ATP induces contraction of cultured brain capillary pericytes via activation of P2Y-type purinergic receptors.

Brain capillary pericytes have been suggested to play a role in the regulation of cerebral blood flow under physiological and pathophysiological conditions. ATP has been shown to cause constriction of capillaries under ischemic conditions and suggested to be involved in the "no-reflow" phenomenon. To investigate the effects of extracellular ATP on pericyte cell contraction, we studied purinergic receptor activation of cultured bovine brain capillary pericytes. We measured intracellular Ca2+ concentration ([Ca2+]i) responses to purinergic agonists with the fluorescent indicators fura-2 and Cal-520 and estimated contraction of pericytes as relative change in cell area, using real-time confocal imaging. Addition of ATP caused an increase in cytosolic calcium and contraction of the brain capillary pericytes, both reversible and inhibited by the purinergic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Furthermore, we demonstrated that ATP-induced contraction could be eliminated by intracellular calcium chelation with BAPTA, indicating that the contraction was mediated via purinergic P2-type receptor-mediated [Ca2+]i signaling. ATP stimulation induced inositol triphosphate signaling, consistent with the notion of P2Y receptor activation. Receptor profiling studies demonstrated the presence of P2Y1 and P2Y2 receptors, using ATP, UTP, ADP, and the subtype specific agonists MRS2365 (P2Y1) and 2-thio-UTP (P2Y2). Addition of specific P2X agonists only caused an [Ca2+]i increase at high concentrations, attributed to activation of inositol triphosphate signaling. Our results suggest that contraction of brain capillary pericytes in vitro by activation of P2Y-type purinergic receptors is caused by intracellular calcium release. This adds more mechanistic understanding of the role of pericytes in vessel constriction and points toward P2Y receptors as potential therapeutic targets.NEW & NOTEWORTHY The study concerns brain capillary pericytes, which have been suggested to play a role in the regulation of cerebral blood flow. We show that extracellular ATP causes contraction of primary brain pericytes by stimulation of purinergic receptors and subsequent release of intracellular Ca2+ concentration ([Ca2+]i). The contraction is mainly mediated through activation of P2Y-receptor subtypes, including P2Y1 and P2Y2. These findings add more mechanistic understanding of the role of pericytes in regulation of capillary blood flow. ATP was earlier suggested to be involved in capillary constriction in brain pathologies, and our study gives a detailed account of a part of this important mechanism.

P2Y14 receptor has a critical role in acute gouty arthritis by regulating pyroptosis of macrophages.

Nod-like receptor protein 3 (NLRP3)-mediated pyroptosis has a causal role in the pathogenesis of gout. P2Y14 receptor (P2Y14R) distributed in immune cells including macrophages is a Gi-coupled receptor that inhibits the synthesis of cAMP, which has been regarded as a potential regulator of inflammatory response. Nevertheless, the role of P2Y14R in MSU-induced pyroptosis of macrophages involved in acute gouty arthritis is still unclear. In our present study, P2Y14R knockout (P2Y14R-KO) disrupted MSU-induced histopathologic changes in rat synoviums, accompanied with a significant inhibition of pyroptotic cell death characterized by Caspase-1/PI double-positive and blockade of NLRP3 inflammasome activation in synovial tissues, which was consistent with that observed in in vitro studies. Owing to the interaction of NLRP3 inflammasome and cAMP, we then investigated the effect of adenylate cyclase activator (Forskolin) on macrophage pyroptosis and gout flare caused by MSU stimulation. The reversal effect of Forskolin verified the negative regulatory role of cAMP in MSU-induced pyroptosis. More importantly, adenylate cyclase inhibitor (SQ22536) intervention led to a reversal of protection attributed to P2Y14R deficiency. Findings in air pouch animal models also verified aforementioned experimental results. Our study first identified the role of P2Y14R/cAMP/NLRP3 signaling pathway in acute gouty arthritis, which provides a novel insight into the pathological mechanisms of pyroptosis-related diseases.

