The nucleobase adenine has previously been reported to activate G protein-coupled receptors in rat and mouse. Adenine receptors (AdeR) thus constitute a new family of purine receptors, for which the designation “P0-receptors” has been suggested. We now describe the cloning and characterization of two new members of the AdeR family from mouse (MrgA10, termed mAde1R) and hamster (cAdeR). Both receptors were expressed in Sf9 insect cells, and radioligand binding studies were performed using [3H]adenine. Specific binding of the radioligand was detected in transfected, but not in untransfected cells, and KD values of 286 nM (mAde1R, Bmax 1.18 pmol/mg protein) and 301 nM (cAdeR, Bmax 17.7 pmol/mg protein), respectively, were determined. A series of adenine derivatives was investigated in competition binding assays. Minor structural modifications generally led to a reduction or loss of affinity, with one exception: 2-fluoroadenine was at least as potent as adenine itself at the cAdeR. Structure–activity relationships at all AdeR orthologs and subtypes investigated so far were similar, but not identical. For functional analyses, the cAdeR was homologously expressed in Chinese hamster ovary (CHO) cells, while the mAde1R was heterologously expressed in 1321N1 astrocytoma cells. Like the previously described AdeRs from rat (rAdeR) and mouse (mAde2R), the mAde1R (EC50 9.77 nM) and the cAdeR (EC50 51.6 nM) were coupled to inhibition of adenylate cyclase. In addition, the cAdeR from hamster expressed in CHO cells produced an increase in intracellular calcium concentrations (EC50 6.24 nM) and was found to be additionally coupled to Gq proteins.
Adenine receptor; G protein-coupled receptor; Sf9 insect cells; Chinese hamster ovary cells; Radioligand binding; Mrg receptor
Inflammation is responsible for secondary organ failure after trauma and hemorrhagic shock (T/HS). Adenosine, acting through four G protein-coupled cell surface receptors, A1, A2A, A2B, and A3, exerts a number of tissue protective and anti-inflammatory effects. The goal of the present study was to test the effect of A2B adenosine receptor stimulation on T/HS-induced organ injury and inflammation in rats. Rats after T/HS were resuscitated with Ringer’s lactate containing the A2B receptor agonist BAY 60–6583 or its vehicle. We found that BAY 60–6583 decreased T/HS-induced lung permeability and plasma creatine kinase levels but failed to affect T/HS-induced lung neutrophil infiltration and IκBα expression and plasma alanine aminotransferase levels. Thus, we conclude that stimulation of A2B receptors protects against T/HS-induced lung and muscle injury.
Trauma; Hemorrhagic shock; A2B adenosine receptor; BAY 60–6583
The rat adenine receptor (rAdeR) was the first member of a family of G protein-coupled receptors (GPCRs) activated by adenine and designated as P0-purine receptors. The present study aimed at gaining insights into structural aspects of ligand binding and function of the rAdeR. We exchanged amino acid residues predicted to be involved in ligand binding (Phe1103.24, Asn1153.29, Asn1734.60, Phe17945.39, Asn1945.40, Phe1955.41, Leu2015.47, His2526.54, and Tyr2687.32) for alanine and expressed them in Spodoptera frugiperda (Sf9) insect cells. Membrane preparations subjected to [3H]adenine binding studies revealed only minor effects indicating that none of the exchanged amino acids is part of the ligand binding pocket, at least in the inactive state of the receptor. Furthermore, we coexpressed the rAdeR and its mutants with mammalian Gi proteins in Sf9 insect cells to probe receptor activation. Two amino acid residues, Asn1945.40 and Leu2015.47, were found to be crucial for activation since their alanine mutants did not respond to adenine. Moreover we showed that—in contrast to most other rhodopsin-like GPCRs—the rAdeR does not contain essential disulfide bonds since preincubation with dithiothreitol neither altered adenine binding in Sf9 cell membranes, nor adenine-induced inhibition of adenylate cyclase in 1321N1 astrocytoma cells transfected with the rAdeR. To detect rAdeRs by Western blot analysis, we developed a specific antibody. Finally, we were able to show that the extended N-terminal sequence of the rAdeR constitutes a putative signal peptide of unknown function that is cleaved off in the mature receptor. Our results provide important insights into this new, poorly investigated family of purinergic receptors.
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The online version of this article (doi:10.1007/s11302-013-9355-6) contains supplementary material, which is available to authorized users.
