Selective P2Y1 Receptor Antagonist Research Articles

Involvement of purinergic P2Y1R in antidepressant-like effects of electroacupuncture treatment on social isolation stress mice.

Depression is a common neuropsychiatric disorder with high incidence and disability. Electroacupuncture (EA) is effective in the treatment of depression. However, the underlying mechanisms are not fully understood. Social isolation stress during post-weaning period can impair purinergic signaling in the brain of rodents and has emerged as a major risk factor for depression. The purpose of this study was to investigate the involvement of P2Y1 receptor (P2Y1R) in the antidepressant-like effects of EA. In this study, C57BL/6 mice were randomly assigned to group-housed (GH) or social isolated (SI) groups at post-natal day 21. After 6weeks of social isolation, EA was performed on acupoints "Bai-hui" (GV20) and "Yin-tang" (GV29), or non-acupoints for 4weeks. The SI mice received either intracerebroventricular injection of a selective P2Y1R agonist, MRS2365 (1nmol); or a selective P2Y1R antagonist, MRS2179 (2μmol), before and after EA. We found that SI mice exhibited depression-like behaviors accompanied with anxiety-like behaviors. The expressions of P2Y1R were well co-localized with GFAP-positive astrocytes and increased in the prefrontal cortex and hippocampus of SI mice. After treated with MRS2179, the depression-like behaviors of SI mice were attenuated, but not with MRS2365. Meanwhile, we found that EA could attenuate social isolation caused depression- and anxiety-like behaviors, and inhibited the up-regulation of P2Y1R in the prefrontal cortex and hippocampus of SI mice. Notably, the positive effects of EA on depression-like behaviors of SI mice could be reversed by MRS2365, while MRS2365 had no effect on the anxiolytic-like effects of EA. Therefore, we provide new evidence that EA could ameliorate depression- and anxiety-like behaviors in social isolation stress mice, and P2Y1R was involved in the antidepressant-like effects of EA.

P2X7 purinergic receptors participate in the expression and extinction processes of contextual fear conditioning memory in mice

The purinergic system consists of two large receptor families – P2X and P2Y. Both are activated by adenosine triphosphate (ATP), although presenting different functions. These receptors are present in several brain regions, including those involved in emotion and stress-related behaviors. Hence, they seem to participate in fear- and anxiety-related responses. However, few studies have investigated the purinergic system in threatening situations, as observed in contextual fear conditioning (CFC). Therefore, this study investigated the involvement of purinergic receptors in the expression and extinction of aversive memories. C57Bl/6 background mice were submitted to the CFC protocol. Wildtype (WT) mice received i.p. injection of either a nonselective P2 receptor (P2R) antagonist, P178 (10 or 30 mg/kg); a selective P2X7 receptor (P2X7R) antagonist, A438079 (10 mg/kg); a selective P2Y1 receptor (P2Y1R) antagonist, MRS2179 (10 mg/kg); or vehicle 10 min prior to or immediately after the extinction session. Additionally, P2X7R KO mice were tested in the CFC protocol. After P2R antagonist treatment, contextual fear recall increased, while acquisition of extinction was impaired. Similar results were observed with the selective P2X7R antagonist, but not with the selective P2Y1R antagonist. Interestingly, P2X7R KO mice showed increased contextual fear recall, associated with impaired acquisition of extinction, in accordance with pharmacologic P2X7R antagonism. Our results suggest that specific pharmacological or genetic blockade of P2X7R promotes anxiogenic-like effects, along with deficits in extinction learning. Thus, these receptors could present an alternative treatment of stress-related psychiatric disorders.

P2Y Receptors Sensitize Mouse and Human Colonic Nociceptors.

Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders. Purine and pyrimidine nucleotides (e.g., ATP and UTP) are strongly implicated in this process following their release from epithelial cells during mechanical stimulation of the gut, and from immune cells during inflammation. Actions of ATP are mediated through both ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptor activation causes excitation of visceral afferents; however, the impact of P2Y receptor activation on visceral afferents innervating the gut is unclear. Here we investigate the effects of stimulating P2Y receptors in isolated mouse colonic sensory neurons, and visceral nociceptor fibers in mouse and human nerve-gut preparations. Additionally, we investigate the role of Nav1.9 in mediating murine responses. The application of UTP (P2Y2 and P2Y4 agonist) sensitized colonic sensory neurons by increasing action potential firing to current injection and depolarizing the membrane potential. The application of ADP (P2Y1, P2Y12, and P2Y13 agonist) also increased action potential firing, an effect blocked by the selective P2Y1 receptor antagonist MRS2500. UTP or ADP stimulated afferents, including mouse and human visceral nociceptors, in nerve-gut preparations. P2Y1 and P2Y2 transcripts were detected in 80% and 56% of retrogradely labeled colonic neurons, respectively. Nav1.9 transcripts colocalized in 86% of P2Y1-positive and 100% of P2Y2-positive colonic neurons, consistent with reduced afferent fiber responses to UTP and ADP in Na(v)1.9(-/-) mice. These data demonstrate that P2Y receptor activation stimulates mouse and human visceral nociceptors, highlighting P2Y-dependent mechanisms in the generation of visceral pain during gastrointestinal disease.

Predominant role of spinal P2Y1 receptors in the development of neuropathic pain in rats

The role of P2X2/3, P2X3, P2X4 or P2X7 and P2Y2, P2Y6, and P2Y12 receptors in neuropathic pain has been widely studied. In contrast, the role of P2Y1 receptors is scarcely studied. In this study we assessed the role of P2Y1 receptors in several neuropathic pain models in the rat. Furthermore, we analyzed the expression of P2Y1 receptors in the ipsilateral dorsal root ganglia (DRG) and dorsal part of the spinal cord during the development and maintenance of neuropathic pain. We also determined the effect of the P2Y1 receptor antagonist on the expression of P2Y1 receptors. Chronic constriction injury (CCI), spared nerve injury (SNI) or spinal nerve ligation (SNL) produced tactile allodynia from 1 to 14 days after nerve injury. CCI, SNI and SNL enhanced expression of P2Y1 receptors in DRG but not in the dorsal part of the spinal cord at 1–3 days after injury. Intrathecal injection of the selective P2Y1 receptor antagonist MRS2500, but not vehicle, reduced tactile allodynia in rats 1–3 days after CCI, SNI, or SNL. Moreover, intrathecal injection of MRS2500 (at day 1 or 3) reduced neuropathy-induced up-regulation of P2Y1 receptors expression. Intrathecal injection of MRS2500 lost most of the antiallodynic effect when injected 14 days after injury. At this time, MRS2500 did not modify nerve-injury-induced P2Y1 receptors up-regulation. Our results suggest that P2Y1 receptors are localized in DRG, are up-regulated by nerve injury and play a pronociceptive role in development and, to a lesser extent, maintenance of neuropathic pain.

Purinergic glio-endothelial coupling during neuronal activity: role of P2Y1 receptors and eNOS in functional hyperemia in the mouse somatosensory cortex.

Impairment of moment-to-moment adjustment of cerebral blood flow (CBF) via neurovascular coupling is thought to play a critical role in the genesis of cognitive impairment associated with aging and pathological conditions associated with accelerated cerebromicrovascular aging (e.g., hypertension, obesity). Although previous studies demonstrate that endothelial dysfunction plays a critical role in neurovascular uncoupling in these conditions, the role of endothelial NO mediation in neurovascular coupling responses is not well understood. To establish the link between endothelial function and functional hyperemia, neurovascular coupling responses were studied in mutant mice overexpressing or deficient in endothelial NO synthase (eNOS), and the role of P2Y1 receptors in purinergic glioendothelial coupling was assessed. We found that genetic depletion of eNOS (eNOS(-/-)) and pharmacological inhibition of NO synthesis significantly decreased the CBF responses in the somatosensory cortex evoked by whisker stimulation and by administration of ATP. Overexpression of eNOS enhanced NO mediation of functional hyperemia. In control mice, the selective and potent P2Y1 receptor antagonist MRS2179 attenuated both whisker stimulation-induced and ATP-mediated CBF responses, whereas, in eNOS(-/-) mice, the inhibitory effects of MRS2179 were blunted. Collectively, our findings provide additional evidence for purinergic glio-endothelial coupling during neuronal activity, highlighting the role of ATP-mediated activation of eNOS via P2Y1 receptors in functional hyperemia.

