Pharmacological characterization of P2Y receptor subtypes - an update.

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. There are eight mammalian P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14). The widely expressed P2Y receptors play important roles in physiology and pathophysiology. This review summarizes the use of pharmacological tools to characterize the P2Y receptor subtypes involved in these responses. MRS2500 is a potent and selective antagonist acting at the P2Y1 receptor. AR-C118925 is useful for the selective antagonism of the P2Y2 receptor. PSB16133 blocks the P2Y4 receptor, MRS2578 is an antagonist at the P2Y6 receptor and NF157 as well as NF340 block the P2Y11 receptor. ADP-induced platelet aggregation is mediated by P2Y1 and P2Y12 receptors. A number of compounds or their active metabolites reduce ADP-induced platelet aggregation by blocking the P2Y12 receptor. These include the active metabolites of the thienopyridine compounds clopidogrel and prasugrel, the nucleoside analogue ticagrelor and the nucleotide analogue cangrelor. PSB0739 is also a potent antagonist at the P2Y12 receptor useful for both in vitro and in vivo studies. MRS2211 and MRS2603 inhibit P2Y13 mediated responses. PPTN is a very potent antagonist at the P2Y14 receptor.

Eighth pharmacologic-historical forum.

SCOPE, HISTORICAL OVERVIEW AND PERSPECTIVES: Athineos Philippu, Department of Pharmacology and Toxicology, University of Innsbruck, Austria The eighth pharmacologic-historical Forum was held online in 2022 in Bonn during the Meeting of the DGPT. In this forum the personalities of Hans Dengler, Paul Martini, Manfred Göthert, and Rudolf Buchheim were honoured by describing their lives and scientific achievements.

Molecular pharmacology of P2Y receptor subtypes.

Professor Geoffrey Burnstock proposed the concept of purinergic signaling via P1 and P2 receptors. P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular adenine and uracil nucleotides. Eight mammalian P2Y receptor subtypes have been identified. They are divided into two subgroups (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11) and (P2Y12, P2Y13, and P2Y14). P2Y receptors are found in almost all cells and mediate responses in physiology and pathophysiology including pain and inflammation. The antagonism of platelet P2Y12 receptors by cangrelor, ticagrelor or active metabolites of the thienopyridine compounds ticlopidine, clopidogrel and prasugrel reduces the ADP-induced platelet aggregation in patients with thrombotic complications of vascular diseases. The nucleotide agonist diquafosol acting at P2Y2 receptors is used for the treatment of the dry eye syndrome. Structural information obtained by crystallography of the human P2Y1 and P2Y12 receptor proteins, site-directed mutagenesis and molecular modeling will facilitate the rational design of novel selective drugs.

Update of P2Y receptor pharmacology: IUPHAR Review 27.

Eight G protein-coupled P2Y receptor subtypes respond to extracellular adenine and uracil mononucleotides and dinucleotides. P2Y receptors belong to the δ group of rhodopsin-like GPCRs and contain two structurally distinct subfamilies: P2Y1 , P2Y2 , P2Y4 , P2Y6 , and P2Y11 (principally Gq protein-coupled P2Y1 -like) and P2Y12-14 (principally Gi protein-coupled P2Y12 -like) receptors. Brain P2Y receptors occur in neurons, glial cells, and vasculature. Endothelial P2Y1 , P2Y2 , P2Y4 , and P2Y6 receptors induce vasodilation, while smooth muscle P2Y2 , P2Y4 , and P2Y6 receptor activation leads to vasoconstriction. Pancreatic P2Y1 and P2Y6 receptors stimulate while P2Y13 receptors inhibits insulin secretion. Antagonists of P2Y12 receptors, and potentially P2Y1 receptors, are anti-thrombotic agents, and a P2Y2 /P2Y4 receptor agonist treats dry eye syndrome in Asia. P2Y receptor agonists are generally pro-inflammatory, and antagonists may eventually treat inflammatory conditions. This article reviews recent developments in P2Y receptor pharmacology (using synthetic agonists and antagonists), structure and biophysical properties (using X-ray crystallography, mutagenesis and modelling), physiological and pathophysiological roles, and present and potentially future therapeutic targeting.

