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1.  Molecular mechanism for opioid dichotomy: bidirectional effect of μ-opioid receptors on P2X3 receptor currents in rat sensory neurones 
Purinergic Signalling  2015;11(2):171-181.
Here, we describe a molecular switch associated with opioid receptors-linked signalling cascades that provides a dual opioid control over P2X3 purinoceptor in sensory neurones. Leu-enkephalin inhibited P2X3-mediated currents with IC50 ~10 nM in ~25 % of small nociceptive rat dorsal root ganglion (DRG) neurones. In contrast, in neurones pretreated with pertussis toxin leu-enkephalin produced stable and significant increase of P2X3 currents. All effects of opioid were abolished by selective μ-opioid receptor antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), nonselective inhibitor naloxone, and by PLC inhibitor U73122. Thus, we discovered a dual link between purinoceptors and μ-opioid receptors: the latter exert both inhibitory (pertussis toxin-sensitive) and stimulatory (pertussis toxin-insensitive) actions on P2X3 receptors through phospholipase C (PLC)-dependent pathways. This dual opioid control of P2X3 receptors may provide a molecular explanation for dichotomy of opioid therapy. Pharmacological control of this newly identified facilitation/inhibition switch may open new perspectives for the adequate medical use of opioids, the most powerful pain-killing agents known today.
doi:10.1007/s11302-015-9443-x
PMCID: PMC4425719  PMID: 25592684
Pain; Sensory neurones; Nociceptive neurones; P2X3 receptors; Opioid receptors; Leu-enkephalin
2.  Shear stress modulates endothelial KLF2 through activation of P2X4 
Purinergic Signalling  2015;11(1):139-153.
Vascular endothelial cells that are in direct contact with blood flow are exposed to fluid shear stress and regulate vascular homeostasis. Studies report endothelial cells to release ATP in response to shear stress that in turn modulates cellular functions via P2 receptors with P2X4 mediating shear stress-induced calcium signaling and vasodilation. A recent study shows that a loss-of-function polymorphism in the human P2X4 resulting in a Tyr315>Cys variant is associated with increased pulse pressure and impaired endothelial vasodilation. Although the importance of shear stress-induced Krüppel-like factor 2 (KLF2) expression in atheroprotection is well studied, whether ATP regulates KLF2 remains unanswered and is the objective of this study. Using an in vitro model, we show that in human umbilical vein endothelial cells (HUVECs), apyrase decreased shear stress-induced KLF2, KLF4, and NOS3 expression but not that of NFE2L2. Exposure of HUVECs either to shear stress or ATPγS under static conditions increased KLF2 in a P2X4-dependent manner as was evident with both the receptor antagonist and siRNA knockdown. Furthermore, transient transfection of static cultures of human endothelial cells with the Tyr315>Cys mutant P2X4 construct blocked ATP-induced KLF2 expression. Also, P2X4 mediated the shear stress-induced phosphorylation of extracellular regulated kinase-5, a known regulator of KLF2. This study demonstrates a major physiological finding that the shear-induced effects on endothelial KLF2 axis are in part dependent on ATP release and P2X4, a previously unidentified mechanism.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9442-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-014-9442-3
PMCID: PMC4336305  PMID: 25563726
Shear stress; Purinergic receptors; Krüppel-like factor 2; ATP
3.  Mutation in the Drosophila melanogaster adenosine receptor gene selectively decreases the mosaic hyperplastic epithelial outgrowth rates in wts or dco heterozygous flies 
Purinergic Signalling  2014;11(1):95-105.
Adenosine (Ado) is a ubiquitous metabolite that plays a prominent role as a paracrine homeostatic signal of metabolic imbalance within tissues. It quickly responds to various stress stimuli by adjusting energy metabolism and influencing cell growth and survival. Ado is also released by dead or dying cells and is present at significant concentrations in solid tumors. Ado signaling is mediated by Ado receptors (AdoR) and proteins modulating its concentration, including nucleoside transporters and Ado deaminases. We examined the impact of genetic manipulations of three Drosophila genes involved in Ado signaling on the incidence of somatic mosaic clones formed by the loss of heterozygosity (LOH) of tumor suppressor and marker genes. We show here that genetic manipulations with the AdoR, equilibrative nucleoside transporter 2 (Ent2), and Ado deaminase growth factor-A (Adgf-A) cause dramatic changes in the frequency of hyperplastic outgrowth clones formed by LOH of the warts (wts) tumor suppressor, while they have almost no effect on control yellow (y) clones. In addition, the effect of AdoR is dose-sensitive and its overexpression leads to the increase in wts hyperplastic epithelial outgrowth rates. Consistently, the frequency of mosaic hyperplastic outgrowth clones generated by the LOH of another tumor suppressor, discs overgrown (dco), belonging to the wts signaling pathway is also dependent on AdoR. Our results provide interesting insight into the maintenance of tissue homeostasis at a cellular level.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9435-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-014-9435-2
PMCID: PMC4336310  PMID: 25528157
Cell competition; Adgf-A; Ent2; LATS1; Warts; Discs overgrown
4.  Cardiac purinergic signalling in health and disease 
Purinergic Signalling  2014;11(1):1-46.
