P2Y Agonist Research Articles

Properties of SK3 channel-expressing PDGFRα (+) cells in the rodent urinary bladder

Localisation of platelet-derived growth factor receptor-α (PDGFRα) (+) cells expressing small-conductance Ca2+-activated K+ (SK3) channels in the urinary bladder was investigated, while putative roles of SK3 (+) PDGFRα (+) cells in suppressing detrusor smooth muscle (DSM) spontaneous activity were explored. In guinea-pig bladder, immunohistochemistry for SK3 channels, PDGFRα or vimentin was examined, as were the effects of purinergic agonists on spontaneous phasic contractions (SPCs). In bladder of PDGFRα-GFP mice, the effects of purinergic agonists on intracellular Ca2+ signaling in PDGFRα (+) cells or DSM cells in situ and SPCs were investigated. SK3 (+) cells co-expressing PDGFRα or vimentin were distributed in DSM bundles but not inter-bundle spaces or lamina propria. SK3 (+) cells had a stellate- or spindle-shape cell body extending processes. MRS2365 (100 nM or 1 μM), a P2Y1 agonist, caused a transient contraction without inhibiting SPCs in both DSM and lamina propria. In PDGFRα-GFP mice bladder, MRS2365, (100 nM), ADP (100 μM) or ATP (100 μM) increased the Ca2+ level of PDGFRα (+) cells without suppressing spontaneous Ca2+ transients in neighboring DSM cells, and also failed to suppress SPCs. Preferential localisation of SK3 positive PDGFRα (+) cells in DSM bundles appears to indicate their functional interaction with DSM cells. However, increases in Ca2+ level of PDGFRα (+) cells upon purinergic stimulation are not associated with the inhibition of Ca2+ or contractile activity in DSM cells. Thus, it is unlikely that the SK3-dependent hyperpolarisation generated in SK3 expressing PDGFRα (+) cells is transmitted to DSMs to suppress their excitability.

Characterisation of P2Y2 receptors in human vascular endothelial cells using AR-C118925XX, a competitive and selective P2Y2 antagonist.

There is a lack of potent, selective antagonists at most subtypes of P2Y receptor. The aims of this study were to characterise the pharmacological properties of the proposed P2Y2 receptor antagonist, AR-C118925XX, and then to use it to determine the role of P2Y2 receptors in the action of the P2Y2 agonist, UTP, in human vascular endothelial cells. Cell lines expressing native or recombinant P2Y receptors were superfused constantly, and agonist-induced changes in intracellular Ca2+ levels monitored using the Ca2+ -sensitive fluorescent indicator, Cal-520. This set-up enabled full agonist concentration-response curves to be constructed on a single population of cells. UTP evoked a concentration-dependent rise in intracellular Ca2+ in 1321N1-hP2Y2 cells. AR-C118925XX (10nM to 1μM) had no effect per se on intracellular Ca2+ but shifted the UTP concentration-response curve progressively rightwards, with no change in maximum. The inhibition was fully reversible on washout. AR-C118925XX (1μM) had no effect at native or recombinant hP2Y1 , hP2Y4 , rP2Y6 , or hP2Y11 receptors. Finally, in EAhy926 immortalised human vascular endothelial cells, AR-C118925XX (30nM) shifted the UTP concentration-response curve rightwards, with no decrease in maximum. AR-C118925XX is a potent, selective and reversible, competitive P2Y2 receptor antagonist, which inhibited responses mediated by endogenous P2Y2 receptors in human vascular endothelial cells. As the only P2Y2 -selective antagonist currently available, it will greatly enhance our ability to identify the functions of native P2Y2 receptors and their contribution to disease and dysfunction.

Purinergic P2Y2 receptors modulate endothelial sprouting.

Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth.