Cardiomyogenesis of embryonic stem cells upon purinergic receptor activation by ADP and ATP.

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.

Contribution of extracellular ATP on the cell-surface F1F0-ATP synthase-mediated intracellular triacylglycerol accumulation.

Cell-surface F1F0-ATP synthase was involved in the cell signaling mediating various biological functions. Recently, we found that cell-surface F1F0-ATP synthase plays a role on intracellular triacylglycerol accumulation in adipocytes, and yet, the underlying mechanisms remained largely unknown. In this study, we investigated the role of extracellular ATP on the intracellular triacylglycerol accumulation. We demonstrated that significant amounts of ATP were produced extracellularly by cultured 3T3-L1 adipocytes and that the antibodies against α and β subunits of F1F0-ATP synthase inhibited the extracellular ATP production. Piceatannol, a F1F0-ATP synthase inhibitor, and apyrase, an enzyme which degrades extracellular ATP, suppressed triacylglycerol accumulation. The selective P2Y1 receptor antagonist MRS2500 significantly inhibited triacylglycerol accumulation, whereas the selective P2X receptor antagonist NF279 has less effect. The present results indicate that cell-surface F1F0-ATP synthase on adipocytes is functional in extracellular ATP production and that the extracellular ATP produced contributes, at least in part, to the cell-surface F1F0-ATP synthase-mediated intracellular triacylglycerol accumulation in adipocytes through P2Y1 receptor.

P2Y13 receptor-mediated rapid increase in intracellular calcium induced by ADP in cultured dorsal spinal cord microglia.

P2Y receptors have been implicated in the calcium mobilization by the response to neuroexcitatory substances in neurons and astrocytes, but little is known about P2Y receptors in microglia cells. In the present study, the effects of ADP on the intracellular calcium concentration ([Ca(2+)]i) in cultured dorsal spinal cord microglia were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescence indicator that could monitor real-time alterations of [Ca(2+)]i. Here we show that ADP (0.01-100 μM) causes a rapid increase in [Ca(2+)]i with a dose-dependent manner in cultured microglia. The action of ADP on [Ca(2+)]i was significantly blocked by MRS2211 (a selective P2Y13 receptor antagonist), but was unaffected by MRS2179 (a selective P2Y1 receptor antagonist) or MRS2395 (a selective P2Y12 receptor antagonist), which suggest that P2Y13 receptor may be responsible for ADP-evoked Ca(2+) mobilization in cultured microglia. P2Y13-evoked Ca(2+) response can be obviously inhibited by BAPTA-AM and U-73122, respectively. Moreover, removal of extracellular Ca(2+) (by EGTA) also can obvious suppress the Ca(2+) mobilization. These results means both intracellular calcium and extracellular calcium are potentially important mechanisms in P2Y13 receptor-evoked Ca(2+) mobilization. However, P2Y13 receptor-evoked Ca(2+) response was not impaired after CdCl2 and verapamil administration, which suggest that voltage-operated Ca(2+) channels may be not related with P2Y13-evoked Ca(2+) response. In addition, Ca(2+) mobilization induced by ADP was abolished by different store-operated Ca(2+) channels (SOCs) blocker, 2-APB (50 μM) and SKF-96365 (1 mM), respectively. These observations suggest that the activation of P2Y13 receptor might be involved in the effect of ADP on [Ca(2+)]i in cultured dorsal spinal cord microglia. Furthermore, our results raise a possibility that P2Y13 receptor activation causes Ca(2+) release from Ca(2+) store, which leads to the opening of SOCs.

