero-oligomerize with the P2X7 and render P2X7 pores inactive.
  4. In normal epithelial cells formation of P2X7 pores occurs preferentially in the apical membrane, and stimulates apoptosis of cells undergoing terminal differentiation.
  5. The expression of P2X7-R in epithelial cells is regulated by three major mechanisms:
    1. Enhancers located at nt +222/+232 and +401/+573 regions downstream of the active P2X7 promoter;
    2. Methylatable cytosines at CpG sites that cluster or co-localize with the enhancers’ sites;
    3. Micro-RNAs miR-186 and miR-150 that activate instability target sites located at the 3′UTR-P2X7.
  6. Expression of P2X7-R is reduced in cancer epithelial cells originating from the ectoderm, the uro-genital sinus, and the distal paramesonephric duct.
  7. Reduced expression of the P2X7-R precedes or coincides with neoplastic changes in those tissues, and may play a role in the development of cancers.
  8. Reduced expression of the P2X7-R in cancer epithelial cells is associated with hypermethylation of CpG sites that cluster the transcription enhancers’ sites (and thereby would tend to reduce transcription); and with increased levels of miR-186 and miR-150 in cancer (that would tend to induce instability of the P2X7 transcript).
  9. Pharmacological activation of P2X7-mediated apoptosis in mice, by local skin application of BzATP, inhibited DMBA/TPA-induced formation of skin papillomas and the transformation of DMBA/TPA-induced papillomas into squamous spindle-cell carcinomas; it tended to prolong life of animals with cancers; and had no local or systemic side effects.

These novel data will be discussed in terms of the physiological role of the P2X7 system in epithelial cells; the value of P2X7-R assays as a potential biomarker of epithelial neoplasia; and the potential novel modality of pharmacological treatment with P2X7 agonists for the prevention and treatment of epithelial neoplasia.

Symposia

S1. Medical chemistry

S1-1

Reactive Blue 2 as a lead structure: potent and selective antagonists for P2 receptor subtypes and ectonucleotidase inhibitors

Christa Müller

PharmaCenter Bonn, University of Bonn, Germany

Reactive Blue 2 (RB-2), a sulfonated anthraquinone derivative, was found to interact with a variety of nucleotide-binding proteins in the human body, including a number of different P2 receptor subtypes and ectonucleotidases. However, RB-2 shows only moderate affinity, is non-selective, and its structure is not drug-like due to its high molecular weight (>800 g/mol) and three negatively charged sulfonate groups. Development of new synthetic methods (1,2) allowed the preparation of a library of more drug-like analogs of RB-2 with modifications in the 2- and 4-position of the anthraquinone core. Recently we developed a simple method to obtain 1-deamino derivatives of RB-2 analogs. The library was screened at various P2Y and P2X receptor subtypes and as inhibitors of ectonucleotidases. Careful analysis of structure-activity relationships at each target and systematic optimization led to new biological tools for several targets (3–7): for example, (i) we developed very potent and selective P2Y12 receptor antagonists (2,6,7); (ii) furthermore, the first potent and selective P2X2 receptor antagonists were obtained (collaboration with R. Hausmann and G. Schmalzing), and (iii) potent and selective inhibitors of ecto-5′-nucleotidase were prepared (3). (1) Baqi Y, Müller CE. Org. Lett. 2007, 9, 1271–1274. (2) Baqi Y, Müller CE. Nat. Protoc. 2010, in press. (3) Baqi Y, Lee SY, Iqbal J, Ripphausen P, Lehr A, Scheiff AB, Zimmermann H, Bajorath J, Müller CE. J. Med. Chem. 2010, 53, 2076–2086. (4) Baqi Y, Weyler S, Iqbal J, Zimmermann H, Müller CE. Purinergic Signal. 2009, 5, 91–106. (5) Weyler S, Baqi Y, Hillmann P, Kaulich M, Hunder AM, Müller IA, Müller CE. Bioorg. Med. Chem. Lett. 2008, 18, 223–227. (6) Baqi Y, Atzler K, Köse M, Glänzel M, Müller CE. J. Med. Chem. 2009, 52, 3784–3793. (7) Hoffmann K, Baqi Y, Morena MS, Glänzel M, Müller CE, von Kügelgen I. J. Pharmacol. Exp. Ther. 2009, 331, 648–655.

S1-2

Synthesis of biologically relevant nucleosides

Sergio Castillón, Maribel Matheu, Yolanda Díaz, Patricia Marcé, David Benito, Fernando Bravo

University Rovira i Virgili, Spain

The study of the biological activity of nucleosides has been a fundamental field of research since the half of the XXth. In this period many nucleoside analogues modified in the carbohydrate moiety or in the pyrimidine and purine bases have been prepared and it have been shown to possess antiviral, antimetabolic, and antibacterial properties. The intense search for clinically useful nucleoside derivatives has resulted in a wealth of new approaches for their synthesis. The main groups of bioactive, modified nucleosides which exhibited a wide range of activities have received great attention from scientists. Our group has worked for more than ten years in developing methodology for synthesising nucleosides with relevant biological properties, such as acyclic nucleosides, possessing acyclic chains instead of a ribose ring, L-derivatives, the counterparts of naturally occurring D-nucleosides, and carbocyclic nucleosides possessing a carbocyclic ring instead of a ribose ring. Other useful modifications include introduction of fluorine atom(s) into the molecule, 2′,3′-dideoxynucleosides, isonucleosides and even more singular structures such as psiconucleosides or disaccharidic nucleosides. The more relevant examples of these families of compounds will be presented, focusing especially in the control of the stereoselectivity in the glycosylation reaction for accessing 2′,3′-dideoxynucleosides, and in methods allowing to prepare both D- and L-nucleosides.

S1-3

New P2X3 receptor ligands useful for pain control

Gloria Cristalli, Rosaria Volpini

Medicinal Chemistry Unit, School of Pharmacy, University of Camerino, Italy

The ionotropic P2X3 receptors, that belong to the large family of purinergic receptors, are integral membrane proteins expressed by sensory ganglion neurons to sense painful stimuli and their activation is believed to be involved in a number of chronic pain conditions including neuropathic pain and migraine. Recently, a series of acyclic-nucleotides, based on the adenine skeleton and bearing in 9-position a phosphorylated four carbon chain, has been synthesized and evaluated for their biological activity on rat P2X3 receptors. The results indicate that they are endowed with modest partial agonism on P2X3 receptors.1 This is an interesting property, as the new acyclic-nucleotides are able to persistently block, by desensitization, P2X3 receptor activity after a brief, modest activation, yet leaving the ability of sensory neurons to mediate responses to standard painful stimuli via a lower level of signalling. Furthermore, new 2,6-diaminopurine derivatives have been designed and synthesized, by utilizing poly-substituted benzyl groups as substituents in 9-position. Biological studies demonstrated that the new purine derivatives behave as P2X3 receptor antagonists, being able to inhibit the alpha,beta-meATP evoked current.

1) Volpini R., Mishra R. M., Kachare D. D., Dal Ben D., Lambertucci C., Antonini I., Vittori S., Marucci G., Sokolova E., Nistri A., Cristalli G. Adenine-Based acyclic nucleotides as novel P2X3 receptor ligands. J Med Chem, 52 (15), 4596–4603, 2009.

S1-4

The crystal structure of the adenosine A2A receptor—implications for drug design

Adriaan Pieter IJzerman

Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands

The adenosine class of G protein-coupled receptors mediates the important role of extracellular adenosine in many physiological processes including antagonism by caffeine, the most widely used pharmacological agent in the world. The crystal structure of the human A2A adenosine receptor in complex with a high affinity antagonist, ZM241385, was determined to 2.6 Å resolution. Four disulfide bridges in the extracellular domain combined with a subtle repacking of the transmembrane helices relative to the beta-adrenergic and rhodopsin receptor structures defines a pocket capable of binding the antagonist in an extended conformation perpendicular to the membrane plane. The binding site highlights an integral role for the extracellular loops, together with the helical core in ligand recognition by this class of GPCRs. These findings question the use of rhodopsin or any other single receptor structure in homology modeling studies. On the other hand the A2A receptor structure itself may constitute a useful starting point for structure-based drug design.

S2. Purines and pain

S2-1

The function of microglial P2 purinergic receptors in neuropathic pain

Kazuhide Inoue

Department of Molecular and System Pharmacology, Kyushu University, Japan

Neuropathic pain is a highly debilitating chronic pain that occurs after nerve injury and is resistant to currently available treatments. We have shown that activating P2X4R upregulated in spinal microglia after nerve injury contributes to neuropathic pain (Nature, 2003), and the stimulation of P2X4Rs causes release of brain-derived neurotrophic factor (BDNF) evoking a collapse of their transmembrane anion gradient and the subsequent neuronal hyper-excitability (Nature, 2005). These receptors are expressed in activated microglia in response to peripheral nerve injury, but the mechanisms underlying activation of microglia following nerve injury remains to be determined. We recently found that a single intrathecal administration of IFN-γ to normal animals produces a long-lasting tactile allodynic behavior and activation of microglia in the spinal cord (PNAS 2009). The expression of IFN-γ receptor mRNA in the spinal cord is localized predominantly in microglia. IFN-γ evoked P2X4 up-regulation through Lin-kinase activation in microglia. Furthermore, IFN-γ receptor-deficient mice (ifngr-/-) exhibited a striking reduction in nerve injury-induced tactile allodynia and activation of microglia. These findings suggest that IFN-γ signaling through IFN-γR plays as a crucial trigger of microglia activation after nerve injury and the subsequent tactile allodynia through P2X4 stimulation.

S2-2

ATP-mediated signalling in trigeminal neurons of an animal model of migraine

Elsa Fabbretti

University of Nova Gorica, Slovenia

Migraine attacks are characterised by persistent pain that originates from sensitisation of nerve terminals of trigeminal ganglion neurons that innervate meninges. Current theories propose that endogenous substances like Calcitonin gene-related peptide (CGRP) and Nerve growth factor (NGF) released during a migraine attack from the trigeminal-vascular system sensitize trigeminal neurons to transmit nociceptive signals to the brainstem. Recent studies indicate that neuronal sensitization might occur via increased function of ATP-gated P2X3 receptors, important in chronic pain. While CGRP induces an up-regulation of P2X3 receptor function by enhancing gene transcription and trafficking, NGF leads to transient receptor activation. Furthermore, we have shown that P2X3 receptors are controlled by tyrosine kinase Csk that exerts a new, important negative modulation on sensory neuron excitability. Using a genetic animal model of migraine pain, namely CACNA1A R192Q KI mice, we have demonstrated that KI trigeminal ganglion neurons display stronger trigeminal responses induced by P2X3 receptor activation. The proposed mechanism is an altered balance between kinase/phosphatase activities, linked especially to CaMKII and calcineurin: the consequence is decreased serine phosphorylation of P2X3 receptors in KI ganglia with functional upregulation in transducing trigeminal pain. Globally, these data show that diverse intracellular elements might affect sensitization of P2X3 receptor function, and how this process highly depends on the integrated action of multiple cellular pathways. Our findings imply that combinatorial strategies to inhibit a chronic pain attack might be the most efficient approaches. Supported by Telethon grant GGP07032, IIT and ARRS grant J3-2376-1540.

S2-3

Peripheral sensitization of nociceptors via P2Y2 mediated potentiation of mechanically activated currents

Stefan Lechner, Gary Lewin

Max-Delbrueck-Centre for Molecular Medicine, Max-Delbrueck-Centre for Molecular Medicine, Germany

Mechanical stimuli impinging on our skin are thought to be converted into electrical signals by mechanically gated ion channels located at the peripheral nerve endings of dorsal root ganglion (DRG) neurons. Under inflammatory conditions sensory neurons are commonly sensitized to mechanical stimuli, so that normally innocuous stimuli become painful (allodynia) and painful stimuli become more painful (hyperalgesia). Here we show that in addition to the well described central mechanisms known to underlie these phenomena, peripheral mechanisms may also play an important role. The algogens UTP and ATP potentiate mechanically activated RA- and IA-currents, which are required for normal mechanotransduction, in peptidergic DRG neurons and consequently also reduce mechanical thresholds for action potential firing in these neurons. Pharmacological characterization strongly suggests that this effect is mediated by the Gq-coupled P2Y2 nucleotide receptor. Moreover, using the in-vitro skin nerve technique, we show that UTP increases the action potential firing rate in response to mechanical stimuli in a subpopulation skin c-fiber nociceptors. Together these findings suggest that UTP sensitizes c-fiber nociceptors via a previously undescribed G-protein dependent potentiation of mechanically activated RA-type currents.

S2-4

Purinergic signaling in neuropathic pain hypersensitivity

Michael Salter

Program in Neurosciences & Mental Health, Hospital for Sick Children, Canada

Increasingly, microglia in the dorsal horn of the spinal cord are recognized as crucial in the pathogenesis of pain hypersensitivity following injury to a peripheral nerve through activation of the P2X4 purinoceptor (Tsuda et al, Nature 424:778–783, 2003; Tsuda et al, TINS 28:101–107, 2005). PNI leads to increased intracellular chloride level and disinhibition of nociceptive neurons in lamina I of the dorsal horn (Coull et al, Nature 424:938–942, 2003). This increase in chloride is due to brain-derived neurotrophic factor (BDNF), released upon P2X4 receptor stimulation from spinal microglia, acting via TrkB receptors on the lamina I neurons (Coull et al, Nature 438:1017–1021, 2005). We have recently identified a unifying mechanism for P2X4 receptor-stimulated BDNF release through an increase in Ca2+ and activation of p38-MAPK leading to the synthesis and SNARE-mediated exocytotic release of BDNF from microglia. Targeting this release pathway may open new therapeutic opportunities for neuropathic pain. Supported by the Canadian Institutes of Health Research, Neuroscience Canada Foundation, and the Howard Hughes Medical Institute.

S3. Caffeine health and behaviour

S3-1

Caffeine effects on cardiovascular and endocrine function

William Lovallo

Department of Psychiatry and Behavioral Sciences, University of Oklahoma Health Sciences Center, USA

Background: Caffeine acts on the central nervous system and cardiovascular system by blocking adenosine receptors. This action impairs adenosine’s ability to regulate central neural activation and cardiovascular function, particularly sympathetic nervous system actions at the blood vessel wall. As a result, caffeine in dietary doses is capable of enhancing stress hormone secretion and particularly in raising blood pressure. Methods: We have studied the effect of caffeine (3.3 mg/kg) in normotensive persons and persons with a family history of caffeine and mildly elevated blood pressures (greater than or equal to 125/80 mmHg) at rest and under mental stress. These basic actions of caffeine make it interesting to examine caffeine’s effects in persons who are at risk for developing hypertension because of a positive family history or mildly elevated blood pressures. Results: Persons at risk for hypertension display enhanced activation of the cortisol response when exposed to a novel experimental environment, indicating increased central nervous system reactivity, and they have exaggerated responses to caffeine in combination with behavioral stress. The increased reactivity to caffeine in persons at risk for hypertension is not subject to complete tolerance even following daily intake. Conclusion: This work suggests that caffeine intake should be restricted in persons who are at risk for developing hypertension.

S3-2

The effects of caffeine on insulin actions in skeletal muscle

Terence Graham

human health and nutr sci, University of Guelph, Canada

When considering methylxanthines and human health, it must be recognized that in many countries most caffeine is consumed as coffee. This is further confounded by the fact that coffee contains many bioactive substances in addition to caffeine; it is rich in phenols (quinides, chlorogenic acid, and lactones) and also has diterpenes (fatty acid esters), potassium, niacin, and magnesium and the vitamin B3 precursor, trigonelline. There is a paradox as consumption of either caffeine or caffeinated coffee results in a marked insulin resistance and yet habitual coffee consumption has been repeatedly been reported to markedly reduce the risk for type 2 diabetes. There is strong evidence that caffeine reduces insulin sensitivity in skeletal muscle and this may be due to a combination of direct antagonism of A1 receptors by caffeine and indirectly beta-adrenergic stimulation as a result of increased sympathetic activity due to caffeine. Caffeine may also induce reduced hepatic glucose output. The responses to caffeinated coffee are very similar. Consumption of caffeine in other beverages, has not been investigated in any detail. Studies in which coffee extracts or decaffeinated coffee have been administered support that diabetics could benefit from this beverage, but more evidence is required before this should be recommended.

S3-3

Caffeine effects on aversive and non aversive memories

Lisiane Porciúncula

Department of Biochemistry, Health and Basic Sciences Institute, Federal University of Rio Grande do Sul, Brasil

The research about the habitual consumption of caffeine has deserved much attention, mainly due to its effects in ameliorating cognitive performance. In rodents, the inhibitory avoidance (IA) task is widely used for studies on the effects of substances in different stages of memory formation. In the IA the nature of stimulus (footshock) allows animals to learn to avoid the footshock after only one training trial. The object recognition task (ORT) deals with the natural motivation of the animals to explore novelties, an innate instinct that drives the animals to learn about their environment. Caffeine was described to impair learning phase in the IA task, but it improved consolidation and retrieval of memory. In the ORT, caffeine improved adult mice recognition memory and prevented aged-cognitive decline. The cholinergic system seems to be involved in the benefits of caffeine on cognition since caffeine was able to prevent scopolamine-induced amnesia in the IA and OR tasks. Besides, caffeine also prevented mnemonic deficits in the ORT in rats submitted to intracerebroventricular infusion of streptozotocin which is a considered a model of sporadic dementia. Supported by CAPES, INCTEN/CNPq, PRONEX/FAPERGS.

S4. P2X7 receptors: neuronal vs glial

S4-1

Physiological role of P2X7 in neurones

M.Teresa Miras-Portugal, Miguel Diaz-Hernandez, Javier Gualix, Rosa Gomez-Villafuertes, Juan Ignacio Diaz-Hernandez, Felipe Ortega, Jesús Sanchez-Nogueiro, Elvira Salas, Esmerilda Delicado, Maria Diaz-Zaera, Miriam Leon-Otegui, Raquel Perez-Sen

Dep. Bioquimica, Facultad Veterinaria, Universidad Complutense de Madrid, Spain

The ionotropic P2X7 receptor is very abundant in neurones, where it plays relevant physiological roles depending on the type and developmental state of the neuron. 1) P2X7 is present in almost half of the isolated synaptic terminals from mature brain where it induces calcium entrance as measured by microfluorimetry in single terminals, and neurotransmitter release measured in synaptic populations. 2) P2X7 in long term cultures of granule neurons colocalizes with the presynaptic marker synaptophysin, inducing calcium entrance followed by glutamate release upon stimulation. At the same time activation of signalling cascades proceeds, inducing cytoskeleton reorganization and neuroprotection through glycogen-syntase-kinase-3 (GSK-3) inhibition. 3) P2X7 plays a leading role controlling axonal growth. In a well established model to study axonal growth cone development as it is the culture of embryonic hippocampal neurons. ATP induces Ca2+ transients in the distal domain of the axon and the concomitant inhibition of axonal growth through P2X7 signalling. Pharmacological inhibition or shRNA silencing of P2X7 receptors induces longer and more branched axons in cultured hippocampal neurons, thus demonstrating that ATP exerts a negative influence on axonal growth through P2X7. Our data suggest that these morphological changes are induced by a signalling cascade in which CaMKII and FAK activity activates PI3-kinase and modifies the activity of its downstream targets. 4) P2X7 control on axonal development can be reproduced in the N2a neuroblastoma model, thus facilitating easier pharmacological approaches to axonal growth and branching pathologies.

S4-2

Functional P2X7 receptors of rodent astrocytes

Peter Illes

Department of Pharmacology, University of Leipzig, Germany

In primary cell cultures of the rat cerebral cortex, astrocytes reacted to the application of ATP or dibenzoyl-ATP (Bz-ATP) with inward current at a holding potential of −80 mV. These current responses considerably increased in an external medium containing no Mg2+ and low Ca2+ concentrations. The use of various receptor-type selective agonists and antagonists suggested the presence of P2X7 receptors. In brain slices of the rat prefrontal cortex, astrocytes were identified on the basis of their small cell diameters, and the absence of action potentials in response to depolarizing current injection. Immunohistochemistry experiments revealed that astrocytes labelled after recording with pipettes filled with the fluorescent dye Lucifer Yellow (LY), exhibited co-localization of LY with the astrocytic markers S100 and GFAP. In addition, P2X7 receptors were also present on the S100- and GFAP-immunoreactive (IR) astrocytes, but not on GSA-B4-IR microglia. All astrocytes reacted to NMDA, ATP, and BzATP at a holding potential of −80 mV. Once again, the current responses to ATP and Bz-ATP were greatly potentiated in a divalent cation-poor medium. The agonist and antagonist profiles of the P2 receptor-ligands investigated equally suggested the existence of P2X7 receptors. Currents through these receptor-channels reversed polarity near 0 mV. There was no response to ATP or BzATP in P2X7−/− mice, when compared with the respective background animals. Hence, electrophysiologically and immunohistochemically identified rodent astrocytes both in cell culture and brain slice preparations exhibit P2X7 receptors. These receptors may be involved in immunological defence reactions, apoptosis/necrosis as well as long-term proliferation such as gliosis.

S4-3

P2X7 receptors trigger the exocytotic release of ATP and modify secretory vesicle dynamics in neuro-2A neuroblastoma cells

Antonio R. Artalejo1, Yolanda Gutiérrez-Martín1, Diego Bustillo-Merino1, Rosa Gómez-Villafuertes2, Luis Miguel Gutiérrez3, Mª Teresa Miras-Portugal2

1Dept. Toxicology and Pharmacology, School of Veterinary Sciences, Universidad Complutense, Spain,2Dept. Biochemistry and Molecular Biology, School of Veterinary Sciences, Universidad Complutense, Spain,3Instituto de Neurociencias, Universidad Miguel Hernández, Spain

Extracellular ATP acting on ionotropic P2X receptors is involved in the control of brain development, maturation, and repair. Previously, we have reported that P2X7 receptors negatively regulate neurite formation in Neuro-2a (N2a) mouse neuroblastoma cells through a Ca2+/calmodulin-dependent kinase II-related mechanism. In the present study, we have used this cell line to investigate a parallel though faster P2X7 receptor-mediated signaling pathway, namely Ca2+-regulated exocytosis. Selective activation of P2X7 receptors evoked exocytosis as assayed by high-resolution membrane capacitance measurements. Using dual-wavelength total internal reflection microscopy we have observed both the increase in near-membrane Ca2+ concentration and the exocytosis of fluorescently labeled vesicles in response to P2X7 receptor stimulation. Moreover, activation of P2X7 receptors also affects vesicle motion in the vertical and horizontal directions, thus involving this receptor subtype in the control of early steps—docking and priming—of the secretory pathway. Immunocytochemical and RT-PCR experiments evidenced that N2a cells express the three neuronal SNAREs as well as nucleotide and monoamine, VMAT-1, VMAT-2, vesicular transporters. Finally, P2X7 receptor stimulation and ionomycin increased the incidence of small transient inward currents (STICs), reminiscent of postynaptic quantal events observed at synapses. STICs were dependent on extracellular Ca2+ and were abolished by brilliant blue G, suggesting they were mediated by P2X7 receptors. Altogether, these results suggest the existence of a positive feedback mechanism mediated by P2X7 receptor-stimulated exocytotic release of ATP that would act on P2X7 receptors on the same or neighbor cells to further stimulate its own release and negatively control N2a cell differentiation.

S5. Purine salvage mechanisms

S5-1

Insights into mechanisms of nucleoside transport, from bacteria to humans

Stephen Allan Baldwin

University of Leeds, United Kingdom

In mammals, nucleoside transporters play key roles in cellular uptake of nucleosides for salvage pathways of nucleotide synthesis, in the regulation of extracellular adenosine concentrations, and in the uptake of anti-cancer and anti-viral nucleoside drugs. Understanding the mechanisms by which these molecules get taken up into cells is therefore both of biological and of clinical interest. Two major families of mammalian transporters are involved in such uptake, the equilibrative nucleoside transporters (ENTs) and the concentrative nucleoside transporters (CNTs). To understand their mechanisms we have employed a combined approach of expression, site-directed mutagenesis and functional assays on human transporters expressed in Xenopus oocytes, together with investigation of their bacterial counterparts, which are more amenable to structural studies. Cysteine-scanning mutagenesis has in particular identified residues likely to be involved in substrate recognition and/or translocation, and in transport-associated conformational changes, in both the mammalian ENT and CNT families. Combined with X-ray crystallographic and solid-state NMR investigations on NupC from Escherichia coli, and other bacterial homologues, the mechanisms of nucleoside transport, while still not fully understood at the molecular level, are becoming clearer.

S5-2

Purine nucleoside and nucleobase transporters in Leishmania parasites

Scott M. Landfear

Department of Molecular Microbiology and Immunology, Oregon Health & Science University, USA

Parasitic protozoa such as Leishmania, that cause important human diseases, are unable to synthesize purines de novo and are completely reliant upon uptake of preformed purines from their hosts, thus providing a potential for novel therapeutic interventions. In Leishmania parasites purines are imported through two nucleoside transporters, NT1 and NT2, and two nucleobase transporters, NT3 and NT4, all members of the SLC29 family of permeases. Extensive site-directed mutagenesis of the NT1 adenosine transporter has revealed multiple residues that play critical roles in transport function. An ab initio computational model of NT1 predicts a structure with similarity to Major Facilitative Superfamily members such as the lactose permease from E. coli. This model fits well with mutagenesis results and predicts that specific residues within several helices serve gating functions that allow the permease to take up its alternate inward-facing and outward-facing conformations. Mutagenesis supports the identification of several of these amino acids as gating residues. Hence, this computational model provides a foundation for further structure-function studies on SLC29 family members.

NT4 is a purine nucleobase transporter that is activated at acid pH. Since the parasites reside within acidified phagolysosomal vesicles of host macrophages during the mammalian stage of their life cycle, this property of NT4 suggests that it may play a particularly central role in purine salvage during infection of the mammalian host. Indeed infections of mice with the [increment]nt4 null mutant of Leishmania major revealed that these mutant parasites were strongly compromised in their ability to form lesions. Null mutants in the NT3 purine nucleobase transporter that functions optimally at neutral pH are less strongly compromised in virulence. However, the inability to generate the double null mutant, [increment]nt3[increment]nt4, indicates that these parasites are reliant upon at least one intact nucleobase transporter for viability.

S5-3

Molecular modelling of substrate recognition at an unusual aminopurine transporter

Catharine Collar1, Mohammed Al Salabi2, Mhairi Stewart2, Michael Barrett2, David Wilson1, Hendrik De Koning2

1Department of Chemistry, Georgia State University, USA,2Institute of Biomedical and Life Sciences, University of Glasgow, United Kingdom

Background: The P2 aminopurine transporter of Trypanosoma brucei displays a very unusual selectivity profile, with similar affinity for adenine, adenosine and several classes of trypanocidal agents. The basis for this phenomenon was investigated. Methods: A large dataset of inhibition constants for adenosine transport by the T. brucei P2 transporter was generated for a highly varied library of 112 potential substrates. Computational biology, specifically Comparative Molecular Field Analysis (CoMFA) and Comparative Molecular Similarity Indices Analysis (CoMSIA) has been employed to investigate compound structure diversity in relation to P2 transporter interaction. Results: Optimised 3D structures were generated for the entire library, and aligned so as to generate a model for optimised interaction with the transporter. This generated a predictive model with correlation coefficients of 0.95 and 0.86 for CoMFA and CoMSIA, respectively, between experimental and predicted values. Contour maps explain the steric and charged interactions between transporter and specific substrates were generated. The main motifs determining high affinity were identified. Conclusion: A new method was developed to study the interactions between purines and transporters, based on computational analysis of competitive inhibition data. This method is equally applicable to receptors and does not require the purification of the protein, or knowledge of its structure.

S5-4

ATP storage and uptake in isolated pancreatic zymogen granules

Kristian Agmund Haanes, Ivana Novak

Department of Biology, University of Copenhagen, Denmark

Imaging studies at our lab have shown that pancreatic acinar cells store ATP in granules. Present study aimed to quantify the ATP content directly and characterize ATP uptake into Zymogen Granules (ZG). ZGs where isolated using a method by Chen et al (2006). ATP content was measured using Luciferin/Luciferase and related to protein in the sample. Estimates of ATP concentration in freshly isolated granules vary between 40 and 120 µM of ATP (n = 6), based on the protein concentrations in ZGs. ATP uptake was characterized by incubating the granules in sucrose, MES, EGTA buffer at pH 6 containing 4 mM KCl and trypsin inhibitor. A Km of 4.9 ± 2.1 mM and a Vmax of 850 ± 250 pg/mg/30 min was calculated based on a Eadie-Hofstee plot (n = 5). DIDS and Evans blue, both at 100 µM, completely blocked ATP uptake. For DIDS an IC50 value of 9.96 ± 1.16 µM was calculated using non-linear regression. Bafilomycin, CCCP and valinomycin did not have significant effect at pH 6. There is a strong pH dependence of the ATP uptake, as uptake at pH 7.2 is more than 2-fold higher than at pH 6 (P < 0.05). In addition, at pH 7.2, CCCP had a significant inhibitory effect (P < 0.05). These experiments show the importance of the pH gradient of ATP uptake into ZGs. This study shows that ZG take up and store ATP and this can be released into the lumen for signalling in pancreatic ducts. Support: DNSRC 272-05-0420, Novo Nordisk Scholarship, Lundbeck Foundation

S6. Purinergic signaling in development and stem cell regulation (May 31-afternoon)

S6-1

Role of the P2Y-like GPR17 receptor in oligodendrogliogenesis

Mariapia Abbracchio

Dept Pharmacol Sci, University Of Milan, Italy

We recently showed that GPR17, a receptor activated by both uracil nucleotides and cysteinyl-leucotrienes (Ciana et al., EMBO J 25(19):4615-27, 2006; Temporini et al., Anal Biochem 384(1):123-9, 2009; Pugliese et al., Am J Physiol Cell Physiol. 297:C1028-40, 2009) is a key regulator of myelination. In vitro, GPR17 decorates a subset of slowly proliferating highly ramified oligodendrocyte precursor cells (OPCs) that also express the oligodendrocyte markers NG2 and Olig2 (Lecca et al., PLoS One 3(10):e3579, 1–15, 2008; Daniele et al, the present meeting). Expression of GPR17 is strictly segregated from that of myelin proteins. In adult brain, GPR17 is found on OPCs of both cortex (Lecca et al., 2008) and spinal cord (Ceruti et al., Brain 132:2206-18, 2009). In developing cortex, GPR17 is transiently upregulated at postnatal day 7 and declines in parallel with myelin maturation. In both developing and adult cortex, actively dividing OPCs never coexpress GPR17, indicating that it is induced at postmitotic stages (Boda et al., Cambridge Centre for Brain Repair Spring School, March 2010). Importantly, the in vitro manipulation of GPR17 with its ligands fosters the maturation of OPCs: conversely, GPR17 downregulation is needed to allow their terminal differentiation (Lecca et al., 2008). Accordingly, GPR17 forced over-expression inhibits OPC differentiation and GPR17 knock out mice show precocious onset of myelination (Chen et al., Nat Neurosci. 12(11):1398-406, 2009). These data suggest that GPR17 orchestrates the transition between immature and myelinating oligodendrocytes and represents a new target for pharmacological manipulation in syndromes characterized by dysfynctional myelination as well as in demyelinating diseases.

S6-2

Non-vesicular release of ATP from axons firing action potentials

Douglas Fields

NICHD, National Institutes of Health, USA

ATP released from synaptic vesicles regulates a wide range of nervous system functions; however, non-vesicular release of ATP from neurons could implicate purinergic signaling in biological processes beyond the synapse and in association with more diverse functions. This could include interactions with glial cells forming the myelin insulation on axons (Schwann cells and oligodendrocytes). Single photon imaging was used to study the release of ATP from mouse dorsal root ganglion neurons in cell culture in response to firing trains of action potentials. The release of ATP occurred in the absence of synapses and under conditions preventing synaptic vesicle fusion, and in the presence of inhibitors of gap junctions. Together with intrinsic optical signaling, live cell imaging, and pharmacological studies, these results show that ATP can be released from DRG axons through specific ion channels in response to action potentials. Previous research has shown that ATP and adenosine released from axons firing action potentials can regulate myelination by Schwann cells and Oligodendrocytes. Regulation of myelination by impulse activity could contribute to nervous system development and plasticity, and this non-vesicular, non-synaptic release of ATP could also participate in signaling between axons and other glial cells, neurons, and vasculature.

S6-3

A role for the A2b adenosine receptor in mesenchymal stem cell differentiation into bone cells

Shannon Carroll H.1, Nathan A. Wigner2, Nitin Kulkarni2, Louis Gerstenfeld2, Katya Ravid3

1Biochemistry, Boston University School of Medicine, USA,2Orthopedic Surgery, Boston University School of Medicine, USA,3Medicine and Biochemistry, Boston University School of Medicine, USA

Background: The A2b adenosine receptor (A2bAR) has a protective role in various models of chronic injury and in inflammation, particularly through its expression in macrophages. Bone fracture healing is a complex process composed of an initial inflammatory response followed by differentiation of mesenchymal stem cells (MSC) into chondrocytes and osteoblasts to form bone, and finally resorption by osteoclasts to remodel the structure. Methods: We examined bone marrow mesenchymal stem cell cultures, known to express the A2bAR, as well as an in vivo model of bone fracture, using control and A2bAR knockout (KO) mice. Results: We describe the role of the A2bAR in these processes and ultimately in bone fracture healing. Bone marrow cells derived from A2bAR KO mice form fewer mineralized nodules when cultured in osteo-inductive media, and have lower expression of osteoblast differentiation genes, as compared to control cells, while induction of MSC lacking the A2bAR to differentiate into osteoclast is being examined. In osteo-inductive cultures, A2bAR is principally expressed by macrophages. A2bAR KO mice display delayed fracture healing with lower expression of genes involved in osteoblast differentiation. Conclusion: These results demonstrate a role for the A2bAR in fracture healing by promoting differentiation of MSC into osteoblasts, while suppressing osteoclast resorption, potentially also through the modulation of macrophage function.

S6-4

Nucleotide signalling in adult neurogenesis

Herbert Zimmermann1, Kristine Gampe1, Ivette Grimm1, Nanette Messemer1, Simon Robson2, Matthias Stanke1

1Institute of Cell Biology and Neuroscience, Goethe-University Frankfurt, Germany,2Department of Medicine, Beth Israel Deaconess Medical Center Harvard medical School, USA

Nucleotide signalling in adult neurogenesis Herbert Zimmermann1, Kristine Gampe1, Ivette Grimm1, Nanette Messemer1, Simon C. Robson2 and Matthias Stanke1 1Institute of Cell Biology and Neuroscience, Biocenter, Goethe-University, 60438 Frankfurt, Germany; 2Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA Background: New neurons are continuously formed in two neurogenic niches of the adult mammalian brain, the subependymal layer (SEZ) of the lateral wall of the lateral ventricles and the dentate gyrus of the hippocampus. Here we analyzed the involvement of the purinergic signalling pathway in the control of adult murine neurogenesis. Methods: The functional expression of nucleotide receptors and their role in cell proliferation and migration was studied in cultured stem cells in vitro and their in vivo impact on progenitor cell proliferation and young neuron survival was analyzed using P2Y1, P2Y2 and NTPDase2 knockout mice. In addition we quantified the effect of in vitro shRNA knockdown of the ectonucleotidases NTPDase2 and tissue nonspecific alkaline phosphatase on progenitor cell proliferation and differentiation. Results: Neural progenitors in vitro express a large variety of P2 receptors and respond to nucleotides with a rapid increase in ERK, CREB, AKT and FAK phosphorylation. They stimulate cell proliferation and migration. The analysis of knockout animals further supports a role of nucleotide signalling in adult neurogenesis. Conclusion: Our results suggest that nucleotides can act as paracrine or autocrine signalling substances in adult neurogenesis and can be of relevance for proliferation, migration and differentiation of progenitors and the survival of young neurons.

S7. Therapeutic potential of purine drugs

S7-1

New frontiers for ligands of adenosine and P2Y receptors

Kenneth A. Jacobson

Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, USA

The development of selective ligands that signal through G protein-coupled purine and pyrimidine receptors has resulted in clinical trials, despite the challenges of this class of GPCRs. Novel drug delivery, prodrug approaches, allosteric modulation, and biased agonism are needed to overcome side effects. The A3 adenosine receptor (AR) agonist IB-MECA has demonstrated efficacy in clinical trials of psoriasis, dry eye, and rheumatoid arthritis by Can-Fite Biopharma. We designed novel A3AR ligands with an improved pharmacological profile containing a methanocarba ribose-ring substitution constrained in the receptor-preferred North (N) conformation, e.g. partial agonist MRS5127. We are systematically studying A3AR efficacy, species dependence, and agonism bias (cAMP vs. arrestin). While A3AR is advanced clinically, we have also introduced selective agonists and antagonists of various P2Y receptors for use as receptor probes, including uncharged or nonhydrolyzable compounds. UDP-activated P2Y6 receptor protects MIN6 pancreatic cells against apoptosis, induces microglial phagocytosis and is a target for neurodegeneration. P2Y6 requires an unusual South (S) conformation, as predicted by receptor docking and shown with enantiomerically pure (S)-methanocarba UDP, MRS2795. Other novel P2Y6 agonists (e.g. N4-MeO-cytidine derivative MRS2957) depend on nucleobase substitution. We introduced GPCR Ligand Dendrimer (GLiDe) conjugates to interact with receptor aggregates and to act as smart drugs. The attachment of GPCR functionalized congeners to tree-like polyamidoamine (PAMAM) dendrimer carriers has greatly increased selectivity and affinity (e.g. A2A, A3, and P2Y14) in novel multivalent and multifunctional probes. Fluorescent and other reporter groups incorporated into ligands can provide new assay and imaging possibilities.

S7-2

Adenosine A2A receptor and neurodegenerative disorders: a promising target and with complex cellular mechanisms as revealed by genetic knockout approaches

Jiang-Fan Chen

Department of Neurology, Boston University School of Medicine, USA

The adenosine A2A receptor (A2AR) has recently emerged as a leading non-dopaminergic therapeutic target in Parkinson’s disease (PD) and a potential target for other neurodegenerative disorders. This is primarily due to (1) clinical phase II–III trials and FDA filing of A2AR antagonists such as KW-6002 (istradefylline) in PD patients and (2) the recent convergence of epidemiological and animal studies suggesting that A2AR ligands also confer a neuroprotective effect in models of PD, stroke, Huntington’s disease, Alzheimer’s disease and traumatic brain injury (TBI). However, the mechanism underlying such a broad spectrum neuroprotection by A2AR ligands remains largely unknown. To address this issue, we have developed a series of neuron- and glial-specific A2AR KO mice and focused our analysis on the A2AR modulation of neuroinflammation. Using forebrain neuron-, microglial- and astrocyte-specific A2AR KO, we demonstrated that A2AR activity exerts motor and neuroprotective effects by distinct cellular mechanisms. Furthermore, our analysis reveals the opposite control of lipopolysaccharide-induced microglial activation by the A2AR in forebrain neurons and by the A2AR in astrocytic and microglial cells. Lastly, selective deletion of A2ARs restricted to striatal neurons revealed for the first time, multiple, selective cognition-enhancing effects, including improved spatial working memory, facilitated reversal learning and enhanced Pavlovian conditioned freezing. Thus, we propose that the striatal A2AR could represent a novel target against the cognitive inflexibility and behavioral rigidity that are characteristic of several major neurodenerative diseases.

S7-3

Treatment of sickle cell anemia with adenosine A2A receptor agonists to suppress iNKT cell activation

Kori L. Wallace1, Joshua J. Field2, Linden Joel3

1Department of Microbiology, University of Virginia, USA,2Department of Medicine, Washington University, USA,3Division of Inflammation Biology, La Jolla Institute for Allergy and Immunology, USA

Background: Ischemia-reperfusion injury (IRI) triggers an inflammatory cascade that is initiated by the activation of CD1d-restricted iNKT cells and is inhibited by activation of adenosine A2AR receptors (A2AR). In sickle cell disease (SCD), misshapen erythrocytes evoke repeated transient bouts of microvascular IRI. In patients with SCD, iNKT cells are increased in number and activated. We reasoned that interfering with iNKT cell activation would reduce SCD pathophysiology. Methods: In order to deplete or prevent activation of iNKT cells, NY1DD mice with SCD were treated with antibodies to deplete or block activation of iNKT cells, crossed with Rag1−/− mice lacing mature T cells, or treated with A2AR agonists. Results: The pulmonary dysfunction that is a hallmark of SCD in NY1DD mice was reduced by: 1) depletion of NKT cells with anti-NK1.1 antibodies; 2) blockade of iNKT TCR activation with anti-CD1d antibodies; 3) blockade of lymphoctye chemotaxis with anti-CXCR3; 4) genetic deletion of lymphocytes; or 5) infusing mice with the A2AR agonist, ATL146e for 3–7 days. ATL146e produced a dose-dependent reduction in pulmonary injury in NY1DD mice that peaked at 30 ng/kg/min. A clinical trial to determine the maximal tolerated dose of an infused A2AR agonist in patients with SCD has received FDA and IRB approval. Conclusions: iNKT cells play a key role in initiating inflammatory cascades in acute IRI and in various chronic inflammatory diseases, including SCD. Blockade of the activation of iNKTs by various means, including A2AR activation, has therapeutic potential if these approaches are well tolerated and effective in man.

S7-4

Denufosol tetrasodium, a P2Y2 receptor agonist for the treatment of Cystic Fibrosis

Jose Luis Boyer, Todd Durham, Matt Barnes, Tomas Navratil, Amy Schaberg

Durham, NC, Inspire Pharmaceuticals, USA

Background: Altered chloride and ion transport resulting from defective CFTR is the cause of Cystic Fibrosis (CF). Denufosol is an ion channel regulator that aims to correct the underlying ion transport defect in the lung and enhance mucociliary clearance by increasing chloride secretion, inhibiting sodium absorption, and stimulating ciliary beat frequency via its agonism of P2Y2 receptors, thus improving lung function. Methods: A 24-week double-blind trial (TIGER-1) was conducted in 352 CF patients to compare inhaled denufosol (60 mg TID) to placebo, followed by an open label extension (OLE) where all received denufosol for an additional 24 weeks. Mean age and mean percent predicted FEV1 was 14.6 years and 92%, respectively. Subjects received standard CF treatment except hypertonic saline. Primary efficacy endpoint was mean change FEV1. Secondary efficacy endpoints were FEF25%–75%, FVC, pulmonary exacerbations, and QOL measures. Results: Treatment with denufosol resulted in an increase in FEV1 of 48 ml compared to 3 ml for placebo at Week 24 endpoint (p = 0.047). In the OLE, patients receiving denufosol continued to improve up to 115 ml over baseline at 48 weeks. Patients who switched from placebo to denufosol in the OLE improved 78 ml in FEV1 at Week 48. Non significant trends for improvement in FEF25%–75% were observed. Denufosol was well tolerated with a growth and adverse event profile similar to placebo. Conclusions: Denufosol is an investigational treatment for CF. The ability of denufosol to improve FEV1 by addressing the underlying ion transport defect makes this a potential promising treatment for CF.

S8. Purines and sensory systems

S8-1

Nucleotides and dinucleotides as natural regulators of intraocular pressure: To see or not to see

Jesus Pintor

Dep. Bioquimica, E.U. Optica, Universidad Complutense de Madrid, Spain

Background: Nucleotides and dinucleotides are present in the aqueous humour inside the eye. These molecules do have some physiological effects within the eye which will be described. Methods: By means of animal models such as New Zealand rabbits, mice or bovine eyes, different approaches will be done to demonstrate the presence of nucleotides, their receptors and their actions. These methodological approaches cover tonometries, immunohistochemical studies, western-blotting, siRNA technologies, ion trafficking and others. Results: Nucleotides and dinucleotides are present at different concentrations in the aqueous humour of animal models as well as in humans. It is of relevance to see that in patients with the pathology termed glaucoma, which produce blindness, the levels of ATP and Ap4A are increased several folds over normal human individuals. This fact correlates well with the presence of P2Y2 receptors in the ciliary body which increase the production of aqueous humour producing an abnormal elevation of intraocular pressure, main reason for the development of glaucoma. This increase is due to the trafficking of aquaporin-1 from the cytoplasm to the cell membrane of the ciliary epithelial cells, therefore facilitating the aqueous humour production. The silencing of the P2Y2 receptor with a siRNA produces a clear return of the pressure to normal values protecting the retina from the damage and the concomitant visual loss. Conclusions: Nucleotides are responsible for the ocular health and good vision. To counteract the negative effects of high nucleotide concentrations and therefore protect the eye, antagonists and siRNA are suggested as therapeutic agents.

S8-2

Contribution of P2X receptors to hearing

Gary Housley1, Tze Ling Loh1, Yogeesan Sivakumaran1, Sherif Tadros1, Allen Ryan2

1School of Medical Sciences, University of New South Wales, Australia,2Surgery & Neuroscience, University of California, San Diego, U.S.A.

Background: P2X2 receptors are broadly expressed in the cells lining the cochlear partition, including the sensory hair cells. P2X receptors are also expressed in the spiral ganglion neurons. This expression is up-regulated with noise exposure. We have tested the hypothesis that ATP-gated ion channels assembled from P2X2 receptors contribute to control of hearing sensitivity. Methods: Wildtype and P2X2 receptor knockout mice were born into two environmental chambers: a) acoustically dampened and b) moderate level (75 dB, 1octave (8–16 kHz)) white noise. The hearing sensitivity in these mice was monitored over 25 weeks using ABR (auditory brainstem response thresholds). Acute noise exposure at a higher sound level was also utilized (95 dB, 8–16 kHz, 30 minutes). Results: Both wildtype and knockout mice in the noisy environment showed significant shifts in puretone ABR thresholds across the octave noise band, compared with the mice in the quiet chamber. However, P2X2 knockout mice exhibited significantly broader hearing loss. In the acute noise study, P2X2 knockout mice developed more permanent threshold shift (hearing loss) than the WT controls. Conclusion: Noise causes hearing loss in P2X2 knockout mice, suggesting that P2X2 receptor signalling in the cochlea is otoprotective. Supported by the National Health & Medical Research Council (Australia)

S8-3

Purinergic signaling in the olfactory system

Colleen Cosgrove Hegg1, Sebastien Hayoz1, Cuihong Jia1, Mary Lucero2

1Pharmacology and Toxicology Department, Michigan State University, USA,2Physiology, University of Utah, USA

In the olfactory epithelium, extracellular ATP is released by neurotransmission, secretion, transporters, and cell damage, activating P2X and P2Y purinergic receptors located on all cell types and evoking inward currents and increases in [Ca2+]i. Purinergics have multiple roles in the nasal olfactory epithelium which will be discussed. (1) ATP opens gap junctions between the glial like sustentacular cells, suggesting a role in intercellular communication. (2) Extracellular ATP modulates sensitivity of neurons to odorants at the level of the olfactory epithelium, thereby contradicting a longstanding dogma that modulation was at the level of the olfactory bulb. (3) Extracellular ATP induces the expression of heat shock proteins via purinergic receptor activation, suggesting a role in stress signaling. (4) Extracellular ATP induces the synthesis and release of multiple growth and trophic factors, including ATP, neuropeptide Y and fibroblast growth factor 2, suggesting a role for the regulation of cell death and survival. (5) ATP increases proliferation of basal progenitor cells via activation of purinergic receptors and subsequent release of growth factors. (6) ATP reduces apoptosis. Collectively, these data suggest purinergic signaling in the olfactory epithelium has broad roles in sensory transduction, initiation of cell turnover and the promotion of neuronal survival.

S8-4

Purinergic signalling in taste

Thomas Finger

Rocky Mountain Taste & Smell Center, Univ. Colorado Medical Sch., USA

In 1999, Bo & Burnstock described the presence of P2X2 and P2X3 receptors on nerve fibers innervating taste buds. It was not until 6 years later that we presented evidence for the crucial role of purinergic signaling in the transmission of signals from taste bud receptor cells to the afferent nerve fibers. In this review, I will discuss recent data from a variety of laboratories showing tastant-induced release of ATP from specific taste receptor cells occurs via a non-vesicular release mechanism that results in activation of the adjacent sensory nerve fibers via P2X2/P2X3 receptors. Mice lacking these receptors exhibit no taste-related activity in the gustatory nerves, yet still retain some food-related learning behaviors mediated by the trigeminal, olfactory, and digestive systems. The ATP signal released from the taste receptor cells apparently activates the nerve fibers but is also degraded by the ectoATPase, NTPDase2, produced by other cells of the taste bud. The resulting ADP and adenosine then may activate P2Y and A2B receptors in the taste buds to modulate further action of the receptor cells. Thus taste buds can be viewed as complex integrative sensory endorgans rather than simple transducers directly converting taste stimuli to a direct neural signal.

S9. Purinergic receptor-receptor interactions

S9-1

Adenosine receptors containing oligomers: relevance for adenosine function

Francisco Ciruela

Departament de Patología i Terapèutica Experimental, Facultat de Medicina-Bellvitge, IDIBELL-Universitat de Barcelona, Spain

Although the G protein-coupled receptor (GPCR) oligomerization has been questioned during the last fifteen years, under some premises the existence of a supramolecular organization of these receptors begins now to be widely accepted by the scientific community. Adenosine receptors are GPCRs that mediate the physiological functions of adenosine and indeed these receptors do also form homo-and heteromers. Thus, adenosine receptors oligomers may enhance the diversity and performance by which extracellular adenosine signal is transferred to the G proteins in the process of receptor transduction. Consequently, these oligomers allow a fine-tuning modulation of adenosinergic function in both normal and pathological conditions. Overall, we underscore here recent significant developments based on adenosine receptors oligomerization that are essential for garnering a better understanding of adenosine function.

S9-2

Receptor heteromerization determines the striatal pre- and postsynaptic profile of adenosine A2A receptor antagonists

Sergi Ferré Gonzalez

NIDA, IRP, NIH, DHHS, National Institute on Drug Abuse, IRP, NIH, DHHS, USA

Receptor heteromer is defined as a macromolecular complex composed of at least two receptor units with biochemical properties demonstrably different from those of its individual components. Striatal adenosine A2A receptors are mostly localized postsynaptically, co-localized with dopamine D2 receptors in the GABAergic enkephalinergic efferent neurons, where they form A2A-D2 receptor heteromers. But striatal A2A receptors are also localized presynaptically, preferentially localized in glutamatergic terminals of cortico-striatal afferents to the GABAergic substance P-dynorphinergic efferent neurons, where they form heteromers with adenosine A1 receptors. Selective striatal postsynaptic A2A receptors should be useful in Parkinson’s disease, while selective striatal presynaptic receptors could be beneficial in dyskinetic disorders, in obsessive-compulsive disorders and drug addiction. We looked for the existence of selective pre- or postsynaptic A2A receptor blocking properties of several A2A receptor antagonists, based on possible differential affinities that A2A receptors could have for those compounds when forming heteromers with either A1 or D2 receptors. Two compounds, SCH-442416 and KW-6002, showed a preferential in vivo profile of striatal pre- and postsynaptic A2A receptor antagonists, respectively. Results obtained with radioligand-binding experiments in cells co-transfected with A2A and D2 or A1 receptors supported a key role of heteromerization in some of the differences observed in vivo. In particular, SCH-442416 was the only screened compound which affinity for the A2A receptor was very significantly decreased upon co-transfection with D2, but not A1 receptor. Receptor heteromers, therefore have to be taken into account when considering screening of compounds binding to G protein-coupled receptors.

S9-3

Complex regulation of CB1-mediated effects by activation and blockade of striatal adenosine A2A receptors

M. Teresa Tebano1, Alberto Martire1, Attila Kofalvi2, Valentina Chiodi1, Antonella Ferrante1, M. Rosaria Domenici1, Samira Cardoso2, Rodrigo Cunha2, Patrizia Popoli1

1Dept. Of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Italy,2Center for Neurosciences and Cell Biology of Coimbra, University of Coimbra, Portugal

Adenosine A2A and cannabinoid CB1 receptors form heteromers and functionally interact in the striatum. Our aim was to investigate by which mechanisms these receptors interact in regulating striatal synaptic transmission.

In extracellular recordings from rat corticostriatal slices (300 µm), the application of the cannabinoid receptor agonist WIN55,212-2 (2 µM) reduced significantly the amplitude of the FP (−45.4 %, p < 0.05 vs basal), an effect which was prevented by the CB1R-antagonist, AM251 (2 µM). Besides the recently reported antagonism elicited by A2AR blockade (Tebano et al., J Neurochem., 110:1921, 2009), also A2AR activation by CGS21680 (100 nM) abrogated CB1-induced effects (−16% FP amplitude, P < 0.05 vs WIN55,212-2 alone). Since CGS21680 prevented WIN-induced increase in paired-pulse facilitation, a presynaptic site of this antagonistic A2A/CB1 interaction was hypothesized. In agreement, in isolated striatal nerve terminal population, CGS21680 (30 nM) inhibited WIN-mediated inhibition of depolarization-induced glutamate release (P < 0.05). Furthermore, immunocytochemical analysis showed that A2ARs preferentially co-localize with CB1Rs in a subset of striatal glutamatergic nerve terminals. Finally, the antagonistic interaction occurring between presynaptic A2A and A1Rs could play a role in the inhibitory effects of CGS21680, since also the A1R, NGR-hTNF antagonist DPCPX (500 nM) significantly attenuated synaptic WIN effects (−12% FP amplitude, P < 0.05 vs WIN55,212-2 alone).

These data indicate that A2ARs may not only enable, but also inhibit CB1 effects in the striatum. Whether these two opposite effects depend on different sub-synaptic sites of interaction, or whether A2ARs forming and not forming heteromers with CB1Rs may differently modulate CB1-dependent effects has to be further explored.

Abs. id: 75

S9-4

Dopamine D2 and adenosine A2A receptors regulate activity of striatopallidal neurons through A2A-D2 receptor heteromerization

Serge Schiffmann1, David Gall1, Sergi Ferré2, Karima Azdad1

1Laboratory of Neurophysiology, Université Libre de Bruxelles, Belgium,2Behavioral Neuroscience Branch, National Institute on Drug Abuse, National Institutes of Health, USA

Electrical activity of striatal medium spiny neurons including membrane potential oscillations between a down- to an upstate could be regulated by G-protein-coupled receptors. Dopamine D2 and adenosine A2A receptors are highly enriched in striatal neurons and are known to exhibit strong interactions whose physiological significance and molecular mechanisms remain partially unclear. In this line, the respective involvement of intracellular signalling cascades commonly and antagonistically targeted by these receptors and A2A-D2 receptor heteromerization remained to be elucidated. By using perforated patch clamp recordings on brain slices and loading of competitive peptides, we showed that D2 and A2A receptors regulate the NMDA-induced transition to depolarized membrane potential plateau through mechanisms relying upon specific protein-protein interactions. Indeed, D2 receptor activation abolished transitions between a hyperpolarized resting potential to a depolarized plateau potential by regulating the CaV1.3a calcium channel activity through interactions with scaffold proteins Shank1/3. Noticeably, A2A receptor activation had no effect per se but fully reversed the effects of D2 receptor activation. This A2A receptor-induced reversal was prevented by specific competitive peptides that block A2A-D2 receptors heteromerization showing that this heteromerization is strictly mandatory. In summary, not only these studies demonstrate that membrane potential transitions and firing patterns in striatal neurons are tightly and antagonistically controlled by D2 and A2A receptors but they also provide a first direct physiological relevance of A2A-D2 receptors heteromerization in striatopallidal neurons.

S10. Purinergic signaling in brain disorders

S10-1

Revisiting A2A receptors and hippocampal excitability: implications for brain dysfunction

Ana Sebastião, Sofia Cristovão-Ferreira, Raquel A Dias, Sandra H Vaz

Institute of Pharmacology and Neurosciences and Unit of Neurosciences, Faculty of Medicine and Institute of Molecular Medicine, University of Lisbon, Portugal

Excitatory actions of adenosine A2A receptors (A2Rs) in the hippocampus have been reported long ago (Sebastião, Ribeiro,1992–Neurosci.Letts,138,41), but their scarce density in the hippocampus (Cunha et al.,1994-Brain Res,649,208) soon raised the hypothesis that the main design of these receptors is fine tuning synapses rather than exerting a direct influence in synaptic transmission (Sebastião, Ribeiro,2000-TIPS,21,341). Recent data shows that the influence of A2Rs upon excitability extends to the tripartite synapse, including modulation of neurotransmitter transport in nerve endings and astrocytes as well as facilitation of extrasynaptic responses to neurotransmitters. The life-span of the predominant inhibitory neurotransmitter in the central nervous system, GABA, is determined by its uptake into nerve endings and astrocytes, through high affinity GABA transporters (GATs). GATs can be regulated by phosphorylation and it is now known that GABA transport is facilitated by A2ARs in nerve endings (Cristovão et al.,2009-J Neurochem 109,336) and astrocytes. A2ARs also facilitate transport of adenosine into nerve endings, limiting its availability to activate inhibitory adenosine A1 receptors (Pinto-Duarte et al.,2005-J. Neurochem 93,595). In what concerns excitatory transmission, A2ARs enhance the availability of membrane located extrasynaptic AMPA receptors, with consequences for synaptic transmission and plasticity. In summary, by enhancing the shut down of a major inhibitory neurotransmitter, GABA, and of a major inhibitory neuromodulator, adenosine, as well as by reinforcing glutamatergic AMPA receptor-mediated responses, A2ARs exacerbate the excitatory tonus, which may aggravate dysfunction in case of over-excitability. Work supported by FCT (Portugal) and EU (Cost B30)

S10-2

Adenosine A2A antagonists are protective in in vivo and in vitro models of brain ischemia

Felicita Pedata, Alessia Melani, Sara Cipriani, Anna Maria Pugliese, Chiara Traini, Francesca Corti, Maria Grazia Giovannini

Department of Pharmacology, University of Florence, Italy

During ischemia adenosine increases in the extracellular milieu. Adenosine A2A receptor expression increases on neurons and microglial cells of striatum and cortex after focal ischemia induced by medial cerebral artery occlusion (MCAo). In this model in the rat, the selective A2A antagonist, 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazole-[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine (SCH58261) (administered 0.01 mg/Kg i.p. 5 min after ischemia) reduces glutamate outflow in the first hrs after ischemia. SCH58261 (subchronically administered after ischemia) protects from neurological deficit, cortical and striatal damage and from myelin disorganization assessed by MAG staining 24 hrs after ischemia. 24 hrs after MCAo, microglia is reactive and oligodendrocytes are clearly stained by OLIG2 antibodies. The A2A antagonist significantly reduces activation of p38 mitogen-activated protein kinase (MAPK) in microglial cells and reduces activation of JNK MAPK in oligodendrocytes (Melani et al. Brain 132:1480-95, 2009. In the acute ischemia model of oxygen and glucose deprivation (OGD) in hippocampal slices, the selective adenosine A2A receptor antagonist 4-2-[7-amino-2-(2-furyl)[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-yl-amino]ethyl phenol (ZM 241385, 100–500 nM, n = 32) significantly prevents or delays anoxic depolarization (AD) appearance and protects against the loss of fEPSP induced by seven min OGD. Moreover it significantly reduces CA1 pyramidal neuronal damage as assessed by cell staining with propidium iodide (5 µg/ml) 3 hrs after the end of OGD. Results indicate that reduced excitoxity in the first hrs after ischemia, delay of AD propagation in the penumbral areas, reduced activation of p38 and JNK MAPK underlie protection by A2A antagonism.

S10-3

Targeting purines in Parkinson’s: From adenosine to caffeine to urate

Michael Schwarzschild

Massachusetts General Hospital, Harvard Medical School, USA

Endogenous purines—from adenosine to urate—have emerged as therapeutic targets for Parkinson’s disease (PD). Adenosine and its actions via its A2A receptor subtype have been intensively explored as a neuromodulator system contributing to the motor dysfunction of PD. Indeed A2A antagonists are now a leading class of non-dopaminergic antiparkinsonian agent with at least 5 distinct antagonists in clinical trials for PD. Contributing to the scientific and industry investment in this target are unique features of the A2A receptor: 1) its remarkably restricted regional and cellular expression in the CNS; 2) its capacity to form functional heteromeric complexes with other GPCRs (e.g., dopamine D2, cannabinoid CB1, and mGlu5 receptors); 3) its possible involvement in other CNS symptoms (e.g., restless legs, dyskinesia, excessive daytime sleepiness, depression); 4) its potential role in the underlying neurodegeneration—based on a convergence of epidemiological data linking caffeine (an adenosine antagonist) to reduced risk of PD, with lab data demonstrating neuroprotection by A2A inactivation. Recently, another purine has been identified as a compelling candidate neuroprotectant in PD. Urate, a major antioxidant as well as the end product of purine metabolism in humans, confers protection in cellular models of PD. Lower levels of urate has been shown to be both a risk factor for developing PD and a prognostic biomarker of its progression. A phase II clinical trial of a urate-elevating strategy using the purine intermediate inosine (http://clinicaltrials.gov/ct2/show/NCT00833690) has been developed in an effort to rapidly translate these findings into disease-modifying therapy for PD.

S10-4

Physiology and pathophysiology of purinergic signalling in neuroglia

Alexei Verkhratsky

The University of Manchester, UK

Purinergic transmission is one of the most ancient and widespread extracellular signalling systems. In the brain, purinergic signalling plays a unique role in integrating neuronal and glial cellular circuits, as virtually every type of glial cell possesses receptors to purines and pyrimidines. These receptors, represented by metabotropic P1 adenosine receptors, metabotropic P2Y purinoceptors and ionotropic P2X purinoceptors, control numerous physiological functions of glial cells and are intimately involved in virtually every form of neuropathology.

S11. Purinergic signaling in the immune system and inflammation

S11-1

Contribution of the fibrinolytic system to the angiogenic effect of adenosine

María Carmen Montesinos Mezquita

Fundación General Universidad de Valencia, Spain

The serine proteases urokinase (uPA) and tissue (tPA) plasminogen activators degrade plasminogen to plasmin, which in turn activates several matrix metalloproteinases, enabling the breakdown of extracellular matrix essential for cell migration at sites of injury. Since adenosine receptor agonists increase plasminogen activator release from rabbit alveolar macrophages, we studied the effect of adenosine A2A receptor activation on wound closure in wild type, tPA deficient and uPA deficient mice, and the effect on the production and secretion of t-PA, u-PA, PAI-1 and annexin II by human dermal microvascular endothelial cells (HMVEC). We found that promotion of wound healing by topical application of a selective adenosine A2A receptor agonist requires the expression of tissue (tPA) but not urokinase plasminogen activator (uPA). Adenosine A2A receptor activation in endothelial cells diminishes the release of the plasminogen activator inhibitor-1 (PAI-1) and promotes the production of annexin II, which acts as a cell membrane co-receptor for plasminogen and its activator tPA. Annexin II also mediates the increased cell membrane-associated plasmin generation in adenosine A2A receptor agonist treated HMVEC, effects that are completally blocked by an antibody against annexin II or a plasmin inhibitor. Similarly, A2A agonist promotion of HMVEC tube formation in a tridimensional matrix, Matrigel, is also blocked by a plasmin inhibitor and antibodies against PAI-1and tPA, but not by an antibody against uPA. Our results showed that tPA participates in the proangiogenic effects of A2A receptor activation through complex mechanisms that involve its main inhibitor PAI-1 and its cell receptor annexin II.

S11-2

Adenosine receptors and regulation of bone metabolism

Bruce Cronstein

NYU School of Medicine, New York University School of Medicine, USA

In prior experiments our laboratory has demonstrated a critical role for adenosine A1 receptors in regulation of multinucleated giant cell formation from human peripheral blood monocytes. We therefore determined whether these receptors play a similar role in regulating osteoclast formation, multinucleated giant cells that form from myeloid precursors and mediate bone turnover. We report here evidence that adenosine A1 receptors play a critical role in osteoclast formation and function in vitro. Blockade or deletion of these receptors leads to osteopetrosis in mice and inhibits ovariectomy-induced bone loss. Interestingly, adenosine A2A receptor ligation diminishes osteoclast formation and function in vitro as well. Because bone erosion due to wear particle-stimulated osteoclast function and formation is a major cause of failure of hip, knee and other orthopedic prostheses we determined whether adenosine A2A receptor agonists prevent wear particle-induced bone erosion. We observed, using a mouse calvarial air pouch model that an adenosine A2A receptor agonist does inhibit wear particle-induced bone loss. These studies demonstrate that adenosine and its receptors play an important role in regulating bone homeostasis and may be novel targets for therapies designed to treat osteoporosis and to diminish prosthesis failure.

S11-3

The P2X7 non-muscle myosin membrane complex: new roles in pore formation and phagocytosis

James Wiley1, Ben Gu1, Bernadette Saunders2, Leanne Stokes3, Kristen Skarratt3, Ronald Sluyter4, Stephen Fuller3

1Florey Neuroscience Institutes, University of Melbourne, Australia,2Centenary Institute, University of Sydney, Australia,3Nepean Clinical School, University of Sydney, Australia,4School of Biological Sciences, University of Wollongong, Australia

Our studies suggest that the mechanism of P2X7 pore dilatation involves the de-coupling of P2X7 from the underlying cytoskeleton. Protein and FRET analysis of the P2X7 membrane complex in monocytic THP-1 cells and P2X7 transfected HEK-293 show the receptor has a tight molecular complex with non-muscle myosin heavy chain (NMMHC) which anchors this receptor to the cytoskeleton. Activation of P2X7 by extracellular ATP slowly dissociates NMMHC from the complex, suggesting that detachment from this anchoring protein is required for P2X7 pore formation. Moreover, S-l-blebbistatin, a specific inhibitor of NMMHC-IIA ATPase, inhibits both pore formation and ATP-induced dissociation of the P2X7-NMMHC complex. P2X7 receptors have a recognized role in the formation of the inflammasome. Our genetic studies have defined two haplotypes of the P2X7 receptor both exhibiting gain of function and enhanced interleukin-1 beta secretion. However, in the absence of ATP, this receptor has a second role in innate immunity. Over expression of the P2X7 receptor in cells greatly augments phagocytosis of beads and bacteria in the absence of serum. Uptake of these particles is inhibited by P2X7 mAb, siRNA to P2X7, or recombinant P2X7 extracellular domain. This novel phagocytic pathway also operates in vivo-evidenced by inhibition of the uptake of latex beads and bacteria by peritoneal macrophages of mice by prior injection of ATP. The role of P2X7 in innate immunity is firstly to regulate the uptake of nonopsonized foreign particles and bacteria. Subsequent tissue damage causes ambient ATP concentrations to rise, activating the P2X7 receptor and thereby promoting inflammasome formation and the secretion of pro-inflammatory cytokines from monocytes, resident macrophages and microglia. Thus the P2X7 receptor has two clearly separate functions in innate immunity.

S11-4

ATP confers tumorigenic properties to dendritic cells

Didier Communi

IRIBHM, ULB, Belgium

Gene profiling identified angiogenic factors such as VEGF and EGF receptor (EGFR) ligands as target genes of ATPγS, a more stable ATP derivative, in human monocyte-derived dendritic cells (MoDCs). We showed that ATPγS particularly in combination with LPS was able to induce the secretion of high levels of VEGF and EGFR ligands by MoDCs. These stimulated MoDCs were able to induce in vitro endothelial tube formation through VEGF secretion and smooth muscle cell proliferation through EGFR ligand secretion. We also observed that LPS + ATPγS-stimulated mouse bone marrow-derived dendritic cells (BMDCs) secrete sufficient concentrations of EGFR ligand to stimulate proliferation of Lewis Lung Carcinoma (LLC) cell growth in vitro. To investigate their effect on tumor progression, we have coinjected subcutaneously stimulated BMDCs or their supernatants with LLC cells in mice. Interestingly, tumor weights were significantly increased after coinjections of LLC cells with LPS + ATPγS-treated BMDCs or their supernatants compared to LPS-treated BMDCs or their supernatants. This study demonstrates that ATPγS is able to confer tumorigenic properties to human and murine DCs.

S12. Trafficking of purine receptors

S12-1

P2X7 receptor signaling towards alternative macrophage polarization: unexpected anti-inflammatory effects

Pablo Pelegrin

Inflammation and Macrophage Group, University Hospital Virgen Arrixaca—FFIS, Spain

In acute inflammation extracellular ATP activates P2X7 ion channel receptors on M1 polarized macrophages to release pro-inflammatory IL-1beta via activation of the caspase-1/NLRP3 inflammasome. In contrast, towards resolution of inflammation macrophages are dynamically polarized to an alternative phenotype with suppression of pro-inflammatory cytokine production. We studied the signaling underlying macrophage phenotype switching and the actions of P2X7 function. We found that extracellular ATP signaling through P2X7 selectively uncouples to the inflammasome over a dynamic polarity gradient as the macrophage progresses towards the alternatively activated state. Under these conditions extracellular ATP acts via its pyrophosphate chains (PPi), independently of all known purine receptors, to inhibit the inflammasome and IL-1beta release. Furthermore, extracellualar PPi was able to attenuate a mouse model of peritonitis and an in vivo model of alternative macrophage activation, we found a lack of innate inflammatory response in P2X7 receptor deficient animals. These findings provide new scope for the use of P2X7 receptor selective antagonist in the treatment of chronic inflammatory diseases and also distinguish an unexpected mechanism of the anti-inflammatory actions of clinically used PPi analogues bisphosphonates.

S12-2

Endolysosomal targeting and function of mammalian P2X4 receptors

Ruth Murrell-Lagnado, Miyyada Boumechache, Omar Qureshi

Department of Pharmacology, University of Cambridge, UK

P2X4 receptors have a widespread distribution throughout the body. In primary cells where we have examined their subcellular distribution, including macrophages, microglia and endothelial cells, the receptors are localized to late endosomes and lysosomes (LEL). Mutational analysis of heterologously expressed receptors has shown that a C-terminal tyrosine based motif and N-terminal dileucine-like motif are both involved in targeting the receptor to LELs. Within lysosomes, P2X4 receptors resist degradation by virtue of N-linked glycans, and they can be delivered from lysosomes to the surface to increase the expression of functional receptors. In activated microglia in culture, a proportion of receptors traffic to and from the cell surface in a dynamic manner and further activation of microglia by LPS was shown to increase the number at the cell surface. In cultured bone marrow derived macrophages, however, receptors were localized exclusively to LELs and no expression was detected at the surface unless lysosome exocytosis was stimulated by incubation with a weak base. We are interested in whether or not P2X4 receptors can function within the acidic environment of the lysosome or phagosome, as well as at the plasma membrane. Intracellular P2X-like receptors have been identified in Dictyostelium and are thought to function within the acidic environment of the contractile vacuole. With the extracellular solution at lysosomal pH (4.6), P2X4 receptors at the cell surface of HEK293 cells can be activated by millimolar extracellular ATP. We are examining the effects of wild type and mutant P2X4 receptor expression on LEL function and calcium signalling.

S12-3

Role of membrane rafts in cell surface distribution and signaling of P2Y1 and P2Y2 receptors in human vascular tissues

Juan Pablo Huidobro-Toro, Andres Norambuena, Ines Poblete, Maria Veronica Donoso

Pontificia Universidad Católica de Chile, Chile

The plasma membrane microregionalization of vascular P2Y1 and P2Y2 receptors (P2Y1R and P2Y2R respectively) remains largely unknown; we aimed therefore at characterizing smooth muscle cell distribution of these receptors derived from human placentae chorionic arteries. Membrane rafts were isolated using sodium carbonate or Optiprep sucrose gradients from endothelium-denuded vessels. We found that 20-30% of the total inactive P2Y1R mass partitioned into raft fractions. Selective P2Y1R activation induced both the receptor dissociation from membrane rafts and its rapid internalization, processes which preceded the receptor-induced vasomotor activity. In contrast, ligand activation of the P2Y2R was required for its partitioning into membrane rafts, an event also followed by rapid receptor internalization. Vessel treatment with methyl beta-cyclodextrin reduced the P2Y1R-raft partitioning and obliterated its functional vasomotor response without affecting those generated by P2Y2R activation, serotonin or KCl. In addition, the P2Y2R-mediated vasomotor activity, the membrane raft partitioning and its internalization were dependent indirectly on the activation of EGF receptors since all these UTP-evoked processed were blunted by AG1478, suggesting that the functional activity of the nucleotide receptors are transregulated by EGF receptors. Therefore, evolutionary related receptors differ in their membrane microregionalization, lipid raft association highlighting differences in signaling denoting functional adjustments. Grant 13980001, PFB 12/2007 and MIFAB funding

S12-4

P2X1 receptor mobility and trafficking: regulation by receptor insertion, activation and GPCR linked pathways

Uliana Lalo, Rebecca Allsopp, Martyn Mahaut-Smith, Richard Evans

CPP, University of Leicester, UK

P2X1 receptors for ATP contribute to signalling in a variety of cell types and following stimulation undergo rapid desensitisation (within 1 s), and require about 5 minutes to recover. In HEK293 cells P2X1 receptors C terminally tagged with enhanced green fluorescent protein (P2X1-eGFP) were predominantly expressed at the cell surface. Following >90% photo-bleaching of P2X1-eGFP fluorescence within a 6 sq.microm circle at the cell surface fluorescence recovery after photo-bleaching (FRAP) was fitted with a time constant of ~60 s and recovered to ~75% of pre-bleach level. Following activation of the P2X1 receptor with alpha, beta-methylene ATP the associated calcium influx doubled the FRAP recovery rate. The protein synthesis inhibitor cycloheximide had only a small effect on repeated FRAP and indicated a limited contribution of new P2X1 receptors to the FRAP. Inhibition of trafficking with brefeldin A (BFA) reduced recovery and this effect could be reversed following receptor activation. In contrast, the dynamin inhibitor dynasore had no effect on FRAP under unstimulated conditions but reduced the level of recovery following agonist stimulation. In functional studies both BFA and dynasore increased the recovery time from desensitisation. We found that P2X1 receptors can be regulated by Galphaq coupled, but not Galpha i and Galpha s coupled GPCRs. PMA treatment increased the FRAP recovery rate with no effect on the immobile fraction suggesting that an effect on receptor trafficking contributes to the P2X1 receptor potentiation. Taken together these studies demonstrate for the first time an important role of receptor recycling on P2X1 receptor responsiveness.

S13. Ectonucleotidase and disease

S13-1

NTPDase1 (CD39) controls nucleotide dependent vasomotion in mouse

Jean Sévigny1, Gilles Kauffenstein1, Cristina Ribas Fürstenau1, Annick Drouin2, Nathalie Thorin-Trescases2, Bernard Robaye3, Pedro D’Orléans-Juste4, François Marceau1, Éric Thorin2

4CHUQ, Université Laval, Canada,2Institut de Cardiologie de Montréal, Université de Montréal, Canada,3Institut de Recherche Interdisciplinaire en Biologie humaine et moléculaire, Université Libre de Bruxelles, Belgium,4CHUS, Université de Sherbrooke, Canada

Background: Extracellular nucleotides are vasoactive molecules which concentrations are regulated by ectonucleotidases. In this study we investigated the role of the blood vessel ectonucleotidase NTPDase1, in the regulation of the vascular tone by nucleotides using Entpd1−/− mice. Methods: Vascular NTPDase expression and activity were evaluated by immuno¬histochemistry, histochemistry, and HPLC analysis. Vascular reactivity was tested in vivo by monitoring mean arterial pressure, and in vitro comparing the reactivity of wild type and Entpd1−/− aortic rings. Expression of nucleotide receptors was determined by RT-PCR. Results: In comparison to controls, Entpd1−/− blood vessels displayed a dramatic deficit of nucleotidase activity in both smooth muscle and endothelial cells. In aortic rings from Entpd1−/− mice, UDP and UTP, via P2Y6, induced a potent constriction contrasting with the weak response obtained in wild type rings. UDP infusion in vivo increased blood pressure and this effect was potentiated in Entpd1−/− mice. Pressurised mesenteric arteries from Entpd1−/− mice displayed an enhanced myogenic response, consistent with higher local concentrations of endogenously released nucleotides. In addition, Entpd1−/− mice also showed an exacerbated decrease in blood pressure after ATP and UTP injection (10 nmole/kg both). In vitro, endothelial P2Y1 and P2Y2 dependant relaxation was facilitated in Entpd1−/− aortic rings. Interestingly, P2Y1, in contrast to P2Y2, was more prone to desensitization in Entpd1−/− aortas. Conclusion: NTPDase1 regulates nucleotide metabolism at the surface of both vascular endothelial and smooth muscle cells, and doing so, contributes to the local regulation of vascular tone modulated by P2Y1, P2Y2 and P2Y6 receptors.