Chronic fructose renders pancreatic ß-cells hyper-responsive to glucose-stimulated insulin secretion through extracellular ATP signaling.

Fructose is widely used as a sweetener in processed food and is also associated with metabolic disorders, such as obesity. However, the underlying cellular mechanisms remain unclear, in particular, regarding the pancreatic ß-cell. Here, we investigated the effects of chronic exposure to fructose on the function of insulinoma cells and isolated mouse and human pancreatic islets. Although fructose per se did not acutely stimulate insulin exocytosis, our data show that chronic fructose rendered rodent and human ß-cells hyper-responsive to intermediate physiological glucose concentrations. Fructose exposure reduced intracellular ATP levels without affecting mitochondrial function, induced AMP-activated protein kinase activation, and favored ATP release from the ß-cells upon acute glucose stimulation. The resulting increase in extracellular ATP, mediated by pannexin1 (Panx1) channels, activated the calcium-mobilizer P2Y purinergic receptors. Immunodetection revealed the presence of both Panx1 channels and P2Y1 receptors in ß-cells. Addition of an ectonucleotidase inhibitor or P2Y1 agonists to naïve ß-cells potentiated insulin secretion stimulated by intermediate glucose, mimicking the fructose treatment. Conversely, the P2Y1 antagonist and Panx1 inhibitor reversed the effects of fructose, as confirmed using Panx1-null islets and by the clearance of extracellular ATP by apyrase. These results reveal an important function of ATP signaling in pancreatic ß-cells mediating fructose-induced hyper-responsiveness.

Natural compounds acting at P2 receptors alleviate peripheral neuropathy.

Neuropathic pain is generally resistant to currently available treatments, and it is often a consequence of nerve injury due to surgery, diabetes or infection. Myocardial ischemic nociceptive signaling increases the sympathoexcitatory reflex to aggravate myocardial injury. Elucidation of the pathogenetic factors might provide a target for optimal treatment. Abundant evidence in the literature suggests that P2X and P2Y receptors play important roles in signal transmission. Traditional Chinese medicines, such as emodin, puerarin and resveratrol, antagonize nociceptive transmission mediated by purinergic 2 (P2) receptors in primary afferent neurons. This review summarizes recently published data on P2 receptor-mediated neuropathic pain and myocardial ischemia in dorsal root ganglia (DRG), superior cervical ganglia (SCG) and stellate ganglia (SG), with a special focus on the beneficial role of natural compounds.

Tools and drugs for uracil nucleotide-activated P2Y receptors.

P2Y receptors (P2YRs) are a family of G protein-coupled receptors activated by extracellular nucleotides. Physiological P2YR agonists include purine and pyrimidine nucleoside di- and triphosphates, such as ATP, ADP, UTP, UDP, nucleotide sugars, and dinucleotides. Eight subtypes exist, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14, which represent current or potential future drug targets. Here we provide a comprehensive overview of ligands for the subgroup of the P2YR family that is activated by uracil nucleotides: P2Y2 (UTP, also ATP and dinucleotides), P2Y4 (UTP), P2Y6 (UDP), and P2Y14 (UDP, UDP-glucose, UDP-galactose). The physiological agonists are metabolically unstable due to their fast hydrolysis by ectonucleotidases. A number of agonists with increased potency, subtype-selectivity and/or enzymatic stability have been developed in recent years. Useful P2Y2R agonists include MRS2698 (6-01, highly selective) and PSB-1114 (6-05, increased metabolic stability). A potent and selective P2Y2R antagonist is AR-C118925 (10-01). For studies of the P2Y4R, MRS4062 (3-15) may be used as a selective agonist, while PSB-16133 (10-06) is a selective antagonist. Several potent P2Y6R agonists have been developed including 5-methoxyuridine 5'-O-((Rp)α-boranodiphosphate) (6-12), PSB-0474 (3-11), and MRS2693 (3-26). The isocyanate MRS2578 (10-08) is used as a selective P2Y6R antagonist, although its reactivity and low water-solubility are limiting. With MRS2905 (6-08), a potent and metabolically stable P2Y14R agonist is available, while PPTN (10-14) represents a potent and selective P2Y14R antagonist. The radioligand [3H]UDP can be used to label P2Y14Rs. In addition, several fluorescent probes have been developed. Uracil nucleotide-activated P2YRs show great potential as drug targets, especially in inflammation, cancer, cardiovascular and neurodegenerative diseases.