Effects of selective agonists of adenosine, P2X, and P2Y receptors on motility of isolated fallopian tube

Background: During reproductive life of women, adenosine causes both contraction (with low concentrations) of fallopian tubes and inhibition of their spontaneous motor activity (with high concentrations). Objective: The aim of this study was to investigate effects of natural agonists of adenosine, P2X and P2Y receptors on motility of isolated fallopian tubes taken from postmenopausal women. Materials and Methods: Isolated preparations of isthmus and ampoule were made from fallopian tubes of 21 women in post-menopause, and then tested for reactivity on increasing concentrations of adenosine and P2X/P2Y selective agonists. Results: Adenosine showed concentration-dependent inhibitory effect on spontaneous contraction of both isthmic and ampullary segments of fallopian tubes, while P2X and P2Y agonists (adenosine-5-diphosphate, adenosine-5-triphosphate, uridine-5-diphosphate, and uridine-5-triphosphate) did not influence motility of the isolated preparations. Contractile effect of adenosine was not observed throughout the concentration range used in the experiments. Conclusions: Fallopian tubes of postmenopausal women are unresponsive to P2X and P2Y agonists, unlike those of women in reproductive period. Only an inhibitory effect of adenosine on spontaneous contractions of fallopian tubes is maintained in post-menopause, while a contractile effect is observed in younger women at low concentrations is lost.

Inorganic Polyphosphate Regulates AMPA and NMDA Receptors and Protects Against Glutamate Excitotoxicity via Activation of P2Y Receptors

Glutamate is one of the most important neurotransmitters in the process of signal transduction in the CNS. Excessive amounts of this neurotransmitter lead to glutamate excitotoxicity, which is accountable for neuronal death in acute neurological disorders, including stroke and trauma, and in neurodegenerative diseases. Inorganic polyphosphate (PolyP) plays multiple roles in the mammalian brain, including function as a calcium-dependent gliotransmitter mediating communication between astrocytes, while its role in the regulation of neuronal activity is unknown. Here we studied the effect of PolyP on glutamate-induced calcium signal in primary rat neurons in both physiological and pathological conditions. We found that preincubation of primary neurons with PolyP reduced glutamate-induced and AMPA-induced but not the NMDA-induced calcium signal. However, in rat hippocampal acute slices, PolyP reduced ion flux through NMDA and AMPA receptors in native neurons. The effect of PolyP on glutamate and specifically on the AMPA receptors was dependent on the presence of P2Y1 but not of P2X receptor inhibitors and also could be mimicked by P2Y1 agonist 2MeSADP. Preincubation of cortical neurons with PolyP significantly reduced the initial calcium peak as well as the number of neurons with delayed calcium deregulation in response to high concentrations of glutamate and resulted in protection of neurons against glutamate-induced cell death. As a result, activation of P2Y1 receptors by PolyP reduced calcium signal acting through AMPA receptors, thus protecting neurons against glutamate excitotoxicity by reduction of the calcium overload and restoration of mitochondrial function.SIGNIFICANCE STATEMENT One of the oldest polymers in the evolution of living matter is the inorganic polyphosphate (PolyP). It is shown to play a role of gliotransmitter in the brain; however, the role of polyphosphate in neuronal signaling is not clear. Here we demonstrate that inorganic polyphosphate is able to reduce calcium signaling induced by physiological or high concentrations of glutamate. The effect of polyphosphate on glutamate-induced calcium signal in neurons is due to the effect of this polymer on the AMPA receptors. The effect of PolyP on glutamate-induced and AMPA-induced calcium signal is dependent on P2Y receptor antagonist. The ability of PolyP to restrict the glutamate-induced calcium signal lies in the basis of its protection of neurons against glutamate excitotoxicity.

Context-Specific Switch from Anti- to Pro-epileptogenic Function of the P2Y1 Receptor in Experimental Epilepsy.