S13-2

Regulatory T cells and CD39 expression in transplantation

Karen Dwyer1, Simon Robson2

1University of Melbourne, Australia,2Beth Israel Deaconess Medical Center, Harvard Medical School, USA

Ectonucleotidases are vascular and regulatory immune cell expressed ecto-enzymes that hydrolyze extracellular nucleotides, in a tandem manner, to adenosine and derivatives. CD39/ectonucleoside triphosphate diphosphohydrolase family members and CD73/ecto-5′-nucleotidase will be the area of focus as these ecto-enzymes are key thromboregulatory factors that also have major effects on inflammation and immunity. The presentation includes an initial explanation of the regulation of purinergic signaling by ectonucleotidases by the scavenging of extracellular nucleotides and generation of nucleosides in both the vasculature and by regulatory immune cells at extravascular sites. CD39 family members together with CD73 are crucial in regulating inflammation and vascular thrombosis as well as modulating immune responses in the vasculature, in transplanted organs and tissues. The prototype CD39 is not only highly expressed by the vascular endothelium but is also co-expressed with CD73 on the surface of CD4+ Foxp3+ regulatory T-cells (Treg), dendritic cells and NKT cells. CD39 and CD73 functions contribute, at least in part to Treg immunosuppressive and vascular protective properties via the generation of extracellular adenosine. Importantly, high levels of CD39 expression can differentiate stable Tregs from CD4+ T-cells with highly reactive T helper (Th) type 17 inflammatory potential, residing within this T memory-cell pool. Inflammatory bowel disease is noteworthy for local tissue injury, high rates of venous thromboembolism, systemic inflammation and aberrant immune responses. This entity will be therefore used as a pertinent clinical area of interest to show how purinergic signaling allows for integration of inflammation, immunity and thrombotic responses. CD39 deficiency in mutant mice exacerbates experimental colitis while genetic polymorphisms in man result in lower levels of CD39 and predispose to disease in a proportion of patients with this condition. In summary, CD39 may be considered a “protective gene” that ameliorates vascular injury, systemic inflammation and those associated immune reactions considered important in multi organ dysfunction.

S13-3

Pathophysiological significance of nucleotide hydrolysing enzymes in urologic disorders

Paulo Correia-de-Sá

Lab Farmacologia e Neurobiologia, ICBAS-Universidade do Porto, Portugal

Inflammation and mechanical perturbations (e.g. shear stress, stretching) stimulate ATP release from many different cell types, including urinary bladder cells. Urodynamic and in vitro studies demonstrated that uroepithelial cells and cholinergic nerves from overactive human bladders (OAB) exhibit a 3-fold increase in ATP release followed by slower inactivation kinetics of the nucleotide compared to cadaveric controls. Urology clinical studies led us to propose that urinary ATP may be a non-invasive biological urinary marker of overactive bladder syndromes, exhibiting higher sensitivity and specificity (AUC: 83.2%, CI 95%: 69.7–96.7) than other experimental markers (e.g. NGF). Increased ATP bioavailability might explain hyperactivity of bladder nerves (via P2X3 receptors) and detrusor contractions (via P2X1); implication of changes in the activity of co-localized inhibitory metabotropic P2Y and P1 receptors by ATP hydrolysing products, ADP and adenosine, will also be discussed. In this respect, immunolocalization studies showed that human urothelial cells are highly enriched in ecto-NTPDase3 enzyme, whereas the detrusor smooth muscle layer exhibits predominantly the ecto-NTPDase1/CD39 marker. These findings were confirmed by HPLC kinetic experiments and may explain the neurochemical differences observed in patients with OAB. Purinergic transmission is also important for initiation and maintenance of penile erection. ATP is a powerful regulator of vascular hemodynamics through the activation of vasoconstrictor P2X and vasodilator P2Y and adenosine receptors. Nucleotide effects may be cut-short by extracellular hydrolysis via the ectonucleotidase cascade. Human corpora cavernosa have a high NTPDase1/CD39 activity converting ATP directly into AMP, without a significant ADP formation. Extracellular ATP hydrolysis is slower in impotent patients with endothelial dysfunction. Decreased NTPDase1/CD39 activity leads to desensitization of P2 purinoceptors and poor adenosine formation regulating tonus of corpora cavernosa in impotent men. Work supported by FCT, Soc.Port.Andrologia and UPorto/SantanderTotta.

S13-4

NTPDase and 5′-nucleotidase activities in physiological and disease conditions: new perspectives for human health

Maria Schetinger, Paula Maldonado, Vera Morsch

Department of Chemistry, Federal University of Santa Maria, Brazil

Background: We intend to report nucleotide hydrolysis in distinct human diseases. Methods: This study emphasized the involvement of NTPDase and 5′-nucleotidase activities in disease processes in several tissues and cell types. Background information is given about the general characteristics of these enzymes in diabetes, pregnancy, hypercholesterolemia and cancer. In addition, the role of these enzymes is considered under clinical conditions such as acute lymphoblastic leukemia (ALL), B-chronic lymphocytic leukemia (B-CLL) and human immunodeficiency virus (HIV). The Human Ethics Committee from the Federal University of Santa Maria approved all protocols. Results: In diabetes, the increased ATP and ADP hydrolysis by NTPDase could be compensatory, avoiding coagulation processes by increasing ADP depletion. In pregnancy, NTPDase and 5′-nucleotidase act directly in thromboregulation. In hypercholesterolemia the increased NTPDase activity is possibly related to a compensatory response to the inflammatory and pro-oxidative state associated with hypercholesterolemia. In breast cancer patients, the enhanced NTPDase activity may be related to changes in platelet activity. The altered activity in lymphocytes from patients with ALL and B-CLL shows that NTPDase may play an important role in the modulation of the disease. Increased NTPDase activity in lymphocytes of HIV patients suggests that it may play an important role in maintaining an adequate balance between the generation and consumption of ATP and preserving cellular integrity and the immune response. Conclusions: It may be suggested that despite advances made toward understanding the molecular structure of ectonucleotidases, much more investigation will be necessary to entirely grasp their role in physiological and pathological conditions.

S14. Purinergic signaling in the cardiovascular system

S14-1

The Role of nucleoside transporters in cardiovascular physiology

Imogen Coe

Department of Biology, York University, Canada

Nucleoside transporters are integral membrane proteins responsible for the flux of nucleosides, nucleobases and nucleoside analog drugs across cellular membranes. Nucleoside transporters exist as two non-homologous families of solute carriers, the SLC 28 family of concentrative nucleoside transporters (CNTs), consisting of 3 isoforms, and the SLC 29 family of equilibrative nucleoside transporters (ENTs), consisting of 4 isoforms. Despite their critically important roles as purine nucleoside transporters, nucleoside analog drug transporters and drug targets, the structure, function and regulation of ENTs and CNTs are poorly understood. We have previously shown that ENTs are highly expressed in the cardiovasculature and are responsible for mediating purine nucleoside flux in cardiomyocytes. We are particularly interested in the role of the ENTs in modulating purinergic signaling in the cardiovasculature and are using a variety of approaches to investigate the role and relative contributions of ENTs nucleoside flux and cardiomyocyte responses to hypoxic challenge. ENTs appears to be subject to regulatory feedback through purinergic signaling pathways and play critical roles in cellular responses to hypoxia. We recently extended our studies into human patient populations and investigated the individual profiles of all NTs in tissues obtained from patients undergoing heart surgery. Our studies suggest that ENTs are an integral component in the modulation of purinergic signaling in the cardiovasculature as a consequence of their pivotal role in facilitating and regulating the flux of purine nucleosides, such as adenosine, across cellular membranes.

S14-2

Hypertension and renal stressors blunt renal microvascular autoregulatory behavior by reducing renal microvascular reactivity to P2X receptor activation

Edward Inscho, Zhengrong Guan, David Osmond

Department of Physiology, Medical College of Georgia, USA

Hypertension induced impairment of autoregulatory control is linked to renal inflammatory processes. We postulated that autoregulatory impairment observed in hypertension or renal stress, reflects reduction in afferent arteriolar reactivity to P2X receptor activation. We’ve shown that autoregulatory responses involve P2X1 receptor activation. More recently, we demonstrated that hypertension impairs autoregulatory behavior. This impairment can be ameliorated by anti-inflammatory treatment. We demonstrated that anti-inflammatory treatment with pentosan polyphosphate or P2Y12 receptor inhibition (Clopidogrel) preserves renal microvascular function in hypertension. When autoregulatory behavior and afferent arteriolar reactivity to P2X receptor activation were assessed in normotensive and Ang II-infused hypertensive rats, both responses were significantly blunted. When rats were treated with clopidogrel or pentosan polysulfate autoregulatory behavior was normalized despite a persistent hypertension with systolic arterial pressure approaching 200 ± 6 mmHg after 13 days of Ang II infusion. Improved autoregulatory behavior coincided with normalized P2X receptor-mediated vasoconstriction in clopidogrel or pentosan polysulfate treated rats. In more recent work, chronic salt loading also impaired autoregulatory behavior and reduced afferent arteriolar reactivity to P2X receptor activation with only a modest increase in systolic arterial pressure. Both autoregulatory behavior and microvascular P2X receptor reactivity were normalized during acute exposure to the antioxidant, Tempol. These observations implicate local inflammatory and oxidant stress as mechanisms for impairing P2X receptor signaling and thereby blunting autoregulatory efficiency. Chronic reductions in autoregulatory efficiency can result inappropriate elevations in glomerular capillary pressure and glomerular injury. Preservation of autoregulatory behavior is supported by preservation of afferent arteriolar reactivity to P2X receptor activation.

S14-3

Adenosine receptor signaling and coronary flow regulation

S. Jamal Mustafa

Department of Physiology & Pharmacology and Center for Cardiovascular and Respiratory Sciences, West Virginia University, USA

Background: Adenosine causes an increase in coronary flow (CF) mostly through A2A adenosine receptor (AR). However, the involvement of other ARs and its signaling in the modulation of CF is not well understood. Using AR knockout (KO) mice, we have investigated the roles for each AR in the regulation of CF. Methods: Mouse Langendorff heart preparation was used for these studies. Results: In A3 AR KO mice, an increase in A2A AR-mediated CF was observed. Also, we found an increase in CF in A1 AR KO mice with A2A selective agonist (CGS-21680). On the other hand, A2A AR KO mice showed a decrease in CF to NECA (non-selective agonist). BAY60-6583 (A2B AR selective agonist) was without an effect on CF in A2B KO mice; however, it increased CF significantly in A2A KO. CGS-21680 also caused a significant increase in CF in A2B KO mice. Adenosine-induced increase in CF in WT, A2A KO, and A2B KO mice was significantly reduced with catalase. Also, BAY60-6583-induced increase in CF in WT mice was significantly inhibited with glibenclamide (KATP channel blocker). Conclusions: These data support stimulatory roles for A2A/A2B and inhibitory roles for A1/A3 ARs in the regulation of CF. We propose, for the first time, that activation of A2B AR may induce the release of H2O2 which then activates KATP channels, leading to hyperpolarization and vasodilation. Therefore, these studies may lead to better understanding of the roles of all four ARs in coronary disease and may lead to better therapeutic approaches.

S14-4

Adenosine A2Areceptor signaling and abnormal calcium handling in human atrial myocytes

Leif Hove-Madsen

Cell Physiology Laboratory, Cardiology Department, Institut Catalá de Ciencies Cardiovasculars, CSIC, Spain

Alterations in the cyclic AMP-dependent regulation of the cardiac ryanodine receptor (RyR2) have been proposed to account for increased spontaneous calcium release and promote arrhythmogenesis in patients with heart failure, ventricular tachyarrhythmias and atrial fibrillation. Although the adenosine A2A receptor (A2AR) also regulates cyclic AMP levels, little is known about the expression and function of this receptor in human cardiac myocytes. We therefore investigated the expression and function of adenosine A2A receptors in human atrial myocytes and their contribution to atrial arrhythmogenesis. Using molecular biological and electrophysiological approaches, we found that the A2AR is expressed in the human right atrium and distributed in a banded pattern along the Z-lines, overlapping with the RyR2. Functionally, A2AR stimulation caused a protein kinase A dependent increase in spontaneous calcium release in isolated human atrial myocytes. Moreover, a comparison of atrial samples from patients with and without atrial fibrillation showed that both A2AR mRNA and protein levels were significantly increased in patients with atrial fibrillation. Functionally, this was linked to a stronger A2AR-mediated stimulation of RyR2 phosphorylation and spontaneous calcium release. Furthermore, A2AR stimulation destabilized the beat-to-beat response in myocytes subjected to rapid stimulation. Importantly, the increased rate of spontaneous calcium release observed in myocytes from patients with atrial fibrillation could be reduced by adenosine A2AR antagonists or adenosine deaminase to levels observed in patients without the arrhythmia, suggesting that excessive A2AR expression and activation by endogenously produced adenosine promote spontaneous calcium release and arrhythmogenesis in patients with atrial fibrillation.

S15. Purinergic and neurotrophic signaling

S15-1

Triggering neurotrophic factor actions through adenosine receptors

Joaquim Alexandre RIBEIRO

Inst Pharmacol. & Neurosci., Fac Med. and Unit Neurosc., Inst Mol Med, Univ Lisbon, Portugal

It has been shown that tonic adenosine receptor activity is a required step to allow synaptic acute actions of neurotrophic factors, namely upon synaptic transmission at both pre- and post-synaptic level as well as upon synaptic plasticity. For example an enhancement of A2A receptor tonus upon ageing may partially compensate the loss of TrkB receptors, rescuing to certain degree the facilitatory action of brain derived neurotrophic factor (BDNF) in aged animals. This might prove to be particularly relevant in the prevention of neurodegeneration upon ageing. A2A receptors also trigger synaptic actions of other neurotrophic factors, such as the glial derived neurotrophic factor (GDNF) at dopaminergic striatal nerve endings. The growing evidence that tonic adenosine A2A receptor activity is a crucial step to allow actions of neurotrophic factors in neurons will be discussed considering the potential interest of these factors in relation to therapeutic strategies for neurodegenerative diseases.

S15-2

A multipurpose and multidirectional way to orchestrate physiopathological conditions in the nervous system: purinergic signalling at the plasma membrane

Cinzia, Volonte1, D'Ambrosi Nadia2, Amadio Susanna2, Apolloni Savina2

1CNR/Santa Lucia Foundation, CNR, Inst. Neurobiology & Molecular Medicine/Santa Lucia Foundation, Italy,2CNR/Santa Lucia Foundation, CNR/Santa Lucia Foundation, Italy

ATP is a multipurpose signaling molecule copiously released in the extracellular environment of the whole nervous system upon cell activation, stress, or damage. Extracellular ATP is also a multidirectional information molecule, due to the concurrent presence at the cell border of various ectonucleotidases, several ATP/adenosine transporters, at least fifteen different P2 receptors for purine/pyrimidine nucleotides, in addition to four metabotropic P1 receptors for nucleosides. All these “purinergic players” are usually co-expressed at the plasma membrane and rarely operate as single units, rather they associate with each other within multi-protein complexes. The resultant multiple purinergic interactions then orchestrate both the magnitude and/or direction of the signals triggered and propagated by each purine/pyrimidine ligand, and the impact of the single ligands on a variety of short- and long-term functions. A stern control system is thus achieved, simultaneously with assorted, even divergent biological outcomes. As a consequence, the purinergic players have biological implications not only as transmitters, modulators, tropic and trophic factors, but also as noxious agents in the insurgence/progression of pathological conditions in the nervous system. In our work, we have shown that purinergic signalling at the plasma membrane depends on finely integrated and cooperative molecular networks; that the occurrence, modulation of expression and interactions especially of purinergic P2 receptors are tightly specialized features in neuronal versus glial cells and, moreover, in different cerebral areas; that extracellular nucleotides can exert throphic but also detrimental effects leading to neurodegeneration and/or neuroinflammation in the nervous system.

S15-3

Chronic caffeine and A2A receptor antagonists afford neuroprotection against hippocampal insults—does the control of bdnf signaling play a role?

Rodrigo Cunha

Center for Neurosciences of Coimbra, University of Coimbra, Portugal

Chronic consumption of caffeine counteracts memory deficits upon aging, an effect mimicked by adenosine A2A receptor (A2AR) antagonist. We report that different insults (icv administration of the β-amyloid fragment present in Alzheimer’s disease patients, a 3 weeks’ protocol of chronic unpredictable stress, the induction of early-life convulsions or the implementation of type 2 diabetes in NONcNZO10/Ltj mice or Goto-Kakizaki rats) all caused decreased memory performance and loss of synaptic markers in the hippocampus. This memory impairment and loss of synaptic markers was prevented by chronic caffeine consumption (1 g/L) and by pharmacological and/or genetic blockade of A2AR. However, the mechanism underlying this A2AR-mediated control of neurodegeneration is still to be determined. We have explored as candidate mechanisms the control of synaptic plasticity (indeed A2AR control long-term potentiation), the control of synaptic viability (indeed A2AR control synaptic mitochondria) and the control of the support and influence of glial cells (indeed A2AR control astrocytic glutamate uptake, the genesis of inflammatory mediators and the impact of inflammatory mediators on neurons). Another possible mechanism relies on the known interaction between A2AR and the function of growth factors, namely BDNF, at the synaptic level, which will be discussed in more detail. The understanding of the mechanism operated by A2AR to control neurodegeneration may be of uppermost importance to gauge the interest, time window of opportunity and safety of A2AR antagonists as novel candidate neuroprotective drugs in different brain neurodegenerative disorders. (Supported by FCT)

S16. Purines and hypoxia

S16-1

The role of CD73 in the adenosinergic response to hypoxia

Linda Thompson

Immunobiology and Cancer, Oklahoma Medical Research Foundation, United States

CD73 knock out mice were used to assess the role of ecto-5?-nucleotidase (CD73) in the physiological response to hypoxia. Three experimental models were used. In the first, mice were subjected to normobaric hypoxia (8% oxygen) for 4 hours. Vascular leak was evaluated by measuring Evan’s blue dye extravasation. Neutrophil migration was assessed by measuring myeloperoxidase activity in tissue extracts. In the second and third models, mice were subjected to alternating 4- to 5-minute periods of cardiac or kidney ischemia and reperfusion followed by a subsequent longer period of ischemia. Infarct sizes were determined by histology. In all three models, CD73 knock out mice suffered more hypoxia-induced damage than wild type mice, suggesting that CD73-generated adenosine is one component of a pathway that is induced to protect organisms from hypoxia. Indeed, the CD73 promoter contains a HIF-1 alpha binding site and CD73 expression is up regulated as a consequence of hypoxia. Adenosine receptor knock out mice, adenosine receptor agonists and antagonists, siRNA, and bone marrow chimeras were used to determine which adenosine receptor ameliorates the effects of hypoxia. All these strategies generated data suggesting that the A2B receptor is the primarily adenosine receptor responsible for regulating hypoxia-induced vascular leak and neutrophil migration, as well as the phenomenon of ischemic preconditioning. Like CD73, the expression of this receptor is also induced by hypoxia. These studies suggest that CD73 and the A2B receptor may be therapeutic targets for the prevention of hypoxia-induced tissue damage.

S16-2

Protective and adverse influences of adenosine on the fetus and newborn

Scott A. Rivkees

Yale Child Health Research Center, Yale University School of Medicine, United States

Few signaling molecules have the potential to influence the developing mammal as the nucleoside adenosine. Adenosine levels are dynamically regulated and increase with increased tissue activity, hypoxia, or stress. Local adenosine concentrations thus provide a humoral barometer of acute changes in cellular physiology. The receptors that transduce adenosine action include A1, A2a, A2b, and A3 adenosine receptors. These receptors differ in their affinities for adenosine and in patterns of tissues expression. During development A1 adenosine receptors (A1ARs) are especially important, and A1ARs are among the earliest receptors expressed in the embryonic brain and heart. In the developing heart, the adenosinergic system is the dominant regulator of fetal cardiac function. Acting in the myocardium, A1ARs play an essential role in protecting the embryo from hypoxia in part by regulating levels of hypoxia-inducible factor 1alpha (HIF1-alpha) (1, 2). Interestingly, exposure to a single dose of caffeine during embryogenesis results in both short-term adverse effects on cardiac development and long-term effects on cardiac function and body composition (3). In comparison with early embryonic protective effects of adenosine, A1ARs play a key role in mediating hypoxia-induced white matter injury at the end of gestation and in the neonatal period. Hypoxia and A1ARs act to adversely affect oligodendrocyte maturation (4–6). Caffeine administration during early postnatal development also has utility in the prevention of brain injury (5). These observations show that adenosine is an important modulator of mammalian development and blockage of adenosine receptors during development may confer deleterious or protective effects depending on the time of exposure.

1.Wendler CC, Amatya S, McClaskey C, Ghatpande S, Fredholm BB, Rivkees SA 2007 A1 adenosine receptors play an essential role in protecting the embryo against hypoxia. Proc Natl Acad Sci U S A 104:9697–9702

2.Wendler CC, Pulson R, Ghatpande S, Greene R, Rivkees S 2010 Identification of the heart as the critical site of adenosine mediated embryo protection. BMC Development (in press)

3.Wendler CC, Busovsky-McNeal M, Ghatpande S, Kalinowski A, Russell KS, Rivkees SA 2009 Embryonic caffeine exposure induces adverse effects in adulthood. FASEB J 23:1272–1278

4.Akundi RS, Rivkees SA 2009 Hypoxia alters cell cycle regulatory protein expression and induces premature maturation of oligodendrocyte precursor cells. PLoS One 4:e4739

5.Back SA, Craig A, Luo NL, Ren J, Akundi RS, Ribeiro I, Rivkees SA 2006 Protective effects of caffeine on chronic hypoxia-induced perinatal white matter injury. Ann Neurol 60:696–705

6.Back SA, Rivkees SA 2004 Emerging concepts in periventricular white matter injury. Semin Perinatol 28:405–414

S16-3

The role of the A2B adenosine receptor in pulmonary fibrosis

Michael Blackburn, Mesias Pedroza

1Department of Biochemistry and Molecular Biology, University of Texas-Houston Medical School, United States,2Department of Biochemistry, University of Texas-Houston Medical School, United States

Fibrosis is a detrimental and poorly treated component of many interstitial lung diseases. We have shown that adenosine levels are elevated in interstitial lung diseases where fibrosis is prominent and that adenosine contributes to fibrotic processes through engagement of the adenosine A2B receptor (A2BR). In pursuit of mechanisms by which A2BR signaling influences pulmonary fibrosis, we found that this pathway promotes the production of interleukin-6 (IL-6) in alveolar macrophages in mice with progressive pulmonary fibrosis. Similarly, engagement of the A2BR drives the production of IL-6 in alternatively activated (M2) macrophages isolated from the lungs of patients with pulmonary fibrosis. Moreover, blockade or genetic deletion of IL-6 signaling in mouse models of adenosine-dependent pulmonary fibrosis leads to diminished disease. Examination of nuclear phosophorylated STAT-3 (P-STAT-3) identified alveolar epithelial cells as potential IL-6 targets in these models. In addition, levels of the soluble IL-6Ralpha (sIL-6Ralpha) were found in lavage fluid of mice and humans with pulmonary fibrosis suggesting that sIL-6Ralpha signaling (IL-6 Trans Signaling) may contribute to the development of adenosine-dependent fibrosis through the process of epithelial mesenchymal transition (EMT). These findings suggest that adenosine contributes to the progression of pulmonary fibrosis by the A2BR-dependent production of IL-6 from alternatively activated macrophages that in turn promote EMT in airway epithelial cells. In addition to these findings, the role of hypoxia and A2BR signaling in acute versus chronic lung injury will be discussed.

S17. Purines and Cancer

S17-1

Nucleoside transporters: role in pharmacokinetics and pharmacodynamics of nucleoside drugs

Jashvant Unadkat

Dept. of Pharmaceutics Univ. of Washington USA

Nucleoside drugs (e.g. gemcitabine and ribavirin) are hydrophilic and, for efficacy and toxicity, they need to be intracellularly phosphorylated to their active metabolites. To gain entry into cells, they need to be transported into cells by the nucleoside transporters. Therefore, nucleoside transporters are often the rate-limiting step in the efficacy and toxicity of nucleoside drugs. Nucleoside transporters consist of two major families, concentrative and equilibrative nucleoside transporters. In many tissues, these two families work in concert to mediate the efficacy and/or toxicity of nucleoside drugs. Using gemcitabine and ribavirin nucleoside drugs as examples, I will illustrate the important role the nucleoside transporters play in mediating the efficacy and toxicity of these drugs. Supported by NIH GM54447.

S17-2

A2B adenosine receptors in tumor-host interaction

Igor Feoktistov, Mikhail Dikov, Sergey Ryzhov, Italo Biaggioni

Medicine, Vanderbilt University, USA

Extracellular concentrations of adenosine increase significantly in metabolically stressful conditions, including inflammation and hypoxia characteristic of solid tumors. Analysis of gene expression in primary human tumors uncovered the overexpression of A2B adenosine receptors, suggesting their potential role in cancer biology. Indeed, we have previously shown that stimulation of A2B receptors in several cancer cell lines upregulates the production of angiogenic factors, suggesting that tumor A2B receptors may benefit tumors by promoting neovascularization. Recently, we have shown that tumor cells can also exploit adenosine-dependent regulation of host cells to their benefit. Differentiation and functionality of immune cells critically depend on the microenvironment and under certain conditions infiltrating immune cells can benefit tumors by producing factors promoting angiogenesis and suppressing immunity. We demonstrated that stimulation of adenosine receptors skews dendritic cell differentiation from normal immune protective phenotype toward immunosuppressive cells with pro-angiogenic and tumor growth promoting properties. We also identified adenosine as an important metabolite inducing secretion of angiogenic factors by tumor immune infiltrates, and identified A2B receptor as a mediator of the observed effects. The profound effect that signaling through A2B receptor has on differentiation and cytokine secretion by tumor-infiltrating immune cells identifies A2B adenosine receptors as an important therapeutic target in cancer biology.

S17-3

Increased sensitivity to thiopurines in methylthioadenosine phosphorylase-deleted cancers

Sally Coulthard1, Christopher Redfern1, Svante Vikingsson2, Malin Lindqvist Appell2, Karin Skoglund2, Ingrid Jakobsen Falk2, Andrew Hall1, Gordon Taylor1, Linda Hogarth1

1Northern Institute for Cancer Research, Newcastle University, UK,2Department of Drug Research and Clinical Pharmacology, Linköping University, Sweden

The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used in the treatment of leukaemia. Incorporation of deoxythioguanosine nucleotides (dGs) into the DNA of thiopurine-treated cells causes cell death but there is also evidence that thiopurine metabolites, particularly the 6-MP metabolite methylthioinosine monophosphate (MeTIMP), inhibit de novo purine synthesis (DNPS).The toxicity of DNPS inhibitors is influenced by methylthioadenosine phosphorylase (MTAP), a gene frequently deleted in cancers. Since the growth of MTAP-deleted tumour cells is dependent on DNPS or hypoxanthine salvage, we would predict such cells to show differential sensitivity to 6-MP and 6-TG. To test this hypothesis, sensitivity to 6-MP and 6-TG was compared in relation to MTAP status using cytotoxicity assays in two MTAP-deficient cell lines transfected to express MTAP: the mismatch repair (MMR) defective T-cell acute lymphoblastic leukaemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a MMR-proficient lung cancer cell line (A549-MTAP-ve) transfected to express MTAP constitutively (A549-MTAP + ve). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intra-cellularly to MeTIMP, was markedly higher in both cell lines under MTAP-ve conditions. However, MTAP loss was associated with increased sensitivity to 6-TG in the MMR-proficient A549 cell line, but not the MMR-deficient Jurkat cell line. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dGs incorporation is the main cause of cytotoxicity with 6-TG. These data suggest that thiopurines, particularly 6-MP, may be more effective in patients with deleted MTAP.

S18. Mechanisms of purine release

S18-1

Release of ATP from peripheral nerves

Carles Solsona Sancho, Ezequiel Mas del Molino, Anna Nualart, Mireia Martin-Satue, Xenia Grandes

Department of Pathology and Experimental Therapeutics, University of Barcelona-IDIBELL-CIBERNED, Spain

Schwann cells of peripheral nerve are decorated with purinergic receptors, which we hypostasize may play an autocrine role. We have therefore investigated if there is a release of ATP from peripheral nerves under some stimulus. Bathing isolated fragments of mouse sciatic nerves with a medium containing luciferin and luciferase mixture (LLM) we have observed an increase of luminescence under electric stimulation, hypotonic and direct mechanical stimulus. The luminescence was detected through a high sensitivity digital camera. Moreover, observing isolated nerve fibers, we found that Ranvier nodes (RN) are hot spots of ATP release. On the other hand connexin 32 (Cx32) is highly expressed in RN. Immunoreactivity for Cx32 was not detected in nerve fibers isolated from Cx32 knockout mice and ATP release from sciatic nerves was impaired. We further analyzed the ATP conducting properties of Cx32 expressed in Xenopus oocytes. Cx32 was activated by depolarizing pulses in oocytes bathed with a solution containing (LLM). A release of ATP was recorded in the tail current activated during repolarization. Either the tail current or the ATP release were altered in the most frequent mutations isolated from patients and expressed in the oocytes. Charcot Marie Tooth linked to chromosome X (CMTX) is due to mutations of Cx32. The results presented here are suggesting that purinergic signaling may be implicated in the CMTX pathogenesis. This work is supported by SAF 2008/732 from the Spanish Government and La Marató de TV3.

S18-2

The origin and mechanism of ATP release in the nervous system

Beáta Sperlágh, Attila Heinrich

Institute of Experimental Medicine, Hungarian Academy of Sciences, Hungary

ATP and its extracellular breakdown product, adenosine are important signaling molecules in the CNS under physiological and pathological conditions. Accordingly, a wide variety of stimuli are known to release ATP and/or adenosine to the extracellular space in the nervous system, which could lead to purine levels sufficiently high to activate either ATP and adenosine receptors. These stimuli mimic physiological neuronal activity such as electrical stimulation and KCl depolarization or pathological challenge, such as combined oxygen-glucose deprivation, bacterial endotoxin or mechanical stimulation. Different stimuli utilize various release mechanisms for both ATP and adenosine. However, less information is available, whether these pathways also serve as a conduit of release for endogenous purines involved in the modulation of synaptic signaling and the source of purines also remained largely enigmatic under these conditions. The microelectrode biosensor technique developed by Dale et al. allows the detection of the efflux of neurotransmitters with better resolution than conventional neurochemical methods and also circumvent the limitations of widely used electrophysiological techniques. Using this technique, we characterized the efflux the ATP, adenosine and glutamate in the in vitro rat hippocampus. To activate the intact glia-neuron ensembles of the hippocampal slice, K+ depolarization was applied, and we demonstrate that ATP, glutamate and adenosine is released under these conditions from glial cells in response to a primary neuronal signal. The release mechanism of ATP and glutamate from glial cells involve connexin hemichannels and P2X7 receptors, whilst adenosine accumulation is partly independent from extracellular ATP breakdown and is mediated by pannexin hemichannels.

S18-3

Auto-inhibition of the ATP release channel Pannexin1

Gerhard Dahl, Feng Qiu

Department of Physiology and Biophysics, University of Miami Medical School, USA

Although originally discovered as a “gap junction” protein, Pannexin1 does not form cell-to-cell channels. Instead Pannexin1 forms channels in the plasma membrane allowing the exchange of molecules between the cytoplasm and the extracellular space. The properties of Pannexin1 channels are consistent with an ATP release function. The Pannexin1 channel is large (500 pS single channel conductance) and highly permeable to ATP. It is mechano-sensitive, can be activated by ATP through purinergic receptors and opens in response to increased cytoplasmic calcium. Pannexin1 is expressed in all (?) cells exhibiting channel-mediated ATP release including erythrocytes. In cells with polar ATP release as in bronchial epithelial cells, Pannexin1 is localized accordingly. ATP release is proportional to Pannexin1 protein content and the pharmacologies of ATP release and that of Pannexin1 channels match. Consistent with the uptake of both cationic and anionic dyes under conditions of channel-mediated ATP release, the Pannexin1 channel is not charge selective. The observation that extracellular ATP inhibits Pannexin1 channels lends further support to the ATP release function of this channel. The channel inhibition is exerted by all ligands of the P2X7 receptor irrespective of the agonist or antagonist function at the receptor by the compounds. The affinity of Pannexin1 for the various compounds is almost two orders of magnitude lower than that of the P2X7 receptor, allowing for initial positive feedback followed by negative control of ATP release by Pannexin1 channels. The binding site for ATP on Pannexin1 is complex and involves moieties in both extracellular loops of the protein.

Oral Communications and Posters

P1. P1 Adenosine

P1-1

Adenosine potentiates human mast cell fibrinolytic activity

Michal Sereda1, Peter Bradding2, Catherine Vial1

1Cell Physiology and Pharmacology, University of Leicester, UK,2Infection, Immunity and Inflammation, University of Leicester, UK

Mast cells are mostly known for their important role in the development of inflammation and allergies. They also contribute to the development of cardiovascular diseases including thrombosis and arthrosclerosis. For example, by secreting tissue-type plasminogen activator (tPA), mast cell can initiate fibrinolysis. The purpose of this study was to determine whether adenosine, a potent mast cell activator, can modulate mast cell fibrinolytic activity. We found that 100 mM adenosine (2 h at 37°C) significantly increased mast cell tPA activity (by 58.6 ± 3.5% for HMC-1(human mast cell line) and 55 ± 6.8% for HLMC (human lung mast cells)) and tPA transcript expression level (by 6.4 folds for HMC-1 and 3.6 folds for HLMC). Adenosine deaminase abolished this potentiation. Real-time PCR revealed that HMC-1 predominantly expressed A2A and A2B receptors while HLMC abundantly expressed A2A, A2B and A3 subtypes. ZM241385 (1 mM), an A2A antagonist, significantly reduced adenosine induced mast cell tPA activity potentiation by 86.2% in HMC-1 (65% in HLMC); an inhibition similar (87.4% in HMC-1) to which obtained with a cocktail of antagonists for A1, A2A, A2B and A3 receptors (each at 1 mM). We also assessed mast cells fibrinolytic properties using a clot lysis assay (measuring fibrin clot lysis 10 hours after the full formation of a tissue factor-induced fibrin clot). Supernatants of HMC-1 treated for 24 h with adenosine (100 μM) significantly increased clot lysis by 92.6%. This effect was abolished by ZM241385. In conclusion, our results show that adenosine potentiates mast cell fibrinolytic activity mainly through the A2A adenosine receptor.

P1-2

Hydrogen peroxide generated from cigarette smoke exposure plays a role in inhibiting adenosine-mediated wound closure in bronchial epithelial cells

Diane Allen-Gipson1, Hui Zhang1, Justin Jarrell1, Jocelyn Jones2, Matthew Zimmerman2

1Pulmonary Critical Care, Sleep & Allergy Division, Department of Internal Medicine, University of Nebraska Medical Center, USA,2Department of Cellular Integrative Physiology, University of Nebraska Medical Center, USA

Background: We hypothesize cigarette smoke exposure generates an imbalance between oxidants and antioxidants in BEC and this imbalance impairs adenosine-mediated wound repair and recovery. Methods: Taqman PCR, Western Blot, total glutathione levels and HPLC studies were conducted in BEAS-2B cells exposed to ±5% cigarette smoke extract (CSE). Cells were then transiently transfected with adenovirus vectors encoding either the superoxide scavenging enzymes (SOD), or hydrogen peroxide scavenging enzymes (catalase), exposed to ±5% CSE and stimulated with A2A receptor agonist (CPCA; 10 microM) and wound closure was assessed. Subsets of control cells were infected with an empty viral vector. Results: Both PCR and Western blot confirmed CSE exposure markly elevated all four subtypes of adenosine receptors. HPLC analysis revealed significant increase in intracellular adenosine accumulation and reduction in glutathione levels in CSE-treated cells. Cells overexpressing mitochondrial catalase completely repealed CSE inhibition of CPCA-stimulated wound closure. On the other hand, cells infected with superoxide had no effect. Conclusion: Collectively, these data demonstrate that CSE-generated hydrogen peroxide play a role in the disturbances of tissue repair processes in the lung. This abstract is funded by: Nebraska Health and Human Services- LB506, Nebraska EPSCoR, and NIH-NHLBI HL084684

P1-3

Adenosine and 2′-deoxyadenosine modified with boron clusters (C2B10H12) as new classes of human blood platelet and neutrophil function modulators

Zbigniew Jan Leśnikowski, Katarzyna Bednarska, Agnieszka Olejniczak, Blażej Wojtczak, Agnieszka Piskała, Zofia Sułowska

IMB PAS, Institute for Medical Biology PAS, Poland

Background: The ability of adenosine to interact with human circulating blood cells, including platelets, lymphocytes and neutrophils is related to its role in regulating aggregatory, immune and inflammatory processes and forms a rationale for search for new adenosine drugs. Methods: Several adenosine-boron cluster conjugates and corresponding phosphates containing modification at C-2′, C-2, C-8 and N-6, have been synthesized and tested for their activity as inhibitors of platelets aggregation after activation with thrombin- or ADP, and production of reactive oxygen species by neutrophils. Results: The adenosine modification with para-carborane at the 2′ position results in the efficient inhibition of platelet function, including thrombin- or ADP-induced aggregation, protein secretion and P-selectin expression. Production of reactive oxygen species by adenosine conjugates depends upon the structure of boron cluster and the length and structure of the linker between the boron and nucleoside components. Conclusions: These preliminary findings, and the new chemistry proposed, form the basis for the development of a new class of adenosine derivatives that modulate human blood platelet activities. Acknowledgment: This work was supported in part by the Polish Ministry of Sciences and Higher Education, funds for science 2007–2010, grant number PBZ K152/H03/2007/10.

P1-4

Activation by guanosine of a putative GPCR

Gabriella Marucci, Michela Buccioni, Carmen Lammi, Catia Lambertucci, Rosaria Volpini, Gloria Cristalli

Medicinal Chemistry Unit, School of Pharmacy, University of Camerino, Italy

G protein coupled receptors (GPCRs), recognizing adenosine and purine and pyrimidine nucleotides as extracellular messengers, have been characterized and classified as purinergic P1 and P2 receptors, respectively. However, some experimental data suggest that other nucleosides as guanosine, inosine and hypoxanthine can also act as signalling molecules via the activation of specific membrane receptors.1 Binding studies, performed on rat brain membranes using [3H]guanosine, have shown that the compound interacts with binding sites, which are distinct from the well-characterized ATP and adenosine receptors. Furthermore, guanosine itself and 6-thioguanosine are equally effective in displacing [3H]guanosine from these sites.2 Starting from these observations and aimed at demonstrating the existence of a putative GPCR for guanosine, Eu-GTP assay was performed in rat brain membranes. This functional assay seems to demonstrate that a GPCR activated by guanosine exists in rat brain. Hence, in order to verify the stability of the nucleoside, it was detected by HPLC. The results showed that guanosine is stable at Eu-GTP assay conditions so confirming the activation by guanosine of a putative GPCR.

1) Caciagli F., Illes P. Progress in Purinergic receptor pharmacology and function PUSI, 1, 297–298, 2005.

2) Traversa U., Bombi G., Camaioni E., Macchiarulo A., Costantino G., Palmieri C., Caciagli F., Pellicciari R. Rat brain guanosine binding site. Biological studies and pseudo-receptor construction Bioorg. Med. Chem. 11(24), 5417–5425, 2003.

P1-5

Importance of selected amino acid residues of the human adenosine A2B receptor for ligand binding and receptor activation

Anke Schiedel1, Dominik T. Thimm1, Sonja Hinz1, Farag Sherbiny2, Thomas Borrmann1, Astrid Maaß2, Christa E. Müller1

1PharmaCenter Bonn, Pharmaceutical Chemistry I, University of Bonn, Germany,2SCAI, Fraunhofer Institute, Germany

Background: In the present study we combined homology modeling of the A2B receptor and exchange of selected amino acids by mutagenesis in order to gain deeper insights into the structure and function of the human A2B receptor. Methods: Wild type and mutant receptors were stably expressed in CHO cells using a retroviral transfection system. Radioligand binding studies were performed with the antagonist radioligand [3H]PSB-603. Activation of the receptors was monitored by measuring stimulation of adenylate cyclase with two structurally diverse agonists. Modeling of the extracellular loop 2 was performed with ModLoop and minimized using the Amber package. The models were evaluated with PROCHECK and PROSAII. Results: The results indicate that the conserved Cys3.25 in helix 3 forms a disulfide bond with Cys45.50 in the second extracellular loop. Trp6.48 and Ser7.42 appear to be involved in binding of NECA, but not in binding of the non-nucleosidic agonist BAY60-6583. The opposite could be observed for Tyr1.35. For antagonist binding, Asn5.42 was found to be particularly important. Leu3.28 and Val6.51 are likely to play a role in agonist as well as in antagonist binding. His7.34 appears to be important for receptor conformation. Conclusion: Based on these results and the X-ray structure of the adenosine A2A receptor, a refined model of the A2B receptor was obtained. This improved model will be used for docking experiments for the design of new drugs, especially antagonists, which have been proposed for the treatment of a number of pathological conditions including bronchial inflammation, asthma, pain, atherosclerosis, and cancer.

P1-6

The second extracellular loop of the adenosine A2A and A2B receptor: role in receptor activation

Benjamin F Seibt, Anke C Schiedel, Christa E Müller

PharmaCenter Bonn, Pharmaceutical Chemistry I, University of Bonn, Germany

Background: The human adenosine A2B receptor, which belongs to the familiy of G protein-coupled receptors (GPCRs), plays an important pathophysiological role in inflammatory processes, especially in respiratory diseases, where it appears to have a proinflammatory role. In contrast to the closely related A2A receptor subtype, which mediates antiinflammatory and immunosuppressive effects at low, nanomolar adenosine concentrations, the A2B receptor is typically only activated by much higher, micromolar concentrations of adenosine. The second extracellular loop (EL2) is known to participate in ligand binding in many GPCRs. The EL2 of the A2B receptor is several amino acids longer then the EL2 of the other adenosine receptor subtypes. Methods: In the present study the complete EL2 of the A2B receptor was replaced by the EL2 of the A2A receptor by overlap extension mutagenesis. The resulting receptor mutant A2B(EL2-A2A) was stably expressed in CHO cells using a retroviral expression system, and characterized by radioligand binding studies using [3H]PSB-603 as a radioligand, and by functional cAMP assays. Results: All agonists investigated (adenosine, NECA, and the non-nucleosidic A2B agonist BAY60-6583) showed increased efficacy at the mutant A2B(EL2-A2A) receptor as compared to the wildtype (wt) A2B receptor (using forskolin as a control). In contrast to the wt A2B receptor, A2B(EL2-A2A) could be activated by the A2A-selective agonist CGS21680 at micromolar concentrations. Conclusion: The EL2 of the adenosine A2 receptors appears to play an important role in receptor activation.

P1-7

CCR2 enhances P2X4 receptor trafficking to the plasma membrane of microglia

Emika Toyomitsu, Makoto Tsuda, Hidetoshi Tozaki-Saitoh, Kazuhide Inoue

Dept. Mol. Syst. Pharmacol., Grad. Sch. Pharm. Sci., Kyushu Univ., Japan

Microglia, resident immune cells in the CNS, sense various extracellular stimuli such as proinflammatory cytokines and chemokines, and nucleotides via their cognate receptors. The P2X4 receptor (P2X4R) is a subtype of the purinergic ionotropic receptors expressed in microglia. P2X4R expression is upregulated under inflammatory or neurodegenerative conditions, and activation of microglial P2X4Rs by extracellular ATP is crucial for a neuropathic pain. It has recently been shown that P2X4Rs are localized predominantly in intracellular lysosomal compartments. It is therefore important to determine how P2X4Rs are recruited from lysosome to the cell surface membrane. In quantitative cell surface biotinylation assay we found that applying CCL2 and CCL12, ligands for CCR2, to microglial cells increased P2X4R expression in a cell surface fraction without changing P2X4 protein level in whole-cell lysate. The effect of CCL2 was inhibited by its neutralizing antibody. The CCR2-mediated P2X4R trafficking was enhanced by the pretreatment with dynasore, an inhibitor of P2X4R endocytosis. We also found that activation of CCR2 increased the release of the lysosomal enzyme beta-hexosaminidase. Finaly, P2X4R-mediated Akt phospholilation is increased by the pretreatment with CCL2. These results suggest that CCR2 signaling may increase the P2X4R expression on the surface membrane of microglia through lysosomal exocytosis.

P1-8

Electrostatic Interactions as key determinants of the quaternary structure of receptor heteromers

(Selected for Oral Poster Presentation)

Gemma Navarro Brugal1, Sergi Ferre2, Arnau Cordomi3, Estefania Moreno1, Josefa Mallol1, Vicent Caçadó1, Antoni Cortés1, Hanne Hoffmann4, Jordi Ortiz4, Enric I. Canela1, Carme Lluís1, Leonardo Pardo3, Rafael Franco1, Amina S. Woods2

1Departament de Bioquímica i Biología Molecular, Universitat de Barcelona, España,2NIDA, NIH, United States,3Laboratori de Medicina Computacional, Unitat de Bioestadística, España,4Universitat Autònoma de Barcelona, Universitat Autònoma de Barcelona, España

The field of G protein-coupled receptor (GPCR) heteromerization is growing by leaps and bound. Little is known about what determines the quaternary structure of GPCR resulting in their unique functional properties. Using Bioluminescence Resonance Energy Transfer (RET) techniques we provide clear evidence of a key role of electrostatic interactions in the determination of the proper quaternary structure of adenosine A2A-cannabinoid CB1-dopamine D2 receptor heteromultimers. These interactions involve hydrophilic intracellular domains that contain evolutionarily conserved arginine-rich epitopes, whose guanidinium groups form salt bridges with a phosphorylated serine or threonine residues from a CK1/2 consensus site. Experiments with selective mutation of these reactive amino acids demonstrated that each protomer in the A2A-CB1-D2 receptor heteromer contains two intracellular key domains that establish selective electrostatic interactions with intracellular domains of the other two protomers. Mutation-induced disruption of the quaternary structure of the A1-CB1-D2 receptor heteromer led to functional modifications (change in MAPK signaling), which allowed their identification in rodent brain tissue.

P1-9

Characterisation of adenosine A2-receptors in HEK293 cells using real-time analysis of intracellular cAMP levels

Joelle Alcock, Lauren May, Stephen Hill

Institute of Cell Signalling, University of Nottingham, UK

We have utilised the GloSensorTM (Promega) technology to study real-time, live-cell analysis of cAMP production in response to the stimulation of the endogenous adenosine receptors in HEK293 cells. GloSensorTM HEK293 cells were seeded in 96-well white-walled plates, left overnight then incubated at 35°C in atmospheric CO2 for 2 hr in 80 μL HBSS containing 4% GloSensorTM cAMP reagent. Antagonist pre-treatment involved exposure to XAC, ZM241385, PSB603 or SCH58261 (10-13 to 10-5 M) for 30 minutes at 35ºC prior to agonist addition. Luminescence was measured on an Envision plate reader (PerkinElmer) for 1 hr at 35ºC following the addition of the non-selective adenosine agonist N-ethylcarboxamidoadenosine (NECA) or the selective adenosine A2a agonist CGS21680 to achieve final concentrations of 10 μM and 1 μM respectively. NECA and CGS21680 both elicited a concentration dependent response in the HEK293 GloSensorTM cell line. The maximal response to CGS21680 was approximately 10% that of NECA. The responses to both NECA and CGS21680 were antagonised by pre-incubation with either the non-selective adenosine antagonist XAC or the A2a-selective ZM241385. Intriguingly, whilst the A2b selective PSB603 was able to antagonise the NECA response there was no effect on the CGS21680 response. Conversely whilst the A2a selective SCH58261 was able to antagonise the CGS21680 response there was no effect on the NECA response. Taken together, these data describe a mixed (A2a and A2b) population of adenosine receptors coupled to cAMP generation in HEK293 cell lines, with the A2b subtype being predominantly expressed.

P1-10

Investigating the function and membrane diffusion of a TM6 toggle switch mutant of the human adenosine A3 receptor

(Selected for Oral Poster Presentation)

Stephen Briddon, Leigh Stoddart, Ross Corriden, Stephen Hill

Institute of Cell Signalling, University of Nottingham, UK

Within TM6 of G-protein coupled receptors, there is a highly conserved tryptophan residue (6.48). This residue is thought to act as a toggle switch promoting the conformational change which forms the active conformation of the receptor. Mutation of this residue (W243) in the human adenosine A3 receptor compromises its ability to activate downstream signalling pathways. The wild type human A3 receptor and an A3 receptor with a W243F point mutation, both tagged on their C-terminus with a fluorescent protein, were expressed in Chinese Hamster Ovary cells and functionally characterised. The adenosine receptor agonist, NECA, showed a similar potency at the W243F mutant receptor compared to the wild-type A3 receptor when measuring inhibition of cAMP accumulation, calcium release or phosphorylation of ERK1/2, but its efficacy was substantially reduced. Expression of the wild type and mutant receptors at the cell surface and their internalisation in response to NECA was confirmed by confocal microscopy. Subcellular diffusion of the receptors in the membrane of living cells was determined using fluorescence correlation spectroscopy (FCS). The diffusion co-efficient of the mutant A3 W243F-GFP receptor and wild type A3-GFP receptor in the membrane was similar. However, specific measurement of the diffusion of agonist-occupied receptors (using the fluorescent agonist ABEA-X-BY630) indicated subtle differences between the two receptors. These results indicate that differences in the membrane organisation of agonist-occupied A3 W243F-GFP as compared to A3-GFP may reflect its reduced ability to couple to downstream effectors. This work was supported by the Medical Research Council Grant G0800006.

P1-11

Synthesis and pharmacological characterization of N6- and 8-substituted adenine derivatives as ligands for the recently discovered adenine receptors

Thomas Borrmann, Maria L. Bauer, Aliaa Abdelrahman, Christa E. Müller

PharmaCenter Bonn, Pharmaceutical Chemistry I, University of Bonn, Germany

Based on structure-activity relationships obtained in our previous studies,(1) we synthesized two series of N6-carbamoyl- and 8-amino-substituted adenine derivatives as adenine receptor ligands. N6-carbamoyl-substituted adenines (urea derivatives) were prepared by reaction of adenine with the appropriate isocyanate, or substitution of an ethyl carbamate intermediate with a variety of different amines. 8-Aminoadenine derivatives were synthesized from 8-bromoadenine, or 8-bromoadenosine, respectively, by nucleophilic substitution of the bromo function and, in the latter case, subsequent hydrolysis of the nucleosidic bond. Adenine receptor affinities were determined in radioligand binding studies at rat brain cortical membranes and membranes of human HEK293 cells. cAMP assays were performed with 1321 N1 astrocytoma cells stably expressing the rat adenine receptor as described.(1) The N6-carbamoylation of adenine with isocyanates resulted in insufficient N6/9-regioselectivity. Alternatively, the N6-substituent was introduced after formation of an ethyl carbamate at position N6 followed by substitution with various aliphatic amines. The direct nucleophilic substitution of 8-bromoadenine with amines required harsh reaction conditions and long reaction times, probably due to deprotonation and thus deactivation of the imidazole moiety. However, 8-bromoadenosine could easily be substituted with a variety of amines in a microwave-assisted reaction, and the products were subsequently hydrolyzed to yield the desired new 8-aminoadenine derivatives in excellent yields. The pharmacological characterization revealed that 8-amino-substitution of adenine is a favorable modification: 8-aminoadenine represents one of the most potent agonists at the human adenine receptor with a Ki value of 34.1 nM.(1) Our results indicate that N6-substituted or unsubstituted 8-aminoadenine derivatives are potent adenine receptor ligands and might serve as both, valuable pharmacological tools and potentially neuroprotective or analgesic drugs.

(1) Borrmann, T.; Abdelrahman, A.; Volpini, R.; Lambertucci, C.; Alksnis, E.; Gorzalka, S.; Schiedel, A. C.; Cristalli, G.; Müller, C. E. Structure-activity relationships of adenine and deazaadenine derivatives as ligands for adenine receptors, a new purinergic receptor family. J. Med. Chem. 2009, 52, 5974–5989.

P1-12

Adenosine A2A and BDNF TrkB receptors have independent roles upon neuronal maturation

(First Poster Presentation Award)

Filipa F. Ribeiro1, Raquel Neves-Tomé1, Natália Assaife-Lopes1, Rui Silva2, Dora Brites2, Joaquim A. Ribeiro1, Ana M. Sebastião1

1Institute of Pharmacology and Neurosciences, Faculty of Medicine and Unit of Neurosciences, Institute of Molecular Medicine, University of Lisbon, Portugal,2iMed.UL-Faculty of Pharmacy, University of Lisbon, Portugal

Adenosine modulates fast synaptic actions of brain-derived neurotrophic factor (BDNF) (Sebastião and Ribeiro, Br. J. Pharmacol. 2009, 158:15). BDNF is involved in neuronal maturation (Dijkhuizen and Ghosh, J. Neurobiol. 2005, 62:278), and its effects are regulated by cAMP (Ji et al., Nat. Neurosci. 2005, 8:164), which can be increased by activation of adenosine A2A receptors (A2AR). Therefore, we hypothesized that A2AR activation could influence the actions of BDNF on neurite outgrowth. E18 forebrain neurons from Sprague-Dawley rats were seeded at 104cells/cm2. At DIV 3, neurons were treated with CGS21680 (10–100 nM), ZM241385 (50 nM) and/or BDNF (20 ng/mL) for three days. BDNF was applied 20 minutes after A2A ligands. Cells were stained with anti-MAP2 and analysed by HCA-Vision software. Fifty neurons were counted from each condition (3–4 independent cultures). The A2AR agonist, CGS21680, increased maximal and total neurite length, and the number of branch points (p0.05) by the presence of CGS21680 (10 nM) or ZM241385 (50 nM). These results suggest that A2AR are involved in the axon elongation process, while BDNF is involved in new primary dendrites formation. Futhermore, the effect of BDNF and A2AR agonists upon neuronal maturation seem to operate independently. BDNF was a gift from Regeneron, USA. Supported by FCT, Portugal.

P1-13

A new small molecule, D2 dopamine receptor agonist, as an allosteric modulator of A2A adenosine receptor signalling

(Selected for Oral Poster Presentation)

Maria Letizia Trincavelli1, Elisabetta Orlandini2, Simona Daniele1, Susanna Nencetti2, Elisa Nuti2, Marco Macchia2, Harald Huebner3, Peter Gmeiner3, Armando Rossello2, Claudia Martini1

1Dept. Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Italy,2Department of Pharmaceutical Sciences, University of Pisa, Italy,3Department of Chemistry and Pharmacy, Emil Fischer Center, Friedrich Alexander University, Germany

Background: In the field of newly pharmacological approaches towards Parkinson’disease (PD), particular emphasis has been placed on A2A adenosine receptor (AR) antagonists, alone or in combined therapy with D2 dopamine receptor (DR) agonists. The A2A ARs and D2 DRs mediate the antagonism between adenosine and dopamine transmission in central areas implicated in PD, and represents a pharmaceutical target for its treatment. We focused our study to the synthesis and the biological evaluation of new D2 DR agonists able to modulate the A2A AR signalling. Methods: The affinities of the synthesized compounds to different G protein coupled receptors were evaluated by radioligand competition studies. Functional activity towards D2Long DR was assessed by [35S]GTPγS binding assay. The effect of cell treatment with the compounds on A2A AR binding parameters and on functional responsiveness were evaluated by Scatchard analysis of [3H]NECA competition binding data and by cAMP assay, respectively, using cells stably expressing A2A AR and D2Long DR. Results: The most promising compound, MM1, showed an appreciable affinity for the D2Long/D4/D3 DRs; in the GTPγS binding assay it proved to be a full agonist at the D2Long DR. Moreover, without binding at ARs, it was able to negatively modulate both the A2A AR affinity and functionality, thus acting as a D2 DR/A2A AR modulator. Conclusion: MM1 may be consider an innovative start point for the development of new drugs for the treatment of PD.

P1-14

Cardioprotection by Adenosine (Ado): role of AMPK and PFK-2 in Ado-mediated inhibition of glycolysis

Mohamed Omar, Sanam Verma, Alexander Clanachan

Department of Pharmacology, University of Alberta, Canada

Background: Adenosine (Ado)-mediated improvements in recovery of post-ischemic left ventricular (LV) function may be due, in part, to inhibition of glycolysis that limits proton (H+) production and Ca2+ overload during reperfusion. This study sought to determine mechanisms responsible for the beneficial effect of Ado on myocardial glucose metabolism. Methods: Isolated working rat hearts were perfused with Krebs solution containing 100 microU/ml insulin and 1.2 mM palmitate and 11 mM [3H/14C]glucose as energy substrates. After an initial baseline perfusion (45 min), hearts were subjected to global ischemia (GI, 17 min) and reperfusion (30 min). Ado (500 microM) or vehicle were added 5 min prior to the onset of GI and were present throughout reperfusion. Hearts were frozen at the end of GI and at the end of reperfusion for biochemical measurements. Results: Relative to vehicle-treated hearts, Ado improved recovery of post-ischemic LV work by 83%. Ado inhibited glycolysis (by 38%), H+ production (by 43%), and glucose uptake (by 35%) during reperfusion, but did not affect glucose oxidation. Ado decreased phosphorylation of 5′-AMP-activated protein kinase (AMPK) by 81% and phosphofructokinase-2 (PFK2) by 42%. Ado also decreased the ratio of fructose-1,6-bisphosphate to fructose-6-phospate by 56%, indicative of inhibition of phosphofructokinase-1 (PFK1). Ado also increased glycogen content at the end of reperfusion (by 50%), which was due to reduced glycogenolysis during ischemia as well as during reperfusion. Conclusion: Our study indicates that Ado-mediated inhibition of glycolysis is due to inhibition of PFK1, an action mediated by inhibition of AMPK and its downstream target PFK2.

P1-15

Synthesis, biological activity and molecular docking analysis of new A2A adenosine receptor agonists

(Selected for Oral Poster Presentation)

Anna M. Rodríguez1, Gloria Rosell2, Jordi Bujons1, Hugo Gutiérrez-de-Terán3, David Rodríguez3, José M. Brea4, M. Isabel Loza4, Ángel Guerrero1, M. Pilar Bosch1

1Dept. of Biological Chemistry and Molecular Modelling, Jordi Girona 18–26, 08034 Barcelona, IQAC-CSIC, Spain,2Dept. Pharmacology and Therapeutic Chemistry (Unity Associated to CSIC), Fac. of Pharmacy, Av. Diagonal s/n, 08028 Barcelona, University of Barcelona, Spain,3Hosp. Clínico Universitario de Santiago, Santiago de Compostela, Fundación Pública Galega de Medicina Xenómica, Spain,4USEF, Dept. Pharmacology, Fac. of Pharmacy, Campus Universitario Sur s/n, 15782 Santiago de Compostela, University of Santiago de Compostela, Spain

Following a previous work from our group (J. Med. Chem. 2004, 47, 4041–4053), we present herein the synthesis, molecular docking analysis and biological evaluation of new 2-alkylaminoadenosine derivatives as potential agonists for the human A2A adenosine receptor (A2AAR). These derivatives contain an ethyl-substituted tetrazole moiety at the ribose ring, a chiral alkyl group at the vicinal position of the amine and different substituted aromatic systems. Synthesis of the chiral amines has been achieved by enzymatic resolution methodologies. Biological activity of the potential agonists has been evaluated by radioligand binding assays using recombinant human ARs, and by determination of the cAMP production in these receptors expressed in transfected CHO (A1AR), HeLa (A2AAR, A3AR) and HEK-293 (A2BAR) cells. Elucidation of the molecular interactions of these potential agonists was achieved through docking studies using the previously solved X-ray crystallography structure of human A2AAR (Science 2008, 322, 1211–1217). Some compounds displayed a remarkable potency and affinity for A2AAR (Ki 5–56 nM, EC50 2–19 nM) and low or negligible affinity for A1AR, A2BAR and A3AR. Computational simulations suggest a ligand binding mode in agreement with the mutagenesis and structural data available for the A2AAR (J. Med. Chem. 2003, 46, 4847–4859), with the phenyl ring oriented in the solvent-exposed extracellular side. New adenosine derivatives with high A2AAR selectivity and potency have been developed. Our results suggest that substitution at the para-position of the phenyl ring by different groups has no significant effect in binding affinity for the human A2AAR.

P1-16

A2A adenosine receptors and ionotropic glutamate receptors are involved on gmp- or guanosine-induced trophic effects in cultured cerebellar granule neurons

Carla Ines Tasca1, Helena Decker1, Claudia Mendes de Aguiar2, Luciana Romao3, Carina Boeck4, Vivaldo Moura-Neto3

1Biochemistry Department, Federal University of Santa Catarina, Brazil,2Cell biology Department, Federal University of Santa Catarina, Brazil,3Anatomy Department, Federal University of Rio de Janeiro, Brazil,4Health Sciences Postgraduation programm, Universidade do Extremo Sul Catarinense, Brazil

The purines, guanosine-5′-monophosphate (GMP) and guanosine (GUO) are implicated in extracellular functions, as modulation of glutamatergic transmission and trophic effects on neural cells. We showed GMP or GUO altered the organization of extracellular matrix proteins, laminin and fibronectin, in astrocytes and increased the number of cocultured cerebellar neurons. Our aim was to analyze the receptors and signaling pathways involved on the trophic effect of these purines in cultured cerebellar granule neurons. Cerebellar granule neurons were cultured with a conditioned medium obtained from GMP- or GUO-treated astrocytes in order to evaluate a neuron-astrocyte interaction. Neurons cultured in Neurobasal medium with GMP or GUO (1–1,000 microM for 24 h) were evaluated for a direct action of purines on neuronal cells. In both culture models, GMP or GUO induced a significant increase in neuronal number and improvement in neuronal adhesion, however the neuritogenesis pattern and cell viability were not altered. Regarding the signaling pathways involved on the increased neuronal number induced by GMP or GUO, the participation of A2A (but not A1) adenosine receptors, and NMDA and Kainate subtypes of glutamate receptors was demonstrated. The blockade of the extracellular-regulated kinase (ERK), calcium-calmodulin dependent kinase II (CaMKII), protein kinase C (PKC), phosphatidilinositol-3´-kinase (PI3-K) and protein kinase A (PKA) activities, inhibited GMP or GUO effect. These results showed trophic effects of GUO or GMP due to the improvement of cerebellar granule cells adhesion, an effect mediated by activation of A2A adenosine receptors, ionotropic glutamate receptors and a complex interaction of signaling pathways.

P1-17

PSB-603—a new A2B antagonist radioligand with subnanomolar affinity and subtype specificity from mouse to man

Sonja Hinz, Thomas Borrmann, Daniela C.G. Bertarelli, Anke C. Schiedel, Christa E. Müller

PharmaCenter Bonn, Pharmaceutical Chemistry I, University of Bonn, Germany

Background: 8-(4-(4-(4-Chlorophenyl)piperazin-1-ylsulfonyl)phenyl)-1-propylxanthine (PSB-603) was identified as a highly selective and potent adenosine A2B receptor antagonist (Borrmann et al., J. Med. Chem. 2009, 3994). Methods: In the present study the new radioligand [3H]PSB-603 was characterized in kinetic, saturation and competition binding experiments at membrane preparations from CHO cells expressing human or mouse A2B receptors. Furthermore, unlabeled PSB-603 was investigated in radioligand binding studies at all adenosine receptor subtypes of various species. PSB-603 was also evaluated in cAMP and calcium mobilization experiments in Jurkat-T cells, a human leukemia cell line which natively expresses the human A2B receptor. Results: From association and dissociation experiments at human recombinant A2B receptors, a kinetic KD-value of 0.652 nM was determined, which was in close agreement with the KD-value obtained in saturation experiments (0.403 nM). Competition experiments of different standard agonists and antagonists versus 0.3 nM [³H]PSB-603 yielded Ki-values which were in accordance with previously published data. In homologous competition experiments at mouse A2B receptors a KD-value of 0.351 nM was determined. cAMP and calcium assays with Jurkat-T cells showed, that PSB-603 was able to antagonize the effects of NECA with a KB value of 0.871 nM (cAMP) and an IC50 value of 1.13 nM ([Ca2+]i). Conclusion: Based on these results we conclude that PSB-603 and its [3H]-labeled form are useful pharmacological tools. In contrast to many other A2B antagonists they are not only selective for human, but also for rodent A2B receptors.

P1-18

Adenosine A2A receptors are mainly coupled to the MAPK rather than cAMP pathway in HEK-293 cells

Rui Sanches, Ricardo Rodrigues, Rodrigo Cunha

Center for Neuroscience of Coimbra, University of Coimbra, Portugal

Adenosine A2A receptors (A2AR) are classified as triggering the cAMP/ PKA pathway. Bolstering the cAMP/PKA pathway abrogates neuroprotection; however, it is mainly A2AR blockade (rather than activation) that affords neuroprotection in adults. This implies that A2AR recruit other transducing systems to control neurodegeneration and MAP kinases (MAPK) emerge as likely candidates (Canas et al., 2009 J.Neurosci. 29:14741). We now transfected HEK293 cells with human A2AR to explore their efficiency in recruiting cAMP and MAPK. CGS21680 (A2AR agonist) caused a larger (P < 0.05) increase of cAMP levels with 300 nM (460 ± 47%) than with 10 nM (283 ± 44%), which was prevented by SCH58261 (50 nM, A2AR antagonist). CGS21680 also triggered ERK1/2 activation with equi-effective effects (P > 0.05, n = 5–6) at 10 nM (181 ± 28%) and 300 nM (194 ± 26%), in a SCH58261-sensitive manner. The same was observed for p38 and for JNK phosphorylation: both increased similarly with 10 nM and 300 nM CGS21680, in a SCH58261-sensitive manner (n = 6) and these effects were unaffected by the PKA inhibitor H89 (1 μM). CGS26180 also decreased Akt activation, with effects larger (P < 0.05, n = 5) for 300 nM (40 ± 7%) than 10 nM (18 ± 3%) that were sensitive to SCH58261 (50 nM) and reverted by H89 (1 μM). These results show that A2AR are more efficaceous to activate MAPK pathways than the cAMP/PKA pathway. Furthermore, the ability of A2AR to activate JNK and p38 and decrease Akt activation, which are hallmarks of greater susceptibility to damage, provide a rationale to understand the neuroprotection afforded by A2AR blockade. (Supported by FCT)

P1-19

Down-regulation and desensitization of adenosine A1 receptors/adenylyl cyclase pathway in immature neurons

(Selected for Oral Poster Presentation)

Maria Angeles Ruiz Gonzalez, David Agustin Leon Navarro, Inmaculada Iglesias Lorenzo, Carlos Alberto Castillo Sarmiento, Jose Luis Albasanz Herrero, Mairena Martin Lopez

Facultad de Químicas, Centro Regional de Investigaciones Biomédicas, Universidad De Castilla-La Mancha, España

Adenosine A1 receptors (A1R) mediate neuroprotective actions of adenosine in brain. We have studied the effect of chronic treatment with (R)-phenylisopropyladenosine (R-PIA), a selective agonist of A1R, on A1R/adenylyl cyclase pathway in cultured immature neurons (4DIV) from cerebral cortex of 18-day-old rat embryos. Incubation with 100 nM R-PIA at 6, 12, 24 and 48 h elicited a time-dependent decrease in total A1R in plasma membranes (93, 58, 43 and 26% of control, respectively) determined by radioligand binding assay using [3H]DPCPX, selective A1R antagonist. This decrease was accompanied by a time-dependent increase in microsomal fraction (21, 56, 124 and 233%, respectively). Real-time RT-PCR assays showed a slight but significant decrease in mRNA level coding for A1R after 12 and 24 h (15 and 11%, respectively) followed by an increase at 48 h (23%). On the other hand, Gi proteins level, determined by Western-blotting, was significantly decreased in plasma membranes at 12 and 24 h of agonist exposure. mRNA level coding alpha-subunit of Gi protein was significantly decreased at 6, 12 and 24 h (20–25%) and increased at 48 h (33%) of treatment. Finally, adenylyl cyclase inhibition elicited by 1 µM CPA (2-Chloro-N6-cyclopentyladenosine), a selective A1R agonist, was significantly decreased after 12, 24 and 48 h of treatment (37, 24 and 23%, respectively) compared with control (54%), suggesting a desensitization of A1/AC pathway. These results suggest that in immature neurons, adenosine A1 receptors shows a similar pattern of desensitization to that described in both full-term fetuses and adult brains.

P1-20

Presynaptic A2A adenosine and CB1 cannabinoid receptors inhibit the function of one another at corticostriatal terminals

Samira Ferreira, Rodrigo Cunha, Attila Köfalvi

Center for Neurosciences of Coimbra, University of Coimbra, Portugal

Background: Δ9-tetrahydrocannabinol (CB1R agonist) and caffeine (A2AR antagonist) are widely used psychoactive substances affecting striatal synaptic plasticity. CB1Rs and A2ARs were recently found to form heteromers in expression systems. Thus, we aimed at exploring the possible interaction between presynaptic striatal CB1Rs and A2ARs. Methods: The following experiments were carried out in the dorsolateral striatum of adult male Wistar rats, and in C57Bl/6 WT, CB1R, and A2AR null-mutant mice: 1) flow cytometric immunoanalysis of purified nerve terminals for the co-localization of presynaptic proteins; 2) competitive binding assays in synaptic and extrasynaptic membranes; 3) [14C]glutamate release assay in purified nerve terminals. Results: CB1Rs and A2ARs co-localize in a subset of vGluT1-positive, i.e. corticostriatal terminals. Specific A2AR and CB1R binding mostly confined to the synaptic membrane fraction. A2AR activation by CGS21680 (30 nM) inhibited synaptic CB1R binding (Bmax) by 29.0%, whereas CB1R activation by WIN55212-2 (1 microM) abolished synaptic A2AR binding. This effect of WIN55212-2 was prevented by the CB1R antagonist, LY320135 (1 microM). The CB1R agonists, WIN55212-2 and O-2545 inhibited the 4-aminopyridine-evoked release of [14C]glutamate and [3H]GABA, which were antagonized both by LY320135 and CGS21680. Conclusions: Presynaptic CB1Rs and A2ARs inhibit the function of one another in the dorsolateral striatum. Since endocannabinoids and adenosine accumulate at high-frequency synaptic discharge, the putative presynaptic CB1R/A2AR heteromers may provide an important relay function in striatal synaptic plasticity, and thus, in the (patho)physiology of the basal ganglia. (Supported by FCT)

P1-21

Absence of synaptic modifications related to adenosine A1 receptor-mediated control of restraint-stress-induced preconditioning

Henrique Silva1, Angelo Tomé1, Manuella Kaster1, Carolina Souza2, Geanne Andrade2, Paula Canas1, Paula Agostinho1, Rodrigo Cunha1

1Center for Neuroscience of Coimbra, University of Coimbra, Portugal,2Laboratory of Neuroscience and Behavior, Department of Physiology and Pharmacology, Federal University of Ceará, Brazil

We recently found that the activation of A1 receptors (A1R) plays a key role in the implementation of preconditioning, whereby a single period (2 hrs) of restraint stress limits neurodegeneration triggered later (24 hrs) by convulsive or ischemic insults. We now tested if this involved a modification of presynaptic A1R function. Male adult (2 month) rats were subject to restraint stress (2 hours) or only handled (control) and sacrificed 24 hours later to evaluate synaptic transmission (Schaffer fiber-CA1 pyramid synapses) and modifications of transducing systems in extracts from whole cerebral cortex or from purified cortical nerve terminals (from the contralateral hemisphere of the same rat). Stressed rats displayed enhanced expression (39.6 ± 4.2%, n = 6) of A1R mRNA and enhanced density of A1R in total membranes (21.4 ± 4.0%, n = 5) but not in synaptosomal membranes (10.5 ± 3.9%, P > 0.05). The potency of 2-chloroadenosine to decrease synaptic transmission (A1R-mediated) was actually decreased in stressed rats (EC50 of 618 ± 28 nM in stressed and 418 ± 13 nM in control, n = 5–7), with no change of tonic A1R-mediated inhibition (9–13% facilitation by 2 U/ml adenosine deaminase or 100 nM DPCPX, n = 3–4). Stressed animals also displayed increased protein levels of PKC epsilon (24.2 ± 4.6, n = 5), phosphorylated ERK1/2 (31.0 ± 6.2, n = 5), and phosphorylated PI3K (53.6 ± 5.7, n = 6), in whole hippocampal extracts, whereas only a mild increase of PKC epsilon was found in purified cortical nerve terminals (12.8 ± 3.7, n = 5). This indicates that preconditioning is mostly associated with extra-synaptic changes of A1R and transducing mediators. (Supported by FCT)

P1-22

Screening in S. cerevisiae for constitutively inactive mutants of the adenosine A2B receptor

(First Oral Poster Presentation Award)

Miriam Peeters, Qilan Li, Adriaan Pieter IJzerman

Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands

Mutagenesis studies have proven to be an informative and solid approach when investigating the activation mechanism of receptors. It is a generally used tool to identify single amino acid changes that lead to a change in activity of the receptor and thus are involved in one of the many steps in the activation mechanism. Here we describe a new experimental approach that combines an unbiased random mutagenesis strategy with a screening method in yeast that allows for fast identification of essential residues in receptor activation. We randomly induced mutations at low frequency in a fragment of the adenosine A2B receptor that includes transmembrane domain 4, the second extracellular loop, and transmembrane domain 5. The resulting mutagenic library was subsequently screened for Constitutively Inactivating Mutants (CIMs) using the S. Cerevisiae MMY24 yeast strain. This strain has been genetically modified that enables us to measure growth as a simple read-out for activation, which is based on the production of histidine, the essential amino acid for yeast growth. By simply changing concentrations of a suppressor of histidine synthesis, 3-aminotriazole, and changing the incubation time after which colonies are selected, we are able to use the same yeast strain to screen for inactivating as well as activating single point mutations.

P1-23

An essential role for the first extracelullar loop in activating the adenosine A2B receptor

Miriam Peeters, Dong Guo, Margot Beukers, Gerard van Westen, Adriaan Pieter IJzerman

Leiden/Amsterdam Center for Drug Research, Leiden University, The Netherlands

Next to the intracellular and transmembrane domains, the highly variable extracellular loops in G protein-coupled receptors (GPCRs) have been reported to be involved in receptor activation. However, the molecular mechanisms of activation are still poorly understood, which is not surprising given the huge diversity of these extracellular regions among GPCR family members. In a random mutagenesis screen on the adenosine A2B receptor (A2BR) using the MMY24 S. Cerevisiae strain as a read-out system we found that two residues in the first extracellular loop (EL1), a phenylalanine and an aspartic acid at positions 71 and 74 respectively, are involved in receptor activation. The potency of the full agonist NECA and the level of constitutive activity were increased in these two receptor mutants, F71L and D74G, respectively. Subsequent site-saturation mutagenesis experiments revealed that the introduction of other mutations at either of the identified positions results in a wide variety of receptor activation profiles. For instance, the substitution for a glutamine at position 74 again resulted in an increase in NECA potency and constitutive activity. Arginine at this position was comparable to the wild-type receptor, whereas the hydrophobic phenylalanine led to a decrease of NECA potency and intrinsic activity. We interpreted these data in the light of the recently revealed structure of the adenosine A2AR, the closest homologue of the A2BR. The two residues are hypothesized to be vital in maintaining the tertiary structure of the extracellular domain of the A2BR.

P1-24

Adenosine deaminase as an allosteric modulator of A2A adenosine receptor

Eduardo Gracia Sánchez, Antonio Cortés Tejedor, Estefania Moreno Guillen, Jana Bakesova, Kamil Pérez-Capote, Josefa Mallol Montero, Enric I. Canela Campos, Rafael Franco Fernández, Carmen Lluís Biset, Vicent Casadó Burillo

Departament de Bioquímica i Biología Molecular, Facultat de Biologia, Universitat de Barcelona, Spain

It has been shown that adenosine deaminase (ADA; EC 3.5.4.4) behaves as an ecto-enzyme anchored to membrane proteins, among them CD26, A1 (A1Rs) and A2B (A2BRs) adenosine receptors. In this study using colocalization, radioligand binding and bioluminescence resonance energy transfer (BRET) techniques, we have demonstrated that ADA interacts with another subtype of ARs, the adenosine A2A receptor, in an activity-independent form. Taking in consideration that A2ARs form homodimers and taking advantage of a new procedure to fit binding data to receptors dimers, here it is demonstrated that ADA markedly enhances agonist binding to sheep striatal A2ARs and also increases the ERK1/2 phosphorylation in Chinese ovary hamster (CHO) cells. The results show that ADA binds to A2ARs behaving as an allosteric effector that markedly enhances agonist affinity and increases receptor functionality. This powerful regulation has important implications for the physiology and pharmacology of neuronal A2ARs.

P2. P2X

P2-1

Hypoxic intensity: a determinant for the role of ATP and adenosine on carotid body chemotransduction

Silvia Vilares Conde1, Emilia C. Monteiro1, Ricardo Rigual2, Ana Obeso2, Constancio Gonzalez2

1CEDOC, Department of Pharmacology, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Portugal,2Instituto de Biología y Genética Molecular, Departamento de Bioquímica y Biología Molecular y Fisiología, Facultad de Medicina, Universidad de Valladolid, CSIC, Spain

Excitatory effects of adenosine and ATP on carotid body (CB) chemotransduction have been described. Our hypothesis is that both, ATP and adenosine are the main responsible for the hypoxic chemotransmission in the CB sensory synapse, depending the contribution of each neurotransmitter on hypoxic intensity. Our hypothesis will be tested measuring carotid sinus nerve (CSN) activity in response to different hypoxic intensities in the absence and in the presence of adenosine and ATP receptors antagonists. Additionally, we investigated how the release of adenosine and ATP and their pathways were modified by the hypoxic intensity (10, 5 and 2%O2). We found that ZM241385, an A2 antagonist, decreased by 27.51 and 69.23% the CSN discharges evoked by 0 and 7%O2, respectively. Suramin, a P2X antagonist, decreased in 83.61 and 31.43% the CSN discharges evoked by 0 and 7%O2, respectively. Simultaneous application of both antagonists inhibited almost completely CSN discharges elicited by both hypoxic intensities. ATP release by the CB increased in parallel to hypoxia intensity while adenosine release increased, preferentially in response to mild hypoxia. We also found that the lower the O2 levels, the higher the percentage of adenosine produced from extracellular catabolism of ATP. Our results demonstrate that ATP and adenosine are the key neurotransmitters involved in hypoxic CB chemotransduction, with a more relevant contribution of adenosine during mild hypoxia while vesicular ATP release constitutes the preferential origin of extracellular adenosine in high intensity hypoxia. Supported by CEDOC/FCT, AI grant (MEC Spain/CRUP Portugal PT2009-0172), BFU2007-61848 and JCyL-GR242.