This review is a historical account about purinergic signalling in the heart, for readers to see how ideas and understanding have changed as new experimental results were published. Initially, the focus is on the nervous control of the heart by ATP as a cotransmitter in sympathetic, parasympathetic, and sensory nerves, as well as in intracardiac neurons. Control of the heart by centers in the brain and vagal cardiovascular reflexes involving purines are also discussed. The actions of adenine nucleotides and nucleosides on cardiomyocytes, atrioventricular and sinoatrial nodes, cardiac fibroblasts, and coronary blood vessels are described. Cardiac release and degradation of ATP are also described. Finally, the involvement of purinergic signalling and its therapeutic potential in cardiac pathophysiology is reviewed, including acute and chronic heart failure, ischemia, infarction, arrhythmias, cardiomyopathy, syncope, hypertrophy, coronary artery disease, angina, diabetic cardiomyopathy, as well as heart transplantation and coronary bypass grafts.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9436-1) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-014-9436-1
PMCID: PMC4336308  PMID: 25527177
ATP; Adenosine; Coronary vessels; Innervation; Cardiomyocytes; Pathophysiology
5.  P2X7 receptor of rat dorsal root ganglia is involved in the effect of moxibustion on visceral hyperalgesia 
Purinergic Signalling  2014;11(2):161-169.
Irritable bowel syndrome (IBS) and inflammatory bowel disease often display visceral hypersensitivity. Visceral nociceptors after inflammatory stimulation generate afferent nerve impulses through dorsal root ganglia (DRG) transmitting to the central nervous system. ATP and its activated-purinergic 2X7 (P2X7) receptor play an important role in the transmission of nociceptive signal. Purinergic signaling is involved in the sensory transmission of visceral pain. Moxibustion is a therapy applying ignited mugwort directly or indirectly at acupuncture points or other specific parts of the body to treat diseases. Heat-sensitive acupoints are the corresponding points extremely sensitive to moxa heat in disease conditions. In this study, we aimed to investigate the relationship between the analgesic effect of moxibustion on a heat-sensitive acupoint “Dachangshu” and the expression levels of P2X7 receptor in rat DRG after chronic inflammatory stimulation of colorectal distension. Heat-sensitive moxibustion at Dachangshu acupoint inhibited the nociceptive signal transmission by decreasing the upregulated expression levels of P2X7 mRNA and protein in DRG induced by visceral pain, and reversed the abnormal expression of glial fibrillary acidic protein (GFAP, a marker of satellite glial cells) in DRG. Consequently, abdominal withdrawal reflex (AWR) score in a visceral pain model was reduced, and the pain threshold was elevated. Therefore, heat-sensitive moxibustion at Dachangshu acupoint can produce a therapeutic effect on IBS via inhibiting the nociceptive transmission mediated by upregulated P2X7 receptor.
doi:10.1007/s11302-014-9439-y
PMCID: PMC4425718  PMID: 25527178
Moxibustion; Dorsal root ganglia; Purinergic 2X7 receptor; Satellite glial cell; Visceral hyperalgesia; Colorectal distension
6.  Rat submandibular glands secrete nanovesicles with NTPDase and 5′-nucleotidase activities 
Purinergic Signalling  2014;11(1):107-116.