Antibody; G protein-coupled receptor; Mutagenesis; Radioligand binding; Rat adenine receptor; Sf9 cells; Signal peptide
Extracellular ATP and related nucleotides promote a wide range of pathophysiological responses via activation of cell surface purinergic P2 receptors. Almost every cell type expresses P2 receptors and/or exhibit regulated release of ATP. In this review, we focus on the purinergic receptor distribution in inflammatory cells and their implication in diverse immune responses by providing an overview of the current knowledge in the literature related to purinergic signaling in neutrophils, macrophages, dendritic cells, lymphocytes, eosinophils, and mast cells. The pathophysiological role of purinergic signaling in these cells include among others calcium mobilization, actin polymerization, chemotaxis, release of mediators, cell maturation, cytotoxicity, and cell death. We finally discuss the therapeutic potential of P2 receptor subtype selective drugs in inflammatory conditions.
ATP; Extracellular nucleotides; P2 receptors; Inflammatory cells; Regulation of immune responses
P2X receptors are expressed on ventrolateral medulla projecting paraventricular nucleus (PVN) neurons. Here, we investigate the role of adenosine 5′-triphosphate (ATP) in modulating sympathetic nerve activity (SNA) at the level of the PVN. We used an in situ arterially perfused rat preparation to determine the effect of P2 receptor activation and the putative interaction between purinergic and glutamatergic neurotransmitter systems within the PVN on lumbar SNA (LSNA). Unilateral microinjection of ATP into the PVN induced a dose-related increase in the LSNA (1 nmol: 38 ± 6 %, 2.5 nmol: 72 ± 7 %, 5 nmol: 96 ± 13 %). This increase was significantly attenuated by blockade of P2 receptors (pyridoxalphosphate-6-azophenyl-20,40-disulphonic acid, PPADS) and glutamate receptors (kynurenic acid, KYN) or a combination of both. The increase in LSNA elicited by L-glutamate microinjection into the PVN was not affected by a previous injection of PPADS. Selective blockade of non-N-methyl-D-aspartate receptors (6-cyano-7-nitroquinoxaline-2,3-dione disodium salt, CNQX), but not N-methyl-D-aspartate receptors (NMDA) receptors (DL-2-amino-5-phosphonopentanoic acid, AP5), attenuated the ATP-induced sympathoexcitatory effects at the PVN level. Taken together, our data show that purinergic neurotransmission within the PVN is involved in the control of SNA via P2 receptor activation. Moreover, we show an interaction between P2 receptors and non-NMDA glutamate receptors in the PVN suggesting that these functional interactions might be important in the regulation of sympathetic outflow.
Sympathetic outflow; ATP; L-Glutamate; Paraventricular nucleus of the hypothalamus
Purinergic pathways are considered important in pain transmission, and P2X receptors are a key part of this system which has received little attention in the horse. The aim of this study was to identify and characterise the distribution of P2X receptor subtypes in the equine digit and associated vasculature and nervous tissue, including peripheral nerves, dorsal root ganglia and cervical spinal cord, using PCR, Western blot analysis and immunohistochemistry. mRNA signal for most of the tested P2X receptor subunits (P2X1–5, 7) was detected in all sampled equine tissues, whereas P2X6 receptor subunit was predominantly expressed in the dorsal root ganglia and spinal cord. Western blot analysis validated the specificity of P2X1–3, 7 antibodies, and these were used in immunohistochemistry studies. P2X1–3, 7 receptor subunits were found in smooth muscle cells in the palmar digital artery and vein with the exception of the P2X3 subunit that was present only in the vein. However, endothelial cells in the palmar digital artery and vein were positive only for P2X2 and P2X3 receptor subunits. Neurons and nerve fibres in the peripheral and central nervous system were positive for P2X1–3 receptor subunits, whereas glial cells were positive for P2X7 and P2X1 and 2 receptor subunits. This previously unreported distribution of P2X subtypes may suggest important tissue specific roles in physiological and pathological processes.