Activation of retinal glial (Müller) cells by extracellular ATP induces pronounced increases in extracellular H+ flux.

Small alterations in extracellular acidity are potentially important modulators of neuronal signaling within the vertebrate retina. Here we report a novel extracellular acidification mechanism mediated by glial cells in the retina. Using self-referencing H+-selective microelectrodes to measure extracellular H+ fluxes, we show that activation of retinal Müller (glial) cells of the tiger salamander by micromolar concentrations of extracellular ATP induces a pronounced extracellular H+ flux independent of bicarbonate transport. ADP, UTP and the non-hydrolyzable analog ATPγs at micromolar concentrations were also potent stimulators of extracellular H+ fluxes, but adenosine was not. The extracellular H+ fluxes induced by ATP were mimicked by the P2Y1 agonist MRS 2365 and were significantly reduced by the P2 receptor blockers suramin and PPADS, suggesting activation of P2Y receptors. Bath-applied ATP induced an intracellular rise in calcium in Müller cells; both the calcium rise and the extracellular H+ fluxes were significantly attenuated when calcium re-loading into the endoplasmic reticulum was inhibited by thapsigargin and when the PLC-IP3 signaling pathway was disrupted with 2-APB and U73122. The anion transport inhibitor DIDS also markedly reduced the ATP-induced increase in H+ flux while SITS had no effect. ATP-induced H+ fluxes were also observed from Müller cells isolated from human, rat, monkey, skate and lamprey retinae, suggesting a highly evolutionarily conserved mechanism of potential general importance. Extracellular ATP also induced significant increases in extracellular H+ flux at the level of both the outer and inner plexiform layers in retinal slices of tiger salamander which was significantly reduced by suramin and PPADS. We suggest that the novel H+ flux mediated by ATP-activation of Müller cells and of other glia as well may be a key mechanism modulating neuronal signaling in the vertebrate retina and throughout the brain.

P2Y receptor regulation of K2P channels that facilitate K+ secretion by human mammary epithelial cells.

The objective of this study was to determine the molecular identity of ion channels involved in K+ secretion by the mammary epithelium and to examine their regulation by purinoceptor agonists. Apical membrane voltage-clamp experiments were performed on human mammary epithelial cells where the basolateral membrane was exposed to the pore-forming antibiotic amphotericin B dissolved in a solution with intracellular-like ionic composition. Addition of the Na+ channel inhibitor benzamil reduced the basal current, consistent with inhibition of Na+ uptake across the apical membrane, whereas the KCa3.1 channel blocker TRAM-34 produced an increase in current resulting from inhibition of basal K+ efflux. Treatment with two-pore potassium (K2P) channel blockers quinidine, bupivacaine and a selective TASK1/TASK3 inhibitor (PK-THPP) all produced concentration-dependent inhibition of apical K+ efflux. qRT-PCR experiments detected mRNA expression for nine K2P channel subtypes. Western blot analysis of biotinylated apical membranes and confocal immunocytochemistry revealed that at least five K2P subtypes (TWIK1, TREK1, TREK2, TASK1, and TASK3) are expressed in the apical membrane. Apical UTP also increased the current, but pretreatment with the PKC inhibitor GF109203X blocked the response. Similarly, direct activation of PKC with phorbol 12-myristate 13-acetate produced a similar increase in current as observed with UTP. These results support the conclusion that the basal level of K+ secretion involves constitutive activity of apical KCa3.1 channels and multiple K2P channel subtypes. Apical UTP evoked a transient increase in KCa3.1 channel activity, but over time caused persistent inhibition of K2P channel function leading to an overall decrease in K+ secretion.