Extracellular nucleotides modulate a wide number of biological processes such as neurotransmission, platelet aggregation, muscle contraction, and epithelial secretion acting by the purinergic pathway. Nucleotidases as NTPDases and ecto-5′-nucleotidase are membrane-anchored proteins that regulate extracellular nucleotide concentrations. In a previous work, we have partially characterized an NTPDase-like activity expressed by rat submandibular gland microsomes, giving rise to the hypothesis that membrane NTPDases could be released into salivary ducts to regulate luminal nucleotide concentrations as was previously proposed for ovarian, prostatic, and pancreatic secretions. Present results show that rat submandibular glands incubated in vitro release membrane-associated NTPDase and ecto-5′-nucleotidase activities. Electron microscopy images show that released membranes presenting nucleotidase activity correspond to exosome-like vesicles which are also present at microsomal fraction. Both exosome release and nucleotidase activities are raised by adrenergic stimulation. Nucleotidase activities present the same kinetic characteristics than microsomal nucleotidase activity, corresponding mainly to the action of NTPDase2 and NTPDase3 isoforms as well as 5′-nucleotidase. This is consistent with Western blot analysis revealing the presence of these enzymes in the microsomal fraction.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9437-0) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-014-9437-0
PMCID: PMC4336312  PMID: 25523180
Submandibular gland; Extracellular nucleotides; NTPDases; Nucleotidases; Nanovesicles; Extracellular vesicles
7.  Pharmacological and molecular characterization of functional P2 receptors in rat embryonic cardiomyocytes 
Purinergic Signalling  2014;11(1):127-138.
Purinergic receptors activated by extracellular nucleotides (adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP)) are well known to exert physiological effects on the cardiovascular system, whether nucleotides participate functionally in embryonic heart development is not clear. The responsiveness of embryonic cardiomyocytes (E) 12 to P2 receptor agonists by measuring Ca2+ influx did not present response to ATP, but responses to P2 agonists were detected in cardiomyocytes taken from E14 and E18 rats. Photometry revealed that the responses to ATP were concentration-dependent with an EC50 of 1.32 μM and 0.18 μM for E14 and E18 cardiomyocytes, respectively. In addition, other P2 agonists were also able to induce Ca2+ mobilization. RT-PCR showed the presence of P2X2 and P2X4 receptor transcripts on E14 cardiomyocytes with a lower expression of P2X3 and P2X7 receptors. P2X1 and a low level of P2X5 receptor messenger RNA (mRNA) were also expressed at E18. Immunofluorescence data indicated that only P2X2 and P2X4 receptor proteins were expressed in E14 cardiomyocytes while protein for all the P2X receptor subtypes was expressed in E18, except for P2X3 and P2X6. Responses mediated by agonists specific for P2Y receptors subtypes showed that P2Y receptors (P2Y1, P2Y2, P2Y4 and P2Y6) were also present in both E14 and E18 cardiomyocytes. Dye transfer experiments showed that ATP induces coupling of cells at E12, but this response is decreased at E14 and lost at E18. Conversely, UTP induced coupling with five or more cells in most cells from E12 to E18. Our results show that specific P2 receptor subtypes are present in embryonic rat cardiomyocytes, including P2X7 and P2Y4 receptors that have not been identified in adult rat cardiomyocytes. The responsiveness to ATP stimulation even before birth, suggests that ATP may be an important messenger in embryonic as well as in adult hearts.
doi:10.1007/s11302-014-9441-4
PMCID: PMC4336309  PMID: 25510459
ATP; P2X receptors; P2Y receptors; P1 receptors; Rat embryo; Heart; Cardiomyocyte; FLIPR; Calcium mobilization
8.  Purine receptors are required for DHA-mediated neuroprotection against oxygen and glucose deprivation in hippocampal slices 
Purinergic Signalling  2014;11(1):117-126.
Docosahexaenoic acid (DHA) is important for central nervous system function during pathological states such as ischemia. DHA reduces neuronal injury in experimental brain ischemia; however, the underlying mechanisms are not well understood. In the present study, we investigated the effects of DHA on acute hippocampal slices subjected to experimental ischemia by transient oxygen and glucose deprivation (OGD) and re-oxygenation and the possible involvement of purinergic receptors as the mechanism underlying DHA-mediated neuroprotection. We observed that cellular viability reduction induced by experimental ischemia as well as cell damage and thiobarbituric acid reactive substances (TBARS) production induced by glutamate (10 mM) were prevented by hippocampal slices pretreated with DHA (5 μM). However, glutamate uptake reduction induced by OGD and re-oxygenation was not prevented by DHA. The beneficial effect of DHA against cellular viability reduction induced by OGD and re-oxygenation was blocked with PPADS (3 μM), a nonselective P2X1–5 receptor antagonist as well as with a combination of TNP-APT (100 nM) plus brilliant blue (100 nM), which blocked P2X1, P2X3, P2X2/3, and P2X7 receptors, respectively. Moreover, adenosine receptors blockade with A1 receptor antagonist DPCPX (100 nM) or with A2B receptor antagonist alloxazine (100 nM) inhibited DHA-mediated neuroprotection. The addition of an A2A receptor antagonist ZM241385 (50 nM), or A3 receptor antagonist VUF5574 (1 μM) was ineffective. Taken together, our results indicated that neuroprotective actions of DHA may depend on P2X, A1, and A2B purinergic receptors activation. Our results reinforce the notion that dietary DHA may act as a local purinergic modulator in order to prevent neurodegenerative diseases.