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Horse; Purinergic P2X receptors; Fore limb; Dorsal root ganglia
The effects of standard adenosine receptor (AR) agonists and antagonists on the proliferation of human T lymphocytes, unstimulated and phytohemagglutinin-stimulated human peripheral blood lymphocytes (PBL), and Jurkat T cells were investigated. Real-time PCR measurements confirmed the presence of all four AR subtypes on the investigated cells, although at different expression levels. A2A ARs were predominantly expressed in PBL and further upregulated upon stimulation, while malignant Jurkat T cells showed high expression levels of A1, A2A, and A2B ARs. Cell proliferation was measured by [3H]-thymidine incorporation assays. Several ligands, including the subtype-selective agonists CPA (A1), BAY60-6583 (A2B), and IB-MECA (A3), and the antagonists PSB-36 (A1), MSX-2 (A2A), and PSB-10 (A3) significantly inhibited cell proliferation at micromolar concentrations, which were about three orders of magnitude higher than their AR affinities. In contrast, further investigated AR ligands, including the agonists NECA (nonselective) and CGS21680 (A2A), and the antagonists preladenant (SCH-420814, A2A), PSB-1115 (A2B), and PSB-603 (A2B) showed no or only minor effects on lymphocyte proliferation. The anti-proliferative effects of the AR agonists could not be blocked by the corresponding antagonists. The non-selective AR antagonist caffeine stimulated phytohemagglutinin-activated PBL with an EC50 value of 104 μM. This is the first study to compare a complete set of commonly used AR ligands for all subtypes on lymphocyte proliferation. Our results strongly suggest that these compounds induce an inhibition of lymphocyte proliferation and cell death through AR-independent mechanisms.
Adenosine receptors; Real-time PCR; Human lymphocytes; Jurkat T cells; Proliferation; [3H]Thymidine incorporation; Receptor-independent mechanisms
Inosine, a naturally occurring purine formed from the breakdown of adenosine, is associated with immunoregulatory effects. Evidence shows that inosine modulates lung inflammation and regulates cytokine generation. However, its role in controlling allergen-induced lung inflammation has yet to be identified. In this study, we aimed to investigate the role of inosine and adenosine receptors in a murine model of lung allergy induced by ovalbumin (OVA). Intraperitoneal administration of inosine (0.001–10 mg/kg, 30 min before OVA challenge) significantly reduced the number of leukocytes, macrophages, lymphocytes and eosinophils recovered in the bronchoalveolar lavage fluid of sensitized mice compared with controls. Interestingly, our results showed that pre-treatment with the selective A2A receptor antagonist (ZM241385), but not with the selective A2B receptor antagonist (alloxazine), reduced the inhibitory effects of inosine against macrophage count, suggesting that A2A receptors mediate monocyte recruitment into the lungs. In addition, the pre-treatment of mice with selective A3 antagonist (MRS3777) also prevented inosine effects against macrophages, lymphocytes and eosinophils. Histological analysis confirmed the effects of inosine and A2A adenosine receptors on cell recruitment and demonstrated that the treatment with ZM241385 and alloxazine reverted inosine effects against mast cell migration into the lungs. Accordingly, the treatment with inosine reduced lung elastance, an effect related to A2 receptors. Moreover, inosine reduced the levels of Th2-cytokines, interleukin-4 and interleukin-5, an effect that was not reversed by A2A or A2B selective antagonists. Our data show that inosine acting on A2A or A3 adenosine receptors can regulate OVA-induced allergic lung inflammation and also implicate inosine as an endogenous modulator of inflammatory processes observed in the lungs of asthmatic patients.
Inosine; Adenosine receptors; Allergy; Ovalbumin
The role of the A2B adenosine receptor (AR) in prostate cell death and growth was studied. The A2B AR gene expression quantified by real-time quantitative RT-PCR and Western blot analysis was the highest among four AR subtypes (A1, A2A, A2B, and A3) in all three commonly used prostate cancer cell lines, PC-3, DU145, and LNCaP. We explored the function of the A2B AR using PC-3 cells as a model. The A2B AR was visualized in PC-3 cells by laser confocal microscopy. The nonselective A2B AR agonist NECA and the selective A2B AR agonist BAY60-6583, but not the A2A AR agonist CGS21680, concentration-dependently induced adenosine 3′,5′-cyclic monophosphate (cyclic AMP) accumulation. NECA diminished lactate dehydrogenase (LDH) release, TNF-α-induced increase of caspase-3 activity, and cycloheximide (CHX)-induced morphological changes typical of apoptosis in PC-3 cells, which were blocked by a selective A2B AR antagonist PSB603. NECA-induced proliferation of PC-3 cells was diminished by siRNA specific for the A2B AR. The selective A2B AR antagonist PSB603 was shown to inhibit cell growth in all three cell lines. Thus, A2B AR blockade inhibits growth of prostate cancer cells, suggesting selective A2B AR antagonists as potential novel therapeutics.