Uridine adenosine tetraphosphate acts as a proangiogenic factor in vitro through purinergic P2Y receptors.

Uridine adenosine tetraphosphate (Up4A), a dinucleotide, exerts vascular influence via purinergic receptors (PR). We investigated the effects of Up4A on angiogenesis and the putative PR involved. Tubule formation assay was performed in a three-dimensional system, in which human endothelial cells were cocultured with pericytes with various Up4A concentrations for 5 days. Expression of PR subtypes and angiogenic factors was assessed in human endothelial cells with and without P2Y6R antagonist. No difference in initial tubule formation was detected between Up4A stimulation and control conditions at day 2 In contrast, a significant increase in vascular density in response to Up4A was observed at day 5 Up4A at an optimal concentration of 5 µM promoted total tubule length, number of tubules, and number of junctions, all of which were inhibited by the P2Y6R antagonist MRS2578. Higher concentrations of Up4A (10 µM) had no effects on angiogenesis parameters. Up4A increased mRNA level of P2YRs (P2Y2R, P2Y4R, and P2Y6R) but not P2XR (P2X4R and P2X7R) or P1R (A2AR and A2BR), while Up4A upregulated VEGFA and ANGPT1, but not VEGFR2, ANGPT2, Tie1, and Tie2. In addition, Up4A increased VEGFA protein levels. Transcriptional upregulation of P2YRs by Up4A was inhibited by MRS2578. In conclusion, Up4A is functionally capable of promoting tubule formation in an in vitro coculture system, which is likely mediated by pyrimidine-favored P2YRs but not P2XRs or P1Rs, and involves upregulation of angiogenic factors.

IL-6 regulates stress-induced REX-1 expression via ATP-P2Y1 signalling in stem cells isolated from human exfoliated deciduous teeth.

OBJECTIVE: To investigate the relationship between ATP and IL-6 in mechanical stress-induced REX-1 expression in SHEDs. METHODS: Cells were stimulated with mechanical stress (0-2.5 gcm(-2)), IL-6 (0.1-5 ng/ml), or ATP (10-100 µM) for 2h in serum-free media. IL-6 and REX-1 expression was examined by qualitative and quantitative polymerase chain reaction. ATP release was measured using a bioluminescence assay. The molecular mechanisms of the signalling pathways were investigated using chemical inhibitors. RESULTS: Mechanical stress induced IL-6 and REX-1 mRNA expression and ATP release. JAK inhibitor I inhibited the increase in REX-1 expression and ATP release but not IL-6 induction. Furthermore, suramin inhibited the upregulation of REX-1 mRNA expression but not ATP release. Exogenous IL-6 promoted both ATP release and REX-1 expression. The IL-6-induced REX-1 expression was attenuated by a P2Y1-specific receptor antagonist. Moreover, REX-1 expression was upregulated in a dose-dependent manner by the addition of ATP or a P2Y1 agonist. This inductive effect was abolished by the P2Y1-specific receptor antagonist. CONCLUSIONS: ATP-P2Y1 signalling is involved in IL-6-regulated stress-induced REX-1 expression in SHEDs. These results imply the participation of mechanical stress, IL-6, and ATP in regulating the expression of REX-1, a pluripotent stem cell marker.

DA-6034-induced mucin secretion via Ca2+-dependent pathways through P2Y receptor stimulation.