doi:10.1007/s11302-014-9438-z
PMCID: PMC4336303  PMID: 25504554
DHA; Adenosine receptors; ATP receptors; Neuroprotection
9.  NTPDase2 and the P2Y1 receptor are not required for mammalian eye formation 
Purinergic Signalling  2014;11(1):155-160.
Eye formation in vertebrates is controlled by a conserved pattern of molecular networks. Homeobox transcription factors are crucially involved in the establishment and maintenance of the retina. A previous study of Massé et al. (Nature, 449: 1058–62, 2007) using morpholino knockdown identified the ectonucleotidase NTPDase2 and the P2Y1 receptor as essential elements for eye formation in embryos of the clawed frog Xenopus laevis. In order to investigate whether a similarly essential mechanism would be active in mammalian eye development, we analyzed mice KO for Entpd2 or P2ry1 as well as double KO for Entpd2/P2ry1. These mice developed normal eyes. In order to identify potential deficits in the molecular identity or in the arrangement of the cellular elements of the retina, we performed an immunohistological analysis using a variety of retinal markers. The analysis of single and double KO mice demonstrated that NTPDase2 and P2Y1 receptors are not required for murine eye formation, as previously shown for eye development in Xenopus laevis.
doi:10.1007/s11302-014-9440-5
PMCID: PMC4336302  PMID: 25504514
NTPDase2; ATP; ADP; P2Y1 receptor; Purinergic signaling; Eye development
12.  Diadenosine tetraphosphate contributes to carbachol-induced tear secretion 
Purinergic Signalling  2014;11(1):87-93.
The purpose of this study is to investigate if the cholinergic stimulation by carbachol on tear secretion is a direct process or if it is also mediated by purinergic mechanisms. Experiments were performed in New Zealand male rabbits. The amount of tear secretion was measured with Schirmer’s test and then analyzed by a HPLC protocol in order to study the nucleotide levels. Animal eyes were instilled with carbachol (a cholinergic agonist), pirenzepine, gallamine and 4-DAMP (muscarinic antagonists), PPADS, suramin and reactive blue 2 (purinergic antagonists), and a P2Y2 receptor small interfering RNA (siRNA). Tear secretion increased with the instillation of carbachol, approximately 84 % over control values 20 min after the instillation and so did Ap4A and ATP release. When we applied carbachol in the presence of muscarinic antagonists, tear volume only increased to 4 % with atropine, 12 % in the case of pirenzepine, 3 % with gallamine, and 8 % with 4-DAMP. In the presence of carbachol and purinergic antagonists, tear secretion was increased to 12 % (all values compared to basal tear secretion). By analyzing tear secretion induced with carbachol in presence of a P2Y2 receptor siRNA, we found that tear secretion was diminished to 60 %. The inhibition of tear secretion in the presence of carbachol and purinergic antagonists or P2Y2 siRNA occurred with no apparent change in the tear amount of Ap4A. These experiments demonstrated the participation of Ap4A in lacrimal secretion process.
doi:10.1007/s11302-014-9434-3
PMCID: PMC4336306  PMID: 25398705
Carbachol; Purinergic; Nucleotides; Tear secretion; P2Y
13.  Enhanced survival of lethally irradiated adenosine A3 receptor knockout mice. A role for hematopoietic growth factors? 
Purinergic Signalling  2014;11(1):79-85.