Extracellular ATP activates inflammatory responses to tissue injury. It is also implicated in establishing lasting network hyperexcitability in the brain by acting upon independent receptor systems. Whereas the fast-acting P2X channels have well-established roles driving neuroinflammation and increasing hyperexcitability, the slower-acting metabotropic P2Y receptors have received much less attention. Recent studies of P2Y1 receptor function in seizures and epilepsy have produced contradictory results, suggesting that the role of this receptor during seizure pathology may be highly sensitive to context. Here, by using male mice, we demonstrate that the metabotropic P2Y1 receptor mediates either proconvulsive or anticonvulsive responses, dependent on the time point of activation in relation to the induction of status epilepticus. P2Y1 deficiency or a P2Y1 antagonist (MRS2500) administered before a chemoconvulsant, exacerbates epileptiform activity, whereas a P2Y1 agonist (MRS2365) administered at this time point is anticonvulsant. When these drugs are administered after the onset of status epilepticus, however, their effect on seizure severity is reversed, with the antagonist now anticonvulsant and the agonist proconvulsant. This result was consistent across two different mouse models of status epilepticus (intra-amygdala kainic acid and intraperitoneal pilocarpine). Pharmacologic P2Y1 blockade during status epilepticus reduces also associated brain damage, delays the development of epilepsy and, when applied during epilepsy, suppresses spontaneous seizures, in mice. Our data show a context-specific role for P2Y1 during seizure pathology and demonstrate that blocking P2Y1 after status epilepticus and during epilepsy has potent anticonvulsive effects, suggesting that P2Y1 may be a novel candidate for the treatment of drug-refractory status epilepticus and epilepsy.SIGNIFICANCE STATEMENT This is the first study to fully characterize the contribution of a metabotropic purinergic P2Y receptor during acute seizures and epilepsy. The findings suggest that targeting P2Y1 may offer a potential novel treatment strategy for drug-refractory status epilepticus and epilepsy. Our data demonstrate a context-specific role of P2Y1 activation during seizures, switching from a proconvulsive to an anticonvulsive role depending on physiopathological context. Thus, our study provides a possible explanation for seemingly conflicting results obtained between studies of different brain diseases where P2Y1 targeting has been proposed as a potential treatment strategy and highlights that the timing of pharmacological interventions is of critical importance to the understanding of how receptors contribute to the generation of seizures and the development of epilepsy.

Alteration of Calcium and Electrical Dynamics in Cerebrovascular Endothelium During Development of Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disease and the most common cause of dementia characterized by a decline in cognitive function among the elderly. Currently, ~5.7 million Americans are living with AD and this number will increase to ~14 million by 2050. Age‐related AD alters blood flow to the brain and is associated with cerebral hypoperfusion, due in part, to impaired vascular endothelial function. However, it is unknown whether AD pathology impacts the biophysical functions of key cerebral vascular G‐protein‐coupled receptors (GPCRs) and K+ channels (KCa2.3 or SKCa, KCa3.1 or IKCa) that govern blood flow via an electrical vasodilatory pathway known as endothelium‐derived hyperpolarization (EDH). Thus, we tested the hypothesis that cerebrovascular endothelial purinergic receptors and Ca2+‐activated K+ channels become functionally dysregulated during progression of AD. We used the triple mutation mouse model of AD (3xTgAD) to examine endothelium isolated from posterior cerebral arteries in young control (YC; 1–2 mo), amyloid‐β plaques (Aβ; 6–8 mo), and plaques + neurofibrillary tangles (Aβ+Tau; ≥ 12 mo); n ≥ 3 male & n ≥ 3 female mice/group. Intracellular calcium concentration ([Ca2+]i) and membrane potential (Vm) were simultaneously measured using Fura‐2 photometry and sharp electrodes (pH 7.4, 37°C). EDH was demonstrated by an increase in [Ca2+]i (ΔF340/F380 ≥ 0.3) concomitant with ΔVm ≥ −10 mV in response to the P2Y agonist ATP (100 μM, 3 min). Membrane hyperpolarization to ATP during Aβ+Tau vs. YC decreased (ΔVm, mV; YC: −20±4, Aβ: −17±2, Aβ+Tau: −13±3) while [Ca2+]i responses were maintained. In contrast, hyperpolarization increased during Aβ and Aβ+Tau (ΔVm, mV; YC: −29±3, Aβ: −36±2, Aβ+Tau: −38±3) during direct activation of SKCa/IKCa alone with SKA‐31 (10 μM, 5 min). Finally, Δ[Ca2+]i and ΔVm responses to hydrogen peroxide (H2O2; 200 μM, 20 min) in young control animals were greater by > 40% as compared to Aβ & Aβ+Tau. These data suggest coupling of endothelial SKCa/IKCa function with Ca2+ signaling is reduced and adaptation to oxidative stress occurs during the development of AD. Altogether, endothelial K+ channel function may be directly calibrated for optimal cerebral blood flow to maintain a healthy brain with aging while helping to prevent neurodegenerative disease.Support or Funding InformationThis research is supported by Loma Linda University School of Medicine new faculty start‐up funds and National Institutes of Health grants R00AG047198 & R56AG062169 (EJB).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Chronic fructose renders pancreatic β-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 β-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 β-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 β-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 β-cells. Addition of an ectonucleotidase inhibitor or P2Y1 agonists to naïve β-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 β-cells mediating fructose-induced hyper-responsiveness.

