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The synaptic event called the inhibitory junction potential (IJP) was arguably one of the more important discoveries made by Burnstock and arguably one of his finer legacies. The discovery of the IJP fundamentally changed how electromechanical coupling was visualised in gastrointestinal smooth muscle. Its discovery also set in motion the search for novel inhibitory neurotransmitters in the enteric nervous system, eventually leading to proposal that ATP or a related nucleotide was a major inhibitory transmitter. The subsequent development of purinergic signalling gave impetus to expanding the classification of surface receptors for extracellular ATP, not only in the GI tract but beyond, and then led to successive phases of medicinal chemistry as the P2 receptor field developed. Ultimately, the discovery of the IJP led to the successful cloning of the first P2Y receptor (chick P2Y1) and expansion of mammalian ATP receptors into two classes: metabotropic P2Y receptors (encompassing P2Y1, P2Y2, P2Y4, P2Y6, P2Y11–14 receptors) and ionotropic P2X receptors (encompassing homomeric P2X1–P2X7 receptors). Here, the causal relationship between the IJP and P2Y1 is explored, setting out the milestones reached and achievements made by Burnstock and his colleagues.

The Gq protein-coupled, ATP- and UTP-activated P2Y2 receptor is a potential drug target for a range of different disorders, including tumor metastasis, inflammation, atherosclerosis, kidney disorders, and osteoporosis, but pharmacological studies are impeded by the limited availability of suitable antagonists. One of the most potent and selective antagonists is the thiouracil derivative AR-C118925. However, this compound was until recently not commercially available and little is known about its properties. We therefore developed an improved procedure for the synthesis of AR-C118925 and two derivatives to allow up-scaling and assessed their potency in calcium mobilization assays on the human and rat P2Y2 receptors recombinantly expressed in 1321N1 astrocytoma cells. The compound was further evaluated for inhibition of P2Y2 receptor-induced β-arrestin translocation. AR-C118925 behaved as a competitive antagonist with pA 2 values of 37.2 nM (calcium assay) and 51.3 nM (β-arrestin assay). Selectivity was assessed vs. related receptors including P2X, P2Y, and adenosine receptor subtypes, as well as ectonucleotidases. AR-C118925 showed at least 50-fold selectivity against the other investigated targets, except for the P2X1 and P2X3 receptors which were blocked by AR-C118925 at concentrations of about 1 μM. AR-C118925 is soluble in buffer at pH 7.4 (124 μM) and was found to be metabolically highly stable in human and mouse liver microsomes. In Caco2 cell experiments, the compound displayed moderate permeability indicating that it may show limited peroral bioavailability. AR-C118925 appears to be a useful pharmacological tool for in vitro and in vivo studies.

The P2X7 receptor (P2X7R) has been implicated in the process of multinucleation and cell fusion. We have previously demonstrated that blockade of P2X7Rs on osteoclast precursors using a blocking antibody inhibited multinucleated osteoclast formation in vitro, but that P2X7R KO mice maintain the ability to form multinucleated osteoclasts. This apparent contradiction of the role the P2X7R plays in multinucleation has prompted us to examine the effect of the most commonly used and recently available P2X7R antagonists on osteoclast formation and function. When added to recombinant RANKL and M-CSF human blood monocytes cultures, all but one compound, decreased the formation and function of multinucleated TRAP-positive osteoclasts in a concentration-dependent manner. These data provide further evidence for the role of the P2X7R in the formation of functional human multinucleated osteoclasts and highlight the importance of selection of antagonists for use in long-term experiments.

This review describing the role of P2Y receptors in neuropathological conditions focuses on obvious differences between results demonstrating either a role in neuroprotection or in neurodegeneration, depending on in vitro and in vivo models. Such critical juxtaposition puts special emphasis on discussions of beneficial and detrimental effects of P2Y receptor agonists and antagonists in these models. The mechanisms reported to underlie the protection in vitro include increased expression of oxidoreductase genes, like carbonyl reductase and thioredoxin reductase; increased expression of inhibitor of apoptosis protein-2; extracellular signal-regulated kinase- and Akt-mediated antiapoptotic signaling; increased expression of Bcl-2 proteins, neurotrophins, neuropeptides, and growth factors; decreased Bax expression; non-amyloidogenic APP shedding; and increased neurite outgrowth in neuronal cells. Animal studies investigating the influence of P2Y receptors in middle cerebral artery occlusion (MCAO) models for stroke prove beneficial effects of P2Y receptor antagonists. In MCAO mice and rats, the application of broad-range P2 receptor antagonists decreased the infarct volume and improved neurological outcome. Moreover, antagonists of the P2Y1 receptor, one of the most abundant P2Y receptor subtypes in brain tissue, decreased neuronal loss and improved spatial memory in rats after traumatic brain injury (TBI). Currently available data show a discrepancy between in vitro and in vivo models concerning the benefits of P2Y receptor activation in pathological conditions. In vitro models demonstrate protection by P2Y receptor agonists, but in vivo P2Y receptor activation deteriorates the outcome after MCAO and controlled cortical impact brain injury, a TBI model. To broaden the scope of the review, we additionally discuss publications that demonstrate detrimental effects of P2Y receptor agonists in vitro and publications showing protective effects of agonists in vivo. All these studies help to better understand the significant role of P2Y receptors especially in stroke models and to develop pharmacological strategies for the treatment of stroke.