Prostate cancer; Cancer; Adenosine receptor; A2B; G protein-coupled receptor (GPCR); Cell proliferation
Adenosine is an important neuromodulator in the central nervous system involved in the regulation of wakefulness, sleep, learning and memory, fear and anxiety as well as motor functions. Extracellular adenosine is synthesized by the cell-surface ectoenzyme ecto-5′-nucleotidase (CD73) from 5′-adenosine monophosphate. While CD73 is widely expressed throughout the mammalian brain, its specific role for behaviour is poorly understood. We examined spatial working memory, emotional responses, motor coordination and motor learning as well as behavioural habituation in mice with a targeted deletion of CD73. CD73 knockout (CD73−/−) mice exhibit enhanced spatial working memory in the Y-maze and enhanced long-term behavioural habituation in the open field. Furthermore, impaired psychomotor coordination on the accelerating rotarod was found in CD73−/− mice. No changes in motor learning and/or anxiety-like behaviour were evident in CD73−/− mice. Our data provide evidence for a role of CD73 in the regulation of learning and memory and psychomotor coordination. Our results might be important for the evaluation of adenosine neuromodulators as possible treatments to ameliorate cognitive and motor deficits associated with neurodegenerative diseases.
Ecto-5'-nucleotidase; Adenosine; CD73 knockout mice; Spatial working memory; Open-field habituation; Psychomotor coordination
Cancer is a chronic disease and its pathogenesis is well correlated with infection and inflammation. Adenosine is a purine nucleoside, which is produced under metabolic stress like hypoxic conditions. Acute or chronic inflammatory conditions lead to the release of precursor adenine nucleotides (adenosine triphosphate (ATP), adenosien diphosphate (ADP) and adenosine monophosphate (AMP)) from cells, which are extracellularly catabolized into adenosine by extracellular ectonucleotidases, i.e., CD39 or nucleoside triphosphate dephosphorylase (NTPD) and CD73 or 5′-ectonucleotidase. It is now well-known that adenosine is secreted by cancer as well as immune cells during tumor pathogenesis under metabolic stress or hypoxia. Once adenosine is released into the extracellular environment, it exerts various immunomodulatory effects via adenosine receptors (A1, A2A, A2B, and A3) expressed on various immune cells (i.e., macrophages, myeloid-derived suppressor cells (MDSCs), natural killer (NK) cells, dendritic cells (DCs), T cells, regulatory T cell (Tregs), etc.), which play very important roles in the pathogenesis of cancer. This review is intended to summarize the role of inflammation and adenosine in the immunopathogenesis of tumor along with regulation of tumor-specific immune response and its modulation as an adjunct approach to tumor immunotherapy.
Adenosine; Inflammation; Tumor; Cancer; DCs; Macrophages; MDSCs; NK cells; T cells; Tregs
The functions of P2X purinoceptors (P2X1-7) in the nervous system of adults have been widely studied. However, little is known about their roles during embryonic development. Our previous work has reported an extensive expression of P2X5 receptors in the adult mouse central nervous system. In the present study, we have examined the expression pattern of P2X5 receptor mRNA and protein during prenatal development of the mouse nervous system (from embryonic day E8 to E17). P2X5 receptors appeared in the neural tube as early as E8 and were gradually confined to new-born neurons in the cortical plate and ventral horn of the spinal cord. Heavy signals for P2X5 receptors were also found in dorsal root ganglia (DRG), retina, olfactory epithelium, and nerve fibers in skeletal muscles. In conclusion, P2X5 receptors were strongly represented in the developing mouse nervous system. The transient high expression pattern of P2X5 receptors in epithelium-like structures suggests a role during early neurogenesis.