A Gs-coupled purinergic receptor boosts Ca2+ influx and vascular contractility during diabetic hyperglycemia.

Elevated glucose increases vascular reactivity by promoting L-type CaV1.2 channel (LTCC) activity by protein kinase A (PKA). Yet, how glucose activates PKA is unknown. We hypothesized that a Gs-coupled P2Y receptor is an upstream activator of PKA mediating LTCC potentiation during diabetic hyperglycemia. Experiments in apyrase-treated cells suggested involvement of a P2Y receptor underlying the glucose effects on LTTCs. Using human tissue, expression for P2Y11, the only Gs-coupled P2Y receptor, was detected in nanometer proximity to CaV1.2 and PKA. FRET-based experiments revealed that the selective P2Y11 agonist NF546 and elevated glucose stimulate cAMP production resulting in enhanced PKA-dependent LTCC activity. These changes were blocked by the selective P2Y11 inhibitor NF340. Comparable results were observed in mouse tissue, suggesting that a P2Y11-like receptor is mediating the glucose response in these cells. These findings established a key role for P2Y11 in regulating PKA-dependent LTCC function and vascular reactivity during diabetic hyperglycemia.

Identification of P2Y receptors involved in oleamide-suppressing inflammatory responses in murine microglia and human dendritic cells

Microglia, a type of immune cell in the central nervous system, are involved in inflammation leading to neurodegenerative diseases. We previously identified oleamide from fermented dairy products as a neuroprotective compound suppressing microglial inflammation. Oleamide is an endocannabinoid and displays anti-inflammatory activity via the cannabinoid-2 (CB2) receptor; however, the mechanism underlying this anti-inflammatory activity has not been fully elucidated. Here, we found that the suppressive effect of oleamide on microglial tumor necrosis factor-α (TNF-α) production was canceled by inhibitors of G-protein-coupled receptor (GPCR) downstream signaling but not by a CB2 antagonist, suggesting that GPCRs other than CB2 are involved in the anti-inflammatory effects of oleamide. An extensive screen for GPCRs using a transforming growth factor-α shedding assay system identified P2Y1, P2Y4, P2Y6, P2Y10, and P2Y11 as candidates for the oleamide target. P2Y1 and P2Y10 agonists suppressed microglial TNF-α production, while a pan P2 receptor antagonist canceled the suppressive effect. Furthermore, we observed a relationship between the P2Y1 agonistic activities and the suppressive activities of oleamide and its analogs. Taken together, our results suggest that, in addition to CB2, P2Y type receptors are the potential targets of oleamide, and P2Y1 plays a role in the suppression of microglial inflammatory responses by oleamide. (200/200 words)

Dystrophic mdx mouse myoblasts exhibit elevated ATP/UTP-evoked metabotropic purinergic responses and alterations in calcium signalling