P2-2

Trafficking and interaction of ATP P2X4 receptor with GABA-gated channels control the excitability of hypothalamic steroidogenic factor 1 neurons

Eric Boue-Grabot1, Emannuelle Donier1, Audrey Martinez1, Maurice Garret1, Estelle Toulmé1, Young-Hwan Jo2

1CNRS UMR 5227, University of Bordeaux, France,2Department of Medicine, Albert Einstein College of Medicine, USA

ATP acts as a cotransmitter with GABA in CNS. Recent reports have demonstrated molecular cross-talk between several P2X and GABA-A receptors; however the existence and function of such crosstalk at central synapses remain to be determined. Here we show that molecular interaction between P2X4 and GABAA receptors is essential for regulating synaptic strength at feeding-related synapses. Immunohistochemistry and single cell RT-PCR studies reveal that SF-1 (steroidogenic factor 1) neurons in the ventromedial nucleus of the hypothalamus express P2X4 subunits that are activated by exogenous ATP. P2X4 subunit is highly and constitutively internalized via a clathrin dependent mechanism. In order to study the function of P2X4 channels, we designed an interference peptide to the intracellular endocytosis motif to block P2X4 internalization. Increased membrane expression of P2X4 channels enhances neuronal excitability of SF-1 neurons, which is further enhanced by molecular interaction between P2X4 and GABAA receptors. Indeed, both receptors reciprocally inhibit each other in an activity-dependent manner in recombinant and intact brain slice preparations. The physical interaction is supported by our studies showing that P2X4 subunits are co-localized with GABAA subunits in SF-1 neurons and that P2X4 co-immunoprecipitate with GABAA subunits in the hypothalamus. Two amino acids in the carboxyl tail of P2X4 appears to be responsible for this functional cross-talk. In fact, our designed competitive peptides containing the identified motif abolish P2X4-induced inhibition of GABAergic synaptic transmission. Our data provide evidence for physical interaction between excitatory and inhibitory receptors that appears to be crucial for determining synaptic strength at central synapses.

P2-3

Development of the first potent P2X2 receptor antagonists

(Second Poster Presentation Award)

Younis Baqi1, Christiane Rosefort2, Ralf Hausmann2, Günther Schmalzing2, Christa E. Müller3

1PharmaCenter Bonn, Pharmaceutical Chemistry I, University of Bonn, Germany,2Department of Molecular Pharmacology, RWTH Aachen University, Germany,3Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Germany

Background: P2X2 receptors belong to the family of purinergic ionotropic receptors gated by ATP. To date there are no known selective or highly potent P2X2 receptor antagonists. So far only TNP-ATP, PPADS, Reactive Blue 2, and suramin were reported as moderately potent, but nonselective antagonists at the P2X2 receptor (IC50 = 4–40 µM). Potent and selective P2X2 antagonists are required as pharmacological tools to elucidate the (patho)physiological roles of the ligand-gated ion channel receptor and to evaluate their potential as drug targets. Methods: A library of anthraquinone derivatives was synthesized by microwave-assisted coupling reactions, some of which required multi-step reaction sequences. The effects of the synthesized anthraquinones on P2X2 receptor-mediated currents were analyzed by using two-electrode voltage-clamp electrophysiology in xenopus laevis oocytes transiently expressing the human P2X2 receptor. Selected compounds were additionally evaluated at other P2 receptor subtypes. Results: We identified the first human P2X2 antagonist with nano-molar potency: disodium 1-amino-4-[3-(4,6-dichloro[1,3,5]triazine-2-ylamino)-4-sulfophenyl¬amino]-9,10-dioxo-9,10-dihydroanthracene-2-sulfo¬nate (IC50 P2X2 = 68 nM). It is highly selective versus the P2Y12 receptor (>200-fold) and the P2X4 receptor subtype (>125-fold). Its analog lacking the sulfonate group on the phenyl ring was even somewhat more potent. Conclusion: New non-nucleotide-derived P2X2 channel blockers showing affinity in the nano-molar range have been developed.

P2-4

Deletion of P2X7 receptor causes genome-wide alterations of genes expression including genes related to depression in mouse amygdale—a whole genome microarray study

Cecilia Katalin Csölle, Beáta Sperlágh

Institute of Experimental Medicine, Hungarian Academy of Science, Hungary

Background: The P2X7 receptor is a ligand-gated ion channel expressed in neuronal and glial cells and implicated in a wide range of pathological conditions, including ischemia and inflammation. P2X7 receptor activation elicits increased release of transmitters in the brain and regulates the maturation / release of proinflammatory cytokine, interleukin-1β (IL-1β). IL-1β is suggested to be involved in the pathophysiology of depressive behavior elicited by peripherally administered bacterial lipopolysaccharide (LPS). Methods: To identify genes affected by the genetic deletion of P2X7 receptors in the mouse amygdala we performed whole mouse genome microarray analysis of mRNA extracted after 6 hours of intaperitoneal LPS injection. For validation of differentially expressed genes, we performed TaqMan real-time RT-PCR. Results: Our microarray studies have identified 8165 transcripts that were significantly affected by the deficiency of P2X7 receptor. Validation experiments revealed that among these genes at least 30 genes could be associated to synaptic signaling, neuroplasticity and thereby to the pathogenesis of depression (e.g. beta and gamma subunits of GABAA receptor, ionotropic glutamate receptors, α2 adrenergic receptor, which were down regulated in KO mice). 287 transcripts were found be more than two-fold over-expressed in the LPS treated groups which can be classified according to their inflammatory biological function: Il4ra, Saa1, chemokine ligands. Conclusion: This study showed that P2X7 receptor deficiency had the most significant effect, indicating that further analysis of results will highlight individual proteins regulated by P2X7 receptors, which could be of potential interest for the study of the neurobiological basis underlying psychiatric diseases like depression.

P2-5

Effects of protons on macroscopic and single-channel currents mediated by the human P2X7 receptor

Fritz Markwardt1, Bente Flittiger1, Manuela Klapperstueck1, Guenther Schmalzing2

1JB Inst f. Physiology ML Univ Halle Germany,2Dep. Mol. Pharmacology RWTH Aachen Germany

Human P2X7 receptors (hP2X7Rs) belong to the P2X family, which opens an intrinsic cation channel when challenged by extracellular ATP. hP2X7Rs are expressed in cells of the inflammatory and immune system. During inflammation, ATP and protons are secreted into the interstitial fluid. Therefore, we investigated the effect of protons on the activation of hP2X7Rs. hP2X7Rs were expressed in Xenopus laevis oocytes and activated by the agonists ATP or benzoyl-benzoyl-ATP (BzATP) at different pH values. The protons reduced the hP2X7R-dependent cation current amplitude and slowed the current deactivation depending on the type and concentration of the agonist used. These effects can be explained by (i) the protonation of ATP, which reduces the effective concentration of the genuine agonist, free ATP4, at the high- and low-affinity ATP activation site of the hP2XR, and (ii) direct allosteric inhibition of the hP2X7R channel opening that follows ATP binding to the low-affinity activation site. Due to the hampered activation via the low-affinity activation site, a low pH (as observed in inflamed tissues) leads to a relative increase in the contribution of the high-affinity activation site for hP2X7R channel opening.

P2-6

Interaction of human P2X7A and B receptor subunits

Manuela Klapperstueck1, Ronja Woltersdorf2, Fritz Markwardt1, Elena Adinolfi3, Francesco Di Virgilio3, Guenther Schmalzing2

1JB Inst. f. Physiology, ML Univ Halle, Germany,2Dep. Mol. Pharmacology, RWTH Aachen, Germany,3Dep. Exp. Diag. Med., Univ Ferrara, Italy

P2X7 receptors are nonselective cation channels opened by extracellular ATP. They are mainly expressed in cells of the immune system and in some epithelia. P2X7 receptors are supposed to assemble as trimers of homomeric P2X7 subunits. A splice variant of the human P2X7 receptor subunit (hP2X7B) was recently identified which lacks the intracellular C-terminal tail of the wt receptor subunit (hP2X7A). In the X. laevis oocyte expression system we investigated the electrophysiological characteristics of hP2X7A and B receptors. hP2X7A and B receptor isoforms mediated typical exponentially saturating and non-desensitizing currents during ATP application. Coexpression of hP2X7A and B, however, resulted in a strongly desensitizing phenotype indicating an interaction between both splice variants. To provide biochemical evidence for the co-assembly human P2X7A and B isoforms, we co-expressed in X. laevis oocytes double-tagged P2X7A-His-StrepII (bearing a nine residue StrepII sequence C-terminal to the C-terminal hexahistidine tag) together with the His-P2X7B as bait and prey, respectively. Proteins were affinity-purified under non-denaturing conditions, resolved by SDS-PAGE, and visualized by autoradiography. Purification by Strep-Tactin chromatography led to the co-isolation of the non-StrepII-tagged His-P2X7B with P2X7A- His-StrepII, and reciprocally P2X7A-His with P2X7B-StrepII. We conclude from these data that P2X7A and P2X7B are capable of coassembling to form P2X7A / P2X7B heterotrimers.

P2-7

The role of protein kinase C and phosphoinositide 3-kinase in P2X7 receptor-mediated oxidation and IL-1beta processing

Malika El Ouaaliti1, Samantha F. Moore2, Amanda B. Mackenzie3

1Departement de biochimie et de biologie moléculaire, Universite Libre De Bruxelles, Belgium,2Department of biochemistry, Bristol University, UK,3Department of Pharmacy and Pharmacology, Bath University, UK

Background: Adenosine 5′ triphosphate (ATP) is a danger-associated molecular pattern detected by macrophage P2X7 receptors leading to NADPH oxidase (NOX2) activation and the formation of bioactive interleukin1 beta (IL-1beta) (Moore & MacKenzie, 2009). This study has investigated the role of Protein Kinase C (PKC) and Phosphoinositide3-kinase (PI3K) in NOX2 activation and subsequent secretion of bioactive IL-1beta. Methods: Lipopolysaccharide (LPS)-primed J774.2 macrophages were loaded with either 2′7′-dichlorodihydrofluorescin diacetate to measure reactive oxygen species (ROS) or fluo-4 AM to detect changes in cytosolic calcium. P2X7-associated membrane permeabilization was detected by ethidium bromide (EtBr) influx. IL-1beta processing and secretion was detected by Western blot analysis of cell lysates and supernatants. Results: Extracellular ATP (1–5 mM) triggered concentration-dependent ROS formation and EtBr uptake. Pre-treatment of cells with the broad spectrum PKC inhibitor; Ro32-0432 (100 nM) or PI3K inhibitor; wortmannin (100 nM) failed to inhibit either of these ATP evoked responses. Moreover, 5 mM ATP evoked increases in intracellular calcium were not altered by pre-treating cells with Ro32-0434 or wortmannin. In control experiments, wortmannin blocked ATP-mediated PKB/AKT phosphorylation. Extracellular ATP (0.8 mM to 5 mM) evokes a concentration-dependent and time-dependent increase in IL-1beta processing. Pre-treatment of cells with 100 nM wortmannin did not significantly alter ATP-mediated IL-1beta processing or secretion. However, 100 nM Ro32-0432 markedly potentiated ATP mediated IL-1beta secretion from LPS-primed macrophages. Conclusion: Here we demonstrate that P2X7 receptors couple to PKC-regulated IL-1beta secretion pathway in murine J774.2 macrophages.

P2-8

Allosteric modulation of human P2X7 receptor by clemastine

Michael Schaefer, Christoph Hempel, Nicole Urban, Helga Sobottka, Wolfgang Nörenberg

Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Germany

To assess the impact of commonly prescribed pharmaceuticals on P2X7 receptor activity, we screened a compound library comprising 1040 approved drugs and 800 natural products in a HEK293 cell line stably expressing human P2X7. A calcium influx assay was established and scaled to the 384-well format. The screening results revealed an as yet unrecognized potentiation of ATP-induced calcium entry by the H1 antihistaminic drug clemastine (5–100 µM) but not by other antihistaminics (e.g. loratadine). Clemastine on its own had no effect at concentrations up to 100 µM. Half-maximal potentiation of ATP-induced Ca2+ entry was observed at about 15 µM clemastine. Since 30 µM clemastine shifted the EC50 of ATP from 1.25 mM to 0.6 mM, we propose a positive allosteric P2X7 modulation. Likewise, clemastine rapidly augmented ATP-induced cation currents recorded in the whole-cell configuration. The potentiation displayed time constants of about 220 ms to develop and was reversible on a similar time scale. At saturating ATP concentrations (10 mM), clemastine produced no further increase in P2X7 current amplitudes. In addition, clemastine markedly accelerated the ATP-induced uptake of the organic cation YoPro-1 and increased the permeability ratio for NMDG with respect to Na+. To assess the biological activity of clemastine on native P2X7 receptors, Ca2+ entry and whole cell currents were measured in human monocyte-derived macrophages. Consistent with the data on recombinant P2X7, clemastine augmented the ATP-induced long-lived cation entry in macrophages. Collectively, these data point to a sensitisation of recombinant and native human P2X7 receptors towards their physiological activator ATP.

P2-9

Pannexin-1 expressed in HEK cells forms a chloride channel but not a hemichannel

Weihong Ma*, Vincent Compan*, Wenxuan Zheng, Elizabeth Martin, Alexei Verkhratsky, Annmarie Surprenant

Faculty of Life Sciences, University of Manchester, United Kingdom

* These authors contributed equally to the study.

Background: Pannexin-1 (Panx1) was recently considered a hemichannel involved in the P2X7 dye uptake; this suggestion was based on the distant topological relationship between Panx1 and the gap-junction family of connexins. We provide electrophysiological and biochemical evidence to demonstrate that Panx1, unlike connexins, is a chloride channel. Methods: Mouse Panx1 was transiently expressed in HEK293 cells. The permeability properties of mPanx1 currents were studied with whole-cell patch-clamp recordings. The stoichiometry of mPanx1 was characterised using native gel electrophoresis and cross-linking reagents on living cells. Results: Replacing extracellular chloride by large anions (aspartate, glutamate or gluconate) resulted in a 40 mV right-shift of the mPanx1 reversal potential, whereas substitution of extracellular cations had no such effect. Replacement of chloride (either extracellularly or intracellularly) with other halides revealed that permeation of mPanx1 is NO3-> I-> Br-> Cl-> F-, as indicated by the reversal potential shift. In addition to its sensitivity to carbenoxolone, mPanx1 currents were inhibited by a number of chloride channel blockers. Native gel electrophoresis revealed that mPanx1 quaternary structure is a trimer but not a hexamer as that for a hemichannel. Finally, cross-linking experiments confirmed this unexpected trimeric state and revealed that mPanx1 is poorly expressed on the plasma membrane. Conclusion: Mouse Panx1 when expressed in mammalian cells forms an anion-selective channel, which does not share functional and quaternary structural similarities with hemichannels.

P2-10

Presence of functional P2X7 receptors in murine neuroblastoma cells

(Selected for Oral Poster Presentation)

Paula García-Huerta1, Rosa Gómez-Villafuertes1, Diego Bustillo2, Emilie Mazoir1, Antonio R. Artalejo2, Mª Teresa Miras-Portugal1

1Dpto. Bioquímica y Biología Molecular, Fac. Veterinaria, Universidad Complutense de Madrid, Spain,2Dpto. Toxicología y Farmacología, Fac. Veterinaria, Universidad Complutense de Madrid, Spain

Clonal neuroblastoma cell lines are commonly used as models to study neuronal differentiation, as they retain the capacity to differentiate into a neuronal-like phenotype, and express a rich repertoire of membrane receptors coupled to the most well-known second messenger systems. In this study, we characterized the presence of native functional P2X7 receptors in non-differentiated Neuro-2a (N2a) neuroblastoma cells. Using both molecular and immunocytochemical techniques, we showed the presence of endogenous P2X1, P2X3, P2X4 and P2X7 subunits in these cells. The functionality of P2X7 receptors in N2a cells was defined on pharmacological and biophysical grounds. BzATP was able to induce an increase in intracellular calcium levels, whereas alpha,beta-meATP and CTP were ineffective, excluding the presence of functional P2X1, P2X3 and P2X4 receptors in these cells. The potency for BzATP was increased by two-fold following the omission of magnesium ions from the extracellular medium, and the P2X7 receptor antagonists KN62, BBG and A438079 inhibited BzATP-evoked calcium responses, thus demonstrating the presence of P2X7 receptors. Interestingly, both KN62 and BBG behaved as slowly reversible antagonists, whereas the inhibitory effect of A438079 was rapidly reversed. In addition, voltage clamp studies showed that BzATP elicits non-desensitizing inward currents sensitive to BBG and A438079 in N2a cells. Together, these data point to the existence of native functional P2X7 receptors in N2a cells (Gómez-Villafuertes et al., 2009).

References:

Gómez-Villafuertes R, del Puerto A, Díaz-Hernández M, Bustillo D, Díaz-Hernández JI, Huerta PG, Artalejo AR, Garrido JJ, Miras-Portugal MT. 2009. FEBS J. 276: 5307-25.

P2-11

Functional three-fold symmetry in the deep pore of the P2X2 receptor assessed by rectification and chloride permeability

Liam E. Browne1, Lishuang Cao2, R. Alan North3

1Faculty of Life Sciences, University of Manchester, UK,2Pain Research Unit, Pfizer Global Research and Development, UK,3Faculty of Medical and Human Sciences, University of Manchester, UK

Background. The P2X2 receptor is selective for cations and displays inward rectification, but the mechanisms underling these properties are unknown. The receptor is a trimer, but it is unclear whether the open channel functions with three-fold symmetry. Methods. We introduced a positive charge in the rat P2X2 receptor pore at T339: substituting with H, K or R; or K in one, two or three of its component subunits by using a concatenated receptor. Whole-cell patch clamp was used to study the effects on rectification and ionic selectivity, after expression in HEK cells. Results. Compared to wild type receptors, outward currents were significantly enhanced for T339H, T339K and T339R mutations. Trimeric concatenated receptors containing no, one, two or three lysines showed step-wise increases in outward current, which was proportional to the number of mutated subunits. To study the effects on ionic selectivity, external chloride ions were substituted by gluconate and the reversal potential was measured. Wild type receptors showed a PCl/PNa value of 0.04, while T339H, T339K and T339R mutations showed values of 0.28, 1.80 and 2.15 respectively. Relative chloride permeability was also dependent on the number of positive charges introduced in the concatemers, such that a significant linear dependence between PCl/PNa and outward rectification was observed (r = 0.96). Conclusion. Positive charge at T339 markedly increases anion permeability and it also reduces rectification. A common molecular mechanism may be involved. Thus, in the deep pore of the open P2X2 receptor each of the three subunits contributes symmetrically to ion permeation.

P2-12

Identification of residues at positions 155 and 348 of the P2X7 receptor as contributors to the functional differences between human and rat species

Helen Bradley, Jocelyn Baldwin, Stephen Baldwin, Lin-Hua Jiang

University of Leeds, University of Leeds, UK

P2X7 receptors are ATP-gated, cation-selective channels which are mainly expressed in immune and glial cells and play an important role in immune responses, inflammation, neuron-glial interactions, pain and cell death. Differences in functional responses of the human P2X7 (hP2X7) and rat P2X7 (rP2X7) receptors have previously been reported, however the molecular basis is unclear. Here we investigated P2X7 receptor function by heterologously expressing wild-type (WT) or mutant P2X7 receptors in HEK293 cells and used whole-cell patch clamp recordings to determine agonist-evoked currents, and biotin labeling to assess surface expression. Peak current responses to ATP are 1.6 times higher for the rat P2X7 than the human P2X7 receptor and the rP2X7 receptor is 2.8 times more sensitive to the agonist. Two non-synonymous single nucleotide polymorphic mutations of the hP2X7 receptor, H155Y and A348T, which change the residues at these positions to those of the WT rP2X7 receptor, increased the agonist-induced peak current amplitude. Conversely, the reciprocal substitutions of the rP2X7 receptor, Y155H and T348A, decreased the agonist-induced peak currents. Surface biotinylation experiments show that the H155Y hP2X7 mutant is more highly expressed at the plasma membrane than wild-type, whilst the rP2X7 receptor with the reciprocal mutation showed reduced surface expression. Structural modeling of the hP2X7 receptor indicates that position 348 is located in the second transmembrane domain. These results show that residues at positions 155 and 348 have a critical role in the functional expression of the P2X7 receptor and are important in determining the species-dependent properties of these receptors.

P2-13

Protein purification, mass spectrometry and DTSSP action at the P2X1 receptor

Jonathan Roberts, Richard Evans

Department of Cell Physiology and Pharmacology, University of Leicester, UK

DTSSP can bind to exposed lysine residues and has been used to cross-link P2X1 receptors. In this study we have used protein purification and mass spectrometry to identify the sites of action of DTSSP. C terminally HisFlag tagged P2X1 receptor expressed in HEK293 cells was purified. Coverage of the P2X1 receptor was increased by deglycosylating the receptors with PNGase F (23.2% vs. 41.3%). High coverage was achieved with trypsin digest though other enzymes were tested (e.g. chymotrypsin, 12.3% coverage). Use of Orbitrap vs. Qtrap mass spectrometer increased coverage further to 59.2%. Initial experiments revealed 100 micromolar DTSSP cross-links human P2X1 receptors to dimers, trimers and at high concentrations (>1 mM) homohexamers. Application of 100 micromolar DTSSP led to functional loss of channel activation by ATP when expressed in oocytes or HEK cells. Radioactive 2AzATP analysis confirmed 100 micromolar DTSSP reduced agonist binding implicating an association of the cross-linker with or near the binding site. Human P2X1 was treated with 100 micromolar DTSSP in intact HEK cells and harvested for protein purification. Purified protein was subsequently treated with PNGase F, isolated by SDS PAGE and run on a mass spectrometer. Under non-reducing conditions no peptides were observed on ms coupled together. Analysis of denatured and reduced protein revealed extracellular DTSSP modifications at 9 out of 19 lysine residues but also at Y274, S130 and S286, showing modification of non-lysine residues. This study highlights the accessibility of certain extracellular residues to DTSSP and inhibition of P2X1 receptors by DTSSP.

P2-14

Cysteine scanning mutagenesis of the region Glu52 to Gly96 of the human P2X1 receptor refines a model of the ATP binding site and identifies residues that contribute to channel gating

Sammy El-Ajouz, Rebecca Allsopp, Richard Evans

Department of Cell Physiology and Pharmacology, University of Leicester, England

Cysteine scanning mutagenesis and methanethiosulfonate (MTS) compounds were used to investigate the contribution of residues Glu52-Gly96 to human P2X1 receptor properties. When expressed in xenopus oocytes only three mutants, K68C, K70C and F92C showed a significant decrease in ATP potency (1000, 10 and 300 fold respectively). The sensitivity of the mutants to partial agonists highlighted further residues that may be also be involved in agonist binding and channel gating. For example, BzATP reduced efficacy at I62C, T75C, G81C, W85C, Y90C, P93C, Q95C and G96 but and increased efficacy at L72C and A73C. Negatively charged MTSES significantly reduced the ATP-evoked response at I62C, S63C, K68C, K70C, L72C, P82C, D86C, V87C, P93C and G96C with a reduction in the ATP potency at the mutants K68C, K70C, V87C, P93C and G96C. Positively charged MTSES potentiated the response at G60C and V91C, increasing the ATP potency at G60C and the response was inhibited at S64C, K68C and K70C, reducing the ATP potency at K68C. Measuring changes in MTS biotinylation showed that the inner cavity of the extracellular portion of the P2X receptor becomes less accessible on channel activation giving an insight into structural re-arrangement upon agonist binding. None of the residues tested showed a reduction in sensitivity to the antagonists suramin and PPADS. For 8 of the mutants (Y55C, K68C, K70C, G71C, T75C, Y90C, P93C, Q95C) there was an increase in sensitivity to suramin, with the greatest inhibition at K70C. These results allow a refinement of our understanding of drug action at the P2X1 receptor.

P2-15

Evidence for functional purinergic P2X7 receptors in astroglial cells of rodent brain slices

Anna Leichsenring, Thomas Riedel, Heike Franke, Claudia Heine, Peter Illes, João Filipe Oliveira

Rudolf Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Germany

The purpose of the present study was to investigate whether astrocytes in the rodent central nervous system (CNS) express functional P2X7 receptors (P2X7R). For our investigations we used freshly prepared 200 micrometer thick slices of rodent brain and spinal cord. By means of whole cell patch-clamp recordings, electrophysiologically identified astrocytes in the prefrontal cortex (PFC), the oriens layer of the hippocampal Ca1 region and the spinal cord substantia gelatinosa (SG) were analyzed. Benzoylbenzoyl ATP (BzATP) - a preferential P2X7R agonist - and ATP caused inward currents in astroglial cells in all investigated tissues. Amplitudes of BzATP and ATP responses were strongly increased in a low divalent cationic external solution and the use of the specific P2X7R antagonist A438079 caused a concentration dependent reduction of the inward current. These results were underlined by excised patch recordings, immunohistochemical detection of the P2X7R protein in astrocytes and recordings in CNS tissues of the respective knockout mice. In conclusion, we were able to show the functional expression of the P2X7R in rodent astrocytes in situ. Our results contrast sharply with the previous view that native astroglial cells do not express purinergic P2X7R. We suggest that P2X7R in astrocytes are not involved in the physiological cross talk with neurons but are sensors of pathologically high ATP concentrations released by brain injury or metabolic limitation.

P2-16

P2X7-mediated currents in human breast cancer cell line have an unusual pharmacology

Zheng Wenxuan1, Gloria Lopez-Castejon2, Alexej Verkhratsky2

1Gloria Lopez-Castejon, Alexei Verkhratsky, University of Manchester, United Kingdom,2Faculty of Life Sciences, University of Manchester, United Kingdom

Background: There is increasing evidence that purinergic signaling plays a pivotal role in cancer cell growth. However, the mechanism through which ATP acts on cancer cells has not been characterized in detail. Therefore, we investigated the expression and functional properties of P2X receptors in human breast cancer cell line HS578T. Methods: The expression of P2X4 and P2X7 receptors in breast cancer cell line HS578T was analyzed by reverse transcription-linked polymerase chain reaction (RT-PCR). The ATP-evoked currents were monitored with whole-cell patch-clamp technique. Results: The expression of mRNA for both P2X4 and P2X7 receptors was detected in human breast cancer cell line HS578T. Extracellular ATP (0.1–5 mM) evoked currents from the breast cancer cell line HS578T; these ATP induced currents that showed certain properties characteristic for P2X7 receptors: (i) Low sensitivity to extracellular ATP with an EC50 of 5.1 mM. (ii) Absence of desensitization in the course of 60 seconds application of 5 mM ATP. (iii) Pronounced facilitation during the initial receptor activation typically seen from the P2X7 receptor. At the same time these P2X7-like currents displayed unique pharmacological features: they could not be activated by BzATP and were blocked neither by the P2X7 specific antagonist A740003 nor by P2X receptors inhibitors BBG, oxATP and suramin. Conclusion: In the human breast cancer cell line HS578T, ATP triggers currents with electrophysical profile similar to P2X7 receptor, although the pharmacology of these currents is not in agreement with that known for P2X7 receptor.

P2-17

A C-terminal calmodulin binding motif differentially controls human and rat P2X7 receptor current facilitation

Sébastien Roger1, Ludovic Gillet1, Alberto Baroja-Mazo2, Annmarie Suprenant3, Pablo Pelegrín2

1Inserm U921, Université François Rabelais de Tours, FRANCE,2Inflammation and Experimental Surgery Group, Hospital Universitario Virgen Arrixaca—Fundación para la Formación e Investigaciones Sanitarias de la Región de Murcia (FFIS), SPAIN,3Faculty of Life Science, University of Manchester, UK

P2X7 receptors (P2X7R) are ATP-gated calcium-permeable cationic channels that show several unusual biophysical properties, in particular marked facilitation of currents and leftward shift in agonist affinity in response to repeated or prolonged agonist applications. We previously found the facilitation at rat P2X7R resulted from a Ca2+-calmodulin dependent process and a distinct calcium-independent process. However, P2X7Rs show striking species differences; thus the present study compared the properties of ATP-evoked facilitation of currents in HEK293 cells transiently expressing the human or rat P2X7R as well as rat/human, human/rat chimeric, and mutated P2X7Rs. For equivalent protein expression and adressing, facilitation at the human P2X7R was 5-fold slower than at the rat P2X7R. We found that human P2X7R shows only calcium-independent facilitation with no evidence for calmodulin-dependent processes, nor does it contain the novel 1-5-16 calmodulin binding domain present in the C-terminus of rat P2X7R. Replacement of the three critical residues of this binding domain from the rat into the human P2X7R (T541I, C552S, G559V) reconstituted the Ca2+-calmodulin-dependent facilitation, leaving the calcium-independent facilitation unaltered. The leftward shift in the ATP concentration-response curve with repeated agonist applications appears to be a property of the calcium-independent facilitation process because it was not altered in any of the chimeric or mutated P2X7Rs. The absence of any Ca2+-dependent facilitation at the human P2X7R may represent a protective adaptation of the innate immune response in which P2X7R plays a significant role.

P2-18

Role of hippocampal P2X7 receptors in the release of NO by ATP and related nucleotides

Ines Maria Poblete Wilson, Juan Godoy, Nibaldo Inestrosa, Juan Pablo Huidobro-Toro

Pontificia Universidad Catolica de Chile, Chile

To assess a link between nucleotide receptors and the release of NO from rat hippocampus, we examined whether ATP and related nucleotides resulted in concentration-dependent NO secretion, an essential transmitter of memory consolidation. Perfusion of hippocampal slices with ACSF allowed the detection of basal NO levels; exogenous ATP elicited a concentration-dependent burst of NO that peaked within 1–2 min and faded 4–5 min later; its EC50 was 30.8 micromolar. The Bz-ATP concentration-response curve was displaced leftwards and resulted more efficacious. This effect was not mimicked by ADP, 2-MeSADP, UTP, UDP alpha, beta methyleneATP nor adenosine, demonstrating a selective agonist profile. 100 nM N-propylarginine blunted NO release revealing nNOS activity. 5–50 micromolar NMDA elicited NO bursts additive with ATP. NMDA effects were blocked by APV or MK-801. The brilliant blue IC50 to half the ATP or BzATP-induced NO production was 130.5 and 9.5 nM respectively; 100 micromolar suramin abolished the ATP effect, supporting P2X7 receptor subtype involvement. Basal NO production was reduced partially by suramin and/or APV/MK-801, implying that both the NMDA as well as the nucleotide receptor regulate basal NO levels. The present results highlight a role of ATP in non-NMDA hippocampal NO production through P2X7 receptor activation. Grant 13980001, PFB 12/2007 and MIFAB funding.

P2-19

Identification and optimization of novel adenine-based antagonists of P2X3 receptors

(Selected for Oral Poster Presentation)

Mayya Sundukova1, Catia Lambertucci2, Rosaria Volpini2, Gloria Cristalli2, Andrea Nistri1

1SISSA, Italy,2University of Camerino, Italy

P2X3 receptors are ATP-activated channels expressed by sensory ganglion neurons, where they play important role in pain transduction afferent pathways. Targeting these receptors with selective, potent antagonists represents an innovative method to treat chronic pain conditions of both neuropathic and inflammatory origin when P2X3 receptor function is enhanced. The current study combined medicinal chemistry and functional approaches to design and test novel adenine-based compounds with various substituents at positions 2, N6, 7 and 9. These compounds were tested for their functional effect on rat P2X3 receptors transiently expressed by HEK 293T cells, by recording whole-cell currents under voltage clamp conditions. New compounds and the P2X3 selective agonist alpha,beta-me-ATP were applied to single cells via a fast perfusion system. None of the tested compounds was able to elicit functional responses even at concentration of 100 microM. In contrast, they exhibited antagonistic activity (with distinct potency) on P2X3 receptors, since their continuous application (20 s) blocked current responses evoked by 100 microM alpha,beta-meATP. The highest potency (IC50 values ~2 microM) was observed with compounds containing a 4-iodo-2-isopropyl-5-methoxybenzyl chain in the 9-position and a methyl group in the N6-position of adenine ring. Preliminary experiments indicated that the most potent antagonists inhibited human P2X3 receptors with a potency similar to that on rat P2X3 receptors. These data suggest these compounds to be interesting candidates for further studies of their action on chronic pain models in vivo. Supported by FIRB grants, by the Friuli Venezia Giulia government, the Telethon Foundation (GGP07032), and IIT.

P2-20

Differential sensitivities of P2X receptors to lipid raft disruption: identification of amino acid residues responsible

Rebecca Allsopp, Ulyana Lalo, Richard Evans

Department of Cell Physiology and Pharmacology, University of Leicester, England

We have shown previously that P2X1 receptors are expressed in lipid rafts and are inhibited by cholesterol depletion. We have extended this work to look at the cholesterol sensitivity of P2X2, 3 & 4 receptors expressed in HEK293 cells. P2X1 receptor mediated currents were inhibited by methyl beta cyclodextrin or filipin treatment (90% ± 3% and 93% ± 2% inhibition respectively). In contrast ATP evoked P2X2, 3 & 4 receptor mediated currents were unaffected following cholesterol depletion. To determine whether the sensitivity of P2X receptors results from differential raft localisation we used sucrose density centrifugation following low pH solubilisation to isolate lipid rafts. P2X1-4 receptors were all detected in flotillin rich lipid raft fractions. The use of high concentration triton X-100 (1%) resulted in detection of only P2X1 in flotillin rich raft fractions, indicating differential sensitivity of the subtypes of raft sensitive to high detergent concentration. Generation of chimeras between P2X1/P2X2 receptors allowed us to determine the region associated with cholesterol sensitivity. Chimeras replaced the N-terminus, trans-membrane domain 1, extracellular cysteine loop, trans-membrane 2 or the C-terminus of P2X1 with the corresponding region from P2X2. Patch clamp analysis of these chimeric receptors transfected into HEK293 cells identified the intracellular N-terminal domain of P2X1 as the region responsible for sensitivity to cholesterol depletion. Construction of further chimeric mutants demonstrated that this sensitivity was associated with the stretch of amino at the end of the N-terminus.

P2-21

Evidence for a role of purinergic P2X4 receptor subtype in ATP-induced depolarization of neurons in supraoptic nucleus

Vojtech Vavra, Anirban Bhattacharya, Irena Svobodova, Hana Zemkova

Cell and Molecular Endocrinology, Institute of Physiology, Czech Republic

Purinergic cation-conducting P2X receptors are expressed throughout the hypothalamus where extracellular ATP appears to be involved in the regulation of hormone secretion. Neurons of the rat supraoptic nuclei (SON) express several P2X receptor mRNAs (P2X2, P2X3, P2X4, P2X6 and P2X7). Immunohistochemistry and functional studies showed that extracellular ATP excites SON neurons through P2X2 receptor activation, but expression of proteins and electrophysiological properties of other P2X receptor channels have been only partially characterized. In this study we show by quantitative RT-PCR that mRNA for the P2X2, P2X7 and P2X4 subunits are the most abundant in the rat hypothalamic tissue containing SON. Single-cell patch-clamp recordings from acutely isolated rat brain slices showed that extracellular ATP induces depolarizing current in a majority of SON cells. ATP application led also to stimulation of electrical activity, promotion of extracellular calcium influx and increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs). The effect of ATP was reversibly inhibited by suramin and PPADS and increased by extracellular pH acidification indicating the presence of P2X2 receptor. Pretreatment with ivermectin, a specific allosteric modulator of P2X4 receptors, caused two-fold increase in the ATP-induced current. This effects resembled the current profile of recombinant P2X4 receptor treated with ivermectin. These results indicate that SON neurons express homomeric and/or heteromeric P2X4 receptors which contribute to ATP-induced depolarization and spontaneous GABA release. This work was supported by the Internal Grant Agency of Academy of Sciences (IAA500110702) and the Centrum for Neuroscience (LC554).

P2-22

Novel properties of the mouse P2X7k splice variant

Xing Jian Xu, Marianela Masin, Viola Marschall, Ruth Murrell-Lagnado

Department of Pharmacology, University of Cambridge, UK

The rodent P2X7k splice variant, which utilizes a different exon 1 and is expressed in both wild type and the Glaxo P2X7−/− mice, has at least a 10-fold greater sensitivity to agonists ATP and BzATP than the full length mouse P2X7a variant, as determined from patch clamp experiments. The mP2X7k receptor also coupled to the rapid uptake of the cationic dye ethidium, and this process was unaffected by the SNP, P451L, that previously was shown to inhibit mP2X7a receptor-mediated dye uptake. In HEK293 cells, activation of mP2X7k also triggered the uptake of the anionic dye Lucifer yellow, the rate of which was dramatically increased by a 5-min pre-incubation with the pannexin-1 inhibitor, carbenoxolone (CBX) at 37°C. CBX also increased mP2X7k-mediated ethidium uptake but the Ca2+ signal evoked by activation of mP2X7k was unaffected, suggesting that CBX selectively affects ‘large pore’ formation. The mechanism of action of CBX is unclear but the results are not consistent with pannexin-1 being the dye uptake pathway. P2X7k is expressed in mice spleen T-lymphocytes where NAD is reported to be a physiologically important agonist. In HEK293 cells, 10 uM NAD stimulated mP2X7k-mediated ethidium uptake whereas mP2X7a did not respond to concentrations up to 300 uM NAD. NAD also stimulated a rapid Ca2+ response in HEK293 cells expressing mP2X7k. The co-expression of mP2X7a and mP2X7k in HEK cells reduced the amplitude of the NAD response compared to cells expressing mP2X7k alone. Our results suggest the expression of P2X7k increases the diversity of P2X7 receptor signaling in rodents.

P2-23

Modulatory effect of point mutations of tyrosine 37 in the first transmembrane domain on P2X3 receptor desensitization

Marie Jindrichova1, Kamil Khafizov2, Andrei Skorinkin3, Hana Zemkova1, Rashid Giniatullin4

1Cell and Molecular Neuroendocrinology, Institute of Physiology, Academy of Sciences Czech, Republic,2Computational Structural Biology group, Max Planck Institute of Biophysics, Germany,3Biochemical and Biophysical Institute, Academy of Sciences, Russia,4Department of Neurobiology, A.I.V.I., University of Kuopio, Finland

The ion channels of purinergic P2X receptor family center around three identical or homologous subunits each containing two transmembrane domains. Although the channels resemble each other, there are many structural and functional differences between each of the specific P2X1-7 receptors. The P2X3 wild-type (WT) receptor is characterized by a very fast activation followed by immediate, rapid and complete desensitization. The aim of this study was to examine the role of highly conserved tyrosine (Tyr37) in the first transmembrane domain in desensitization properties of the rat P2X3 receptor. The tyrosine residue was substituted to alanine or phenylalanine and mutated receptors were expressed in HEK293 cells. Mutant Y37A showed enhanced sensitivity to agonists and distinct biphasic desensitization with very slow second time constant. In contrast to P2X3-WT which completely desensitized during 2 seconds, the Y37A mutant showed incomplete desensitization even during 2 minutes of ATP application. The same slow desensitization was observed during stimulation with various ATP agonists (alfa,beta-meATP; beta,gama-meATP; 2-Me-SATP) and differences in agonist properties were evident only as differential rates of current decay after agonist washout. On the other hand, recovery from desensitization was much faster for the Y37A mutant compared to WT receptor. Substitution of phenylalanine for tyrosine did not rescue native desensitization properties of P2X3 receptor. Using cyclic kinetic model of P2X3 receptor we suggest that conserved tyrosine 37 in the first transmembrane domain has an unique regulatory role in promotion of the long living desensitized states via dual mechanism including accelerated desensitization onset and slow channel ungating.

P2-24

Preventing gating of the P2X7 ion channel in vitro and in vivo with monoclonal antibodies and recombinant single domain Nanobodies

Welbeck Danquah1, Björn Rissiek1, Catelijne Stortelers2, Toon Laeremans2, Tanja Schoknecht1, Gudrun Dubberke1, Friedrich Haag1, Friedrich Koch-Nolte1

1Institute of Immunology, University Medical Centre, Hamburg, Germany,2Ablynx Ablynx, Ghent, Belgium

The purpose of this study was to generate and characterize functional antibodies directed against murine P2X7. P2X7 allows T cells to sense nucleotides released during inflammation. Gating of P2X7 is induced by the soluble ligand ATP or by NAD-dependent ADP-ribosylation of Arg 125, a covalent modification catalyzed by ADP-ribosyltransferase, ART2. We raised 6 rat monoclonal antibodies (mAbs) and 14 recombinant llama single domain antibodies (Nanobodies) from animals immunized with a P2X7 expression vector. These were tested for interference with P2X7 function using Yac-1 lymphoma cells that endogenously express P2X7 and ART2. The readout used was shedding of CD62L induced by treatment of cells with NAD or ATP in the presence of P2X7-specific or control antibodies. mAb Hano43 blocked NAD but not ATP induced shedding of CD62L, most likely by sterically blocking the ADP-ribosylation site. Consistent with this interpretation, Hano43 recognized native but not ADP-ribosylated P2X7. Nanobodies 8G11 and 13A7 blocked NAD- and ATP-induced shedding of CD62L. Recloning into a bivalent format markedly increased P2X7-binding and P2X7-blocking potencies of these Nanobodies. Intravenous injection of bivalent 13A7 into mice efficiently blocked P2X7 on T cells in spleen, lymph node and liver, as assessed by resistance of these cells to NAD- and ATP-induced shedding of CD62L. These Nanobodies provide new tools for specifically blocking P2X7 function in vitro and in vivo and pave the way for testing the therapeutic potential of P2X7-specific Nanobodies in models of autoimmune diseases.

P2-25

Characterisation of C-terminal-truncated P2X7 receptor splice variants

Sara J Barnes1, Marianela Masin1, Viola Marschall1, Elizabeth Mooney1, Christopher Young2, KoiNi Lim2, Dariusz C Gorecki2, Ruth Murrell-Lagnado1

1Department of Pharmacology, University of Cambridge, United Kingdom,2School of Pharmacy and Biomedical Sciences, University of Portsmouth, United Kingdom

Contributing to the diversity of human and rodent P2X7 receptors are alternatively spliced variants. Here, we describe two murine splice variants with C-terminal truncations (ΔC) that arise from the use of alternative exon 13 s. P2X7(13b) terminates at residue 430, whereas P2X7(13c) possesses an additional 11-amino acid stretch after residue 430. The tissue distribution and structural and functional characteristics of the ΔC variants were investigated and compared to the full-length variant, P2X7(a). RT-PCR analysis using tissue from the wild-type C57BL strain of mice indicated that the ΔC variants are expressed in a range of tissues including brain, salivary gland and spleen. The recombinant receptors expressed in HEK293 cells were analysed by BN-PAGE and cross-linking studies and both ΔC splice variants formed stable homotrimers. Whole-cell patch-clamp recordings revealed that BzATP-induced currents were at least an order of magnitude smaller for the ΔC variants compared to P2X7(a) and the EC50 value was 1.3 mM. Co-expression of P2X7(13b) with P2X7(a) reduced the amplitude of 1 mM BzATP-evoked currents by ~3-fold compared to P2X7(a) alone and shifted the EC50 value from 320 µM to 410 uM, suggesting that the ΔC variant exerts a dominant negative effect. Expression of P2X7 receptor complexes in tissue from wild type and Pfizer P2X7−/− mice was analyzed by BN-PAGE and using cross-linkers. Blots using an antibody to the extracellular domain of P2X7 showed trimers of a size consistent with the ΔC variants in spleen and salivary gland from Pfizer P2X7−/− mice, suggesting that these mice are not null for P2X7 expression.

P2-26

Pore-formation induced haemolysis requires P2-receptor activation

(Selected for Oral Poster Presentation)

Marianne Skals, Julie Hejl, Jens Leipziger, Helle Praetoius

Dept. of Physiology and Biophysics Aarhus University Denmark

Recently, we showed that the pore forming α-haemolysin form Escherichia coli (HlyA) uses P2X-receptor activation (Skals et al PNAS 2009) to induce lysis of human, murine and equine erythrocytes. This result was surprising, as one previously regarded pore-formation as sufficient for cell lysis. We discovered that HlyA-induced haemolysis is a sequential process of shrinkage and swelling (Skals et al, JBC2010) and that the final haemolysis is prevented by blockers of P2X receptors and pannexin channels. It is essential to know whether this is specific to HlyA-induced cell damage or if other pore-formers in general involve the same mechanism. Here we investigate if the haemolysis produced both by complement activation and α-toxin from Staphylococcus aureus also requires purinergic signalling for full haemolysis. In murine erythrocytes, both unselective P2-receptor antagonists and selective blockers of P2X1 and P2X7 receptors completely blocked the α-toxin induced haemolysis in a concentration dependent fashion. In P2X7 receptor KO mice α-toxin induced haemolysis was blunted. Similarly, P2-receptor antagonists concentration-dependently inhibited complement-induced haemolysis in sheep erythrocytes. Inhibitors of pannexins completely abolish complement-induced haemolysis, whereas they exert a partially effect on the α-toxin-induced lysis. Thus, we can conclude that pore-formers in general (haemolysin A from E. Coli, α-toxin from staph. aureus and the membrane attack complex of the complement system) all produce cell damage by specific activation of a purinergic signalling cascade. These findings may have therapeutic potential in treatments have clinically relevance, as it pinpoints a way to ameliorate cell damage inflicted by pore-formation and by pharmacological means dampen intravascular haemolysis.

P2-27

P2X - Pannexin-1 interactions in Xenopus laevis oocytes

Ronja Woltersdorf1, Manuela Klapperstück2, Mazyar Mahmoudi1, Fritz Markwardt2, Günther Schmalzing1

1Molecular Pharmacology, RWTH Aachen University, Germany,2Julius-Bernstein-Institute for Physiology, Martin Luther University of Halle, Germany

Background: Pannexin-1 is considered a candidate for the pore-forming protein that permeabilizes cells upon sustained P2X7 receptor activation. Here, we investigated the nature of the physical interaction between pannexin-1 and the P2X7 receptor. Methods: Pannexin-1 and StrepII-tagged P2X receptors were recombinantly co-expressed in X. laevis oocytes as prey and bait, respectively. Proteins were purified by Strep-Tactin chromatography, resolved by SDS-PAGE and visualized in their 35S-methionine-labeled and plasma membrane-bound forms. We also recorded whole-oocyte currents by two-electrode voltage clamping (TEVC). Results: Human pannexin-1 could be co-purified with the human P2X7 receptor as an intermolecular disulfide-bonded complex. Cell surface labeling and deglycosylation experiments revealed that only the core-glycosylated immature form of pannexin-1 interacted with the P2X7 receptor, but not its complex-glycosylated mature form. Interactions between mature, plasma membrane-bound forms of both proteins were never detected, neither in non-stimulated cells nor in cells treated with chemical cross-linkers to stabilize weak protein-protein interactions with and without sustained ATP-activation. We observed a similar non-specific and DTT-sensitive interaction of human pannexin-1 with several other human P2X subunits (P2X1 to P2X4) as well as with the mouse P2X7 receptor and mouse pannexin-1. We neither detected pannexin-induced changes of the ATP-dependent oocyte currents nor a formation of large pores in oocytes co-expressing P2X7 receptors and pannexin-1. Conclusion: We conclude that pannexin-1 and the P2X7 receptor interact physically only at the level of the endoplasmic reticulum in X. laevis oocytes. Our electrophysiological data do not support the view that pannexin-1 represents the pore-forming principle activated through P2X7 receptors.

P2-28

Heteromerization between P2X7(a) and P2X7(k) splice variants

Conny Neblung1, Jens Rabenstein2, Viola Marschall2, Miriam León-Otegui3, Annette Nicke1

1MBNS, MPI for Experimental Medicine, Germany,2Neurochemistry, MPI for Brain Research, Germany,3Veterinary Faculty Complutense, University of Madrid, Spain

We have previously characterized a P2X7 splice variant, P2X7(k), with an alternative intracellular N-terminus and transmembrane domain 1 in rodent tissues. In comparison to the P2X7(a) variant, this variant shows increased agonist sensitivity and a higher propensity to form large cation permeable pores. Analysis by RT-PCR showed overlapping expression of both subunits in a variety of tissues. Here we coexpressed hexahistidyl-tagged and myc-tagged P2X7(a) and P2X7(k) variants in Xenopus laevis oocytes to investigate if they can form heteromeric complexes. Upon solubilization with digitonin, protein complexes containing His-tagged P2X7 subunits were purified under non-denaturing conditions via a Ni2+-NTA agarose resin and separated by SDS-PAGE. Following western blotting, co-purified myc-tagged subunits were detected with an anti-myc antibody. Our results show that myc-tagged P2X7(a) subunits can be co-purified with His-tagged P2X7(k) subunits and vice versa indicating that both variants can assemble into heteromeric receptors which might contribute to the diversity of P2X7 receptor signalling. To investigate the tissue distribution of both subunits on a cellular level, subtype-specific probes for in situ hybridization are currently generated. To this aim, the 5´non-translated region of the P2X7(k) variant is analyzed by 5´RACE.

P2-29

Role of the domain encompassing Arg304-Ile328 in rat P2X2 receptor activation

(Selected for Oral Poster Presentation)

Mark Young1, YiHong Zhang2, Lishuang Cao3, Helen Broomhead3, LinHua Jiang4

1School of Biosciences, Cardiff University, United Kingdom,2Department of Physiology and Pharmacology, University of Bristol, United Kingdom,3Faculty of Life Sciences, University of Manchester, United Kingdom,4Faculty of Biological Sciences, University of Leeds, United Kingdom

Binding of ATP to the ecto-domain of P2X receptors causes a conformational change which is transmitted from the ATP binding site to the transmembrane channel gate, leading to channel opening. It is thought that the final 25 amino acids of the ecto-domain (pre-TM2) play a critical role in this signal transduction process. In order to understand the role of pre-TM2 in ion channel function and protein expression, we examined the effects of cysteine substitutions in the pre-TM2 region of rat P2X2 (Arg304-Ile328). Mutations within the upper half of pre-TM2 (Arg304-Ile314) severely perturbed ion channel function, whereas mutations within the lower half had little effect. We also observed a decrease in apparent molecular mass for the majority of mutants in the upper half of pre-TM2, due to loss of complex N-glycosylation at nearby Asn298, reflecting a subtle alteration in the conformation of the receptor, and suggesting that protein-protein interactions within the upper half of pre-TM2 are vital to enable efficient sensing of the conformational change induced by ATP binding. Interpretation of these results using a molecular model of rat P2X2 supports this conclusion; the upper half of pre-TM2 forms a beta-strand which is involved in key protein-protein interactions, and situated beside the putative ATP binding site. In contrast, the lower half of pre-TM2 is involved in relatively few interactions, explaining why cysteine mutants were well-tolerated in this region. Our results are consistent with ATP binding causing a vertical movement of pre-TM2, which is subsequently transmitted to TM2 and the channel gate.

P2-30

BzATP activates P2X7and P2Y11receptors in lacrimal gland myoepithelial cells

Darlene A. Dartt, Kaori Ohtomo, Marie Shatos, Robin R. Hodges

Schepens Eye Research Institute, Harvard Medical School, USA

Purpose: To determine the types of purinergic receptors activated by the P2X7 receptor agonist BzATP in lacrimal gland myoepithelial cells. Methods: Rat lacrimal glands were dissociated by collagenase digestion and cultured until myoepithelial cells appeared. Immunofluorescence experiments were performed using antibodies to P2X2, P2X7, P2Y11 and P2Y13 receptors. For intracellular calcium ([Ca2+]i) measurements, myoepithelial cells were loaded with the calcium indicator dye fura2. Calcium measurements were obtained using the InCyt Im2TM ratio Imaging System. The purinergic receptor agonists ATP (P2Y and P2X agonist), UTP (P2Y agonist), and 2’(3’)-O-(4-benzoylbenzoyl) adenosine 5’-triphosphate (BzATP, P2X7 agonist) were used. A438079 and Brilliant blue G (BBG) were used as P2X7 receptor antagonist and NF157 as a P2Y11 receptor antagonist. Results: Immunohistochemistry confirmed the cellular presence of P2X2, P2X7, P2Y11 and P2Y13 receptors. ATP increased [Ca2+]i with maximum increase of 360±32nM at 10-3M. UTP increased [Ca2+]i with maximum increase of 351±7nM at 10-4M. BzATP (10-4M) increased [Ca2+]i in the presence of Mg2+ by 160±54nM and the absence of Mg2+ by 191±9nM. ATP (10-4M) increased [Ca2+]i by 342±25nM in the presence of Mg2+ and by 289±85nM in the absence of Mg2+. Pretreatment with A438079 (10-4M), BBG (10-4M), and NF157(10-4M) inhibited the BzATP stimulated increase in [Ca2+]i by 40%, 36% and 60% respectively. Addition of BzATP completely desensitized the ATP Ca2+ response as did ATP for the BzATP response. Conclusions: We conclude that in lacrimal gland myoepithelial cells BzATP appears to activate both P2X7 and P2Y11 receptors, the latter being the major response. Supported by NIH grant EY06177.

P3. P2Y

P3-1

Microglia P2Y6 receptors mediate an inhibition of astroglia proliferation in cultures of rat brain cortex

Gloria Queiroz, Clara Quintas, João Magalhães, Jorge Gonçalves

REQUIMTE, Laboratory of Pharmacology, Faculty of Pharmacy, University of Porto, Portugal

Background: Microglia P2Y receptors trigger chemotaxis and phagocytosis but their roles on the regulation of astrogliosis are largely unknow. We investigated the influence of P2Y pyrimidine receptors on astroglial proliferation in cultures of astrocytes with activated microglia. Methods: Primary cultures of astrocytes with 4% activated microglia were obtained by treatment with LPS 100 ng/mL since DIV1. Effects of nucleotides on cell proliferation were measured by methyl-[3H]-thymidine incorporation and its metabolism was evaluated by HPLC-UV. Pyrimidine receptors were detected by western blot and immunocytochemistry double labeling of microglia and P2Y2,4,6 receptors with antibodies anti-CD11b (1:50) and anti-P2Y2,4,6 receptors (1:200), respectively. Results: UTP and UDP (0.001–1 mM) inhibited astroglial proliferation up to 43 ± 2% (n = 10,P < 0.05). UTP (0.1 mM) was metabolized into UDP in 1h. The effect of UDP (1 mM) was prevented by the P2Y6 antagonist MRS2578 (1 µM), mimicked by the P2Y6 agonist PBS0474 (0.1 µM; 31 ± 3%, n = 5, P < 0.05) and abolished by the PLC inhibitor U73122 (1 µM) and the PKC inhibitor RO 32,0432 (1 µM). Immunocytochemistry revealed that P2Y6 receptors were co-localized with microglia. When the NTPDase inhibitor ARL67156 (0.1 mM) was present, the inhibitory effect of 0.1 mM UTP was reversed into a increase in proliferation of 44 ± 8% (n = 6,P < 0.05) that was mimicked by UTPγS (0.1 mM; 37 ± 6%, n = 4, P < 0.05) and prevented by 100 µM suramin or 10 µM RB2.

Conclusion: P2Y2/4 receptor-induced astroglial proliferation is prevented by a high rate of UTP metabolism with UDP formation and activation of P2Y6 receptors coupled to the PLC-PKC pathway. These receptors are highly expressed in activated microglia and mediate an indirect inhibition of astroglial proliferation.

P3-2

Airway epithelial cell P2Y6R-Expression is up-regulated during acute and chronic allergic inflammation and contributes via modulating of CXC8 and IL-6 secretion to development of airway remodelling

Rodolfo de Paula Vieira1, Tobias Müller1, Johann Christian Virchow Jr2, Volker von Gernler1, Melanie Grimm1, Sanja Cicko1, Thorsten Dürk1, Stephan Sorichter1, Robert Zeiser3, Marco Idzko1

1Department of Pneumology, University Hospital Freiburg, Germany,2Department of Pulmonary Medicine, University Hospital Rostock, Germany,3Division of Hematology and Oncology, University Hospital Freiburg, Germany

ATP binding to purinergic receptors (P2R) have been recognized as inflammatory mediator, involved in the development asthma. Here we investigated the involvement of P2Y6R-subtype in acute (AAAI) and chronic (CAAI) allergic airway inflammation in mouse and humans. We detected an up-regulation of the P2Y6R on the airway epithelial cells of animals with AAAI and CAAI, which was followed by increased eosinophilic airway inflammation and remodeling, Th2 cytokine production and airway hyperresponsiveness (AHR), which were abrogated when sensitized animals were treated with selective P2Y6R-antagonist (MRS2578) prior to allergen challenge. MRS2578 was also effective in reducing airway inflammation, remodelling and AHR in house dust mite model of allergic airway inflammation. Mechanistically we observed that MRS2578 inhibit the release of IL-6 and CXCL8 by lung epithelial cells in vivo, while intrapulmonary application of P2Y6R agonist UDP in animals with AAAI and CAAI results in increase of IL-6 and KC/CXCL8 content in BALF. In addition selective activation of the P2Y6R by UDP induces release of IL-6 and CXCL8 in mouse (LA-4) and human lung epithelial (BEAS-2B) cells in vitro. Finally experiments with P2Y6R−/− and WT chimera animals confirmed the importance of P2Y6R on the allergic airway inflammation. In summary we demonstrated for the first time that P2Y6R expression on airway epithelial cells in vivo is up-regulated during acute and chronic allergic airway inflammation, and that selective blocking of the P2Y6R reduced the cardinal features of acute and chronic airway inflammation.

P3-3

High glucose and free fatty acids induce beta-cell apoptosis via autocrine effects of ADP acting on the P2Y13 receptor

Chanyuan Tan, Siv Svensson, Björn Olde, David Erlinge

Department of Cardiology, Lund University, Sweden

High levels of glucose and saturated fatty acids have detrimental effects on beta-cell function and survival. However, the signaling pathways mediating these effects are not entirely known. In a previous study, we found that ADP regulates beta-cell insulin secretion and beta-cell apoptosis. We have now investigated if autocrine/paracrine mechanisms of ADP and purinergic receptors are involved in this process. ATP release in MIN6c4 cells was measured by bioluminescence. High glucose (16.6 mM) and palmitate (100 μM) potently elevated the extracellular ATP levels within seconds of the exposure, and both effects could be inhibited by the calcium-channel blocker nifedipine. This rapid response of high glucose is specific to glucose, as equimolar concentration of mannitol was without effect. Exposure of the cells to the VRAC-channel inhibitor NPPB, or the pannexin-1 blocker carbenoxolone, also resulted in a substantial decrease in high glucose/palmitate induced ATP release. These results indicate that, calcium-channels and VRAC-channels might be involved in the ATP release mechanism. High glucose and palmitate inhibited cAMP production, reduced cell proliferation and increased Caspase-3 activity in MIN6c4 cells. All these effects could be diminished by the P2Y13 antagonist MRS2211. Further studies show that blocking the P2Y13 receptor results in enhanced CREB phosphorylation, which is known to be involved in beta-cell survival. These findings provide further support for the concept that P2Y13 plays an important role in beta-cell apoptosis and suggest that autocrine/paracrine mechanisms, related to ADP and P2Y13 receptors, are involved in the proapoptotic effects of high glucose and free fatty acids.

P3-4

Involvement of PKD in UDP-stimulated microglial phagocytosis

Ayako Kataoka, Hidetosi Tozaki-Saitoh, Yui Koga, Ayumi Uesugi, Makoto Tsuda, Kazuhide Inoue

Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Japan

The clearance of tissue debris by microglial phagocytosis is one of the crucial components in the maintenance of the brain homeostasis. Recently, it was reported that extracellular UDP stimulates phagocytosis activity of microglia through P2Y6 receptor activation. However, the intracellular mechanism underlying these phenomena has not been identified. First, we examined the effects of some PKC inhibitors on UDP-stimulated phagocytosis of fluorescent microsphere. As a result, we found that the facilitation of phagocytosis by UDP was effectively inhibited by Gö6976, a PKC and PKD (PKCμ) inhibitor and CID755673, a selective PKD inhibitor, but not by Gö6983 which is less sensitive to PKD. Then, we observed rapid phosphorylation and membrane translocation of PKD after UDP stimulation. The phosphorylation of PKD at Ser916 was partially suppressed by Gö6976 and CID755673, but the translocation to the plasma membrane was little affected. On the other hand, these inhibitors clearly suppressed large vacuole formation which was visible after UDP stimulation and contained phagosytosed microsphere. Furthermore, we found that these vacuoles were surrounded by PKD immunostaining. These data suggest that PKD control the microglial phagocytosis by regulating vacuole formation.

P3-5

Presence of functional P2 receptors in human SK-N-MC neuroblastoma cells

(Second Oral Poster Presentation Award)

Miriam León-Otegui1, Javier Gualix1, Maria Recuero2, Maria Jesus Bullido2, Fernando Valdivieso2, Maria Teresa Miras-Portugal1

1Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain,2Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Spain

SK-N-MC human neuroblastoma cells have been used as a parental cell line with which generate lines that stably overexpress proteins of interest in different neurodegenerative disorders. In this study we have analyzed the presence of functional P2 receptors in the SK-N-MC cell line. If such receptors were present, SK-N-MC cells could serve as a model with which analyze different aspects of the nucleotide signalling in neural tissues, including their putative interaction with proteins involved in neurodegenerative diseases. Western blotting and immunocytochemical analysis indicated the presence of several P2 proteins in the SK-N-MC cells. Functional analyses were performed by single-cell imaging after loading cells with the calcium dye Fura-2. SK-N-MC cells showed transient [Ca2+]i increases upon application of ADP, 2MeSADP or ADPbetaS. Responses to these agonists are likely mediated by a P2Y1 receptor, as demonstrated by the almost complete blockade exerted by the MRS2179 antagonist. ATP was also able to induce [Ca2+]i increases in the SK-N-MC cells. Responses to ATP were partially blocked by both MRS2179 and TNP-ATP, this suggesting that ATP could interact with two different P2 receptors: a P2Y1 and a P2X receptor. To further characterize the P2X receptor we analyze the effect of several agonists. No cells showed responses to either alpha,beta-meATP or Bz-ATP, although calcium transients could be observed when cells were challenged with CTP. Moreover, both the CTP response and the MRS2179-resistant component of the ATP response were potentiated in the presence of ivermectin, such pharmacology profile suggesting the presence of a functional P2X4 receptor.

P3-6

Roles of highly conserved amino acid residues of the human platelet P2Y12-receptor in receptor function and ligand recognition

Kristina Hoffmann, Emanuele Marine, Ivar von Kügelgen

University of Bonn, Germany

Background: The P2Y12-receptor plays an important role in platelet aggregation. In previous studies, we identified several amino acid residues involved in the interaction of the receptor protein with agonists and antagonists. In the present study, we searched for other residues possibly involved in the interaction of the human P2Y12-receptor with the nucleotide agonist 2-methylthio-ADP (2-MeSADP) and antagonists including the irreversible antagonist p-chloromercuribenzene-sulfonic acid (pCMBS). Methods: Wild-type or mutant receptor proteins were stably expressed in CHO Flp-In cells. Receptor function was assessed by measuring changes in cellular cAMP or the cAMP-response-element (CRE) directed luciferase expression in cells stimulated by forskolin [10 or 1 µM]. Results: In cells expressing the wild-type receptor, 2-MeSADP [0.01 nM–1 µM] inhibited cellular cAMP levels and the CRE-directed luciferase expression concentration-dependently with IC50-values of about 1 nM. When the highly conserved amino acid residue N43 in transmembrane region 1 (TM1) was replaced by serine, no change in the IC50-concentration was observed, indicating that N43 is not involved in the interaction with the agonist. The same was true when C248 in TM6 was replaced by the neutral alanine. In contrast, no responses to the agonist were observed in cells expressing C97R-, C175D-, C248S- and P251A-mutant receptors. In cells expressing wild-type receptors or C248A-mutant receptors pCMBS inhibited receptor function with almost identical potencies. Conclusions: These results show that the residues C97 in TM3, C175 in EL2 and P251 in TM6 play crucial roles for receptor structure and function, whereas C248 may play an intermediate role.

P3-7

Differential control of desensitization and internalization by C-terminal phosphorylation sites of the human P2Y1-receptor

Carsten Hoffmann1, Susanne Reiner1, Nicole Ziegler1, Cathrine Leon2, Kristina Lorenz1, Kathrin von Hayn1, Christian Gachet2, Martin Lohse1

1Pharmacology, University of Wuerzburg, Germany,2UMR S949 INSERM, University of Strasbourg, France

The P2Y1-receptor is involved in the regulation of a variety of physiological processes, among them is platelet aggregation. Receptor desensitization is often highly regulated to control those processes. To study the role of potential phosphorylation sites in receptor desensitization, internalization and beta-arrestin-2 translocation of the P2Y1-receptor, we constructed three P2Y1-clustermutants, each lacking potential phosphorylation sites in the 3rd intracellular loop (P2Y1 Gr3), or either the proximal-(P2Y1 Gr1) or distal C-terminus (P2Y1 Gr2). The receptor constructs were C-terminally fused to a fluorescent protein and expressed in HEK-293 cells. Quantification of ADP-induced internalization provided by either a cell surface ELISA or confocal microscopy indicated that P2Y1 Gr1 and Gr3, like the wild-type P2Y1-receptor, underwent rapid internalization while internalization for the P2Y1 Gr2 receptor was fully impaired. ADP-induced beta-arrestin-2-YFP translocation to membrane P2Y1-receptors followed the same pattern as receptor internalization. Hence, no beta-arrestin-2-YFP translocation was observed with the P2Y1 Gr2-construct. Individual mutations indicate that residues Ser352 and Thr358 are essential for receptor internalization and beta-arrestin-2-YFP translocation. In contrast, analysis of the desensitization of Calcium signalling by measuring fura-2 in cells pre-treated with or without PMA revealed receptor desensitization for the P2Y1 Gr2-construct that was identical to wild-type P2Y1-receptor. In contrast receptor desensitization was prevented by the lack of potential phosphorylation sites in the P2Y1 Gr1-construct. While PMA did induce receptor desensitization, incubation with PKC and CaM-Kinase inhibitors did not block P2Y1-receptor internalization in HEK-293 cells. Taken together these results indicate that different phosphorylation sites and kinases regulate P2Y1-receptor desensitization and internalization.

P3-8

Mechanisms of protein kinase D activation in response to P2Y2 and P2X7 receptors in primary astrocytes

Esmerilda G. Delicado1, Luz María G. Carrasquero2, Lucía Sánchez-Ruiloba2, Teresa Iglesias2, Mª Teresa Miras-Portugal1

1Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain,2Instituto de Investigaciones Biomédicas, "Alberto Sols", CSIC-UAM, Spain

Protein kinase D (PKD) is a family of serine/threonine kinases that can be activated by many stimuli via protein kinase C. The present work focuses on the study of the activation of protein kinase D, PKD, by nucleotides due to the fact that, just as the P2 receptors, it also is expressed in immune and nervous system and it is involved in apoptosis, neurite growth, growth cone organization and cellular proliferation. We demonstrate that P2Y2 and P2X7 receptor stimulation of primary rat cerebellar astrocytes rapidly increases PKD1/2 phosphorylation and activity. P2Y2 receptor response evokes a PKD1/2 activation that is dependent on a pertussis toxin-insensitive G protein, phospholipase C (PLC)-mediated generation of diacylglycerol and protein kinase C. This mechanism is similar to the one described for other G-protein coupled receptors. In contrast, the way the ionotropic P2X7 receptor activates PKD1/2 is significantly different. Importantly, this response is not dependent on calcium entry, but depends on the activity of several phospholipases, including phosphoinositide-phospholipase C (PI-PLC), phosphatidylcholine-phospholipase C (PC-PLC) and also phospholipase D (PLD). Immunoblot and confocal microscopy analysis show that PKD1/2 activation by nucleotides is transient. The active kinase first moves to and concentrates in certain plasma membrane domains. Then, phosphorylated-PKD1/2 translocates to intracellular vesicles, where it remains active. All together, our results open the perspective of PKD1/2 being involved in many physiological functions where nucleotides play important roles not only in astrocytes but in other cell types bearing these receptors.

P3-9

Synthesis and structure-activity relationships of a new class of P2Y2 receptor antagonists

Mario Funke, Stefanie Rinker, Petra Hillmann, Christa E. Müller

Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, Germany

P2Y2 receptor antagonists have been proposed as anti-inflammatory, neuroprotective and anti-hypertensive agents. However, potent and selective P2Y2 antagonists are currently not available. In a screening campaign, we identified 3-nitrophenyl 4-(3-methylbenzamido)benzenesulfonate (PSB-0402-1) as a relatively potent antagonist at human P2Y2 receptors with an IC50 value of 3.64 µM. In the present study we investigated the properties and structure-activity relationships of this new class of P2Y2 receptor antagonists. The stability of PSB-0402-1 was assessed by LCMS. A series of phenyl 3- and 4-benzamidobenzenesulfonates and -sulfonamides was synthesized. Furthermore, the sulfonate function was replaced by amide and urea groups. The compounds were investigated in calcium assays at human P2Y2, P2Y4 and rat P2Y6 receptors expressed in 1321N1 astrocytoma cells. The ester function of the lead structure was shown to be highly stable at different pH values. Depending on the substitution of the benzamido moiety, the synthesized phenyl sulfonates showed antagonistic activity at P2Y2 receptors mostly in the very low micromolar range and selectivity versus P2Y4 and P2Y6 receptors. In contrast to the sulfonates the corresponding sulfonamides were inactive at the receptors. A novel class of relatively potent and selective P2Y2 receptor antagonists has been identified. They might be useful as pharmacological tools to elucidate the (patho)physiological roles of the P2Y2 receptor and have potential as lead structures for drug development.

P3-10

Essential role of P2Y6 receptor in vascular contraction—lessons from P2Y6 knockout mice

Gilles Kauffenstein1, Laurent Loufrani1, Jean Sévigny2, Bernard Robaye3, Daniel Henrion1

1UMR CNRS 6214—INSERM 771, Université Angers, France,2CRRI, Université Laval, Canada,3IRIBHM, Université libre de Bruxelles, Belgium

Background: Extracellular nucleotides exert their effect trough cell membrane bound P2 receptors. Seven ligand-gated P2X (P2X1-7) and eight G protein-coupled P2Y receptors (P2Y1,2,4,6,11-14) have been cloned. P2X receptors are activated by ATP while P2Y receptors are differentially activated by ATP, ADP, UTP, UDP or UDP-glucose. Activation of vascular smooth muscle cells (VSMC) P2 receptors promotes vasoconstriction via P2X1 and pyrimidine-sensitive (UDP, UTP) P2Y receptors and takes part to the neurogenic response of resistance arteries. The nature of the receptor(s) mediating the effect of uracyl nucleotides remains uncertain. Based on expression pattern and functional data P2Y2, P2Y4 and P2Y6 receptors where proposed to be involved. However, the lack of specific antagonist makes it difficult to point their respective contribution. Purpose: In the present work we took advantage of P2Y6−/− mice to assess the contribution of P2Y6 in vascular contraction. Results: We found that P2Y6 receptor is essential for UDP, UDPbetaS and UTP mediated contraction of mouse aorta, mesenteric and cerebral arteries. P2Y6 is not the receptor for the EDCF Up4A from which the receptor remains unknown. The contraction to non-nucleotides vasoactive agents was not modified. In future work, we will investigate the potential role of P2Y6 receptor in myogenic response. Conclusion: We report here that P2Y6 is the exclusive receptor involved in vascular contraction to uracyl nucleotides. These results suggest that this receptor could be a target for new anti-hypertensive molecules.

P3-11

P2Y11 receptors: novel aspects of receptor pharmacology and receptor trafficking

Georg Reiser, Michael Haas

Institut für Neurobiochemie, Otto-von Guericke Univerität, Germany

Background: We have shown that P2Y11 receptors stably expressed in HEK293 cells form hetero-oligomers with P2Y1 receptors (Ecke et al. Biochem. J. (2008) 409, 107–116). The hetero-oligomers possess pharmacological properties clearly distinct from the non-oligomeric receptors. Furthermore, non-hetero-oligomeric P2Y11-R, which is incapable of undergoing endocytosis if expressed without P2Y1-R in 1321N1 astrocytoma cells, show internalization after treatment with nucleotides known to be P2Y1-R agonists. Methods: P2Y11-R (wild-type and mutant) were expressed in different cell types with different P2Y1 receptor background. The receptor was analyzed pharmacologically by measuring the phospolipase C-coupling induced responses. Results: The Ala-87-Thr single nucleotide polymorphism of the P2Y11-R, which was reported to be linked to increased risk of acute myocardial infarction and C-reactive protein levels (Amisten et al. Eur Heart J. (2007) 28, 13–18), is not able to alter the pharmacological properties of the P2Y11-R, when expressed in 1321N1 cells. However, if stably expressed in HEK293 cells it shows a remarkable decrease in responsiveness regarding rise of intracellular calcium concentration towards P2Y1-R and P2Y11-R agonists compared to the wild-type P2Y11 receptor and even un-transfected HEK293 cells expressing endogenous P2Y1-R. P2Y1-R specific agonists also were unable to trigger P2Y11-A87T internalization in HEK293 cells. Conclusion: These results indicate a relevance of the Ala-87 residue for hetero-oligomeric receptor function and possibly in inflammation-related diseases, since both receptors are co-expressed in the brain, smooth muscle tissue, and immune cells. The hetero-oligomers may represent the physiological condition in cells expressing both receptor subtypes.

P3-12

P2Y1 receptors activates nitric oxide synthase in rat pineal gland

(Selected for Oral Poster Presentation)

Zulma Ferreira, Camila Petrilli, Regina Markus

Instituto de Biociências—Dep. Physiology, University of São Paulo, Brazil

Background: We have previously shown that ATP is coreleased with noradrenaline from sympathetic neurons that innervate rat pineal gland. The activation of P2Y1 receptors potentiates noradrenaline-induced melatonin synthesis via an increase in intracellular calcium mediated by activation of phospholipase C (Mortani-Barbosa et al., Eur. J. Pharmacol. 401:59–62, 2000; Ferreira et al., Eur. J. Pharmacol. 415:151, 2001; Pharmacology 69:33–37, 2003). Here we investigated whether activation of P2Y1 triggers the production of NO, cGMP and activation of NFKB pathway in the pineal. Methods: NO and cGMP were measured in AMP-PNP-stimulated pinealocytes by fluorometric assay, and RIA, respectively. NFkB nuclear content was determined by EMSA in cells stimulated by ADP. Results: AMP-PNP (0.1–1 mM) increased in a dose-dependent manner pinealocytes NO content (maximal increase 15.2 ± 2.6% in 3 different preparations). This response was mediated by P2Y1 receptors, as it was abolished by the A3P5P (0.3 mM, n = 3). The non-selective nitric oxide synthase (NOS) antagonist L-NAME (0.1 mM, n = 3) also blocks NO production. As a matter of fact, activation of P2Y1 receptors by AMP-PNP (0.3 mM) increased cGMP content 5 times over basal, while ADP (0.3 mM, 1 min) increased NFKB nuclear translocation in comparison to non-stimulated glands (n = 4 per treatment). Conclusion: These data suggest that the synthesis of NO triggered by P2Y1 receptors in pineal glands result in the formation of cGMP. In addition it might depend on the activation of calcium-dependent constitutive NOS, due to increasing in intracellular calcium, as well as the expression of iNOS, which is mediated by NFKB. Support: FAPESP, CNPq.

P4. Purine/Pyrimidine transport

P4-1

Gamma-irradiation induces P2X7 receptor-dependent ATP release from B16 melanoma cells

Yasuhiro Ohshima1, Mitsutoshi Tsukimoto1, Takato Takenouchi2, Hitoshi Harada3, Akina Suzuki1, Mitsuru Sato2, Hiroshi Kitani2, Shuji Kojima1

1Faculty of Pharmaceutical Sciences, Tokyo University Of Science, Japan,2Transgenic Animal Research Center, National Institute of Agrobiological Sciences, Japan,3Faculty of Pharmaceutical Sciences, Suzuka, University of Medical Science, Japan

Background: Ionizing irradiation causes not only growth arrest and cell death, but also release of growth factors or signal transmitters, which promote cancer malignancy. Extracellular ATP controls cancer growth through activation of purinoceptors. However, there is no report of radiation-induced ATP release from cancer cells. Here, we examined gamma-irradiation-induced ATP release and its mechanism in B16 melanoma. Methods: Extracellular ATP was measured by luciferin-luciferase assay. To investigate mechanism of radiation-induced ATP release, we pharmacologically inhibited the ATP release and established stable P2X7 receptor-knockdown B16 melanoma cells using two short hairpin RNAs targeting P2X7 receptor. Results: Cells were exposed to 0.5–8 Gy of gamma-rays. Extracellular ATP was increased, peaking at 5 min after 0.5 Gy irradiation. A selective P2X7 receptor channel antagonist, but not anion transporter inhibitors, blocked the release of ATP. Further, radiation-induced ATP release was significantly decreased in P2X7 receptor-knockdown cells. Conclusion: Our results indicate that gamma-irradiation evokes ATP release from melanoma cells, and P2X7 receptor channel plays a significant role in mediating the ATP release. We suggest that extracellular ATP could be a novel intercellular signaling molecule released from cancer cells when cells are exposed to ionizing radiation.