Adenosine A3 receptor knockout (A3AR KO) mice and their wild-type (WT) counterparts were compared from the point of view of their abilities to survive exposures to lethal doses of γ-radiation belonging to the range of radiation doses inducing the bone marrow acute radiation syndrome. Parameters of cumulative 30-day survival (experiment using a midlethal radiation dose) or cumulative 11-day survival (experiment using an absolutely lethal radiation dose), and of mean survival time were evaluated. The values of A3AR KO mice always reflected their higher survival in comparison with WT ones, the P values being above the limit for statistical significance after the midlethal radiation dose and standing for statistical significance after the absolutely lethal radiation dose. This finding was considered surprising, taking into account the previously obtained findings on defects in numbers and functional properties of peripheral blood cells in A3AR KO mice. Therefore, previous hematological analyses of A3AR KO mice were supplemented in the present studies with determination of serum levels of the granulocyte colony-stimulating factor, erythropoietin, and thrombopoietin. Though distinct differences in these parameters were observed between A3AR KO and WT mice, none of them could explain the relatively high postirradiation survival of A3AR KO mice. Further studies on these mice comprising also those on other than hemopoietic tissues and organs can help to clarify their relative radioresistance.
doi:10.1007/s11302-014-9432-5
PMCID: PMC4336304  PMID: 25358454
Adenosine A3 receptor; Postirradiation survival; Hematopoiesis; Granulocyte colony-stimulating factor; Erythropoietin; Thrombopoietin
14.  Comparative genomic and expression analysis of the adenosine signaling pathway members in Xenopus 
Purinergic Signalling  2014;11(1):59-77.
Adenosine is an endogenous molecule that regulates many physiological processes via the activation of four specific G-protein-coupled ADORA receptors. Extracellular adenosine may originate either from the hydrolysis of released ATP by the ectonucleotidases or from cellular exit via the equilibrative nucleoside transporters (SLC29A). Adenosine extracellular concentration is also regulated by its successive hydrolysis into uric acid by membrane-bound enzymes or by cell influx via the concentrative nucleoside transporters (SLC28A). All of these members constitute the adenosine signaling pathway and regulate adenosine functions. Although the roles of this pathway are quite well understood in adults, little is known regarding its functions during vertebrate embryogenesis. We have used Xenopus laevis as a model system to provide a comparative expression map of the different members of this pathway during vertebrate development. We report the characterization of the different enzymes, receptors, and nucleoside transporters in both X. laevis and X. tropicalis, and we demonstrate by phylogenetic analyses the high level of conservation of these members between amphibians and mammals. A thorough expression analysis of these members during development and in the adult frog reveals that each member displays distinct specific expression patterns. These data suggest potentially different developmental roles for these proteins and therefore for extracellular adenosine. In addition, we show that adenosine levels during amphibian embryogenesis are very low, confirming that they must be tightly controlled for normal development.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9431-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-014-9431-6
PMCID: PMC4336307  PMID: 25319637
Adenosine metabolism; Adenosine receptor; Embryogenesis; Extracellular adenosine; Nucleotide transporter; Xenopus
15.  Effects of NAD at purine receptors in isolated blood vessels 
Purinergic Signalling  2014;11(1):47-57.
Nicotinamide adenine dinucleotide (NAD) belongs to the family of naturally occurring adenine dinucleotides, best known for their various intracellular roles. However, there is evidence that they can also be released from cells to act as novel extracellular signalling molecules. Relatively little is known about the extracellular actions of NAD, especially in the cardiovascular system. The present study investigated the actions of NAD in the rat thoracic aorta, porcine coronary artery and porcine mesenteric arteries, mounted in organ baths for isometric tension recording. In the rat thoracic aorta and porcine coronary artery, NAD caused endothelium-independent concentration-dependent vasorelaxations which were unaffected by palmitoylCoA, a P2Y1 receptor antagonist, but which were blocked by CGS15943, a non-selective adenosine receptor antagonist. In the porcine coronary artery, NAD-evoked relaxations were abolished by SCH58261, a selective A2A receptor antagonist. In the rat thoracic aorta, NAD-evoked relaxations were attenuated by A2A receptor antagonism with SCH58261 but were unaffected by an A2B receptor antagonist, MRS1754. In contrast, in the porcine mesenteric artery, NAD-evoked endothelium-independent contractions, which were unaffected by a P2 receptor antagonist, suramin, or by NF449, a P2X1 receptor antagonist, but were attenuated following P2X receptor desensitisation with αβ-meATP. In conclusion, the present results show that NAD can alter vascular tone through actions at purine receptors in three different arteries from two species; its molecular targets differ according to the type of blood vessel.
doi:10.1007/s11302-014-9428-1
PMCID: PMC4336311  PMID: 25315718
NAD; P2 purine receptors; Adenosine receptors; Artery; Vasorelaxation
17.  The vesicular nucleotide transporter (VNUT) is involved in the extracellular ATP effect on neuronal differentiation 
Purinergic Signalling  2015;11(2):239-249.