Pathophysiology of Duchenne Muscular Dystrophy (DMD) is still elusive. Although progressive wasting of muscle fibres is a cause of muscle deterioration, there is a growing body of evidence that the triggering effects of DMD mutation are present at the earlier stage of muscle development and affect myogenic cells. Among these abnormalities, elevated activity of P2X7 receptors and increased store-operated calcium entry myoblasts have been identified in mdx mouse. Here, the metabotropic extracellular ATP/UTP-evoked response has been investigated. Sensitivity to antagonist, effect of gene silencing and cellular localization studies linked these elevated purinergic responses to the increased expression of P2Y2 but not P2Y4 receptors. These alterations have physiological implications as shown by reduced motility of mdx myoblasts upon treatment with P2Y2 agonist. However, the ultimate increase in intracellular calcium in dystrophic cells reflected complex alterations of calcium homeostasis identified in the RNA seq data and with significant modulation confirmed at the protein level, including a decrease of Gq11 subunit α, plasma membrane calcium ATP-ase, inositol-2,4,5-trisphosphate-receptor proteins and elevation of phospholipase Cβ, sarco-endoplamatic reticulum calcium ATP-ase and sodium‑calcium exchanger. In conclusion, whereas specificity of dystrophic myoblast excitation by extracellular nucleotides is determined by particular receptor overexpression, the intensity of such altered response depends on relative activities of downstream calcium regulators that are also affected by Dmd mutations. Furthermore, these phenotypic effects of DMD emerge as early as in undifferentiated muscle. Therefore, the pathogenesis of DMD and the relevance of current therapeutic approaches may need re-evaluation.

Stimulation of murine P2Y11-like purinoreceptor protects against hypoxia/reoxygenation injury and decreases heart graft rejection lesions

ObjectiveMyocardial ischemia reperfusion is a major cause of cell injury during cardiac transplantation and is responsible for increased graft rejection. Several in vitro studies demonstrated the protective effect of P2Y11-like purinoreceptor stimulation in the context of myocardial ischemia/reperfusion. In this study, we hypothesized a possible cardioprotective role of P2Y11R stimulation against ischemia/reperfusion lesions and validated its clinical effect in vivo in a heart transplantation model. MethodsWe subjected H9c2 rat cardiomyocyte-derived cell line to 5 hours of hypoxia and 1 hour of reoxygenation. P2Y11R selective agonist NF546 and antagonist NF340 were added at the onset of reoxygenation. Cell injuries were assessed by microculture tetrazolium reduction and intracellular adenosine triphosphate level. Clinical effect of P2Y11R stimulation was further investigated in vivo. Hearts from BALB/c mice were transplanted intra-abdominally into allogenic C57BL/6 mice (n = 104). Recipient mice were injected with P2Y11R agonist. Mice in the sham group were injected with saline solution. In the control group, hearts from C57BL/6 were transplanted into syngeneic C57BL/6 mice. Rejection lesions were investigated using histology and immunohistochemistry at days 3, 5, and 7 after transplantation. We measured caspase activities to quantify apoptosis. Production of proinflammatory and anti-inflammatory cytokines was investigated. ResultsP2Y11R stimulation at the onset of reoxygenation significantly reduced in vitro hypoxia/reoxygenation injuries. This protection was suppressed with P2Y11R antagonist. In vivo, cardiac allograft survival was significantly prolonged after P2Y11R stimulation. Rejection lesions, classified according to the International Society of Heart Lung Transplantation guidelines and quantified using the mean number of inflammatory cells per field, were significantly reduced in the treated group. At day 5 after transplantation, P2Y11R agonist pretreated allografts also demonstrated less apoptotic lesions. ConclusionsOur data suggest a novel cardioprotective role of P2Y11R at the onset of reoxygenation/reperfusion against reperfusion injuries. Pharmacologic conditioning using P2Y11 agonist may be beneficial after cardiac transplantation in improving myocardial ischemia/reperfusion outcomes and decreasing graft rejection lesions.

Inhibitory Neural Regulation of the Ca 2+ Transients in Intramuscular Interstitial Cells of Cajal in the Small Intestine.