ATP; P2X receptors; P2X5 receptors; Neurogenesis
Schwann cells (SCs) are peripheral myelinating glial cells that express the neuronal Ca2+-dependent cell adhesion molecule, neural cadherin (N-cadherin). N-cadherin is involved in glia–glia and axon–glia interactions and participates in many key events, which range from the control of axonal growth and guidance to synapse formation and plasticity. Extracellular UTP activates P2Y purinergic receptors and exerts short- and long-term effects on several tissues to promote wound healing. Nevertheless, the contribution of P2Y receptors in peripheral nervous system functions is not completely understood. The current study demonstrated that UTP induced a dose- and time-dependent increase in N-cadherin expression in SCs. Furthermore, N-cadherin expression was blocked by the P2 purinoceptor antagonist suramin. The increased N-cadherin expression induced by UTP was mediated by phosphorylation of mitogen-activated protein kinases (MAPKs), such as Jun N-terminal kinase, extracellular-regulated kinase and p38 kinase. Moreover, the Rho kinase inhibitor Y27632, the phospholipase C inhibitor U73122 and the protein kinase C inhibitor calphostin C attenuated the UTP-induced activation of MAPKs significantly. Extracellular UTP also modulated increased in the expression of the early transcription factors c-Fos and c-Jun. We also demonstrated that the region of the N-cadherin promoter between nucleotide positions −3698 and −2620, which contained one activator protein-1-binding site, was necessary for UTP-induced gene expression. These results suggest a novel role for P2Y purinergic receptors in the regulation of N-cadherin expression in SCs.
N-cadherin; Schwann cells; UTP; P2Y receptors; Mitogen-activated protein kinases; Peripheral nervous system
ATP is a ligand of P2X family purinoceptors, and exogenous ATP administration evokes pain behaviors. To date, there is a lack of systematic studies to address relationships between endogenous ATP and neuropathic pain. In this report, we took advantage of a mouse model of resiniferatoxin (RTX)-induced neuropathic pain to address the role of endogenous ATP in neuropathic pain. After RTX administration, endogenous ATP markedly increased in dorsal root ganglia (DRGs) (p < 0.01) and skin tissues (p < 0.001). The excessive endogenous ATP was removed by apyrase, an ATP hydrolyzing enzyme, administration via either a lumbar puncture route (p < 0.001) or an intraplantar injection (p < 0.001), which led to the normalization of neuropathic pain. In addition, intraplantar treatment with apyrase caused mechanical analgesia. Linear analyses showed that the densities of P2X3(+) neurons (r = −0.72, p < 0.0001) and P2X3(+) dermal nerves (r = −0.72, p < 0.0001) were inversely correlated with mechanical thresholds. Moreover, the contents of endogenous ATP in skin tissues were linearly correlated with P2X3(+) dermal nerves (r = 0.80, p < 0.0001) and mechanical thresholds (r = −0.80, p < 0.0001). In summary, this study demonstrated that enhanced purinergic signalling due to an increase in endogenous ATP after RTX-induced nerve injury contributed to the development of neuropathic pain. The data in this report provide a new therapeutic strategy for pain control by targeting the endogenous ligand of purinergic signalling.
ATP; Purinergic signalling; P2X3 purinoceptor; Resiniferatoxin; Neuropathic pain; Apyrase
This study continues our earlier findings on the hematopoiesis-modulating effects of adenosine A1 and A3 receptor agonists that were performed on committed hematopoietic progenitor and precursor cell populations. In the earlier experiments, N6-cyclopentyladenosine (CPA), an adenosine A1 receptor agonist, was found to inhibit proliferation in the above-mentioned hematopoietic cell systems, whereas N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA), an adenosine A3 receptor agonist, was found to stimulate it. The topic of this study was to evaluate the possibility that the above-mentioned adenosine receptor agonists modulate the behavior of early hematopoietic progenitor cells and hematopoietic stem cells. Flow cytometric analysis of hematopoietic stem cells in mice was employed, as well as a functional test of hematopoietic stem and progenitor cells (HSPCs). These techniques enabled us to study the effect of the agonists on both short-term repopulating ability and long-term repopulating ability, representing multipotent progenitors and hematopoietic stem cells, respectively. In a series of studies, we did not find any significant effect of adenosine agonists on HSPCs in terms of their numbers, proliferation, or functional activity. Thus, it can be concluded that CPA and IB-MECA do not significantly influence the primitive hematopoietic stem and progenitor cell pool and that the hematopoiesis-modulating action of these adenosine receptor agonists is restricted to more mature compartments of hematopoietic progenitor and precursor cells.