Su2037 TRPV2 Knockout Mice Exhibit Enhanced Gastric Emptying

this is the case in patients with more severe and/or complicated disease. OBJECTIVE: To test the hypothesis that inhibitory neurotransmission to sigmoid colon CSM is impaired in patients with complicated diverticulitis. METHODS: Standard intracellular recordings were performed from sigmoid colon CSM of patients who underwent sigmoid resection because of complications of diverticulitis (n=4) and compared to control tissue obtained from patients having resection for cancer. Tissue adjacent to recording sites was also examined histologically for muscle layer thickness and density of innervation using SNAP-25 immunohistochemistry. Quantitative analysis of electrophysiological recordings, histology and immunohistochemistry were performed by ‘blinded' observers. Statistical significance was determined using t-tests with a p-value of <0.05 considered significant. RESULTS: Resting membrane potential was significantly more depolarized in CSM from diverticulitis patients vs controls (-39.0±0.9 vs -46.0±1.8 mV). In control tissue electrical stimulation (4 pulses, 0.5 ms) produced an inhibitory junction potential (IJP) of 11.0±1.3 mV that was markedly inhibited (to 1.5±0.7 mV) by application of the selective P2Y1 receptor antagonist MRS-2500. The magnitude of the IJP was markedly attenuated in diverticulitis patients vs controls (0.7±0.7 vs 11.0±1.3 mV). The CSM layer was on average 50% thicker in complicated diverticulitis patients versus controls (2.07 ± 0.65 mm vs 1.39 ± 0.23 mm) and this appeared to be due to muscular hypertrophy, as the average CSM cell number/length of tissue was significantly decreased in diverticulitis (0.84 ± 0.08 cells/100 μm in diverticulitis vs 1.6 ± 0.2 cells/100 μm in controls). Axon density was similar in the two groups (0.23 ± 0.06 axon intersections/CSM cell in controls vs 0.22 ± 0.03 axon intersections/CSM cell in diverticulitis). CONCLUSIONS: Purinergic inhibitory neurotransmission to sigmoid CSM appears markedly impaired in patients with complicated diverticulitis and resting membrane potential is also relatively depolarized. This is associated with hypertrophy of the CSM layer, but no apparent alteration in innervation density. Further studies are required to determine the mechanisms underlying this abnormality and how it may relate to the pathophysiology of diverticular disease.

Histamine Induces ATP Release from Human Subcutaneous Fibroblasts, via Pannexin-1 Hemichannels, Leading to Ca2+ Mobilization and Cell Proliferation

Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca(2+) ([Ca(2+)]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca(2+)]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca(2+)]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with (10)Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca(2+)]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADP-sensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca(2+)]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.

Functional role of vasoactive intestinal polypeptide in inhibitory motor innervation in the mouse internal anal sphincter

There is evidence that vasoactive intestinal polypeptide (VIP) participates in inhibitory neuromuscular transmission (NMT) in the internal anal sphincter (IAS). However, specific details concerning VIP-ergic NMT are limited, largely because of difficulties in selectively blocking other inhibitory neural pathways. The present study used the selective P2Y1 receptor antagonist MRS2500 (1 μm) and the nitric oxide synthase inhibitor N(G)-nitro-l-arginine (l-NNA; 100 μm) to block purinergic and nitrergic NMT to characterize non-purinergic, non-nitrergic (NNNP) inhibitory NMT and the role of VIP in this response. Nerves were stimulated with electrical field stimulation (0.1-20 Hz, 4-60 s) and the associated changes in contractile and electrical activity measured in non-adrenergic, non-cholinergic conditions in the IAS of wild-type and VIP(-/-) mice. Electrical field stimulation gave rise to frequency-dependent relaxation and hyperpolarization that was blocked by tetrodotoxin. Responses during brief trains of stimuli (4 s) were mediated by purinergic and nitrergic NMT. During longer stimulus trains, an NNNP relaxation and hyperpolarization developed slowly and persisted for several minutes beyond the end of the stimulus train. The NNNP NMT was abolished by VIP6-28 (30 μm), absent in the VIP(-/-) mouse and mimicked by exogenous VIP (1-100 nm). Immunoreactivity for VIP was co-localized with neuronal nitric oxide synthase in varicose intramuscular fibres but was not detected in the VIP(-/-) mouse IAS. In conclusion, this study identified an ultraslow component of inhibitory NMT in the IAS mediated by VIP. In vivo, this pathway may be activated with larger rectal distensions, leading to a more prolonged period of anal relaxation.