Gastrointestinal motility is coordinated by enteric neurons. Both inhibitory and excitatory motor neurons innervate the syncytium consisting of smooth muscle cells (SMCs) interstitial cells of Cajal (ICC) and PDGFRα+ cells (SIP syncytium). Confocal imaging of mouse small intestines from animals expressing GCaMP3 in ICC were used to investigate inhibitory neural regulation of ICC in the deep muscular plexus (ICC-DMP). We hypothesized that Ca2+ signaling in ICC-DMP can be modulated by inhibitory enteric neural input. ICC-DMP lie in close proximity to the varicosities of motor neurons and generate ongoing Ca2+ transients that underlie activation of Ca2+-dependent Cl− channels and regulate the excitability of SMCs in the SIP syncytium. Electrical field stimulation (EFS) caused inhibition of Ca2+ for the first 2–3 s of stimulation, and then Ca2+ transients escaped from inhibition. The NO donor (DEA-NONOate) inhibited Ca2+ transients and Nω-Nitro-L-arginine (L-NNA) or a guanylate cyclase inhibitor (ODQ) blocked inhibition induced by EFS. Purinergic neurotransmission did not affect Ca2+ transients in ICC-DMP. Purinergic neurotransmission elicits hyperpolarization of the SIP syncytium by activation of K+ channels in PDGFRα+ cells. Generalized hyperpolarization of SIP cells by pinacidil (KATP agonist) or MRS2365 (P2Y1 agonist) also had no effect on Ca2+ transients in ICC-DMP. Peptidergic transmitter receptors (VIP and PACAP) are expressed in ICC and can modulate ICC-DMP Ca2+ transients. In summary Ca2+ transients in ICC-DMP are blocked by enteric inhibitory neurotransmission. ICC-DMP lack a voltage-dependent mechanism for regulating Ca2+ release, and this protects Ca2+ handling in ICC-DMP from membrane potential changes in other SIP cells.

Diabetes-induced colonic slow transit mediated by the up-regulation of PDGFRα+ cells/SK3 in streptozotocin-induced diabetic mice.

A major complication related to gastrointestinal (GI) symptoms in diabetic patients is chronic constipation. Constipation has serious negative impacts on quality of life; however, without a comprehensive understanding of the disease, currently available treatments cannot provide a cure. Platelet-derived growth factor receptor alpha-positive cells (PDGFRα+ cells), which form the SIP syncytium with interstitial cells of Cajal and smooth muscle cells, play important roles in GI motility. In the present study, the contributions of PDGFRα+ cells to diabetes-induced colonic slow transit were investigated in streptozotocin (STZ)-induced diabetic mice. Western blotting, quantitative PCR, contractile experiments, and intracellular recording were used in the present study. The results demonstrated that the colon length was increased in STZ-treated mice. The colonic transit of artificial fecal pellets in vitro was significantly delayed in STZ-treated mice. The mRNA and protein expression of PDGFRα, small-conductance Ca2+ -activated K channels (SK3), and P2Y1 receptors were increased in the colons of STZ-treated mice. In contractile experiments, the colonic smooth muscles were more sensitive to the SK3 agonist and antagonist (CyPPA and apamin) and the P2Y1 agonist and antagonist (MRS2365 and MRS2500) in STZ-treated mice. Intracellular recordings showed the responses of membrane potentials in colonic smooth muscle cells to CyPPA, apamin, MRS2365, and MRS2500 were more sensitive in STZ-treated mice. The electric field stimulation-induced P2Y1/SK3-dependent fast inhibitory junctional potentials (fIJPs) of colonic smooth muscles were more significantly hyperpolarized in STZ-treated mice. These results suggest that the purinergic neurotransmitters/P2Y1/SK3 signaling pathway is up-regulated in the diabetic colons, thereby mediating diabetes-induced colonic slow transit.

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.

Coupling of P2Y receptors to Ca2+ mobilization in mesenchymal stromal cells from the human adipose tissue

The purinergic transduction was examined in mesenchymal stromal cells (MSCs) from the human adipose tissue, and several nucleotides, including ATP, UTP, and ADP, were found to mobilize cytosolic Ca2+. Transcripts for multiple purinoreceptors were detected in MSC preparations, including A1, A2A, A2B, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y13, P2Y14, P2X2, P2X4, and P2X7. Cellular responses to nucleotides were insignificantly sensitive to bath Ca2+, pointing at a minor contribution of Ca2+ entry, and were suppressed by U73122 and 2-APB, implicating the phosphoinositide cascade in coupling P2Y receptors to Ca2+ release. While individual cells were sensitive to several P2Y agonists, responsiveness to a given nucleotide varied from cell to cell, suggesting that particular MSCs could employ different sets of purinoreceptors. Caged Ca2+ stimulated Ca2+-induced Ca2+ release (CICR) that was mediated largely by IP3 receptors, and resultant Ca2+ transients were similar to nucleotide responses by magnitude and kinetics. A variety of findings hinted at CICR to be a universal mechanism that finalizes Ca2+ signaling initiated by agonists in MSCs. Individual MSCs responded to nucleotides in an all-or-nothing manner. Presumably just CICR provided invariant Ca2+ responses observed in MSCs at different nucleotide concentrations. The effects of isoform specific agonists and antagonists suggested that both P2Y1 and P2Y13 were obligatory for ADP responses, while P2Y4 and P2Y11 served as primary UTP and ATP receptors, respectively. Extracellular NAD+ stimulated Ca2+ signaling in each ATP-responsive MSC by involving P2Y11. The overall data indicate that extracellular nucleotides and NAD+ can serve as autocrine/paracrine factors regulating MSC functions.