An Agonist Radioligand for the Proinflammatory Lipid-Activated G Protein-Coupled Receptor GPR84 Providing Structural Insights.

The orphan G protein-coupled receptor (GPCR) GPR84 is expressed on immune cells mediating proinflammatory and immunostimulatory effects. In this study, we prepared the fully efficacious, nonbiased GPR84 agonist 6-hexylamino-2,4(1H,3H)-pyrimidinedione (6) in tritium-labeled form ([3H]PSB-1584) by hydrogenation of a hexenyl-substituted precursor with tritium gas. The radioligand was characterized by kinetic, saturation, and competition assays using membranes of Chinese hamster ovary cells recombinantly expressing the human GPR84. [3H]6 reversibly labeled the receptor with high affinity (KD 2.08 nM). Structurally diverse orthosteric and allosteric ligands, including newly designed and synthesized compounds, were studied in competition binding assays. A homology model of GPR84 was generated to perform docking studies rationalizing the experimental data. The radioligand was additionally used for labeling GPR84 in native cells and tissues. [3H]6 constitutes the first GPR84 agonist radioligand representing a powerful tool for this poorly investigated GPCR, which has potential as a future drug target.

Pharmacology of P2Y receptors.

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. There are eight mammalian P2Y receptor subtypes divided into two subgroups (P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11) and (P2Y12, P2Y13, and P2Y14). The P2Y receptors are expressed in various cell types and play important roles in physiology and pathophysiology including inflammatory responses and neuropathic pain. The antagonism of P2Y12 receptors is used in pharmacotherapy for the prevention and therapy of cardiovascular events. The nucleoside analogue ticagrelor and active metabolites of the thienopyridine compounds ticlopidine, clopidogrel and prasugrel inhibit platelet P2Y12 receptors and reduce thereby platelet aggregation. The P2Y2 receptor agonist diquafosol is used for the treatment of the dry eye syndrome. The P2Y receptor subtypes differ in their amino acid sequences, their pharmacological profiles and their signaling transduction pathways. Recently, selective receptor ligands have been developed for all subtypes. The published crystal structures of the human P2Y1 and P2Y12 receptors as well as receptor models will facilitate the development of novel drugs for pharmacotherapy.

6-(Ar)Alkylamino-Substituted Uracil Derivatives: Lipid Mimetics with Potent Activity at the Orphan G Protein-Coupled Receptor 84 (GPR84).

GPR84, a Gi protein-coupled receptor that is activated by medium-chain (hydroxy)fatty acids, appears to play an important role in inflammation, immunity, and cancer. Recently, 6-octylaminouracil (4) has been reported to act as an agonist at GPR84. Here, we describe the synthesis of 69 derivatives and analogs of 4, 66 of which represent new compounds. They were evaluated in (a) cyclic adenosine monophosphate accumulation and (b) ß-arrestin assays in human GPR84-expressing cells. Potent nonbiased as well as G protein-biased agonists were developed, e.g., 6-hexylamino-2,4(1H,3H)-pyrimidinedione (20, PSB-1584, EC50 5.0 nM (a), 3.2 nM (b), bias factor: 0) and 6-((p-chloro- and p-bromo-phenylethyl)amino)-2,4(1H,3H)-pyrimidinedione (47, PSB-16434, EC50 7.1 nM (a), 520 nM (b), bias factor: 1.9 = 79-fold Gi pathway-selective; 48, PSB-17365, EC50 2.5 nM (a), 100 nM (b), bias factor 1.3 = 20-fold selective), which were selective versus other free fatty acid-activated receptors. Compounds 20 and 48 were found to be metabolically stable upon incubation with human liver microsomes. A pharmacophore model was created on the basis of structurally diverse lipidlike GPR84 agonists.

Structure, Pharmacology and Roles in Physiology of the P2Y12 Receptor.