Progress in understanding of inhibitory purinergic neuromuscular transmission in the gut

Recent studies with genetic deletion of P2Y1 receptor (P2Y1-/-) have clinched its role in enteric purinergic inhibitory neurotransmission and suggested that β-NAD may be the purinergic inhibitory neurotransmitter in the colon. In this issue of the Journal, Gil and colleagues extend their earlier observations to the cecum and gastric antrum, showing that P2Y1 receptor mediated purinergic inhibition may be a general phenomenon in the gut. However, the authors made an unexpected observation in contrast with their earlier findings in the colon that neither the selective P2Y1 receptor antagonist MRS2500, nor P2Y1 receptor deletion, blocked the hyperpolarizing action of β-NAD in the cecum. These observations suggest that β-NAD may be the purinergic inhibitory neurotransmitter in the colon, but not in the cecum. This group had previously reported that the selective P2Y1 receptor antagonist MRS 2179 suppressed the hyperpolarizing action of ATP or ADP. Further studies are now needed to determine whether the hyperpolarizing actions of ATP and ADP are suppressed by the more potent P2Y1 antagonist MRS2500, and in P2Y1-/- mutants to test the intriguing possibility that different purines serve as purinergic inhibitory neurotransmitters in the colon and cecum and perhaps in different parts of the gut. Studies in P2Y1-/- mice will resolve other issues in purinergic neurotransmission including cellular localization of the β-NAD or ATP-activated P2Y1 receptors on either smooth muscle cells or PDGFRα+ fibroblast-like cells, relationship of purinergic to nitrergic neurotransmission and understanding the physiological and clinical importance of purinergic transmission in gastrointestinal motility and its disorders.

Parallel pathways of glutamate and ATP-mediated excitotoxicity cause significant neural cell death during ischaemia: potential for novel neuroprotective strategies

Neural cell death during cerebral ischaemia is correlated with various human pathological conditions leading to significant levels of mortality and morbidity. Despite decades of intense research there remains a paucity of clinically effective non-vascular neuroprotective measures. We present data supporting the theory that parallel pathways of ATP and glutamate excitotoxicity operate during ischaemia, which require the cooperation of both astrocytes and neurons, and that concomitant blockade of both pathways at the receptor level provides synergistic neuroprotection by preventing both astrocyte and neuronal death during the initial phase of a severe ischaemic insult.Investigations were carried out with primary neuron and astrocyte cell cultures obtained from embryonic mice. Astrocytes and neurons expressed a range of functional P2 and glutamate receptors. 90 min of oxygen-glucose deprivation (OGD), used to simulate ischaemia, induced significant release of glutamate from both monocultures of neurons or astrocytes while astrocytes alone were responsible for ATP release. OGD induced significant amounts of both astrocyte and neuronal death, with neuronal death being enhanced in the presence of astrocytes. In co-cultures of astrocytes and neurons AMPA/kainate and NMDA receptor antagonist prevented only neuronal death, whereas broad-spectrum P2 receptor antagonists were significantly protective of both neurons and astrocytes. Astrocytes and neurons were also protected by a selective P2Y1 receptor antagonist. Combining AMPA/kainate, NMDA, and P2 receptor antagonists, even at greatly reduced concentrations, was significantly protective of all cell types during OGD.These results provide further evidence for the existence of both ATP and glutamate mediated excitotoxicity during ischaemia in the brain and highlight the important role of astrocytes in this process. Furthermore, the synergistic protective effect of inhibiting both pathways suggests that future effective neuroprotective strategies may benefit from a multipronged approach aimed at the large number of cell death pathways known to lead to eventual cell death and dysfunction. FundingNone.

Bradykinin activation of fibroblasts of the rat subcutaneous tissue triggers the release of ATP and P2 purinoceptors activation