PURPOSE: We evaluated whether DA-6034 is involved in mucin secretion via P2Y receptor activation and/or intracellular Ca2+ concentration ([Ca2+]i) change. Also, we investigated the effect of P2Y receptor inhibitors or Ca2+ chelators on the DA-6034-induced mucin secretion and [Ca2+]i increases. METHODS: Effects of DA-6034 on mucin expression in primary, cultured, conjunctival epithelial cells was studied using RT-PCR, Western blot analysis, and periodic acid-schiff (PAS) staining. To evaluate thin film layer thickness generated by mucin and fluid secretion, cells were incubated in DA-6034 with/without P2Y antagonists or extracellular/intracellular Ca2+ chelators, and were imaged with confocal microscope using Texas Red-dextran dye. In addition, DA-6034-induced Ca2+-dependent Cl- channels opening was evaluated using perforated patch clamp. Fluo-4/AM was used to measure changes in [Ca2+]i induced by DA-6034 in Ca2+-free or Ca2+-containing buffered condition, as well as P2Y antagonists. RESULTS: DA-6034 induced the expression of mucin genes, production of mucin protein, and increase of number of mucin-secreting cells. P2Y antagonists inhibited DA-6034-induced mucin and fluid secretion, which was also affected by extracellular/intracellular Ca2+ chelators. DA-6034 stimulated Cl- channel opening and [Ca2+]i elevation. Further, [Ca2+]i increases induced by DA-6034 were lacking in either P2Y antagonists or Ca2+-free buffered condition, and diminished when endoplasmic reticulum Ca2+ was depleted by cyclopiazonic acid in Ca2+-free buffered condition. CONCLUSIONS: This study demonstrated that DA-6034 has a potential to induce mucin secretion via Ca2+-dependent pathways through P2Y receptors in multilayer, cultured, human conjunctival epithelial cells.

ATP acting through P2Y receptors causes activation of podocyte TRPC6 channels: role of podocin and reactive oxygen species.

Extracellular ATP may contribute to Ca(2+) signaling in podocytes during tubuloglomerular feedback (TGF) and possibly as a result of local tissue damage. TRPC6 channels are Ca(2+)-permeable cationic channels that have been implicated in the pathophysiology of podocyte diseases. Here we show using whole cell recordings that ATP evokes robust activation of TRPC6 channels in mouse podocyte cell lines and in rat podocytes attached to glomerular capillaries in ex vivo glomerular explants. The EC50 for ATP is ~10 µM and is maximal at 100 µM, and currents were blocked by the P2 antagonist suramin. In terms of maximal currents that can be evoked, ATP is the strongest activator of podocyte TRPC6 that we have characterized to date. Smaller currents were observed in response to ADP, UTP, and UDP. ATP-evoked currents in podocytes were abolished by TRPC6 knockdown and by pretreatment with 10 µM SKF-96365 or 50 µM La(3+). ATP effects were also abolished by inhibiting G protein signaling and by the PLC/PLA2 inhibitor D-609. ATP effects on TRPC6 were also suppressed by knockdown of the slit diaphragm scaffolding protein podocin, and also by tempol, a membrane-permeable quencher of reactive oxygen species. Modulation of podocyte TRPC6 channels, especially in foot processes, could provide a mechanism for regulation of glomerular function by extracellular nucleotides, possibly leading to changes in permeation through slit diaphragms. These results raise the possibility that sustained ATP signaling could contribute to foot process effacement, Ca(2+)-dependent changes in gene expression, and/or detachment of podocytes.

Permeation of fluorophore-conjugated phalloidin into live hair cells of the inner ear is modulated by P2Y receptors.

Phalloidin, a toxin isolated from the death cap mushroom, Amanita phalloides, binds to filamentous actin with high affinity, and this has made fluorophore-conjugated phalloidin a useful tool in cellular imaging. Hepatocytes take up phalloidin via the liver-specific organic anion transporting polypeptide 1b2, but phalloidin does not permeate most living cells. Rapid entry of styryl dyes into live hair cells has been used to evaluate function, but the usefulness of those fluorescence dyes is limited by broad and fixed absorption spectra. Since phalloidin can be conjugated to fluorophores with various spectra, we investigated whether it would permeate living hair cells. When we incubated mouse utricles in 66 nM phalloidin-CF488A and followed that by washes in phalloidin-free medium, we observed that it entered a subset of hair cells and labeled entire hair bundles fluorescently after 20 min. Incubations of 90 min labeled nearly all the hair bundles. When phalloidin-treated utricles were cultured for 24 h after washout, the label disappeared from the hair cells and progressively but heterogeneously labeled filamentous actin in the supporting cells. We investigated how phalloidin may enter hair cells and found that P2 receptor antagonists, pyridoxalphosphate-6-azophenyl-2', 4'-disulfonic acid and suramin, blocked phalloidin entry, while the P2Y receptor ligands, uridine-5'-diphosphate and uridine-5'-triphosphaste, stimulated uptake. Consistent with that, the P2Y6 receptor antagonist, MRS 2578, decreased phalloidin uptake. The results show that phalloidin permeates live hair cells through a pathway that requires metabotropic P2Y receptor signaling and suggest that phalloidin can be transferred from hair cells to supporting cells in culture.