P4-2

Purinergic regulation of the nucleoside transporter rCNT2 in rat neuronal model

(Selected for Oral Poster Presentation)

Lorena Medina Pulido, Miriam Molina Arcas, F. Javier Casado, Ferran Burgaya, Marçal Pastor Anglada

Departament de Bioquímica i Biología Molecular, Facultat de Biología, Universitat de Barcelona, Spain

PURINERGIC REGULATION OF THE NUCLEOSIDE TRANSPORTER rCNT2 IN RAT NEURONAL MODEL Lorena Medina-Pulido1, Miriam Molina-Arcas1, F. Javier Casado1, Ferran Burgaya2, Marçal Pastor-Anglada1 1Departament de Bioquímica i Biologia Molecular, Institut de Biomedicina (IBUB), Universitat de Barcelona. CIBER EHD, Barcelona 2Departament de Biologia Cel•lular, Universitat de Barcelona, Barcelona High extracellular adenosine levels have neuroprotective effects and they are likely to be regulated by either extracellular synthesis or membrane transport processes. Previous studies from our laboratory (MCB 24:2710, 2004) had proven purinergic up-regulation of the high affinity Na+ -coupled adenosine transporter CNT2 by A1R activation in epithelia. Distribution studies of nucleoside transporters in CNS revealed CNT2 expression in CNS (J.Neurochem.90:883, 2004). The present study aimed at characterising the purinergic regulation of CNT2 in the rat pheocromocytoma cell line PC12. PC12 cells were differentiated with nerve growth factor (NGF) into a neuronal model. Western blot, immunocytochemistry, real-time PCR, in vitro tests of substrate uptake and pharmacological regulation were performed at different stages of differentiation. PC12 differentiation induced CNT2 activity and associated with an increase in A1R mRNA levels whereas A2aR, A2bR and A3R receptor mRNA amounts were down regulated. A transient increase of CNT2 activity was observed by a mechanism consistent with A1R activation. CNT2 activity in this model was sensitive to inhibition by methylxanthines, such as caffeine, natural antagonists of adenosine. This study suggests that CNT2 expressed in CNS may be target of methylxanthines and regulated by P1 type receptors, thus contributing to modulate extracellular adenosine levels.

P4-3

The high-affinity adenosine transporter, rCNT2, requires dual sorting signals for its trafficking to plasma membrane

Itziar Pinilla Macua, Ivette Aymerich, Isabel Huber, F. Javier Casado, Marçal Pastor-Anglada

Departament de Bioquímica i Biología Molecular, Facultat de Biología, Universitat de Barcelona, Spain

The high-affinity adenosine Na+ dependent transporter, CNT2, is expressed in both polarized and non polarized cells. However, there is no information about the domains involved in CNT2 sorting to the plasma membrane. This work aimed at identifying structural and functional patterns for CNT2 trafficking. Several CNT2 constructs with partial deletions of the intracellular N-terminal and extracellular C-terminal tails have been generated, as well as site-directed mutagenesis of a potential Casein Kinase 2 (CK2) domain, several highly conserved glutamates, and some extracellular cysteines, as well. The comprehensive topological mapping of the N-tail revealed the following features: 1) A glutamate enriched region (NPGLELME) between residues 21 and 27 seems to be implicated in ER export, since side-directed mutagenesis of these conserved glutamates resulted in ER accumulation. 2) Mutation of a potential CK2 domain leads to a loss of brush border-specific insertion whereas the simulation of the phosphate negative charge by acidic amino acids results in a wild type phenotype recovery. The analysis of the deleted constructs of the C-terminus tail and their sorting to the plasma membrane anticipates a role in ER export and trafficking, especially between residues G591 and T642. Site-directed mutagenesis of Cys649, Cys650 and Cys658 revealed the importance of Cys650 in the correct trafficking of rCNT2. In conclusion, CNT2 possesses dual sorting signals. Within the N-terminus tail an acidic region is responsible for its proper insertion into the plasma membrane whereas the putative CK2 domain is implicated in the apical insertion of the transporter protein. C-terminus Cys residues may also be involved in CNT2 trafficking.

P4-4

All- trans-retinoic acid promotes trafficking of the human concentrative nucleoside transporter 3 (hCNT3). Implications for purine-based anticancer therapy

Paula Fernández Calotti1, Dolors Colomer2, Marçal Pastor Anglada1

1IBUB, CIBERehd, University Of Barcelona, Spain,2IDIBAPS, Hospital Clinic, Spain

Background: Concentrative nucleoside transporter (hCNT3) transports efficiently some adenosine-derivatives used in anticancer therapy such as fludarabine (Flu), a major choice in the treatment of chronic lymphocytic leukaemia (CLL). Although CLL cells lack this transport activity, Flu-resistant populations of CLL have high levels of intracellular hCNT3 protein. Objective: Our study aimed at determining the effect of all-trans-retinoic acid (ATRA) on hCNT3 activity as a suitable mechanism to improve Flu-based CLL therapy. Methods: Nucleoside transport was measured using [5,6-3H]-nucleosides. The amount of hCNT3 mRNA and protein were analyzed by real-time PCR and western blot. The mechanism was evaluated by using specific inhibitors and verified by western blotting or by specific gene silencing. Apoptosis was evaluated by flow cytometry analysis. Results: ATRA increased hCNT3-related activity in a CLL cell line and other non lymphoid cell types. This effect requires trafficking of already synthesized hCNT3 proteins to the plasma membrane and is mediated by TGF-β1 and by activation of p38, ERK1/2 and the small GTPases RhoA. Interestingly co-culture of ATRA and Flu in primary CLL cells from Flu-resistant patients significantly restored sensivity to the analog. Preliminary data indicates that this is due to an increased entrance of Flu through the newly located hCNT3 at the plasma membrane. Conclusion: We describe for the first time a mechanism of hCNT3 trafficking to the plasma membrane with a concomitant reversion of Flu insensitivity that might lead to improvement of CLL treatment.

P5. Purine/Pyrimidine release

P5-1

ATP induce ATP release in hepatoma cells

Maria Victoria Espelt1, German Sanchez Alberti1, Osvaldo Chara2, Pablo Julio Schwarzbaum1

1Conicet, IQUIFIB, Argentina,2Conicet CIC, UNLP, Argentina

Background: In hepatic cells extracellular nucleotides act via P receptors regulating different physiological responses as: glucogenolysis, gluconeogenesis, canalicular contraction, etc. The aim of our study was to analyze how extracellular ATP (ATPe) activates ATP release in HepG2 cells. Methods: ATPe was quantified using the luciferin-luciferase method. Intracellular calcium was measured by fluorescence microscopy. Results: After addition of exogenous ATP, ATPe rised to a maximum followed by an exponential decay. The maximum reached was 43.1 ± 3.01 nM (with 50 nM ATP added; n = 6), 135.0 ± 19.25 nM (100 nM ATP; n = 7), 269.0 ± 26.78 nM (200 nM ATP; n = 7), and 555.8 ± 39.70 nM (400 nM, n = 5). When cells were preincubated with cibacron blue and suramin (P2 blockers), 400 nM ATP induced 426.0 ± 33.02 nM ATPe. ATP release increased to 823.4 ± 36.16 nM when cells were stimulated with 400 nM ATP (n = 4) and preincubated with PI3K inhibitors, being this release inhibited with P2 inhibitors (640.2 ± 71.45 nM). Regarding potential 2nd messengers activating ATP release, we determined the percentage of cells showing at least one spike in Ca2+ after incubation with different ATPe concentrations. The spike frequency augmented hyperbolically with ATPe concentration with a K0.5 = 3.8 uM. Conclusion: ATP induce ATP release in HepG2 cells in concentrations between 100–400 nM. The release is further stimulated with PI3K inhibitors and inhibited with cibacron blue and suramin. ATPe increases Ca2+ spike frequency, which in principle could lead to ATP secretion.

P5-2

Adenosine A2A receptors increase sympathetic co-transmitter release

Maria Veronica Donoso Gomien, Juan Pablo Huidobro-Toro

Pontificia Universidad Catolica de Chile, Chile

To assess A2A adenosine receptors as presynaptic modulators of sympathetic transmission, we characterized selective ligands on the electrically evoked release of noradrenaline (NA) and ATP from the rat vas deferens nerve endings. Ex-vivo prostatic halves were maintained in Krebs buffer gassed with a mixture of 95/5% O2/CO2; tissues were electrically depolarized with 16 Hz pulses (70 V, 1 msec for 1-min); the tissue superfusate was collected to quantify NA and ATP overflow. Depolarization of the tissue nerve endings in the presence of 30 nM CGS21680 (CGS) elicited an increase in NA overflow from 21 ± 2.6 (n = 12) to 37 ± 8.7 pmol (n = 6, p < 0.05) and total purines from 326 ± 25 (n = 12) to 489 ± 74 pmol (n = 6, p < 0.05). Consonant with the previous findings, 10 nM of A2A antagonists, either ZM241385 (ZM) or SCH58261 (SCH), significantly halved the electrical stimulation elicited transmitter release overflow. Parallel experiments revealed that the joint addition of ZM plus CGS or SCH plus CGS abolished the increment of transmitter release evoked by CGS. Functional correlates of these findings indicate that CGS or 10 nM adenosine increased the motor action of the electrically driven nerve activity supporting a role of presynaptic A2A receptors in the sympathetic co-transmitter tone maintenance. Moreover, CGS reduced the tissue tension induced by exogenous NA or ATP, revealing in addition, a postjunctional inhibitory action. Supported by grant 13980001, PFB 12/2007 and the MIFAB.

P5-3

Microvesicles shed from microglial cells upon ATP activation: a new pathway of glia-to-neuron communication

Flavia Antonucci1, Loredana Riganti1, Elena Turola1, Paola Giussani2, Paola Viani2, Michela Matteoli1, Claudia Verderio3

1Institute of Neuroscience and Dept. of medical Pharmacology, University of Milano, Italy,2Department of Medical Chemistry, Biochemistry and Biotechnology, University of Milano, Italy,3Institute of Neuroscience and Dept. of medical Pharmacology, CNR, Italy

In the course of inflammation, microglia shed microvesicles (MVs) from the plasma membrane. MVs,together with exosomes, are extracellular vesicles, known to function as shuttles for the delivery of cargoes between different cells (Ratajczak et al., 2006; Simons and Raposo 2009. Aim of the study was to investigate whether microglia-derived MVs affect neuronal exocytosis. We first analyzed miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons exposed to MVs and found that MVs increased by 2 fold the frequency of mEPSCs without changes in their amplitude. MV-induced stimulation of synaptic vesicles fusion was confirmed by monitoring the internalization of antibodies against the luminal domain of synaptotagmin I and by analyzing the distribution of synapsin I at the level of growth cones. Recent data suggest that the fusion process is enhanced by the sphingolipid sphingosine (Darios et al., 2009); also our previous evidence indicates that acid sphingomyelinase, a key enzyme in sphingolipid metabolism, is associated to MVs shed from microglia (Bianco et al., 2009). Based on this evidence, we tested the possibility that MVs potentiate synaptic vesicle exocytosis by favouring neuronal sphingosine availability. Direct measurements of sphingolipid metabolism in neurons exposed to MVs revealed a 1.68 fold increase in sphingosine synthesis. Furthermore, pretreatment of cultured neurons with N-oleoylethanolamine NOE (60 microM), which prevents sphingosine synthesis, strongly inhibited presynaptic stimulation induced by MVs. These data provide the first evidence that MVs contribute to microglia-to-neuron signalling and underlie the role of the sphingolipid system in the modulation of neuronal activity by glial cells.

P5-4

In vivo bioluminescence imaging of ATP release in rat cerebral cortex

Takahiro Takano, Weiguo Peng, Maiken Nedergaard

Center for Translational Neuromedicine, University of Rochester, USA

Purinergic signaling plays prominent roles in most pathological events in CNS, such as onsets of microglial movements and other inflammatory responses. However, the spatiotemporal pattern of ATP release in acute CNS pathologies is largely unknown. Direct imaging of ATP release has the potential to reveal the dynamics of purinergic signaling in real time, as well as dissect the mechanisms involved in ATP release. Here we employed a highly sensitive liquid-cooled CCD camera to capture bioluminescence signals as a consequence of ATP efflux from the exposed tissue. Essentially no ATP release was detected from control, uninjured spinal cord or cortex. Traumatic injury of the spinal cord inflected by weight-drop induced a prominent increase of ATP in not infarct itself, but peri-traumatic regions, which sustained for more than 6 hours (Wang et al., 2004). In contrast, little or only moderate ATP was observed in the peri-infarct of cerebral cortex. Moreover, focal ischemia induced by permanent MCA occlusion was not associated with any sustained increases in ATP release from the exposed cortex. Instead, spontaneous point source bursts of ATP release were frequently observed in the ischemic hemisphere. The apparently random pattern of ATP burst release was reminiscent of ATP burst releases in cultured astrocytes via opening of connexin hemichannels (Arcuino et al. 2002). Combined, we revealed two distinct patterns of purinergic signaling in traumatic versus ischemic injury. The excessive ATP release evoked by traumatic injury of spinal cord compared to cortex may partly explain why inflammatory responses are greater in spinal cord.

P5-5

Acupuncture induces release of adenine nucleotides and adensoine

Takumi Fujita, Weiguo Peng, Takahiro Takano, Kim Tieu, Maiken Nedergaard

Center for Translational Neuromedicine, University of Rochester, USA

Acupuncture is an invasive procedure in which fine needles are inserted and manipulated in patients, so as to relieve pain. Although the analgesic effect of acupuncture is well documented, the mechanism by which the procedure reduces pain is poorly understood. It is in this regard of interest that adenosine is an analgesic agent that suppresses pain through Gi-coupled A1-adenosine receptors. To determine whether the extracellular concentration of adenosine changes during acupuncture, we measured the release of purines from muscle tissue using microdialysis samples collected in close proximity to the acupuncture point. The purines were quantified using high-performance liquid chromatography with UV absorbance before, during and after acupuncture. At baseline, the concentrations of ATP, ADP, AMP, and adenosine were in the low nM range. Acupuncture applied by gentle manual rotation of the acupuncture needle sharply increased the extracellular concentration of all purines. Adenosine increased ~24-fold during the 30 min acupuncture session. The extracellular concentration of ATP returned to baseline after acupuncture, whereas adenosine, AMP, and ADP remained significantly elevated at 60 min. Pharmacological inhibition of adenosine deaminase activity potentiated the acupuncture-elicited increase in adenosine (3.7 fold), and prolonged the increase in adensoine to 2 hrs. These results show that acupuncture triggers an increase in the extracellular concentration of adenosine, which can be further increased by suppression of deaminase activity. The time course of acupuncture-mediated release of adenosine and anti-nociception overlaps, suggesting that adenosine may contribute to the clinical benefits of acupuncture.

P5-6

Taste buds of P2X2/P2X3 double knockout mice fail to release ATP

(Selected for Oral Poster Presentation)

Sue Kinnamon1, Yijen Huang2, Leslie Stone1, Elizabeth Pereira2, Thomas Finger1, Stephen Roper2

1Rocky Mountain Taste and Smell Center, University of Colorado Denver, USA,2Dept. Physiol. Biophys., University of Miami Sch. of Medicine, USA

Taste buds are the transducing elements of gustatory sensation. A typical taste bud comprises approximately 100 individual cells, whose apical processes protrude through a taste pore and encounter sapid stimuli in the oral cavity. The basolateral regions of taste cells release transmitter to activate gustatory afferent nerve fibers. Recent data strongly implicate ATP as the primary transmitter that couples taste transduction to activation of the sensory afferents. These data include the presence of P2X2 and P2X3 on gustatory afferent nerve fibers, the release of ATP in response to taste stimuli, and most importantly, the complete absence of taste responses in gustatory afferent nerves of P2X2/P2X3 double knockout mice. Further, recent data suggest that ATP release from taste cells occurs by non-vesicular mechanisms, most likely via hemichannels composed of Pannexin-1. In this study we have asked whether ATP release is normal in the P2X2/P2X3 double knockout mice. Two complimentary approaches, biosensor cells expressing purinergic receptors and a luciferin/luciferase-based assay were used to assess taste-evoked ATP release in the knockouts compared to their wildtype counterparts. Although taste cells of the knockout mice generated normal increases in intracellular Ca2+ in response to taste stimuli, they failed to release ATP. Preliminary experiments showed similar expression of Pannexin-1 in knockout and wildtype mice, suggesting that this aspect of the release mechanism is intact. Future experiments are directed at understanding how the absence of P2X receptors leads to a failure to release ATP.

P5-7

ATP release and autocrine signalling following gentle pipetting of HeLa cell media

Juan Pablo Huidobro-Toro, Javiera Lopez, Andrea Hinojosa, Sonja Buvinic, Alfonso Gonzalez

Pontificia Universidad Catolica de Chile, Chile

To assess autocrine actions of ATP, we examined whether pipetting the culture media of HeLa cells elicited the release of nucleotides to the media and ascertained if the extracellular ATP generated an intracellular response. Gentle pipetting the cell media, elicited within 5–10 seconds a rise in ATP that decayed with an estimated half life of 15 minutes; ATP was hydrolyzed to ADP, AMP and adenosine. The rise in extracellular ATP was proportional to the times the cells were pipetted, reaching with three rapid manoeuvres a maximal secretion of 359.4 ± 55.2 pmolATP per million cells (n = 44). Pipetting in the presence of apyrase obliterated the nucleotides rise. ATP secretion was not dependent on connexion/pannexin hemichannels, nor on vesicular transport, but on extracellular and intracellular calcium pools. The ATP released by pipetting operated as an autocrine signal since it was paralleled by intracellular calcium transients obliterated by apyrase or 10 µM suramin; 1 µM MRS 2179 reduced only partially these transients; 2-MeSADP or ATP mimicked the transients. Pipetting caused a rapid EGF receptor phosphorylation; receptor internalization was observed within 15–30 minutes. EGFR activity was followed by ERK activation by phosphorylation, which was eliminated by apyrase and reduced by suramin. In sum, ATP elicits an autocrine action exerted via nucleotide receptors which involve EGF receptor transmodulation. Funded by PFB 12/2007, MIFAB and grant13980001.

P5-8

ATP release and [Ca2+]i oscillations by fibroblasts in response to stressful conditions

Ana Pinheiro1, Célia Soares1, Maria Adelina Costa2, Paulo Correia-de-Sá1

1Laboratório de Farmacologia e Neurobiologia, UMIB, ICBAS, Portugal,2Departamento de Química, ICBAS, Portugal

Pain related to the musculoskeletal system is very common. ATP has been claimed to excite or sensitize myofascial receptors. However, the mechanism(s) by which purinergic signals mediate myofascial pain is still speculative. Therefore, we decided to investigate the response of fibroblasts to mechanical stimuli and inflammatory mediators, in order to evaluate the mechanisms involved on ATP influence of neuronal activity. Experiments were performed in the first subcultures of rat and human fibroblasts isolated from subcutaneous connective tissue. Single-cell imaging was performed by confocal microscopy (Fluoview 1000, Olympus) in the time-lapse mode. Cytosolic free calcium was monitored on cells loaded with Fluo-4NW. Mitochondrial calcium was examined using Rhod-5N-AM. ATP release was inferred from destaining of cells loaded with quinacrine fluorescent dye. To examine membrane cell dynamics, fibroblast cultures were superfused with the FM4-64 dye and scanned synchronously for ATP release measurements. The morphology of the cells was continuously monitored by Hoffman contrast microscopy. The results showed that inflammatory mediators, like bradykinin and histamine, significantly increased ATP release possibly via a mechanism related to intracellular calcium oscillations. Data also suggest that activation of P2X7 purinoceptors mediate a positive feedback mechanism regulating ATP release from fibroblasts. Via this mechanism, fibroblasts of the subcutaneous tissue may amplify purinergic signaling between neighboring cells (e.g. sensory neurons) under painful situations. In view of this, we hypothesized that fibroblasts may be a target for therapeutic manipulation in chronic painful conditions (e.g. fibromyalgia). Work supported by FCT (FEDER funding, PTDC/SAU-FCF/108263/2008 and UMIB-215/94). A.R. Pinheiro received a PhD Scholarship from FCT (SFRH/BD/47373/2008). C. Soares received a Young Investigator Fellowship from FCT (BII/UMIB-ICBAS/2009-1).

P5-9

Rho-dependent pannexin 1-mediated ATP release from airway epithelia

(Selected for Oral Poster Presentation)

Eduardo Lazarowski, Lucia Seminario-Vidal, Silvia Kreda, Juliana Sesma, Seiko Okada, Richard Boucher

Cystic Fibrosis Center, University of North Carolina at Chapel Hill, USA

Extracellular ATP and its metabolite adenosine activate airway epithelial purinergic receptors that regulate the mucociliary clearance (MCC) process that removes inhaled pathogens from the airways. Mechanical stresses are ubiquitous stimuli promoting epithelial ATP release-mediated MCC functions, but the mechanisms regulating stress-promoted ATP release are not well understood. In the present study, we investigated mechanisms potentially regulating ATP release in osmotic and mechanic stress-stimulated airway epithelial cells. Well-differentiated primary cultures of human primary bronchial epithelial (WD-HBE) cells subjected to a 30% hypotonic challenge displayed enhanced ATP release, which was paralleled with overlapping kinetics with the uptake of the hemichannel fluorescence probe propidium iodide. ATP release and dye uptake were inhibited by the hemichannel blocker carbenoxolone and by the pannexin 1-selective blocking peptide 10Panx1. Pannexin 1 siRNA oligonucleotides reduced ATP release and dye uptake in siRNA-transfected A549 lung epithelial cells. WD-HBE cells displayed enhanced RhoA activation and myosin light chain (MLC) phosphorylation in response to hypotonic challenge. Importantly, inhibitors of Rho and MLC kinases as well as transfecting cells with the RhoA dominant negative mutant RhoA(T19N) markedly reduced ATP release and abolished dye uptake. Lastly, applying phasic motion that imparts shear stress similar to that caused by tidal breathing resulted in enhanced ATP release, which was inhibited by carbenoxolone and 10Panx1, and by Rho kinase and MLC kinase inhibitors. Collectively, our data suggest that pannexin 1 acts as ATP release pathway in mechanically-stimulated airway epithelial cells, and that pannexin hemichannel opening is regulated by RhoA/Rho kinase and MLC phosphorylation.

P5-10

Thrombin and neutrophil elastase promote nucleotide release from mucin granules in airway epithelial cells

Silvia Kreda, Lucia Seminario, Catherina van Heusden, Wanda O'Neal, Lisa Jones, Richard Boucher, Eduardo Lazarowski

Cystic Fibrosis Center, Univ. North Carolina at Chapel Hill, USA

Airway epithelial cell nucleotide release is crucial for purinergic regulation of airway innate defence. We have previously demonstrated that ATP release from airway goblet cells is associated with Ca2+-regulated mucin exocytosis, suggesting that ATP is released as a co-cargo molecule from mucin-containing granules. Here, we describe a receptor-mediated mechanism that promotes nucleotide release from mucin granules that may be relevant in airway inflammation. Thrombin and neutrophil elastase are abundant in lumenal airway secretions during inflammation. Both proteases stimulated the simultaneous apical release of mucins and ATP from human bronchial epithelial cells. Protease-mediated mucin and ATP release were dependent on intracellular-Ca2+ and actin cytoskeleton reorganisation since BAPTA-AM, cytochalasin-D, and Rho and myosin-light-chain kinase inhibitors blocked both responses. To test whether ATP is co-released with mucin from mucin granules, we measured the nucleotide composition of purified mucin granules. Mucin granules contained ATP, but the levels of ADP and AMP were ten-fold higher than that of ATP. Accordingly, apical secretions from protease-stimulated cells contained relative high levels of ADP/AMP, which could not be accounted for solely based on ATP release and hydrolysis. Thus, mucin granules contribute to ATP release and are a source of extracellular ADP and AMP. Direct release of ADP/AMP from mucin granules provides a source of airway surface adenosine to signal ciliated cell A2b-receptors, therefore, promoting ion/water secretion and mucin hydration. Nucleotide release from mucin granules may be an important contributing mechanism in protease-mediated epithelial inflammation in obstructive lung diseases. Supported by NIHP01-HL34322.

P5-11

Characterization of K+ depolarization evoked ATP, adenosine and glutamate release in rat hippocampus: a microelectrode biosensor study

Attila Heinrich, Beáta Sperlágh

Institute of Experimental Medicine, Hungarian Academy of Science, Hungary

Glial cells are active players in information processing in the brain and both adenine nucleotides and nucleosides as well as glutamate play a key role in glia-neuron interactions. In our study rat hippocampal slices were depolarized with 25 mM K+ and the release of ATP, adenosine and glutamate were recorded with enzymatic microelectrode biosensors. ATP and glutamate sensor exhibited a rapidly increasing current during depolarization. Blockade of the metabolism of extracellular ATP with ARL67156 enhanced the extracellular level of ATP and inhibited the detected long lasting adenosine efflux. Ca2+-free medium abolished both ATP and adenosine accumulation in response to K+ depolarization. ATP, adenosine and glutamate release was reduced in the presence of the tetrodotoxin. Extracellular concentration of ATP and adenosine were decreased in the presence of D-AP-5. High concentration of carbenoxolone, inhibited the ATP, adenosine and glutamate efflux, while in pannexin—selective concentration CBX decreased only the adenosine efflux. Perfusion with the glia-selective fluoroacetate inhibited the elevation of ATP, adenosine and glutamate levels. The selective P2X7 receptor antagonist BBG abolished ATP release and decreased adenosine efflux. Our results demonstrate that ATP and adenosine efflux in response to K+ depolarization are mediated by axonal activation via NMDA receptors and probably derived from glial cells through different hemichannels. Although the efflux of adenosine did arise from previously released ATP, through the actions of ectonucleotidases, it is also released directly via pannexin hemichannels. In contrast, ATP is released through gap junction hemichannel and/or via P2X7 receptor into extracellular space, before it is rapid breakdown to adenosine.

P5-12

ATP release from mice sciatic nerve

Anna Nualart Martí, Ezequiel Mas del Molino, Xenia Grandes, Carles Solsona

Pathology and Exp Therapeutics Dept, Universitat de Barcelona, Spain

Little is known about the stimuli which trigger regulated ATP release from different cell types. It has been suggested that ATP can be released from cells in a regulated manner, via protein channels that cross the plasma membrane. Previous works have suggested that Schwann cells which wrap the axons in peripheral nerves, may be involved in this process. ATP release from sciatic nerve trunks from connexin 32 Knock-out (Cx32 KO) and wild type (WT) mice was investigated using a source of stimuli that are described to regulate connexin channel opening: electrical stimuli, divalent free solution perfusion and mechanical stimuli as hypotonic shock. ATP release was detected in response to electrical trains of stimuli and during the perfusion of divalent free or hypotonic solutions into a chamber containing a sciatic nerve trunk. ATP released in response these stimuli was inhibited by Octanol 1.5 mM and Flufenamic Acid 50 μM, which are well-known connexin blockers. Identical experiments were performed in sciatic nerve trunks from Cx32 KO mice. ATP released was not appreciated during divalent free solution perfusion. However, it was detected in response to hypotonic shock, but the amount of ATP released was decreased and showed different kinetics when compared to the WT nerves. These results suggest that ATP in mice sciatic nerve may be released trough connexin channels present in the Schwann cell membrane and probably Cx32 has a great importance in this process. This work is supported by grants SAF2008/732 and Fundació La Marató de TV3 06/3033.

P5-13

The pattern of ATP release from rat hippocampal nerve terminals is different from classical neurotransmitters

Ângelo Tomé, Ricardo Rodrigues, Teresa Almeida, Rodrigo Cunha

Center for Neurosciences of Coimbra, University of Coimbra, Portugal

ATP is released in a frequency-dependent manner from nerve terminals and its release is disproportionately larger at higher frequencies of nerve stimulation (Cunha et al., 1996, J.Neurochem. 67:2180). We have now confirmed in rat hippocampal nerve terminals that the evoked release of ATP was also disproportionately larger at higher concentrations of extracellular potassium, in contrast to what is observed for classical neurotransmitters such as glutamate, GABA or acetylcholine. This evoked release of ATP was blunted by removal of extracellular calcium, by bafilomycin A1 (100 nM) and by botulinum toxin E (100 nM), indicating a vesicular release. Also, the evoked release of ATP was insensitive to N-type calcium channel inhibition with 500 nM omega-conotoxin GVIA, slightly inhibited (24 ± 4%) by the P-type calcium channel inhibitor, omega-agatoxin IVA (200 nM), and mostly sensitive to the L-type calcium channel inhibitors nitrendipine (10 µM, 46 ± 11% inhibition) or nifedipine (10 µM, 51 ± 9% inhibition). This contrasts with the predominant sensitivity of the evoked release of acetylcholine to omega-agatoxin IVA (200 nM, 43 ± 6% inhibition) and omega-conotoxin GVIA (500 nM, 33 ± 6% inhibition) and insensitivity to nifedipine (10 µM), as is known to occur also for the evoked release of glutamate or GABA. These results indicate that the pattern of ATP release from nerve terminals is different from that of classical neurotransmitters, ATP being mostly released in a manner sensitive to L-type channels that are recruited at high intensities of nerve stimulation. (Supported by FCT).

P5-14

Human erythrocytes release ATP in a cyclic AMP-regulated manner

Montalbetti Nicolas1, Lazarowski Eduardo2, Schwarzbaum Pablo1

1IQUIFIB, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, University of Buenos Aires-CONICET, Argentina,2University of North Carolina at Chapel Hill, University of North Carolina at Chapel Hill, USA

Human erythrocytes release ATP in response to various physiologically relevant stimuli, but the underlying mechanisms enabling ATP release from red blood cells are not well defined. Exposure of human erythrocytes (4% hematocrit) to forskolin resulted in enhanced ATP release, which was dependent on the presence of the phosphodiesterase inhibitor papaverine (vehicle, 12  ± 1 nM; 100 microM papaverine, 20 ± 2 nM; 100 microM papaverine and 30 microM forskolin, 42 ± 3 nM) or 3-isobutyl-1-methylxanthine (IBMX) (vehicle, 13 ± 2 nM; 30 microM IBMX, 22 ± 3 nM; 30 microM IBMX and 30 microM forskolin, 49 ± 5 nM). The beta-adrenergic receptor agonist isoproterenol dose-dependently (EC50  = 2.9 ± 0.7 nM) promoted ATP release, and this release was comparable in magnitude with that induced by forskolin. However, ATP release was dramatically potentiated (5- to 10-fold increase) when isoproterenol and forskolin were combined. Since erythrocytes lack vesicles, we hypothesized that cyclic AMP-stimulated ATP release reflects a conductive mechanism. Consistently with this hypothesis, ATP release from forskolin-stimulated erythrocytes was markedly reduced by preincubating the cells with 10 microM carbenoxolone, a non-selective hemichannel inhibitor. ATP release also was inhibited by the pannexin 1-selective blocking peptide (30 microM 10Panx1) but not by its scramble control peptide. Our study suggests that agonists promoting cyclic AMP formation stimulate ATP release from human erythrocytes via a pannexin 1-mediated pathway. 1- Supported by UBA, CONICET and ANPCyT (1432). 2- Supported by NIH.

P5-15

Connexins and ATP release

Ezequiel Mas del Molino1, Xènia Grabdes1, Anna Nualart1, Luis C. Barrio2, Luc Leybaert3, Carles Solsona1

1Pathology and Experimental Therapeutics Dpt, Univesity of Barcelona, University of Barcelona, Spain,2Hospital Ramón y Cajal, Madrid, Hospital Ramón y Cajal, Madrid, Spain,3Dept. Basic Medical Sciences, Ghent University, Ghent, Belgium

Twenty one genes for connexins have been identified until now in humans. Each connexin (Cx) is expressed in specific tissues or cell types. On the other hand, many cell types express more than one connexin. Connexins form hexameric structures in the plasma membrane called hemichannels or connexons, harbouring a central pore that permit the passage of ions and small molecules between cytoplasm and extracellular surroundings. Recent findings indicate that non-junctional hemichannels can open under both physiological and pathological conditions, depending on cell context. In the present work we have studied the currents generated by hemichannels formed by these human connexins Cx26, Cx32 and Cx43, expressed in Xenopus laevis oocytes and the simultaneous recording of ATP release. The activation of Cxs was done by depolarization pulses. The release of ATP is generally associated with tail currents. Connexin 26 is expressed in many tissues, and one of them is cochlea. Mutations in Cx26 cause nonsyndromic hearing loss and other syndromes affecting ectoderm-derived tissues. X-linked Charcot Marie Tooth (CMTX) is an inherited neurodegenerative disease affecting both motor and sensory peripheral nerves. CMTX is due to mutations in Cx32 and more than 200 mutations have been so far described. Schwann cells express Cx32 in the Schmidt-Lanterman incisures and in the paranodes in which may form hemichannels. Connexin 43 is found in astrocytes and is the major protein of cardiac ventricular gap junctions which are crucial to cell-cell communication and cardiac function. The protein level of Cx43 is reduced in patients with heart failure or dilated cardiomyopathy, pathophysiological conditions often associated with arrhythmias. Acknowledgments: This work is supported by grant SAF 2008/00732 of the Ministerio de Ciencia e Innovacion (MICINN) of the Spanish Government and by La Fundació La Marató de TV3. EMM is recipient of a Felloship of the MICINN.

P6. Purine extracellular metabolism

P6-1

Adenosine deaminase activity in intact trophozoites of Trichomonas vaginalis

Marina Weizenmann1, Patrícia Vieira1, Geraldo De Carli2, Maurício Bogo3, Carla Bonan3, Tiana Tasca1

1Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Brazil,2Instituto de Geriatria e Gerontologia, Pontifícia Universidade Católica do Rio Grande do Sul, Brazil,3Faculdade de Biociências Pontifícia, Universidade Católica do Rio Grande do Sul, Brazil

Background: To characterize the adenosine deaminase (ADA) activity from intact trophozoites of Trichomonas vaginalis, the etiologic agent of trichomonosis. Methods. ADA activity was characterized by a colorimetric assay carried out at 635 nm for measuring the ammonia released. The ADA protein sequences obtained from BLASTP function via the GenBank database were aligned with ClustalX program and a phylogenetic tree was built with MEGA 4.0 program using statistical Neighbor-Joining method with proportional (p) distance. Results. Considering adenosine as substrate, the protein curve was linear between 50 to 150 ug protein/mL, and the time course was linear up to 40 minutes. The optimal pH for deamination was 7.5. Adenosine and 2-deoxyadenosine were substrates for ADA, while guanosine and 2-deoxyguanosine were not deaminated. The apparent values for KM and Vmax were, respectively, 1.13 ± 0.07 mM and 2.61 ± 0.054 nmol NH3/min/mg of protein. Adenosine deamination was strongly inhibited in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Calcium and magnesium also inhibited the activity; effect prevented by EDTA. Furthermore, when ecto-5′-nucleotidase was inhibited, there also was no ADA activity, strongly suggesting the cascade association between these enzymes. The phylogenetic tree revelead four well-resolved terminal clades supported by high bootstrap values, confirming the presence of two ADA orthologues for T. vaginalis, which composed the second clade. Conclusion. Our data suggest the presence of an ecto-ADA in the parasite surface. The occurrence of ADA activity in T. vaginalis may represent important implications for the purinergic system in the immune response during trichomonosis. Financial support: CNPq (Brazil).

P6-2

Chronic ethanol treatment alters purine nucleotide hydrolysis and nucleotidase gene expression pattern in zebrafish brain

Eduardo Pacheco Rico1, Denis Broock Rosemberg1, Andrei da Silveira Langoni2, André Arigony Souto3, Renato Dutra Dias2, Maurício Reis Bogo2, Carla Denise Bonan2, Diogo Onofre Souza1

1Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Brazil,2Departamento de Biologia, Celular e Molecular Pontifícia Universidade Católica do Rio Grande do Sul, Brazil,3Faculdade de Química, Pontifícia Universidade Católica do Rio Grande do Sul, Brazil

The zebrafish is a model for the study of human diseases and the use of this species in biochemical and behavioral studies on alcoholism has increased recently. However, there are no data concerning the effects of chronic ethanol exposure on the purinergic system. The aim of this study was to evaluate nucleotide hydrolysis by NTPDases and ecto-5′-nucleotidase after long-term ethanol exposure. Additionally, the gene expression patterns of NTPDases1-3 and 5′-nucleotidase were determined. Animals were exposed to 0.5% ethanol for 7, 14, and 28 days. Zebrafish were euthanized, their brains were removed by dissection and brain membranes were prepared. NTPDase and 5′-nucleotidase activity were determined by the measurement of inorganic phosphate released from nucleotide hydrolysis. There were no significant changes in ATP and GTP hydrolysis after all treatments. However, a decrease in ADP (46% and 34%) and GDP (48% and 36%) hydrolysis was verified after 7 and 14 days, respectively. After 7 and 14 days of ethanol exposure, a significant decrease in AMP hydrolysis (48% and 36%) was also observed, whereas GMP hydrolysis was inhibited only after 7 days (46%). NTPDase2_mv and NTPDase3 mRNA transcript levels decreased after 7 and 14 days, respectively. In contrast, ethanol increased NTPDase1, NTPDase2_mq and NTPDase3 transcript levels after 28 days of exposure. NTPDase2_mg and 5′-nucleotidase gene expression was not altered. The ecto-nucleotidase pathway may be a target of chronic ethanol toxicity. Therefore, regulation of the purinergic system may play a role in the mechanisms underlying the effects of ethanol on the central nervous system.

P6-3

Antipsychotic drugs alter adenine nucleotide hydrolysis in zebrafish (Danio rerio) brain

Carla Denise Bonan1, Kelly Juliana Seibt1, Renata Luz Oliveira1, Eduardo Pacheco Rico2, Tathyana Mar Amorim Franco1, Renato Dutra Dias1, Mauricio Reis Bogo1

1Departamento de Biologia Celular e Molecular, Pontifícia Universidade Católica do Rio Grande do Sul, Brazil,2Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Brazil

Zebrafish is an ideal vertebrate model system to study human diseases and drug screening. We evaluated the in vitro and in vivo effects of typical (haloperidol) and atypical (olanzapine and sulpiride) antipsychotic drugs on NTPDase and 5′-nucleotidase activities from zebrafish brain followed by a gene expression pattern analysis. For in vitro experiments, antipsychotics (1–250 uM) were added to reaction medium and maintained throughout the enzyme assays. For in vivo exposure, zebrafish were submitted to haloperidol (9 uM), olanzapine (100 uM), or sulpiride (250 uM) treatment for 2 hours. Zebrafish were euthanized, their brains were removed by dissection, and brain membranes were obtained. Ectonucleotidase activities were determined by the measurement of inorganic phosphate released from nucleotide hydrolysis. The in vitro experiments demonstrated that ATP hydrolysis was inhibited at 250 uM haloperidol (28.9%) and olanzapine (60.7%) whereas ADP hydrolysis was decreased at 250 uM haloperidol (26.5%) and sulpiride (25.6%). After 2 h-exposure, only haloperidol was able to inhibit ATP hydrolysis (35%). Haloperidol was also able to decrease NTPDase3 and two isoforms NTPDase2 (NTPDase 2_mv and NTPDase2_mq) mRNA transcript levels. These findings demonstrated that antipsychotic drugs inhibited NTPDases whereas did not change 5´-nucleotidase activity. The evaluation of the effects of antipsychotics on purinergic system in zebrafish may contribute to a better understanding of mechanism of action of these drugs as well as discover potential pharmacological targets and accelerate the pace of psychiatric drug discovery.

P6-4

Characterization of ATP and ADP hydrolysis activity in plasma membranes isolated from rat uterus

Maja Milosevic, Ivana Stanojevic, Snjezana Petrovic, Dunja Drakulic, Natasa Velickovic, Anica Horvat

POBox 522 Vinca Institute of Nuclear Sciences Serbia

Extracellular nucleotides modulate female reproductive functions, fertilization and pregnancy. The aim of this study was to characterize ecto-nucleotidase activity in uterine plasma membranes isolated from Wistar albino rats. Enzyme activities were determined by measuring the amount of liberated inorganic phosphate using a colorimetric assay. Magnesium and calcium ions stimulated ATP and ADP hydrolysis in a similar way. Significant decreases in hydrolysis of both substrates were observed in presence of 1 mM EDTA, confirming cation dependence. The pH optimum was found to be 8.0 for ATP and 8.5 for ADP. Kinetic parameters were determined using substrates in range 0.05–5 mM. The apparent Km for ATP and ADP were 460.7 ± 8.0 and 574.5 ± 10.5 microM, respectively. The Vmax values were 3726.2 ± 64.5 and 2656.7 ± 48.1 nmol/min/mg for ATP and ADP, correspondingly. Chevillard plot demonstrated that one enzyme site was responsible for ATP and ADP hydrolysis. A significant ATP and ADP hydrolysis inhibition (P < 0.05) were observed in the presence of 1–10 mM sodium azide, 1 mM sodium fluoride, 0.3 mM suramin and 0.1 mM sodium orthovanadate, while no inhibition with ouabain, oligomycine or theophylline were detected. Minor alkaline phosphatases contamination was confirmed by levamisole. Western blot verified presence of NTPDase1, while NTPDase2 and NTPDase3 were not detected.

According to our results, ATP and ADP hydrolysis observed in plasma membranes from rat uterus corresponded to NTPDase1 activity. These findings are consistent with literature data about NTPDase1 presence on the surface of myometrial smooth muscle cells and blood vessel endothelium.

P6-5

The NTPDase family in zebrafish

(Selected for Oral Poster Presentation)

Mauricio Bogo1, Denis Rosemberg2, Eduardo Rico2, Andrei Langoni1, Jonathan Spinelli1, Talita Pereira1, Renato Dias1, Diogo Souza2, Carla Banan1

1Faculdade de Biociencias, PUCRS, Brazil,2Departamento de Bioquimica, UFRGS, Brazil

The nucleoside triphosphate diphosphohydrolase (NTPDase) family cleaves tri- and diphosphonucleosides to monophosphonucleosides and is responsible for terminating purinergic transmission. Since the NTPDase family in zebrafish is poorly understood, in the present study we evaluated the nucleotide hydrolysis in three tissues of adult zebrafish (brain, liver, and heart), confirmed the presence of distinct NTPDase members by a phylogenetic analysis and verified their relative gene expression profiles in the respective tissues. A different profile of ATP and ADP hydrolysis in the brain, liver, and heart as a function of time (0–60 min) and protein amount (3–15 ug) was observed. Sodium azide (20 mM), ARL 67156 (300 uM) and Suramin (300 uM) differently altered the nucleotide hydrolysis in zebrafish tissues, suggesting the contribution of distinct NTPDase activities. Homology-based searches identified the presence of NTPDase1–6 and NTPDase8 orthologs. In addition, the phylogeny also grouped three NTPDase2 and two NTPDase5 paralogs. The deduced amino acid sequences share the apyrase conserved regions (ACRs), conserved cysteine residues, putative N-glycosylation, phosphorylation, N-acetylation sites, and different numbers of transmembrane domains. RT-PCR experiments revealed the existence of a distinct relative entpd1–6 and entpd8 expression profile in brain, liver, and heart. Taken together, our results indicate that several NTPDase members might contribute to a tight regulation of nucleotide hydrolysis in zebrafish tissues.

P6-6

Ecto-5′-nucleotidase (e-5NT) switches from neurons to astrocytes after cortical stab injury in rat

Nadezda Nedeljkovic1, Ivana Bjelobabam2, Ana Parabucki2, Irena Lavrnja2, Sanja Pekovic2, Mirjana Stojiljkovic2

1Faculty of Biology, University Belgrade, Faculty of Biology, University Belgrade, Serbia,2Institute for Biological Research Sinisa Stankovic, University Belgrade, Serbia

Background: Ecto-5′-nucleotidase (e-5NT) is ectoenzyme that controls extracellular levels of ATP and adenosine and thus, neuronal, astrocytic, and microglial function. We demonstrated in the rat model of cortical stab injury, that activity and expression of e-5NT changed in a biphasic manner, with an immediate decrease, followed by prominent up-regulation two weeks after the injury. In the present study we describe cellular distribution of e-5NT after the injury. Methods. Animals were submitted to stab injury to the left cortex according to stereotaxic coordinates. Another group of animals were processed as sham control. Animals were allowed to survive for 0 hr–15 days. Brains were processed for immunocytochemical analysis. Results. Double immunofluorescence staining against e-5NT and NeuN or GFAP revealed that in intact controls e-5NT is mainly present at neuronal cell bodies and apical dendrites, while rare e-5NT +-GFAP+ structures were highly scattered. At sections obtained at different post-injury times, the number of e-5NT+ neuronal cell bodies gradually decrease, whereas number of e-5NT+-GFAP+ structures increase, providing almost completely overlapping signal at sections obtained 7 days postinjury. Conclusions. Our data demonstrate that e-5NT changes cellular localization after the injury, from predominant neuronal to almost exclusive astrocytic localization in the perilesioned area. Although certain level of neuronal cell loss is observed, it does not account for the total decrease in neuronal e-5NT expression. Functional significance of switch in e-5NT cellular localization imply that neurons and astrocytes play different roles in the extracellular metabolism of purine nucleotides and cellular responses induced by them.

P6-7

Extracellular nucleotides and ectonucleotidases control IL-8 production in human colon HT-29 cells

Fariborz Bahrami1, Filp Kukulski2, Joanna Lecka2, Julie Pelletier2, Jean Sévigny2

1Centre de Recherche en Rhumatologie et Immunologie, Department of Microbiology-Infectology and Immunology, Faculty of Medicine, Université Laval, Canada,2Centre de Recherche en Rhumatologie et Immunologie, Department of Microbiology, Université Laval, Canada

Background: Toll-like receptor 3 (TLR3) recognizing double-stranded viral RNA plays an important role in the etiology of inflammatory bowel disease (IBD). Here we demonstrate that TLR3-induced IL-8 release by intestinal epithelial HT-29 cell line requires a concomitant activation of P2 receptors. Methods: To investigate the role of P2 receptors in TLR3-induced IL-8 release by HT-29 cells, the cells were stimulated with TLR3 agonist poly (I:C), in the presence or absence of P2 receptor antagonists and apyrase (a nucleotide scavenger) and their supernatants were analyzed by ELISA for detection of IL-8. The expression of ectonucleotidases was detected by immunoblotting and RT-PCR and their activity was analyzed by RP-HPLC. Results: Considerable amounts of IL-8 were produced by HT-29 cells in response to the stimulation of TLR3 with poly (I:C) which were markedly diminished by apyrase, general P2 receptor antagonists and a selective P2Y11 antagonist (NF157). P2Y11 receptor was dominantly expressed in HT-29 cells and its ligands (ATPgammaS and ATP), potentiated IL-8 release by a low concentration of poly (I:C), causing suboptimal activation of TLR3. Among the ectonucleotidases affecting IL-8 secretion, HT-29 dominantly expressed mRNA and activity of NTPDase2 that hydrolysed ATP to ADP, and also exhibited high activity of adenylate kinase (ADK) that produced high amounts of ATP from ADP as a substrate. Conclusion: Extracellular nucleotides mediate TLR3-induced IL-8 release from HT-29 cells, mostly via P2Y11 receptor. The tandem of NTPDase2 and ADK at the surface of HT-29 appears to affect the activation of ATP-dependent P2 receptors, especially P2Y11.

P6-8

Structural studies on ecto-nucleotidases

(Selected for Oral Poster Presentation)

Norbert Sträter, Matthias Zebisch, Karen Yates, Ulrike Krug

Institute of Bioanalytical Chemistry, University of Leipzig, Germany

In many tissues, ecto-nucleosid triphosphate diphosphohydrolases (NTPDase) and 5′-nucleotidase are the major activities which specifically terminate the extracellular signalling action of ATP or other nucleotides. In addition to the receptors, also the enzymes are promising pharmaceutical targets to interfere with purinergic signalling. In order to understand the structure and function of these enzymes and to participate in the structure-guided development of novel nucleotidase inhibitors, we set out to determine X-ray crystal structures of mammalian and microbial NTPDases as well as 5′-nucleotidases. For the mammalian enzymes, the preparation of mg amounts of protein for structural studies from bacterial expression involved the development of optimized refolding procedures of the disulfide-linked eukaryotic proteins that are expressed into inclusion bodies. Also, a careful design of the expression construct and of different variants is necessary to achieve useful refolding yields and crystallizable protein. In case of the NTPDases, only the ectodomains are expressed and in order to obtain crystals of the NTPDase1 enzyme, we also removed a putative membrane interaction loop that was discovered by the structure analysis of rat NTPDase2. Our results show that NTPDases as well as 5′-nucleotidase undergo a significant domain motion in catalysis, which also needs to be taken into account for the structure-based development of novel inhibitors. Compared to the large 90° hinge-bending domain rotation of the 5′-nucleotidases, the domain movement of the NTPDases is smaller with rotations of up to 17°.

P6-9

NTPDases of microbial pathogens

Ulrike Krug1, Matthias Zebisch1, Michael Johnson2, Norbert Sträter1

1Institute of Bioanalytical Chemistry, University of Leipzig, Germany,2Proteomics Technology Centre of Expertise, University of Technology Sydney, Australia

Nucleoside triphosphate diphosphohydrolases (NTPDase) catalyze the hydrolysis of extracellular nucleotides. These enzymes are found in mammals as well as in microbial pathogens where they are thought to modulate the host-response to an infection by hydrolysis of host ATP. We aim to determine crystal structures of microbial NTPDases to use them for structure-guided drug design. For the production of NTPDases from Legionella pneumophila, Schistosoma mansoni, Toxoplasma gondii, Trichomonas vaginalis and Trypanosoma cruzi we have established E. coli expression systems. NTPDases of Toxoplasma gondii and Trichomonas vaginalis were expressed as insoluble inclusion bodies. Using rapid dilution systems it was possible to refold the denatured protein to an active form. NTPDase from Legionella pneumophila was expressed in soluble form by secretion to the periplasm. Subsequently the proteins were purified by chromatographic methods and applied to crystallization screens. We were able to determine the crystal structures by different phasing methods, namely SAD and MIRAS. Furthermore, the proteins could be crystallized in complex with substrate analogs. These crystal structures provide insight into the domain motion of the enzyme and broaden our knowledge about the catalytic mechanism and substrate specificity (as well as promiscuity) already derived from the crystal structure of rat NTPDase2.

P6-10

Role of the purinergic system in controlling blood-brain barrier functions: focus on ATP-metabolizing enzymes

Agnes Kittel1, Laura Colombo2, Giulia Magna2, Francesca Vigano2, Marta Boccazzi2, Beata Sperlagh1, Maria Deli3, MariaPia Abbracchio2, Stefania Ceruti2

1Laboratory of Molecular Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Hungary,2Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission – Department of Pharmacological Sciences, Università degli Studi di Milano, Italy,3Institute of Biophysics, Biological Research Center of the Hungarian Academy of Science, Hungary

The blood-brain barrier (BBB) is composed of endothelial cells, pericytes and astrocytes, and serves as interface between the blood flow and the central nervous system (CNS). Because of the role of BBB in different brain pathologies, understanding the molecular mechanisms modulating its functions might help unveiling new therapeutic targets. To date the role of purinergic transmission in controlling BBB functions is not fully understood. Therefore, we used a new in vitro cell culture model of BBB to investigate the expression and distribution of ectonucleotidases (NTPDases), which hydrolyze extracellular nucleotides, and purinergic P2Y receptors, either in control conditions or following exposure to ischemia in vitro. RT-PCR analysis showed that astrocytes and pericytes expressed all the cloned P2Y receptors, and that endothelial cells showed only the P2Y1,2,4 subtypes. NTPDase1 and 2 were expressed by all the three types of cell. Endothelial cells were extremely susceptible to cell death when oxygen-glucose deprivation (OGD) was applied to mimic cytotoxicity induced by ischemia in vivo, whereas astrocytes and pericytes were more resistant. A semi-quantitative assay highlighted increased ecto-ATPase activity following exposure to OGD in all three types of cell. This new in vitro model proved its usefulness to demonstrate a role for extracellular nucleotides in modulating BBB responses to ischemic events, and to determine if the purinergic system could represent a new target for the development of effective therapies to brain pathologies.

P6-11

NTPDases role in the patophysiology of behavioral impairment in rats induced by seizure in early age

Giana Cognato1, Bruna Bellaver1, Fernanda Cenci Vuaden1, Luiz Eduardo Baggio Savio1, Maurício Reis Bogo2, Márcia Wink3, Diogo Souza1, Carla Denise Bonan2

1Universidade Federal do RS, Brazil,2Pontifícia Universidade Católica do RS, Brazil,3Fundação Universidade Federal de Ciências Médicas de Porto Alegre, Brazil

Seizures occur more frequently in the neonatal period than in any other time in life and many data have shown that seizures in young animals lead to later cognitive impairment. There are several evidence that long-term potentiation (LTP) can contribute to the neural basis for learning and memory mechanisms and could be modulated by ATP or adenosine. ATP may be hydrolyzed to adenosine by Ecto-nucleotidases. Thus, we have investigated if hippocampal ecto-nucleotidases activities are altered at different time periods (PN7 to PN90) after an episode of seizure induced by kainic acid (KA) in 7 day-old rats (PN7). We also evaluated if 90 day-old rats (PN90) previously submitted to seizure at PN7 presented behavioral impairment in Y-maze task. Our results have shown memory impairment of PN90 rats previously submitted to one single seizure episode at PN7, which is accompanied by an increased ATP hydrolysis in hippocampal synaptosomes. Furthermore, the relative expression of NTPDases responsible for ATP hydrolysis was not altered by the KA administration in early age. Since we have found an augmented hydrolysis of ATP at PN90 rats and this nucleotide seems to be important to LTP induction, we could assume that impairment of memory observed in adult rats which have experience a convulsive episode in neonatal period may be related to the increased ATP hydrolysis activity. These findings correlate the purinergic signaling to the cognitive deficits induced by neonatal seizures and contribute to a better understanding about the mechanisms envolved in seizure-induced memory dysfunction.

P6-12

Blood homocysteine, ADP and ADO levels together with ecto-nucleotidases activities may reflect vascular damage induced by cyclosporine administration in rats

Jean Pierre Oses1, Ana Elisa Böhmer1, Liz Marina Brum2, André Corrêa3, Giordano Viola1, Paulo Saraiva4, David Driemeier3, Luis Valmor Portela1, Diogo Onofre Souza1

1Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Brasil,2Instituto de Cardiologia do Rio Grande do Sul, FUC, Brasil,3Departamento de Medicina Veterinária Preventiva, Universidade Federal do Rio Grande do Sul, Brasil,4Escola de Farmácia, Universidade Federal do Rio Grande do Sul, Brasil

Vascular disease is a major cause of morbidity and mortality among transplanted recipients and cyclosporine (CsA) treatment has been consistently implicated in this event. In this study we proposed to assess blood total homocysteine (tHcy), ecto-nucleotidases activities and adenine nucleotide/nucleoside levels searching for putative markers/mechanisms of vascular damage induced by chronic CsA treatment in non-transplanted rats. Thirty male Wistar rats were divided in three groups: control group treated with corn oil, CsA 5 mg/Kg and CsA 15 mg/Kg. The oil or CsA were administered by daily gastric gavage during 8 weeks. CsA 15 mg/Kg treatment increased blood levels of tHcy. Both CsA treatments (5 and 15 mg/Kg) decreased adenine nucleotides hydrolysis by ecto-nucleotidases in serum, which negatively correlated with tHcy levels (r: −0.74, r: −0.63 and r: −0.63, p < 0.004, for ATP, ADP and AMP, respectively). CsA 15 mg/Kg induced a statistically significant increase in ADP and decrease in adenosine (ADO) plasma levels compared to control group. tHcy levels positively correlated with plasma ADP levels and negatively correlated with adenosine levels (r: 0.84, p < 0.0001 and r: −0.68, p < 0.0001, respectively). Rats under CsA 15 mg/Kg treatment presented cell injury and inflammatory responses in the endothelium and intima layer of the aorta artery. In conclusion, blood ecto-nucleotidases activity, tHcy, and ADP and ADO levels from may have a potential role in vascular damage induced by CsA.

P6-13

Evidence for the role of Ecto-NTPDase expression in the transport of the growth hormone auxin in Arabidopsis

(Selected for Oral Poster Presentation)

Elizabeth Henaff, Jian Wu, Janak Zalawadia, Trieu Pham, Greg Clark, Stanley Roux

Molecular Cell & Developmental Biology, The University of Texas at Austin, USA

Two very similar ecto-NTPDases (apyrases), AtAPY1 and AtAPY2, help control the concentration of extracellular ATP (eATP), and play major roles in the regulation of plant growth and development. The growth of most plant tissues requires the hormone auxin, and inhibition of auxin transport inhibits growth. We tested whether the expression of APY1/APY2 could influence auxin transport. A model system for assessing auxin transport is the gravity-directed downward bending of roots (gravitropism), which requires the asymmetric transport and accumulation of auxin preferentially on the lower flank of the root. Roots release ATP as they grow, and the concentration of this eATP is limited in part by APY1 and APY2 activity. Root gravitropism was measured in single knockouts (apy1 or apy2) and in apy2 mutants that could be induced by estradiol to suppress the expression of APY1 by RNAi. Root bending angles were assayed by a digital video system. The apy1 and wild-type bending rates were not statistically different, but apy2 mutants showed statistically different rates of gravitropism 30–40% lower than wild-type controls (p < 0.05), and the RNAi-suppressed mutants were inhibited >90%. RNAi mutants expressing a promoter:GFP construct (DR5-GFP) that indirectly reports auxin levels were assayed to show that APY suppression prevented the normal asymmetric expression pattern of GFP after the gravitropic stimulus. These data imply that ecto-NTPDase control of the [eATP] in the elongation zone of roots may be crucial for polar auxin transport and root gravitropism.

P6-14

Changes in ecto-nucleotidases in murine and human endometria along the female cycle

Elisabet Aliagas Marin1, M.R. Taco-Sánchez2, A. Vidal2, J. Ponce3, É.G. Lavoie4, B. Torrejón-Escribano5, J. Sévigny4, Mireia Martín Satué1

1Dept. Patologia i terepèutica Experimental, Facultat de Medicina, Universitat de Barcelona, Spain,2Servei d’Anatomia Patològica, IDIBELL, Spain,3Servei de Ginecologia, IDIBELL, Spain,4Centre de Recherche en Rhumatologie et Immunologie, Université Laval, Canada,5Serveis Cientificotècnics, Universitat de Barcelona, Spain

Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions, such as those requiring contraction, hormone synthesis, sperm motility and maintenance of fluid composition. Extracellular nucle¬otide levels are affected by cell surface ectonucleotidases, amongst which the ecto¬-nucleoside triphosphate diphosphohydrosase (E-NTPDase) family regroups the most abundant and effective enzymes to hydrolyse ATP and ADP to AMP. Ecto-5′-nucleotidase (CD73) hydrolyses AMP to adenosine. The aim of the present work was to characterize the cellular expression and enzymatic activity of NTPDases and ecto-5′-nucleotidase (CD73) in the murine female repro¬ductive tract, giving special attention to possible changes in the endometria throughout the cycle. This was addressed by immunological techniques and by in situ enzymatic assays. NTPDase3 and ecto-5′-nucleotidase were identified in several epithelia, such as that of oviducts, uterus and endometrial glands, suggesting a coordinate activity of both enzymes. Marked changes in expression and activity of endometrial ecto-nucleotidases were detected along the female cycle pointing to hormone influence. This study suggests close regulation of extracellular nucleoside and nucleotide levels in the female tract by ectonucleotidases that may influence fertility.

P6-15

Extracellular purine metabolism and signalling of CD73-derived adenosine in murine Treg and Teff cells

Michael Romio, B. Reinbeck, S. Burghoff, Jürgen Schrader

Department of Cardiovascular Physiology, Heinrich-Heine University Düsseldorf, Germany

Extracellular adenosine formed by CD73 acts as potent inhibitor of inflammation. Since regulatory T-cells (Treg) have been shown to express CD73 as a novel marker enzyme, the present study explored the role of CD73-derived adenosine in modulating NF-κB activity and cytokine/chemokine release from murine Treg and Teff cells. Stimulating splenocytes and CD4+ T-cells from CD73-/-mice with anti-CD3/anti-CD28, significantly upregulated activated NF-κB (WT: 4.36 ± 0.21, CD73-/-: 6.58 ± 0.75; n=4; p=0.029). This was associated with an augmented release of the pro-inflammatory cytokines IL-2 and IFN-γ. Treatment of stimulated CD4+ T-cells with ADO [25 µM] and AMP [50µM] potently reduced IL-2 and IFN-γ release, mediated by adenosine A2A receptors (A2AR). A2aR activation of Teff with CGS21680 significantly inhibited the release of IL-1, IL-2, IL-3, IL-IL-4, IL-12, IL-13, IFN-γ, TNF-α and GM-CSF. Teff also released of high amounts of CCL3 and CCL4, previously not described, which was strongly inhibited by CGS21680. A2AR activation in Treg did not alter cytokine/chemokine release. Expression of CD73 is the most prominent of all ATP and adenosine metabolising enzymes on Treg. Antigenic stimulation potently reduced the expression of the ligand-gated ion channel P2X7 (-90%) and the nucleoside transporter CNT2 (-70%) both in WT and CD73-/- Treg. In summary our data show that in CD4+ T cells, CD73-derived adenosine tonically inhibits active NF-κB, thereby modulating the release of a broad spectrum proinflammatory cytokines and chemokines. Downregulation of P2X7 and CNT2 in WT and CD73-/- Tregs may be a important mechanism to prevent ATP-induced apoptosis thereby maintaining the ability of Tregs to generate immunosuppressive adenosine.

P7. Inflammation and cancer

P7-1

P2X7 receptor activation in human cancer cells increases SK3-dependent migration and enhances extracellular matrix invasion via the release of mature cysteine cathepsins

Sébastien Roger1, Aurélie Chantome1, Bilel Jelassi1, Pablo Pelegrin2, Annmarie Surprenant3

1Inserm U921, Université De Tours, France,2Inflammation and Experimental Surgery Group, University Hospital “Virgen de la Arrixaca”-FFIS, Spain,3Faculty of Life Science, University of Manchester, United Kingdom

ATP-gated P2X7 receptors (P2X7R) are unusual plasma membrane calcium-permeable ion channels extensively studied in immune cells where their activation leads to rapid release of pro-inflammatory cytokines. More recently, several tumours have been shown to express P2X7R at unusually high levels and these receptors have been proposed as potential cancer cell biomarkers. However, their functionality and involvement in cancer cell processes such as proliferation or apoptosis is debated. Here, we report the functionality of ATP-gated P2X receptors in the highly aggressive human breast cancer cell line MDA-MB-435s. While RT-PCR experiments show the mRNA expression of P2X4, P2X5, P2X6 and P2X7 receptors, patch-clamp recordings and pharmacology (KN62 and A740003) strongly suggest the only functionality of P2X7R. Stimulating the cells with millimolar concentrations of ATP was found to trigger ethidium uptake and to increase intracellular calcium concentration. These effects were inhibited by KN62. While P2X7 receptor activation had a very weak effect on cell viability, it increases cell migration by 35% and markedly increases cell invasion through extracellular matrix by 150%. The increased migration was due to the activation of Ca2+-activated SK3 potassium channels while the increased invasion was due to the release of cysteine cathepsins independently of SK3 channel activity. Our results demonstrate a novel mechanistic link between P2X7R and cancer cell invasion, identify P2X7R as a potentially important trigger for cathepsin release from cancer cells, and suggest a new therapeutic role for the newly developed P2X7R antagonists.

P7-2

P2X7 receptor-mediated release of cathepsin is involved in extracellular matrix degradation

Gloria Lopez-Castejon1, Pablo Pelegrin2, Annmarie Surprenant1

1Faculty of Life Science, University of Manchester, United Kingdom,2Inflammation and Experimental Surgery Group, University Hospital-Virgen Arrixaca, Spain

Cathepsins, classically considered intracellular lysosomal proteases, are now known to play additional roles, like extracellular matrix (ECM) degradation and bone remodelling, when released into the extracellular media. Their presence in the extracellular compartment has been associated with their participation in joint diseases like arthritis. It is well established that ATP activation of the P2X7 receptor (P2X7R) triggers rapid release of pro-inflammatory cytokines, like interleukine (IL)-1beta, in activated macrophages. ATP can also induce the release of cathepsins, however little is known about this process. Here we have studied the involvement of P2X7R activation in cathepsin release in an IL-1beta free scenario and its biological significance. ATP induced a rapid release of cathepsins from resting (non LPS-primed) human and mouse macrophages independently of the presence of IL-1beta. Enzyme activities measured in supernatants were confirmed to relate to cathepsins by sensitivity to cathepsin inhibitors. P2X7R was responsible for this release as shown by the lack of secretion in P2X7R deficient macrophages and inhibition of the response by P2X7R antagonists. This ATP induced release was dose and time dependent and dissociated from ATP-induced cell death, as indicated by low levels of LDH release. Furthermore, this cathepsin release correlated with an increase in gelatinase activity in the supernatants that was not present in P2X7R deficient macrophages. These results suggest that cathepsin release may act as a novel danger signal upon P2X7R activation during inflammation, degrading the ECM and inducing tissue damage independently of the pro-inflammatory actions via IL-1 cytokines.

P7-3

Adenosine modulates HIF-1alpha, VEGF, IL-8 and foam cells formation in a human model of hypoxic foam cells

(Selected for Oral Poster Presentation)

Stefania Gessi1, Eleonora Fogli1, Valeria Sacchetto1, Stefania Merighi1, Katia Varani1, Delia Preti2, Edward Leung3, Stephen MacLennan3, Pier Andrea Borea1

1Department of Clinical and Experimental Medicine Pharmacology Section, University of Ferrara, Italy,2Department of Pharmaceutical Sciences, University of Ferrara, Italy,3King Pharmaceuticals Research and Development, Inc, R&D, USA

Background: Foam cells formation by oxidized low-density lipoprotein (oxLDL) accumulation in macrophages is crucial for development of atherosclerosis. Hypoxia has been demonstrated in atherosclerosis and hypoxia-inducible factor-1 (HIF-1) has been shown to promote intraplaque angiogenesis and foam cells (FC) development. As hypoxia induces HIF-1α stabilization and adenosine (ado) accumulation, we investigated whether this nucleoside regulates HIF-1alpha in FC. Methods: Evaluation of ado receptors expression through real-time RT-PCR, binding and western blot experiments. Evaluation of HIF-1alpha, VEGF, IL-8 and foam cells modulation by ado receptors through western blot analysis, ELISA studies and oil red O staining. Results: Ado, under hypoxia, stimulates HIF-1alpha accumulation by activating all adenosine receptors (ARs). HIF-1alpha modulation involved extracellular signal-regulated kinase 1/2 (ERK 1/2), p38 mitogen-activated protein kinase (p38 MAPK) and protein kinase B (Akt) phosphorylation in the case of A1, A2A, A2B and ERK 1/2 phosphorylation in the case of A3 receptors. Ado, through the activation of A3 and A2B receptors, stimulates vascular endothelial growth factor (VEGF) secretion in a HIF-1alpha dependent way. Furthermore ado, through the A2B subtype, induces an increase of Interleukin-8 (IL-8) secretion in a ERK 1/2, p38 and Akt kinases-dependent but not HIF-1alpha-mediated way. Finally, ado stimulates FC formation and this effect is strongly reduced by A3 and A2B blockers and by HIF-1alpha silencing. Conclusions: This study provides the first evidence that A3, A2B or mixed A3/A2B antagonists may be useful to block important steps in the atherosclerotic plaque development ado-induced.

P7-4

Adenosine A2A receptor signaling switches macrophages from an M1 to an M2-like phenotype

Samuel Joseph Leibovich, Grace Pinhal-Enfield, Stan Grinberg

UMDNJ, New Jersey Medical School, USA

Macrophages are activated by Toll-like Receptor (TLR) agonists to express a “classical” (M1) inflammatory phenotype, characterized by production of iNOS and cytokines such as TNF-alpha and IL-12. In contrast, macrophages are activated by IL-4, IL-13 or glucocorticoids to express an “alternatively activated” anti-inflammatory, wound healing phenotype, characterized by production of arginase, VEGF and IL-10. We have shown previously that agonists of A2ARs switch macrophages from an M1 to an M2-like phenotype. Unstimulated macrophages express low levels of A2ARs, and TLR agonists strongly induce the expression of A2ARs in an NF-kappaB-dependent manner. Using Q-RT-PCR and Western blot analyses, we find that TLR agonists strongly suppress phospholipase-Cbeta2 (PLCbeta2) expression in macrophages. This suppression is mediated at the mRNA stability level by TLR-dependent regulation of specific mi-RNAs, including miR-466l, which bind to sites in the PLCbeta2 3′UTR. Suppression of PLCbeta2 was found to play an important role in A2AR-mediated induction of VEGF expression by macrophages. We propose that regulated expression of A2ARs plays a key role in determining macrophage phenotype in inflammation, wound healing and tumorigenesis. While unstimulated macrophages respond to TLR agonists by rapidly adopting an inflammatory M1 phenotype, TLR-dependent up-regulation of A2ARs primes these M1 macrophages, rendering them susceptible to stimulation by adenosine, which then switches them to an M2-like phenotype that mediates angiogenesis and wound healing. This pathway for the generation of M2-like macrophages is independent of IL-4 and IL-13, and depends simply on the micro-environmental regulation of extracellular adenosine levels in response to cell stress, inflammation or ischemia.

P7-5

ATP confers tumorigenic properties to dendritic cells

Bles Nathalie1, Larissa Di Pietrantonio1, Jean-Marie Boeynaems2, Didier Communi1

1IRIBHM, Université Libre de Bruxelles, Belgium,2IRIBHM, Erasme, Belgium

The importance of dendritic cell (DC) functions in the regulation of tumor growth has been previously reported. ATP is known as a regulator of DC activities and is present at high concentrations in tumors. Using microarray technology, we identified ATP target genes displaying tumorigenic properties in DCs. We demonstrated that angiogenic factors like VEGF and some EGF receptor (EGFR) ligands are target genes of ATPgS, a more stable ATP derivative, in human monocyte-derived dendritic cells (MoDCs). We have shown that MoDCs were able to secrete high levels of VEGF and EGFR ligands in response to ATPgS particularly in combination with LPS. Moreover, these stimulated MoDCs were able to induce in vitro endothelial tube formation through VEGF secretion and smooth muscle cell proliferation through EGFR ligand secretion. We have also observed that LPS+ATPgS-stimulated mouse bone marrow-derived dendritic cells (BMDCs) secrete sufficient concentrations of EGFR ligand to stimulate proliferation of Lewis Lung Carcinoma (LLC) cell growth in vitro. To study the implication of nucleotide-treated DCs in tumor progression, we have coinjected stimulated BMDCs or their supernatants with LLC cells in mice. Interestingly, tumor weights were significantly increased after coinjection of LLC cells with LPS+ATPgS-treated BMDCs or their supernatants compared to LPS-treated BMDCs or their supernatants. The present study demonstrates that ATPgS is able to confer tumorigenic properties to human and murine DCs. Antagonists of ATP receptors expressed on DCs and blocking antibodies directed against ATP target genes in DCs could have a therapeutic potential as anti-tumor agents.

P7-6

A2B and A3 adenosine receptors modulate vascular endothelial growth factor and interleukin-8 expression in human melanoma cells treated with etoposide and doxorubicin

Merighi Stefania1, Simioni Carolina1, Mirandola Prisco2, Gessi Stefania1, Varani Katia1, Tabrizi Mojgan Aghazedeh3, Baraldi Pier Giovanni3, Borea Pier Andrea1

1Department of Clinical and Experimental Medicine, Pharmacology Section, University Of Ferrara, Italy,2Department of Human Anatomy, Pharmacology and Forensic Medicine, Institute of Normal Human Anatomy, University of Parma, Italy,3Department of Pharmaceutical Sciences, University of Ferrara, Italy

Background - Cancer patients undergoing treatment with systemic cancer chemotherapy drugs often have abnormal growth factor and cytokine profiles. Thus, serum levels of interleukin-8 (IL-8) are elevated in patients with malignant melanoma. In addition to IL-8, aggressive melanoma cells secrete, through its transcriptional regulator Hypoxia-inducible Factor 1 (HIF-1), vascular endothelial growth factor (VEGF), which promotes angiogenesis and metastasis of human cancerous cells. Whether these responses are related to adenosine, a ubiquitous mediator expressed at high concentrations in cancer and implicated in numerous inflammatory processes, is not known and is the focus of this study. Methods - We have examined whether the DNA-damaging agents etoposide (VP-16) and doxorubicin can affect IL-8, VEGF and HIF-1 expression in human melanoma cancer cells by means of western blot and ELISA assays. In particular, we have investigated whether these responses are related to the modulation of the adenosine receptor subtypes, named A1, A2A, A2B and A3 through siRNA assays. Results - We have demonstrated that A2B receptor blockade can modulate IL-8 production while blocking A3 receptors it is possible to further decrease VEGF reduction due to VP-16 and doxorubicin in melanoma cells. Conclusions - This understanding may present the possibility of using adenosine antagonists to reduce chemotherapy-induced inflammatory cytokine production and to improve the ability of chemotherapeutic drugs to block angiogenesis. Consequently, we conclude that adenosine receptor modulation may be useful for refining the use of chemotherapeutic drugs to treat human cancer more effectively.

P7-7

CD73 promotes tumor immune escape and metastasis

(Selected for Oral Poster Presentation)

John Stagg, Upulie Divisekera, Nicole McLaughlin, Mark Smyth

Peter MacCallum Cancer Centre University of Melbourne, Australia

Extracellular adenosine is a potent immunosuppressor. A crucial component in the production of extracellular adenosine is the membrane-bound enzyme CD73, which catalyzes the dephosphorylation of adenosine monophosphate to adenosine. Using RNA-interference, anti-CD73 monoclonal antibody (mAb) and CD73-deficient mice, we demonstrated that CD73 plays an important role in promoting tumor immune escape in different mouse models of cancer. Knockdown of CD73 in 4T1.2 breast cancer cells significantly decreased tumour growth in immunocompetent mice, but had no impact on primary tumour growth in severe-combined immunodeficient (SCID) mice. However, CD73 knockdown significantly inhibited spontaneous lung metastasis in SCID mice, even when SCID mice were further depleted of natural killer (NK) cells. Similar outcomes were observed when tumor-bearing mice were treated with anti-CD73 mAb. Our observation that CD73 enhances lung metastasis independently of its immunosuppressive effect led us to further investigate its role in tumour cell migration. Using in vitro transwell assays, we demonstrated that CD73 inhibition significantly suppressed tumour cell chemotaxis, most likely via activation of A2B adenosine receptors. Treatment of 4T1.2 tumor cells with BAY-606583, a selective A2B agonist, significantly enhanced the expression of the chemokine receptors CCR4, CCR5 and CCR6. Because CD73 is also expressed on non-transformed cells, we next investigated the role of host CD73 using CD73-deficient mice. We observed that CD73-deficient mice were significantly protected against a tumorigenic injection of MC38 colon cancer cells and EG7 lymphoma cells in a CD8+ T cell-dependent manner. In conclusion, our studies identified tumor-derived CD73 as an important mechanism of tumor immune escape and metastasis, and established the proof-of-concept that targeted therapy against CD73 can generate adaptive anti-tumor immunity and inhibit tumor metastasis.

P7-8

A3AR modulations in response to chronic treatment with CF101: data from experimental inflammatory animal models

Sara Bar Yehuda1, Avivit Ochaion2, Shira Cohen2, Renana Patoka2, Faina Barer2, Pnina Fishman2

1Can-Fite BioPharma, Israel,2Can-Fite BioPharma, Can-Fite BioPharma, Israel

Background: A3 adenosine receptor (A3AR) is over-expressed in inflammatory cells whereas normal cells show low or no receptor expression. CF101, a highly selective agonist at the A3AR, acts as an anti-inflammatory agent via the de-regulation of the NF-κB signaling pathway. The aim of the present study is to look at A3AR fate upon chronic treatment with CF101 in different experimental animal models. Methods The following experimental inflammatory animal models were used: Myelin Basic Protein induced Experimental Auto-immune Encephalomyelitis, Dextran Sulfate Sodium induced colitis, Mono Iodoacetate induced osteoarthritis, Collagen induced Arthritis and adjuvant induced arthritis (AIA). CF101 (10 or 100 µg/kg) was administered orally upon onset of disease two/three times per day. Disease severity was evaluated utilizing clinical score. A3AR, NF-κB and GATA1 expression levels were tested by Western blot analysis in protein extracts derived from inflammatory tissues and peripheral blood mononuclear cells (PBMCs). Results A3AR and NF-κB expression levels were directly correlated to disease severity. In all experimental animal models, A3AR expression levels were down-regulated in the inflammatory cells shortly after CF101 administration. CF101 exerted an anti-inflammatory effect manifested by a significant decreased clinical score. In the AIA model, A3AR and GATA1 expression levels in PBMCs were decreased 2 hours but were up-regulated 12 hours after treatment with CF101, demonstrating that receptor is recovered. Conclusions CF101 is a potent disease modifying drug in various inflammatory animal models. A3AR is down-regulated shortly after agonist treatment followed by receptor recovery, suggesting the receptor as an anti-inflammatory target for chronic treatment.