Purinergic signaling may be involved in embryonic development of the heart. In the present study, the effects of purinergic receptor stimulation on cardiomyogenesis of mouse embryonic stem (ES) cells were investigated. ADP or ATP increased the number of cardiac clusters and cardiac cells, as well as beating frequency. Cardiac-specific genes showed enhanced expression of α-MHC, MLC2v, α-actinin, connexin 45 (Cx45), and HCN4, on both gene and protein levels upon ADP/ATP treatment, indicating increased cardiomyogenesis and pacemaker cell differentiation. Real-time RT-PCR analysis of purinergic receptor expression demonstrated presence of P2X1, P2X4, P2X6, P2X7, P2Y1, P2Y2, P2Y4, and P2Y6 on differentiating ES cells. ATP and ADP as well as the P2X agonists β,γ-methylenadenosine 5'-triphosphate (β,γ-MetATP) and 8-bromoadenosine 5'-triphosphate (8-Br-ATP) but not UTP or UDP transiently increased the intracellular calcium concentration ([Ca(2+)](i)) as evaluated by the calcium indicator Fluo-4, whereas no changes in membrane potential were observed. [Ca(2+)](i) transients induced by ADP/ATP were abolished by the phospholipase C-β (PLC-β) inhibitor U-73122, suggesting involvement of metabotropic P2Y receptors. Furthermore, partial inhibition of [Ca(2+)](i) transients was achieved in presence of MRS2179, a selective P2Y1 receptor antagonist, whereas PPADS, a non-selective P2 receptor inhibitor, completely abolished the [Ca(2+)](i) response. Consequently, cardiomyocyte differentiation was decreased upon long term co-incubation of cells with ADP and P2 receptor antagonists. In summary, activation of purinoceptors and the subsequent [Ca(2+)](i) transients enhance the differentiation of ES cells toward cardiomyocytes. Purinergic receptor stimulation may be a promising strategy to drive the fate of pluripotent ES cells into a particular population of cardiomyocytes.

Enhanced sensitivity to noxious stimuli and the perception of non-noxious stimuli as painful are hallmark sensory perturbations associated with chronic pain. It is now appreciated that ATP, through its actions as an excitatory neurotransmitter, plays a prominent role in the initiation and maintenance of chronic pain states. Mechanistically, the ability of ATP to drive nociceptive sensitivity is mediated through direct interactions at neuronal P2X3 and P2X2/3 receptors. Extracellular ATP also activates P2X4, P2X7, and several P2Y receptors on glial cells within the spinal cord, which leads to a heightened state of neural-glial cell interaction in ongoing pain states. Following the molecular identification of the P2 receptor superfamilies, selective small molecule antagonists for several P2 receptor subtypes were identified, which have been useful for investigating the role of specific P2X receptors in preclinical chronic pain models. More recently, several P2X receptor antagonists have advanced into clinical trials for inflammation and pain. The development of orally bioavailable blockers for ion channels, including the P2X receptors, has been traditionally difficult due to the necessity of combining requirements for target potency and selectivity with suitable absorption distribution, metabolism, and elimination properties. Recent studies on the physicochemical properties of marketed orally bioavailable drugs, have identified several parameters that appear critical for increasing the probability of achieving suitable bioavailability, central nervous system exposure, and acceptable safety necessary for clinical efficacy. This review provides an overview of the antinociceptive pharmacology of P2X receptor antagonists and the chemical diversity and drug-like properties for emerging antagonists of P2X3, P2X2/3, P2X4, and P2X7 receptors.

There is considerable evidence that purines are vasoactive molecules involved in the regulation of blood flow. Adenosine is a well known vasodilator that also acts as a modulator of the response to other vasoactive substances. Adenosine exerts its effects by interacting with adenosine receptors. These are metabotropic G-protein coupled receptors and include four subtypes, A(1), A(2A), A(2B) and A(3). Adenosine triphosphate (ATP) is a co-transmitter in vascular neuroeffector junctions and is known to activate two distinct types of P2 receptors, P2X (ionotropic) and P2Y (metabotropic). ATP can exert either vasoconstrictive or vasorelaxant effects, depending on the P2 receptor subtype involved. Splanchnic vascular beds are of particular interest, as they receive a large fraction of the cardiac output. This review focus on purinergic receptors role in the splanchnic vasomotor control. Here, we give an overview on the distribution and diversity of effects of purinergic receptors in splanchnic vessels. Pre- and post-junctional receptormediated responses are summarized. Attention is also given to the interactions between purinergic receptors and other receptors in the splanchnic circulation.

F. Kukulski, S.A. Levesque, E.G. Lavoie, J. Lecka, F. Bigonnesse, A.F. Knowles, S.C. Robson, T.L. Kirley & J. Sevigny (2005) Comparative hydrolysis of P2 receptor agonists by NTPDases 1, 2, 3 and 8. Purinergic Signalling 1(2): 193–204. On page 194 of the above-mentioned article, in ‘Materials and methods’ under ‘Plasmids,’ second paragraph, the mouse NTPDase2 accession number AY37674 is incorrect and should read {"type":"entrez-nucleotide","attrs":{"text":"AY376711","term_id":"36312788","term_text":"AY376711"}}AY376711.