Structure-based approaches to ligands for G-protein-coupled adenosine and P2Y receptors, from small molecules to nanoconjugates.

Adenosine receptor (ARs) and P2Y receptors (P2YRs) that respond to extracellular nucleosides/nucleotides are associated with new directions for therapeutics. The X-ray structures of the A2AAR complexes with agonists and antagonists are examined in relationship to the G-protein-coupled receptor (GPCR) superfamily and applied to drug discovery. Much of the data on AR ligand structure from early SAR studies now are explainable from the A2AAR X-ray crystallography. The ligand-receptor interactions in related GPCR complexes can be identified by means of modeling approaches, e.g., molecular docking. Thus, molecular recognition in binding and activation processes has been studied effectively using homology modeling and applied to ligand design. Virtual screening has yielded new nonnucleoside AR antagonists, and existing ligands have been improved with knowledge of the receptor interactions. New agonists are being explored for central nervous system and peripheral therapeutics based on in vivo activity, such as chronic neuropathic pain. Ligands for receptors more distantly related to the X-ray template, i.e., P2YRs, have been introduced and are mainly used as pharmacological tools for elucidating the physiological role of extracellular nucleotides. Other ligand tools for drug discovery include fluorescent probes, radioactive probes, multivalent probes, and functionalized nanoparticles.

Modulation of P2 receptors on pancreatic ß-cells by agonists and antagonists: a molecular target for type 2 diabetes treatment.

Morbidity and mortality from diabetes mellitus (DM) are serious worldwide concerns. By the year 2030, the estimated number of diabetic patients will reach a staggering 439 million worldwide. Diabetes mellitus type 2 (DM2), which involves disturbances in both insulin secretion and resistance, is the most common form of diabetes and affects approximately 5 to 7% of the world's population. When a patient with DM2 cannot regulate his or her blood glucose levels through diet, weight loss, or exercise, oral medications, such as hypoglycemic agents (i.e., sulphonylureas, biguanides, alpha glucosidase inhibitors and thiazolidinediones), are crucial. Here, we discuss some physiological aspects of P2 receptors on pancreatic ß-cells, which express a variety of P2 receptor isoforms. These receptors enhance glucose-dependent insulin release. In addition, we speculate on the potential of purinergic compounds as novel or additional treatments for Type 2 Diabetes mellitus.

P2Y purinoceptors as potential emerging therapeutical target in vascular disease.

In the last two decades a tremendous development has been noticed in our understanding of the purinergic system, consisting of heterogeneously expressed purinoceptor subtypes and its classical agonists: e.g., adenosine triphosphate, uridine triphosphate or complex dinucleoside polyphosphates. These agonists exert multiple effects on the vascular system: they regulate the relaxation and constriction of arterial blood vessels, lead to proliferation and migration in endothelial cells and vascular smooth muscle cells, and mediate potent proinflammatory responses or phenotypic cell changes. This review focuses on the P2 purinoceptor subtype P2Y and its pleiotropic effects in the vascular wall under physiological and pathophysiological condition. Various experimental and clinical studies provide evidence that pharmacological targeting of P2Y might be effective in reducing vascular alterations under disease conditions.

P2Y receptors in the mammalian nervous system: pharmacology, ligands and therapeutic potential.