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Adenosine receptor agonists; CPA; IB-MECA; Hematopoietic stem cells; Long-term repopulating ability
Extracellular ATP and its hydrolysis product, adenosine, acting through specific receptors collectively named purinergic receptors, regulate female fertility by influencing the endometrial fluid microenvironment. There are four major groups of ecto-nucleotidases that control the levels of extracellular ATP and adenosine and thus their availability at purinergic receptors: ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-nucleotide pyrophosphatase/phospho-diesterases (E-NPPs), ecto-5′-nucleotidase (5′NT), and alkaline phosphatases (APs). The aim of the present work is to characterize the expression and distribution of ecto-nucleotidases in human endometrium along the menstrual cycle and after menopause, to evaluate their potential utility as fertility markers. We examined proliferative, secretory and atrophic endometria from women without endometrial pathology undergoing hysterectomy. We show that the ecto-nucleotidases are mainly present at endometrial epithelia, both luminal and glandular, and that their expression fluctuates along the cycle and also changes after menopause. An important result was identifying NPP3 as a new biological marker of tubal metaplasia. Our results emphasize the relevance of the study of purinergic signaling in human fertility.
Ecto-nucleotidases; Endometrium; Purinergic signaling; Fertility; CD39; CD73; NPP
Purinergic signaling plays a major role in the regulation of phagocytosis in microglia. Interplay between P2 and P1 receptor activation is controlled by a cascade of extracellular enzymes which dephosphorylate purines resulting in the formation of adenosine. The ATP- and ADP-degrading capacity of cultured microglia depends on the expression of ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) and is several times higher when compared to astrocytes which lack this enzyme. In brain slices, deletion of CD39 resulted in a 50 % decrease of ADP-degrading ability, while the degradation of ATP was decreased to about 75 % of the values measured in wild-type brain tissue. Microglia in acute slices from cd39−/− animals had increased constitutive phagocytic activity which could not be further enhanced by ATP in contrast to control animals. Pharmacological blockage of P2 receptors decreased the constitutive phagocytic activity to a similar base level in wild-type and cd39−/− microglia. Activation of P1 receptors by non-hydrolysable adenosine analog significantly decreased phagocytic activity. Deletion of CD73, an enzyme expressed by microglia which converts AMP to adenosine did not affect phagocytic activity. Taken together, these data show that CD39 plays a prominent role in controlling ATP levels and thereby microglial phagocytosis.
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The online version of this article (doi:10.1007/s11302-012-9339-y) contains supplementary material, which is available to authorized users.
Phagocytosis; Microglia; Purinergic signaling; cd39; cd73
Adenosine plays an important role in neuromodulation and neuroprotection. Recent identification of transient changes in adenosine concentration suggests adenosine may have a rapid modulatory role; however, the extent of these changes throughout the brain is not well understood. In this report, transient changes in adenosine evoked by one second, 60 Hz electrical stimulation trains were compared in the caudate–putamen, nucleus accumbens, hippocampus, and cortex. The concentration of evoked adenosine varies between brain regions, but there is less variation in the duration of signaling. The highest concentration of adenosine was evoked in the dorsal caudate–putamen (0.34 ± 0.08 μM), while the lowest concentration was in the secondary motor cortex (0.06 ± 0.02 μM). In all brain regions, adenosine release was activity-dependent. In the nucleus accumbens, hippocampus, and prefrontal cortex, this release was partly due to extracellular ATP breakdown. However, in the caudate–putamen, release was not due to ATP metabolism but was ionotropic glutamate receptor-dependent. The results demonstrate that transient, activity-dependent adenosine can be evoked in many brain regions but that the mechanism of formation and release varies by region.
Carbon-fiber microelectrode; Striatum; Hippocampus; Cortex; Electrical stimulation; Fast-scan cyclic voltammetry
Hypoxic injury occurs when the blood supply to an organ is interrupted; subsequent reperfusion halts ongoing ischemic damage but paradoxically leads to further inflammation. Together this is termed ischemia–reperfusion injury (IRI). IRI is inherent to organ transplantation and impacts both the short- and long-term outcomes of the transplanted organ. Activation of the purinergic signalling pathway is intrinsic to the pathogenesis of, and endogenous response to IRI. Therapies targeting the purinergic pathway in IRI are an attractive avenue for the improvement of transplant outcomes and the basis of ongoing research. This review aims to examine the role of adenosine receptor signalling and the ecto-nucleotidases, CD39 and CD73, in IRI, with a particular focus on renal IRI.