Before being released, nucleotides are stored in secretory vesicles through the vesicular nucleotide transporter (VNUT). Once released, extracellular ATP participates in neuronal differentiation processes. Thus, the expression of a functional VNUT could be an additional component of the purinergic system which regulates neuronal differentiation and axonal elongation. In vitro expression of VNUT decreases neuritogenesis in N2a cells differentiated by retinoic acid treatment, whereas silencing of VNUT expression increases the number and length of neurites in these cells. These results highlight the role of VNUT in the neuritogenic process because this transporter regulates the ATP content in neurosecretory vesicles.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-015-9449-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-015-9449-4
PMCID: PMC4425722  PMID: 25847073
VNUT; Neuritogenesis; Retinoic acid differentiation; ATP; Neuroblastoma cells; SLC17A9
18.  PQ-69, a novel and selective adenosine A1 receptor antagonist with inverse agonist activity 
Purinergic Signalling  2014;10(4):619-629.
Potent and selective adenosine A1 receptor (A1AR) antagonists with favourable pharmacokinetic properties used as novel diuretics and antihypertensives are desirable. Thus, we designed and synthesized a series of novel 4-alkylamino substitution-2-arylpyrazolo[4,3-c]quinolin-3-one derivatives. The aim of the present study is to characterize the biological profiles of the optimized compound, PQ-69. In vitro binding assay revealed a Ki value of 0.96 nM for PQ-69 in cloned hA1 receptor, which was 217-fold more selective compared with hA2A receptors and >1,000-fold selectivity for hA1 over hA3 receptor. The results obtained from [35S]-GTPγS binding and cAMP concentration assays indicated that PQ-69 might be an A1AR antagonist with inverse agonist activity. In addition, PQ-69 displayed highly inhibitory activities on isolated guinea pig contraction (pA2 value of 8.99) induced by an A1AR agonist, 2-chloro-N6-cyclopentyl adenosine. Systemic administration of PQ-69 (0.03, 0.3, 3 mg/kg) increased urine flow and sodium excretion in normal rats. Furthermore, PQ-69 displayed better metabolic stability in vitro and longer terminal elimination half-life (t1/2) in vivo compared with 1,3-dipropyl-8-cyclopentylxanthine. These findings suggest that PQ-69 exhibits potent antagonist effects on A1AR in vitro, ex vivo and in vivo, it might be a useful research tool for investigating A1AR function, and it could be developed as a potential therapeutic agent.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9424-5) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-014-9424-5
PMCID: PMC4272367  PMID: 25248972
4-butylamino-2-(3-fluorophenyl)[4,3-c]quinolin-3-one; Non-xanthine adenosine A1 receptor (A1AR) antagonist; Inverse agonism; Diuresis
19.  Raised tone reveals ATP as a sympathetic neurotransmitter in the porcine mesenteric arterial bed 
Purinergic Signalling  2014;10(4):639-649.
The relative importance of ATP as a functional sympathetic neurotransmitter in blood vessels has been shown to be increased when the level of preexisting vascular tone or pressure is increased, in studies carried out in rat mesenteric arteries. The aim of the present study was to determine whether tone influences the involvement of ATP as a sympathetic cotransmitter with noradrenaline in another species. We used the porcine perfused mesenteric arterial bed and porcine mesenteric large, medium and small arteries mounted for isometric tension recording, because purinergic cotransmission can vary depending on the size of the blood vessel. In the perfused mesenteric bed at basal tone, sympathetic neurogenic vasocontractile responses were abolished by prazosin, an α1-adrenoceptor antagonist, but there was no significant effect of α,β-methylene ATP, a P2X receptor-desensitizing agent. Submaximal precontraction of the mesenteric arterial bed with U46619, a thromboxane A2 mimetic, augmented the sympathetic neurogenic vasocontractile responses; under these conditions, both α,β-methylene ATP and prazosin attenuated the neurogenic responses. In the mesenteric large, medium and small arteries, prazosin attenuated the sympathetic neurogenic contractile responses under conditions of both basal and U46619-raised tone. α,β-Methylene ATP was effective in all of these arteries only under conditions of U46619-induced tone, causing a similar inhibition in all arteries, but had no significant effect on sympathetic neurogenic contractions at basal tone. These data show that ATP is a cotransmitter with noradrenaline in porcine mesenteric arteries; the purinergic component was revealed under conditions of partial precontraction, which is more relevant to physiological conditions.
doi:10.1007/s11302-014-9426-3
PMCID: PMC4272366  PMID: 25231507
ATP; Cotransmission; Noradrenaline; P2X receptor; Porcine mesenteric arteries; Sympathetic nerves
20.  Expression of mediators of purinergic signaling in human liver cell lines 
Purinergic Signalling  2014;10(4):631-638.