P7-9

Blockade of A2A receptor leads to delayed tumor appearance in carcinogen-induced tumor model

(Selected for Oral Poster Presentation)

Hadar Eini1, Oshri Naamani1, Eli Lewis1, Cidio Chaimovitz1, Amos Douvdevani2

1Ben-Gurion University, Israel,2Soroka Medical Center & Ben-Gurion University of the Negev, Israel

Background: Epidemiological studies show reduced cancer rate in coffee drinking subjects. Adenosine accumulates in solid tumors and has been shown to stimulate angiogenesis and to inhibit the activity of immune cells mainly by the A2A receptor (A2AR). Here, we examined the involvement of the A2AR in the escape of carcinogen-induced tumor from immune surveillance. Methods: Tumors were induced in WT, heterozygote and A2AR-KO mice by injection of the carcinogen 3-methylcholanthrene (3-MCA). Several cell lines were developed by isolating cells from excised tumors. Established cell lines were injected into mouse footpad, and tumor growth and cytokine profile were monitored. Results: Following inoculation of 3-MCA, both A2AR-KO and heterozygote mice showed delayed tumor development compared to their WT littermates (day of tumor appearance: 127 and 110 Vs 83, respectively, p = 0.035 for WT Vs KO). Accordingly, mice given caffeine (0.1% in drinking water), had a significantly decreased rate of tumor compared to control (14% Vs 53%, p = 0.0286). Injection of a weakly tumorigenic cell line which was found to be rejected by WT mice showed elevated IL-15, IL-15 Receptor-alpha and IFN-gamma mRNA levels compared to the intact tissues. These cytokine levels were further increased following caffeine treatment. Conclusion: Our data support findings that indicate a beneficial effect of caffeine on the suppression of tumor development, and show that adenosine may suppress immune response towards developing tumor through the A2A receptor. A2AR pharmacological antagonism might therefore represent a potential treatment for cancer.

P7-10

Blockade of murine T cell activation by antagonists of P2Y6 and P2X7 receptors

Mitsutoshi Tsukimoto1, Akihiro Tokunaga1, Hitoshi Harada2, Shuji Kojima1

1Faculty of Pharmaceutical Sciences, Tokyo University of Science, Japan,2Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Japan

BACKGROUND: Recent reports have shown that T cell receptor (TCR)-dependent ATP release from T cells is involved in production of interleukin-2 (IL-2) through activation of P2 receptors. Here, we investigated in detail the mechanisms of ATP release from activated T cells and the involvement of P2 receptor-mediated signaling in TCR-dependent T cell activation. METHODS&RESULTS: We studied the mechanisms of ATP release during TCR-dependent T cell activation by investigating the effects of various inhibitors on TCR-dependent ATP release from murine T cells. We found that ATP release from murine T cells is regulated by various mechanisms, including anion channel and gap junction hemichannel. Next, we examined the involvement of P2 receptors in murine T cell activation. Murine T cells were activated by stimulation of TCR, and both CD25 expression and IL-2 production were observed in activated T cells. Ecto-nucleotidase apyrase and P2Y6 antagonist MRS2578 significantly blocked the increases of both CD25 expression and IL-2 production, whereas P2X7 antagonists A438079 and oxidized ATP inhibited IL-2 production rather than CD25 expression, suggesting the involvement of P2Y6 and P2X7 receptors in different processes of T cell activation. MRS2578 also blocked TCR-dependent elevation of cytosolic Ca2+ in T cells. The P2X7 and P2Y6 receptors were expressed in murine CD4 T cells. CONCLUSION: our results indicate that activation of P2Y6 and P2X7 receptors contributes to T cell activation via TCR.

P7-11

Involvement of P2Y6 receptor in induction of cyclooxygenase-2 after ultraviolet B-irradiation

Erina Takai1, Mitsutoshi Tsukimoto1, Hitoshi Harada2, Shuji Kojima1

1Faculty of Pharmaceutical Sciences, Tokyo University of Science, Japan,2Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Japan

BACKGROUND: We investigated the involvement of extracellular nucleotides and P2 receptors in ultraviolet B (UVB)-induced inflammation in human keratinocyte HaCaT cells. METHODS: Concentration of extracellular ATP was measured by Luciferin-Luciferase assay. Activation of p38 MAPK and expression of cyclooxygenase-2 (COX-2) were examined by immune blotting. RESULTS: The UVB-irradiation induced rapid ATP release in HaCaT cells and it was significantly inhibited by pretreatment with anion transporter blockers or the maxi-anion channel blockers. Since the activation of p38 MAPK is involved in the expression of pro-inflammatory cytokines and proteins, such as COX-2, we examined the effect of UVB-irradiation on activation of p38 MAPK and expression of COX-2. The UVB-irradiation induced activation of p38 MAPK in HaCaT cells, and it was significantly blocked by pretreatment with ecto-nucleotidase, apyrase and P2Y6 receptor antagonist, MRS2578. The expression of COX-2 was induced by UVB-irradiation, and it was also inhibited by p38 MAPK pathway inhibitor or MRS2578. These results suggest that P2Y6 receptor would play a role in UVB-induced COX-2 expression, which is mediated by activation of p38 MAPK. Moreover, knockdown of P2Y6 receptor by transfection with siRNA in HaCaT cells attenuated the induction of COX-2 in response to UVB-irradiation. CONCLUSION: Our results suggest that UVB-irradiation would evoke ATP release from keratinocytes and also suggest that activation of P2Y6 receptor would play an important role in the UVB-induced expression of COX-2, which is a downstream of p38 MAPK-dependent pathway. We conclude the involvement of P2Y6 receptor in a mechanism of UVB-induced inflammation in keratinocytes.

P7-12

The ectonucleotidase NTPDase1 controls IL-8 production by human neutrophils

(Selected for Oral Poster Presentation)

Filip Kukulski, Fariborz Bahrami, Fethia Ben Yebdri, Joanna Lecka, Mireia Martín-Satué, Sébstien Lévesque, Jean Sévigny

Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Université Laval, Canada

Background: Recent works demonstrate that NTPDase1 deficit exacerbates inflammation. In line with these results, we show that NTPDase1 inhibition increases the production of proinflammatory interleukin 8 (IL-8) by human neutrophils. Methods: NTPDase1 protein expression on freshly isolated human blood neutrophils or neutrophil-like HL60 cells was determined by FACS. Extracellular ATP hydrolysis by neutrophils was assessed by RP-HPLC. NTPDase1 and P2Y2 expression knockdown were achieved by nucleofection with specific siRNAs. MIP-2 and KC (rodent counterparts of human IL-8) production in vivo was investigated using murine air pouch model. IL-8, MIP-2 and KC release were measured by ELISA. Results: Both human blood neutrophils and neutrophil-like HL60 cells express NTPDase1 protein and activity. NTPDase1 expression and activity inhibition with a specific siRNA and a potent inhibitor NF279, respectively, markedly increased IL-8 production by neutrophils stimulated with LPS (TLR4 agonist) and Pam3CSK4 (TLR1/2) but not with flagellin (TLR5) and gardiquimod (TLR7 and 8). Both primary neutrophils and neutrophil-like HL60 cells produced IL-8 also in response to exogenous nucleotides. As ATP was the most potent agonist of this response, we hypothesized that it was mediated by P2Y2 receptor which mediates the majority of ATP-induced responses in human neutrophils. In agreement, P2Y2 knockdown in neutrophil-like HL60 cells markedly decreased LPS-and Pam3CSK4-induced IL-8 productions. In line with these in vitro results, LPS-treated NTPDase1 deficient mice exhibited an increased production of chemokines MIP-2 and KC compared to wild type mice. Conclusion: NTPDase1 controls IL-8 production by human neutrophils via the regulation of P2Y2 activation.

P7-13

Changes in forward light scatter underlying P2X7 receptor-mediated T cell death

Hitoshi Harada1, Machiko Maehata2, Ko Misaka2, Mitsutoshi Tsukimoto3, Masakuni Degawa2

1Faculty of Pharmaceutical Sciences Suzuka, University Of Medical Science, Japan,2School of Pharmaceutical Sciences, University of Shizuoka, Japan,3Faculty of Pharmaceutical Sciences, Tokyo University of Science, Japan

Background: We investigated the critical event underlying P2X7 receptor—-mediated T cell death. Methods: Forward light scattering property and fluorescence intensity of dye were analyzed by flow cytometry. Morphological changes were observed with a confocal laser scanning microscope. Cell death was assessed by quantification of lactate dehydrogenase release. Results: Activation of P2X7 receptor by ATP leads to multiple downstream events including influx of ions, membrane blebbing, pore formation to allow the passage of larger molecular weight species and cell death. We detected that activation of P2X7 receptor by ATP induced membrane blebbing, pore formation, decrease in forward light scattering property (FS) and cell death in murine splenic T cells. Decreased extracellular Cl-suppressed ATP-induced decrease in FS and cell death, without inhibiting pore formation. On the other hand, a Rho kinase inhibitor, Y-27632 suppressed ATP-induced membrane blebbing, but did not affect ATP-induced pore formation and decrease in FS and cell death. Moreover, assay for the duration-dependency of ATP-treatment revealed that decrease in FS is essential for induction of cell death. Conclusion: These observations indicate that decrease in FS is a critical event of P2X7 receptor-mediated cell lysis in murine T lymphocytes.

P7-14

CF102 exerts a differential effect in various pathological liver conditions: protection from inflammation damage and anti-tumor activity

Sara Bar Yehuda1, Pnina Fishman1, Salomon Stemmer2, Galina Zozulya1, Avivit Ochaion1, Renan Patoka1, Faina Barer1, Lea Rath-Wolfson3, Shira Cohen1

1Can-Fite BioPharma, Can-Fite BioPharma, Israel,2Institute of Oncology, Davidoff Center, Rabin Medical Center, Sackler School of Medicine, Tel Aviv University, Israel,3Department of Pathology, Rabin Medical Center, Golda Campus, Sackler Faculty of Medicine, Tel-Aviv University, Israel

Backgrounds: A3AR found to be highly expressed in a broad spectrum of cancerous and inflammatory tissues. Selective agonists at the A3AR, such as CF101 and CF102 were found to induce anti-inflammatory and anti-cancer effects. In this study we examined the differential effect of CF102 in pathological conditions of the liver. Methods: The anti-inflammatory protective effect of CF101 was tested in a model of liver inflammation induced by Concanavalin A (Con. A) and an anti-cancer effect of CF102 was examined by a xenograft model of hepatocellular carcinoma (HCC). The mechanism of action was explored by following the expression levels of key signaling proteins in liver and tumor extracts, utilizing Western blot analysis. Results: In Con. A-induced liver injury CF102 (100μg/kg) markedly reduced secretion of glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) in comparison to the vehicle-treated group. In addition, CF102 treatment inhibited the Nuclear Factor κB (NF-κB)-Tumor necrosis factor-α (TNF-α) signaling pathway and prevented apoptosis in the liver as was demonstrated by decreased expression levels of Fas receptor (FasR) and the pro-apoptotic proteins Bax and Bad in liver tissues. CF102 was also found to induce up regulation of the apoptotic signaling pathway in the HCC model, resulting in tumor growth inhibition. Conclusions: CF102 protects the liver against Con. A-induced hepatitis and inhibits HCC tumor growth in experimental models. These results suggest that CF102, through its differential effect, is a potential drug candidate to treat various pathological liver conditions.

P7-15

Tuning of T cell immunosuppressive function by ATP

Fabio Grassi1, Ursula Schenk2, Michela Frascoli1, Michele Proietti1

1T cell development lab Institute for Research in Biomedicine, Switzerland,2Pharmanalytica SA, Pharmanalytica SA, Switzerland

T cell stimulation by cognate antigen determines a functional response, the nature of which depends on concomitant stimuli provided by accessory molecules and cytokine milieu. T cell receptor (TCR) triggering of CD4+ T cells is followed by increased oxidative synthesis and release of ATP from stimulated cells through pannexin hemichannels. Consequent stimulation of P2X receptors sustains mitogen-activated protein kinase (MAPK) activation and implements T cell effector functions. High expression of P2X7 is part of the transcriptional signature of immunosuppressive regulatory T cells (Tregs) expressing the transcription factor Foxp3. We show that P2X7 activation by ATP inhibits Tregs suppressive potential. In inflammatory conditions, Tregs displayed increased ATP synthesis and P2X7 mediated signaling, which determined lineage instability and Tregs differentiation to pro-inflammatory IL-17 secreting T helper (TH17) effector cells. Moreover, pharmacological P2X antagonism promoted cell autonomous conversion of naïve CD4+ T cells into Tregs upon TCR stimulation. Thus, ATP has the so far unique property to integrate extracellular cues and cellular energetics to tune the T cell immunosuppressive program as an external soluble factor.

P7-16

Adenosine receptor activation augments IL-10 production by murine microglial cells

Balazs Koscso1, Zsolt Selmeczy2, Leonora Himer2, Balazs Csoka1, Sylvester E. Vizi2, György Haskó1

1Department of Surgery, University of Medicine and Dentistry - New Jersey Medical School, USA,2Department of Pharmacology, Institute of Experimental Medicine, HAS, Hungary

Microglia, the intrinsic macrophages of the central nervous system produce the anti-inflammatory cytokine IL-10 following activation with the bacterial cell wall product peptidoglycan (PGN), which is recognized by Toll-like receptor 2 (TLR2). Adenosine is an endogenous purine nucleoside that binds to specific G protein-coupled receptors (A1, A2A, A2B, and A3), and is well recognized as an effective modulator of the immune system. In this study we investigated the effect of adenosine on IL-10 production by primary microglia and the murine microglial cell line BV-2. Cells were treated with adenosine, or selective adenosine receptor agonists and antagonists, in conjunction with 20 μg/ml PGN for 6 or 24 hours. Adenosine treatment augmented IL-10 production by both primary microglia and BV-2 cells activated with PGN. The non-selective adenosine receptor agonist NECA was the most potent IL-10 inducer, and its effect was prevented by pretreatment with the A2B antagonist MRS-1754. Adenosine receptor activation augmented IL-10 mRNA levels, and this effect was prevented by blocking transcription with actinomycin D. The stimulatory effect of adenosine on IL-10 production was mediated by p38 and not ERK1/2. IL-10 promoter analysis, chromatin immunoprecipitation (CHIP) experiments and lentiviral delivery of shRNA against CREB suggested that CREB activation is necessary for the stimulatory effect of adenosine on IL-10 transcription. These results demonstrate that A2B adenosine receptor activation augments IL-10 production by PGN-activated microglial cells through a p38- and CREB-mediated pre-transcriptional mechanism.

P7-17

A2B adenosine receptors and growth inhibition in cancer cells

Kristina Lorenz, Marie Vidal, Rahul Yadav, Daniela Bieber, Karsten Rübeling, Lena Sumski, U-Ju Heinlein, Sonja Kachler, Karl-Norbert Klotz

Department of Pharmacology, University of Wuerzburg, Germany

Adenosine receptors subtypes may play a role in various aspects of cancer development and tumor growth. Screening of cancer cell lines shows differential expression of adenosine receptor subtypes. A total of four ovary and five endometrial cancer cell lines were tested: two ovary cancer cell lines expressed low levels of A2A (OAW 42 cells) and A2B (SKOV 3 cells) receptors while very low levels of A1 and A2A receptors were identified in the endometrial HEC-1A and Ishikawa cancer cells. Both, the human A375 and MV3 melanoma cell lines as well as the non-tumorigenic human keratinocyte cell line HaCaT express A2B adenosine receptors, but no other subtypes were detected. The breast cancer cell line MCF-7 does not express detectable levels of any adenosine receptor subtypes as opposed to the MDA-MB-231 breast cancer cells which express A2B adenosine receptors as the sole subtype at very high levels. It was previously shown that stimulation of A2B receptors in MDA-MB-231 mediates an activation of adenylyl cyclase and, most likely via activation of Gq, a Ca2+ signal. In addition, activation of A2B adenosine receptors with NECA causes a time-dependent inhibition of ERK1/2 phosphorylation. Presumably as a consequence of MAPK inhibition an inhibition of MDA-MB-231 cell proliferation was observed in the presence of the receptor agonist NECA. Therefore, the A2B adenosine receptor might serve as a target for the inhibition of cell proliferation and, thus, may provide a novel target for co-therapies in cancer treatment.

P7-18

Interaction between the adenosine A3 receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA) and the extrinsic cell death pathway in human leukemia cells

Petr Mlejnek, Petr Dolezel

Department of Biology, Faculty of Medicine and Dentistry, Palacky University Olomouc, Czech Republic

BACKGROUND: The A3 adenosine receptor (A3AR) agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (Cl-IB-MECA) has been implicated in modulation of the extrinsic cell death pathway in cancer cells. Cl-IB-MECA was reported to induce an increase in expression of Fas in HL-60 and MOLT-4 cells (Kim et al. 2002). In contrast, Morello and co-workers demonstrated that Cl-IB-MECA enhanced TRAIL-mediated apoptosis via the modulation of NF-kappaB signalling pathway in thyroid cancer cells (Morello et al. 2009). METHODS: Human leukemia K562 cells were cultivated in a growth medium containing micromolar concentrations of Cl-IB-MECA in combination with cell death-inducing cytokines (FasL, TRAIL, or TNFalfa) and the number of apoptotic cells was evaluated by examination of nuclear morphology, measurement of caspase-3 activity, and by estimation of the hypoploid population of cells. RESULTS: We observed that Cl-IB-MECA augmented TRAIL-induced apoptosis in K562 cells. Effect of TRAIL seemed to be specific as no similar synergism was observed for other tested ligands, including FasL or TNFalfa. A detailed analysis revealed that Cl-IB-MECA induced an increase in expression of TRAIL receptors in K562 cells. CONCLUSION: Cl-IB-MECA enhances TRAIL-induced apoptosis via elevated expression of TRAIL receptors in human leukemia K562 cells. Acknowledgement: This work was supported by grant MSM 6198959216 (Ministry of Education, Youth and Sports).

P7-19

Inhibitory effects of benzodiazepines on the adenosine A2B-receptor-mediated production of interleukin-8 in human mast cells

Rosa Altarcheh Xifró, Kristina Hoffmann, Petra Spitzlei, Ivar von Kügelgen

Dept. of Pharmacology, University of Bonn, Germany

Background: The activation of adenosine A2B-receptors mediates pro-inflammatory responses in human mast cells including the production of interleukin-8 (IL-8). There is evidence for an inhibitory action of benzodiazepines on immune responses of mast cells. Therefore, we analyzed effects of benzodiazepines on responses of mast cells to activation of A2B-receptors. Methods: IL-8 levels in the supernatant of cultured human mast (HMC1) cells were determined by means of a sandwich ELISA. Cellular cAMP levels were measured by a radioaffinity assay and the activity of transcription factors by a reporter gene assay. Results: NECA (N-ethylcarboxamido-adenosine; 0.3 to 10 µM) increased the IL-8 concentration in the supernatant about 20-fold above basal. These responses to NECA were markedly reduced by the A2B-receptor antagonist MRS 1754 (8-(4-[{(4-cyanophenyl)carbamoylmethyl}oxy]phenyl)-1,3- di(n-propyl)xanthine, 1 µM). Moreover, diazepam (3 to 30 µM), flunitrazepam (3 to 30 µM), and the agonist of the peripheral-type benzodiazepine receptors 4´-chlorodiazepam (3 to 30 µM) markedly inhibited the NECA-induced IL-8 production, whereas clonazepam (3 to 30 µM) caused a modest reduction. Flunitrazepam also reduced NECA-induced increases in luciferase expression controlled by the cAMP response element (CRE)-promotor and the Nuclear factor of activated T-cells (NFAT)-promotor. Neither diazepam nor flunitrazepam changed the NECA-induced increases in cellular cAMP concentration in Chinese hamster ovary cells expressing recombinant human adenosine A2B-receptors. Conclusions: The data demonstrate that benzodiazepines reduce the A2B-receptor-mediated IL-8 production as well as the A2B-receptor-mediated activity of transcription factors in human mast cells downstream of the adenosine receptor. The rank order of potency indicates the involvement of peripheral-type benzodiazepine receptors.

P7-20

Increase of myeloid microvesicles in the cerebrospinal fluid as biomarker of microglia/macrophage activation in neuroinflammatory disorders

Claudia Verderio1, Alessandra Bergami2, Francesca Ruffini2, Luisa Novellino3, Loredana Riganti3, Elena Turola3, Irene Corradini3, Chiara Maiorino2, Maura Francolini3, Gianvito Martino2, Michela Matteoli3, Luca Muzio2, Roberto Furlan2

1Institute of Neuroscience and Dept. of medical Pharmacology, CNR, Italy,2Clinical Neuroimmunology Unit, Department of Neuroscience, Institute of Experimental Neurology (INSPE), San Raffaele Scientific Institute DIBIT, Italy,3Institute of Neuroscience and Dept. of medical Pharmacology, CNR and University of Milan, Italy

Cerebrospinal fluid represents an easily accessible source of material for diagnosis and monitoring of neurological diseases. As a direct recipient of cell shedding products, it is a potential indicator of abnormal CNS states such as inflammation, infection, neurodegenerative processes and tumor growth (Zougman A, 2008). Here we show that rodent CSF contains microvesicles (MVs) of microglial origin, similar in size and content to MVs released in vitro from microglia exposed to ATP, a mediator of intercellular communication and a danger signal (Bianco et al., 2005; Bianco et al., 2009). Of note, flow cytometry analysis of microglia-derived MVs indicates increased levels of MVs in the CSF of mice intracerebrally injected with lentiviral vectors codifying for IFNγ or TNFα, a protocol known to induce dramatic activation of microglia/macrophages. These results represent the proof of principle that the amount of microglial MVs in the CSF reflects the activation state of microglia in vivo and thus the extent of the inflammatory condition. In line with these results microglia-derived MVs also increase in rodent CSF in the course of Experimental Autoimmune Encephalomyelitis (EAE), a widely used model for human Multiple Sclerosis, reflecting disease activity. In both chronic and relapsing EAE, the number of microglia-derived MVs is closely associated to disease course, increasing with EAE severity and decreasing with recovery. These findings link ATP-induced microglia activation to the propagation of MVs in vivo and identify CSF MVs as a novel biomarker for diagnosis and monitoring of neuroinflammatory diseases.

P7-21

Activation of P2X7 by ADP-ribosylation renders T cells sensitive for phagocytosis by macrophages

Nicole Schwarz, Felix Scheuplein, Friedrich Haag, Friedrich Koch-Nolte

Institute of Immunology, University Hospital Hamburg-Eppendorf, Germany

Here we address the question whether gating of P2X7 by T cells influences the interaction of these cells with macrophages. P2X7 allows T cells to sense nucleotides released during inflammation. Gating of P2X7 is induced by the soluble ligand ATP or by NAD-dependent ADP-ribosylation, a covalent modification catalyzed by ADP-ribosyltransferases ART2. Intravenous injection of NAD results in depletion of naive T cells in wild type mice but not in ART2KO mice. Using FACS and fluorescence microscopy analyses we show that NAD and ATP induce naive T cells to externalize phosphatidylserine (PS) immediatly, which is regarded as a common „eat me“ signal of apoptotic cells. Further, we show that P2X7 activation renders T cells sensitive for binding to peritoneal macrophages. Indeed, these cells efficiently phagocytose T cells following brief exposures either to ATP or NAD. PS-externalization induced by P2X7-activation proceeds with much faster kinetics than that induced by classical inducers of T cell apoptosis, i.e. ligation of death receptors, UV irradiation or treatment with staurosporine. The finding that P2X7 activation renders T cells sensitive for phagocytosis by macrophages provides an explanation for the rapid disappearance of T cells in vivo after injection of NAD. Moreover, in the context of inflammation, the phagocytic clearance of apoptotic T cells may display an important mechansim to enhance physiological immune responses and to prevent autoimmune diseases.

P7-22

Adenosine A2A receptor activation protects CD4+ T lymphocytes against activation-induced cell death

Leonóra Himer1, Balázs Csóka2, Balázs Koscsó2, E. Sylvester Vizi1, Bruce N. Cronstein4, Gyorgy Hasko2

1Institute of Experimental Medicine, Hungarian Academy of Sciences, Hungary,2New Jersey Medical School, UMDNJ, USA,3Institute of Experimental Medicine, Hungarian Academy of Sciences, Hungary,4Department of Medicine School of Medicine, New York University School of Medicine, USA

Background: Activation-induced cell death (AICD) is initiated by T-cell receptor (TCR) restimulation of already activated and expanded peripheral T cells and is mediated through Fas/Fas ligand (FasL) interactions. Adenosine is a purine nucleoside signaling molecule, and its immunomodulatory effects are mediated by 4 G-protein-coupled receptors: A1, A2A, A2B, and A3. Methods: In this study, we investigated the role of A2A receptors in regulating CD4+ T lymphocyte AICD using pharmacological and molecular approaches. Results: The selective A2A receptor agonist CGS21680 (EC50 15.2–32.6 nM) rescued mouse CD4+ hybridomas and human Jurkat cells from AICD and that this effect was reversed by the selective A2A receptor antagonist ZM241385 (EC50 2.3 nM). CGS21680 decreased phosphatidylserine exposure on the membrane, as well as the cleavage of caspase-3, caspase-8 and poly(ADPribose) polymerase indicating that A2A receptor stimulation blocks the extrinsic apoptotic pathway. In addition, CGS21680 attenuated both Fas and FasL mRNA expression. This decrease in FasL expression was associated with decreased activation of the transcription factor systems NF-kappaB, NF-ATp, early growth response (Egr)-1, and Egr-3. The antiapoptotic effect of A2A receptor stimulation was mediated by protein kinase A. Conclusion: These results demonstrate that A2A receptor activation suppresses the AICD of peripheral T cells.

P8. Neuropsychiatric disease

P8-1

Two haplotypes of the P2X7 receptor containing the Ala-348 to Thr polymorphism exhibit a gain-of-function effect and enhanced interleukin-1beta secretion

Leanne Stokes1, Stephen Fuller1, Ronald Sluyter2, Kristen Skarrat1, Ben Gu3, James Wiley3

1Sydney Medical School, University of Sydney, Australia,2School of Biological Sciences, University of Wollongong, Australia,3Florey Neurosciences Institute, University of Melbourne, Australia

The P2X7 receptor is an ATP-gated cation channel expressed in immune cells and plays a role in pro-inflammatory cytokine release from monocytes and macrophages. This study investigated the co-inheritance of twelve functionally relevant single nucleotide polymorphisms (SNPs) in the human P2X7 gene (P2RX7) and the functional effect of each singly and in combination was assessed by measurements of ATP-induced currents and ethidium+ uptake. Genotyping of 3430 Caucasian subjects identified four common haplotypes in addition to the common (wild-type) P2X7-1. Two haplotypes (denoted P2X7-2 and P2X7-4) contained various combinations of gain-of-function SNPs. P2X7-4 was uniquely identified by the Gln-460 to Arg polymorphism (rs2230912). When expressed in HEK-293 cells, recombinant P2X7-2 and P2X7-4 haplotypes displayed a three-fold and five-fold increase respectively in receptor function compared to the wild-type P2X7-1. Both P2X7 haplotypes contained the Ala-348 > Thr polymorphism (rs1718119) and this mutation was critical for the gain-of-function effect. Peripheral blood monocytes and erythrocytes from subjects homozygous for gain-of-function P2X7 haplotypes exhibited increased ATP-induced ethidium+ uptake and 86Rb+ efflux respectively and this correlated with increased IL-1beta secretion from LPS-primed monocytes. Inheritance of these P2X7 haplotypes predisposing to increased pro-inflammatory cytokine secretion may be important in genetic association studies of inflammatory, infectious and psychiatric disorders.

P8-2

Caffeine prevents memory recognition impairment in a rat model of sporadic dementia

Janaína Espinosa1, Marcelo Silveira da Costa2, Vanessa Kazlauckas2, Micheli Figueiró2, Eduardo Kalinine2, Diogo Onofre Souza2, Lisiane de Oliveira Porciúncula2

1Graduate Program in Neuroscience, UFRGS, Brazil,2Department of Biochemistry, UFRGS, Brazil

Cerebral infusion of streptozotocin (STZ) disturbs brain insulin signaling leading to impairment on learning and memory being considered a model of sporadic dementia in rats. Caffeine consumption prevents cognitive aging decline and mnemonic deficits in two animal models of Alzheimer's disease. However, in this model of dementia the effects of caffeine are unknown. Thus, we tested whether caffeine could prevent memory dysfunction in this model. Wistar male adult rats were treated for 2 weeks with drinking water or caffeinated drinking water (1 mg/mL, p.o) prior to intracerebroventricular (i.c.v) administration of STZ, followed by additional 4 weeks of caffeinated or drinking water. After treatments, animals performed novel object recognition task and the exploratory behavior and locomotor activity were evaluated in an open-field apparatus (OF). Caffeine treatment prevented the impairment of memory recognition on the novel object caused by STZ. There was no significant difference on exploratory behavior and locomotor activity between groups. Thus, caffeine prevented memory deficits produced by STZ without affecting exploratory behavior and locomotor activity. Since STZ exerts its effects by disturbing insulin signaling, our results suggest that adenosine receptors may play a role on memory deficits under brain insulin resistant state. Finally, caffeine-mediated effects on cognition suggest that adenosine receptors blockade may be useful under impaired insulin metabolism. Supported by CAPES, INCT/CNPq.

P8-3

Systemic administration of GMP induces anxiolytic-like behavior in rats

Marcelo Ganzella, Roberto F. Almeida, Victor H.C. Junior, Rafael B. Faraco, Ana E. Bohmer, Diogo O. Souza

UFRGS, Brasil

Anxiety disorders are common psychiatric diseases in medical practice. The glutamatergic system has received considerable attention over recent years as potential target for anxiolytic drugs. A number of studies have demonstrated that guanine based purines (GBPs) play an important role in the modulation of the glutamatergic system, with several studies indicating the ability of the GBPs to reduce glutamatergic activity. In the present study, we investigated the effect of GBPs (by GMP administration) in behavioral models of anxiety. Male adult Wistar rats were pretreated with anintraperitoneal (i.p.) injection, of saline (NaCl 0.9%); GMP 10, 25, 50, 100 and 150 mg/kg, and diazepam 2 mg/kg (positive control). One hour after the injection, rats were subjected to the light/dark test, elevated plus-maze test, and open field test. Additionally, purines concentration in the cerebralspinal fluid (CSF) was verified. The administration of GMP at dose of 25 and 50 mg/kg promoted anxiolytic like behaviors, like diazepam, in the light/dark and plus-maze test. However, in the open field test, as well as in the analysis of CSF purine concentration, neither GMP, nor diazepam differed from saline group one hour after administration. Thus, this is the first study showing that acute administration of GMP produce anxiolytic-like effect.

P8-4

Motor effects of adenosine A2A receptors in the MAM model of schizophrenia

Rosa Luisa Potenza, Monica Armida, Antonella Ferrante, Antonella Pèzzola, Patrizia Popoli

Dept. Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Italy

Adenosine A2A and dopamine D2 receptors physically and functionally interact in the ventral striatum. Since D2Rs are involved in the antipsychotic effects of neuroleptics, A2AR agonists provide a potential new approach to schizophrenia. Treatment of rats with methylazoxymethanol acetate (MAM) in utero yields an adult pattern of neuropathology resembling schizophrenia. We have characterized MAM and control animals in a series of behavioural tests related to schizophrenia (motor activity, Morris Water Maze and pre-pulse inhibition of the acoustic startle response) to establish the validity of prenatal MAM treatment in our condition (20 mg/kg i.p. on gestational day 17). We analysed the motor effects elicited by A2AR activation or blockade in MAM and control offspring, measuring spontaneous motor activity by autotomated cages, which allow to measure "total" motor activity as well as to discriminate among horizontal, vertical and stereotyped activity. As compared with the saline-exposed rats, MAM rats exhibited spontaneous hyperactivity that persisted into adulthood. The administration of CGS 21680 (0.5 mg/kg i.p.) a selective adenosine A2AR agonist, significantly reduced the number of rearings only in MAM-treated animals (-84% P<0.05 vs. VEH). Conversely, the selective A2A antagonist SCH 58261 (2 mg/kg i.p.) induced hypermotility in both groups, but tended to be less effective in MAM rats (+121% e +481% in MAM and control rats respectively, P<0.05 vs. VEH). These experiments suggest a different state of activation of A2ARs in the stereotyped activity of MAM-exposed offspring. The possible relevance of these data to schizophrenia needs to be further explored.

P8-5

Genetic or pharmacological blockade of adenosine A2A receptors prevents behavioral and morphological effects of chronic stress in mice

(Selected for Oral Poster Presentation)

Manuella Kaster1, Paula Canas1, Jörg Hockemeyer2, Christa Muller2, Ana Lúcia Rodrigues3, Rodrigo Cunha1

1Center for Neuroscience of Coimbra, University of Coimbra, Portugal,2Pharmazeutische Chemie I, Pharmazeutisches Institut, University of Bonn, Germany,3Department of Biochemistry, Federal University of Santa Catarina, Brazil

Chronic stress is known to trigger spine loss leading to cognitive impairment. Since caffeine and A2A receptors (A2ARs) antagonists prevent memory dysfunction upon noxious conditions (beta-amyloid, diabetes, convulsions), we now tested the effect of A2AR blockade on behavioural and morphological effects of chronic stress. Chronic unpredictable stress (CUS) was applied to male C57B6 mice. CUS enhanced plasmatic corticosterone, reduced weight gain, increased immobility time in the forced swim test, reduced time spent in open arms in the elevated plus-maze and reduced the spontaneous alternation in the Y-maze, indicating that CUS induces a depressive- and anxiogenic-like state associated with memory deterioration. Western blot or histological analysis of CUS mice hippocampus revealed lack of neuronal damage (FluoroJade-C staining) and microglia activation (CD11b staining) but presence of astrogliosis (GFAP staining) and decreased immunoreactivity of synaptic proteins (SNAP-25 and syntaxin) Caffeine (1 g/L, non-selective adenosine receptor antagonist) or KW6002 (3 mg/kg, selective A2AR antagonist), applied through drinking water 4 weeks before and throughout CUS, prevented all these behavioural and morphological modifications (albeit caffeine was anxiogenic per se). Likewise, all these CUS-induced behavioural and morphological changes were abrogated in A2AR knockout (KO) mice (albeit A2AR-KO mice displayed anxiogenic-like behaviour). In conclusion, both pharmacological and genetic approaches support the neuroprotective potential of A2AR antagonists against chronic noxious stimuli which involve synaptic deterioration and memory dysfunction.(Supported by CAPES, FCT)

P8-6

Key role of adenosine A1 receptors in the control of restraint-stress-induced preconditioning against subsequent ischemic or convulsive insults

Geanne Andrade1, Manuela Kaster1, Carolina Souza2, Ailton Fonteles2, Paula Canas1, Rodrigo Cunha1

1Center for Neuroscience of Coimbra, Portugal,2Federal University of Ceará, Brasil

The activation of A1 receptors (A1R) constitutes a hurdle for spreading of brain damage and is involved in preconditioning, whereby a submaximal noxious stimulus limits subsequent neurodegeneration triggered by a more intense noxious stimulus. We now tested if a single period of restraint stress could afford preconditioning against subsequent ischemic or convulsive insults and if this involved bolstering A1R function. Male adult (2month) rats subject to restraint stress (2hrs) displayed 24hrs later a 34-29% increased expression (real-time PCR) and 21–28% enhanced density (binding and Western blot analysis) of A1R in their hippocampus and cerebral cortex (n = 5–6). When rats were subject either to kainate (10 mg/kg, ip)-induced convulsions or focal ischemia upon distal occlusion of the middle cerebral artery, the rats subject to restraint stress 24hrs earlier displayed an extent of kainate-induced hippocampal lesion (FluoroJadeC-labeling) 64–73% lower (in CA1 and CA3 regions, n = 4) than injured rats that were not previously subjected to restraint stress and an extent of ischemia-induced cortical damage (2,3,5-triphenyltetrazolium chloride, TTC-labeling) similar to rats not subject to ischemia (n = 8–12). Notably, treatment with the A1R antagonist DPCPX (1 mg/kg, ip) abrogated this ability of restraint stress to reduce subsequent neuronal damage by kainate (n = 4) or abolish subsequent neuronal damage by ischemia (n = 8–16). This indicates that the activation of increased levels of A1R by endogenous extracellular adenosine plays a key role in stress-induced preconditioning against subsequent convulsions- or ischemia-induced cortical damage. (Supported by CNPq and FCT)

P8-7

Gender and dose-dependent changes in the purinergic-glutamatergic systems interactions in triple-transgenic 3xTg-AD for Alzheimer’s disease and its modulation by postnatal handling

Ismael Alvarez Montón1, Raquel Baeta-Corral2, Frank M. LaFerla3, Lydia Giménez-Llort2

1Autonomous University of Barcelona, Institute of Neuroscience·Autonomous University of Barcelona, Spain,2Institute of Neuroscience and Dept. Psychiatry and Forensic Medicine, Autonomous University of Barcelona, Spain,3Dept. Neurobiology and Behavior and Institute for Brain Aging, University of California Irvine, USA

Background: Glutamate excitotoxicity has been related to the mechanisms underlying neurodegenerative disorders and is a key target for some succesful therapeutical strategies. There, purinergic signalling is considered to play a relevant modulatory role. We have studied the functional interplay of glutamate-adenosine systems at the behavioral level in a triple-transgenic animal model for Alzheimer’s disease as well as the putative effects of postnatal handling, an early-life stimulation that seems to be able to exert long-term modulation of cAMP. Methods: Male and female 3xTg-AD mice were studied at different ages modeling different stages of the disease: asymptomatic, mild (intra-neuronal beta-amyloid) and severe amyloid/tau pathologies. A multicage activity system recorded the time-course of the motor effects induced by a wide range of doses of NMDA (25–200 mg/kg, i.p., from non-convulsant to excitotoxic profiles) and those by theophylline (3–30 mg/kg, i.p.). Mice receiving early postnatal handling twice a day (PND1-PND21) were studied to assess the effects of sensorial stimulation on these responses. Results: Gender- and dose-dependent differences were found in the initial adenosine-related motor depressant response induced by NMDA and its convulsive thershold as well as in the dose-effect range induced by theophylline. The results suggest differential functional interactions between glutamatergic and purinergic systems in the progress of the disease in 3xTg-AD mice. Early-life stimulation seems to be able to modulate some of the motor-activity patterns. Conclusion: Glutamate-adenosine systems interactions are modified in 3xTg-AD mice from early stages of the disease and early-life interventions known to modulate cAMP may help to ameliorate them.

P8-8

Genetical deletion and pharmacological antagonism of P2X7 receptors results in antidepressant phenotype in mice

Romeo D. Ando1, Cecilia Csölle1, Ágnes Kittel1, Flóra Gölöncsér1, Soproni Krisztina1, Zelena Dóra1, József Haller1, Tamás Németh2, Attila Mócsai2, Beáta Sperlágh2

1Institute of Experimental Medicine, Hungary,2Semmelweis University, School of Medicine, Hungary

Introduction: We investigated the role of P2X7 receptor (P2X7R) in mice models of depressive-like behavior in the wild-type and P2X7 knockout mice. Methods: The effect of genetic deletion of P2X7R and the administration of P2X7R antagonists were investigated in tail suspension test (TST), forced swim test (FST) and in sucrose preference test (SPT) under basal conditions and after bacterial lipopolysaccharide (LPS) challenge. Depressive behavior was studied in CD45.1 mice transplanted with P2X7R+/+ and P2X7R−/− bone marrow. Results: P2X7R knockout mice displayed antidepressant phenotype in the TST and FST. The pro-depressive effects of LPS challenge were attenuated in the FST by P2X7R knockout and in the TST by the acute pretreatment with the P2X7 antagonist BBG. Subacute (but not acute) treatment with BBG resulted in dose-dependent antidepressive effects (one-week, 25–50 mg/kg i.p.) in the TST. No change in baseline depressive behavior was observed in P2X7R−/− bone marrow transplanted mice in the TST and FST. In the SPT, the depressive-like behavior after LPS challenge was attenuated in P2X7R knockout mice. The acute and subacute (4 days) administration of the P2X7R negative allosteric modulator AZ-10606120 had antidepressant properties in the wild-type mice which was counteracted by P2X7R knockout, similarly to the effects of citalopram. Conclusions: The antidepressant phenotype of P2X7R KO mice and the attenuation of LPS-induced depressive behavior by the inhibition of P2X7R is an indication for the role of P2X7 receptors in the pathophysiology of depressive disorders, and suggest that P2X7R antagonists may represent a novel target in the therapeutics of depression.

P9. Neurodegeneration/Repair/Neuroprotection

P9-1

Multimodal evaluation of the neuroprotective potential of an adenosine A2A antagonist in a rodent model of focal cerebral ischemia

Karen Nieber1, Fronz Ulrike1, Weidlich Sarah1, Bolze Johannes2

1Institute of Pharmacy, University of Leipzig, Germany,2Neuro- / Cardiorepair Unit, Fraunhofer Institut für Zelltherapie und Immunologie, Germany

Background: Adenosine A2A antagonists has been shown to be neuroprotective when administered intraperitonially after permanent right middle cerebral artery occlusion (MCAO) in Wistar rats (Melani et al. Brain Res 2006). However, the STAIR (Stroke Therapy Academic Industry Round Table) quality assurance criteria were not implemented in these studies. The current study investigated the neuroprotective effect of continuous administration of 8-(3-chlorostyryl)caffeine (CSC) under conditions simulating a clinically relevant scenario. Treatment was initiated after a 2-hour time window and rigorous implementation of STAIR criteria. Methods: Spontaneously hypertensive rats were randomly assigned to two groups and underwent MCAO. CSC (5 mg/kg/d) or the vehicle was applied for three days using an osmotic minipump. Correct pump function was controlled by residual volume.Treatment efficacy was monitored by magnetic resonance imaging. For histological examination, Hematoxylin & Eosin staining as well as Luxol Fast Blue/Cresyl Violet staining were used Results: Edema-corrected lesion volume did not differ significantly after continuous application of CSC under STAIR conditions and was neither increased nor decreased significantly between 24 and 72 hours post MCAO. Extensive hepatic biotransformation of CSC leads to metabolites which may probably have lost their A2A antagonistic character, resulting in decreased effectiveness. This metabolite was dechlorinated and exhibited a lower molecular weight then the parent compound. Conclusion: The results suggest that A2A antagonists in general seem to have a very narrow time window of less than 2 hours, depending on the early incidence of excitotoxicity after stroke. This could considerably limit their benefit in models of focal cerebral ischemia.

P9-2

Altered nucleotide receptors expression pattern in a cerebral malaria murine model

(Selected for Oral Poster Presentation)

Jesús Sánchez Nogueiro, Patricia Marín-García, Marta Bautista, Míriam León-Otegui, María Linares, Darío Méndez, Carlos Monériz, Amalia Diez, José Manuel Bautista, María Teresa Miras-Portugal

Dpto. Bioquímica y Biología Molecular, Fac. Veterinaria, Universidad Complutense de Madrid, Spain

Background: Cerebral malaria is a non-traumatic encephalopathy caused by P. falciparum infection in humans and P. berghei in mice. The severe neurological complications of this infection may result from several coexisting pathological processes: hypoxia, hypoglycaemia, cerebral swelling, haemorrhage and inflammation. These pathological processes occur with cellular damage that might cause a massive release of different neurotransmitters, including nucleotides. Nucleotides and their hydrolytic products act through specific nucleotide receptors, which are divided in: ionotropic P2X and metabotropic P2Y receptors coupled to G proteins. Seven different P2X receptors (P2X1-7) and eight P2Y receptors (P2Y1,2,4,6,11,12,13,14) have been cloned in mammals. Since high levels of ATP can participate in the cellular damage reparation or can induce neurological and neurodegenerative alterations through its receptors, we studied the purinergic receptors expression in a murine model of cerebral malaria. Methods: Using Q-PCR and immunochemical assays we have analyzed the expression pattern of nucleotide receptors in different brain regions (cerebellum, frontal cortex, olfactory bulbs, hippocampus and thalamus-hypothalamus) from mice infected by P. berghei (ANKA). Results: We reported an altered expression pattern at P2X1,2,3,4,7 and P2Y1,2,12,13 receptors, which depends on both P2 receptor and analyzed cerebral region. Thalamus-hypothalamus showed the most modified P2 expression pattern, and P2Y2 receptor was found up-regulated in most of cerebral regions. Conclusion: This altered expression suggests a probable participation of these receptors in the pathogenesis of cerebral malaria and/or in the fatal neurological consequences of this infection. References: Marín-García et al. (2009) Altered nucleotide receptor expression in a murine model of cerebral malaria. J. Infect. Dis. 200: 1279–1288.

P9-3

Axodendritic fibres of mouse cerebellar granule neurons exhibit a diversity of functional P2X receptors

Jesús Sánchez-Nogueiro1, Patricia Marín-García1, David León2, Miriam León-Otegui1, Elvira Salas1, Rosa Gómez-Villafuertes1, Francisco Javier Gualix Sanchez1, Maria Teresa Miras-Portugal1

1Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain,2Área de Bioquímica, Facultad de Química, Universidad de Castilla-La Mancha, Spain

Distribution and functional expression of P2X receptors were analyzed in mouse cerebellum axodendritic fibres, using different experimental approaches such as RT-PCR, western blot, immunochemistry, microfluorimetric experiments and exocytotic studies. RT-PCR and western blot assays demonstrated the presence of P2X1,2,3,4,7 subunits in both the whole cerebellum and cultured cerebellar granule neurons. Immunochemical analysis of tissular and cellular location of P2X1,2,3,4,7 receptors confirmed their presence and unequal distribution between somas and axodendritic prolongations. Calcium measurement microfluorimetric experiments using a variety of modulators of the P2X subunits revealed the presence of different functional P2X receptors in the axodendritic fibres. Activation of functional P2X1 and P2X3 receptors was demonstrated by the use of the synthetic agonist alpha,beta-meATP and the selective antagonist Ip5I. Responses mediated by P2X1 subunits were also confirmed by using ZnSO4. Activation of functional P2X4 receptors was observed when granule cells were stimulated in the presence of ivermectin. Exocytotic studies confirmed the role of most P2X subunits in the induction of neurotransmitter release in axodendritic fibres from mouse cerebellar granule neurons.

P9-4

Epigenetic modulation of ADORA2A: a putative therapeutical tool for Parkinson’s disease treatment

(Selected for Oral Poster Presentation)

Marta Barrachina1, Sandra P. Buira1, José Luis Albasanz2, Guido Dentesano1, Jesús Moreno1, Salvador Juvés1, Mairena Martín2, Isidre Ferrer1

1Departament de Patología i Terapèutica Experimental, Facultat de Medicina, IDIBELL-Universitat de Barcelona, Spain,2Facultad de Químicas. CRIB., Universidad de Castilla-La Mancha, Spain

Pharmacological modulation of A2ARs is particularly useful in Parkinson’s disease (PD). However, little is known about the regulation of A2AR gene (ADORA2A). A bioinformatic analysis revealed the presence of three CpG islands in the 5′ UTR region of human ADORA2A. Next, HeLa, SH-SY5Y and U87-MG cells were treated for 48 h with 5 microM 5-azacytidine (Aza). Increased A2AR levels were demonstrated in HeLa and SH-SY5Y cells when compared with non-treated cells. No modifications were seen in U87-MG cells. The increased A2AR mRNA and protein levels were accompanied by a loss of DNA methylation pattern in HeLa and SH-SY5Y cells, as measured with the SEQUENOM MassArray platform. The Aza treatment also reduced the affinity of a methyl-CpG-binding protein (MeCP2) for ADORA2A by quantitative chromatin immunoprecipitation in HeLa cells. In parallel, we show that A2ARs are increased in the putamen post-mortem of PD patients non pharmacologically-treated with respect to age-matched controls, although it has not been associated with DNA methylation lost in the 5′UTR region of ADORA2A. However, the DNA methylation profile of ADORA2A in human brain is similar to SH-SY5Y and U87-MG. Interestingly, A2AR levels were reduced by S-adenosylmethionine (SAM) treatment in SH-SY5Y and U87-MG. Therefore, these results show for the first time that DNA methylation plays a role in ADORA2A transcription and, subsequently, in constitutive A2AR cell surface levels. It is proposed that the design of DNA methylating drugs combined with A2AR antagonists might be useful for future pharmacological intervention in the reduction of A2AR activity in PD patients.

P9-5

P2X7 receptor activation induces CXCL2 production in microglia through NFAT and PKC/MAPK pathways

Miho Shiratori, Hidetoshi Tozaki-Saitoh, Mai Yoshitake, Makoto Tsuda, Kazuhide Inoue

Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Japan

Microglia play an important role in many neurodegenerative conditions. ATP, which damaged cells can release or leak triggers microglial activation through P2 receptors, and stimulates the release of oxygen radicals, proinflammatory cytokines and chemokines from activated microglia. However little is known about mechanisms of ATP-induced chemokine release from microglia. In this study, we found that high concentration of ATP induces the mRNA expression and the release of CXCL2 from microglia. The similar effect was observed in treatment with a P2X7 receptor (P2X7R) agonist, 2′-and 3′-O-(4-benzoylbenzoyl) ATP (BzATP), and inhibited by pretreatment with a P2X7R antagonist, Brilliant blue G (BBG). ATP induced both activation of NFAT and MAPK (p38, ERK, JNK) through P2X7R. ATP-induced mRNA expression of CXCL2 was inhibited by INCA-6 (NFAT inhibitor), SB203580 (p38 inhibitor), U0126 (MEK-ERK inhibitor) and JNK inhibitor. MAPK inhibitors, however, did not inhibit activation of NFAT. In addition, PKC inhibitors suppressed ATP-induced ERK and JNK activation, and also inhibited ATP-induced CXCL2 expression in microglia. These results suggest that ATP increased CXCL2 production via both NFAT and PKC/MAPK signaling pathways through P2X7 receptor stimulation in microglia.

P9-6

Administration of P2X7 receptor antagonists attenuates the neuronal apoptosis, body-weight loss and motor-coordination deficits that characterize Huntington’s disease

María Diez Zaera1, Miguel Díaz Hernández2, Jesús Sánchez Nogueiro2, Rosa Gómez Villafuertes2, Josep M. Canals3, Jordi Alberch3, María Teresa Miras Portugal2, José J. Lucas4

1Departamento de Bioquímica y Biología Molecular IV, Universidad Complutense de Madrid, Spain,2Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain,3Departament de Biologia Cellular i Anatomia Patologica, Facultat de Medicina, Institu d'Investigacions Biomediques August Pi i Sunyer-Universitat de Barcelona, Spain,4Centro de Biología Molecular "Severo Ochoa", Consejo Superior de Investigaciones Científicas, Spain

The precise mechanism by which mutant huntingtin elicits its toxicity remains unknown. However, synaptic alterations and increased susceptibility to neuronal death are known contributors to Huntington’s disease (HD) symptomatology. While decreased metabolism has long been associated with HD, recent findings have surprisingly demonstrated reduced neuronal apoptosis in Caenorhabditis elegans and Drosophila models of HD by drugs that diminish ATP production. Interestingly, extracellular ATP has been recently reported to elicit neuronal death through stimulation of P2X7 receptors. These are ATP-gated cation channels known to modulate neurotransmitter release from neuronal presynaptic terminals and to regulate cytokine production and release from microglia. We hypothesized that alteration in P2X7-mediated calcium permeability may contribute to HD synaptic dysfunction and increased neuronal apoptosis. Using mouse and cellular models of HD, we demonstrate increased P2X7-receptor level and altered P2X7-mediated calcium permeability in somata and terminals of HD neurons. Furthermore, cultured neurons expressing mutant huntingtin showed increased susceptibility to apoptosis triggered by P2X7-receptor stimulation. Finally, in vivo administration of the P2X7-antagonist Brilliant Blue-G (BBG) to HD mice prevented neuronal apoptosis and attenuated body weight loss and motor-coordination deficits. These in vivo data strongly suggest that altered P2X7-receptor level and function contribute to HD pathogenesis and highlight the therapeutic potential of P2X7 receptor antagonists.

P9-7

Dopamine D2 and adenosine A2A receptors regulate activity of striatopallidal neurons through A2A-D2 receptor heteromerization

Serge Schiffmann1, David Gall1, Sergi Ferré2, Karima Azdad1

1Laboratory of Neurophysiology, Université Libre de Bruxelles, Belgium,2Behavioral Neuroscience Branch, National Institute on Drug Abuse, National Institutes of Health, USA

Electrical activity of striatal medium spiny neurons including membrane potential oscillations between a down- to an upstate could be regulated by G-protein-coupled receptors. Dopamine D2 and adenosine A2A receptors are highly enriched in striatal neurons and are known to exhibit strong interactions whose physiological significance and molecular mechanisms remain partially unclear. In this line, the respective involvement of intracellular signalling cascades commonly and antagonistically targeted by these receptors and A2A-D2 receptor heteromerization remained to be elucidated. By using perforated patch clamp recordings on brain slices and loading of competitive peptides, we showed that D2 and A2A receptors regulate the NMDA-induced transition to depolarized membrane potential plateau through mechanisms relying upon specific protein-protein interactions. Indeed, D2 receptor activation abolished transitions between a hyperpolarized resting potential to a depolarized plateau potential by regulating the CaV1.3a calcium channel activity through interactions with scaffold proteins Shank1/3. Noticeably, A2A receptor activation had no effect per se but fully reversed the effects of D2 receptor activation. This A2A receptor-induced reversal was prevented by specific competitive peptides that block A2A-D2 receptors heteromerization showing that this heteromerization is strictly mandatory. In summary, not only these studies demonstrate that membrane potential transitions and firing patterns in striatal neurons are tightly and antagonistically controlled by D2 and A2A receptors but they also provide a first direct physiological relevance of A2A-D2 receptors heteromerization in striatopallidal neurons.

P9-8

P2X7 receptor regulates alpha- and beta-secretase activity through GSK3. Therapeutic efficacy of P2X7 antagonist administration in mice model of AD

Juan Ignacio Díaz Hernández1, Rosa Gómez-Villafuertes1, Miriam León-Otegui1, Lourdes Hotecillas-Prieto2, Javier Gualix1, Jose Luis Trejo2, Jose Javier Lucas3, Juan Jose Garrido3, Maria Teresa Miras-Portugal1, Miguel Díaz-Hernández1

1Dpto. Bioquímica y Biología Molecular IV, Universidad Complutense de Madrid, Spain,2Dpto. Neurobiología Molecular, Celular y del Desarrollo, Instituto Cajal. CSIC, Spain,3Centro de Biología Molecular “Severo Ochoa”, CSIC-UAM, Spain

A large number of evidences have implicated A-beta peptide derived from amyloid precursor protein (APP) as central player to the pathogenesis of Alzheimer’s disease (AD). Abnormal production of A-beta peptide causes an important synaptic dysfunction and cognitive deficits in transgenic mice models. Previous works have shown an increase in purinergic receptor P2X7 expression in late stages of AD, but its implication in early stages of AD has not been established. In this work, we treated 4-months-old transgenic mice model for APP, J20 line, with specific P2X7 antagonist, Brilliant Blue G (BBG). Histochemical analysis of hippocampus from treated and un-treated mice showed a significant decrease in number of amyloid plaques. This decrease was linked to a change in the proteolytic pattern of APP; decreasing beta-carboxy-terminal fragment (beta-CTF) produced by beta-secretase and increasing production of alpha-CTF by alpha-secretase. Interestingly, the high catalitiyc activity of GSK-3 enzyme observed in J20 transgenic mice was also reverted by BBG treatment. Nevertheless, no changes in P2X7 expression or distribution were found between genotypes or treatments. Finally, the pharmacological inhibition of P2X7 receptor improves the cognitive deficits characterized in J20 transgenic mice model. These results strongly suggest the therapeutic potential role of P2X7 receptor antagonists.

P9-9

P2X7, NMDA and BDNF receptors converge on GSK3 phosphorylation and cooperate to promote survival in cerebellar granule neurons

Raquel Pérez-Sen, Felipe Ortega, Verónica Morente, Esmerida G. Delicado, MªTeresa Miras-Portugal

Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain

GSK3 is a key player in the regulation of neuronal survival. In previous work we have reported that P2X7 receptors were coupled to GSK3 phosphorylation and inhibition in cerebellar granule neurons. This signalling mechanism was responsible for the protective effect that the P2X7 nucleotide agonist, BzATP, exhibited against cell death induced by the inhibition of the PI3K/Akt survival route. Here we examine P2X7-mediated GSK3 signalling and analyze their possible interaction with NMDA and BDNF receptors at the level of intracellular mechanisms and neuroprotective effects. We demonstrate that these receptors exhibited a similar pattern of sustained GSK3 phosphorylation that was mainly dependent on PKC. While BzATP and NMDA cooperate to increase GSK3 phosphorylation, BDNF was the most potent inducing GSK3 phosphorylation, which was also dependent on the PI3K pathway. BzATP exhibited additive effects with both NMDA and BDNF to rescue granule neurons from cell death induced by PI3K pharmacological inhibition, which simulates conditions of trophic deprivation. This survival effect was demonstrated to be mediated by the PKC-dependent GSK3 pathway. In addition, BDNF protective effect also involved ERK-1,2 proteins, indicating that, in conditions of sustained PI3K inactivation, BDNF can switch its signalling requirements to the ERK pathway to maintain GSK3 inhibition and elicit neuroprotection. In conclusion the present results give evidence for the role played by nucleotides as relevant signals amplifying neuroprotective actions of glutamate and neurotrophins, and support the role of GSK3 as an important convergence point for survival promoting factors. (OrtegaF (2009) NeurotoxRes 15:193; OrtegaF (2010) CellMolLifeSci Feb10).

P9-10

P2Y13 and P2X7 act in combination to promote GSK3 signalling and neuroprotection in cerebellar granule neurons

Verónica Morente Rodríguez, Raquel Pérez-Sen, Felipe Ortega, MªTeresa Miras-Portugal

Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain

GSK3 plays a key role in neuronal survival regulation, being the PI3K/Akt/GSK3 axis triggered by insulin/IGF-I related factors the main survival route against apoptosis induced by trophic withdrawal. Nucleotides were also described to be coupled to GSK3 signalling in cerebellar granule neurons. The activation of both P2Y13 and P2X7 receptors induced significant increases in GSK3 phosphorylation on Ser9/21 residues, which is associated with the inhibition of its catalytic activity, being always the responses elicited by P2X7 receptors more robust. Additionally, while P2Y13 signalling followed the classical PI3K/Akt pathway and induced the nuclear translocation of beta-catenin, which is a GSK3 substrate and transcriptional regulator, P2X7 receptor responses were only dependent on calcium-dependent PKCs. These different signalling requirements suggested a possible cooperation between these two receptors. As expected, an additive effect was observed on GSK3 phosphorylation upon co-stimulation with both 2MeSADP and BzATP, agonists for P2Y13 and P2X7 receptors, respectively. In addition, cooperation between P2Y13 and P2X7 receptors was reproduced at the level of survival. Although 2MeSADP and BzATP were almost ineffective in protecting granule neurons against apoptotic death induced by trophic support withdrawal, they were able to exhibit a rescue effect of around 30% when they were added together. In addition, they both cooperated with the survival factor IGF-I in this protective action. These results give evidence of nucleotides as relevant survival signals acting alone or in combination with trophic factors, being GSK3 signalling involved in this effect. (OrtegaF (2008) Neurochem104:62; OrtegaF (2009) NeurotoxRes15:193).

P9-11

Species differences in expression and behaviour of P2X7 receptors in primary cerebellar astrocytes

Elvira Salas1, Luz María G. Carrasquero2, Esmerilda G. Delicado1, Mª Teresa Miras-Portugal1

1Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain,2Instituo de Investigaciones Biomédicas "Alberto Sols", CSIC-UAM, Spain

P2X7 receptor is one of the receptors that more controversy has arisen in the last years. Whereas P2X7 receptor in cell of hematopoietic lineage exhibits cytotoxic activity, it modulates neurotransmission and exerts neuroprotective actions in the nervous system. Furthermore, studies carried out with KO models suggested that there could be a different expression of this protein in the nervous system. Since changes in P2X7 expression have been observed in pathological situations using different experimental rodent models, we decided to approach the characterization of P2X7 receptor in mouse cerebellar astrocytes to compare with that in rat astrocytes (1). The functionality of P2X receptors was studied by recording nucleotide-elicited responses calcium in individual cells. Mouse astrocytes responded to ATP evoking calcium responses, which displayed different kinetic dependent on the ATP concentration used. The stimulation of astrocytes with micromolar ATP concentrations elicited transient responses. However, millimolar ATP concentrations triggered calcium responses that exhibited two components; the first one was metabotropic in nature and the second one was sustained and dependent on extracellular calcium. The latter component was modulated by extracellular Mg2+ and sensitive to P2X7 antagonists. In contrast to that found in rat astrocytes, prolonged stimulation of P2X7 receptor lead to cell death, which was independent of calcium entry. This was corroborated by the use of cerebellar astrocytes derived from KO mice. Data reported here reveal species differences in P2X7 expression level, affinity for the endogenous agonist and different behaviour upon prolonged receptor stimulation. 1) Carrasquero et al. (2009) J Neurochem 110: 879-889.

P9-12

Role of purinergic P2 receptors on neuronal survival and ERK1/2 activation after oxygen-glucose deprivation in the CA1 region of the rat hippocampus

Anna Maria Pugliese, Traini Chiara, Marangoni Martina, Cipriani Sara, Pedata Felicita

Dept. Pharmacology, University of Florence, Italy

Background: The role of purinergic P2 receptors during ischemia, induced by oxygen and glucose deprivation (OGD) in the CA1 region of rat hippocampal slices was investigated. Methods: We conducted extracellular recordings of CA1 field excitatory post-synaptic potentials (fEPSPs) followed by histochemical analysis (propidium iodide, PI) coupled to Western blot. Results: Seven-min OGD elicited an irreversible loss of fEPSP and the appearance of anoxic depolarization (AD), an unambiguous sign of neuronal damage. Seven-min OGD was applied in the presence of pyridoxalphosphate-6-azophenyl-2′,4′-disulfonate (PPADS, P2 unselective, 30 µM, n = 19), 2′-deoxy-N6-methyladenosine 3′-5-bisphosphate (MRS2179, selective for P2Y1 receptor, 10 µM, n = 20) and brilliant blue G (BBG, selective for P2X7 receptor, 1 µM, n = 16). All these antagonists prevented the irreversible failure of neurotransmission and AD appearance induced by 7-min OGD. As assessed by PI staining, the extent of CA1 cell injury induced by 7-min OGD was significantly attenuated by PPADS (30 µM, n = 8), MRS2179 (10 µM, n = 7) and BBG (1 µM, n = 5). Western blot analysis indicated that 7-min OGD significantly increased ERK1/2 activation in comparison to control and all P2 antagonists tested significantly reduced this activation. The ERK1/2 inhibitor 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126, 10 µM, n = 13) induced a significant fEPSP recovery and prevented AD appearance after 7-min OGD. Conclusion: Our data show that P2Y1 and P2X7 receptors play a detrimental role in the CA1 hippocampal region during OGD by mediating the activation of ERK1/2.

P9-13

Molecular mechanisms of purine-mediated neuroprotection. Role of protein kinase C-related kinase (PRK1) in the formation and stability of neurites during hypoxia

Bettina Thauerer, Stephanie zur Nedden, Gabriele Maria Baier

Biocenter/Neurobiochemistry, Medical University of Innsbruck, Austria

Background: Multiple signaling pathways regulate the critical balance between cell death and survival in ischemia–reperfusion. Hypoxic cells release soluble nucleotide and nucleoside pools. We and others have previously observed the protective capacity of purine nucleosides (1,2) under hypoxia, whereby guanosine gained our special attention for its neurite stimulating capacity (3–6). Likewise earlier data (7) indicated a special role of the protein kinase C-related kinase 1 (PRK1/PKN alpha), a lipid-activated serine/threonine protein kinase and a member of the protein kinase C (PKC) superfamily in cellular stress response, which stimulated our further interest in the study of a potential role for PRK1 and guanosine in the endogenous mechanisms involved in the protection of neuronal cells that are subjected to hypoxic insult. Methods: To study the role of PRK1 in cellular stress response and neurotrophin-mediated signaling, PC12 cells were transfected with small interfering RNA (siRNA) directed against PRK1. Results: Loss of functional PRK1 initiated a significant loss of viability and inhibited neurite formation. SiRNA-mediated knockdown of PRK1 also completely stalled guanosine-mediated neuroprotective effects. Additionally, the F-actin-associated cytoskeleton and the expression of the plasticity protein GAP-43 were disturbed upon PRK1 knockdown. Conclusion: Based on these data, a putative role of PRK1 as a key-signaling element for the successive NGF- and purine nucleoside-mediated protection of hypoxic neuronal cells was hypothesized (8). 1) Fredholm, B. B., AP, I. J., Jacobson, K. A., Klotz, K. N., and Linden, J. (2001) Pharmacol Rev 53(4), 527–552. 2) Sitkovsky, M. V., Kjaergaard, J., Lukashev, D., and Ohta, A. (2008) Clin Cancer Res 14(19), 5947–5952. 3) Bocklinger, K., Tomaselli, B., Heftberger, V., Podhraski, V., Bandtlow, C., and Baier-Bitterlich, G. (2004) Eur J Cell Biol 83(2), 51–54. 4) Tomaselli, B., Nedden, S. Z., Podhraski, V., and Baier-Bitterlich, G. (2008) Mol Cell Neurosci 38(4), 559–568. 5) Tomaselli, B., Podhraski, V., Heftberger, V., Bock, G., and Baier-Bitterlich, G. (2005) Neurochem Int 46(7), 513–521. 6) Zur Nedden, S., Tomaselli, B., and Baier-Bitterlich, G. (2008) J Neurochem. 7) Tomaselli, B., Podhraski, V., Böck, G., and Baier-Bitterlich, G. (2005) American Journal of Biochemistry and Biotechnology 2(3), 161–167. 8) Thauerer, B., Zur Nedden, S., and Baier-Bitterlich, G. J Neurochem.

P9-14

Modulation of the rat neuromuscular transmission by adenosine upon ageing

Paula Pousinha, Alexandra M. Correira, Ana M. Sebastião, Joaquim A. Ribeiro

Institute of Pharmacol. and Neurosc, Neurosc. Unit, Faculty of Medicine and Institute of Molecular Medicine, University of Lisbon, Portugal

In infant rats, adenosine A2A receptors (A2ARs) predominate over A1 receptors (A1Rs) to module neuromuscular transmission (NMT) (Pousinha et al., 2010, JPET 332:153–163). We now investigated whether this also occurs throughout ageing. Evoked (EPPs) and miniature end plate potentials (MEPPs) were recorded from single fibres of the phrenic-diaphragm from (weeks old) infant (3–4), young adult (12–16), older adult (36–38) and aged (80–90) rats. The phrenic nerve was stimulated every 2 s. Muscle twitches were prevented with high Mg2+ concentration. The A1R agonist, CPA (100 nM) decreased EPPs amplitude in all age groups, indicating that the inhibitory role of adenosine was maintained upon ageing. On the contrary, the excitatory effect of the A2AR agonist, CGS21680 (10 nM), decreased throughout age and was not observed in aged rats. The A1R/A2AR mixed agonist, CADO (30 nM) as well as the adenosine kinase inhibitor (ITU, 10 µM, to promote adenosine release) increased EPP amplitude in infant, young adult and older adult rats, an effect prevented by the A2AR antagonist. There was a concomitant increase in MEPPs frequency and quantal content of EPPs, without change in MEPPs amplitude, consistent with pre-synaptic modulation. The A2AR antagonist, ZM241385 (50 nM), converted the excitatory effects of these drugs into A1R-mediated inhibitory ones, confirming their ability to activate A1Rs and A2ARs, with predominance of A2ARs. In aged rats, CADO or ITU caused only a slight decrease in EPPs amplitude, which was not modified by the A2AR antagonist. The results suggest that the net modulation of NMT by A1Rs and A2Rs changes with age.

P9-15

Tripartite myenteric synapse: crosstalk between excitatory A2A, NK1 and M3 receptors

Isabel Silva1, Margarida Duarte-Araújo2, Dominique Fernandes2, Fátima Ferreirinha2, Cátia Vieira2, Paulo Correia-de-Sá2

1Laboratório de Farmacologia e Neurobiologia, Instituto de Ciências Biomédicas Abel Salazar - Universidade do Porto, Portugal,2Laboratório de Farmacologia e Neurobiologia, Instituto de Ciências Biomédicas Abel Salazar, Portugal

Interstitial cells of Cajal (ICC) contribute to normal motility in the gastrointestinal tract, an action that depends on their ability to mediate mechanosensitive stimuli, to generate electric slow waves, to amplify nitrergic and cholinergic inputs from enteric nerves to smooth muscle, and to set the membrane potential gradients across muscle wall through gap junctions. ICC loss is associated with a number of gastrointestinal motility disorders. Therefore, there is a considerable interest in understanding the factors that regulate their interaction with neighboring nerve terminals and smooth muscle fibers—tripartite myenteric synapse. In this study, we investigated the crosstalk between adenosine A2A, muscarinic M3 and tachykinergic NK1 facilitatory receptors in the rat longitudinal muscle—myenteric plexus. Agonists of muscarinic M3 (oxotremorine, 300 µM, 34 ± 4%, n = 5) and of adenosine A2A (CGS21680C, 3 nM, 53 ± 10%, n = 5) receptors increased [3H]-ACh and adenosine outflow from stimulated nerve terminals in control (C) rats, but they were devoid of effect when capsaicin-sensitive tachykinergic nerve fibers were destroyed (CAP rats). Activation of NK1 receptors with s,m-SP (300 nM) restored CGS21680C facilitation (46 ± 17%, n = 6) in CAP rats, but the effect of oxotremorine was unchanged. Synergism between A2A and NK1 receptors was confirmed using selective antagonists of NK1 (L732138, 20 nM) and A2A (ZM241385, 50 nM) receptors. Muscarinic M3 receptors co-localize with NK1 receptors on intramuscular ICC (IM-ICC), while adenosine A2A receptors are mainly expressed on cholinergic nerve terminals. CAP rats exhibit increased translocation of NK1 receptors to the cell membrane and decreased M3 receptor immunoreactivity at IM-ICC, along with a reduction in the number substance P positive nerve fibers. Data demonstrate that NK1 receptors interact synergistically with A2A, but not with M3 receptors, to amplify ACh release from myenteric nerve terminals. Work supported by FCT (FEDER funding, PTDC/CVT/74462/2006 and UMIB-215/94). D. Fernandes received a Young Investigator Fellowship from FCT (BII/UMIB-ICBAS/2008-3).

P9-16

Caffeine prevents disruption of memory consolidation in the inhibitory avoidance and novel object recognition tasks by scopolamine in adult mice

Paulo Henrique Saldanha Botton, Marcelo Silveira da Costa, Ana Paula Ardais, Sabrina Mioranzza, Diogo Onofre de Souza, Lisiane de Oliveira Porciúncula

Department Of Biochemistry, UFRGS, Brasil

Caffeine is a psychostimulant with positive effects on cognition. The cholinergic system seems to participate of the effects of caffeine on wakefulness but there are few studies about its participation in the cognitive enhancer properties of caffeine. In this study, the effects of caffeine were investigated on scopolamine-induced impairment of memory in adult mice in the inhibitory avoidance (IA) and novel object recognition (NOR) tasks. Caffeine (10 mg/kg, i.p) was administered during 4 consecutive days and the treatment was interrupted twenty-four hours before scopolamine administration (2 mg/kg, i.p). Scopolamine was administered immediately after training sessions. Short- and long-term memory was evaluated in both tasks. Pre treatment with caffeine prevented the disruption of short- and long-term memory recognition by scopolamine. Besides, caffeine was also able to prevent scopolamine-induced amnesia in the inhibitory avoidance task. Thus, acute treatment with caffeine followed by its withdrawal was effective against cholinergic-induced disruption of memory consolidation assessed in an aversive and non-aversive task. Our results suggest that the cholinergic system may be involved in the positive effects of caffeine on cognitive functions. Supported by CAPES, INCT-EN/CNPq, FAPERGS.

P9-17

Increased adenosine deaminase activity favours neuromuscular impairment in toxin-induced myasthenia gravis

Cristina Costa, Tiago Morais, Diogo Trigo, José Noronha-Matos, Diogo Paramos, Maria Alexandrina Timoteo, Maria Teresa Magalhães-Cardoso, Laura Oliveira, Paulo Correia-de-Sá

Lab. Farmacologia e Neurobiologia, UMIB, Instituto de Ciências Biomédicas Abel Salazar - Universidade do Porto (ICBAS-UP), Portugal

Adenosine plays a key role in adjusting the modulatory pattern of neuromuscular transmission to the nerve stimulation conditions. While adenosine acts as an inhibitory signal (via A1 receptors) under resting conditions, amplification of neuromuscular transmission due to A2A receptors activation becomes evident at high levels of synaptic adenosine. The concentration of adenosine at the neuromuscular junction is a function of adenosine formation from the catabolism of released adenine nucleotides (e.g. ATP), adenosine equilibrative transport and extracellular deamination. Recently, we showed a decline of adenosine tonus at motor endplates from rats with α-bungarotoxin-induced myasthenia gravis (TIMG), which could be partially due to reduced adenosine outflow from paralyzed skeletal muscle fibers. In this study, we investigated the contribution of adenosine inactivation mechanisms to neuromuscular impairment in myasthenic rats.

While adenosine formation from the catabolism of adenine nucleotides was unchanged, the kinetics of extracellular deamination of adenosine into inosine (measured by HPLC) was significantly enhanced in TIMG rats, i.e. the half-degradation time of adenosine (30 μM) was 32 ± 11 min (n = 5) and 59 ± 13 min (n = 3) in TIMG and control rats, respectively. Experiments measuring nerve-evoked [3H]-ACh release and exocytosis by video-microscopy in the presence of the selective A2A receptor antagonist, ZM 241385 (50 nM), confirmed that tonic facilitation of transmitter release through A2A receptors was significantly (P < 0.05) decreased in TIMG rats as compared to controls. Impairment of the A2A-receptor-mediated tonus was particularly evident during high-frequency (50 Hz) nerve stimulation.

Data indicate that the fast kinetics of adenosine deamination may account for failure of A2A receptors activation, which might contribute to preserve neuromuscular transmission in myasthenic animals.

P9-18

Activation of adenosine A1 receptors inhibits extrasynaptic GABAergic currents in hippocampus

Diogo M. Rombo, Raquel A. Dias, Joaquim A. Ribeiro, Ana M. Sebastião

Faculdade de Medicina de Lisboa, Instituto de Medicina Molecular, Instituto de Farmacologia e Neurociências, Portugal

Adenosine, through inhibitory A1 receptors modulates neuronal excitability in the hippocampus by inhibiting the glutamatergic transmission at the pre- and postsynaptic level. The role of adenosine on GABAergic transmission in the hippocampus is much less known. We now investigated whether adenosine, through A1 receptors, modulates postsynaptic currents mediated by GABAA receptors. Muscimol (30 µM, GABAA receptor agonist)-evoked postsynaptic currents (PSCs), afferent-evoked inhibitory postsynaptic currents (IPSCs) and miniature inhibitory postsynaptic currents (mIPSCs), were recorded (whole-cell configuration, Vh = −80mV) from CA1 pyramidal cells and interneurons of rat (Wistar, 3–5w) hippocampal slices.

At CA1 pyramidal cells, the activation of A1 receptors with CPA (10–30 nM) decreased (to 54 ± 3.9%,p < 0.0001,n = 12) the peak amplitude of PSCs and this inhibition was reversed by DPCPX (50 nM), an adenosine A1 receptor antagonist, to 83 ± 2.5% of baseline (n = 4,p < 0.05). DPCPX per se unmasked the inhibition of PSCs by endogenous extracellular adenosine since it increased the inhibitory currents by 13 ± 1.0% (n = 5,p < 0.001). Neither the amplitude nor the frequency of mIPSCs (n = 12), or the amplitude of IPSCs (n = 10), were affected by CPA indicating an action of A1 receptors on extrasynaptic GABAA receptors.

At stratum radiatum interneurons, activation of A1 receptors with CPA attenuated GABAA PSCs in 4 out of 9 cells tested to 57 ± 2.3% (p < 0.001). The PSCs in the other 5 cells tested were not changed by CPA superfusion, suggesting the existence of two populations of interneurons differently sensitive to adenosine.

Together, these results indicate that extrasynaptic GABAA receptor-mediated currents are modulated by adenosine A1 receptors in pyramidal hippocampal neurons and in a subset of interneurons. Support:FCT.

P9-19

Modification of the levels of adenosine receptors in the rat hippocampus induced by maternal separation

Vânia L. Batalha1, Ana M. Sebastião1, Joaquim A. Ribeiro1, Luisa V. Lopes2

1Institute of Pharmacology and Neurosciences, Faculty of Medicine and Neurosciences Unit, Instituto de Medicina Molecular and Faculty of Medicine Lisbon, Portugal,2Neurosciences Unit and Institute of Pharmacology and Neurosciences, Instituto de Medicina Molecular and Faculty of Medicine Lisbon, Portugal

A role for adenosine A2A receptors in stress has been suggested on the basis of increased aggressiveness of A2A-knockout mice. We now investigated if the induction of chronic stress, through maternal separation, could have an impact on adenosine receptors and related proteins in the brain.