P2Y receptors for extracellular nucleotides are coupled to activation of a variety of G proteins and stimulate diverse intracellular signaling pathways that regulate functions of cell types that comprise the central nervous system (CNS). There are 8 different subtypes of P2Y receptor expressed in cells of the CNS that are activated by a select group of nucleotide agonists. Here, the agonist selectivity of these 8 P2Y receptor subtypes is reviewed with an emphasis on synthetic agonists with high potency and resistance to degradation by extracellular nucleotidases that have potential applications as therapeutic agents. In addition, the recent identification of a wide variety of subtype-selective antagonists is discussed, since these compounds are critical for discerning cellular responses mediated by activation of individual P2Y receptor subtypes. The functional expression of P2Y receptor subtypes in cells that comprise the CNS is also reviewed and the role of each subtype in the regulation of physiological and pathophysiological responses is considered. Other topics include the role of P2Y receptors in the regulation of blood-brain barrier integrity and potential interactions between different P2Y receptor subtypes that likely impact tissue responses to extracellular nucleotides in the CNS. Overall, current research suggests that P2Y receptors in the CNS regulate repair mechanisms that are triggered by tissue damage, inflammation and disease and thus P2Y receptors represent promising targets for the treatment of neurodegenerative diseases.

Pharmacogenetics of the antiplatelet effect of aspirin.

The concept of "pharmacogenetics" addresses genetically determined variation in how individuals respond to drugs. Accordingly, specific genetic variants have been suggested as contributors to a reduced response to various antiplatelet drugs. Aspirin is a cornerstone in secondary cardiovascular prevention and has been thoroughly investigated. The efficacy of aspirin is well documented, although with considerable interindividual variation. According to meta-analyses, a reduced antiplatelet effect of aspirin confers an increased risk of cardiovascular events. The platelet response to aspirin is assessed by in vitro evaluation of thromboxane-dependent platelet function. A reduced antiplatelet effect of aspirin can be explained by several mechanisms, which are largely determined by clinical, pharmacodynamic, biological and genetic factors. During the past decade, numerous studies have identified genetic polymorphisms significantly associated with cardiovascular events and modulating the antiplatelet effect of aspirin. However, results have been contradictory allowing only few firm conclusions to be drawn. Polymorphisms in genes encoding glycoproteins (IIb/IIIa, Ia/IIa, VI and Ibα), cyclooxygenases (1 and 2), adenosine diphosphate receptors (P2Y1 and P2Y12) and proteins of importance for haemostasis (thromboxane A2 receptor, coagulation factor XIII, etc.) have been investigated. In particular, a polymorphism in the gene encoding glycoprotein IIb/IIIa has been associated with a reduced antiplatelet effect of aspirin. The additive value of an individual's genetic makeup in predicting the antiplatelet effect of aspirin and the risk of cardiovascular events remains controversial. The present review outlines the pharmacology of aspirin and provides an overview of specific genetic variations considered to influence the antiplatelet effect of aspirin.

Purinergic activation of rat skeletal muscle membranes increases Vmax and Na+ affinity of the Na,K-ATPase and phosphorylates phospholemman and α1 subunits.

Muscle activity is associated with an increase in extracellular purines (ATP, ADP), which are involved in signalling mechanisms. The present study investigates the effect of purines on the function of Na,K-ATPase (Na,K-pump) in rat skeletal muscle. Na,K-ATPase activity was quantified by measuring the release of inorganic phosphate in the presence of ATP and variable Na(+) concentrations. In membranes purified from glycolytic muscle fibres, purinergic stimulation increases V (max) and decreases the K (m) (higher Na(+) affinity) of the Na,K-ATPase. Stimulatory effects were obtained using ATP, ADP, 2-methylthio-ADP and UPT, but not UDP and adenosine. The effect of ADP on V (max) can be inhibited by the non-specific P2Y receptor antagonists, suramin and PPADS. Moreover, the P2Y(13) receptor antagonist MRS 2211 strongly inhibited the response to ADP, whereas the specific P2Y(1) receptor antagonist MRS 2500 had less effect. Based on results from these agonists and antagonists, we conclude that P2Y(13) receptors mediate the main effects observed, that P2Y1 receptors are also involved and that some P2Y(2)/P2Y(4) receptors also appear to be involved. Receptor antagonists had no effect on ADP-induced subunit (phospholemman and α1) phosphorylation and changes in K (m) (Na(+) affinity). Thus, the stimulatory effects of purines are mediated by two independent mechanisms: P2Y receptor-mediated increase in Na,K-ATPase capacity (increased V (max)) and P2Y receptor-independent phosphorylation of Na,K-ATPase phospholemman and α1 subunits, which induce changes in ion affinity. These mechanisms may contribute to up-regulation of Na,K-ATPase during muscle activity.