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. The platelet ADP-receptor which has been denominated P2Y12 receptor is an important target in pharmacotherapy. The receptor couples to Gαi2 mediating an inhibition of cyclic AMP accumulation and additional downstream events including the activation of phosphatidylinositol-3-kinase and Rap1b proteins. The nucleoside analogue ticagrelor and active metabolites of the thienopyridine compounds ticlopidine, clopidogrel and prasugrel block P2Y12 receptors and, thereby, inhibit ADP-induced platelet aggregation. These drugs are used for the prevention and therapy of cardiovascular events such as acute coronary syndromes or stroke. The recently published three-dimensional crystal structures of the human P2Y12 receptor in complex with agonists and antagonists will facilitate the development of novel therapeutic agents with reduced adverse effects. P2Y12 receptors are also expressed on vascular smooth muscle cells and may be involved in the pathophysiology of atherogenesis. P2Y12 receptors on microglial cells operate as sensors for adenine nucleotides released during brain injury. A recent study indicated the involvement of microglial P2Y12 receptors in the activity-dependent neuronal plasticity. Interestingly, there is evidence for changes in P2Y12 receptor expression in CNS pathologies including Alzheimer's diseases and multiple sclerosis. P2Y12 receptors may also be involved in systemic immune modulating responses and the susceptibility to develop bronchial asthma.

Diindolylmethane Derivatives: Potent Agonists of the Immunostimulatory Orphan G Protein-Coupled Receptor GPR84.

The Gi protein-coupled receptor GPR84, which is activated by (hydroxy)fatty acids, is highly expressed on immune cells. Recently, 3,3'-diindolylmethane was identified as a heterocyclic, nonlipid-like GPR84 agonist. We synthesized a broad range of diindolylmethane derivatives by condensation of indoles with formaldehyde in water under microwave irradiation. The products were evaluated at the human GPR84 in cAMP and ß-arrestin assays. Structure-activity relationships (SARs) were steep. 3,3'-Diindolylmethanes bearing small lipophilic residues at the 5- and/or 7-position of the indole rings displayed the highest activity in cAMP assays, the most potent agonists being di(5-fluoro-1H-indole-3-yl)methane (38, PSB-15160, EC50 80.0 nM) and di(5,7-difluoro-1H-indole-3-yl)methane (57, PSB-16671, EC50 41.3 nM). In ß-arrestin assays, SARs were different, indicating biased agonism. The new compounds were selective versus related fatty acid receptors and the arylhydrocarbon receptor. Selected compounds were further investigated and found to display an ago-allosteric mechanism of action and increased stability in comparison to the lead structure.

Pharmacology and structure of P2Y receptors.

P2Y receptors are G-protein-coupled receptors (GPCRs) for extracellular nucleotides. There are eight mammalian P2Y receptor subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14). P2Y receptors are widely expressed and play important roles in physiology and pathophysiology. One important example is the ADP-induced platelet aggregation mediated by P2Y1 and P2Y12 receptors. Active metabolites of the thienopyridine compounds ticlopidine, clopidogrel and prasugrel as well as the nucleoside analogue ticagrelor block P2Y12 receptors and thereby platelet aggregation. These drugs are used for the prevention and therapy of cardiovascular events. Moreover, P2Y receptors play important roles in the nervous system. Adenine nucleotides modulate neuronal activity and neuronal fibre outgrowth by activation of P2Y1 receptors and control migration of microglia by P2Y12 receptors. UDP stimulates microglial phagocytosis through activation of P2Y6 receptors. There is evidence for a role for P2Y2 receptors in Alzheimer's disease pathology. The P2Y receptor subtypes are highly diverse in both their amino acid sequences and their pharmacological profiles. Selective receptor ligands have been developed for the pharmacological characterization of the receptor subtypes. The recently published three-dimensional crystal structures of the human P2Y1 and P2Y12 receptors will facilitate the development of therapeutic agents that selectively target P2Y receptors. This article is part of the Special Issue entitled 'Purines in Neurodegeneration and Neuroregeneration'.

Modeling ligand recognition at the P2Y12 receptor in light of X-ray structural information.