BACKGROUND. Pain related to musculoskeletal system is increasingly common and purines, acting through distinct signaling pathways, are described to be involved in nociception (Schleip & Klingler, 2008). Considering that fibroblasts from subcutaneous connective tissue can release adenine or uridine nucleotides, triggering intracellular Ca 2+ waves in response to stressful conditions, these cells may play a key role in the propagation/amplification of signals to primary afferent neurons (Furuya et al., 2005). Thus, we decided to investigate the involvement of purinoceptors on bradykinin (BK)-induced activation of fibroblasts of the rat subcutaneous tissue. METHODS. Experiments were performed in the first subculture of fibroblasts isolated from rat subcutaneous connective tissue. Intracellular ATP release was inferred from destaining of cells loaded with quinacrine (an ATP-binding intracellular fluorescent dye) by confocal microscopy (Orriss et al., 2009). Intracellular Ca 2+ oscillations were monitored using a microplate reader after loading the cells with the fluorescent Ca 2+ dye, Fluo-4NW. The expression of P2 purinoceptors on cultured fibroblasts of the rat subcutaneous tissues was evaluated by immunofluorescence confocal microscopy. RESULTS. Exposure to BK (30 μM) significantly enhanced fluorescence destaining of cultured fibroblasts loaded with quinacrine, suggesting that fibroblasts release intracellular ATP when stimulated with BK. In the presence of brefeldin A, which is a potent inhibitor of vesicular trafficking and secretion, BK (30 μM) was unable to increase intracellular Ca 2+ accumulation. Moreover, we observed a decrease of the BK-induced Ca 2+ response in the presence of the Cx36 and Cx50 inhibitor, mefloquine, and of the selective pannexin-1 inhibitor, 10 Panx. The intracellular Ca 2+ accumulation induced by BK was also partially attenuated by apyrase, an enzyme that hydrolyses ATP and ADP directly to AMP. Interestingly, the ecto-NTPDase inhibitor, POM-1, which prevents sequential dephosphorylation of ATP to ADP leading to ATP accumulation and ADP depletion, also prevented BK-induced intracellular Ca 2+ increase. These results point towards the participation of ADP generated from the extracellular catabolism of released ATP on BKinduced fibroblast Ca 2+ rises. Fibroblast Ca 2+ oscillations caused by BK were partially attenuated in the presence of the selective P2Y1 receptor antagonist, MRS 2179, but not upon the selective blockade of P2Y12 and P2Y13 receptors, respectively with AR-C66096 and MRS 2211. The expression of P2Y1 receptors on cultured fibroblasts from rat the subcutaneous tissue was confirmed by immunofluorescence confocal microscopy. CONCLUSIONS. Data suggest that BK-evoked responses may be partially mediated by endogenous released adenine nucleotides via a mechanism involving hemichannels and vesicle exocytosis. Extracellular hydrolysis of released ATP into ADP may facilitate the inflammatory responses of BK through the activation of P2Y1 receptors. Understanding how fibroblasts of the subcutaneous tissue trigger the release of signal amplification molecules, such as adenine nucleotides, which may ultimately contribute to activate neighboring cells, including sensory neurons, enlightens the hypothesis that fibroblast may be a novel target for therapeutic intervention in chronic painful conditions.

Nucleotides control the excitability of sensory neurons via two P2Y receptors and a bifurcated signaling cascade

Nucleotides contribute to the sensation of acute and chronic pain, but it remained enigmatic which G protein-coupled nucleotide (P2Y) receptors and associated signaling cascades are involved. To resolve this issue, nucleotides were applied to dorsal root ganglion neurons under current- and voltage-clamp. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), and uridine triphosphate (UTP), but not uridine diphosphate (UDP), depolarized the neurons and enhanced action potential firing in response to current injections. The P2Y2 receptor preferring agonist 2-thio-UTP was equipotent to UTP in eliciting these effects. The selective P2Y1 receptor antagonist MRS2179 largely attenuated the excitatory effects of ADP, but left those of 2-thio-UTP unaltered. Thus, the excitatory effects of the nucleotides were mediated by 2 different P2Y receptors, P2Y1 and P2Y2. Activation of each of these 2 receptors by either ADP or 2-thio-UTP inhibited currents through KV7 channels, on one hand, and facilitated currents through TRPV1 channels, on the other hand. Both effects were abolished by inhibitors of phospholipase C or Ca2+-ATPase and by chelation of intracellular Ca2+. The facilitation of TRPV1, but not the inhibition KV7 channels, was prevented by a protein kinase C inhibitor. Simultaneous blockage of KV7 channels and of TRPV1 channels prevented nucleotide-induced membrane depolarization and action potential firing. Thus, P2Y1 and P2Y2 receptors mediate an excitation of dorsal root ganglion neurons by nucleotides through the inhibition of KV7 channels and the facilitation of TRPV1 channels via a common bifurcated signaling pathway relying on an increase in intracellular Ca2+ and an activation of protein kinase C, respectively.