Ischemia–reperfusion injury; Renal transplant; Purinergic signalling; Adenosine receptor; Ectonucleotidase
To investigate whether fetal endothelial cell proliferation and migration are modulated by the A2A adenosine receptor (A2AAR), nitric oxide (NO) and the vascular endothelial growth factor (VEGF) signaling pathway, we isolated human umbilical vein endothelial cells from normal pregnancy (n = 23), preterm delivery (n = 4), and late-onset (LOPE, n = 10) and early-onset preeclampsia (EOPE, n = 8). We used the non-selective adenosine receptor agonist (NECA) and the selective agonist (CGS-21680) and/or selective antagonist (ZM-241385) for A2AAR. Also, the nitric oxide synthase (NOS) inhibitor, l-NAME, was used in co-incubation with CGS-21680. Compared to normal pregnancy, EOPE exhibited low cell proliferation and migration associated with reduced expressions of A2AAR and VEGF and NO synthesis (i.e., total and phosphorylated serine1177 endothelial NOS and nitrite formation). In contrast, LOPE exhibited the opposite behavior in all these markers compared to normal pregnancy or EOPE. Cell proliferation and migration were increased by CGS-21680 (or NECA) in all analyzed groups (EOPE>LOPE>normal pregnancy) compared to their respective basal conditions, an effect that was associated with high NO and VEGF synthesis and blocked by ZM-241385 with significantly different IC50 for each group (EOPE>LOPE>normal pregnancy). The differences seem independent of gestational age. l-NAME blocked the CGS-21680-mediated cell proliferation and migration in normal pregnancy and LOPE (IC50 = 36.2 ± 2.5 and 8.6 ± 2.2 nM, respectively) as well as the VEGF expression in normal pregnancy. Therefore, the A2AAR/NO/VEGF signaling pathway exhibits a pro-angiogenic effect in normal pregnancies and LOPE, whereas impairment in this pathway seems related to the reduced angiogenic capacity of the fetal endothelium in EOPE.
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Adenosine; Adenosine receptor; Nitric oxide; Angiogenesis; Placental; Early- and late-onset preeclampsia
Gold nanoparticles (AuNPs) allow the tuning of pharmacokinetic and pharmacodynamic properties by active or passive targeting of drugs for cancer and other diseases. We have functionalized gold nanoparticles by tethering specific ligands, agonists and antagonists, of adenosine receptors (ARs) to the gold surface as models for cell surface interactions with G protein-coupled receptors (GPCRs). The AuNP conjugates with chain-extended AR ligands alone (PEGylated nucleosides and nonnucleosides, anchored to the Au via thioctic acid) were found to be insoluble in water due to hydrophobic entities in the ligand. Therefore, we added a second, biologically inactive pendant moiety to increase the water solubility, consisting of a PEGylated chain terminating in a carboxylic or phosphate group. The purity and stability of the immobilized biologically active ligand were examined by ultrafiltration and HPLC. Pharmacological receptor binding studies on these GPCR ligand-derivatized AuNPs (2–5 nm in diameter), performed using membranes of mammalian cells stably expressing human A1, A2A, and A3ARs, showed that the desired selectivity was retained with Ki values (nanomolar) of A3AR agonist 21b and A2AAR antagonists 24 and 26a of 14 (A3), 34 (A2A), and 69 (A2A), respectively. The corresponding monomers displayed Ki values of 37, 61, and 1,420 nM, respectively. In conclusion, we have synthesized stable, water-soluble AuNP derivatives of tethered A3 and A2AAR ligands that retain the biological properties of their monomeric ligands and are intended for therapeutic and imaging applications. This is the first prototypical application to gold carriers of small molecule (nonpeptide) GPCR ligands, which are under investigation for treatment of cancer and inflammatory diseases.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-012-9338-z) contains supplementary material, which is available to authorized users.