Male Wistar rats were assigned to control (CTR) or maternal separated (MS) group. The MS were separated from their mothers for 3h/day (2–14 postnatal days). At 7–8 weeks of age the hippocampi, cortices and striata were dissected. Saturation binding curves with the selective antagonist for A1 receptors, [3H]DPCPX (0–7 nM) were performed. The levels of A2A, glucocorticoid (GR), mineralocorticoid (MR), CRH (CRH-R1) and TrkB receptors, GABAA-b3 and GluR1 subunits and BDNF were evaluated by immunoblot analysis.

In the hippocampus the A2A receptor levels were increased by 56.2 ± 9.4% (n = 3; P < 0.05) and A1 slightly decreased (~11%; n = 4; P < 0.05) in MS animals, but no differences were found in the cortex or striatum. The MR/GR ratio was more affected by MS in the hippocampus than in cortex or striatum. A decrease of 26.1 ± 4.5% in the levels of GR was observed in the hippocampus, compared to a decrease of 16.3 ± 3.4% in cortex and of 9.6 ± 2.6% in striatum (n = 3–4, P < 0.05). MS also altered hippocampal markers of synaptic plasticity. Thus, GluR1 and GABAA-b3 subunits plus TrkB receptors were decreased (n = 3–4, P < 0.05) while BDNF and CRH-R1 were increased (n = 3–4, P < 0.05).

Overall these data show that stress induces long-term changes in the hippocampus, impacting on the levels of adenosine receptors and related synaptic plasticity markers, in an “early-ageing”-like phenomenon. These changes may underlie the synaptic plasticity modifications observed upon stress in this brain area.

Supported by FCT grant PTDC/SAU-NEU/099853/2008

P9-20

Adenosine originated from adenine nucleotides rehabilitates neuromuscular transmission in toxin-induced myasthenia gravis

Diogo Paramos, Cristina Costa, José Bernardo Noronha-Matos, Maria Alexandrina Timóteo, Maria Teresa Magalhães-Cardoso, Laura Oliveira, Paulo Correia-de-Sá

Laboratório de Farmacologia e Neurobiologia, Instituto de Ciências Biomédicas Abel Salazar, Portugal

We provided evidence that, under physiological conditions, adenosine originating from the catabolism of adenine nucleotides activates preferentially facilitatory A2A receptors. Tonic activation of presynaptic A2A receptors allows a near complete recovery of neurotransmitter release from the decline occurring after a previous tetanic train (tetanic depression) by increasing Ca2+ influx via Cav1 (L-type) channels. In view of this, manipulation of A2A receptors activation might be of clinical interest to preserve neuromuscular transmission in compromised motor endplates, such as that observed in myasthenia gravis. Thus, we decided to evaluate the effect of exogenously applied adenosine, as well as of the nucleoside precursor, AMP, on [3H]-ACh release from stimulated (5 Hz, 750 pulses) motor nerve terminals of rats with α-bungarotoxin-induced myasthenia gravis (TIMG) as compared to control littermates. Real-time measurements of exocytosis were also performed by video-microscopy using the FM4-64 fluorescence dye. Adenosine (0.010–1 mM) concentration-dependently decreased [3H]-ACh release in TIMG rats, but its inhibitory (A1) potency was shifted to the right as compared to control animals. Adenosine failed to facilitate (via A2A receptors) nerve-evoked transmitter release in myasthenic animals. This effect contrasts with that caused by the adenosine precursor, AMP (100 µM), which increased [3H]-ACh release by 35 ± 14% (n = 5) in TIMG rats. A similar activity profile was observed measuring real-time exocytosis by video-microscopy. Data suggests that adenosine originated from the catabolism of extracellular adenine nucleotides rehabilitates neuromuscular transmission in myasthenic individuals through the activation of facilitatory A2A-receptors. This work was supported by FCT (FEDER funding, PTDC/SAU-FCF/108462/2008 and UMIB-215/94).

P9-21

The P2Y-like receptor GPR17 as a sensor of damage and a new potential target in spinal cord injury

Stefania Maria Ceruti1, Giovanni Villa1, Tiziana Genovese2, Emanuela Mazzon2, Renato Longhi3, Patrizia Rosa4, Placido Bramanti5, Salvatore Cuzzocrea2, Maria P. Abbracchio1

1Department of Pharmacological Sciences, Universita' Degli Studi Di Milano, Italy,2Department Of Clinical And Experimental Medicine And Pharmacology, Universita' Di Messina, Italy,3Institute of Biocatalysis And Molecular Recognition, CNR, Milano, Italy,4Institute of Neuroscience, CNR, Milano, Italy,5IRCCS Centro Neurolesi "Bonino Pulejo", Messina, Italy

Traumatic spinal cord injury (SCI) is a devastating condition with an annual incidence of 15–40 cases per million and enormous social and health costs. Due to the compelling need for the discovery of new pharmacological targets whose modulation could effectively improve outcome after SCI, we focused on a new P2Y-like receptor, GPR17. It is activated by uracil nucleotides (e.g, UDP-glucose) and cysteinyl-leukotrienes (cysLTs), whose concentrations are markedly increased following injury, suggesting their possible role as “danger signals” to alert responses to tissue damage and start repair. In intact spinal cord, neurons, oligodendrocytes and ependymal cells lining the central canal express GPR17. Following SCI, a marked death of GPR17+ neurons and oligodendrocytes inside the lesion was observed, followed by the appearance of proliferating GPR17+ microglia/macrophages migrating to and infiltrating into the lesion. Moreover, 72 hours after SCI, GPR17+ ependymal cells started to proliferate and to express GFAP (taken here as a marker of pluripotency), suggesting their reaction to injury and their “de-differentiation” to pluripotent progenitors. The in vivo knock-down of GPR17 by anti-sense oligonucleotides markedly reduced SCI-induced cell death and related histological and motor deficits. Taken together, our findings suggest that GPR17 may act as a “sensor” of damage, that is activated by nucleotides and cysLTs released in the lesioned area, and plays a crucial role in the early phases of tissue damage development. Moreover, its presence on pre-oligodendrocytes and precursor-like cells suggests GPR17 as a novel target for therapeutic manipulation to foster remyelination and functional repair in SCI.

P9-22

Treadmill running causes anxiety-related behavior and changes in the adenosine receptors immunocontent in the hippocampus of adult rats

Marcelo Silveira da Costa, Gabriela Tondolo Fioreze, Paulo Henrique Saldanha Botton, Sabrina Mioranzza, Diogo Onofre Souza, Luís Valmor Portela, Lisiane de Oliveira Porciúncula

Department of Biochemistry, UFRGS, Brazil

The practice of physical exercise improves brain functions and causes modifications in many neurotransmitters. As a neuromodulator, adenosine controls the release of many neurotransmitters. However, effects of exercise training on adenosinergic system are still unknown. In this study, behavioral analysis and immunocontent of adenosine receptors (A1R and A2AR) were investigated in rats submitted to exercise training (20 minutes/day; 1, 3 or 7 times/week, during 2 months) in a motorized treadmill. The frequency of exercise did not alter locomotor activity, memory and recognition memory. However, when compared to sedentary group all exercise groups showed a decrease on the time spent in open arms in the elevated plus maze, suggesting an anxiety-like behavior. No differences were found for hippocampal adenosine A1 receptors immunocontent. There was a decrease (30%) on adenosine A2A receptors immunocontent in rats trained 1 time/week and an increase (25%) in rats trained 7 times/week. Our results indicate that hippocampal adenosine A2A receptors change differently its immunocontent according to the frequency of treadmill running. Besides, treadmill running may trigger anxiety-like behavior without affecting learning and memory. Supported by CAPES, CNPq/INCT, FAPERGS.

P9-23

The P2 receptor antagonist ppads differentialy affects kainate-and NMDA-induced neurotoxicity in cultured hippocampal neurons

Ana Patrícia Simões, Daniela Pochmann, Rodrigo Cunha

Center for Neuroscience of Coimbra, University of Coimbra, Portugal

We have previously observed that the density of ATP receptors (P2R) changes upon noxious brain insults and that the blockade of P2R prevents glutamate-induced neurotoxicity in cultured hippocampal neurons. We now evaluated if a P2R antagonist (PPADS) was equi-effective to prevent neurotoxicity triggered by different agonists of different ionotropic glutamate receptors in cultured hippocampal neurons. Cultured hippocampal neurons (7 days in vitro) were obtained from Wistar rats embryos and were exposed to kainate (100 microM), NMDA (100 microM) or quinolinic acid (3 mM) in the absence or presence of PPDAS (20 microM, added 30 min before the glutamate receptor agonists), and neuronal damage was estimated by modifications of the reduction of MTT. Exposure of cultured neurons to kainate (6h), NMDA (12h) or quinolinic acid (24h) decreased neuronal viability by circa 40%, 50% and 30%, respectively. PPADS attenuated neurotoxicity caused by NMDA or by quinolinic acid, whereas it failed to modify kainate-induced neurotoxicity. These results suggest that P2R blockade only affords effective neuroprotection against particular noxious stimuli in cultured hippocampal neurons and suggest that P2R might be particularly effective for less acute insults that mainly involve the activation of NMDA receptors for expression of neurotoxicity. (Supported by FCT).

P9-24

Blockade of adenosine A2A receptor prevents beta amyloid-induced inhibition of glutamate uptake in mice brain cortical astrocytes

Marco Matos1, Alexandre S. Rodrigues2, Rodrigo A. Cunha1, Paula M. Agostinho1

1Center for Neuroscience of Coimbra, Universidade de Coimbra, Portugal,2Laboratory of Cellular Neurobiology, Federal Fluminense University, Brazil

Alzheimer’s disease (AD) is characterized by a progressive cognitive impairment resulting from an initial synaptic dysfunction in cortical regions, associated with the accumulation of beta-amyloid peptides (Aβ, mainly Aβ1-42). In accordance with the hypothesis that glutamatergic dysfunction may be associated with the early phases of AD, we previously showed that Aβ decreases astroglial glutamate uptake (Matos et al., 2008, Neuroscience 156: 898). Since we also found that antagonists of adenosine A2A receptors (A2AR) prevent memory impairment caused by Aβ (Canas et al., 2009, J Neurosci 29: 14741), we now tested if A2AR affected Aβ-induced decrease of astrocytic glutamate uptake. CGS21680 (A2AR agonist, 5–100 nM) added acutely or sub-chronically (for 24hrs) decreased D-3H-aspartate uptake in primary cultures of CB57-BL6 mice astrocytes by 57.1 ± 3.0% (n = 3) or 29.3 ± 3.8% (n = 5), respectively. SCH58261 (A2AR antagonist, 50 nM, added acutely or during 24hrs) was devoid of effects alone, but prevented D-aspartate uptake (n = 3). Confirming previous results, we observed that Aβ1-42 (1–5 µM, 24 hrs) inhibited D-aspartate uptake by 35.6 ± 5.8% alone (n = 5). Notably, this inhibitory effect of Aβ1-42 was inhibited by SCH58261 (14.4 ± 4.4% inhibition, n = 3). These results show that: 1) A2AR inhibit the activity of glutamate transporters in astrocytes; 2) the tonic activation of A2AR is involved in the inhibitory effects of Aβ on glutamate uptake by astrocytes. (Supported by FCT).

P9-25

Blockade of adenosine A2A receptors prevents the exacerbation of glutamate excitotoxicity by interleukin-1 beta in cultured hippocampal neurons

Ana Patrícia Figueiredo Rocha Simões, Carla Gomes Silva, João André Duarte, Paula Margarida Canas, Rodrigo Cunha

Center for Neuroscience of Coimbra, University of Coimbra, Portugal

Interleukin-1beta (IL1B) is a major pro-inflammatory cytokine known to aggravate excitotoxicity. Blockade of A2A receptors (A2AR) prevented IL1B-induced activation of JNK and p38 in hippocampal neurons. Thus, we now tested if the blockade of A2AR could also prevent IL1B-induced exacerbation of excitotoxicity. Hippocampal neurons, 7–8 DIV, were exposed to IL1B (100 ng/ml) 5 minutes before adding glutamate (100 µM, 25 minutes). Cell viability was evaluated 24 hours later by syto-13/propidium iodide staining and LDH assay. Intracellular calcium deregulation was also studied using single cell calcium imaging using Fura-2. The blockade of A2AR was achieved by adding antagonist SCH58261 (50 nM) 15 minutes before the stimuli and prevailing throughout the experiment. Syto-13/propidium iodide staining showed a reduction (17 ± 6%, n = 6) of viable cells by glutamate, which was aggravated by IL1B (51 ± 13%, n = 7). Blockade of A2AR attenuates this effect (11 ± 6%, n = 4) but exacerbates glutamate-induced neurotoxicity (31 ± 18%, n = 5). Also, glutamate with IL1B induced LDH leakage (155 ± 14% of control, n = 6) which was prevented by SCH58261 (121 ± 16% of control, n = 6). Single cell calcium imaging of cultured neurons showed that glutamate with IL1B induced a larger and sustained increase of intracellular calcium than glutamate alone, which was prevented by SCH58261; again, the A2AR antagonist did not prevent and even exacerbated glutamate-induced effects. Our results suggest that the neuroprotection afforded by the blockade of A2AR may involve the particular control of IL1B direct effects on neurons. (Supported by FCT)

P9-26

Blockade of synaptic adenosine A2A receptor prevents convulsion-induced damage

Elisabete Augusto1, Paula M. Canas1, Lisiane O. Porciúncula2, Tiago M. Alfaro1, Geanne M. Andrade3, Attila Köfalvi1, Paula M. Agostinho1, Rodrigo A. Cunha1

1Center for Neuroscience of Coimbra, University of Coimbra, Portugal,2Dept. Biochemisty, Univ. Federal Rio Grande do Sul, Brazil,3Dept. Physiology and Pharmacology, Univ. Federal Ceará, Brazil

Adenosine A2AR blockade control synaptic plasticity and affords robust neuroprotection. Thus, we now tested if and how adenosine A2A receptors (A2AR) might control seizures and associated neuronal damage. Both chronic caffeine consumption (1 g/L through the drinking water) or the A2AR antagonist SCH58261 (0.05 mg/kg, ip, 30min before or 2hrs after insult) afforded a robust and sustained (1–7 days) protection against kainate (10 mg/kg, ip) neurotoxicity (cresyl violet or FluoroJade), astrogliosis (GFAP) and microgliosis (CD11b or tomato lectin staining) in the rat hippocampus, without affecting the severity of tonic/clonic convulsions. Likewise, although the severity of convulsions was not modified, there was no kainate (35 mg/kg, sc)-induced toxicity in A2AR knockout mice. Hippocampal A2A receptor expression was up-regulated after kainate administration (after 2 hours) and A2AR density was increased mainly in nerve terminals. Notably, real-time PCR showed the presence of A2AR mRNA in purified hippocampal nerve terminals. Accordingly, there is a gain of function of presynaptic A2ARs, which tonically control the evoked release of glutamate (but not GABA) after direct kainate exposure to hippocampal slices. These results question the role of adenosine as an anti-epileptic agent since A2AR-mediated control of neurodegeneration is a key event in the evolving pathogenesis of epilepsy and suggest that the blockade of synaptically-regulated A2AR may provide a novel strategy to control neuronal damage in temporal lobe epileptic conditions. (Supported by FCT)

P9-27

Different modulation of adenosine receptors in human brain from dementia with Lewy body

(Selected for Oral Poster Presentation)

Jose Luis Albasanz Herrero1, Sandra Buira2, David León1, Carlos Alberto Castillo1, Isidre Ferrer2, Marta Barrachina2, Mairena Martín1

1Facultad de Químicas. CRIB., Universidad De Castilla-la Mancha, Spain,2Institut de Neuropatologia, Servei d’Anatomia Patològica, IDIBELL-Hospital Universitari de Bellvitge, Spain

Dementia with Lewy Bodies (DLB) is a neurodegenerative disease characterized by the presence of Lewy bodies, also present in Parkinson disease (PD). These neural inclusions are composed of abnormally phosphorylated neurofilament proteins aggregated with ubiquitin and alpha-synuclein. Pure form of DLB (DLBp) share clinical and pathological characteristics with PD and minimal AD pathology, while common form of DLB (DLBc) is characterized by abundant senile plaques and neurofibrillary tangles. Adenosine receptor /adenylyl cyclase (AC) stimulatory and inhibitory pathways were studied by radioligand binding, real-time PCR, Western blotting and AC activity assays in post mortem human frontal cortex from DLB patients in both DLBp and DLBc as compared with age matched non-demented controls. Total adenosine A1 receptors number were significantly increased in DLBp (177% of control cases) and no significantly altered in DLBc neither in early nor advanced AD changes. However, adenosine A2A receptors were increased in both pure and common form of DLB. Gene expression of these receptors was also differently modulated, while A2A mRNA level were significantly increased in DLBp, in agreement with protein level detected, no changes were observed in A1 mRNA levels. There were no significant variations on inhibitory or stimulatory effect on adenylyl cyclase activity exhibited by selective A1 or A2A receptor agonists in pure form of DLB. However, AC responses mediated by A1 and A2A were both potentiated in common form of DLB. These results show that stimulatory A2A and inhibitory A1 adenosine receptors are differentially impaired in DLB and suggest a potential diagnostic role for these receptors.

P9-28

Guanosine is neuroprotective against oxygen/glucose deprivation in hippocampal slices via potassium channels (BK), PI3K/Akt pathway activation and glutamate uptake

Tharine Aparecida Dal-Cim, Wagner Martins, Carla Inês Tasca

Universidade Federal de Santa Catarina, Brasil

Guanine derivates (GD) have been implicated in many relevant extracellular roles, such as modulation of glutamate transmission, protecting neurons against excitotoxic damage. uring oxygen/glucose deprivation (OGD). The aim of this study has been to evaluate the involvement of potassium channels and phosphatidilinositol-3 kinase (PI3K) pathway, in the mechanisms related to the neuroprotective role of guanosine in rats hippocampal slices subjected to oxygen/glucose deprivation (OGD). The addition of guanosine (100 μM) to hippocampal slices, subjected to 15 minutes of OGD and followed by 2 hours of re-oxygenation, has been neuroprotective. The presence of K+ channel blockers, glibenclamide (20 μM) or apamin (300 nM) has revealed neuroprotective effect of guanosine was not dependent on ATP-sensitive K+ channels and small conductance Ca2+-activated K+ channels. The presence of charybdotoxin (100 nM), a large conductance Ca2+-activated K+ channels (BK) blocker, has inhibited the neuroprotective effect of guanosine. Hippocampal slices subjected to OGD and re-oxygenation have showed a significant reduction of glutamate uptake. Addition of guanosine in the re-oxygenation period has blocked the reduction of glutamate uptake. This guanosine effect has been inhibited when hippocampal slices were pre-incubated with charybdotoxin or wortmanin (a PI3K inhibitor, 1 μM) in the re-oxygenation period. Guanosine promoted an increase on Akt protein phosphorylation. However, the presence of charybdotoxin has changed such effect. In conclusion, the neuroprotective effect of guanosine involves augmentation of glutamate uptake, which is modulated by BK channels and the activation of PI3K pathway. Moreover, neuroprotection caused by guanosine depends on the expression increase of phospho-Akt protein.

P9-29

Blockade of P2 receptors protects against neuronal damage and memory deficts induced by permanent focal brain ischemia

Carolina Melo de Souza1, Marta R. S. do Carmo2, Ana P. F. Menezes2, Analu A. Fonteles2, Rodrigo A. Cunha1, Geanne M. de Andrade2

1Centre for Neuroscience of Coimbra, University of Coimbra, Portugal,2Laboratory of Neuroscience and Behavior, Federal University of Ceará, Brazil

ATP has been proposed to act as a danger signal upon noxious insults since it controls neuronal death through P2 receptors (P2R). We now tested the impact of PPADS (P2R antagonist) on neuronal injury caused by permanent middle cerebral artery occlusion (MCAO). Male Swiss mice received PPADS (1.0 nmols/1 μL, icv) 15 min before MCAO (induced by electrocoagulation) and mice were evaluated behaviourly (locomotion, sensorimotor function, working and aversive memory), morphologically (2,3,5-triphenyltetrazolium chloride, TTC, staining) and biochemically (myeloperoxidase activity) after 24 hours. PPADS prevented MCAO-induced infarcted area (control: 0.89 ± 0.18%; MCAO: 9.06 ± 1.20%: MCAO+PPADS: 1.86 ± 0.18%) and MCAO-induced myeloperoxidase (MPO) activity (U/mg tissue) in striatum (control: 3.60 ± 0.63; MCAO: 16.24 ± 4.86; MCAO+PPADS: 6.13 ± 0.65) and temporal cortex (control: 6.16 ± 1.23; MCAO: 22.33 ± 4.98; MCAO+PPADS: 6.28 ± 0.38). Moreover, PPADS prevented MCAO-induced decreased exploratory activity (number of rearings, MCAO: 9.5 ± 1.8; MCAO+PPADS: 20.6 ± 3.7) and MCAO-induced memory deficits (percentage spontaneous alternations in the Y-maze: control: 73.8 ± 1.9%; MCAO: 56.7 ± 2.9%; MCAO+PPADS: 72.6 ± 4.0%), with similar conclusion being obtained when evaluating early aversive memory (passive avoidance test). Our data provide evidence for a key role of P2 receptors in the control of cerebral ischemia pathophysiology, albeit the P2R involved and the underlying mechanisms remain to be unraveled. (Supported by CNPq, FUNCAP, FCT).

P9-30

The parkinson’s disease-associated receptor GPR37 oligomerizes with the adenosine A2A receptor

Jorge Gandía1, Víctor Fernández-Dueñas1, Victoria Wong2, Igor Stagljar2, Francisco Ciruela1

1Departament de Patología i Terapèutica Experimental, Facultat de Medicina-Bellvitge, IDIBELL-Universitat de Barcelona, Spain,2Faculty of Medicine, University of Toronto, Canada

The substrate of parkin, the orphan G protein-coupled receptor 37 (GPR37), form insoluble aggregates that accumulate in brain tissue of Parkinson's disease patients (e.g. Lewy bodies and neurites). By means of membrane yeast two-hybrid (MYTH) screen, GPR37, was identified as a binding partner for the adenosine A2A receptor (A2AR). Co-expression and co-immunoprecipitation experiments showed a close and specific interaction between GPR37 and A2AR in transfected HEK-293. Furthermore, bioluminescence resonance energy transfer (BRET) experiments showed that GPR37 and A2AR are in close proximity, thus allowing us the detection the GPR37-A2AR oligomers in living cells. Interestingly, the interaction of GPR37 with A2AR modulated the cell surface expression of A2AR. Also, the functionality of the A2AR was affected by the coexpression of the GPR37. Overall, these results show that A2AR interacts with GPR37 in living cells and that the interaction is relevant for A2AR cell surface expression and function. This work was supported by grants SAF2008-01462 and Consolider-Ingenio CSD2008-00005 from Ministerio de Ciencia e Innovación.

P9-31

Nimodipine inhibits Interleukin 1-beta production induced by beta-amyloid or ATP on microglia

Juana Maria Sanz Molina, Paola Chiozzi, Renato Fellin, Giovanni Zuliani, Francesco Di Virgilio

Dep. of Clinical and Experimental Medicine, Sec. of Internal Medicine, University of Ferrara, Italy

Background: The activation of microglia and the increased production of interleukin 1-beta are considered two early phenomenon in the pathogenesis of Alzheimer’s disease. We have recently demonstrated in vitro that beta- amyloid, triggers ATP and interleukin 1-beta release by binding to the P2X7 receptor (Sanz et al., J Immunol, 2009, 182, 4378). The aim of the present study was to evaluate in vitro the effect of Nimodipine, a dihydropyridine L-type calcium channel blocker, on interleukin 1-beta production induced by ATP or beta-amyloid. Methods. Microglial cells were incubated with LPS for 5 hours in presence or absence of Nimodipine (Bayer); then different concentrations of ATP or beta-amyloid were added. After 30 minutes supernatant was used to measure interleukin 1-beta. For chronic experiments, primary mouse microglial cell were incubated for 7 days with beta-amyloid in absence or presence of Nimodipine. Intracellular interleukin-1-beta was measured in the cellular lysate. Results. Nimodipine decreased interleukin 1-beta release triggered by ATP or beta-amyloid on LPS-pretreated microglia. Chronic treatment of microglia with beta-amyloid induced an accumulation of intracellular interleukin 1-beta. These effects were inhibited in presence of Nimodipine. Conclusions. We demonstrated that Nimodipine significantly inhibited microglial activation and interleukin 1-beta production in both acute and chronic conditions. In patients with Alzheimer disease Nimodipine might contribute to decrease the inflammatory state characteristic of this pathology.

P9-32

Behavioral and biochemical characterization as antiparkinsonian drug of the new adenosine A2A antagonist 8-ethoxy-9-ethyladenine

Annalisa Pinna1, Silvia Pontis2, Nicola Simola2, Rosaria Volpini3, Nicoletta Schintu2, Gloria Cristalli3, Micaela Morelli2

1Institute of Neuroscience, National Reseach Council, Italy,2Department of Toxicology, University of Cagliari, Italy,3Department of Chemical Sciences, University of Camerino, Italy

Background: Adenosine A2A receptor antagonists have emerged as an attractive non-dopaminergic target in clinical trials aimed at evaluating improvement in motor deficits in Parkinson’s disease (PD). Moreover, preclinical studies suggest that A2A receptor antagonists may slow the course of the underlying neurodegeneration of dopaminergic neurons. Previous studies assessing the antiparkinsonian activity of a few 9-ethyladenine derivatives, demonstrated that the 8-ethoxy-9-ethyladenine (ANR 94) has high selectivity and affinity for the human adenosine A2A receptor subtype and high antiparkinsonian activity in two in vivo rodent model of PD. In line with this evidence, we have confirmed the in vivo antiparkinsonian activity of the ANR 94 by assessing its role on limb akinesia, gait impairment and sensory-motor deficits produced by 6-hydroxydopamine (6-OHDA) lesion and by verifying its neuroprotective role in MPTP mouse model. Results. In 6-OHDA-lesioned rats, acute administration of ANR 94, similarly to L-DOPA, counteracted impairments in the initiation of stepping movements, in the adjusting step and in the vibrissae-evoked forelimb placing induced by the lesion. Additionally, administration of ANR 94 to subchronic MPTP-treated mice: 1) counteracted dopamine neuron degeneration in the substantia nigra pars-compacta (SNc), 2) partially prevented the astroglial response (GFAP-positive cells) in SNc and in striatum, and 3) totally antagonized microglial activation (CD11b-positive cells) in SNc and partially in striatum. Conclusion. These results, showed that the A2A receptor antagonist ANR 94 besides being beneficial in improving akinesia, gait deficits and sensory-motor impairments in PD also relieve astrogliosis and microgliosis, suggesting potential therapeutic application for this new synthesized A2A antagonist.

P9-33

Interleukin-6-type cytokines in neuroprotection and neuromodulation: OSM, but not LIF, requires neuronal adenosine A1 receptor function

Shamsudheen Moidunny1, Erik Boddeke1, Raquel Dias2, Evelyn Wesseling1, Yuko Sekino3, Ana Sebastiao2, Knut Biber1

1Rijksuniversiteit Groningen, University Medical Center Groningen, The Netherlands,2University of Lisbon, Faculty of Medicine, Portugal,3Gunma University Graduate School of Medicine, Department of Neurobiology and Behavior, Japan

Neuroprotection induced by the family of interleukin (IL)-6-type cytokines has been widely discussed. However, the underlying mechanism(s) involved are not fully understood. We have previously shown that the neuroprotective effect of IL-6 is based upon induced expression of the adenosine A1 receptors (A1Rs). As the members of IL-6-type cytokine family share gp130 receptor subunit in their signaling, we hypothesized that they all use a similar mechanism to that of IL-6 to induce neuroprotection. In order to investigate this, we studied the effects of two closely related members of the family, oncostatin M (OSM) and leukemia inhibitory factor (LIF), on the survival of cultured cortical and hippocampal neurons against glutamate toxicity. Surprisingly, we found that only OSM, but not LIF-induced neuroprotection is based upon induced expression of A1Rs. These findings were also confirmed in A1R deficient (A1R–/–) neurons. In addition, OSM but not LIF, enhanced the expression of A1Rs in wild-type mouse cortical neurons. Furthermore, we tested the effects of pretreatment with OSM and LIF on A1R mediated inhibition of excitatory post-synaptic currents (EPSC) in hippocampal slice cultures. We observed that OSM pretreatment potentiated the A¬1R mediated EPSC inhibiton, both under normal and hypoxic conditions. Whereas, LIF did not show any effect. Our results strongly suggest that, despite known structural and functional similarities, OSM and LIF use different mechanisms to achieve neuroprotection and neuromodulation. These findings could lead to a better understanding of cytokine biology and could have consequences for the putative use of IL-6-type cytokines in treatment of neurodegenerative disorders.

P10. Cardiovascular/Renal dysfunctions

P10-1

Effect of acute and chronic hypoxia on ecto-nucleotidase activities in arterial vasa vasorum endothelial cells: implications for angiogenesis

Gennady Yegutkin1, Mikko Helenius1, Sirpa Jalkanen1, Evgenia Gerasimovskaya2, Kurt Stenmark2

1Medicity Research Laboratory, University of Turku, Finland,2Department of Pediatrics, UCD, Aurora, USA

Background: Extracellular ATP and ADP have long been known as important regulators of vascular tone, permeability and homeostasis, however contribution of purinergic signaling to endothelial cell growth and proliferation under various hypoxic and inflammatory states remains poorly understood. Methods: Using vasa vasorum endothelial cells (VVEC) isolated from pulmonary artery adventitial compartments of control and chronically hypoxic calves, these studies were aimed to characterize biochemical properties of microvascular endothelial network at sites of active angiogenesis. Nucleotide-converting activities were assayed by thin-layer chromatography using 3H-labelled ATP, ADP and AMP as appropriate substrates. Results: Specific ATPase, ADPase and ecto-5′-nucleotidase activities were decreased in VVEC from hypoxic calves by 30, 50 and 75% respectively, as compared with control cells. This diminished nucleotide-inactivating capability of chronically hypoxic VVEC is consistent with potent pro-angiogenic effects of ATP and ADP in these cells. By contrast, in vitro challenging of cultured VVEC to acute hypoxia (4–24 hours, 1% 02) was accompanied by moderate 15–20% up-regulation of ecto-nucleotidase activities. Conclusion: These results suggest that unique pattern of nucleotide-mediated angiogenic activation of VVEC from chronically hypoxic vessels is defined by down-regulation of key inactivating ectoenzymes, nucleoside triphosphate diphosphohydrolase (NTPDase) and ecto-5′-nucleotidase/CD73. Furthermore, different molecular mechanisms are presumably involved in regulation of purinergic signaling cascade during acute and chronic hypoxia.

P10-2

Involvement of adenosine A2B receptors and Epac in the early induction of nuclear factor NR4A1 in primary human coronary smooth muscle cells

Hinze Annette Viktoria, Harst Anja, Mayer Peter, von Kügelgen Ivar

Federal Institute for Drugs and Medical Devices, University Bonn, Germany

Background: Extracellular adenosine and adenine nucleotides play important roles in the regulation of the blood vessel tonus and thrombosis. Less is known about the effects of these ligands on gene expression. Thus we now searched for adenosine-induced early gene expression in cultured human coronary smooth muscle cells (HCASMCs). Methods: HCASMCs were incubated for one hour with agonist or antagonists to be studied. Changes in gene expression were detected by DNA array hybridization and confirmed by real-time RT-PCR; cAMP was quantified by a radioaffinity assay. Results: Adenosine induced a set of early genes including the nuclear receptor subfamily 4, group A, member 1 (NR4A1/Nur77/TR3) which has been proposed to be involved in anti-atherogenic, protective effects in the vasculature. The pattern of adenosine-induced genes strongly resembles the changes caused by the adenylate cyclase activator forskolin. The analogue N-ethyl-carbox¬amido¬adenosine (NECA) also elicited a strong induction of NR4A1 and increased cellular cAMP levels. The effects of adenosine and NECA were blocked by the potent A2B receptor antagonists 8-[4-(4-benzylpiperazide-1-sulfonyl)phenyl]-1-propylxanthine (PSB-601) and 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine (MRS 1754). Experiments with protein kinase A inhibitor KT-5720 (hexahydro-hydroxy-methyl-oxo-epoxy-diindolo-pyrrolo-benzodiazocine-carboxylic acid-hexyl ester) and the Epac selective cAMP analogue 8-(4-chlorophenylthio)-2′-O-methyl-cAMP demonstrated that the induction of NR4A1 was virtually completely mediated via the Epac pathway. Conclusion: In summary, our results demonstrate the operation of adenosine A2B receptors in HCASMCs which mediate marked effects of adenosine and analogues on the early induction of NR4A1 via the Epac pathway.

P10-3

On the mechanism of UTP-induced inotropic effects in the heart

Joachim Neumann1, Andreas Simm2, Hasan Bushnaq2, Rolf-Edgar Silber2, Ulrich Gergs1

1Depart. Pharmacology, Martin-Luther-University Halle-Wittenberg, Germany,2Clinic for Heart and Thoracic surgerey, Martin-Luther-University Halle-Wittenberg, Germany

UTP acts via G protein-coupled P2-receptors, which are further divided into P2Y- and P2X-receptors. We were interested in cardiac effects of UTP. In electrically driven mouse left atrial preparations, we noted a positive inotropic effect (PIE) of UTP. The effect started at 1 µM UTP and leveled off at 100 µM UTP (30% of pre-drug value, n = 11), the highest concentration used. The effect was also observed after pre-treatment of mice with 5 mg/kg body weight reserpine, to empty endogenous catecholamine stores. This argues that effects of UTP occur independently of adrenoceptor stimulation by release of catecholamines. In addition, a PIE was observed in the presence of 100 µM UTPgS, a derivative of UTP stable to hydrolysis. This argues that UTP and not a degradation product mediates the PIE. Next, contractile effects of UTP were studied in electrically driven (1 Hz) atrial preparations from patients undergoing cardiac bypass surgery. Also in these preparations, UTP (cumulatively applied as 10, 30, 100 µM) exerted a PIE. At 100 µM UTP (the highest concentration investigated) force of contraction was increased by 27 ± 5% of pre-drug value (n = 15, p < 0.05). This PIE of UTP in human preparations was not attenuated by pre-incubation with SQ22536 (10 µM, an adenylate cyclase inhibitor) or U73122 (10 µM, a phospholipase C inhibitor) and could not be blocked by pre-incubation with P-receptor antagonists suramin (100 µM) or reactive blue (up to 500 µM). In summary, the inotropic effects of UTP on force of contraction in the human atrium, probably occur independently of cAMP and IP3. The exact receptor subtype(s) need(s) to be elucidated.

P10-4

Preconditioning with adenosine A1 receptor agonist attenuates allogeneic mixed lymphocyte reaction (MLR)

Oshri Naamani1, Hadar Eini1, Cidio Chaimovitz1, Amos Douvdevani2

1Ben-Gurion University of the Negev, Israel,2Soroka Medical Center & Ben-Gurion University of the Negev, Israel

Background: In a previous model of bacterial peritonitis, we demonstrated that activation of the adenosine A1 receptor (A1R) 24 hours before inoculation reduces inflammation through upregulation of the anti-inflammatory A2A receptor (A2AR). In our present study, we examined whether preconditioning with A1R agonist attenuates cellular immune response to allogeneic challenge. Methods: 24 h and 12 h before splenectomy C57/Bl and BALB/c mice were treated with two injections of the A1R agonist (CCPA 0.1 mg/kg) for preconditioning or vehicle. Mixed lymphocyte reaction (MLR) assays were conducted to assess splenocyte reactivity. Proliferation was measured by 3H-thymidine uptake. Results: In two-way MLR, a decrease of 65 ± 7 (N = 6, p = 0.001) in proliferation was observed in allogeneic MLR's performed with cells from preconditioned C57/BL spleens compared to proliferation of cells from vehicle-treated animals. Similarly, in one-way MLR (radiated BALB/c splenocytes) using cells from preconditioned C57/BL mice, a significant decrease (P < 0.05) was observed compared to vehicle cells. Preconditioned cells showed (~100 fold) increase sensitivity to inhibition by the A2AR agonist CGS 21680. In two-way MLR's, double side preconditioning (P < 0.001) compared to untreated allogeneic MLR's.

Conclusion: Our data indicate that preconditioning with A1R-agonist is able to remarkably suppress allogeneic reaction, probably by increasing A2AR sensitivity to agonists. This beneficial effect of A1R preconditioning may be useful in new therapeutic treatment aimed at reducing the immune response to allograft transplantation.

P10-5

Extracellular GTP modulates aquaporin membrane insertion on M1-CCD cell line

Rosa Mancinelli1, Rita La Rovere1, Stefania Fulle1, Giorgio Fanò1, Paola Lanuti2, Laura Pierdomenico2, Marco Marchisio2, Tiziana Pietrangelo1

1Dept. BAMS, University G. d'Annunzio Chieti-Pescara, Italy,2Dept. of Biomorphology, University G. d'Annunzio Chieti-Pescara, Italy

Background: Extracellular nucleotides, mainly ATP, regulate solute and water transport processes in renal epithelia. We observed that extracellular guanosine 5′-triphosphate (GTP), never considered in previous studies, shows a physiological effect on the nephron. In particular we studied the GTP effect on aquaporin (AQP) modulation. Methods. Cell Culture: M1-CCD cell line (ecacc) were cultured in Ham’s F-12 medium with 5% FBS. Cytometric assay: M1 cells were detached and immunolabeled by appropriate method to detect intracellular or surface AQP2, using different antibodies: E-loop, C-loop and pre C-terminus antibodies (kindly gifted by Profs G. Procino and M. Svelto) and C-terminus antibody (epitope 254–271, #AQP-002, Alomone). Cells were acquired on a FACSCalibur flow cytometer using CellQuest™ software. Data were analysed using FlowJo™ software. [Ca2+]i measurement: M1 cells were seeded in 96 well plates with optical membrane (Corning) and grown in GM for 3–5 days, incubated for 45 min at 37°C in NES and 5 μm Fura-2AM. The samples were treated and images collected as described in Pietrangelo et al., 2006. Results. Calcium imaging experiments carried out on single living cells, showed that GTP increased [Ca2+]i in two different kinetics: a transient and oscillatory one that has been described for the first time on these cells. Immunolabeling revealed that 3 days of incubation with 500 μM GTP increases the number of cells expressing AQP2 in the plasmamembrane. Conclusion. These results demonstrated that GTP promote AQP2 insertion in the M1 plasmamembrane via intracellular calcium signaling. We propose GTP as a new physiological agent in kidney.

P10-6

Streptozotocin-induced diabetes alters ATP and ADP hydrolysis in rat heart left ventricle

(Selected for Oral Poster Presentation)

Cristina Ribas Fürstenau1, Bárbara Rücker1, Daniela Pochmann1, Gustavo Vieira2, Lívia B. Bischoff2, João José F. Sarkis1, Luiz F. Zerbini3, Emerson A. Casali2, Márcia R. Wink4

1Departamento de Bioquímica, Universidade Federal do Rio Grande do Sul, Brazil,2Laboratório de Fisiologia e Nutrição Experimental, Centro Universitário Metodista do Sul – IPA, Brazil,3Department of Medicine, BIDMC Genomics Center and Harvard Medical School, United States,4Departamento de Ciências Básicas da Saúde, Universidade Federal de Ciências da Saúde de Porto Alegre - UFCSPA, Brazil

Background: Diabetes represents an important public health issue and the cardiac alterations observed in this pathology remain controversial. Considering the increasingly importance attributed to purinergic system in cardiovascular regulation, the present study aims to investigate the nucleotide metabolism in heart left ventricle of rats submitted to experimental diabetes mellitus by streptozotocin (STZ). Methods: Male adult Wistar rats received a single intraperitoneal injection of STZ (65 mg/Kg of body weight)or 0.1 M citrate (control group). The effect of short-term insulin treatment was also evaluated after 30 days of diabetes induction plus 6 days of insulin subcutaneous administration (NPH insulin 2U in the morning and 4U in the afternoon) or saline (control group). After 30 or 36 days, glucose levels, body weight (BW), heart rate (HR), and systolic blood pressure (SBP) were analyzed. Ectonucleotidases activities were colorimetrically determined in cardiac synaptosomes using ATP, ADP, AMP and 5′TMP as substrates. Moreover, ATP metabolism was followed by HPLC in different groups. Results: STZ injection increased glucose levels, reduced body weight, and diminished HR and SBP. The insulin treatment was able to reverse all these parameters. ATP and ADP hydrolysis were decreased in cardiac synaptosomes of STZ rats, and were completely recovered with insulin treatment. The HPLC analysis showed that ATP was slower hydrolyzed in diabetic groups in comparison to their controls. Conclusion: Taken together, these data suggest a possible involvement of purinergic system in the cardiac alterations observed in diabetes and its manipulation may be of interest in the treatment of this disease.

P10-7

Mechanism of purinergic cardioprotection in the ENT1-null mouse

Jennifer Rose1, Zlatina Naydenova2, Andrew Bang2, Almut Grenz3, Tobias Eckle3, Holger Eltzschig3, Doo-Sup Choi4, James Hammond5, Imogen Coe1

1Department of Biology, Muscle Health Research Centre, York University, Canada,2Department of Biology, York University, Canada,3Center for Hypoxia and Ischemia Research, University of Colorado, Denver, USA,4Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, USA,5Department of Physiology and Pharmacology, University of Western Ontario, Canada

Background: We recently showed that the ENT1-null mouse heart is cardioprotected from myocardial infarction (Rose et al. 2010; AJP). While this demonstrates an essential role for ENT1 in purinergic cardioprotection, the molecular and biochemical mechanisms underlying this effect are not well understood. Using the established ENT1 global knock-out mouse model, we investigated the role of ENT1 in purinergic signalling at the cellular level. Methods: We used a variety of techniques (HPLC, qRT-PCR, cAMP assays, microarrays) to study intracellular and extracellular levels of purine nucleotides, nucleosides and nucleobases plus expression of purinergic ecto-enzymes and hypoxia markers in ENT1-null and wild-type littermate cardiomyocytes under hypoxic and normoxic conditions. Results: Absence of ENT1 increases extracellular adenosine under hypoxia. The source of this adenosine is not due to up-regulation of CD39 or CD73. In addition, cAMP levels are significantly higher in ENT1-null hypoxic cardiomyocytes than wild-type littermates. Conclusions: These data demonstrate a central role of ENT1 as an upstream mediator of adenosine-dependent cardioprotection in the heart. It is apparent that ENT1 is an integral component of a complex pathway involving extracellular adenosine, adenosine receptor signalling, altered nucleotide pools and changes in gene expression in cardioprotection. However, while we have also shown that ENT1 is the predominant NT in human cardiovascular tissues (Marvi et al. submitted), we also observe the presence of variable levels of ENT2. Therefore, future studies will use transgenic animals with conditional cardiac-tissue specific ablation of ENT1 and/or ENT2 to provide further insight into the role of ENTs in cardiovascular physiology.

P10-8

Detrimental role of adenosine signaling in sickle cell disease and novel therapies

(Selected for Oral Poster Presentation)

Yang Xia1, YuJin Zhang1, Yingbo Dai1, Jiaming Wen1, Almut Grenz2, Holger Eltzchig2, Rodney Kellems1, Michael Blackburn1, Harinder Juneja3

1Biochemistry and Molecular Biology, University of Texas, Health Science Center at Houston-Medical School, USA,2Department of Anesthesiology, The University of Colorado, USA,3Department of Internal Medicine, University of Texas Health Science Center at Houston-Medical School, USA

Introduction-Sickle cell disease (SCD) is a devastating inherited hemolytic disorder due to a single missense mutation in the β-globin gene resulting in the production of hemoglobin S (HbS). Despite our knowledge of the molecular defect in HbS, we remain unable to control HbS polymerization and erythrocyte sickling under hypoxic conditions, which are central to the pathophysiology of the disease. Methods and Results- Multiple factors and metabolites are altered in response to hypoxia and may contribute to the pathogenesis of the disease. Using metabolomic profiling, we discovered that adenosine is highly elevated in the blood circulation of SCD transgenic mice. Next, we demonstrated that lowering adenosine concentrations by treatment with polyethylene glycol-modified adenosine deaminase (PEG-ADA) significantly reduced sickling, hemolysis and multiple organ damage in the mice. Mechanistically, we identified that elevated adenosine signaling through the A2B adenosine receptor promotes sickling by the induction of 2,3-diphosphoglycerate (2,3-DPG), an erythroid specific molecule that binds the deoxygenated form of Hb. Similar to mouse studies, we demonstrated that both adenosine and 2,3-DPG are increased in the blood circulation of SCD patients. Lastly, we provide compelling evidence that PEG-ADA and an A2BR antagonist decrease 2,3-DPG levels and subsequent reduce sickling in primary erythrocytes from SCD patients. Conclusion and Significance-Overall, we have identified that elevated adenosine induces 2,3-DPG via A2BR signaling and contributes to sickling in both human and mouse with SCD. Our findings reveal a novel therapeutic possibility to treat and prevent sickling and progression to multiple life-threatening complications by targeting on the adenosine signaling pathway.

P10-9

P2 Purinoceptor subtype changes in patients with vasculogenic erectile dysfunction

Miguel Faria1, Maria Alexandrina Timóteo1, José Maria Lafuente-de-Carvalho2, Paulo Correia-de-Sá3

1Lab.Farmacologia e Neurobiologia, UMIB, ICBAS-Universidade do Porto, Portugal,2Lab.Farmacologia e Neurobiologia, UMIB and Serv. Urologia, Centro Hospitalar do Porto (CHP), ICBAS-Universidade do Porto, Portugal,3Lab.Farmacologia e Neurobiologia, UMIB, CBAS-Universidade do Porto, Portugal

ATP is a potent relaxant agent in the human corpus cavernosum (HCC) acting either directly, through P2 receptors located on endothelium and smooth muscle fibres, or indirectly, via adenosine generated by ecto-nucleotidases. In this study, we aimed at characterizing the P2 purinoceptors responsible for HCC tone in samples collected from organ donors (controls) and from erectile dysfunction (ED) patients submitted to penile prosthesis implantation.

In the control group, the rank potency order for relaxation of HCC strips was (IC50 µM): ADP(30) ~ ADPbetaS(30) > ATPgammaS(100)~2-MeSATP(100)>ATP(300). In the presence of adenosine deaminase (0.5 U/ml), relaxations caused by ADP and ATP were significantly (P < 0.05) attenuated in ED patients. Likewise, the stable adenine nucleotide analogues ADPbetaS (30 µM) and ATPgammaS (30 µM) had their relaxation effects decreased to a maximum of 20%. The concentration-response curve of ADP (0.3–3000 µM) was significantly (P < 0.05) shifted to the right in the presence of the selective P2Y12 receptor antagonist, MRS 2395 (10 µM), in both groups. Selective blockade of P2Y1 (with MRS 2179, 0.3 µM) and P2Y13 (with MRS 2211, 10 µM) receptors were virtually ineffective in control individuals, but these drugs attenuated the relaxation caused by ADP and ADPbetaS in ED patients.

Data indicate that relaxation of the HCC by P2 purinoceptor agonists is severely attenuated in ED patients. Relaxation of the HCC from control subjects may be mediated by ADP-sensitive P2Y12 receptors, while we observed a yet unexplained shift towards the activation of P2Y1 (and perhaps P2Y13) receptors in ED patients.

Work supported by FCT (FEDER funding, UMIB-215/94 and SFRH/BD/25470/2005), Univ. Porto/Santander Totta and Soc. Port. Andrologia.

P10-10

Remodeling of atrial adenosine A2A receptors occurs in patients with atrial fibrillation and is linked to abnormal sarcoplasmic reticulum calcium handling

Carlos Barrera Hernandez1, Cristina Prat2, Anna Llach2, Cristina E. Molina2, Vicent Casadó1, Francisco Ciruela3, Carme Lluis1, Rafael Franco1, Leif Hove-Madsen2, Juan Cinca2

1Departament de Bioquímica i Biología Molecular, Facultat de Biologia, Universitat de Barcelona, Spain,2Institut Català de Ciencies Cardiovasculars, Spain,3Departament de Patologia i Terapèutica Experimental, Facultat de Medicina-Bellvitge, IDIBELL-Universitat de Barcelona, Spain

Background: Atrial fibrillation (AF) is associated with remodeling of membrane receptors and sarcoplasmic reticulum (SR) calcium handling, but the relationship between expression of adenosine A2A receptors (A2AR) and SR calcium handling in AF is not known. Also unknown is the distribution of said adenosine receptors in pig heart. Methods and results: Immunolabelling, real time PCR, and western blotting on human atrial tissue samples from patients with and without AF revealed a four-fold higher expression of the functional dimeric form of the A2AR in patients with AF. CGS21680 increased phosphorylation of the ryanodine receptor at residue 2809 in patients with AF while patients without AF had lower baseline levels of ryanodine receptor phosphorylation. Real time PCR and western blotting of pig heart samples revealed a similar pattern of expression in control pigs. Conclusions: Patients with AF have higher expression of the A2AR and the agonist CGS21680 increases ryanodine receptor phosphorylation, suggesting that up-regulation of A2AR expression may contribute to abnormal calcium handling in the fibrillating human atrium. Pig can be a viable model for adenosine cardiovascular effects studies.

P10-11

Role of P2 receptors in a cardiomyocyte ischemic hypoxia model

Simona Cosentino1, Simona Barcella2, Haijian Luo1, Joachim C. Burbiel3, Christa E. Müller3, Elena Tremoli2, Cristina Banfi2, Maria P. Abbracchio1

1Department of Pharmacological Sciences, University of Milan, Italy,2Monzino Cardiologic Center IRCCS, Monzino Cardiologic Center IRCCS, Italy,3Pharmaceutical Institute Pharmaceutical Chemistry I, University of Bonn, Germany

Blood levels of extracellular nucleotides are greatly increased during heart ischemia, but, despite the presence of both P2X and P2Y receptors on cardiomyocytes, their effects on the subsequent myocardial damage are still unknown. In this study, we aimed at investigating the role of ATP and specific P2 receptors in the appearance of cell injury in a cardiac model of ischemic hypoxia (murine HL1 cardiomyocytes). Cells were maintained in a modular incubator chamber in a controlled humidified atmosphere of 95% N2 for 16 hr in a glucose-free medium. In this condition, we detected a significant increase (+52% ± 21) in the release of ATP in the culture medium, and, in cultured cells, a 300% increase of cytoplasmic histone-associated-DNA-fragments, which represent an early marker of apoptosis. To dissect the role of specific P2 receptors we used a combined approach by both adding different P2 antagonists to cardiomyocytes before hypoxia and by silencing selected P2 receptors genes via specific small interfering RNAs. For the latter, gene silencing was checked by real-time quantitative PCR. Both selective pharmacological inhibition with ARC-118925 (10 µM) and KN-62 (1 µM) and gene silencing indicated that P2Y2 and P2X7 receptors are directly involved in the induction of cell death during ischemic hypoxia, whereas the P2Y4 receptor has a protective effect. These findings may have important therapeutic implications for the development of novel cardioprotective agents that specifically target P2 receptors.

P10-12

ATP inhibits NaCl absorption via basolateral P2X receptors in mouse medullary thick ascending limb (mTAL)

Rita Delgado Marqués1, Markus Bleich2, Helle Praetorius1, Jens Leipziger1

1Department of Physiology and Biophysics, Aarhus University, Denmark,2Inst. of Physiology, Christian Albrechts University, Kiel, Germany

Extracellular nucleotides regulate epithelial transport via luminal and basolateral P2 receptors. Renal epithelia express multiple P2 receptors which mediate significant inhibition of solute absorption. Recently, we found several P2 receptors in the medullary thick ascending limb (mTAL) including luminal and basolateral P2Y2 receptors. In addition we found evidence for a basolateral P2X receptor. It is currently not known if extracellular nucleotides influence transport in this segment. Objective: Investigate if extracellular ATP influences NaCl absorption in mTAL. Methods: We used isolated, perfused mouse mTAL to electrically measure Na+ absorption. By microelectrodes we determined the transepithelial voltage (Vte) and the transepithelial resistance (Rte) and via these the transepithelial Na+ absorption (equivalent short circuit current, Isc). Results: Non-stimulated mTALs show the following transport characteristics: Vte: +9.03 ± 0.44 mV, (lumen-positive), Rte: 8 ± 1.1 Ω cm2, Isc: 1404 ± 21.4 µA/cm2 (n = 16). As expected, luminal furosemide (100 µM) completely blocked transport. Basolateral ATP (100 µM) acutely (within 1 minute) and reversibly reduced the absorptive Isc. After 2 minutes a maximal reduction was measured and amounted to 19.6 ± 2.8% (n = 8). In the presence of ATP transport inhibition was sustained. Suramin blocked the ATP effect. Basolateral UTP, a P2Y2/P2Y4 receptor agonist was without effect as was adenosine. Conclusion: These data define that basolateral ATP exerts a significant inhibition of Na+ absorption in mouse mTAL and point to a P2X receptor-mediated mechanism. Intriguingly, these data add yet another example of P2 receptor mediated inhibition of tubular transport in intact renal epithelium.

P10-13

Pharmacological characterisation of P2Y receptor subtypes that mediate vasoconstriction of rat pulmonary artery

Charles Kennedy1, Callum Mitchell1, Alison Gurney2

1SIPBS, University of Strathclyde, Scotland,2Faculty of Life Sciences, University of Manchester, England

The aim of this study was to use recently developed, subtype-selective P2Y receptor agonists and antagonists to characterise the P2Y receptor subtypes that mediate the vasoconstrictor actions of nucleotides in pulmonary vascular smooth muscle. Small, intrapulmonary arteries were dissected from rats. The endothelium was removed and the arteries mounted on intraluminal wires under isometric conditions. UTP and UDP evoked slowly developing, tonic contractions, which reached a peak within 5 min. The selective P2Y6 antagonist MRS2578 inhibited the peak amplitude in a concentration-dependent manner, with IC50 values of 39 nM and 90 nM respectively. The inhibition curves were noticeably shallow. MRS2578 had no significant effect on the amplitude of contractions evoked by KCl. MK571 an antagonist of CysLT1 receptors, also acted in a concentration-dependent manner to inhibit the contractions evoked by UTP and UDP, but contractions induced by KCl were depressed by a similar extent. The endogenous agonist of P2Y14 receptors UDP-glucose did not evoke contractions. The selective P2Y1 receptor agonist MRS2365 induced small, concentration-dependent contractions that reached a peak within 1–2 min. The selective P2Y1 antagonist MRS2179 significantly inhibited the contractions to MRS2365, but had no effect on the contractions elicited by UDP, UDP or KCl. Thus, functional, contractile P2Y6 receptors, but not P2Y14 receptors, are present in rat small intrapulmonary arteries and are a site of action of UTP and UDP. The contribution of CysLT1 receptors is, however, unclear. Functional P2Y1 receptors are also present, but only mediate small contractions.

P11. Respiratory dysfunction

P11-1

P2Y2 receptor regulates eosinophil accumulation in inflamed lungs of ovalbumin-treated mice

(Selected for Oral Poster Presentation)

Gilles Vanderstocken1, Benjamin Bondue1, Michael Horckmans1, Larissa Di Pietrantonio1, Bernard Robaye1, Jean-Marie Boeynaems2, Didier Communi1

1IRIBHM, Université Libre de Bruxelles, Belgium,2IRIBHM, ERASME, Belgium

Asthma is a Th2 lymphocyte-associated inflammatory airway disease characterized by airway eosinophilia, airway obstruction and bronchial hyper-responsiveness. ATP is considered as an important mediator in this disease and are found in the airways of ovalbumin-sensitized mice after allergen exposure. Asthma features are suppressed using P2 receptor antagonists or ATP neutralizing apyrase. The P2Y2 receptor is an ubiquitous G-protein coupled receptor that is fully activated by ATP and UTP. We evaluated lung inflammation in mice deficient for the P2Y2 purinergic receptor. First we observed comparable infiltration of macrophages and neutrophils in the bronchoalveolar lavage fluids of LPS-aerosolized P2Y2+/+ and P2Y2−/− mice. Secondly, we decided to generate asthmatic P2Y2+/+ and P2Y2−/− mice using the ovalbumin (OVA) model. We observed that eosinophil accumulation, a distinctive feature of lung allergic inflammation, was defective in OVA-treated P2Y2-deficient mice compared to OVA-treated wild type animals. Interestingly the expression of membrane and soluble forms of endothelial adhesion molecules was reduced in OVA-treated P2Y2−/− mice compared to OVA-treated wild type animals. Our data defines P2Y2 as a regulator of eosinophil accumulation during lung inflammation.

P11-2

Breathing through the maturation of purinergic signaling

(Selected for Oral Poster Presentation)

Maryse Picher, Brian Button, Richard Boucher

CF Center University of North Carolina, USA

Background: Lung maturation is highly dependent on the mechanical stress imposed by normal rhythmic breathing. Given the extensive ramifications of purinergic signaling in airway homeostasis, we postulated that modifications in the regulation of P2Y2 and A2B receptor agonists are part of the maturation process. Methods. Primary cultures of human bronchial epithelial cells at various stages of differentiation (day 7 to 28) were exposed 0–7 days to cyclic compressive stress (CCS) mimicking breathing, then assayed for surface nucleotide concentrations, ectonucleotidase activities and mRNA levels. These CCS conditions accelerated the establishment of epithelial polarity without causing significant inflammation (IL-8 secretion) or remodeling (mucus cell metaplasia). Results. Static differentiation (day 7 to 14) down-regulated NSAP, NPP1 and NPP3, up-regulated NTPDase3 and adenosine deaminase 1 (ADA1), while NPP2, NTPDase1 (CD39) and ecto 5′-nucleotidase (CD73) remained stable. During this period, CCS selectively up-regulated the high-affinity tandem CD39–CD73 >5-fold, which was prevented by apyrase. Differentiated cultures (day 28) exposed 3 days to CCS experienced a milder, but significant, up-regulation of the high-affinity tandem, indicating that breathing is essential to complete the maturation process of the enzymatic network. The CCS-exposed day-28 cultures exhibited higher activity ratios for NTPDase3/NSAP (high-capacity ecto-ATPases), NTPDase1/NPPs (high-affinity ecto-ATPases) and CD73/NSAP (ecto-AMPases) than day-7 and day-28 static cultures. They also maintained higher ATP (6-fold) and ADO (3-fold) surface concentrations, which is known to significantly enhance mucus clearance. Conclusion. This study reveals the critical role of breathing-induced mechanical stress in the maturation of purinergic signaling on airway surfaces (Supported by the NIH PPG-P01-HL034332).

P11-3

Opposite deregulations of NTPDase1 (CD39) and NTPDase3 by cystic fibrosis

Maryse Picher1, Michel Fausther2, Julie Pelletier2, Carla M. Ribeiro1, Jean Sevigny2

1CF Center, University of North Carolina, USA,2Centre de Recherche en Rhumatologie et Immunologie, Université Laval, CA

Background: Cystic fibrosis (CF) is an inherited obstructive disease aggravated by damaging inflammatory responses to persistent infection. Since the purinergic regulation of mucociliary clearance is essential for airway defenses, we tested the impact of CF on the ectonucleotidases regulating ATP on the epithelial surfaces. Methods. Primary cultures of human bronchial epithelial cells (HBEs) from healthy donors and CF patients were compared for the functional polarities and expression levels of NTPDase1 and NTPDase3. The in vitro model of the disease was completed by exposing CF HBEs to supernatant of the mucopurulent material (SMM) collected from CF airways. These data were validated in vivo by immunolocalization in human airway tissue. Results. On normal HBEs, NTPDase1 was restricted to the apical surface, while NTPDase3 exhibited a bilateral distribution. Aseptic CF HBEs presented normal polarities, but NTPDase1 was down-regulated >50%, while NTPDase3 was up-regulated 3-fold. Chronic infection and inflammation mediated by SMM induced mucus cell metaplasia and shifted the polarity of both enzymes, as NTPDase1 moved to basolateral surfaces and NTPDase3 became predominantly apical. The SMM-exposed CF HBEs accurately reproduced the impact of the disease on their tissue distribution and expression level. Furthermore, the closely-related NTPDases exhibited distinct tissue distributions. While NTPDase1 was widely expressed (fibroblasts, epithelial, endothelial, alveolar and inflammatory cells), NTPDase3 was restricted to non-alveolar epithelial cells and particularly concentrated on mucus-secreting cells. Conclusion. Such remarkable disparities in distribution, polarity and responses to CF strongly suggest that NTPDase1 and NTPDase3 regulate distinct purinergic events in human airways. Supported by the NIH (PPG-P01-HL034332).

P11-4

Purinergic regulation of alveolar surface liquid by human type 2 cells

Maryse Picher, Peter F. Bove, Barbara R. Grubb, Troy D. Rogers, Seiko F. Okada, Carla M. Ribeiro, Brian Brighton, Scott H. Randell, Wanda K. O’Neal, Richard C. Boucher

CF Center University of North Carolina, USA

Background: It is well established that airway surfaces are maintained hydrated by the complex interplay of ion channels activated by P2Y2 (ATP, UTP) and A2B (adenosine) receptors. However, the purinergic regulation of ion/liquid transport within the alveoli remains poorly understood. Methods: Cultures of human alveolar Type 2 (AT2) cells and transgenic mice expressing secreted Clara Cell 10 (CC10) fused to eGFP were utilized to investigate this question. The AT2 cultures were used within 5–10 days of seeding, during which they highly express the selective markers proSPC and non-specific alkaline phosphatase, but not AT1 marker aquaporin5. Results: The purinergic receptors expressed by AT2 cells are: A2A, A2B,P2Y2,4,6 and P2X4. They absorb liquid when the surface is flooded with saline, and secrete liquid in response to UTP, forskolin (adenylate cyclase activator) or cyclic compressive stress mimicking breathing. The cultures maintain an alveolar surface liquid height of ~4 microm, which is reduced to ≤ 2 microm by apyrase. As in bronchial epithelia, AT2 cells mediate Na+/liquid absorption through the epithelial Na+ channel (ENaC), and Cl-/liquid secretion through the cystic fibrosis transmembrane conductance regulator (CFTR). However, AT2 cells regulate these channels differently, as UTP activates CFTR and inhibits ENaC, without apparent CFTR-mediated ENaC inhibition. The eGFP-CC10 transgenic mice exhibited evidence of bulk solute flow movement from the alveolus to tracheal surfaces. Conclusion: This study provides evidence for a unique purinergic regulation of ion/liquid transport across the alveolar barrier, and liquid secreted within the alveoli in vivo communicates with airway surfaces. Supported by NIH (PPG-P01-HL034332).

P11-5

Role of P2X7 receptors in murine silicosis

Leonardo Monção-Ribeiro1, Sulamita Borges de Avelar1, Radovan Borojevic1, Christina Takiya1, Robson Coutinho-Silva2

1Bioengineering Program, Institute of Biomedical Sciences Federal University of Rio de Janeiro, Brasil,2Biophysics Institute Carlos Chagas Filho, Immunobiology Program, Federal University of Rio de Janeiro/Biophysics Institute Carlos Chagas Filho, Brasil

P2X7 receptor (P2X7R) activation is involved in a number of pro-inflammatory responses in macrophages and other immune cells. Silicosis is an occupational lung disease resulting from the inhalation of silica particles over prolonged periods of time, which causes chronic inflammation and progressive nodular pulmonary fibrosis. The aim of this study was to characterize the importance of P2X7 activation in lung inflammatory response of silicotic animals. C57BL/6 wild-type and P2X7R knockout mice were intratracheally instillated with silica (20 mg–50 uL/animal) (WT-SIL/KO-SIL) or saline (50 uL-CTRL-WT/CTRL-KO). After 14 days of silica instillation, the animals were sacrificed and lungs collected for histological, immunohistochemistry and histomorphometry analysis. The silica instillation increased significantly the P2X7R immunoexpression in WT animals. The immunoreactivity was presented in inflammatory cells mostly, bronchiolar cells, and alveolar epithelial cells. Animals WT-SIL and KO-SIL showed alveolitis and formation of nodular structures. However, there was a significant decrease in the number of inflammatory cells (mononuclear cells, and polymorphonuclear cells) both in the in nodules in animals KO-SIL when compared to WT-SIL animals. Collagen fibers amount was similar between all genotypes. The nodular area was significantly diminished in KO-SIL animals when compared to WT-SIL animals. The KO-SIL animals exhibited a significant decrease in iNOS and NFkB/p65 immunoreactivity. The immunoreactivity for p-smad2/3 (TGF-beta-signaling activator) was diminished when compared with WT-SIL animals. Therefore, the P2X7 receptor appears to modulate the pulmonary inflammatory response in experimental silicosis. Supported by CNPq and FAPERJ.

P12. Liver/gastrointestinal dysfunction

P12-1

Extracellular ATP modulates natural killer cell function during liver regeneration

Nadine Graubardt1, Markus Trochsler1, Alyssa Rellstab1, Yara Banz1, Alexander Kroemer2, Daniel Candinas1, Simon C. Robson2, Guido Beldi1

1Inselspital Bern, University of Bern, Switzerland,2Beth Israel Deaconess Medical Center, Harvard Medical School, USA

Cell stress and injury causes the release of nucleotides into the extracellular milieu where they activate specific purinergic receptors. The levels of extracellular ATP are controlled not only by release, but also by enzymatic catabolism by the ectonucleotidase CD39. Natural killer cells have been shown to both increase liver injury and to mediate hepatocellular proliferation after partial hepatectomy in mice. We aimed to investigate the impact of elevated extracellular nucleotides on NK cell effector functions in a murine model of liver regeneration. Expression of purinergic receptors and ectonucleotidases on NK cells was assessed by RT-PCR. Effects of extracellular nucleotides and specific antagonists on secretion of IFNγ and cytotoxicity of sorted NK cells were measured in vitro. Parameters of cytotoxicity and hepatocellular proliferation were assessed post-partial hepatectomy in vivo. NK cells exhibit a restricted repertoire of P2 receptors and high expression of CD39. In vitro experiments reveal that ATPγS and ADPβS inhibit secretion of IFNγ by NK cells in response to IL-12 and IL-18. Further, cytotoxicity of purified NK cells was decreased after incubation with ATPγS, UTPγS and to a less extent with ADPβS. This effect was reversed by specific P2 receptor antagonists. In vivo, treatment with apyrase 30 min prior to partial hepatectomy resulted in an increase in cytotoxicity at 4 hours and hepatocellular proliferation at 48 hours post partial hepatectomy. Extracellular ATP modulates NK cell effector functions such as secretion of IFNγ and cytotoxicity. Control of extracellular nucleotide levels by CD39 impacts on cytotoxicity via the P2 receptor activation.

P12-2

CD39 overexpression on donor liver protects against ischemia reperfusion injury in a murine model of liver transplantation

Sandra Pommey1, Bo Lu1, Simon Robson2, Anthony d'Apice1, Peter Cowan1, Karen Dwyer1

1St. Vincent's Hospital, University of Melbourne, Australia,2Beth Israel Deaconess Medical Centre, Harvard Medical School, USA

Background: Adenosine signalling has protective effects in models of warm hepatic ischemia reperfusion injury (IRI) through a targeted action on CD4+ T cells and/or NKT cells. Both warm and cold ischemia are inherent to organ transplantation and contribute to IRI. Warm ischemic times are relatively short, however cold ischemia can be prolonged and adversely affects subsequent graft function. Human CD39 transgenic (hCD39tg) mice have an increased capacity to produce adenosine and are deficient in CD4+ T cells and invariant NKT (iNKT) cells.

Hypothesis: Donor livers from hCD39tg mice are protected in a liver transplant model characterised by prolonged cold ischemia.

Methods: Cold IRI was examined in an orthotopic mouse liver transplantation model. Livers from wild-type (WT) or hCD39tg mice were transplanted into WT recipients (n = 6 per group) after 18hrs of cold storage in UW solution. Serum alanine transaminase (ALT) and IL-6 were measured following 6 hours of reperfusion.

Results: Following prolonged cold storage and syngeneic transplantation, hCD39tg liver grafts functioned significantly better than WT grafts (ALT 10018 ± 653 U/L versus 15638 ± 1513 U/L, p < 0.01), in accordance with histological findings of less extensive necrosis. Systemic IL-6 was also significantly lower in recipients of hCD39tg grafts (2396 ± 438 pg/mL versus 4336 ± 647 pg/mL, p < 0.05).

Conclusion: CD39 overexpression in the donor liver protects against cold IRI in a model of liver transplantation. This could be mediated by (1) increased adenosine generation within the graft, conferring anti-inflammatory effects, and/or (2) fewer “passenger” or donor-derived iNKT cells and CD4+ T cells within the graft, reducing the inflammatory response.

P12-3

P2Y1 receptors mediate purinergic neuromuscular transmission in the human colon

Diana Gallego1, Víctor Gil2, Míriam Martínez-Cutillas2, Maite Martín2, Pere Clavé3, Marcel Jiménez4

1Centro De Investigacion Biomedica En Red De Enfermedades Hepaticas Y Digestivas (CIBERehd) Instituto De Salud Carlos III, Spain,2Departament de Biologia Cel·lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, Spain,3Hospital de Mataró -CIBERehd, Instituto de Salud Carlos III, Spain,4Departament de Biologia Cel·lular, Fisiologia i Immunologia-CIBERehd, Universitat Autònoma de Barcelona, Spain

Background: P2Y1 receptors are probably responsible of the purinergic neurotransmission in the human gastrointestinal tract. The aim of the present study is to characterize the inhibitory neuromuscular transmission in the human colon using three different P2Y1 antagonists: MRS 2179, MRS 2279 and MRS 2500. Methods: We performed 1- Electrical field stimulation (EFS) causing mechanical relaxation using conventional muscle bath technique; 2- EFS-induced Inhibitory Junction Potential (IJP) using intracellular microelectrodes technique and 3- Calcium imaging (Fluo-4 AM) with cultured human colonic smooth muscle cells (HCSMC). Results: ADPßS (1 µM) induced calcium transients in HCSMC. The response was abolished by MRS2179 (10 µM), MRS2279 (1 µM) and MRS2500 (1 µM). All the antagonists tested inhibited the non-nitrergic relaxation in a concentration-dependent manner: MRS2179 (0.87 µM; logIC50 = −6.06 ± 0.21; n = 14); MRS2279 (0.26 µM; logIC50 = −6.59 ± 0.07; n = 7 ); MRS2500 (0.09 µM; logIC50 = −7.06 ± 0.09; n = 5). Similarly the IJP was concentration dependently inhibited by MRS2179 (1.22 µM; logIC50 = −5.95 ± 0.11; n = 5), MRS2279 (0.28 µM; logIC50 = −6.55 ± 0.05; n = 5) and MRS2500 (0.07 µM; logIC50 = −7.15 ± 0.02; n = 5). Conclusion: Our data demonstrate that P2Y1 receptors mediate the purinergic inhibitory neurotransmission in the human colon. The activation of the receptor probably causes an intracellular calcium increase that activates potassium channels. The rank order of potency of the antagonists is MRS2500 > MRS2279 > MRS2179. Financial support: BFU2009-11118, V.G AP2007-01583 and D.Gallego (CIBERehd Instituto de Salud Carlos III)

P12-4

Involvement of P2Y1 receptors and nitric oxide in the inhibitory neural tone in the rat colon

Diana Gallego1, Miriam Martínez-Cutillas2, Maite Martín3, Marcel Jimenez3

1Centro De Investigacion Biomedica En Red (Ciberehd), Instituto de Salud Carlos III, Spain,2Departament de Biologia Cel·lular, Fisiologia i Immunologia, Universitat Autònoma de Barcelona, Spain,3Departament de Biologia Cel·lular, Fisiologia i Immunologia- CIBERehd, Universitat Autònoma de Barcelona, Spain

Background: Nitric oxide (NO) and ATP are inhibitory neurotransmitters in the gastrointestinal tract. However the relative contribution of each neurotransmitter in the neural tone is still unknown. The aim of the present work was to study the “spontaneous” neuromuscular transmission in the rat colon. Methods: Smooth muscle resting membrane potential (RMP) from rat mid colon was recorded using microelectrode technique. Electrophysiological data were correlated to “in vitro” (muscle bath) and “ex vivo” (strain gauges) motility studies. Results: Spontaneous inhibitory junction potentials (sIJP) were recorded in the presence of nifedipine (1 µM). sIJP were evaluated using the mean standard deviation (SD) and the frequency distribution (0.5 mV bin) of RMP. When recordings showed sIJP an increase in SD was observed. Briefly our data show that: 1- TTX 1 µM depolarized 4.7 ± 0.4 mV (n = 5) smooth muscle cells and abolished sIJP (SD 0.14 ± 0.03 vs 0.97 ± 0.12 mV in control, P < 0.01, n = 5). 2-MRS2500 1 µM did not modify membrane potential but dose dependently abolished sIJP (IC50: 3.1 nM; n = 5). 2- L-NNA 1 mM depolarized 5.3 ± 0.7 mV (n = 5) smooth muscle cells but did not modify sIJP. 3- These results correlate with “in-vitro” and “ex-vivo” data where L-NNA but not P2Y1 antagonists tonically increased the spontaneous motility. Conclusion: NO and ATP have different inhibitory functions in the rat colon. NO release modulates smooth muscle RMP and tonically regulates spontaneous motility. In contrast, ATP acting on P2Y1 receptors is responsible of sIJP and probably mediates fast and transient relaxation. Financial support: BFU2009-11118, V.G AP2007-01583, D.Gallego (CIBERehd Instituto de Salud Carlos III)

P12-5

ATP effects on HCT8 human intestinal cell line and on human colon cells from biopsy samples

Carolina Souza1, Giani Santoro2, Heitor Souza3, Morgana Castelo-Branco4, Alessandra Abalo5, Robson Coutinho-Silva6, Claudia Mara Lara Melo Coutinho Coutinho7

1Lab. de Inovações em Terapias, Ensino e Bioprodutos- LITEB, Instituto Oswaldo Cruz - IOC, FIOCRUZ, Brazil,2Lab. de Inovações em Terapias, Ensino e Bioprodutos / Programa de Imunobiologia, Instituto de Biofísica Carlos Chagas Filho, Instituto Oswaldo Cruz, FIOCRUZ / Universidade Federal do Rio de Janeiro, Brazil,3Depto. de Clinica Médica, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro, Brazil,4Depto. de Histologia e Embriologia, Universidade Federal do Rio de Janeiro, Brazil,5Lab. de Inovações em Terapias, Ensino e Bioprodutos- LITEB, Instituto Oswaldo Cruz, FIOCRUZ, Brazil,6Programa de Imunobiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Brazil,7Fiocruz, Brazil

In the present study, we investigated nitric oxide (NO) production and morphological changes of HCT8 cells in response to ATP treatment. We also studied ATP effects, combined or not with IFN-gamma, on human colonic mucosal explants obtained from endoscopic biopsies. Transmission electron microscopy analysis revealed that 2 mM ATP treatment for 24h can result in apoptosis or autophagy of HCT8 cells. HCT8 cells did not produce NO in response to different concentrations of ATP or LPS, as assayed by Griess reagent. Colonic biopsy specimens from normal human colonic samples placed on organ culture dishes were treated with IFN-gamma (2 ng/ml) for 24 h, and/or ATP (2 mM/ml) for the last 8 h of culture. We found that ATP induced apoptosis on human intestinal explants showing TUNEL-positive cells in the epithelium and in the lamina propria. The percentage of apoptotic cells significantly increased both in the epithelium and in the lamina propria after treatment with IFN-gamma and ATP, as compared to control cells, without stimulus or stimulated either with IFN-gamma or ATP. In another set of experiments, human colonic epithelial cells were isolated from biopsy specimens to analyze caspase-3 activity. We observed a significant increase of caspase-3 activity when the colonic epithelial cells were incubated with IFN-gamma followed by ATP, as compared to control cells. Taken together, our data suggest that P2X7 receptors on human epithelial cells are modulated by IFN-gamma and, once activated, induces apoptosis and autophagy. Financial support: PETROBRAS, CNPq, PRONEX-FAPERJ.

P12-6

Involvement of adenosine signaling in controlling the release of ghrelin from the mouse stomach

(Selected for Oral Poster Presentation)

Gary Kaiyuan Yang1, Linda Yip2, Bertil B. Fredholm3, Timothy J. Kieffer4, Yin Nam Kwok4

1University of British Columbia, Canada,2Department of Medicine - Division of Immunology and Rheumatology, Stanford University, United States of America,3Department of Physiology and Pharmacology, Karolinska Institute, Sweden,4Department of Cellular and Physiological Sciences, University of British Columbia, Canada

The orexigenic hormone, ghrelin, is predominately located in the stomach. Alterations of ghrelin levels are associated with various eating disorders, obesity and diabetes. We have previously shown that adenosine alters the release of gastric somatostatin and gastrin. In this study, the involvement of adenosine in regulating the release of ghrelin was examined in adenosine A1 (A1RKO) and A2A receptor knockout mice (A2ARKO) using the isolated vascularly perfused stomach. Total ghrelin was measured by commercially available radioimmunoassay kit. Adenosine stimulated ghrelin release concentration-dependently. The percentage change of ghrelin release in the presence of 0.1, 1.0, 10 µM adenosine was −2 ± 10, 40 ± 13 and 132 ± 26, respectively. The adenosine-induced increase was abolished by the A2A receptor-selective antagonist, ZM 241385, but not by the A1 receptor-selective antagonist, DPCPX. In addition, the A2A receptor-selective agonist, CGS 21680, augmented ghrelin release concentration-dependently, whereas the A1 receptor-selective agonist, CCPA, inhibited ghrelin release. In A2ARKO mice, adenosine inhibited ghrelin release and DPCPX blocked this inhibition. The adenosine deaminase inhibitor, EHNA, enhanced ghrelin release in wild type and A1RKO but not in A2ARKO mice. Immunohistochemical studies revealed that immunoreactivities of ghrelin were co-localized with A1 and A2A receptor in the gastric nerve fibers. Immunoreactivities of ghrelin were also found to be co-localized with A1 receptors in the gastric mucosa. Blockade of neural activities with tetrodotoxin abolished the stimulatory effect of adenosine on ghrelin release. In conclusion, adenosine stimulates tonically ghrelin release through A2A receptor activation and inhibits ghrelin release through A1 receptor activation.