Impaired P2X and P2Y receptor-mediated signaling in HPRT-deficient B103 neuroblastoma cells.

Defect of hypoxanthine phosphoribosyl transferase (HPRT) causes Lesch-Nyhan disease (LND), but the link between HPRT deficiency and the self-injurious behavior of LND is unknown. In a previous study (Pinto et al., J. Neurochem. 72 (2005) 1579-1586) we reported on a decrease in nucleotidase activity in membranes of several HPRT(-) cell lines and fibroblasts from LND patients. Since nucleotidases are involved in ATP-induced signal transduction, in the present study, we tested the hypothesis that P2X and P2Y receptor-mediated signal transduction is impaired in HPRT deficiency. As model we studied rat B103 neuroblastoma cells. Compared to control cells, in HPRT(-) cells, NTP and NDP-induced Ca(2+) influx across the membrane and Ca(2+) mobilization from intracellular stores were impaired. Both P2X and P2Y receptors were involved in the responses. Quantitative real-time PCR revealed reduced expression of receptors P2X(3), P2X(5), P2Y(2), P2Y(4), P2Y(12), P2Y(13) and P2Y(14) in HPRT deficiency. Collectively, HPRT deficiency is associated with abnormal purinergic signaling, encompassing P2X and P2Y receptors and nucleotidases.

Uridine adenosine tetraphosphate-induced contraction is increased in renal but not pulmonary arteries from DOCA-salt hypertensive rats.

Uridine adenosine tetraphosphate (Up(4)A) was reported as a novel endothelium-derived contracting factor. Up(4)A contains both purine and pyrimidine moieties, which activate purinergic (P2)X and P2Y receptors. However, alterations in the vasoconstrictor responses to Up(4)A in hypertensive states remain unclear. The present study examined the effects of Up(4)A on contraction of isolated renal arteries (RA) and pulmonary arteries (PA) from DOCA-salt rats using isometric tension recording. RA from DOCA-salt rats exhibited increased contraction to Up(4)A versus arteries from control uninephrectomized rats in the absence and presence of N(G)-nitro-l-arginine (nitric oxide synthase inhibitor). On the other hand, the Up(4)A-induced contraction in PA was similar between the two groups. Up(4)A-induced contraction was inhibited by suramin (nonselective P2 antagonist) but not by diinosine pentaphosphate pentasodium salt hydrate (Ip(5)I; P2X(1) antagonist) in RA from both groups. Furthermore, 2-thiouridine 5'-triphosphate tetrasodium salt (2-ThioUTP; P2Y(2) agonist)-, uridine-5'-(γ-thio)-triphosphate trisodium salt (UTPγS; P2Y(2)/P2Y(4) agonist)-, and 5-iodouridine-5'-O-diphosphate trisodium salt (MRS 2693; P2Y(6) agonist)-induced contractions were all increased in RA from DOCA-salt rats. Protein expression of P2Y(2)-, P2Y(4)-, and P2Y(6) receptors in RA was similar between the two groups. In DOCA-salt RA, the enhanced Up(4)A-induced contraction was reduced by PD98059, an ERK pathway inhibitor, and Up(4)A-stimulated ERK activation was increased. These data are the first to indicate that Up(4)A-induced contraction is enhanced in RA from DOCA-salt rats. Enhanced P2Y receptor signaling and activation of the ERK pathway together represent a likely mechanism mediating the enhanced Up(4)A-induced contraction. Up(4)A might be of relevance in the pathophysiology of vascular tone regulation and renal dysfunction in arterial hypertension.