Purinergic signaling regulates a diverse and biologically relevant group of processes in the liver. However, progress of research into functions regulated by purinergic signals in the liver has been hampered by the complexity of systems probed. Specifically, there are multiple liver cell subpopulations relevant to hepatic functions, and many of these have been effectively modeled in human cell lines. Furthermore, there are more than 20 genes relevant to purinergic signaling, each of which has distinct functions. Hence, we felt the need to categorize genes relevant to purinergic signaling in the best characterized human cell line models of liver cell subpopulations. Therefore, we investigated the expression of adenosine receptor, P2X receptor, P2Y receptor, and ecto-nucleotidase genes via RT-PCR in the following cell lines: LX-2, hTERT, FH11, HepG2, Huh7, H69, and MzChA-1. We believe that our findings will provide an excellent resource to investigators seeking to define functions of purinergic signals in liver physiology and liver disease pathogenesis.
doi:10.1007/s11302-014-9425-4
PMCID: PMC4272373  PMID: 25194703
Liver cell line; Purinergic signaling; Hepatic stellate cell; Hepatocyte; Cholangiocyte
21.  Characterization of circulating microparticle-associated CD39 family ecto-nucleotidases in human plasma 
Purinergic Signalling  2014;10(4):611-618.
Phosphohydrolysis of extracellular ATP and ADP is an essential step in purinergic signaling that regulates key pathophysiological processes, such as those linked to inflammation. Classically, this reaction has been known to occur in the pericellular milieu catalyzed by membrane bound cellular ecto-nucleotidases, which can be released in the form of both soluble ecto-enzymes as well as being associated with exosomes. Circulating ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1/CD39) and adenylate kinase 1 (AK1) activities have been shown to be present in plasma. However, other ecto-nucleotidases have not been characterized in depth. An in vitro ADPase assay was developed to probe the ecto-enzymes responsible for the ecto-nucleotidase activity in human platelet-free plasma, in combination with various specific biochemical inhibitors. Identities of ecto-nucleotidases were further characterized by chromatography, immunoblotting, and flow cytometry of circulating exosomes. We noted that microparticle-bound E-NTPDases and soluble AK1 constitute the highest levels of ecto-nucleotidase activity in human plasma. All four cell membrane expressed E-NTPDases are also found in circulating microparticles in human plasma, inclusive of: CD39, NTPDase 2 (CD39L1), NTPDase 3 (CD39L3), and NTPDase 8. CD39 family members and other ecto-nucleotidases are found on distinct microparticle populations. A significant proportion of the microparticle-associated ecto-nucleotidase activity is sensitive to POM6, inferring the presence of NTPDases, either −2 or/and −3. We have refined ADPase assays of human plasma from healthy volunteers and have found that CD39, NTPDases 2, 3, and 8 to be associated with circulating microparticles, whereas soluble AK1 is present in human plasma. These ecto-enzymes constitute the bulk circulating ADPase activity, suggesting a broader implication of CD39 family and other ecto-enzymes in the regulation of extracellular nucleotide metabolism.
doi:10.1007/s11302-014-9423-6
PMCID: PMC4272360  PMID: 25165006
Ecto-nucleotidase; CD39; ATP; ADP; Adenosine; Purinergic signaling; TLC
22.  Contact lenses: new devices for nucleotide delivery in ocular pathologies 
Purinergic Signalling  2014;10(3):419-420.
doi:10.1007/s11302-014-9422-7
PMCID: PMC4152449  PMID: 25138143
23.  Adenosine enhances progenitor cell recruitment and nerve growth via its A2B receptor during adult fin regeneration 
Purinergic Signalling  2014;10(4):595-602.