P12-7

Regional distribution of adenosine receptors at the longitudinal muscle—myenteric plexus of the rat ileum: functional implications

Margarida Duarte-Araújo, Cátia Vieira, Maria de Fátima Ferreirinha, Paulo Correia-de-Sá

Laboratório de Farmacologia e Neurobiologia, UMIB, ICBAS, Universidade do Porto, Portugal

Adenosine exerts its actions through A1, A2A, A2B and A3 receptors. We demonstrated that adenosine plays a dual role on acetylcholine (ACh) release from myenteric motoneurons via high-affinity A1 inhibitory and A2A facilitatory receptors. The therapeutic potential of adenosine-related compounds for controlling intestinal motility disorders prompted us to investigate the role of low-affinity adenosine receptors, A2B and A3, on electrically-evoked (5 Hz, 200 pulses) [3H]-ACh release. We used confocal microscopy to explore the regional distribution of adenosine receptor subtypes at the longitudinal muscle-myenteric plexus of the rat ileum. The A3 receptor agonist, 2-Cl-IB MECA (1-10 nM), concentration-dependently increased the release of [3H]-ACh from stimulated myenteric neurons. The facilitatory effect of 2-Cl-IB MECA (3 nM, 17 ± 3%, n = 5) was attenuated by MRS 1191 (10 µM) and by ZM 241385 (50 nM), which block selectively A3 and A2A receptors, respectively. PSB 603 (10 nM), a highly selective A2B receptor antagonist, failed to modify the inhibitory effect of the most likely A2B receptor agonist, NECA (300 nM, −21 ± 6%, n = 9); on its own, PSB 603 (10 nM) was devoid of effect on [3H]-ACh release. Data using selective antagonists, DPCPX (10 nM, +17 ± 4%, n = 4), ZM 241385, 50 nM, −37 ± 10%, n = 6), and MRS 1191 (10 µM, −28 ± 8%, n = 7), indicate that modulation of evoked [3H]-ACh release is balanced through tonic activation of inhibitory (A1) and facilitatory (A2A and A3) receptors by endogenous adenosine. Immunolocalization studies showed that A2B receptors co-localize with GFAP on myenteric glial cells. The A1 and A3 receptors are mainly distributed to cell bodies of ganglionic myenteric neurons, whereas A2A receptors are localized predominantly on cholinergic nerve terminals. Tandem localization of excitatory A2B and A2A receptors on myenteric neurons might explain why cell body stimulation of A2B receptors (with 2-Cl-IB MECA (3 nM) is no longer apparent upon maximal activation of prejunctional A2A receptors with the selective agonist, CGS 21680C (3 nM). Work supported by FCT (FEDER funding, PTDC/CVT/74462/2006 and UMIB-215/94).

P12-8

The relationship between diabetes-induced hepatic localization change in adenosine 3′-monophosphate forming enzyme activity and mitochondrial permeability transition

(Selected for Oral Poster Presentation)

Akihiro Miyamoto, Hiroyuki Fujimori

Department of Toxicology, Faculty of Pharmaceutical Sciences, Setsunan University, Japan

To elucidate the pathophysiolosical importance of adenosine 3′-monophosphate (3′-AMP) forming enzyme, one of the ribonuclease, the effect of streptozotocin (STZ)-induced diabetes on the enzyme activities was investigated in the cytosol and mitochondrial fractions of ICR mouse liver. Furthermore, we examined whether mitochondrial permeability transition (MPT) might cause release of 3′-AMP forming enzyme activity from isolated mitochondria to cytosol. 3′-AMP forming enzyme activity was evaluated by the sum of the amount of produced 3′-AMP and adenosine from poly(A) as substrate. 3′-AMP and ATP were determined by forming their 1-N6-etheno derivatives and analyzing by a reverse phase/HPLC with fluorescence detection. MPT-induced mitochondrial swelling was determined as the decrease in the absorbance at 540 nm of the mitochondrial suspension. After 2 weeks, mouse treated with a single dosage of STZ (100, 150 or 200 mg/kg i.p.) was induced a dose-dependent hyperglycemia and hypoinsulinemia. In the diabetic liver, ATP levels decreased, indicating that mitochondrial ability to synthesize of ATP declined. In the STZ 200 mg/kg group, the activities of 3′-AMP forming enzyme in hepatic mitochondria reduced, inversely, the cytosolic enzyme activity increased. Next, phenylarsine oxide caused the release of the cytocrome c and 3′-AMP forming enzyme activity and the release of the enzyme activity was prevented by cyclosporin A, an inhibitor of the MPT. These results suggested that the enhancement of the 3′-AMP forming enzyme activity in the hepatic cytosol fraction of diabetic mouse might be partly due to the release of the enzyme from mitochondria via MPT.

P12-9

Involvement of P2Y1 receptors and nitric oxide in the inhibitory neural tone in the rat colon

Víctor Gil1, Diana Gallego2, Míriam Martínez1, Maite Martín3, Marcel Jiménez3

1Department of Cell Biology, Physiology and Immunology, Universitat Autònoma de Barcelona, Spain,2CIBERehs, Instituto de Salud Carlos III, Spain,3Department of Cell Biology, Physiology and Immunology - CIBER ehd Universitat Autònoma de Barcelona, Spain

Background: Nitric oxide (NO) and ATP are inhibitory neurotransmitters in the gastrointestinal tract. However the relative contribution of each neurotransmitter in the neural tone is still unknown. Methods: Smooth muscle resting membrane potential (RMP) from rat mid colon was recorded using microelectrode technique. Electrophysiological data were correlated to "in vitro" (muscle bath) and "ex vivo" (strain gauges) motility studies.Results: Spontaneous inhibitory junction potentials (sIJP) were recorded in the presence of nifedipine (1 µM). sIJP were evaluated using the mean standard deviation (SD) and the frequency distribution (0.5 mV bin) of RMP. When recordings showed sIJP an increase in SD was observed. Briefly our data show that: 1- TTX 1 µM depolarized 4.7 ± 0.4 mV (n = 5) smooth muscle cells and abolished sIJP (SD 0.14 ± 0.03 vs 0.97 ± 0.12 mV in control, P < 0.01, n = 5). 2-MRS2500 1 µM did not modify membrane potential but dose dependently abolished sIJP (IC50: 3.1 nM; n = 5). 2- L-NNA 1mM depolarized 5.3 ± 0.7 mV (n = 5) smooth muscle cells but did not modify sIJP. 3- These results correlate with "in-vitro" and "ex-vivo" data where L-NNA but not P2Y1 antagonists tonically increased the spontaneous motility. Conclusion: NO and ATP have different inhibitory functions in the rat colon. NO release modulates smooth muscle RMP and tonically regulates spontaneous motility. In contrast, ATP acting on P2Y1 receptors is responsible of sIJP and probably mediates fast and transient relaxation. Financial support: BFU2009-11118,V.G AP2007-01583, D.Gallego (CIBERehd Instituto de Salud Carlos III)

P13. STEM cells/development

P13-1

Nucleotides affect neurogenesis and catecholaminergic differentiation of mouse fetal midbrain-derived neural precursor cells

Jasmin Delic, Herbert Zimmermann

Institute of Cell Biology and Neuroscience Goethe, University Frankfurt, Germany

Background: Production of dopaminergic neurons from neural stem cells (NSCs) in vitro may provide a tool for cell therapeutic approaches to Parkinson’s disease. Dopaminergic neurons are formed from precursor cells during early fetal development. Here we analysed the potential of nucleotides to support cell survival and to drive neuronal and dopaminergic differentiation of fetal midbrain-derived NSCs. Methods: Precursor cells were isolated from the ventral midbrain of mouse embryos at stages E13.5 and E10.5, plated for adherent culture and cultured in differentiation media. Nucleotides were added to the culture at defined time intervals and the NSCs were analyzed for differentiation into neurons after 7 days. Results: There were significant differences regarding survival and neuron formation between cells harvested at days 10.5 and 13.5. Cells harvested at E10.5 revealed increased neurogenic potential. RT-PCR analysis indicated expression of a variety of P2X and P2Y receptors as well as the ecto-nucleotidases NTPDase2 and tissue non-specific alkaline phosphatase. In E13.5-derived cells ADPbetaS reduced the number of neurons whereas UTP and 2MeSATP increased it twofold. 2MeSATP specifically enhanced catecholaminergic differentiation. In E10.5-derived cells neuron formation was increased by ADPbetaS and 2MeSATP whereas 2ClATP specifically enhanced catecholaminergic differentiation. Conclusion: Our results demonstrate that nucleotides can be applied to enhance the formation of dopaminergic neurons from fetal mouse midbrain-derived neural precursors.

P13-2

In vitro and in vivo analysis of the role of purinergic signalling in adult neurogenesis

(Selected for Oral Poster Presentation)

Kristine Gampe1, Simon Robson2, Herbert Zimmermann1

1Institute of Cell Biology and Neuroscience, Goethe University Frankfurt, Germany,2Department of Medicine, Harvard Medical School, USA

Background: The subgranular layer of the hippocampus (SGL) and the subependymal zone (SEZ) at the lateral ventricles harbour progenitor cells providing new neurons for the granule cell layer and the olfactory bulb, respectively. Here we analyzed adult neurogenesis in vitro and in vivo in P2Y1 and P2Y2 receptor knockout mice and in nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) knockout mice. Methods: Cell proliferation was quantified in vitro after isolation of neural stem cells from the SEZ. For in vivo analysis animals were injected with the cell proliferation marker BrdU and the number of BrdU-labelled cells in the dentate gyrus, the SEZ or the olfactory bulb was analyzed after short term and long term survival. Results: Proliferation of neural stem cells from the SEZ of P2Y1 and P2Y2 receptor-deficient mice was reduced in vitro. In contrast, in vivo proliferation remained unaltered in both the SEZ and the SGL but long-term survival of young neurons was increased in P2Y2 knockout mice. Deletion of NTPDase2 did not affect in vitro proliferation. Conclusion: The data further support a role of nucleotide signalling in adult neurogenesis. They reveal significant differences between in vitro and in vivo analysis and furthermore suggest the existence of compensatory mechanisms in knockout animals.

P13-3

Kinetic properties of ecto-5′-nucleotidase during postnatal development in rat cerebral cortical and hippocampal synaptic plasma membranes

Ivana Stanojevic, Maja Milosevic, Natasa Velickovic, Dunja Drakulic, Snjezana Petrovic, Anica Horvat

POBOX522, Vinca Institute of Nuclear Science, Serbia

Ecto-5′-nucleotidase, an extracellular enzyme of the purine catabolic pathway, catalyzes the breakdown of AMP to adenosine. The aim of the present study was to analyze kinetic properties of ecto-5′-nucleotidase in synaptic plasma membranes (SPM) isolated from cerebral cortex and hippocampus of Wistar female rats in different stages of postnatal development (postnatal days 7, 15, 20, 30 and 90). Enzymatic and kinetic assays were performed with Malachite green method, using increasing AMP concentrations (0.05–1.20 mM) and Vmax and Km were calculated by Eadie-Hofstee transformation. The enzyme activities of ecto-5′-nucleotidase were increased steadily from birth, reaching maximum values at day 90 in both areas, but more efficient and higher in hippocampal SPMs. Immunoblot analysis of ecto-5′-nucleotidase relative protein abundance had the same profile in both areas. Kinetic analysis revealed that enzyme exhibited the highest specific activity at day 90. The Km values in cortical SPMs indicated lowest affinity of the enzyme at day 7, highest in days 15 and 20, after that it slightly decreased to day 90, but still significantly higher than Km of day 7. In hippocampal SPMs, Km values indicated that affinity was lowest at day 7, increased to day 20 with plateau until day 90. These results imply that in the postnatal cortex and hippocampus, ecto-5′-nucleotidase activity depends of synaptic contacts maturation and could affect stage-specific developmental processes, while in the adults hydrolyzing activity was dominant feature. The increased ecto-5′-nucleotidase activities with age suggest more efficient production of P1 receptor agonist and potent neuromodulator, adenosine.

P13-4

P2 receptors influence differentiation in human mesenchymal and ectomesenchymal stem cells

Edda tobiasch1, Nina Zippel1, Nadine Scholze2, Christian Limbach2, Christian Urban3, Claudio Luparello4, Andreas Pansky2, Matthias Kassack3

1Hochschule Bonn-Rhein-Sieg, Germany,2Bonn-Rhein-Sieg, Germany,3University of Duesseldorf, Germany,4University of Palermo, Italy

Background: Purinergic receptors are well known to participate in important cellular processes, such as proliferation and migration, but the role of these receptors during mesenchymal differentiation has been deciphered only fragmentarily so far. Human adult mesenchymal stem cells (MSCs) are of major interest for Regenerative Medicine, in particular due to their ability to differentiate into cells forming muscle, cartilage or bone. In contrast to this, the ectomesenchymal stem cells derived from dental follicle are further committed towards hard tissues. To evaluate the potential influence of P2 receptors in stem cell differentiation, these two stem cell types with their differences in lineage potential have been compared for the receptor expression. Methods: Using semiquantitative RT-PCR and Western blotting, the role of metabotropic P2Y and ionotropic P2X receptor subtypes has been examined in mesenchymal and ectomesenchymal stem cells during differentiation. Calcium imaging following agonist stimulation was used to demonstrate the functional activity of P2 receptors. Results: Both cell types expressed several functionally active P2X and P2Y receptor subtypes, but with differences in the expression level. Interestingly, some particular P2 receptor subtypes were found to be differently regulated during adipogenic and osteogenic differentiation. Moreover, the administration of agonists and antagonists of P2 receptors had a direct influence on those differentiations. Conclusions: Taken together, purinergic receptors play an important role during the differentiation towards the adipogenic and osteogenic lineage. Thus, in the future, artificial P2 receptor ligands might be used to control mesenchymal stem cell fate.

P13-5

Characterization of P2X receptors in adult adherent neural progenitor cells (NPCs)

Nanette Messemer, Christin Kunert, Heike Franke, Peter Illes, Patrizia Rubini

Rudolf Boehm Institute of Pharmacology and Toxicology, University of Leipzig, Germany

The aim of this project was to elucidate whether cultured adherent neural progenitor cells of the subventricular zone (SVZ) of adult mice express functional P2X-receptors (P2XR). NPCs were prepared from the SVZ of adult mice. After 7 days, neurospheres were dissociated and brought into adherent culture. Whole-cell patch clamp recordings and calcium imaging were used to characterize the functional expression of P2XR, which was further supported by immunocytochemical stainings. In these adherent cultures we identified the P2X7R as the predominantly expressed member of the P2X family. Inward currents of ATP and the preferential P2X7R agonist Bz-ATP were largely increased in a reduced divalent cation environment. The responses evoked by Bz-ATP and ATP decreased with the age of the adherent cultures. This current reduction could be prevented when growth factors (EGF and FGF-2) were reapplied every day in the culture medium. The use of specific antagonists like A438097 and BBG (microM range), reduced the answer of the P2X7R in patch clamp and Ca2+ imaging experiments. P2XR agonists with selectivity for other subtypes (P2X1,2: alpha,beta-meATP) and antagonists (P2X1-3: TNP-ATP; P2X1: NF449, non-selective P2XR: PPADS) did not alter the calcium signal caused by Bz-ATP. Experiments with NPCs derived from P2X7R knockout mice, indicated that the Bz-ATP or ATP induced responses are basically due to the activation of P2X7R. These results demonstrate that the P2X7R is functionally expressed in adherent cultures of NPCs from the SVZ. Growth factors may play a pivotal role in the expression of this receptor in the NPCs.

P13-6

Modulation of gabaergic and glutamatergic synaptic events by adenosine A1 and A2A receptors in the imature hippocampus

Carla Gomes da Silva1, Monique Esclapez2, Rodrigo Cunha1, Christophe Bernard2

1Center for neuroscience and cell biology, Coimbra, Portugal,2U751 Epilepsie et Cognition INSERM, France

The role of adenosine in the immature nervous system remains poorly explored. Thus, we now explored the localization and role of A1 receptors (A1R) and A2AR in the hippocampus from P4-P7 mice pups. Upon sub-synaptic fractionation, we observed that A1R are located, as expected, at pre- and post-synaptic sites; in contrast, A2AR are confined to the post-synaptic and extra-synaptic fractions. Both receptors modulate the properties of miniature GABAergic (mGABA) and glutamatergic currents recorded in CA3 region from P4-P7 hippocampal slices. Blocking both receptors with SCH58261 (100 nM, A2AR antagonist) and DPCPX (100 nM, A1R antagonist) decrease the frequency and area of mGABA currents. Furthermore, SCH58261 increases the evoked response to locally applied GABA (50 µM), a concentration expected to mainly activate extrasynaptic GABAA receptors. In contrast, DPCPX always decreases the amplitude of evoked GABAergic responses. Frequencies from neurons displaying low basal mAMPA currents are increased upon exposure to A1R or A2AR antagonists, although there is some heterogeneity in response of different neurons. The amplitude of AMPA events is unchanged in the presence of TTX and may be decreased in some neurons after electrical stimulation, which suggest that some of the modulation mediated by adenosine receptors is activity dependent. These results give some hint to the existence of novel roles for adenosine A1 and A2A receptors during the development of hippocampal circuits. (Supported by FCT)

P13-7

A1 adenosine receptors play an active role in proliferation and osteogenic differentiation of human dental pulp-derived mesenchymal stem cells

Renata Mara Ciccarelli

Department of Biomedical Sciences, University of Chieti, Italy

Mesenchymal stem cells (MSC) have been isolated from dental tissues and are largely studied for future application in regenerative dentistry. In this study, we used MSC obtained from dental pulp (DPMSC) of normal human impacted third molars. These cells, under undifferentiating conditions, were negative for hematopoietic (CD45, CD14, CD34) and positive (>95%) for mesenchymal (CD29, CD73, CD90, CD105, CD166, CD146, STRO-1) markers. Moreover, when cultured in lineage-specific inducing media, they differentiated into osteoblasts and adypocytes (evaluated by Alizarin Red and Red Oil stainings, respectively), thus showing a multipotency. By RT-PCR we observed that DPMSC possess all four types of adenosine receptors, the expression of which is not altered following differentiation. Exposure of undifferentiated DPMSC to the A1 adenosine receptor (A1R) agonist CCPA significantly increased the number of cells in G1 phase of cells cycle and selectively up-regulated CD90 expression/cell, as assessed by flow cytometry. These effects were abolished by cells pre-treatment with the selective A1R antagonist DPCPX. When undifferentiated DPMSC were supplemented with a commercial osteogenic medium (Lonza), cells previously exposed to CCPA maintained CD90 up-regulation and showed an increased expression of osteogenic markers (runx-2, alkaline phosphatase, osteopontin, osteocalcin) and mineralization evaluated as extracellular calcium deposition, in comparison with controls. These findings indicate that the stimulation of A1R enhances the osteogenic differentiation efficiency of DPMSC. CCPA-induced CD90 up-regulation may have clinical implications being possibly related to an increased suppression of immune response by MSC that could facilitate tissue repair and remodeling.

P13-8

The P2Y-like GPR17 receptor orchestrates the transition between immature and myelinating oligodendrocytes and is a new target for myelin repair

Simona Daniele1, Marta Fumagalli2, Philip R. Lee3, Maria Letizia Trincavelli1, Davide Lecca2, R. Douglas Fields3, Claudia Verderio4, Maria Pia Abbracchio2, Claudia Martini1

1Dept. Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Italy,2Department of Pharmacological Sciences, University of Milan, Italy,3Nervous System Development and Plasticity Section, National Institutes of Health, USA,4CNR Institute of Neuroscience, University of Milan, Italy

Background: In the central nervous system, oligodendrocytes progenitors cells (OPC) differentiate into mature myelinating oligodendrocytes. Identifying the mechanisms that regulate oligodendrocyte differentiation and myelination are of great interest in the search of therapeutic treatments for CNS demyelinating diseases. A recent study showed that OPCs natively express the GPR17 receptor, and that this receptor, activated by both uracil nucleotides and cysteinyl-leucotrienes, plays a crucial role in oligodendrogliogenesis. In the present study we characterized GPR17 in primary cultured OPCs and investigated the expression changes occurring OPC differentiation to mature oligodendrocytes. Methods: GPR17 mRNA expression was evaluated in parallel to the mRNAs of some mayor myelin proteins, by Real time PCR analysis. Levels of the GPR17 protein and of specific oligodendrocyte markers at different stages of differentiation was detected by immunocytochemistry. The functional activity of GPR17 in OPCs was evaluated by calcium and cAMP measurement assays. GPR17 silencing was achieved by small interfering RNAs. Results: In primary rat embryonic OPC cultures, GPR17 expression is restricted to the first culture days and segregated from that of myelin proteins. In primary rat postnatal OPCs (p-OPC), GPR17 decorates a specific subset of slowly proliferating highly ramified NG2+ pre-oligodendrocytes and is turned down in morphologically mature myelinating cells. In p-OPC, GPR17 agonists are able to inhibit forskolin-stimulated cAMP production whereas no coupling to the calcium pathway was detected. After GPR17 silencing, response to GPR17 agonists were abolished. Conclusion: Our data suggest that GPR17 orchestrates the transition between immature and myelinating oligodendrocytes and may serve as a new potential therapeutic target for CNS myelin repair in demyelinating diseases.

P13-9

Expression of ATP-gated P2X7 receptors in mouse embryonic stem cells

(Selected for Oral Poster Presentation)

Amanda MacKenzie, Melanie Welham, Belinda Thompson

Department of Pharmacy and Pharmacology, University of Bath, UK

Background: P2X7 receptors are ligand-gated ion channels classically associated with expression in immune and epithelial cells. Adenosine 5′triphosphate (ATP) activated P2X7 receptors couple to an intrinsic ion channel followed by activation of second permeation state that leads to the influx of large organic compounds such as fluorescent ethidium bromide (EtBr). In the current study, we investigate the expression of ATP-gated P2X7 receptors in undifferentiated mouse embryonic stem (ES) cells. Methods: PCR detection of mRNA, whole-cell patch clamp recordings of ATP-evoked ionic currents and EtBr influx measurements were used to evaluate P2X7 receptor expression. ES cell self-renewal was assessed in measurements of alkaline phosphatase activity while cell necrosis is measured by the release of lactate dehydrogenase. Results: We demonstrate that mouse ES cells express mRNA encoding five P2X7 variants (P2X7a-d, P2X7k). Extracellular ATP evokes ionic currents in whole-cell patch clamp recordings and EtBr influx, both inhibited by A438079, a P2X7 receptor antagonist. ATP-mediated EtBr influx is also inhibited by small interfering (si)RNA knockdown of P2X7 expression. In assays of alkaline phosphatase activity, siRNA knockdown of P2X7 expression did not alter the percentage of self-renewing colonies of ES cells cultured in the presence of leukemia inhibitor factor. In immune cells, P2X7 receptors are associated with necrotic/apoptotic cell death. Extracellular ATP (1–5 mM) triggers rapid cell necrosis inhibited by either A438079 or siRNA knockdown of P2X7 expression. Conclusion: Together these data demonstrate the functional expression of ATP-gated P2X7 receptors in mouse ES cells where receptor stimulation leads to necrotic cell death.

P13-10

P2 receptors involvement in cell differentiation and proliferation in human osteoblasts

José Noronha Matos1, J. Coimbra1, A. Sá-e-Sousa1, A. Barbosa1, R. Freitas2, J.M. Neves2, F. Ferreirinha1, M.A. Costa1, P. Correia-de-Sá1

1Laboratório Farmacologia e Neurobiologia, Instituto Ciências Biomédicas Abel Salazar, Portugal, 2Serv. Ortopedia e Traumatologia, CHVNGaia-Espinho, Portugal

Bone turnover takes place at discrete sites in the remodelling skeleton that are subject to mechanical loading forces. Released nucleotides working through P2 receptors regulate various aspects of bone biology. Here, we investigated the P2 purinoceptors activity in human osteoblast cells as differentiation and maturation proceed. Bone marrow was harvested from patients submitted to orthopaedic surgery. Patients were divided into age groups: 30–40 and 60–83 years old. Osteoblast cell primary-cultures were characterized for proliferation (MTT assay), total protein content (method of Lowry) and alkaline phosphatase (ALP) activity during 30 days in vitro. Single-cell [Ca2+]i dynamics was evaluated by confocal-microscopy. Cells from young adults (30–40 years old) were the most proliferative and exhibit early differentiation, i.e. ALP activity was maximum (1.3 ± 0.2 nmol/minute.mg protein) at culture day 7. Cells from older patients were less proliferative and differentiate at later stages (21–28 days in culture). The rank agonist potency order to increase osteoblast ALP activity at day 7 was UTP > UDP ~ BzATP > ADPβS. At this stage, UDP (100 µM) also increased cell proliferation, while ADPβS, UTP and BzATP kept cells proliferating beyond day 21. The rank potency order to increase osteoblast [Ca2+]i was UDP > UTP > BzATP ~ ADPβS at day 7, but it changed to ADPβS > BzATP > UDP ~ UTP at day 28. P2Y6 immunoreactivity decreases with time, while at day 28 osteoblasts express predominantly P2Y1, P2Y2 and P2Y4 receptors. Data show that heterogeneity of P2 purinoceptors expression on human osteoblasts depend on the maturation stage. Participation of these receptors to switch “on/off” bone formation may prompt for new targets to control bone disorders.

P13-11

A1 adenosine receptors play an active role in proliferation and osteogenic differentiation of human dental pulp-derived mesenchymal stem cells

Iolanda D'Alimonte1, Eleonora Nargi1, Marco Marchisio2, Paola Lanuti3, Stefano Tete'4, Emanuela Torelli5, Francesco Caciagli5, Renata Ciccarelli1

1Department of Biomedical Sciences & StemTech Group, University of Chieti, Italy,2Department of Biomorphology & StemTech Group, University of Chieti, Italy,3Department of Biomorphology, University of Chieti, Italy,4Department of Oral Sciences & StemTech Group, University of Chieti, Italy,5Department of Biomedical Sciences, University of Chieti, Italy

Mesenchymal stem cells (MSC) have been isolated from dental tissues and are largely studied for future application in regenerative dentistry. In this study, we used MSC obtained from dental pulp (DPMSC) of normal human impacted third molars. These cells, under undifferentiating conditions, were negative for hematopoietic (CD45, CD14, CD34) and positive for mesenchymal (CD29, CD73, CD90, CD105, CD166, CD146, STRO-1) markers. Moreover, when cultured in lineage-specific inducing media, they differentiated into osteoblasts and adypocytes (evaluated by Alizarin Red and Red Oil stainings, respectively), thus showing a multipotency. By RT-PCR we observed that DPMSC possess all four types of adenosine receptors, the expression of which is not altered following differentiation. Exposure of undifferentiated DPMSC to the A1 adenosine receptor (A1R) agonist CCPA significantly increased the number of cells in G1 phase of cells cycle and selectively up-regulated CD90 surface expression/cell, as assessed by flow cytometry. These effects were abolished by cells pre-treatment with the selective A1R antagonist DPCPX. When undifferentiated DPMSC were supplemented with a commercial osteogenic medium (Lonza, Milan), cells exposed to CCPA showed an increased mineralization evaluated as extracellular calcium deposition, in comparison with controls. These findings indicate that the stimulation of A1R restrains DPMSC duplication enhancing their osteogenic differentiation efficiency. CCPA-induced modulation of CD90 expression and mineralization may have clinical implications possibly facilitating tissue repair and remodeling.

P13-12

Roles of enpp4 during vertebrate kidney development

Karine Masse1, Junichi Kyono2, Surinder Bhamra2, Elizabeth Jones2

1UMR-CNRS 5164, University of Bordeaux, France,2University of Warwick, UK

The ENPP gene family, a subfamily of the ectonucleotidases can hydrolyze purines and also can catalyse the generation of bioactive lipids, placing the ENPP enzymes as key regulators of two major physiological signalling pathways and also important players in several pathological conditions. We have cloned and characterized all the members of this family in the frog. Each gene displays a distinct specific expression pattern during embryogenesis, especially enpp4 being specifically expressed in the pronephros, the functional amphibian embryonic kidney. Enpp4 has been conserved during evolution, but its roles in vertebrates remain unknown. We have investigated the functional roles of enpp4 in kidney development, using Xenopus as a vertebrate model, which is particularly well suited for kidney formation studies. Gain-of-function of enpp4 activity (mRNA injection) results in enlarged and ectopic pronephros formation. Catalytically dead enpp4 mRNA injection abolishes these phenotypes. Translational inhibition of endogenous enpp4 (morpholino injection) specifically causes reduced pronephric structures. Co- expression of enpp4 and S1P receptor mRNAs gives additive effects on pronephros development. This synergy is not observed following co-injection of enpp4 with LPA or purines receptors mRNAs. These data suggest that the pronephric phenotypes induced by enpp4 misexpression are due to the bioactive lipids, S1P, generated by enpp4 activity. (Wellcome Trust 082071)

P13-13

Purinergic stimulation of human mesenchymal stem cells potentiates their chemotactic response to CXCL12, increases the homing capacity and the production of pro-inflammatory cytokines

Davide Ferrari1, Sara Gulinelli1, Valentina Salvestrini2, Giovanna Lucchetti2, Roberta Zini3, Rossella Manfredini3, Luisa Caione4, Wanda Piacibello4, Marilena Ciciarello2, Lara Rossi2, Marco Idzko5, Sergio Ferrari3, Francesco Di Virgilio1, Roberto Lemoli2

1University of Ferrara, Italy,2University of Bologna, Italy,3University of Modena, Italy,4University of Torino, Italy,5University of Freiburg, Germany

Nucleotides binding to specific plasma membrane receptors (P2Rs) modulate a wide variety of biological responses. Here, we show that bone marrow-derived human mesenchymal stem cells (hMSCs) express several functional P2R subtypes at the molecular and protein level. hMSCs were resistant to the cytotoxic effects of ATP, as demonstrated by the lack of morphological and mitochondrial changes or release of intracellular markers of cell death. Gene expression profiling revealed that hMSCs stimulated with ATP underwent a down-regulation of genes involved in cell proliferation, whereas those involved in cell migration were strongly up-regulated. Functional studies confirmed the inhibitory activity of ATP on proliferation of hMSCs and clonogenic stromal progenitors. ATP potentiated the chemotactic response of hMSCs to the chemokine CXCL12, and increased their spontaneous migration. In vivo, xenotransplant experiments demonstrated that the homing capacity of hMSCs to murine bone marrow was significantly increased by pre-treatment with ATP. Moreover, ATP increased the production of the pro-inflammatory cytokines IL-2, IFN-gamma, and IL-12p70, while decreasing the anti-inflammatory cytokine IL-10 and this finding was associated with the reduced ability of MSCs of inhibiting T-cell proliferation. Thus, our data show that purinergic signaling modulates hMSCs functions and highlight a role for extracellular nucleotides in hMSCs biology.

P13-14

The purinergic system controls the axonal growth and branching of hippocampal neurons

Miguel Díaz Hernández1, Juan Ignacio Díaz Hernández1, Maria Diez-Zaera1, Rosa Gómez-Villafuertes1, Elena Hernandez Alvarez1, Paula Garcia-Huerta1, Ana del-Puerto2, Jose Javier Lucas2, Herbert Zimmermann3, Juanjo Jose Garrido2, Maria Teresa Miras-Portugal1

1Dep. Bioquimica, Facultad Veterinaria, Universidad Complutense de Madrid, Spain,2CBMSO, CSIC, Spain,3Department of Neurochemistry, Johann Wolfgang Goethe-University, Germany

During establishment of neural circuits or in the regenerative processes, neurons grow axons toward their target regions in response to both positive and negative stimuli. However, the extracellular signals controlling axonal growth are poorly understood. Here we report that ATP has a negative effect in axonal growth and concomitantly induces calcium transients at most distal region of the axon. This effect is mediated by P2X7 receptors (P2X7R) that we have identified at axonal growth cones, and which activation induced modifications in CAMKII and FAK activities, which were able to activate PI3-kinase and to modify the activity of its downstream targets. Moreover, we demonstrated that extracellular ATP levels in the growth cone are regulated by the tissue nonspecific alkaline phosphatase (TNAP). Thus, selective blockade of TNAP activity inhibited the axonal growth whereas addition of alkaline phosphatase promoted. Contribution of adenosine was excluded from effects induced by TNAP. In the axonal growth cones we found co-localization of P2X7 receptors and TNAP in microdomains. Additional pharmacological and molecular biological analyses suggested a close functional interrelation of TNAP and P2X7 receptors whereby TNAP prevents P2X7 receptor activation by hydrolyzing ATP in the immediate environment of the receptor. Furthermore, inhibition of the P2X7 receptor reduced TNAP expression whereas addition of alkaline phosphatase enhanced P2X7 receptor expression. Our results suggest that purinergic signalling mechanisms involving TNAP and the P2X7 receptor critically control axonal growth in hippocampal neurons whereby TNAP controls the local availability of extracellular ATP.

P13-15

Maternal glutamate intake during gestation and lactation modulates adenosine A1 and A2A receptors in rat brain from mothers and neonates

Antonio Lopez Zapata, Mairena Martin Lopez, Carlos Alberto Castillo Sarmiento, Jose Luis Albasanz Herrero, David Agustin Leon Navarro

Facultad de Químicas. CRIB., Universidad De Castilla-la Mancha, España

Pregnant rats were daily treated with 1 g/L of L-glutamate in their drinking water during pregnancy and/or lactation and the effect on adenosine A1 and A2A receptors in brains from both mothers and 15 days-old neonates was assayed using binding and real time PCR assays. Mothers receiving L-glutamate during gestational period showed a significant decrease on total adenosine A1 receptor number (75 %). A similar decrease was detected in mothers treated with L-glutamate during lactation (70 %) and throughout gestation and lactation (66 %). No variation in mRNA level coding adenosine A1 receptor was detected in mothers in any case. In both male and female neonates, adenosine A1 receptor was also decreased after chronic glutamate exposure during gestation (86 % and 88 %, respectively), lactation (69 % and 36 %, respectively) and gestation plus lactation (70 % and 82 %, respectively). No variations were found in mRNA level coding adenosine A1 receptor in neonatal brain in response to chronic glutamate intake throughout gestation and/or lactation. Concerning to adenosine A2A receptor, binding assays revealed that this receptor remains unaltered in maternal brain in response to glutamate exposure whereas a significant increase was detected in male and female neonates (283 % and 667 %). No variations were found in mRNA level coding adenosine receptor neither maternal nor neonatal brain in response to chronic glutamate intake throughout gestation and/or lactation. These results suggest that maternal L-glutamate intake during gestation and /or lactation modulates adenosine A1 and A2A receptors in both mother and neonates.

P13-16

P2X signaling in T cell lineage choice during development in the thymus

Michela Frascoli1, Ursula Schenk2, Michele Proietti1, Fabio Grassi1

1T cell development lab, Institute for Research in Biomedicine, Switzerland,2Pharmanalytica SA, Pharmanalytica SA Switzerland

In immature T cells, lineage choice during development in the thymus is dictated by T cell receptor (TCR) signal strength with increasing strength resulting in induction of the gamma/delta differentiation program. In fact, artificial reduction of gamma/deltaTCR signalling was shown to divert gamma/deltaTCR expressing cells toward the alpha/beta fate. We investigated whether pericellular ATP were controlling signal strength in thymocytes as observed in mature T cells. Analysis of transcriptional regulation of purinergic receptors in thymocyte subsets by real time (RT)-PCR revealed expression of P2X4, P2X7 and P2Y14 receptors in the precursors of the alpha/beta and gamma/delta lineage. Interestingly, expression of P2X7 was selectively increased in immature gamma/delta CD25+ cells. Analogously to mature T cells, these cells released ATP following TCR stimulation. Treatment of E14 fetal thymus organ cultures (FTOC) with oxidized ATP (oATP) as a P2X7 receptor antagonist, determined the generation of aberrant CD4+8+ cells (e.g. alpha/beta committed) expressing gamma/deltaTCR. This phenomenon correlated with impaired ERK phosphorylation in p2x7 knock-out immature gamma/delta thymocytes upon gamma/deltaTCR stimulation. Our results indicate that extracellular ATP influences T cell lineage choice during thymic development by regulating TCR signal strength.

P14. Animal models of purine dysfunction

P14-1

CD39/CD73 double knockout mice are protected against bleomycin-induced dermal fibrosis

(Selected for Oral Poster Presentation)

Edwin Chan1, Patricia Fernandez1, Hailing Liu1, Simon Robson2, Bruce Cronstein1

1Medicine/Clinical Pharmacology, New York University School of Medicine, USA,2Hepatology, Harvard University, USA

BACKGROUND: Adenosine may be formed intracellularly from adenine nucleotides or extracellularly through sequential dephosphorylation by nucleoside triphosphate diphosphohydrolase (CD39) and ecto-5′nucleotidase (CD73). Deficiency in either or both results in vastly decreased adenosine levels in tissues. The resultant decrease in tissue adenosine protects against bleomycin-induced pulmonary fibrosis. To further characterize the contribution of endogenous adenosine to skin fibrosis, we determined whether changes in nucleoside levels regulate dermal fibrosis.

METHODS: Male CD39/CD73-double deficient mice (CD39/73 KO) were injected with the fibrosing agent bleomycin (0.1U sc qod) for 18 days and compared to wild-type littermates. After sacrifice, dermal morphometric measurements were assessed on freshly excised skin and 6 mm skin punch biopsies (4 biopsies from the dorsum of each animal). Dermal collagen content was measured by hydroxyproline determination and skin adenosine levels were assessed by HPLC.

RESULTS: CD39/CD73 KO mice showed lower dermal thickness (0.29 ± 0.05 vs. 0.36 ± 0.10 mm), skin-fold thickness (0.79 ± 0.16 vs. 0.97 ± 0.37 mm), tensile strength (198.2 ± 7.3 vs. 248.7 ± 7.0 g) and hydroxyproline content (21.7 ± 1.2 vs. 26.5 ± 1.1 μg/mg tissue) (n = 6, p < 0.01 for each) compared to control mice after bleomycin treatment; correlating with a two-third decrease in adenosine levels compared to wild-type controls.

CONCLUSIONS: The skin is relatively resistant to the fibrosing effects of the well-known sclerosant, bleomycin, in an animal model of low tissue adenosine. Adenosine is necessary for the full sclerosing effects to occur in response to a toxic insult such as bleomycin. Blockade of adenosine or its downstream effectors may be useful in the treatment of diseases such as scleroderma where dermal fibrosis is a prominent manifestation.

P14-2

Transgenic expression of human CD39 protects against chemically induced diabetes and IBMIR following intraportal islet transplantation

(Selected for Oral Poster Presentation)

Joanne Chia1, Tanya Craig1, Tharun Mysore1, Lisa Murray-Segal1, Simon Robson, Anthony d'Apice1, Peter Cowan1, Karen Dwyer1

1St. Vincent's Hospital, University of Melbourne, Australia,2Beth Royal Deaconess Medical Centre, Harvard Medical School, United, States of America

Background: Islet transplantation is a potential cure for type 1 diabetes mellitus; however graft function is lost post transplantation due to blood-mediated inflammatory reaction (IBMIR) and recurrent autoimmunity. CD39 together with CD73 hydrolyses extracellular nucleotides to anti-inflammatory adenosine. Human CD39 expression on islets protects against IBMIR ex vivo. Aim: To examine the effect of hCD39 expression in mouse models of diabetes and syngeneic islet transplantation. Methods: Diabetes was induced in wild-type (WT), CD39KO, hCD39 transgenic (TG), A2ARKO and TGxA2ARKO mice using multiple low dose streptozotocin (MLDS). 400 WT or TG islets were transplanted intraportally into spontaneously diabetic mice. Results: WT mice became diabetic at day 42 (BGL 21.2 ± 2.4 mM) following MLDS. CD39KO mice developed diabetes earlier (BGL 24.5 ± 1.6 mM at day 8). In contrast, TG mice showed only a small rise in BGL (13.3 ± 0.8 mM at day 90). A2ARKO mice had heightened susceptibility to MLDS similar to CD39KO mice, whereas TGxA2ARKO mice were similar to WT mice. Irradiated TG mice reconstituted with WT, CD39KO or A2ARKO bone marrow remained euglycemic following MLDS (BGL 10.2 ± 1.2 mM; 10.8 ± 0.6 mM; and 13.8 ± 1.4 mM, respectively). 400 WT islets transplanted into diabetic syngeneic recipients led to a partial and transient fall in BGL, whereas mice receiving 400 TG islets were restored to long term (>120 days) euglycemia. Conclusions: The level of hCD39 expression correlates inversely with severity of hyperglycemia in the MLDS model, and hCD39 expression on tissues alone is sufficient for protection. Overexpression of hCD39 protects islet grafts from IBMIR in vivo and preserves islet function.

P14-3

CD39 protects in kidney ischaemia reperfusion injury (IRI) via adenosine 2B receptor signalling

Siddharth Rajakumar1, Bo Lu1, Sandra Crikis1, Simon Robson2, Anthony d'Apice1, Peter Cowan1, Karen Dwyer1

1Immunology Research Centre, St Vincent's Health, Melbourne, Australia,2Beth Israel Deaconess Medical Center, Harvard Medical School, USA

Background: IRI is a complex pathophysiological process implicated in numerous clinical settings including acute kidney injury and organ transplantation. The adenosine 2A (A2AR) and 2B receptor (A2BR) are important in the pathogenesis of renal IRI while CD39 is integral to the phenomenon of ischaemic preconditioning. In this study we examine the effects of CD39 over-expression in warm and cold renal IRI and the role of A2AR and A2BR.

Methods: Wild-type (WT), CD39-transgenic (CD39tg), adenosine 2A receptor-deficient (A2ARKO) and CD39tg x A2ARKO mice underwent unilateral renal ischaemia reperfusion injury (18 minutes ischaemia, 24 hour reperfusion) and renal function (serum creatinine, umol/l) and histological renal injury (scored S 0-9) assessed. Mice were pre-treated with an adenosine 2B receptor (A2BR) inhibitor (PSB1115). Kidneys from WT and CD39tg mice were transplanted into WT recipients with 5 hours cold ischaemia time (4 degrees) and assessed after 24 hours of reperfusion.

Results: In this model of IRI, WT mice exhibit severe renal injury [Cr 81.5, S 4.1] and CD39tg mice are protected [Cr 43.3, S 1.6, p < 0.05]. CD39tg x A2ARKO mice are protected to a similar extent as CD39tg mice. CD39tg mice pre-treated with PSB115 lose their protection [Cr 96.2, S 3.8] Compared to recipients of WT donor kidneys [Cr 163, S 4.2], recipients of CD39tg kidneys have improved renal function and tubular injury score at 24 hours [Cr 134, S 1.7, p < 0.05].

Conclusion: Over-expression of CD39 protects against warm and cold IRI. In warm IRI this protection is dependent on A2BR signalling.

P14-4

P2X7 receptor regulates the inflammatory response in Trichinella spiralis infected mice

Chris Keating1, Carlos Martínez2, Annmarie Surprenant3, David Grundy1, Pablo Pelegrín2

1Biomedical Sciences, University of Sheffield, Sheffield, S10 2TN, United Kingdom;2Inflammation and Experimental Surgery Group, University Hospital “Virgen de la Arrixaca”-FFIS, Carretera Palmar, 30120 Murcia, Spain;3Faculty of Life Science, Michael Smith Building D3315, University of Manchester, Manchester, M13 9PT, United Kingdom

Trichinella spiralis infection in mice is associated with enteric inflammation followed by post-infectious sensory and motor dysfunction. Here we use P2X7 knockout (KO) mice to investigate the role of P2X7 receptors in regulating the mechanisms underlying the generation of sensory dysfunction in this model. P2X7-deficient mice show an unbalanced inflammatory infiltrate into the lamina propia, with an increase of T lymphocytes and a decrease on macrophages and neutrophils. Following infection, peritoneal LDH levels were significantly increased in WT animals but remained unchanged in the P2X7-KOs. Enteric infection with T. spiralis is accompanied by a P2X7 dependent increase in IL-1beta in serum, spleen and peritoneal lavage; and a P2X7 independent increase in TNF-alpha. The inability of P2X7-deficient mice to develop visceral hypersensitivity suggests that the P2X7 mediated pathway is crucial in implementing this behavioral response to inflammation. This study has been supported by grants from isciii (EMER07/049) and CIBERhed (IP52).

P14-5

Antagonism of adenosine A2A receptors delays neurodegeneration in a lentiviral mouse model of Machado-Joseph disease

Nélio Da Mota Gonçalves1, Ana T. Simões2, Rodrigo A. Cunha2, Luis Pereira De Almeida2

1Centro de Neurociências e Biologia Celular, University of Coimbra, Portugal,2Center for Neuroscience and Cell Biology, Faculty of Pharmacy, University of Coimbra, Portugal

Machado-Joseph Disease (MJD) is a dominantly-inherited neurodegenerative disorder associated with an abnormal CAG expansion, which is translated into an expanded polyglutamine tract within ataxin-3, whose physiological function has been linked to ubiquitin-mediated proteolysis. Since adenosine A2A receptors (A2AR) afford neuroprotection, we now tested their ability to control MJD neurodegeneration. Male adult C57Bl6 mice were injected in the striatum either unilaterally with lentiviral vectors encoding mutant ataxin-3 or bilaterally with lentiviral vectors encoding mutant ataxin-3 in one hemisphere and wildtype ataxin-3 in the other hemisphere (Alves et al., Human Mol Gen 17:2071). Mice were sacrificed at different time points (from 2–12 weeks) for striatal immunohistochemical analysis of ataxin-3 inclusions, cresyl violet (neurodegeneration), DARPP32 and NeuN (neuronal markers), MAP2 (dendritic marker), synaptophysin (synaptic marker), CD11b (microgliosis) and GFAP (astrogliosis), which was compared between both hemispheres to evaluate MJD-induced modifications. Treatment with the non-selective adenosine receptor antagonist caffeine (1 g/L, applied through drinking water) from 3 weeks before insult onwards, increased the number of mutant ataxin-3 inclusions, reduced the loss of neuronal, dendritic and synaptic markers, microgliosis and astrogliosis over time in the mutant ataxin-3-treated hemisphere. Furthermore, A2AR knockout mice also displayed increased number of ataxin-3 inclusions and a decreased and delayed neurodegeneration in the striatum of mutant ataxin-3-treated hemisphere at 8–12 weeks. This indicates that caffeine delays MJD-associated striatal pathology likely through antagonism of A2AR.(Supported by: FCT)

P14-6

The zebrafish as a model to study nucleotide signalling upon neuronal injury

Dirk Sieger, Francesca Peri

EMBL, Germany

Microglia are the “professional” phagocytes of the brain. These cells constantly scan the healthy brain with their long and dynamic branches, which in response to lesion are rapidly redirected towards the site of damage. Microglia express several P2X and P2Y receptors and in recent years some have been associated with the response of microglia to neuronal injury. It has been shown that microglia are attracted and phagocytose damaged neurons through the nucleotide-evoked activation of P2Y(12) and P2Y(6) receptors, respectively. Knowing the molecules that control these complicated cell behaviors is only the beginning ultimately, we need to understand how these factors work both in space and time, to influence the behavior of the entire microglial population. We are establishing the zebrafish larvae as a powerful model system to investigate the role of nucleotide signaling upon neuronal injury in vivo. Lesions in the zebrafish brain are generated with a pulsed 532 nm laser and the response of microglia is monitored live by in vivo confocal imaging. By specific inhibition of P2X and P2Y receptors we can show which receptors are required for cell polarity and migration upon injury. A genetically encoded ATP sensor will allow us to show the distribution of ATP upon injury. Furthermore by expressing a P2Y(12)-GFP fusion it will be possible to identify the cells that are directly activated by ATP. These approaches will lead to a better understanding how nucleotides are organizing the response of microglia upon injury in vivo.

P14-7

Effect of P2X7 receptor knockout on exocrine secretion of pancreas, salivary and lacrimal glands

Ivana Novak, Ida M. Jans, Louise Wohlfahrt

Department of Biology, University of Copenhagen, Denmark

The P2X7 receptor is expressed in exocrine glands, but it is not clear whether the receptor has long-term effects related to regulation of cell survival, or whether it regulates epithelial transport and thus secretion. The aim of our study was to determine whether the P2X7 receptor affects fluid secretion in pancreas, salivary glands and tear glands. We used wild-type mice and P2X7−/− mice (courtesy of Pfizer) that were anaesthetized and stimulated with pilocarpine (10 mg/kg, I.P.) and secretion was collected from respective glands. In cell preparations from these glands we also measured ATP release and intracellular calcium activity using fura-2. The data shows that in P2X7−/− mice pancreatic secretion was reduced by 30% and salivary secretion by 50%, but tear secretion was increased by 30–40%. In all cell preparations ATP release could be elicited by carbachol and other agonists, and there was no difference between +/+ and−/− mice. ATP (0.1 mM) elicited 50% lower fura-2 peak in pancreatic cells from P2X7−/− animals. On the other hand, carbachol (0.05 mM) increased fura-2 peak responses in parotid and lacrimal cells of P2X7−/− animals. In some physiological parameters monitored, the P2X7 receptor function was suppressed females. In conclusion, these results demonstrate that cholinergic stimulation releases ATP that can via P2X7 receptors up-regulate pancreatic and salivary secretion but down-regulate tear secretion. Our data indicate that there is an interaction between purinergic, cholinergic and possibly estrogen signals. We conclude that the P2X7 receptor is important in short-term regulation of exocrine gland secretion.

P14-8

Generation of BAC transgenic mice expressing fluorescent P2X3 receptors

(Selected for Oral Poster Presentation)

Ralf Hausmann1, Tanja Nußbaum1, Markus Grohmann2, Heike Franke2, Günther Schmalzing1

1Molecular Pharmacology, RWTH Aachen University, Germany,2Rudolf-Boehm-Institute of Pharmacology and Toxicology, University of Leipzig, Germany

Background: P2X3 receptors are known to be crucially involved in sensory and pain transmission. Accordingly, information about the precise distribution of P2X3 receptors on neurons in health and disease is of significant interest in relation to physiological and pathophysiological pain states. Methods. To facilitate the morphological and functional identification of neurons carrying P2X3 receptors, we have generated a bacterial artificial chromosome (BAC) construct encoding the mouse P2X3 subunit C-terminally fused in frame with EGFP under the control of the endogenous mouse P2RX3 gene promoter. The BAC recombineering was performed by a galK selection-based protocol according to Warming et al. (http://recombineering.ncifcrf.gov/). Results. After pronuclear injection, four founder mice were tested positive for successful integration of the P2X3-GFP transgene. Confocal microscopy of freshly isolated dorsal root ganglia (DRG) neurons of one BAC strain showed moderate GFP fluorescence. In the three other BAC strains, only very weak fluorescence was detected that was difficult to distinguish from background. Immunostaining with a monoclonal GFP antibody and counterstaining with an ILB4 antibody verified the presence of GFP in the cultured DRG neurons of all four P2X3-GFP BAC strains. Conclusion. The P2X3-GFP transgene is expressed at least at low levels in all four positively tested P2X3-GFP BAC strains. The overall low fluorescence of our transgenic mice may in part originate from direct EGFP-tagging of the P2X3 receptor, which as a membrane protein might be synthesized at significantly lower levels than soluble EGFP.

P15. Novel purine-based therapeutics

P15-1

Effects of chronic adenosine A2A receptor blockade on Huntington’s disease pathophysiology

(Selected for Oral Poster Presentation)

Stéphane Mievis1, Cécile Duru2, Clémence Simonin3, Claire-Marie Dhaenens4, Sylvie Burnouf4, Gilbert Vassart1, Luc Buée4, Florence Richard5, Michael Genin6, Bernard Sablonniere4, Alexandra Durr7, Anne-Catherine Bachoud-Levi8, Pierre Krystkowiak2, Catherine Ledent1, David Blum4

1IRIBHM, Université Libre de Bruxelles, Belgium,2Service Neurologie, UMR CNRS 8160, CHU Amiens, France,3Service de Neurologie et Pathologie du Mouvement, EA 2683, CHRU Lille, France,4UDSL, Inserm U837, France,5Unité d'Epidémiologie Pôle de Santé Publique, CHRU Lille, France,6Unité de Biostatistiques, Pôle de Santé Publique, CHRU Lille, France,7AP-HP, Departement de Genetique et Cytogenetique, Groupe Hospitalier Pitié-Salpetriere, Paris, France,8AP-HP, Centre de référence maladie rare/maladie de Huntington, France

Background: Several lines of evidence, including from genetic study, involve A2A receptors (A2ARs) in Huntington’s disease (HD) pathophysiology. However, whether their chronic blockade is of therapeutical interest remains unclear.

Methods: We have genetically invalidated A2ARs in the N171-82Q transgenic model of HD and monitored survival, motor performances and histopathological changes. In addition, in a preliminary study, we have evaluated whether caffeine consumption is a modifier of age at onset in a cohort of French HD patients.

Results: Our data demonstrate that A2AR invalidation significantly decreased the survival of N171-82Q mice by 16% and worsens their motor performances by more than 30% while it did not impact on gross anatomy. Using in situ hybridization, we observed that mRNA expressions of enkephalin, substance P and DARPP32 were significantly decreased in N171-82Q mice but that A2AR invalidation only further decreased enkephalin expression by 25%. Supporting the deleterious influence exerted by A2A receptor blockade in HD, in a small cohort of HD patients, we also demonstrated that the consumption of the non-specific A2A receptor antagonist caffeine significantly influenced age at onset with an advanced age at onset in high consumers (>250 mg/d) as compared to low consumers (<100 mg/d).

Conclusion: Altogether, our data support that the chronic blockade of A2AR exerts a deleterious effect on HD pathophysiology and do not support A2A receptor blockade as therapeutical option to explore in HD.

P15-2

Glial overexpression of Adenosine Kinase is associated with seizure occurrence in a transgenic model of AD-like Tau pathology

Humez Sandrine1, Anisur Rahman2, Cyril Laurent1, Luc Buée1, Nicolas Sergeant1, David Blum1, Detlev Boison2

1UDSL, Inserm U837, France,2Robert Stone Dow Neurobiology Laboratories, Legacy Research, Portland, USA

Background: Astrogliosis is a pathological hallmark of Alzheimer’s disease (AD). Previous work has demonstrated that astrogliosis can lead to increased expression of the adenosine-removing enzyme adenosine kinase (ADK), which can cause adenosine deficiency and subsequently trigger “silent” seizures. Interestingly, patients with AD frequently suffer from episodes of silent seizures in deep brain structures. So far, seizure occurrence in AD has only been related to the amyloid side of the disease. Whether AD-related seizures involve adenosine and whether Tau may play a role remains unknown. We hypothesized that hippocampal Tau pathology may lead to astrogliosis-related-adenosine deficiency through adenosine kinase over-expression and seizure occurrence.

Methods: We have evaluated seizures and ADK expression in the THY-Tau22 transgenic model of AD-like Tau pathology. THY-Tau22 mice and littermate controls were equipped with electro-encephalographic (EEG)-recording electrodes in CA1. After recovery, animals were subjected to at least 24h of continuous video-EEG-monitoring. After completion of monitoring, the hippocampal formation of the animals was subjected to immunohistochemical analysis.

Results: THY-Tau22 mice exhibited progressive development of astrogliosis and cognitive impairment that paralleled hippocampal Tau pathology. At the age of 6–7 months, all recorded animals experienced frequent recurrent electrographic seizures (typically around 4 seizures per h with each seizure lasting about 1 min) as compared to littermate controls. In contrast to littermate controls, all animals with seizures displayed profound astrogliosis and overexpressed astrocytic ADK throughout the hippocampal formation.

Conclusion: Our data establish that overexpression of ADK in astroglia and hippocampal seizures are a pathological hallmark related to Tau pathology.

P15-3

Membrane vesicles released by microglial cell may be involved in purinergic signaling

Agnes Kittel1, Petra Misják2, Dániel Boncz3, Bence György2, Krisztina Pálóczy2, Beáta Sperlágh1, Edit Buzás2

1Laboratory of Molecular Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Hungary,2Department of Genetics, Cell- and Immunobiology, Semmelweis Medical School, Hungary,3Department of Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Hungary

Membrane vesicles (MVs) are small (50–1000 nm) membrane-bound structures released by many kinds of cells in physiological/pathophysiological conditions. They are considered as important mediators of intercellular communication. Our goal was to investigate whether MVs can play a role in purinergic signaling. As a model system we used the BV2 microglia cell line, since microglia cells are very sensitive to changes in the concentration of extracellular ATP, and express both NTPDase1 (an ectonucleotidase) and purinergic receptors. Among P2 receptors, P2Y12 was found on the leading microglial cell process moving toward the higher ATP or nucleotide concentrations. We worked out a protocol for the preparation of MVs from tissue culture supernatants. Enzyme cytochemistry was applied for demonstration of ecto-ATPase activity, immunocytochemistry for NTPDase1, P2Y12 and CD63 (to identify exosomes), quantitative real time RT-PCR for investigation of changes in the expression of NTPDase1 and P2Y12 after LPS treatment. FACS experiments proved significant increase in the proportion of annexin and CD11b positive MVs. Our results show that BV2 microglial cell line is able to produce a wide range of MVs, different in size, morphology and in molecular composition. Furthermore, LPS treatment can modify their composition and the ratio of MV subpopulations. Immunostaining revealed that mainly CD63 positive exosomes (50–100 nm in diameter) and irregular shaped membrane vesicles (100–1000 nm) contain P2Y12 and NTPDase1, and the proportion of the latter is increased after LPS treatment. These data suggest the involvement of MVs in purinergic signaling and may support their potential use as diagnostic tools.

P15-4

Essential role for ATP in the pathogenesis of COPD and emphysema

Marco Idzko1, Sanja Cicko1, Monica Lucattelli2, Marek Lommatzsch3, Tobias Müller1, Giovanna de Cunto2, Silvia Cardini2, Melanie Grimm1, Thorsten Dürk1, Gernot Zissel1, Stephan Sorichter1, Davide Ferrari4, Francesco di Virgilio4, J. Christian Virchow3, Giuseppe Lungarella2

1Department of Pneumolgoy, University Hospital Freiburg, Germany,2Department of Pneumolgoy, University Siena, Italy,3Department of Pneumolgoy, University Hospital Rostock, Germany,4Department of Pneumolgoy, University Hospital Ferrara, Italy

Background: Chronic obstructive pulmonary disease (COPD) is an inflammatory disease often associated with cigarette smoke inhalation. Extracellular ATP induces inflammation by binding to purinergic receptors, but its role in COPD is unknown. Methods: The influence of cigarette smoke on endobronchial ATP was studied in healthy adults. In addition, endobronchial ATP concentrations were studied in smokers and ex-smokers with COPD of different severity. The expression of purinergic receptors on immune cells and its functional implications were studied in patients with COPD and in controls. Finally, the role of ATP was investigated in a mouse model of acute and chronic smoke-induced lung inflammation where neutralisation of ATP or purinergic receptor blockage, as well as purinergic receptor deficient animals were studied. Results: Endobronchial ATP concentrations were elevated in smokers and even further elevated following acute smoke exposure. Endobronchial ATP was also increased in patients with COPD and in ex-smokers. Highest levels were observed in patients with severe COPD and there was a correlation with endobronchial neutrophil numbers and FEV1-reduction. Purinergic receptors were upregulated on neutrophils and macrophages from patients with COPD, which was shown to have pro-inflammatory effects (such as recruitment and elastase release/MMP-9 secretion). In the mouse model, we showed that the development of smoke-induced lung inflammation and emphysema was markedly reduced by neutralizing ATP or blocking purinergic receptors. Finally in P2X7−/− knockout animals ATP failed to induce COPD associated changes. Conclusion: Endogenous ATP, via activation of P2Y2R and P2X7R, contributes to the development of chronic airway inflammation and emphysema. Thus targeting P2R might be a new therapeutic option for COPD.

P15-5

A highly potent nucleotide-based drug for reducing intraocular pressure

Eliahu Shay1, Martín-Gil Alba2, Perez de Lara María Jesús2, Pintor Jesús2, Camden Jean3, Weisman Gary A.3, Lecka Joanna4, Sévigny Jean4

1Department of Chemistry, Bar-Ilan University, Israel,2Dep. Bioquímica, Universidad Complutense de Madrid, Spain,3Biochemistry Department, University of Missouri-Columbia, Columbia, US,4Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Université Laval, QC, Canada

Extracellular nucleotides can modify the production or drainage of the aqueous humour via activation of P2 receptors, and therefore affect the intraocular pressure (IOP). We have synthesized slowly-hydrolysable nucleoside di- and tri-phosphate analogues, 1, and 8–14. Analogues 8–14 were completely resistant to hydrolysis by alkaline phosphatase over 30 min at 37°C. In human blood serum, analogues 8–14 exhibited high metabolic stability, e.g., analogues 9 and 10–14 were only 15% and 0% degraded after 24 h, respectively. Moreover, analogues 8–14 were highly stable at pH 1.4 with t1/2 values for hydrolysis of 1 h–30 days. Analogues 8–14 were agonists of the P2Y1 receptor (EC50 0.57–9.54 µM, vs.0.0025 µM for 2-MeS-ADP). Ocular administration of analogues 1 and 9 into rabbits reduced IOP by about 32%, (EC50 95.5 nM and 30.2 µM for analogues 9 and 1, respectively). To date, analogue 9 is the most potent and stable nucleotide reported to reduce IOP. Moreover, this analogue was more effective at reducing IOP than several common glaucoma drugs. In addition, although slightly less efficacious than timolol maleate, analogue 9 represents a promising alternative to timolol maleate, which cannot be used for the treatment of patients suffering from asthma or cardiac problems.

P15-6

Inhibition of P2X7 receptor promotes differentiation in neuroblastoma cells

Rosa Gómez-Villafuertes1, Ana del Puerto2, Miguel Díaz-Hernández1, Juan I. Díaz-Hernández1, Paula G. Huerta1, Juan J. Garrido2, Mª Teresa Miras-Portugal1

1Dpto. Bioquímica y Biología Molecular, Fac. Veterinaria, Universidad Complutense de Madrid, Spain,2Centro de Biologı´a Molecular ‘Severo Ochoa’, CSIC-UAM, Spain

ATP, via purinergic P2X receptors, acts as a neurotransmitter and neuromodulator in both systems, and is also involved in many biological processes, including cell proliferation, differentiation and apoptosis. Previously, we have reported that P2X7 receptor inhibition promotes axonal growth and branching in cultured hippocampal neurons (Díaz-Hernández et al., 2008). Here, we demonstrate that P2X7 receptors negatively regulate neurite formation in murine Neuro-2a (N2a) neuroblastoma, a cell line that express native P2X7 receptors (Gómez-Villafuertes et al., 2009). Our results showed that either pharmacological inhibition of P2X7 receptor or its knockdown by small hairpin RNA interference resulted in increased neuritogenesis in N2a cells cultured in low serum-containing medium, whereas P2X7 overexpression significantly reduced the formation of neurites. Interestingly, P2X7 receptor inhibition modified the phosphorylation state of Ca2+/calmodulin-dependent kinase II (CaMKII), focal adhesion kinase (FAK), Akt and glycogen synthase kinase 3 (GSK3), all protein kinases that participate in signalling cascades related to neuronal differentiation. Together, our results support a role of P2X7 receptors in the regulation of neuritogenesis and in the fine control of the balance between proliferation/differentiation of neuroblastoma cells. Moreover, our data point to P2X7 receptor antagonists as future potential therapeutic agents in the treatment of neuroblastoma tumour cells.

References:

Díaz-Hernandez M, del Puerto A, Díaz-Hernandez JI, Diez-Zaera M, Lucas JJ, Garrido JJ, Miras-Portugal MT. 2008. J Cell Sci. 121: 3717-28. Gómez-Villafuertes R, del Puerto A, Díaz-Hernández M, Bustillo D, Díaz-Hernández JI, Huerta PG, Artalejo AR, Garrido JJ, Miras-Portugal MT. 2009. FEBS J. 276: 5307-25.

P15-7

Functional characterisation of adenine and the new adenine receptor ligands PSB-09073 and PSB-08162 in the rat cingulate cortex

Frauke Berger1, Kathrin Sichardt1, Thomas Borrmann2, Christa E. Müller2, Karen Nieber1

1Institute of Pharmacy, University Leipzig, Germany,2PharmaCenter Bonn, Pharmaceutical Sciences Bonn (PSB), Pharmaceutical Institute, University Bonn, Germany

The purine nucleobase adenine was found to be the endogenous ligand of an orphan rat G-protein coupled receptor. PSB-09073 and PSB-08162 were synthesised as new ligands for the adenine receptor. Biological activity of these compounds has been shown in cAMP assays. PSB-09073 was characterised as an agonist and PSB-08162 as an antagonist at the adenine receptor (personal communication C.E. Müller, Bonn). The aim of the present study was to evaluate the effect of adenine, PSB-09073, and PSB-08162 on the synaptic transmission in the rat cingulate cortex. Therefore, the mRNA of the adenine receptor was detected in tissue probes of the rat cingulate cortex. Functional investigations were done using intracellular recordings with glass microelectrodes on pyramidal cells in layer V of rat brain slices containing parts of the cingulate cortex. Postsynaptic potentials (PSPs) were evoked by electrical field stimulation with a concentric bipolar electrode. Adenine decreased the amplitude of the PSPs reversibly and concentration dependently (10 µM–1 mM) but had no influence on membrane potential and input resistance. NMDA and non-NMDA component of the PSPs were inhibited in the same range suggesting an influence on the presynaptic glutamate release. The adenine receptor agonist PSB-09073 (500 µM) caused an inhibition of the PSPs to a lower extent in comparison to adenine. The adenine receptor antagonist PSB-08162 (100 µM) completely blocked the adenine induced inhibition of the PSPs. The current results confirm the functional expression of adenine receptors in the rat cingulate cortex. The new ligands PSB-09073 and PSB-08162 show the expected effects as agonist or antagonist.

P15-8

Renal effects of adenosine on diabetic Wistar and SHR rats

Manuela Sofia Rodrigues Morato1, Daniela Patinha2, Joana Afonso2, Teresa Sousa2, António Albino-Teixeira2

1Pharmacology Department, Faculty of Pharmacy of Porto and REQUIMTE-FARMA, University of Porto, Portugal,2Institute of Pharmacology and Therapeutics Faculty of Medicine of Porto and IBMC, University of Porto, Portugal

Background: The purpose was to investigate the effect of 2-chloroadenosine (2CADO) and 1,3-dipropyl-8-sulfophenylxanthine (DPSPX) on renal function, renin-angiotensin system (RAS) and oxidative stress of diabetic rats. Methods: Fourteen days after i.p. injection with streptozotocin (65 mg/Kg; STZ) or vehicle, male Wistar and SHR (12 weeks) rats were i.p. infused with 2CADO (5 mg/Kg/d) or DPSPX (90µg/Kg/h) for 7 days. Systolic blood pressure (SBP) was measured with intra-aortic catheters. Glucose, proteins, angiotensinogen (AGT), H2O2, glomerular filtration rate (GFR), catalase activity and TBARS were quantified by standard methods in plasma, 24h-urine and kidneys. Statistical analysis by Student t-test or ANOVA-Dunnett's test. Results: STZ increased plasma glucose, urinary glucose and protein excretion in both strains, and GFR in SHR rats. 2CADO attenuated glucose excretion. DPSPX attenuated glucose and protein excretion in Wistar rats and GFR in SHR rats. Diabetes increased urinary excretion of AGT; 2CADO decreased this effect in Wistar-STZ rats. Diabetes increased catalase activity and H2O2 production in the medula of Wistar rats; 2CADO attenuated this increase. In SHR rats, cortical production of H2O2 decreased with diabetes and more with 2CADO. SBP was maintained in Wistar rats; in SHR rats, paralleled the results on H2O2. Conclusion: STZ-induced diabetes alters renal function, increases renal RAS and oxidative stress in Wistar and SHR rats. Adenosine seems more relevant in Wistar- than in SHR-diabetic rats. Supported by FCT (PTDC/SAUFCF/67764/2006; SFRH/BD/43187/2008) and Abbott Diabetes Care.

P15-9

P2X7 receptor activation during seizure-induced cell death

Tobias Engel1, Rosa Gomez-Villafuertes2, Katsuhiro Tanaka1, Paula Garcia-Huerta2, Maria Teresa Miras-Portugal2, David Henshall1, Miguel Diaz-Hernandez2

1Royal College of Surgeons in Ireland, Ireland,2Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain

Epilepsy is a common and disabling neurologic disorder which affects about 1% of the population. Seizures are the result of abnormal electrical activity in the brain and they can be profoundly disabling. In addition, experimental modelling and clinical neuroimaging has shown that seizures are capable of causing neuronal death and this may contribute to epileptogenesis, impairments in cognitive function or the epilepsy phenotype. In at least 30–40 % of patients’ treatment is inadequate, with patients continuing to experience seizures; therefore, there is an important need to develop new antiepileptogenic drugs. Prolonged single seizures (status epilepticus) and repeated seizures over time are damaging to the brain. ATP acts as a fast neurotransmitter via cationic ligand-gated P2X receptors. P2X7 receptors gate Na+, K+ and Ca2+, and are present in microglia as well as presynaptic terminals in CNS regions, including the hippocampus, where they may enhance glutamate release. P2X7 receptors are up-regulated in animal models of epilepsy and P2X7-mediated currents are increased after seizures in mice. Here, we investigated whether inhibition of P2X7 receptors has effects on cell death using an in vivo mouse model of focal-onset status epilepticus triggered by intra-amygdala kainic acid. P2X7 receptor levels were increased after KA injection and in chronic epileptic mice. Intracerebroventricular injection of the specific P2X7 inhibitor BBG or anti-P2X7 antibodies significantly reduced hippocampal and cortical damage after status epilepticus. These data demonstrate that the P2X7 receptor contributes to seizure-induced neuronal death in vivo. P2X7 receptors may represent a target for neuroprotection and perhaps anti-epileptogenesis.

P15-10

Synthesis of nucleoside-5′-monophosphates as prodrugs of adenosine A2A Receptor agonists activated by ecto-5′-nucleotidase

Ali El-Tayeb1, Ajiroghene Thomas1, Jürgen Schrader2, Christa E. Müller11

1University of Bonn, Germany,2Department of Cardiovascular Physiology, Heinrich-Heine University Düsseldorf, Germany

Background: The systemic application of adenosine A2A receptor-selective agonists as anti-inflammatory agents is limited by their potent hypotensive activity.1 Therefore we developed phosphorylated A2A agonists as prodrugs which are preferably activated at the site of inflammation by ecto-5′-nucleotidase.1 To optimize our lead compound 2 cyclohexylethylthio-AMP, we synthesized analogs, in which the cyclohexyl ring is replaced by saturated heterocyclic rings. Methods: The target adenosine derivatives were prepared by alkylation of substituted hetero-cyclic rings with bromoethanol in alkaline medium followed by mesylation and subsequent coupling with 2-thioadenosine. Alternatively, amides were obtained via reaction of bromoace-tylbromide with substituted nitrogen-containing heterocyclic rings followed by coupling with 2-thioadenosine. The obtained compounds were purified and the adenosine receptor affinity and selectivity of the new nucleosides was determined by radioligand binding studies. Results: Many of the 25 new nucleosides showed A2A affinity of <100 nM; two compounds showed A2A affinity <25 nM with enhanced selectivity against other adenosine receptor sub-types. 2-(2-(4-(4-Fluorophenyl)piperazin-1-yl)ethylthio)adenosine (Ki A2A 9.5 nM) was further phosphorylated at the 5´-position. Conclusion: We obtained very potent and selective A2A agonists in a series of 2-thioadenosine derivatives. Subsequent phosphorylation was performed for selected compounds in order to study their properties as substrates of ecto-5′-nucleotidase.

1. El-Tayeb, A. et. al. J. Med. Chem., 2009, 52, 7669–7677.

P15-11

Anti-inflammatory action of extracellular pyrophosphate produced by macrophages ENPP activity

Alberto Baroja-mazo1, Gloria Lopez-castejón2, Maria Barberà-cremades1, Ana I. Gómez1, Pablo Pelegrin1

1Inflammation and Experimental Surgery Group, Hostpial Universitario Virgen de la Arrixaca -Fundación para la Formación e Investigación Sanitarias (FFIS), Spain,2Faculty of Life Science, University of Manchester, United Kingdom

Plasticity is a well-known property of macrophages and is controlled by different signaling pathways induced by changes in environmental signals. Macrophage polarization is a gradient of activation states adjusted from the extremes called classic (M1) and alternative (M2) activation, this gradient on polarization results to be crucial for the immune response. Here we show that both, during in vitro and in vivo, M1 and M2 macrophage polarization was accompanying with an opposite pattern of up- and down-regulated genes, being genes that control the extracellular metabolism of ATP a clear hallmark. In M1 macrophages ectonucleotidases from the NTPDase family where up-regulated, whereas in M2 macrophages were from the ENPP family, suggesting the generation of pyrophosphates (PPi) in M2 alternative macrophages. We found that extracellular PPi signals through M2 macrophages to damp both, NLRs and TLRs signaling to adjust cytokine production in response to resolving microenvironments. In this context extracellular PPi was able to enhance the resolution phase of a peritonitis mouse model though the decrease of pro-inflammatory cytokines without affecting to the total peritoneal leukocyte infiltration. This dynamic regulation of macrophage activation by extracellular PPi led to the suppression of pro-inflammatory responses. Our study reveals another level of plasticity modulating innate immunity to resolve inflammation. This study has been supported by grant from isciii (EMER07/049 and FIS09/00120) and Fundación Séneca (11922/PI/09).

P15-12

Adenosine A2A receptor occupancy promotes dermal fibrosis by modulating Fli1 and CTGF expression

Hailing Liu, Edwin Chan, Patricia Fernández, Rina Karuvelil, Bruce Cronstein

Clinical Pharmacology NYU Medical Center, USA

Background: We have previously reported that adenosine, acting at A2A receptors, enhances dermal collagen production although the mechanism by which adenosine receptor stimulation promotes collagen production is not clear. Fli1 is a known transcriptional repressor of fibrillar collagen genes and connective tissue growth factor (CTGF/CCN2) in dermal fibroblasts. To further clarify the mechanism by which A2A receptor stimulation induces dermal collagen accumulation, we explored the effects of A2A receptor occupancy on Fli1 and downstream mediators of fibroblast matrix production. Methods: We stimulated cultured primary human dermal fibroblasts with the selective adenosine A2AR agonist CGS21680 (1mM) for varying time periods and measured message levels (real time-RTPCR) for fli1 and CTGF. In addition we measured cell associated and supernatant levels of collagen I and nuclear levels of fli1 by Western Blot. Results: Adenosine A2A receptor stimulation for 4 hours reduced Fli1 mRNA expression by 47 fold (vs. control) 4hrs) and reduced nuclear protein levels of Fli1 by 31.9 CGS21680 (24hrs). Because diminished nuclear Fli1 should increase CTGF levels we next examined the effect of A2A receptor stimulation on CTGF mRNA and protein secretion. CTGF mRNA level was increased following A2A receptor stimulation 8 hours by 3.68-fold (vs. control) by A2AR stimulation. In keeping with the change in mRNA CGS21680 stimulated a 4.69-fold increase in CTGF protein secretion (24 hours) as well (p<0.03, n=4). As expected A2A receptor stimulation increased collagen I secretion (1.82-fold, vs. control, p<0.05, n=3) and the increase in collagen production was completely abrogated by an antibody to CTGF (1.07+0.04 fold, vs. CGS, p<0.05, n=3) but not by control antiserum (1.57+0.027, Vs. CTGF-Ab, p<0.05, n=3). Conclusion: A2AR occupancy promotes dermal matrix production by suppressing expression of the transcriptional repressor Fli1 leading to an increase in CTGF expression which acts in an autorcrine fashion to stimulate collagen production. These findings further suggest that modulation of A2AR function may be a novel therapeutic target for limiting fibrosis in such conditions as scleroderma.