The G protein-coupled P2Y12 receptor (P2Y12R) is an important antithrombotic target and of great interest for pharmaceutical discovery. Its recently solved, highly divergent crystallographic structures in complex either with nucleotides (full or partial agonist) or with a nonnucleotide antagonist raise the question of which structure is more useful to understand ligand recognition. Therefore, we performed extensive molecular modeling studies based on these structures and mutagenesis, to predict the binding modes of major classes of P2Y12R ligands previously reported. Various nucleotide derivatives docked readily to the agonist-bound P2Y12R, but uncharged nucleotide-like antagonist ticagrelor required a hybrid receptor resembling the agonist-bound P2Y12R except for the top portion of TM6. Supervised molecular dynamics (SuMD) of ticagrelor binding indicated interactions with the extracellular regions of P2Y12R, defining possible meta-binding sites. Ureas, sulfonylureas, sulfonamides, anthraquinones and glutamic acid piperazines docked readily to the antagonist-bound P2Y12R. Docking dinucleotides at both agonist- and antagonist-bound structures suggested interactions with two P2Y12R pockets. Thus, our structure-based approach consistently rationalized the main structure-activity relationships within each ligand class, giving useful information for designing improved ligands.

Adenosine activates brown adipose tissue and recruits beige adipocytes via A2A receptors.

Brown adipose tissue (BAT) is specialized in energy expenditure, making it a potential target for anti-obesity therapies. Following exposure to cold, BAT is activated by the sympathetic nervous system with concomitant release of catecholamines and activation of ß-adrenergic receptors. Because BAT therapies based on cold exposure or ß-adrenergic agonists are clinically not feasible, alternative strategies must be explored. Purinergic co-transmission might be involved in sympathetic control of BAT and previous studies reported inhibitory effects of the purinergic transmitter adenosine in BAT from hamster or rat. However, the role of adenosine in human BAT is unknown. Here we show that adenosine activates human and murine brown adipocytes at low nanomolar concentrations. Adenosine is released in BAT during stimulation of sympathetic nerves as well as from brown adipocytes. The adenosine A2A receptor is the most abundant adenosine receptor in human and murine BAT. Pharmacological blockade or genetic loss of A2A receptors in mice causes a decrease in BAT-dependent thermogenesis, whereas treatment with A2A agonists significantly increases energy expenditure. Moreover, pharmacological stimulation of A2A receptors or injection of lentiviral vectors expressing the A2A receptor into white fat induces brown-like cells-so-called beige adipocytes. Importantly, mice fed a high-fat diet and treated with an A2A agonist are leaner with improved glucose tolerance. Taken together, our results demonstrate that adenosine-A2A signalling plays an unexpected physiological role in sympathetic BAT activation and protects mice from diet-induced obesity. Those findings reveal new possibilities for developing novel obesity therapies.

Central P2Y12 receptor blockade alleviates inflammatory and neuropathic pain and cytokine production in rodents.

In this study the role of P2Y12 receptors (P2Y12R) was explored in rodent models of inflammatory and neuropathic pain and in acute thermal nociception. In correlation with their activity to block the recombinant human P2Y12R, the majority of P2Y12R antagonists alleviated mechanical hyperalgesia dose-dependently, following intraplantar CFA injection, and after partial ligation of the sciatic nerve in rats. They also caused an increase in thermal nociceptive threshold in the hot plate test. Among the six P2Y12R antagonists evaluated in the pain studies, the selective P2Y12 receptor antagonist PSB-0739 was most potent upon intrathecal application. P2Y12R mRNA and IL-1ß protein were time-dependently overexpressed in the rat hind paw and lumbar spinal cord following intraplantar CFA injection. This was accompanied by the upregulation of TNF-α, IL-6 and IL-10 in the hind paw. PSB-0739 (0.3mg/kg i.t.) attenuated CFA-induced expression of cytokines in the hind paw and of IL-1ß in the spinal cord. Subdiaphragmatic vagotomy and the α7 nicotinic acetylcholine receptor antagonist MLA occluded the effect of PSB-0739 (i.t.) on pain behavior and peripheral cytokine induction. Denervation of sympathetic nerves by 6-OHDA pretreatment did not affect the action of PSB-0739. PSB-0739, in an analgesic dose, did not influence motor coordination and platelet aggregation. Genetic deletion of the P2Y12R in mice reproduced the effect of P2Y12R antagonists on mechanical hyperalgesia in inflammatory and neuropathic pain models, on acute thermal nociception and on the induction of spinal IL-1ß. Here we report the robust involvement of the P2Y12R in inflammatory pain. The anti-hyperalgesic effect of P2Y12R antagonism could be mediated by the inhibition of both central and peripheral cytokine production and involves α7-receptor mediated efferent pathways.