A major issue in regenerative medicine is the control of progenitor cell mobilisation. Apoptosis has been reported as playing a role in cell plasticity, and it has been recently shown that apoptosis is necessary for organ and appendage regeneration. In this context, we explore its possible mode of action in progenitor cell recruitment during adult regeneration in zebrafish. Here, we show that apoptosis inhibition impairs blastema formation and nerve growth, both of which can be restored by exogenous adenosine acting through its A2B receptor. Moreover, adenosine increases the number of progenitor cells. Purinergic signalling is therefore an early and essential event in the pathway from lesion to blastema formation and provides new targets for manipulating cell plasticity in the adult.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9420-9) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-014-9420-9
PMCID: PMC4272362  PMID: 25084769
Apoptosis; Stem cells; Adenosine; A2B receptor; Nerve growth; Proliferation; Zebrafish; Regeneration
24.  P2X receptors regulate adenosine diphosphate release from hepatic cells 
Purinergic Signalling  2014;10(4):587-593.
Extracellular nucleotides act as paracrine regulators of cellular signaling and metabolic pathways. Adenosine polyphosphate (adenosine triphosphate (ATP) and adenosine diphosphate (ADP)) release and metabolism by human hepatic carcinoma cells was therefore evaluated. Hepatic cells maintain static nanomolar concentrations of extracellular ATP and ADP levels until stress or nutrient deprivation stimulates a rapid burst of nucleotide release. Reducing the levels of media serum or glucose has no effect on ATP levels, but stimulates ADP release by up to 10-fold. Extracellular ADP is then metabolized or degraded and media ADP levels fall to basal levels within 2–4 h. Nucleotide release from hepatic cells is stimulated by the Ca2+ ionophore, ionomycin, and by the P2 receptor agonist, 2′3′-O-(4-benzoyl-benzoyl)-adenosine 5′-triphosphate (BzATP). Ionomycin (10 μM) has a minimal effect on ATP release, but doubles media ADP levels at 5 min. In contrast, BzATP (10–100 μM) increases both ATP and ADP levels by over 100-fold at 5 min. Ion channel purinergic receptor P2X7 and P2X4 gene silencing with small interference RNA (siRNA) and treatment with the P2X inhibitor, A438079 (100 μM), decrease ADP release from hepatic cells, but have no effect on ATP. P2X inhibitors and siRNA have no effect on BzATP-stimulated nucleotide release. ADP release from human hepatic carcinoma cells is therefore regulated by P2X receptors and intracellular Ca2+ levels. Extracellular ADP levels increase as a consequence of a cellular stress response resulting from serum or glucose deprivation.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-014-9419-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s11302-014-9419-2
PMCID: PMC4272363  PMID: 25059924
ADP; ATP; Nucleotide release; P2X receptor; P2X4; P2X7
25.  UTP is not a biased agonist at human P2Y11 receptors 
Purinergic Signalling  2014;10(4):581-585.
Biased agonism describes a multistate model of G protein-coupled receptor activation in which each ligand induces a unique structural conformation of the receptor, such that the receptor couples differentially to G proteins and other intracellular proteins. P2Y receptors are G protein-coupled receptors that are activated by endogenous nucleotides, such as adenosine 5′-triphosphate (ATP) and uridine 5′-triphosphate (UTP). A previous report suggested that UTP may be a biased agonist at the human P2Y11 receptor, as it increased cytosolic [Ca2+], but did not induce accumulation of inositol phosphates, whereas ATP did both. The mechanism of action of UTP was unclear, so the aim of this study was to characterise the interaction of UTP with the P2Y11 receptor in greater detail. Intracellular Ca2+ was monitored in 1321N1 cells stably expressing human P2Y11 receptors using the Ca2+-sensitive fluorescent indicator, fluo-4. ATP evoked a rapid, concentration-dependent rise in intracellular Ca2+, but surprisingly, even high concentrations of UTP were ineffective. In contrast, UTP was slightly, but significantly more potent than ATP in evoking a rise in intracellular Ca2+ in 1321N1 cells stably expressing the human P2Y2 receptor, with no difference in the maximum response. Thus, the lack of response to UTP at hP2Y11 receptors was not due to a problem with the UTP solution. Furthermore, coapplying a high concentration of UTP with ATP did not inhibit the response to ATP. Thus, contrary to a previous report, we find no evidence for an agonist action of UTP at the human P2Y11 receptor, nor does UTP act as an antagonist.
doi:10.1007/s11302-014-9418-3
PMCID: PMC4272372  PMID: 25015314
Biased agonism; P2Y11 receptor; Inositol phosphates; Intracellular Ca2+

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