P15-13

Adenosine A2A receptors control the levels of BDNF in resting and LPS-stimulated microglial cells in culture

Catarina Vale Gomes, Rui Sanches, Rodrigo A. Cunha

Centre for Neuroscience of Coimbra, University of Coimbra, Portugal

Blockade of adenosine A2A receptors (A2AR) affords neuroprotection in different neurodegenerative conditions, where microglia activation and neuroinflammation are present. Since microglia cells release neurotrophic factors and A2AR control their actions, we now explored if A2AR directly control the levels of BDNF in resting microglia cells and upon their activation with lipopolysaccharide (LPS). We tested the impact of either LPS (100 ng/ml, 6 hours) and/or SCH58261 (50 nM; A2AR antagonist, added 20 min before LPS) in the levels of BDNF (mature protein, 13 kDa, assessed by Western blot analysis normalised according to beta-tubulin levels) in a microglia cell culture line (N9). We first confirmed that A2AR were present in N9 cells and exposure to LPS tended to decrease A2AR density (70 ± 7%, n = 7, P < 0.05). LPS (100 ng/ml) decreased BDNF immunoreactivity to 74 ± 7% (n = 9, P < 0.05) of control and SCH58261 prevented the LPS-induced decrease of BDNF levels (143 ± 29%, n = 7, P < 0.05). Neither SCH58261 nor LPS modified the viability of N9 cells, as assessed by a preservation of MTT reduction. These results provide the first demonstration that A2AR control BDNF levels in a microglia cell line, where A2AR counteract the effects of LPS. This suggests that the interaction between A2ARs and neurotrophic factors may impact on neuroinflammation and thus, on degeneration and neuronal repair. (Supported by FCT; the generous help of Raquel Ferreira is greatly acknowledged)

P15-14

Adenosine receptors partially prevent excitotoxic cell death in cortical neurons

Carlos Alerto Castillo Sarmiento, David León, Ballesteros-Yañez Inmaculada, Albasanz José Luis, Martín Mairena

Facultad de Químicas. CRIB., University of Castilla La Mancha, Spain

Background: Adenosine is a neuromodulator which acts through adenosine receptors regulating functions as inhibition of glutamate release. Adenosine A1 and A2A receptors activation most often regulate opposite actions. Methods: Primary rat cortical neurons and rat C6 cells, an astrocytic derived cell line, were exposed to 100 µM L-glutamate and cell viability and transduction pathways mediated by both A1 and A2A receptors were analyzed using radioligand binding assays, real time PCR and enzymatic assays. Results: Glutamate induced excitotoxic damage only in cortical neurons being C6 cells preserved. In C6 cells, adenosine A1 and A2A receptors were increased and decreased respectively. Consequently, A1-mediated adenylyl cyclase (AC) inhibition and A2A-mediated AC stimulation were, respectively, increased and decreased after glutamate exposure. In cortical neurons, glutamate treatment increased both A1 and A2A receptors. Moreover, AC responsiveness to A1 or A2A receptors agonists was potentiated in these cells, where pharmacological activation of AC induced cell death. Finally, activation of A1 receptor or blockade of A2A receptor during glutamate treatment partially prevented the glutamate-induced cell death detected in cultured cortical neurons. Conclusion: Results show that adenosine receptors are regulated by glutamate being this regulation dependent on the cell type and suggesting that adenosine receptors could be promising targets in the therapy against excitotoxic cell death.

P15-15

Structure-activity relationship of (N)-methanocarba phosphonate analogues of 5′-AMP as cardioprotective agents acting through a cardiac P2X receptor

T. Santhosh Kumar1, Si-Yuan Zhou2, Bhalchandra V. Joshi1, Ramachandran Ramachandran1, Tiehong Yang2, Bruce T. Liang2, Kenneth A. Jacobson1

1Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, USA,2Pat and Jim Calhoun Cardiology Center, University of Connecticut Health Center, USA

Background: Cardiac P2X receptors represent a novel and potentially important therapeutic target for the treatment of heart failure. MRS2339, is an (N)-methanocarba monophosphate derivative of 2-chloro-AMP that contains a rigid bicyclic ring system (bicyclo[3.1.0]hexane) in place of ribose, activates this cardioprotective channel. This ring system impedes hydrolysis of the 5′-phosphate in a model compound by its nucleotidase. Methods: A more chemically and biologically stable linkage than the phosphate group in MRS2339 has been introduced in the form of the phosphonate groups using Michaelis–Arbuzov and Wittig reactions, which provided various short chain, saturated or unsaturated long chain phosphonate and short chain methyl phosphonate analogues, which were chronically administered via mini-osmotic pump (Alzet). Results: Certain analogues significantly increased intact heart contractile function in calsequestrin-overexpressing mice (genetic model of heart failure) compared to vehicle-infused mice. Two phosphonates, (1′S,2′R,3′S,4′R,5′S)-4′-(6-amino-2-chloropurin-9-yl)-2′,3′-(dihydroxy)-1′-(phosphonomethylene)-bicyclo[3.1.0]hexane, MRS2775, and its homologue MRS2935, both 5′-saturated, containing a 2-Cl substitution, improved echocardiography-derived fractional shortening (20.25% and 19.26%, respectively, versus 13.78% in controls), while unsaturated 5′-extended phosphonates, all 2-H analogues, and a methylphosphonate were inactive. However, it is worth noting that all these phosphonate analogues were inactive at P2Y1 receptor, which excludes the possibility that the observed cadiovascular effects of the phosphonate derivatives were a result of activation of an endothelial P2Y1 receptor. Conclusion: Thus, chronic administration of nucleotidase-resistant phosphonates conferred a beneficial effect, likely via cardiac P2X receptor activation. Therefore, we have greatly expanded the range of carbocyclic nucleotide analogues that represent potential candidates for the treatment of heart failure.

P15-16

Multivalent dendrimeric and monomeric adenosine agonists attenuate cell death

Athena M. Keene1, Ramachandran Balasubramanian1, John Lloyd1, Asher Shainberg2, Kenneth A. Jacobson1

1Laboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, USA,2Faculty of Life Sciences, Bar-Ilan University, Israel

Background: Multivalent GPCR Ligand-Dendrimer (GLiDe) conjugates have increased potency or selectivity in comparison to monomeric ligands. The pharmacokinetics and pharmacodynamics can be tuned and reporter or targeting moieties can be introduced. Methods: The applicability of a novel multivalent adenosine receptor (AR) agonist to A3AR-mediated cardioprotection was tested in mouse HL-1 cardiomyocytes, in which the expression of AR subtypes could be measured and manipulated. Results: Quantitative RT-PCR indicated high expression levels of endogenous A1 and A2A, but not of A2B and A3ARs. Results: Activation of the heterologously expressed human A3AR in HL-1 cells significantly attenuated cell damage following 4 h exposure to H2O2 (750 µM) but not in untransfected cells. A3 agonist IB-MECA (EC50 3.8 µM) and non-selective agonist NECA (EC50 3.9 µM) protected A3 AR-transfected cells against H2O2 in a concentration-dependent manner, as determined by LDH release. A generation 5.5 PAMAM (polyamidoamine) dendrimeric conjugate of a N6-chain-functionalized adenosine agonist (N-2-aminoethyl-ADAC) was newly synthesized and characterized chemically and pharmacologically. Its mass indicated an average of 60 amide-linked nucleoside moieties out of 256 theoretical carboxylic acid attachment sites. It nonselectively activated the A3AR expressed in CHO cells (IC50 66 nM in cAMP inhibition) and protected A3-transfected HL-1 cells from apoptosis-inducing H2O2 with greater potency (IC50 35 nM) than monomers. Conclusion: This potent cardioprotective conjugate retained A3AR interaction. While the precise mechanism of A3AR-induced protection and participation of simultaneous binding of multiple receptors are uncertain, this research provides the groundwork for the use of multivalent adenosine drugs in treating cardiac diseases.

P16. New technical developments

P16-1

Long-term caffeine intake prevents diet-induced insulin resistance and hypertension in rats through a mechanism involving a decrease in plasma catecholamines

Tiago Nunes da Silva1, Miguel Mota Carmo2, Emília Carreira Monteiro3, Silvia Vilares Conde3

1CEDOC, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Portugal,2CEDOC, Department of Pathophysiology, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Portugal,3CEDOC, Department of Pharmacology, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Portugal

Background: Caffeine can acutely increase blood pressure and lower insulin sensitivity, however several epidemiological studies suggest no association between long-term coffee consumption and diabetes and hypertension. We tested the hypothesis that chronic caffeine intake prevents the development of insulin resistance and hypertension in high-fat diet (HF) rats and high-sucrose diet rats (HSu). Methods: Six groups of animals were used: control, caffeine-treated (Caff), HF, caffeine-treated HF (HFCaff), HSu and caffeine-treated HSu (HSuCaff). The control group was fed a sham diet; the HSu and HF models were obtained by the administration of 35% sucrose in drinking water during 28 days; or of a lipid rich diet during 21 days. Caffeine was administered in drinking water (1 g/l) during the last 15 days of the experimental protocol. Insulin sensitivity was assessed by means of an insulin tolerance test. Blood pressure, weight gain, visceral fat, insulinemia, serum free fatty acids, plasma catecholamines and plasma NOx were also measured. Results: The chronic ingestion of caffeine prevented the development of insulin resistance and hypertension in both HF and HSu rats. Caffeine reversed the increase in visceral fat, weight gain and hyperinsulinemia induced by the HF diet. Also, caffeine decreased free fatty acids to control values in HSu rats. Caffeine did not modify plasma NOx but it significantly decreased plasma catecholamines to control levels both in HSu and HF animals. Conclusions: Chronic caffeine administration prevents the development of diet-induced insulin resistance and hypertension through a mechanism that involves the decrease in plasmatic catecholamines.

P16-2

Imaging exocytotic ATP release from A549 cells with TIRF microscopy

Ryszard Grygorczyk1, Irina Akopova2, Sabina Tatur3, Mariusz Grygorczyk3, Ignacy Gryczynski2, Zygmunt Gryczynski2, Julian Borejdo2

1Department Medicine, University of Montreal, Canada,2Health Science Center, University of North Texas, USA,3Research Center CHUM, University of Montreal, Canada

Release of nucleotides constitutes the first step of purinergic signaling cascade, but the underlying mechanisms of release remain incompletely understood. Regulated calcium-dependent exocytosis of ATP-loaded vesicles and ATP-conducting channels were suggested as possible pathways of nucleotide release. The goal of this study was to examine ATP release from lung epithelial A549 cells by total internal reflection fluorescence (TIRF) microscopy in an attempt to directly visualize ATP-loaded vesicle movement, recruitment and fusion with the plasma membrane. Cells were loaded with quinacrine or Bodipy-ATP, a fluorescent markers of ATP intracellular storage sites, and time-lapse imaging was performed using through the objective TIRF system. The time-course of fluorescence intensity changes of individual fluorescently-stained vesicles was evaluated. Following hypoosmotic stimulation approximately 20%–50% of vesicles visible by TIRF at the cell base showed a quasi-instantaneous disappearance during the first minute, as expected for vesicle fusion and dispersal of their content. The rate of exocytotic events increased significantly (up to ~4-fold) in stimulated cells compared to spontaneous events observed in unstimulated cells. This was accompanied by enhanced recruitment of new vesicles into the evanescent field. Results support exocytotic ATP release mechanism, consistent with luciferin-luciferase luminescence measurements. Agents known to disrupt exocytosis (brefeldin, monensin), or cytoskeleton (nocodazole, cytocholasin) reduced ATP release significantly (by up to 80%), while the release was completely blocked by N-ethylmaleimide (1 mM), and low (10°C) temperature. Thus, Ca2+-dependent exocytosis is a major pathway of hypotonic shock-induced ATP secretion from A549 cells. (Supported by CIHR and CCFF (RG), ETFG Tx. (Z.G.), NIH-HL090786 (JB)).

P16-3

Optimization of a new method to quantify cAMP level applied to adenosine receptors

Buccioni Michela, Diego Dal Ben, Carmen Lammi, Catia Lambertucci, Gabriella Marucci, Rasaria Volpini, Gloria Cristalli

Medicinal Chemistry Unit, School of Pharmacy, University of Camerino, Italy

Although a number of assays already exists for monitoring G protein coupled receptor (GPCR) functions, the development of new technologies will undoubtedly be an advantage in drug discovery. Promega recently developed a biosensor technology called GloSensor™ cAMP Assay, which provides a simple and powerful new way to measure cAMP level in cells. The assay uses a genetically modified form of firefly luciferase containing a cAMP-binding protein moiety. Binding of cAMP causes a conformational change that leads to increased light output. Following pre-equilibration with the substrate, cells transiently or stably expressing biosensor variants are used to monitor the cAMP level after the GPCR activation by agonists. The luminescence output allows to evaluate the activity of ligands at the receptor under study. The aim of the present work was the optimization and validation at all adenosine receptors of two new improved plasmids containing the gene for the modified form of firefly luciferase. In particular, the experiments were carried out by transiently expressing these new vectors at CHO cell line stably transfected with adenosine A2A and A3 receptors in order to verify the two plasmids efficacy at Gs and Gi system, respectively. Preliminary results of determining cAMP level at adenosine A2A and A3 receptors with this new method showed that both plasmids can be used at Gs and Gi coupled receptor system.

P16-4

Analysis of microglial activation in tissue slice using two-photon imaging

(Selected for Oral Poster Presentation)

Junya Masuda, Hidetoshi Tozaki-Saitoh, Makoto Tsuda, Kazuhide Inoue

Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Japan

Microglia, immune cells in the central nervous system (CNS), play various important roles in most CNS diseases. Transformation of microglial morphology from ramified (resting) to amoeboid (activated) is often used as their activation marker. Recent studies have shown that ramified microglia are highly dynamic in the intact brain and rapidly respond to local injury by extending their processes toward the injury site. In addition, this response was regulated by P2Y12 receptor and was mimicked by local injection of ATP, suggesting that microglia sense extracellular nucleotides as damage signals. Although two-photon microscopy has become a major tool for in vivo real time imaging, the effect of experimental procedure on microglial activity remains to be carefully assessed especially in ex vivo tissue preparation. Therefore, to conduct a detailed analysis of microglial dynamics in ex vivo, we employed acute brain slices and tracked each identical cell by two-photon imaging. After slice preparation, microglia gradually retracted their processes, reduced process branching, and became hypertrophic, whereas their process motility rate, a dynamic indication of microglial sensing, immediately started to increase. Moreover, these activation states were also affected by the depth of their location from sliced surface. These results suggest that the increase of sensing activity preceding morphological transformation could be another activation marker of microglia. Our present finding thus provides new information that there is time- and depth-restriction in ex vivo experiments for the study of native microglia.

P16-5

Rapid chromatography quantification of adenine e guanine purines

Gisele Hansel, Denise Barbosa Ramos, Luís Valmor Portela, Diogo Souza, Ana Elisa Bohmer

Biochemistry Department, Universidade Federal do Rio Grande do Sul-UFRGS, Brazil

Introduction: Purines play important roles in physiological and pathological conditions. Although there are several methods for identifying and measuring these substances, none of them can measure all adenine and guanine purines in a single and rapid HPLC running. The establishment of such methodology would be a valuable tool in biological studies Methods: High-performance liquid chromatography (HPLC) was performed to measure ATP, ADP, AMP, adenosine (ADO), GTP, GDP, GMP, guanosine (GUO), IMP, inosine (INO), hypoxanthine (HIPOX, xanthine (XANT)and uric acid (UA). It was used a C18 column at 24ºC, with UV detector (254 nm). Two phosphate buffers pH 6.0 were used, one being with acetonitrila 15%. The mobile phase flow rate was 1.2 mL/min during 17 minutes. The analytical method was validated through the analysis of accuracy, precision, quantification limit, linearity, and stability. Results: Standard solutions remained stable for up to 5 months at −20º C. Linearity range was observed: from 0.5 to 2000 pmoles at AMP, GTP, GDP, GMP, IM; from 0.5–100 pmoles at UA;from 1–2000 pmoles at ATP, GUO, INO, ADO, HIPOX, XANT; and from 5–2000 pmoles at ADP. The precision results showed ranged from 0.05 % to 3.9 % for intra-day and from 1.39 % to 4.92 % for inter-day for all the purines. The accuracy ranged from 89.3 % to 105.2% for intra-day and from 86.1 % to 111.8 % for inter-day for all the purines. Conclusion: This method was simple, rapid, stable, sensitive, specific, and accurate, and allows measuring all these purines in a single running.

P16-6

Novel fluorescent biological tools for studying adenosine receptors

Sabrina Gollos, Fabian Heisig, Andrea Behrenswerth, Christa E. Müller

Pharmaceutical Institut, Pharmaceutical Chemistry I, University Bonn, Germany

NOVEL FLUORESCENT BIOLOGICAL TOOLS FOR STUDYING ADENOSINE RECEPTORS Sabrina Gollos, Fabian Heisig, Andrea Behrenswerth and Christa E. Müller Pharma-Center Bonn, Pharmaceutical Chemistry I, University of Bonn, D-53121 Bonn, Germany. presenting author: sgollos@uni-bonn.de Background 4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-s-indacene (BODIPY) dyes are useful tools to image processes in living cells due to their high photochemical stability, their exceptional spectral properties, their high absorption coefficients and their small size. BODIPY dyes show high fluorescence quantum yields (theta greater than 0.5) and high peak intensities (1,2). Hence they are highly suitable for biological investigations. Methods In the present study we synthesized new, functionalized BODIPY derivatives, which fluoresce at approximately 500 nm and therefore do not interfere with biological fluorophores. Various functional groups (Br, I, NH2, SH, OH) were attached via alkyl spacers of different lengths to the BODIPY core in order to allow for their attachment to receptor ligands, or their integration into the pharmacophore of adenosine receptor ligands. All new, functionalized BODIPY derivatives showed high fluorescence quantum yields and absorption and emission wavelengths near 500 nm. In a subsequent step we coupled these BODIPY dyes to adenosine and xanthine derivatives, respectively. The affinities of the obtained derivatives were determined in radioligand binding studies at membrane preparations at A1, A2A, A2B and A3 receptors. Agonists were additionally characterized in functional assays. Conclusion Fluorescent A1-agonists and A2B-antagonists were obtained, some of which show high potency (Ki values at low nanomolar concentrations) combined with receptor subtype-selectivity. The new fluorescent adenosine receptor ligands will be useful tools for studying adenosine receptor binding and function.

(1) Loudet, A., Burgess, K. BODIPY dyes and their derivatives: Synthesis and Spectroscopic properties. Chem. Rev. 2007, 107, 4891–4932.

(2) Middleton RJ, Briddon SJ, Cordeaux Y, Yates AS, Dale CL, George MW, Baker JG, Hill SJ, Kellam B. New Fluorescent Adenosine A1-Receptor Agonists That Allow Quantification of Ligand-Receptor Interactions in Microdomains of Single Living Cells. J. Med. Chem. 2007, 50, 782–793.

P16-7

Frontal affinity chromatography-mass spectrometry useful for characterization of new ligands for GPR17 receptor

Stefania Maria Ceruti1, Enrica Calleri2, Gloria Cristalli3, Claudia Martini4, Caterina Temporini2, Chiara Parravicini1, Rosaria Volpini3, Simona Daniele4, Gabriele Caccialanza2, Davide Lecca1, Catia Lambertucci3, Maria Letizia Trincavelli4, Gabriella Marucci3, Irwing W. Wainer5, Graziella Ranghino6, Piercarlo Fantucci7, Maria P. Abbracchio1, Gabriella Massolini2

1Department of Pharmacological Sciences, Universita' Degli Studi Di Milano, Italy,2Department of Pharmaceutical Chemistry, University of Pavia, Italy,3Department of Chemical Sciences, University of Camerino, Italy,4Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Italy,5Gerontology Research Center, National Institutes of Health, USA,6Delos SRL, Italy,7Department of Biotechnology and Biosciences, Universita' Di Milano-Bicocca, Italy

We here report the application of the frontal affinity chromatography-mass spectrometry (FAC-MS) approach, along with a molecular modeling study, to the screening of potential drug candidates towards the recently deorphanized G protein-coupled receptor GPR17, which is dually activated by uracil nucleotides and cysteinyl-leukotrienes. GPR17 was found to be highly expressed in organs typically undergoing ischemic damage (i.e., brain, heart and kidney), thus representing a potential target for new therapeutic approaches to acute and chronic neurodegenerative diseases. GPR17 was entrapped on the surface of an immobilized artificial membrane and the stationary phase was used to screen a library of nucleotide derivatives by FAC-MS in order to select high affinity GPR17 ligands. The chromatographic results obtained have been successfully validated with a reference functional assay (i.e., the [35S]GTPγS binding assay). The simplicity of the method allowed us to explore the binding mode of the nucleotide site of the receptor by extending this approach to a column where an artificial GPR17 receptor bearing a mutation in a key amino acid for ligand recognition (Arg255) has been immobilized. Comparison of the different chromatographic behaviour of the nucleotide derivatives in the library together with in silico studies has been used to gain insight into the structure requirement of GPR17 ligands. With this innovative multidisciplinary approach we were able to identify several new and potent GPR17 agonists and antagonists to be subsequently tested in biological assays in vitro and in vivo for their pharmacological activity towards known paradigms of neurodegeneration.

P16-8

Lentivirus-mediated regulation of protein expression as a tool to study the effects of ethanol on P2X4 receptors in microglia

Liana Asatryan, Sneha Inamdar, Letisha Wyatt, Olga Ostrovskaya, Ronald Alkana, Daryl Davies

School of Pharmacy, University of Southern California, USA

Ethanol inhibits ATP-induced currents in P2X4 receptors (P2X4Rs) recombinantly expressed in oocytes and HEK293 cells. Moreover, using a lentiviral delivery system, we recently over-expressed P2X4Rs in cultured embryonic neurons and found that the degree of ethanol inhibition was similar to our findings in oocytes and HEK293 cells. Building evidence suggests that P2X4Rs play a role in alcohol consumption. Presently, little is known regarding the role of P2X4Rs in ethanol action expressed in microglia. The current study represents the first step to develop methods to manipulate P2X4Rs expression in microglia. We produced lentivirus encoding P2X4R-GFP fusion protein and shRNA oligonucleotides targeting P2X4Rs. Microglial BV-2 cells were transduced with lentivirus and overexpression/knockdown of P2X4Rs tested using fluorescence imaging and Western immunoblotting. Infection of BV-2 cells with lentivirus (MOI of 10(6)) containing P2X4-GFP resulted in a significant increase in P2X4R protein expression with 50–60% transduction efficiency. Lentiviral infection of BV-2 cells with 2 different shRNA oligonucleotides resulted in 50% reduction of P2X4R protein expression per shRNA tested. Combining the two shRNAs led to an 80% knockdown of P2X4R expression. We demonstrate successful use of lentivirus-mediated regulation of P2X4Rs in microglia. Patch-clamp electrophysiology is currently underway to test the effects of ethanol on wildtype and lenitvirally transduced BV-2 cells. This work will set the stage for the use of lentiviral technology to investigate the roles of neuronal and microglial P2X4Rs in ethanol-induced behaviors.

P17. Caffeine

P17-1

Chronic caffeine treatment during adolescence improves behavioral flexibility in an animal model of attention deficit hyperactivity disorder

Pablo Pandolfo1, Nuno Miguel de Jesus Machado2, Attila Kofalvi3, Reinaldo N. Takahashi1, Rodrigo A. Cunha3

1Dept. Pharmacology, Centre Biological Sciences, Federal Univ. Santa Catarina, Brazil,2Center Neuroscience Coimbra, Univ. Coimbra, Portugal,3Center Neurosciences Coimbra, Fac. Medicine, Univ. Coimbra, Portugal

Attention deficit hyperactivity disorder (ADHD) involves dopaminergic dysfunction affecting prefrontal cortex and striatal circuits, resulting in cognitive and motor abnormalities. Since adenosine and dopamine (DA) neuromodulation is tightly inter-twinned, we now tested if caffeine (non-selective adenosine receptor antagonist) prevented behavioral and neurochemical modifications in Spontaneously Hypertensive Rats (SHR, compared with control Wistar-Kyoto rats, WKYR), a validated ADHD model. Male SHR and WKYR were treated with caffeine (2 mg/kg, i.p., twice a day) during adolescence (P25–46) and tested in adulthood (P50–60) in the attentional set-shifting task before sacrifice for neurochemical analysis of DA uptake by nerve terminals and levels of DA transporter (DAT), D1 and D2 receptors. SHR made significantly more errors in the attentional set-shifting tasks compared with the WKYR; caffeine treatment improved behavioral flexibility selectively in SHR, while having no effect in WKYR (n = 7–8). SHR displayed increased levels of DAT and increased DA uptake in prefrontal cortex and striatal terminals of corticostriatal circuits responsible for this type of executive functioning; chronic caffeine treatment eliminated strain differences in DAT levels and DA uptake. Finally, A2A, D1 and D2 receptor densities were increased in prefrontal cortex terminals of SHR. These findings show that chronic caffeine treatment during adolescence confers long-term cognitive benefits in acquisition and maintenance of novel behavioral strategies, suggesting that adenosine receptor antagonists might represent a potentially interesting drug to treat behavioral flexibility dysfunction in ADHD. (Supported by FCT)

P17-2

Chronic caffeine consumption prevents chronic unpredictable stress-induced memory deficits but does not alter hippocampal BDNF levels

Ana Paula Ardais1, Marcelo Silveira da Costa2, Sabrina Mioranzza2, Paulo Henrique Botton2, Daniela Pochmann1, Rodrigo A. Cunha1, Lisiane de Oliveira Porciúncula2

1Center for Neuroscience of Coimbra, University of Coimbra, Portugal,2Biochemistry Department, Universidade Federal do Rio Grande do Sul, Brasil

Caffeine has been found to prevent memory deficits caused by different noxious stimuli, an effect mimicked by antagonists of adenosine A2A receptors (A2AR) through mechanisms still unraveled. Since A2AR control the signaling of BDNF-operated Trk-B receptors, we now tested if the ability of caffeine to prevent memory impairment in a model of chronic unpredictable stress (CUS) was accompanied by changes in the hippocampal levels of BDNF. Adult male Wistar rats were exposed or not (control) to the CUS protocol for 15 days and some of the rats in each of the groups were allowed to consume caffeine (0.3 g/L or 1.0 g/L) in the drinking water. Rats were then evaluated behaviorly before being sacrificed for BDNF quantification by Western blot analysis of hippocampal extracts. Rat subjected to CUS displayed a 56% deficit of spatial memory measured in the Y maze task, compared to control (i.e. non-stressed rats). Consumption of caffeine did not modify the Y maze performance of control rats, but prevented the CUS-induced memory impairment. In contrast, the levels of hippocampal BDNF remained constant both in rats subject to CUS irrespective of whether or not they consumed caffeine. The present results show that the CUS-induced memory impairment can be prevented by caffeine, and suggest that both CUS-induced memory impairment and caffeine neuroprotection are not related to modifications of the levels of BDNF in the hippocampus. (Supported by FCT)

P17-3

Caffeine improves attention defficit in neonatal 6-OHDA lesioned rats

Fabiana Núnez, Miguel Caballero, Silvia Sánchez, Víctor Fernández-Dueñas, Francisco Ciruela

Departament de Patología i Terapèutica Experimental, Facultat de Medicina-Bellvitge, IDIBELL-Universitat de Barcelona, Spain

Nowadays the pharmacological treatment of the attention deficit hyperactivity disorder (ADHD) is based on amphetamine derivatives (p.e. methylphenidate) which carry a large array of adverse side effects. For this reason less aggressive psychostimulant drugs are being proposed as an ADHD alternative treatment. Following this tendency, we decided to study the possible therapeutic use of caffeine on an animal model of ADHD, namely the neonatal 6-OHDA-lesioned rat. Thus, postnatal day (PND) 7 rats were lesioned at the left striatum with 6-OHDA (n = 7) or with saline (n = 5). Then, at PND25 hyperactivity and attention was measured with the Olton maze before caffeine was administered ad libitum in the drinking water. Next, we repeated these measurements at PND40 to asses the effect of caffeine in hyperactivity and attention. Interestingly, while no changes in the hyperactivity measurements were observed before and after caffeine administration a significant improvement in the attention of the 6-OHDA lesioned rats was achieved after caffeine treatment. In addition, the expression of adenosine A2A receptors (A2ARs) in the striatum of these animals was analyzed by western-blot. Thus, under these experimental conditions a significant decrease of the A2ARs in the left striatum of the 6-OHDA lesioned animals was observed. Overall, our results led us to hypothesize that caffeine might be useful to manage the attention deficit in ADHD. This work was supported by grants SAF2008-01462 and Consolider-Ingenio CSD2008-00005 from Ministerio de Ciencia e Innovación.

P17-4

Long-term caffeine intake prevents diet-induced insulin resistance and hypertension in rats through a mechanism involving a decrease in plasma catecholamines

Maria P. Guarino1, Tiago Nunes da Silva1, Miguel Mota Carmo1, Emilia C. Monteiro2, Silvia V. Conde2

1CEDOC, Department of Pathophysiology, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Portugal,2CEDOC, Department of Pharmacology, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Portugal

Background: Caffeine can acutely increase blood pressure and lower insulin sensitivity, however several epidemiological studies suggest no association between long-term coffee consumption and diabetes and hypertension. We tested the hypothesis that chronic caffeine intake prevents the development of insulin resistance and hypertension in high-fat diet (HF) rats and high-sucrose diet rats (HSu). Methods: Six groups of animals were used: control, caffeine-treated (Caff), HF, caffeine-treated HF (HFCaff), HSu and caffeine-treated HSu (HSuCaff). The control group was fed a sham diet; the HSu and HF models were obtained by the administration of 35% sucrose in drinking water during 28 days; or of a lipid rich diet during 21 days. Caffeine was administered in drinking water (1 g/l) during the last 15 days of the experimental protocol. Insulin sensitivity was assessed by means of an insulin tolerance test. Blood pressure, weight gain, visceral fat, insulinemia, serum free fatty acids, plasma catecholamines and plasma NOx were also measured. Results: The chronic ingestion of caffeine prevented the development of insulin resistance and hypertension in both HF and HSu rats. Caffeine reversed the increase in visceral fat, weight gain and hyperinsulinemia induced by the HF diet. Also, caffeine decreased free fatty acids to control values in HSu rats. Caffeine did not modify plasma NOx but it significantly decreased plasma catecholamines to control levels both in HSu and HF animals. Conclusions: Chronic caffeine administration prevents the development of diet-induced insulin resistance and hypertension through a mechanism that involves the decrease in plasmatic catecholamines.

P18. Infections and rare diseases

P18-1

Disordered lymphoid purine metabolism contributes to pathogenesis of persistent microbial infection and arthritis development in mice

(Selected for Oral Poster Presentation)

Gennady Yegutkin1, Jukka Hytönen2, Sirpa Jalkanen1, Matti Viljanen2

1MediCity Laboratory, University of Turku, Finland,2Department of Medical Microbiology and Immunology, University of Turku, Finland

Extracellular ATP and adenosine are important regulators of immune responses, however contribution of purinergic signalling to host defence during persistent microbial infections remains obscure. Lyme borreliosis is a common arthropod-borne infection caused by Borrelia burgdorferi sensu lato. Here we investigated whether lymphoid purinergic signalling contributes to the mechanisms by which borreliae species evade the immune system and trigger joint inflammation. Inoculation of Borrelia garinii to C3H/He mice induced symptomatic infection, as ascertained by elevated levels of IgG antibodies, enlargement of draining lymph nodes with hyperplasia of B-cell follicle area, persistent spirochete dissemination into the tissues and joint swelling. Purine catabolism was also activated in lymph nodes but not spleen and blood of acutely infected C3H/He mice, particularly manifested in transient up-regulations of ATPase/NTPDase and ecto-5′-nucleotidase/CD73 on T-lymphocytes and adenosine deaminase on B-lymphocytes. Unlike C3H/He strain, infected C57Bl/6 mice efficiently eradicated spirochetes at more chronic stage without any signs of arthritis. Lymphocytes from borrelia-resistant C57Bl/6 mice also displayed markedly enhanced adenosine-generating capability due to ~3-times higher ratio of ecto-5′-nucleotidase to adenosine deaminase. Strikingly, deletion of key adenosine-generating enzyme, ecto-5′-nucleotidase/CD73, was accompanied by significant joint swelling in borrelia-infected CD73-deficient C57Bl/6 mice. Collectively, these data suggest that insufficient basal adenosine level and/or pathogen-induced disordered lymphoid purine homeostasis may serve as important pre-requisite for promotion of inflammatory responses and further host’s commitment to persistence of bacterial infection and arthritis development.

P18-2

Adenosine deaminase-induced costimulation potentiates the generation of effector, memory and regulatory CD4+ T-cells

Victor Casanova Güell1, Isaac Naval Macabuhay1, José M. Martínez Navio1, Rodrigo Pacheco2, Nuria Climent Vidal3, Felipe García4, José M. Gatell4, Josefa Mallol1, Teresa Gallart5, Carme Lluis Biset1, Rafael Franco Fernández1

1Departament Bioquímica i Biologia Molecular / IDIBAPS, Universitat de Barcelona, Spain,2Fundación Ciencia para la Vida and Instituto Milenio de Biología Fundamental y Aplicada, Universidad San Sebastián, Chile, Chile,3Services of Immunology and Infectious Diseases & AIDS Unit, Hospital Clinic/IDIBAPS, Spain,4Infectious Diseases & AIDS Unit/ HIVACAT, Hospital Clinic, Spain,5Services of Immunology and Infectious Diseases & AIDS Unit, Hospital Clinic, Spain

Efficient antigen-specific activation of naïve CD4+ T-cells by dendritic cells (DCs) results in their proliferation and differentiation toward effector T-cells (Teffs), which drive immune response. This process also generates regulatory T-cells (Tregs), which attenuate the function of Teffs. After effector response, most T-cells undergo apoptosis and the few antigen-specific T-cells remaining alive differentiate into memory T-cells. When reactivated, memory T-cells coordinate a faster, stronger and more prolonged and efficient immune response. We have previously described that adenosine deaminase (ADA), by interacting with CD26 in the CD4+ T-cell surface and with the adenosine receptor A2B on the DC-surface, triggers a costimulatory signal for human T-cells. This interaction is impaired in T-cells from HIV-infected patients, resulting in a decreased T-cell proliferation and thus contributing to the immunodeficiency. In this work, we have addressed the question of whether ADA-mediated costimulatory effect may modulate the differentiation of naïve T-cells toward Teffs, Tregs and memory T-cells. Our results obtained from autologous coculture experiments show that, by a mechanism dependent on the coordinate action of TNF-α, IFN-γ and IL-6, ADA potentiates the differentiation of naïve T-cells toward Teffs and also the generation of memory T-cells. In addition, ADA evokes an enhanced production of Tregs. Interestingly, ADA-mediated potentiation on the generation of Teffs, Tregs and memory T-cells occurred not only in CD4+ T-cells from healthy individuals, but also in CD4+ T-cells from HIV-infected patients. These data suggest that action of ADA-mediated interaction between DCs and T-cells is a relevant signal for CD4+ T-cell differentiation.

P18-3

The neonatal adenosine system acts via adenosine 3 receptor to enhance TLR2-mediated IL-6 production in vitro and in vivo

Melanie Coombs1, Taiese Bingham1, Leighanne Gallington1, Liat Stoler-Barak1, Mirjam Belderbos2, Victoria Philbin1, Palanivel Velupillai1, John Kelly3, Enes Karabalut1, Khoa Ngyuen4, Louis Bont2, James Wynn5, Marlene Jacobsen6, Bruce Cronstein4, Taiese Bingham7

1Children's Hospital Boston, U.S.A.,2University Medical Centre, Utrecht, NL,3Boston University, U.S.A.,4New York University, U.S.A.,5Duke University, U.S.A.,6Merck, U.S.A.,7Children's Hospital Boston/Harvard, Medical School, U.S.A.

Background: Skewed neonatal cord blood mononuclear cell cytokine production to TLR2 agonists in vitro, characterized by low Th1-polarizing TNF but high Th2-polarizing IL-6, is mediated by increased CBMC sensitivity to adenosine (Levy et al J Immunol 2006).

Objective: To characterize the impact of adenosine signaling on TLR2-mediated neonatal monocyte cytokine production in vitro and assess the role of neonatal adenosine 3 receptor (A3R) in TLR2-mediated cytokine production in vivo.

Methods: AR expression of human neonatal CB-derived Mos and adult monocytes was determined by RT-PCR and flow cytometry. Mos cultured in autologous plasma and pre-treated (30 min) with adenosine (0.1–100 µM), were stimulated with fibroblast-stimulating lipopeptide (FSL-1; TLR2) or lipopolysaccharide (LPS; TLR4). Human neonatal cord blood or adult peripheral blood was pre-incubated +A3R antagonist MRS1220 prior to stimulation with FSL or live Staphyloccocus epidermidis (SE). Wild type and A3R-deficient newborn and adult mice were injected i.v. with TLR agonists. Cytokines were measured by ELISA.

Results: Compared to adult PBMos, neonatal CBMos demonstrated impaired FSL-induced TNF but preserved IL-6 production that was enhanced by adenosine (p < 0.05). The A3R antagonist MRS1220 inhibited SE-induced IL-6 production by human neonatal cord blood (p = 0.05) but not in adult peripheral blood. CBMos expressed similar basal levels of A3R as adult Mos. A3R was inducible by both FSL and live SE. A3R-deficient neonatal mice demonstrated a selective deficiency in TLR2-mediated IL-6 production after i.v. injection of FSL (p < 0.05).

Conclusions: A3R on neonatal CBMos is induced by TLR2-agonists and selectively increases TLR2-mediated IL-6 production in vitro and in vivo.

P18-4

A2A and CB1 receptors form heterodimers and functionally interact in human dermal fibroblasts: a new target for modulating collagen production in systemic sclerosis?

Pier Leopoldo Capecchi, Pietro Enea Lazzerini, Mariarita Natale, Sauro Lorenzini, Enrico Selvi, Estrella Garcia-Gonzalez, Epifania Balistreri, Monica Castrichini, Elena Gianchecchi, Cinzia Montilli, Gerarda Pompella, Mauro Galeazzi, Franco Laghi Pasini

Dept. of Clinical Medicine and Immunological Science, University of Siena, Italy

Background: Adenosine promotes the development of tissue fibrosis in different organs, via the interaction with the A2A receptor. Also cannabinoids are able to modulate fibrogenesis, with an opposite effect of receptor stimulation, with the CB1 promoting and the CB2 inhibiting the fibrotic process, respectively. Moreover, recent evidence in the central nervous system suggests physical and functional interactions between A2A and CB1 receptors, resulting in a strict dependence for CB1 signalling on A2A receptor co-activation. The present study on dermal fibroblasts from patients with systemic sclerosis (SSc) is aimed at: evaluating the extent of A2A expression and its role in the regulation of collagen biosynthesis; confirming the formation of A2A-CB1 heteromers also in these cells; testing the putative synergistic anti-fibrotic effect of the concomitant modulation of purinergic and cannabinoid systems. Methods and Results. SSc fibroblasts expressed A2A, and both the CB1 and CB2 cannabinoid receptors, also displaying a fibrogenetic phenotype when compared with controls. A2A and CB1 co-immunoprecipitate in both healthy and SSc cells, thereby suggesting their physical interaction as a heterodimer. A2A activation induced an increase in collagen production in TGF-beta-stimulated SSc fibroblasts and human dermal fibroblasts (HDFa cells), which was in turn inhibited by cell stimulation with a non-selective cannabinoid agonist. Finally, the co-incubation with an A2A antagonist and a non-selective cannabinoid agonist produced additive effects markedly suppressing the synthesis of collagen. Conclusion. Our findings support a role for both the purinergic and the cannabinoid systems in sclerodermic fibroblasts, thus suggesting a multi-target therapeutic approach to systemic sclerosis.

P18-5

A2B adenosine receptors protect against sepsis-induced mortality by dampening excessive inflammation

(Selected for Oral Poster Presentation)

Balázs Csóka1, Zoltán H. Németh2, Peter Rosenberger3, Holger K. Eltzschig4, Balázs Koscsó1, Michael R. Blackburn5, György Haskó1

1Department of Surgery, UMDNJ-NJMS, USA,2Department of Surgery, Morristown Memorial Hospital, Morristown, USA,3Department of Anesthesiology and Intensive Care Medicine, University Hospital Frankfurt am Main, Johann Wolfgang Goethe University, Germany,4Mucosal Inflammation Program, Department of Anesthesiology, University of Colorado, Denver, USA,5Department of Biochemistry and Molecular Biology, University of Texas-Houston Medical School, USA

Despite intensive research, efforts to reduce the mortality of septic patients have failed. Adenosine is a potent extracellular signaling molecule and its levels are elevated in sepsis. Adenosine signals through G-protein coupled receptors and can regulate the host’s response to sepsis. Here we studied the role of A2B adenosine receptors in regulating the mortality and inflammatory response of mice following polymicrobial sepsis. Genetic deficiency of A2B receptors increased the mortality of mice suffering from cecal ligation and puncture-induced sepsis. The increased mortality of A2B knockout mice was associated with increased levels of inflammatory cytokines and chemokines, and augmented NF-kappaB and p38 activation in the spleen, heart, and plasma in comparison to wild-type animals. In addition, A2B receptor knockout mice showed increased splenic apoptosis and phosphatase and tensin homolog (PTEN) activation, and decreased Akt activation. Experiments using bone-marrow chimeras revealed that it is the lack of A2B receptors on non-hematopoietic cells that is primarily responsible for the increased inflammation of septic A2B receptor-deficient mice. These results indicate that A2B receptor activation may offer a new therapeutic approach for the management of sepsis.

P18-6

CD73 dependent extracellular adenosine in immunoglobulin class switch recombination

Elisabetta Traggiai1, Francesca Schena2, Stefano Volpi2, Ursula Schenk3, Marco Gattorno2, Alberto Martini2, Marco Canossa4, Fabio Grassi3

1Department of Pediatric Science, Institute G. Gaslini, Hospital, Italy,2Department of Pediatric Science, Institute G Gaslini, Italy,3Institute for Research in Biomedicine, Institute for Research in Biomedicine, Switzerland,4Department of Human and general Physiology, University of Bologna, Italy

Peripheral human B lymphocytes synthesize and release ATP upon coordinate stimulation through B cell receptor (BCR) and Toll-like receptor 9 (TLR9). In B cells ATP was detected in TI-VAMP positive vesicles and released by exocytose in a calcium dependent fashion. Autocrine stimulation of P2X receptors exerted a crucial effect on proliferation and differentiation of human B cells to plasma cell. However, in IgM memory B cells ATP activity in the extracellular milieu was modulated by ectoapyrase CD39 and ecto-5′-nucleotidase CD73, which are selectively expressed in this B cell subset. Since IgM memory B cells are characterized by intense class switch recombination activity we investigated whether extracellular adenosine and A2A receptors expressed in B lymphocytes contributed in inducing class switch recombination concomitantly to a slower rate of proliferation. Interestingly, we show that lack of CD73 expression on the surface of lymph-mononuclear cells in a patient displaying autoimmunity and lymphoadenopathy correlated with reduced class switch recombination.

P18-7

Extracellular ATP activates a pro-inflammatory response through P2Y-receptors in the human urinary tract

Susanne Säve1, Katarina Persson2

1School of Natural Sciences, Linneaus University, Sweden,2School of Health and Medical Sciences, Örebro University Hospital, Sweden

Extracellular ATP can be released by many cell types under conditions of cellular stress. The role of ATP in the host response during urinary tract infection has not previously been addressed. In the present study, we investigated ATP release from bacteria-infected uroepithelial cells and the effect of ATP on the host cell pro-inflammatory IL-8 response. The human kidney epithelial cell line A498 and the human uroepithelial cell line UROtsa, were grown in cell culture and stimulated by the uropathogenic E. coli (UPEC) strain IA2 or the stable ATP analogue ATP-gamma-S. ATP and IL-8 levels were measured in cell culture medium with a luciferin-luciferase assay and ELISA, respectively. The results showed that UPEC-infection of uroepithelial cells for one hour significantly increased (p < 0.01) the extracellular ATP levels. ATP-gamma-S (10 and 100 uM) stimulated release of IL-8 from UROtsa and A498 cells after 6 and 24 hours. Experiments with different purinoceptor agonists suggested that P2Y receptors, and not P2X receptors, were responsible for the ATP-gamma-S-induced IL-8 release. The potency profile further suggested involvement of P2Y1, P2Y2 and/or P2Y11 receptors, and RT-PCR studies confirmed that the cells expressed these receptors. The IL-8 release increased 12-fold in UPEC-infected cells and apyrase, an enzyme that degrades ATP, reduced this increase by approximately 50%. The present study suggest that enhanced ATP release and P2Y receptor activation during urinary tract infection may represent a novel, non-TLR4-mediated mechanism for production of pro-inflammatory IL-8 in human urinary tract epithelial cells.

P18-8

P2X7 receptor-mediated killing of Toxoplasma gondii by murine macrophages

Stephen Fuller1, Michael Lees2, Nicola Boulter2, Catherine Miller2, Alana Zakrzewski2, James Wiley1, Nicholas Smith2

1Department of Medicine, The University of Sydney, Australia,2Institute for the Biotechnology of Infectious Diseases, University of Technology, Sydney, Australia

Background: The P2X7 receptor (P2X7R) is highly expressed on the macrophage cell surface and activation of infected cells by extracellular ATP has been shown to kill intracellular Mycobacterium, Chlamydia, and Leishmania species. Toxoplasma gondii is also able to infect and survive in cells of the monocyte/macrophage lineage and in this study we show that P2X7R activation is a factor in the host-response to T. gondii infection. Methods P2X7R-dependent killing of T.gondii was studied using murine macrophages from BALB/c, C57BL/6J and P2X7R−/− mice on a C57BL/6J background. Macrophages were infected with T.gondii tachyzoites and activated using 1 mM ATP. T.gondii viability was assessed using flow cytometry and fluorescent microscopy. Serum from infected mice was collected and assayed for nitric oxide using the Griess Assay. Apoptosis of macrophages was assessed using Annexin-V-FITC/propidium iodide staining. Results ATP treatment of macrophages from wild-type mice resulted in a marked reduction in T. gondii viability. In contrast, ATP treatment of P2X7R−/− murine macrophages did not affect T. gondii viability. NO production is known to effect parasite killing and although there was a trend towards higher serum NO levels in P2X7R−/− infected mice, this did not achieve statistical significance. Intracellular T. gondii death occurred in parallel to host cell apoptosis Conclusion In this paper we show that macrophages from P2X7R−/− mice kill T. gondii less effectively compared to wild-type mice. We show that P2X7R-mediated T. gondii killing occurs in parallel with host cell apoptosis and is independent of nitric oxide production.

P18-9

QSAR model on adenosine derivatives as antifungal agents against candida albicans: a data mining approach

Alice Borghini1, Marcello Imbriani2, Daniele Pietra3, Mario Campa4, Giovanna Batoni4, Maria Cristina Breschi5, Anna Maria Bianucci1

1Department of Pharmaceutical Chemistry, University of Pisa, Italy,2Fondazione S. Maugeri, IRCCS, Fondazione S. Maugeri, IRCCS, Italy,3Centro Interdipartimentale di Farmacologia Clinica e Terapia Sperimentale, University of Pisa, Italy,4Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, University of Pisa, Italy,5Dipartimento di Psichiatria, Neurobiologia, Farmacologia e Biotecnologie, University of Pisa, Italy

Candida species cause many pathologies, ranging from non-invasive mucocutaneous infections to more serious invasive processes. Particularly, Candida albicans is the most common pathogen responsible for oropharyngeal, cutaneous and genital infections. It is known from the literature that some known adenosine derivatives such as cryscandine, sinefungin, cordycepin and 5′-deoxy-5′-methylthio-adenosine possess anticandidal activity. Thus, in this work, adenosine derivatives were collected from the literature and analyzed for their potential anticandidal activity. This dataset was used to develop a quantitative structure-activity relationships model able to predict the antifungal activity of adenosine derivatives against Candida albicans. Such a model was built using a data mining approach based on a classification tree. The results of this study suggest that adenosine derivatives should have a free NH2 at the 6 position of the purine system and a free OH at the 2′ position of the ribose ring. A COOH group may be directly bound at the 4′ position of ribose, if the 3′ position bears an aminoacid residue. The optimal substituent at the 3′ position of ribose is indeed an aminoacid residue, with L configuration of Cβ and possibly bearing an aromatic or S-aromatic substituent, which should be not significantly hindered from the steric point of view; it can be optionally substituted with not significantly polar groups. Chains bearing not significantly polar substituents are also allowed at 5′ position. These characteristics supposedly reflect the need for the molecule to have a proper value of logP, in order to better cross the cell membranes of the microorganism and be active as anticandidal agents.

P19. Sensory systems

P19-1

Ap4A and Ap5A are released by the corneal epithelium

Gonzalo Carracedo Rodriguez, Ana Guzman-Aranguez, Patricia Loma Lozano, Jesus Pintor Just

Universidad Complutense de Madrid, Spain

Background: To assess whether the release of Ap4A and Ap5A to corneal epithelial cells is due to mechanical stress. Methods: The study was designed with an experiment In Vitro and other In Vivo. For In Vivo experiments, Schirmer trips were located at the temporal tarsal conjunctiva of the lower lid of 16 anesthetized human eyes with normal tear secretion. The patients were instructed to blink at 0, 12, 30, and 60 blinks per minute during 5 minutes. In Vitro experiments were performed in stratified human corneal epithelial cells in culture. The cells were stimulated with several rinses and the supernatant was collected before and after stimulation. All samples collected were analyzed with HPLC. Results: The concentrations of Ap4A and Ap5A in closed-eyes individuals was 0.19 + 0, 60 µM and 0.11 + 0, 70 µM, respectively. With 60 blink per minute values raised to 14.82 + 1.77 µM for Ap4A and 13.76 + 3.43 µM for Ap5A. For In Vitro experiments the levels of Ap4A and Ap5A before mechanical stimulus were 3.18 + 0.43 µM and 0.81 + 0.13 nM, respectively. After stimulation with several rinses dinucleotides increased their values to 12.01 + 2.19 nM for Ap4A and 2.83 + 0.41 nM for Ap5A. Conclusion: Dinucleotides are released by applying a mechanical stimulus in vivo and in vitro from the corneal epithelium, this explaining the origin of these compounds which are relevant for the ocular surface biochemistry and physiology.

P19-2

P2Y2 nucleotide receptors increase the presence of aquaporin-1 in rabbit non pigmented ciliary epithelial cells

Alba Martin Gil, Jesus Pintor

Dto. Bioquimica Y Biología Molecular Iv. Eu Óptica., Universidad Complutense De Madrid, Spain

Purpose: To investigate the role of P2Y2 purinergic receptors in the raise of aquaporin-1 levels in NPE cell membranes and cytoplasm. Methods: Immortalized NPE cells were seeded in high glucose DMEM until they were subconfluent. Immunocytochemical studies were performed by means of an antibody against aquaporin-1 (AQP1) from Santa Cruz. Previous to this immunostaining cells were challenged with 100 uM of the dinucleotide Ap4A, at 15, 30, 60, 90 and 120 min. Antagonists of the P2Y receptors were pre-incubated 30 min before Ap4A was added. Results: The application of Ap4A to the NPE cells demonstrated a gradual increase in the presence of AQP1 which was time-dependent from the beginning to 60min, when the maximal expression of the protein is reached (200 % of increase when compared to control) (n = 8). Times longer than 60 min, gradually returned the fluorescence to initial values (at 120 min). At 60 min there was an increase of AQP1 presence in the plasma membrane. P2Y receptor antagonists demonstrated the involvement of this receptor in the action of Ap4A. In particular reactive blue 2 was the best reversing the effect of Ap4A. In the case of this antagonist, it was able to inhibit up to 80 % of the effect depicted by Ap4A. Conclusion: Diadenosine tetraphosphate, Ap4A, was able to mobilize AQP1 which was more visible after 60 min incubation with this dinucleotide. This increase in AQP1 expression can be the explanation to what was found in the whole rabbit in which P2Y2 agonists increase intraocular pressure.

P19-3

P2Y4 purinergic signaling in Reissner's membrane of the cochlea

Jun Ho Lee, Chang-Hee Kim, Sun O. Chang, Seung-Ha Oh

Department of Otolalryngology, Seoul National University Hospital, Korea

It has been known that the epithelial cells of Reissner’s membrane are capable of transporting Na+ out of endolymph via epithelial Na+ channel (ENaC). We have demonstrated the developmental change of Na+ absorption in Reissner's membrane during early neonatal period of rodent animals (Kim et al., 2009). It is known that there is P2X2 receptor expressed in Reissner's membrane. And also, we have recently found that P2Y4 receptor contribute to regulate Na+ absorption in Reissner's membrane possibly by decrease of phosphatidylinositol 4,5-biphosphate, PI(4,5)P2, in the plasma membrane through PLC activation (Kim et al., 2010). We will discuss the physiologic significance of this regulation in the inner ear.

P19-4

Autocrine stimulation of neurons following mechanosensitive strain requires pannexin and P2X7 channels

(Selected for Oral Poster Presentation)

Claire Mitchell1, Jingsheng Xia2, Jason Lim2, Wennan Lu2, Alan Laties3

1University of Pennsylvania, USA,2Anatomy and Cell Biology, University of Pennsylvania, USA,3Ophthalmology, University of Pennsylvania, USA

Mechanical strain produces complex pathological effects on neuronal systems which can lead to neuronal disfunction and ultimately death. While astrocytes may contribute, it is not clear whether neurons respond directly to mechanical forces. In the glaucomatous eye, retinal ganglion cells are killed following a rise in intraocular pressure. An increase in pressure can lead to enhanced extracellular ATP levels in the retina, while stimulation of P2X7 receptors is known to raise calcium and kill ganglion cells. As such, we examined the mechanosensitive ATP release from isolated retinal ganglion cells and explored the physiological response to this release. Immunopurified ganglion cells swollen by exposure to mild hypotonicity were found to release ATP into the bath. This release was blocked by 10 µM carbenoxelone and 1 mM probenicid, implicating pannexins as a conduit. Ganglion cells attached to a silicone substrate were stretched and this strain also triggered a carboxelone-sensitive release of ATP. Whole cell patch clamp recording was used to determine how ganglion cells responded to this released ATP. Mild swelling induced the activation of a non-specific cation current. This current was inhibited by removal of extracellular ATP with apyrase, by inhibition of the P2X7 receptor with A438079 and by both carbenoxelone and probenicid. Together, these data suggest mechanical strain triggers ATP release from retinal ganglion cells and that this released ATP autostimulates P2X7 receptors. It remains to be determined whether the autocrine stimulation of purinergic receptors is a general response to a mechanical strain on neurons.

P19-5

Rapid cytokine release following stimulation of the P2X7 receptor on RPE cells

Thor Eysteinsson1, L-A Tu2, S. Guha3, J. Lim2, A.M. Laties4, C.H. Mitchell3

1Physiology, University of Iceland, Iceland,2Anatomy and Cell Biology, University of Pennsylvania, USA,3Physiology, University of Pennsylvania, USA,4Ophthalmology, University of Pennsylvania, USA

Retinal pigmented epithelial (RPE) cells lie between retinal photoreceptors and the choroidal blood supply. In macular degeneration, the release of growth factors and cytokines from distressed RPE cells may contribute to the choroidal neovascularization that ultimately leads to a loss of sight. Choroidal neovascularization is enhanced by the cytokine IL-6, and inhibition of IL-6 can prevent inflammation and new vessel production. Although message for the IL-6 gene is elevated in RPE cells from eyes with macular degeneration, the signals initiating IL-6 release from RPE cells are not known. We asked whether stimulation of the P2X7 receptor triggers IL-6 release from RPE cells. Exposure of cultured human ARPE-19 cells to BzATP increased intracellular calcium, as measured with the dye fura-2. The rise was sustained, blocked by Brilliant Blue G, A438079 and KN-62, dependent upon the presence of extracellular calcium, and inhibited by extracellular magnesium: together these characteristics are most consistent with the activation of a P2X7 receptor by BzATP. BzATP also increased extracellular levels of IL-6, as measured with an ELISA. Extracellular IL-6 levels increased 1, 15 and 60 min after BzATP was presented. This release was blocked by 10 µM carbenoxolone (10 µM), possibly implicating pannexins. Together these findings suggest RPE cells express P2X7 receptors whose activation leads to a rapid release of IL-6. The contribution of this purinergic pathway in choroidal neovascularization remains to be determined. EY-013434, EY-015537, EY-001583 (CHM); Research to Prevent Blindness, the Paul and Evanina Bell Mackall Foundation Trust (AML), Jody Sack Fund.

P19-6

The treatment of noise-induced hearing loss by A1 adenosine receptor agonists

(Selected for Oral Poster Presentation)

Srdjan Vlajkovic1, Ann Chi Yan Wong1, Cindy Guo1, Kyu Hyun Lee1, Gary Housley2, Peter Thorne3

1Department of Physiology, The University of Auckland, New Zealand,2Department of Physiology, University of New South Wales, Australia,3Discipline of Audiology, The University of Auckland, New Zealand

Here, we report that the activation of adenosine receptor signalling can mitigate cochlear injury after exposure to noise. In this study, Wistar rats (8–10 weeks) were exposed to broadband noise (110 dBSPL for 24 hours) to induce permanent threshold shift. Adenosine and selective adenosine receptor agonists (CCPA, CGS-21680 and Cl-IB-MECA) were applied to the round window membrane of the cochlea six hours post-exposure. Hearing function was assessed by auditory brainstem responses (ABR) before and 48 hours after exposure. A partial threshold recovery (up to 20dB) was observed in the cochleae treated with adenosine and the selective A1 adenosine receptor agonist CCPA. No threshold recovery was observed with CGS-21680 or Cl-IB-MECA, the selective A2A and A3 adenosine receptor agonists respectively. Free radical damage generated in the noise-exposed cochlea, as demonstrated by nitrotyrosine immunoreactivity, was reduced by adenosine and CCPA administration. We further investigated the effect of adenosine amine congener (ADAC), a selective adenosine A1 receptor agonist devoid of peripheral side effects, on noise-induced cochlear injury. ADAC was administered intraperitoneally (100 µg/kg/day) at time intervals after noise exposure (8–12 kHz, 110 dB SPL for 2 or 24 hours, n = 8/group). Hearing thresholds were assessed by ABRs and hair cell loss evaluated by quantitative histology. ADAC administration led to substantial threshold recovery (25–30dB), supported by increased sensory hair cell survival and reduced nitrotyrosine immunoreactivity. Our studies pinpoint A1 adenosine receptors as promising pharmacological target to mitigate noise-induced cochlear injury. Supported by RNID (UK), Deafness Research Foundation (NZ) and Auckland Medical Research Foundation.

P20. Bone and joints

P20-1

Expression and functional role of adenosine receptors in regulating inflammatory responses in human synoviocytes

Katia Varani1, Fabrizio Vincenzi1, Alice Tosi1, Martina Targa1, Federica Masieri2, Alessia Ongaro2, Monica De Mattei2, Leo Massari3, Pier Andrea Borea1

1Dep. of Clinical and Experimental Medicine, Pharmacology Section, University of Ferrara, Italy,2Department of Morphology and Hystology, University of Ferrara, Italy,3Department of Biomedical Sciences and Advanced Therapies, Orthopaedic Clinic, University of Ferrara, Italy

Background: Adenosine is an endogenous modulator, interacting with four G-protein coupled receptors named as A1, A2A, A2B and A3, which acts as a potent inhibitor of inflammatory processes in several tissues. Nowadays functional effects modulated by adenosine receptors (ARs) on human synoviocytes have not been investigated in detail. We evaluated mRNA, the protein levels, the functional role of ARs and their pharmacological modulation in human synoviocytes isolated from patients with osteoarthritis. Methods: mRNA, western blotting, saturation and competition binding experiments, cyclic AMP, p38 mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-kB activation, tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) release were performed in human synoviocytes. Results: A1, A2A, A2B and A3ARs mRNA and protein are expressed in human synoviocytes. Well-known adenosine agonists and antagonists showed affinity values in the nanomolar range and were coupled to stimulation or inhibition of adenylyl cyclase. A2A and A3ARs activation inhibited p38 MAPK and NF-kB pathway, an effect abolished by selected adenosine antagonists. A2A and A3 agonists were able to diminish TNF-α and IL-8 production. The involvement of PI3K or Gs pathway in A3 or A2AARs functional responses have been demonstrated. A1 and A2BARs were not implicated in the inflammation downstream whereas the stimulation of A2A and A3ARs was closely associated with a down-regulation of the inflammatory status. Conclusion: These results indicate that A2A and A3ARs may represent a potential target in therapeutic anti-inflammatory joint interventions.

P20-2

Bone status of P2X7 knockout mice in two different strains

Susanne Syberg1, Peter Schwarz1, Solveig Petersen1, Jens-Erik Beck Jensen2, Thomas H. Steinberg3, Alison Gartland4, Iain Chessell5, Niklas Rye Jorgensen6

1The Research Centre for Ageing and Osteoporosis, Copenhagen University Hospital Glostrup, Denmark,2Osteoporosis and Bone Metabolic Unit, Depts. of Endocrinology and Clinical Biochemistry, Copenhagen University Hospital Hvidovre, Denmark,3Dept. of Internal Medicine, Washington University School of Medicine and the St. Louis Veterans Affairs Medical Centre, USA,4Mellanby Centre for Bone Research, University of Sheffield, UK,5NeuroScience, MedImmune, Milstein Building, Cambridge, UK,6Dept. of Clinical Biochemistry, Copenhagen University Hospital Glostrup, Denmark

In this study we wanted to investigate the importance of the genetic background by comparing bone status of a new BALB/cJ P2X7−/− strain with the one made by Glaxo (Chessel, Pain 114, 2005). Female mice were DEXA scanned on a PIXImus, femurs were collected for bone strength measurements and serum for bone marker analysis. The expression of the recently described splice variant of P2X7 (P2X7-k) was assessed by RT-PCR and the functionality in osteoblasts were assayed by measuring BzATP-induced calcium responses. Bone related parameters altered only slightly or not significant in the B6 P2X7−/− became significant in the BALB/cJ P2X7−/− when compared to wild type. The BALB/cJ P2X7−/− showed reduced levels of serum C-telopeptide fragment, higher total and regional bone mineral density, and increased bone strength again compared to the wild type littermates. We also measured the level of IL-1b in the bone marrow from B6 P2X7−/− and the level was lower in P2X7−/− than in the wild type mice. P2X7-k was expressed in bone marrow besides in cultured osteoclasts and osteoblasts, and in osteoblasts no calcium response could be induced by application of BzATP. In conclusion, we have shown that the genetic background of mice with ablation of the P2X7 receptor has underestimated the bone phenotype of the P2X7−/− mice, giving P2X7 a more significant regulatory role in bone remodeling than seen in previous studies. We have also shown that P2X7-k is expressed in the BALB/c P2X7−/− model, but that the splice variant is non-functional in osteoblasts in vitro.

P20-3

Nucleoside-5′-monophosphates as antiinflammatory prodrugs in collagen-induced arthritis (CIA)

Juergen Schrader1, Michael Romio1, Ulrich Flögel1, Zhaoping Ding1, L. Galbarz2, Ali El-Tayeb2, Christa Müller2

1Dept. Physiology, University of Duesseldorf, Germany,2PharmaCenter, Univ. Bonn, Germany

We have recently developed nucleoside-5′-monophosphates to serve as prodrugs of adenosine A2A receptor agonists activated by ecto-5′-nucleotidase (CD73) (El-Tayeb et al. 2009). Because ecto-5′-NT is upregulated in inflamed tissue, the A2A agonists are expected to be released from their prodrug at the site of inflammation. To test this concept under in-vivo conditions, arthritis was induced in DBA mice by immunization with collagen CII. Local inflammation of joints of the lower limb was measured in-vivo with 19F-MRI which sensitively detects the accumulation of monocytes/macrophages (Flögel et al. 2008). Osmotic pumps were used to treat mice continuously with cyclohexylethylthio-adenosine (chet-Ado) and the prodrug chet-AMP. Concentrations of the compounds were 0.5 mg/100 µl and infusion rate was 0.5 µl/h. Degree of arthritis was measured at day 8 and 11 after booster injection of CII. Eight days after booster injection control animals showed serve signs of CIA, as indicated by strong localized fluorine signals. Treating mice with chet-AMP reduced the degree of arthritis by about 50%. These results were confirmed by quantifying CIA by visual scoring of paw oedema formation. Measurement of cytokines/chemokines in plasma of chet-AMP treated and untreated mice revealed that in the prodrug-treated group IFN-γ, IL-6, IL-1β, MIP-1α were decreased. IL-10, which is known to inhibit activation and effector function of T cells, monocytes, and macrophages was found to be increased. Hemodynamic studies revealed that upon acute i.v. infusion chet-AMP caused less vasodilatation compared to chet-Ado. Collectively this study provides first in-vivo evidence which establishes the prodrug concept as a site specific anti-inflammatory therapy in a mouse arthritis model.

P20-4

Adenosine A1 receptors regulates RANKL-induced osteoclastogenesis through JNK pathway

Wenjie He, Tuere Wilder, Bruce Cronstein

NYU, New York University, USA

Adenosine is a purine molecule necessary for normal cell metabolism and growth. Recent work from our laboratory using adenosine A1-deficient mice has demonstrated that adenosine A1 receptors play a critical role in regulating bone turnover, raising the intriguing possibility of targeting A1 receptors for therapeutic advancement in osteoporosis and other bone diseases. In the present study, we investigated the mechanism by which A1 receptors regulate osteoclastogenesis in vitro using bone marrow-derived osteoclasts (osteoclast precursors derived from fresh marrow and exposed to M-CSF and RANKL).

As we have previously demonstrated, treatment with the specific A1-receptor antagonist DPCPX (1 μM) inhibits (50 ± 1.2% inhibition, p < 0.001 vs. RANKL alone, n = 3) the differentiation of osteoclast precursors into TRAP+ multinucleated cells expressing specific osteoclast markers (MMP9, Integrin αv, Integrin β3, Cathepsin K and TRAP). Western blot analysis of cell extracts demonstrated that DPCPX diminished activation (phosphorylation) of Jun N-terminal kinase (JNK) (31 ± 8.5% inhibition p < 0.05 vs. RANKL alone, n = 3) and c-Jun (64 ± 8.8% inhibition p < 0.05 vs. RANKL alone, n = 3), signaling intermediates critical for osteoclast differentiation. Furthermore, real time RT-PCR demonstrated that DPCPX treatment reduced message for key osteoclastogenic transcription factors, c-fos (40 ± 2% inhibition p < 0.001 vs. RANKL alone, n = 3), c-Jun (30 ± 4% inhibition p < 0.001 vs. RANKL alone, n = 3) and NFATc1 (90 ± 8% inhibition p < 0.001 vs. RANKL alone, n = 3).

Collectively, these data suggest that DPCPX has an antiosteoclastogenic effect by inhibiting RANKL-induced activation of the JNK/c-Jun pathway, thereby suppressing the expression of c-fos and NFATc1 in osteoclast precursors. These results are consistent with the hypothesis that endogenously released adenosine, acting at A1 receptors, is critical for the expression and activation of critical signaling intermediates required for osteoclastogenesis. Moreover, these results further support the notion that blockade of adenosine A1 receptors may be useful in the treatment and prevention of osteoporosis and premature bone loss.

P20-5

Adenosine A2A receptor agonists: can they prevent/treat joint prosthesis loosening?

Aranzazu Mediero Munoz1, Sally R. Frenkel2, Igor Immerman2, Scott Hadley2, Damani Howell2, Bruce N. Cronstein1

1Department of Medicine, NYU Medical Center, USA,2Department of Orthopaedic Surgery, Nyu Hospital for Joint Diseases, USA

We and others reported that adenosine A2A receptors suppress inflammation in vitro and in vivo. We have also observed that activated receptors suppress osteoclast formation in vitro and that mice lacking these receptors have increased numbers of osteoclasts in their bones.Survival of joint implants depends on biological fixation, and is undermined by prosthesis loosening. Inflammation and osteoclast-mediated bone resorption in response to wear particles near prostheses contribute to loosening.. We hypothesized that adenosine A2A receptor agonist (CGS21680) might also prevent osteoclast-mediated bone resorption at the site of prosthesis wear. If so, site-specific delivery of the agonist could enhance implant survival, delaying or eliminating the need for revision arthroplastic surgery.This study examined the effect of A2A agonists on bone resorption in a calvarial model of wear particle-induced bone resorption.

18 C57 mice received, directly on their calvaria: polyethylene particulate plus A2A agonist injection every other day, particulate plus saline injection, or no particles (sham). Sacrifice was at day 14. We also determined the signaling pathways involved in A2A receptor-mediated suppression of osteoclast differentiation in mouse osteoclast cultures treated with a range of doses of CGS21680 for 3–7 days. TRAP staining and Western blots were performed.

Histology of calvarial bone showed inflammation in both particle-exposed groups. μCT showed pitting and increased porosity in both particle-exposed groups; pitting was absent in controls. Bone mineral density was significantly greater in controls than in either particulate group (p < 0.05), with no difference in density between the particulate groups. Control bone volume/trabecular volume was significantly greater (p < 0.005) than in either particulate group. However, A2A agonist -treated specimens had a significantly greater mean volume than did the untreated group (p < 0.0005). Trabecular thickness was significantly greater in controls than in the particulate-only group (p < 0.05), with no difference in thickness between controls and A2A agonist -treated specimens.

TRAP staining of GM-CSF/RANKL-stimulated osteoclast precursors revealed that in vitro, the A2A agonist CGS21680 inhibited osteoclast differentiation by as much as 38 ± 1% with an IC50 of approximately 50 nM (p < 0.05, n = 4). As we and others have previously demonstrated, CGS21680 induced activation (phosphorylation) of erk1/2 and JAK1 while downregulating NFkB activation (n = 2).

It appears that the A2A receptor agonist may reduce the destructive effects of polyethylene wear debris on bone. In the presence of inflammation induced by the particulate, bone volume and trabecular thickness may have been protected by treatment with the agonist, as demonstrated by the greater values for these two parameters when compared to untreated specimens. Therapeutic potential of the A2A receptor agonist merits further study.

P21. Pain

P21-1

Role of interferon regulatory factor-8 in the pathogenesis of neuropathic pain

(Selected for Oral Poster Presentation)

Takahiro Masuda1, Makoto Tsuda1, Ryohei Yoshinaga1, Tomohiko Tamura2, Kazuhide Inoue1

1Dept. Mol. Syst. Pharmacol., Grad. Sch. Pharma., Kyushu University, Japan,2Dept. Immunol., Grad. Sch. Med., Yokohama City University, Japan

Neuropathic pain is a debilitating pain condition that arises as a consequence of excessive excitability of neurons in spinal dorsal horn after peripheral nerve injury (PNI). This pathological abnormality of neurotransmission involves signaling from activated spinal microglia that induce or enhance expression of various genes including purinoceptors and proinflammatory cytokines. However, a key transcription factor regulating gene expression and neuropathic pain states is not identified. In the present study, we examined the role of interferon regulatory factor-8 (IRF-8), a member of IRF family transcription factor, in regulating tactile allodynia (hypersensitivity to innocuous mechanical stimuli). We found that PNI increased the expression of IRF-8 in the spinal microglia in a cell-specific manner. Furthermore, mice lacking IRF-8 (irf8−/−) exhibited a marked reduction in allodynia after PNI compared with wild-type mice without affecting normal pain sensitivity or responses to cold stimuli. In contrast, these mice showed a similar pain behavior in an inflammatory pain model. Interestingly, irf8−/− mice failed to increase the expression of genes crucial for producing pain hypersensitivity (such as P2X7 and P2Y12) in the spinal cord following PNI. Together, our present findings suggest that IRF-8 critically contributes to the pathogenesis of neuropathic pain but not of inflammatory pain.

P21-2

CGRP-mediated enhancement of purinergic neuron/glia communication by the algogenic factor bradykinin in trigeminal mouse ganglia: implications for migraine pain

(Selected for Oral Poster Presentation)

Stefania Maria Ceruti1, Giovanni Villa1, Marta Fumagalli1, Laura Colombo1, Matteo Zanardelli1, Elsa Fabbretti2, Claudia Verderio3, Arn M.J.M. van Den Maagdenberg4, Andrea Nistri5, Maria P. Abbracchio1

1Department of Pharmacological Sciences, Universita' Degli Studi Di Milano, Italy,2University of Nova Gorica, Slovenia,3Institute of Neuroscience, CNR, Italy,4Department of Human Genetics and Neurology, Leiden University Medical Centre, The Netherlands,5Neurobiology Sector and Italian Institute of Technology Unit, International School for Advanced Studies (SISSA), Italy

Purinergic signaling plays a special role in neurons-to-satellite glial cells (SGCs) communication within trigeminal ganglia (TG), a key relay station for pain. We previously showed that, in primary TG cultures, the algogenic mediator bradykinin (BK) potentiates purinergic glial P2Y-receptors. Here we investigated the molecular basis of this effect in wild type (wt) and in CACNA1A-R192Q KI mice expressing a human mutation causing familial hemiplegic migraine type 1. We utilized several different approaches, including single cell calcium imaging, immunocytochemistry, RT-PCR analysis, and CGRP measurements with an ELISA kit. Calcium imaging of wt cultures revealed functional BK receptors in neurons only, suggesting a paracrine action by BK to release a soluble mediator responsible for the observed effects on glial cells. We identified this mediator as the peptide CGRP whose ELISA-assayed levels were markedly increased by BK (61.5 ± 6.7 pg/ml vs 36.2 ± 3.7 pg/ml *p < 0.01), while the CGRP antagonist CGRP8-37 inhibited the BK action. Unlike CGRP, BK was ineffective on neuron-free SGCs cultures, confirming the CGRP neuronal source. Interestingly, we demonstrated the CGRP-mediated glial P2Y receptor potentiation occurs via activation of ERK1/2 pathways. Importantly, we also show, for the first time, that both the basal and BK-stimulated trigeminal release of CGRP are increased (by 49.6% and 80.2%, respectively) in the CACNA1A-R192Q KI mouse. This suggests that the detected changes are of relevance to migraine pathophysiology in patients, and that P2Y receptors on glial cells may represent new targets for the development of innovative therapeutic agents for migraine pain. Sponsored by the Italian Comitato Telethon.

P21-3

The analgesic effect of acupuncture is mediated by adenosine A1 receptors

Nanna Goldman-Nedergaard1, Michael Chen1, Takumi Fujita1, Qiwu Xu1, Jiang-Fan Chen2, Jurgen Schnermann3, Takahiro Takano1, Lane Bekar1, Kim Tieu1, Maiken Nedergaard1

1Center for Translational Neuromedicine, University of Rochester, USA,2Department of Neurology, Boston University, USA,3NIDDK, NIH, USA

Acupuncture is an invasive intervention commonly used to relieve pain. Acupuncture is practiced worldwide, despite difficulties reconciling its principles with evidence-based medicine. Although the analgesic effect of acupuncture is well documented, surprisingly little is understood about its biological basis. Insertion of the acupuncture needles is in itself not sufficient to relieve pain and the pain threshold is reported to slowly increase, and to outlast acupuncture (1). Because purines are released in response to tissue injury and the anti-nociceptive effects of peripheral adenosine A1 receptors are well documented (2), we here evaluated the effect of deletion of A1 receptor on the effect of acupuncture in mice models of chronic inflammatory or neurophatic pain. Deletion of A1 receptors significantly reduced the anti-nociceptive effect of acupuncture in both mice models (p < 0.01, n = 5–12). To specifically address whether acupuncture acted directly on ascending nerve tracks, we recorded in vivo responses of the left anterior cingulate cortex (ACC) to painful stimulation of the right foot (3). Acupuncture consistently reduced the field excitatory postsynaptic potentials (fEPSP) in the ACC in WT mice, but not in mice with deletion of A1 receptors. Direct injection of the A1 receptor agonist CCPA in the acupoint replicated the anti-nociceptive effect of acupuncture. These observations suggest that adenosine A1 receptors on ascending nerves, at least in part, mediate the analgesic effects of acupuncture. Thus, interference with A1 receptors or with adenosine metabolism may prolong the clinical benefit of acupuncture.

1. Zhao ZQ (2008) Neural mechanism underlying acupuncture analgesia. Prog Neurobiol 85(4):355–375.

2. Sawynok J (1998) Adenosine receptor activation and nociception. Eur J Pharmacol 347(1):1–11.

3. Wei F & Zhuo M (2001) Potentiation of sensory responses in the anterior cingulate cortex following digit amputation in the anaesthetised rat. J Physiol 532(Pt 3):823–833.

P21-4

The oral reversibly-binding antiplatelet agent, Ticagrelor, acts as an antagonist at the P2Y-like receptor GPR17

Claudia Martini1, Simona Daniele1, Maria Letizia Trincavelli1, Davide Lecca2, Anna Panighini1, Maria Pia Abbracchio2

1Dept. Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Italy,2Dept. Pharmacological Science, University of Milan, Italy

Background: Ticagrelor (formerly known as AZD6140) is a novel reversibly-binding P2Y12 receptor antagonist, currently in clinical trial as an antiplatelet agent in the prevention of cardiac death, myocardial infarction and stroke after acute coronary syndromes. In this work, we investigated the ticagrelor activity on GPR17, a newly deorphanized P2Y-like receptor located at an intermediate phylogenetic position between P2Y and CysLT receptor families, and responsive to both uracyl nucleotides and cysteinyl-leukotrienes. Methods: Ticagrelor activity towards GPR17 was evaluated by means of GTPγS binding in 1321N1 cells transfected with human GPR17, by assessing the effect of various compound concentrations on GPR17-G protein coupling. Results: In 1321N1 cells transfected with GPR17, when tested alone, ticagrelor (0.1 nM–1 µM) increased GTPγS binding, suggesting a partial agonist activity. When tested in the presence of nucleotide and cysteinyl-leucotriene agonists, ticagrelor was able to counteract GTPγS binding stimulation with a potency in the nanomolar range. Ticagrelor thus behaved as a partial agonist, showing intrinsic activity when utilized alone, and as an antagonist in the presence of GPR17 agonists. Antagonism was competitive at GPR17 nucleotide binding site and non-competitive at the cysteinyl-leukotriene binding site. Conclusion: As excessive GPR17 activation has been involved in brain damage progression during ischemia, besides its peripheral activity as an anti-platelet agent, ticagrelor may also be useful as a centrally-acting anti-ischemic agent, due to its antagonistic activity on GPR17. Funded by Italian COFIN-MIUR projects and by AstraZeneca Pharmaceuticals LP.


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