P2X1 receptor-mediated inhibition of the proliferation of human coronary smooth muscle cells involving the transcription factor NR4A1.

Adenine nucleotides acting at P2X1 receptors are potent vasoconstrictors. Recently, we demonstrated that activation of adenosine A2B receptors on human coronary smooth muscle cells inhibits cell proliferation by the induction of the nuclear receptor subfamily 4, group A, member 1 (NR4A1; alternative notation Nur77). In the present study, we searched for long-term effects mediated by P2X1 receptors by analyzing receptor-mediated changes in cell proliferation and in the expression of NR4A1. Cultured human coronary smooth muscle cells were treated with selective receptor ligands. Effects on proliferation were determined by counting cells and measuring changes in impedance. The induction of transcription factors was assessed by qPCR. The P2X receptor agonist α,ß-methylene-ATP and its analog ß,γ-methylene-ATP inhibited cell proliferation by about 50 % after 5 days in culture with half-maximal concentrations of 0.3 and 0.08 µM, respectively. The effects were abolished or markedly attenuated by the P2X1 receptor antagonist NF449 (carbonylbis-imino-benzene-triylbis-(carbonylimino)tetrakis-benzene-1,3-disulfonic acid; 100 nM and 1 µM). α,ß-methylene-ATP and ß,γ-methylene-ATP applied for 30 min to 4 h increased the expression of NR4A1; NF449 blocked or attenuated this effect. Small interfering RNA directed against NR4A1 diminished the antiproliferative effects of α,ß-methylene-ATP and ß,γ-methylene-ATP. α,ß-methylene-ATP (0.1 to 30 µM) decreased migration of cultured human coronary smooth muscle cells in a chamber measuring changes in impedance; NF449 blocked the effect. In conclusion, our results demonstrate for the first time that adenine nucleotides acting at P2X1 receptors inhibit the proliferation of human coronary smooth muscle cells via the induction of the early gene NR4A1.

Characterization of new G protein-coupled adenine receptors in mouse and hamster.

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 [(3)H]adenine. Specific binding of the radioligand was detected in transfected, but not in untransfected cells, and K D values of 286 nM (mAde1R, B max 1.18 pmol/mg protein) and 301 nM (cAdeR, B max 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.

The rat adenine receptor: pharmacological characterization and mutagenesis studies to investigate its putative ligand binding site.

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 (Phe110(3.24), Asn115(3.29), Asn173(4.60), Phe179(45.39), Asn194(5.40), Phe195(5.41), Leu201(5.47), His252(6.54), and Tyr268(7.32)) for alanine and expressed them in Spodoptera frugiperda (Sf9) insect cells. Membrane preparations subjected to [(3)H]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, Asn194(5.40) and Leu201(5.47), 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.

Inhibitory effects of benzodiazepines on the adenosine A(2B) receptor mediated secretion of interleukin-8 in human mast cells.

The activation of adenosine A(2B) receptors in human mast cells causes pro-inflammatory responses such as the secretion of interleukin-8. There is evidence for an inhibitory effect of benzodiazepines on mast cell mediated symptoms in patients with systemic mast cell activation disease. Therefore, we investigated the effects of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast cell leukaemia (HMC1) cells by an enzyme linked immunosorbent assay. The adenosine analogue N-ethylcarboxamidoadenosine (NECA, 0.3-3 µM) increased interleukin-8 production about 5-fold above baseline. This effect was attenuated by the adenosine A(2B) receptor antagonist MRS1754 (N-(4-cyanophenyl)-2-{4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy}-acetamide) 1 µM. In addition, diazepam, 4'-chlorodiazepam and flunitrazepam (1-30 µM) markedly reduced NECA-induced interleukin-8 production in that order of potency, whereas clonazepam showed only a modest inhibition. The inhibitory effect of diazepam was not altered by flumazenil 10 µM or PK11195 (1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide) 10 µM. Diazepam attenuated the NECA-induced expression of mRNA encoding for interleukin-8. Moreover, diazepam and flunitrazepam reduced the increasing effects of NECA on cAMP-response element- and nuclear factor of activated t-cells-driven luciferase reporter gene activities in HMC1 cells. Neither diazepam nor flunitrazepam affected NECA-induced increases in cellular cAMP levels in CHO Flp-In cells stably expressing recombinant human adenosine A(2B) receptors, excluding a direct action of benzodiazepines on human adenosine A(2B) receptors. In conclusion, this is the first study showing an inhibitory action of benzodiazepines on adenosine A(2B) receptor mediated interleukin-8 production in human mast (HMC1) cells. The rank order of potency indicates the involvement of an atypical benzodiazepine binding site.