G protein-coupled receptor; Nanoparticle; Nucleoside; Adenosine; Radioligand binding
The P2X4 receptor is an ATP-gated ion channel expressed in neurons, endothelia and immune cells. Plasma membrane expression of P2X4 is regulated by dynamin-dependent endocytosis, and this study identifies a Rab5-dependent pathway of receptor internalisation. Expression of Rab5 constructs altered the distribution of P2X4 in HEK-293 cells, and both constitutive internalisation and agonist-induced desensitisation of P2X4 were increased by co-expression of wild-type Rab5 or constitutively active Rab5 (Q79L). Expression of inactive dynamin K44A and Rab5 S34N constructs abolished agonist-induced desensitisation, suggesting internalisation as the underlying mechanism. Blocking P2X4 internalisation in this way also abolished potentiation of ATP-induced currents by the allosteric modulator ivermectin. This suggests that the dynamin-Rab5 internalisation pathway is essential for the ivermectin potentiation effect. In agreement with this hypothesis, the co-expression of wild-type dynamin, wild-type Rab5 or active Rab5 (Q79L) could increase the potentiation of the ATP-induced P2X4 response by ivermectin. These findings highlight Rab5 GTPase as a key regulator of P2X4 receptor cell surface expression and internalisation.
P2X4 receptor; Dynamin; Rab5; Ivermectin
The pancreas is an organ with a central role in nutrient breakdown, nutrient sensing and release of hormones regulating whole body nutrient homeostasis. In diabetes mellitus, the balance is broken—cells can be starving in the midst of plenty. There are indications that the incidence of diabetes type 1 and 2, and possibly pancreatogenic diabetes, is rising globally. Events leading to insulin secretion and action are complex, but there is emerging evidence that intracellular nucleotides and nucleotides are not only important as intracellular energy molecules but also as extracellular signalling molecules in purinergic signalling cascades. This signalling takes place at the level of the pancreas, where the close apposition of various cells—endocrine, exocrine, stromal and immune cells—contributes to the integrated function. Following an introduction to diabetes, the pancreas and purinergic signalling, we will focus on the role of purinergic signalling and its changes associated with diabetes in the pancreas and selected tissues/organ systems affected by hyperglycaemia and other stress molecules of diabetes. Since this is the first review of this kind, a comprehensive historical angle is taken, and common and divergent roles of receptors for nucleotides and nucleosides in different organ systems will be given. This integrated picture will aid our understanding of the challenges of the potential and currently used drugs targeted to specific organ/cells or disorders associated with diabetes.
Adenosine 5′-triphosphate; β cells; Insulin; Obesity; Nephropathy; Pain; Exocrine pancreas; Cardiovascular disease
Syntheses and biological activities of imidazo-, pyrimido- and diazepino[2,1-f]purinediones containing N-alkyl substituents (with straight, branched or unsaturated chains) are described. Tricyclic derivatives were synthesized by the cyclization of 8-bromo-substituted 7-(2-bromoethyl)-, 7-(3-chloropropyl)- or 7-(4-bromobutyl)-theophylline with primary amines under various conditions. Compound 22 with an ethenyl substituent was synthesized by dehydrohalogenation of 9-(2-bromoethyl)-1,3-dimethyltetrahydropyrimido[2,1-f]purinedione. The obtained derivatives (5–35) were initially evaluated for their affinity at rat A1 and A2A adenosine receptors (AR), showing moderate affinity for both adenosine receptor subtypes. The best ligands were diazepinopurinedione 28 (Ki = 0.28 μM) with fivefold A2A selectivity and the non-selective A1/A2A AR ligand pyrimidopurinedione 35 (Ki A1 = 0.28 μM and Ki A2A = 0.30 μM). The compounds were also evaluated for their affinity at human A1, A2A, A2B and A3 ARs. All of the obtained compounds were docked to the A2A AR X-ray structure in complex with the xanthine-based, potent adenosine receptor antagonist—XAC. The likely interactions of imidazo-, pyrimido- and diazepino[2,1-f]purinediones with the residues forming the A2A binding pocket were discussed. Furthermore, the new compounds were tested in vivo as anticonvulsants in maximal electroshock, subcutaneous pentylenetetrazole (ScMet) and TOX tests in mice (i.p.). Pyrimidopurinediones showed anticonvulsant activity mainly in the ScMet test. The best derivative was compound 11, showing 100 % protection at a dose of 100 mg/kg without symptoms of neurotoxicity. Compounds 6, 7, 8 and 14 with short substituents showed neurotoxicity and caused death. In rat tests (p.o.), 9 was characterized by a high protection index (>13.3). AR affinity did not apparently correlate with the antiepileptic potency of the compounds.
Electronic supplementary material
The online version of this article (doi:10.1007/s11302-013-9358-3) contains supplementary material, which is available to authorized users.
Tricyclic xanthine derivatives; Adenosine A1, A2A, A2B and A3 receptor affinity; Anticonvulsant activity; Molecular modelling studies