P2Y1 Receptors Mediate Apamin-Sensitive and -Insensitive Inhibitory Junction Potentials in Murine Colonic Circular Smooth Muscle

Purinergic inhibitory neuromuscular transmission plays an important role in the control of intestinal motility. In most tissues this neurotransmission is apamin-sensitive, but recent studies in human colonic circular smooth muscle (CSM) suggest the presence of apamin-insensitive purinergic inhibitory junction potentials (IJPs). The current studies used conventional intracellular recordings on colonic CSM strips to characterize the purinergic IJPs in murine colonic CSM. P2Y1 receptor expression was examined by using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. The IJP induced by nerve stimulation (NS) of one and four pulses in neuronal nitric-oxide synthase knockout mice consists of an apamin-sensitive and a dominant apamin-resistant component. These are identical to the IJPs in wild-type and CD1 mice in the presence of N(omega)-nitro-l-arginine methyl ester (200 microM) and were significantly inhibited by alpha,beta-methylene ATP (50 microM), an analog of ATP. IJPs were not affected by the P2X receptor antagonist 2',3'-o-(2,4,6-trinitrophenyl)-ATP (10 microM). Furthermore, apamin-resistant IJPs induced by single-pulse NS were abolished by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (100 microM), a P2 receptor antagonist; 2'-deoxy-N6-methyl adenosine 3,5-diphosphate (MRS-2179; 10 microM), a selective P2Y1 receptor antagonist; and tetrodotoxin (1 microM). Aboral NS induced apamin-sensitive purinergic IJPs, whereas oral and circumferential NS produced apamin-sensitive and -resistant IJPs, with the latter predominating. RT-PCR and immunohistochemistry confirmed the presence of P2Y1 receptors on smooth muscle and in the myenteric plexus. These data suggest that, depending on stimulus location, activation of P2Y1 receptors produces both apamin-sensitive and apamin-resistant IJPs in murine colonic CSM.

Low-intensity pulsed ultrasound-dependent osteoblast proliferation occurs by via activation of the P2Y receptor: Role of the P2Y1 receptor

Low-intensity pulsed ultrasound (LIPUS) is commonly used in the treatment of fractures and nonunion-promoting acceleration of healing fractures. In this report, we investigated the implication of the P2 receptors in osteoblast proliferation induced with LIPUS treatment. We observed that ADP, ATP, UTP, and UDP promote osteoblast increase and an increase of intracellular Ca2+, through activation of P2Y receptors. Osteoblasts' expression of the P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, and P2Y13 receptors was confirmed. In addition, the participation of the P2Y1 receptor in osteoblast increase and the ADP-dependent increase of Ca2+ concentration were shown. Furthermore, release of ATP/purines was induced by LIPUS treatment. Finally, LIPUS-dependent osteoblast increase was abolished in the presence of the Ca2+ chelator (BAPTA), the inositol 1,4,5-trisphosphate receptor antagonist (2-APB), and the selective P2Y1 receptor antagonist (MRS2179). In conclusion, LIPUS treatment induces osteoblastogenesis via the release of purines, such as ATP, activating P2Y receptors, mainly the P2Y1 receptor.

Diguanosine pentaphosphate: an endogenous activator of Rho-kinase possibly involved in blood pressure regulation

Rho-kinase activity is increased in cardiovascular disease and in the pathophysiology of hypertension. Few endogenous factors are known that activate the Rho-kinase pathway. Stimulation of P2Y receptors activates the Rho-kinase pathway. Recently identified diguanosine pentaphosphate (Gp5G) possibly activates P2Y receptors. In this study, Gp5G was identified and quantified in human plasma. The influence of Gp5G on vascular tone was studied. Gp5G in human plasma was purified to homogeneity by several steps. Gp5G was quantified and identified by matrix-assisted laser desorption/ionization mass spectrometry and enzymatic analysis. The vasoactive effects of Gp5G were studied in the isolated perfused rat kidney and after intra-aortic application. Activation of Rho-kinase was measured using western blot analysis. The plasma level of Gp5G in healthy donors is 9.47 +/- 4.97 nmol/l. Gp5G increases contractile responses induced by angiotensin II in a dose-dependent way [ED50 (-log mol) angiotensin II: 10.9 +/- 0.1; angiotensin II plus Gp5G (100 nmol/l): 11.5 +/- 0.1]. P2 receptor antagonists inhibited the Gp5G-induced increase in angiotensin II vasoconstriction. MRS2179, a selective P2Y1 receptor antagonist, had no effect on Gp5G-mediated angiotensin II potentiation. Rho-kinase inhibition by Y27632 abolished the Gp5G-induced increase of contractile responses to angiotensin II. Concentrations of 10 nmol/l Gp5G activated the translocation of RhoA from the cytosolic to the membranous fraction indicating the activation of Rho-kinase. The intra-aortic application of 100 pmol Gp5G significantly increased mean arterial blood pressure by 13.5 +/- 4.2 mmHg. Gp5G is an endogenous activator of Rho-kinase, which might affect vascular tone control by Rho-kinase at physiological levels. Gp5G activates P2Y4&6 receptors, and might play a role in physiological and pathophysiological vascular tone control.