Expression and function of the metabotropic purinergic P2Y receptor family in experimental seizure models and patients with drug-refractory epilepsy.

ATP is released into the extracellular space during pathologic processes including increased neuronal firing. Once released, ATP acts on P2 receptors including ionotropic P2X and metabotropic P2Y receptors, resulting in changes to glial function and neuronal network excitability. Evidence suggests an involvement of P2Y receptors in the pathogenesis of epilepsy, but there has been no systematic effort to characterize the expression and function of the P2Y receptor family during seizures and in experimental and human epilepsy. Status epilepticus was induced using either intra-amygdala kainic acid or pilocarpine to characterize the acute- and long-term changes in hippocampal P2Y expression. P2Y expression was also investigated in brain tissue from patients with temporal lobe epilepsy. Finally, we analyzed the effects of two specific P2Y agonists, ADP and UTP, on seizure severity and seizure-induced cell death. Both intra-amygdala kainic acid and pilocarpine-induced status epilepticus increased the transcription of the uracil-sensitive P2Y receptors P2ry2 , P2ry4 , and P2ry6 and decreased the transcription of the adenine-sensitive P2Y receptors P2ry1 , P2ry12 , P2ry13 . Protein levels of P2Y1 , P2Y2 , P2Y4 , and P2Y6 were increased after status epilepticus, whereas P2Y12 expression was decreased. In the chronic phase, P2ry1 , P2ry2 , and P2ry6 transcription and P2Y1 , P2Y2 , and P2Y12 protein levels were increased with no changes for the other P2Y receptors. In hippocampal samples from patients with temporal lobe epilepsy, P2Y1 and P2Y2 protein expression was increased, whereas P2Y13 levels were lower. Demonstrating a functional contribution of P2Y receptors to seizures, central injection of ADP exacerbated seizure severity, whereas treatment with UTP decreased seizure severity during status epilepticus in mice. The present study is the first to establish the specific hippocampal expression profile and function of the P2Y receptor family after experimental status epilepticus and in human temporal lobe epilepsy and offers potential new targets for seizure control and disease modification.

Effects of 4(1H)-quinolinone derivative, a novel non-nucleotide allosteric purinergic P2Y2 agonist, on cardiomyocytes in neonatal rats

Purinergic P2Y2 receptors, G-protein coupled receptors that primarily couple with Gαq/11-proteins, are activated equipotently by adenosine-5′-triphosphate (ATP) and uridine-5′-triphosphate. Evidence suggests that P2Y2 agonists make potential drug candidates for the treatment of cardiovascular diseases. However, selective non-nucleotide, small-molecule P2Y2 agonists have yet to be developed. In this report, we discuss Compound 89, a novel non-nucleotide allosteric P2Y2 agonist that was active in signal transduction and gene induction, and in our in vitro cardiac hypertrophy model. Compound 89 exhibited selective P2Y2 agonistic activity and potentiated responses to the endogenous agonist ATP, while exhibiting no agonistic activities for four other Gαq/11-coupled human P2Y (hP2Y) receptors and one representative Gαi/o-coupled hP2Y12 receptor. Its P2Y2 agonistic effect on mouse P2Y2 receptors suggested non-species-specific activity. Compound 89 acted as a pure positive allosteric modulator in a Ca2+ mobilization assay of neonatal rat cardiomyocytes; it potentiated ATP-induced expression of genes in the nuclear receptor 4A family (negative regulators of hypertrophic stimuli in cardiomyocytes). Additionally, Compound 89 attenuated isoproterenol-induced cardiac hypertrophy, presumably through dose-dependent interaction with pericellular ATP. These results indicate that Compound 89 is potentially efficacious against cardiomyocytes and therefore a good proof-of-concept tool for elucidating the therapeutic potential of P2Y2 activation in various cardiovascular diseases.