Molecular pharmacology, physiology, and structure of the P2Y receptors.

The P2Y receptors are a widely expressed group of eight nucleotide-activated G protein-coupled receptors (GPCRs). The P2Y(1)(ADP), P2Y(2)(ATP/UTP), P2Y(4)(UTP), P2Y(6)(UDP), and P2Y(11)(ATP) receptors activate G(q) and therefore robustly promote inositol lipid signaling responses. The P2Y(12)(ADP), P2Y(13)(ADP), and P2Y(14)(UDP/UDP-glucose) receptors activate G(i) leading to inhibition of adenylyl cyclase and to Gßγ-mediated activation of a range of effector proteins including phosphoinositide 3-kinase-γ, inward rectifying K(+) (GIRK) channels, phospholipase C-ß2 and -ß3, and G protein-receptor kinases 2 and 3. A broad range of physiological responses occur downstream of activation of these receptors ranging from Cl(-) secretion by epithelia to aggregation of platelets to neurotransmission. Useful structural models of the P2Y receptors have evolved from extensive genetic analyses coupled with molecular modeling based on three-dimensional structures obtained for rhodopsin and several other GPCRs. Selective ligands have been synthesized for most of the P2Y receptors with the most prominent successes attained with highly selective agonist and antagonist molecules for the ADP-activated P2Y(1) and P2Y(12) receptors. The widely prescribed drug, clopidogrel, which results in irreversible blockade of the platelet P2Y(12) receptor, is the most important therapeutic agent that targets a P2Y receptor.

A2B receptors mediate the induction of early genes and inhibition of arterial smooth muscle cell proliferation via Epac.

AIMS: Extracellular adenosine and adenine nucleotides play important roles in the regulation of the blood vessel tonus and platelet aggregation. Less is known about the effects of these extracellular signalling molecules on gene expression in vascular smooth muscle cells involved in long-term vascular effects. In the present study, we therefore searched for adenosine-induced changes in the expression of early genes in cultured human coronary artery smooth muscle cells (HCASMCs). METHODS AND RESULTS: Whole-genome DNA array hybridization revealed that adenosine induced a set of early genes including the nuclear receptor subfamily 4, group A, member 1 (NR4A1/Nur77/TR3). The pattern of the effects of adenosine on gene expression resembles the change in expression induced by the direct activator of adenylate cyclase forskolin. Real-time reverse-transcriptase PCR confirmed that adenosine and its analogue N-ethyl-carboxamidoadenosine elicited a strong induction of NR4A1. These effects were markedly attenuated by A(2B) receptor antagonists including 8-[4-(4-benzylpiperazide-1-sulfonyl)phenyl]-1-propylxanthine (PSB-601) and were mimicked by a cyclic AMP (cAMP) analogue [8-(4-chlorophenylthio)-2'-O-methyl-cAMP, 8CPT] acting on the exchange protein activated by cAMP (Epac). Long-term experiments over 5 days showed that 2-chloroadenosine decreased cell proliferation in the presence of platelet-derived growth factor. This effect of 2-chloroadenosine was also attenuated by PSB-601 and mimicked by 8CPT. Treatment with small interfering RNA directed against NR4A1 attenuated the inhibitory effect of 8CPT on proliferation. CONCLUSIONS: In summary, our results demonstrate the operation of adenosine A2(B) receptors mediating an early induction of NR4A1 and a decrease in cell proliferation via the cAMP/Epac pathway in HCASMCs.