Enhanced food intake after stimulation of hypothalamic P2Y1 receptors in rats: modulation of feeding behaviour by extracellular nucleotides

The present study was aimed to clarify the role of purinergic signalling in the regulation of ingestion behaviour. The ATP/ADP analogues 2-methylthioATP (2-MeSATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS) increased the food intake after intracerebroventricular infusion in 18-h food-deprived rats. This effect was abolished by pretreatment with the non-selective P2X/P2Y receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) or the selective P2Y1 receptor antagonist MRS 2179, respectively. The stimulation of food intake mediated by ADPbetaS was also blocked by pretreatment with the nitric oxide synthase (NOS) inhibitor Nw-nitro-L-arginine methylester (L-NAME), as well as with the inhibitor of the soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), suggesting that the orexigenic effect seems to be closely related with the ensuing formation of nitric oxide. The immunohistochemical staining indicating a co-localization of P2Y1 receptor- and nNOS-immunoreactivities in a population of neurons in the ventromedial hypothalamic nucleus (VMH) agrees with this assumption. Further experiments with the direct local application of these compounds into the VMH and lateral hypothalamic nucleus (LH) show that the stimulation of P2Y1 receptors in these functionally antagonistic brain regions exerts an increased food intake. Hence, different signal transduction mechanisms may operate in the VMH and LH. Our assumption is supported by distinct effects of the NOS inhibitor L-NAME in these two hypothalamic nuclei. The present data suggest that ATP/ADP, acting as extracellular signal molecules in the rat brain, are involved in the regulation of food intake, possibly depending on P2Y1-receptor-mediated nitric oxide production.

Agonist concentration-dependent differential responsivity of a human platelet purinergic receptor: pharmacological and kinetic studies of aggregation, deaggregation and shape change responses mediated by the purinergic P2Y1 receptor in vitro

Platelet shape change (SC), aggregation and deaggregation responses are integral components of hemostasis that are elicited and modulated in vivo by the simultaneous activation of several membrane receptors. Selective activation of the purinergic P2Y1 receptor in vitro elicits a sustained SC and a small, transient aggregation response that is reversed rapidly by a robust deaggregation response (Platelets 2003; 14: 89). Using a kinetics-based turbidimetric approach to study the modulation of these concurrent components of human platelet responses, we demonstrate that these P2Y1 receptor-related responses and a number of their kinetic and steady-state characteristics are differentially elicited and modulated. P2Y1 receptor agonist concentrations that elicited aggregation (pEC50 for ADP, 2-MeSADP; 5.88, 6.69) were 10-fold greater than those that elicited SC (7.33, 7.67). The magnitude of the aggregation response was agonist concentration-dependent, saturable and was associated with an agonist concentration-dependent deceleration of the deaggregation response. Gi-coupled receptor (α2A-adrenoceptor, EP3 and P2Y12 receptors) agonists also enhanced aggregation through deceleration of the deaggregation response, and an inhibitor of PI3K activity (wortmannin) inhibited aggregation through acceleration of the deaggregation response. Neither treatment affected the extent or the kinetics of the SC response. The aggregation but not the SC response was rapidly desensitized by P2Y1 receptor activation by ADP. The affinity of the selective P2Y1 receptor antagonist, A3P5P, was independent of the response measured and is consistent with the presence of a single P2Y1 receptor subtype. The differential characteristics and modulation of the SC and aggregation responses by a single receptor support the idea that different signaling pathways activated at different occupancy states of the same receptor underlie the two responses. P2Y1 receptor-mediated platelet aggregation and SC responses provide a convenient model for studying the phenomenon of agonist-directed signaling by differential occupancy of the same membrane receptor.