Lipopolysaccharide (LPS) induced pulmonary neutrophil recruitment and platelet activation is mediated via the P2Y1 and P2Y14 receptors in mice

Platelet activation occurs during host defence and in various inflammatory disorders. In animal models of infection and inflammation, experimental depletion of platelets leads to significantly reduced leukocyte recruitment and impaired clearance of pathogens from the lung. It is now appreciated that purinergic receptor activation is required for leukocyte activation, motility and adhesion, and platelet interactions with leukocytes can be modulated by purinergic stimulation of platelets. Here, we have investigated the role of platelet P2Y1, P2Y12, P2Y14, and P2X1 receptors on leukocyte recruitment and chemotaxis.Mice were administered either vehicle controls or selective P2Y1, P2Y12, P2Y14, or P2X1 antagonists intravenously before intranasal administration of lipopolysaccharide (LPS) to investigate the effect of these drugs on pulmonary leukocyte recruitment, peripheral platelet counts, bleeding times, and ex vivo platelet aggregation. Separately, platelets were incubated with P2Y1, P2Y12, P2X1 antagonists, or P2Y14 agonists to assess effects on platelet-induced neutrophil chemotaxis in vitro.Pulmonary neutrophil recruitment induced by intranasal LPS administration was inhibited in mice administered either with P2Y1 or P2Y14 antagonists, but not with P2Y12 or P2X1 antagonists. Furthermore, the administration of either a P2Y1 or a P2Y14 antagonist reversed the incidence of peripheral thrombocytopaenia associated with LPS exposure. Bleeding times were significantly increased in mice administered P2Y1, P2Y12, or P2X1 antagonists, whilst ex vivo platelet aggregation to ADP was significantly reduced. These haemostatic responses remained unaltered following antagonism of P2Y14. In vitro chemotaxis assays revealed direct antagonism of platelet P2Y1, but not P2Y12 or P2X1 receptors suppressed platelet-dependent neutrophil motility towards Macrophage derived chemokine (MDC, CCL22). Furthermore, the stimulation of platelets with selective P2Y14 agonists (UDP-glucose, MRS2690) resulted in significant platelet-dependent neutrophil chemotaxis.These results reveal a role for P2Y1 and P2Y14 activation of platelets following exposure to LPS, whilst haemostatic indices were unaffected by inhibition of platelet function with the P2Y14 antagonist in response to LPS.

BPTU, an allosteric antagonist of P2Y1 receptor, blocks nerve mediated inhibitory neuromuscular responses in the gastrointestinal tract of rodents

P2Y1 receptors mediate nerve mediated purinergic inhibitory junction potentials (IJP) and relaxations in the gastrointestinal (GI) tract in a wide range of species including rodents and humans. A new P2Y1 antagonist, with a non-nucleotide structure, BPTU, has recently been described using X-ray crystallography as the first allosteric G-protein-coupled receptor antagonist located entirely outside of the helical bundle. In this study, we tested its effect on purinergic responses in the gastrointestinal tract of rodents using electrophysiological and myographic techniques. BPTU concentration dependently inhibited purinergic inhibitory junction potentials and inhibition of spontaneous motility induced by electrical field stimulation in the colon of rats (EC50 = 0.3 μM) and mice (EC50 = 0.06 μM). Mechanical inhibitory responses were also concentration-dependently blocked in the stomach of both species. Compared to MRS2500, BPTU displays a lower potency. In the rat colon nicotine induced relaxation was also blocked by BPTU. BPTU also blocked the cessation of spontaneous contractility elicited by ADPβS and the P2Y1 agonist MRS2365. We conclude that BPTU is a novel antagonist with different structural and functional properties than nucleotidic antagonists that is able to block the P2Y1 receptor located at the neuromuscular junction of the GI tract.