Extracellular ATP and cAMP signaling promote Piezo2-dependent mechanical allodynia after trigeminal nerve compression injury.

Trigeminal neuralgia (TN) is a type of severe paroxysmal neuropathic pain commonly triggered by mild mechanical stimulation in the orofacial area. Piezo2, a mechanically gated ion channel that mediates tactile allodynia in neuropathic pain, can be potentiated by a cyclic adenosine monophosphate (cAMP)-dependent signaling pathway that involves the exchange protein directly activated by cAMP 1 (Epac1). To study whether Piezo2-mediated mechanotransduction contributes to peripheral sensitization in a rat model of TN after trigeminal nerve compression injury, the expression of Piezo2 and activation of cAMP signal-related molecules in the trigeminal ganglion (TG) were detected. Changes in purinergic P2 receptors in the TG were also studied by RNA-seq. The expression of Piezo2, cAMP, and Epac1 in the TG of the TN animals increased after chronic compression of the trigeminal nerve root (CCT) for 21 days, but Piezo2 knockdown by shRNA in the TG attenuated orofacial mechanical allodynia. Purinergic P2 receptors P2X4, P2X7, P2Y1, and P2Y2 were significantly up-regulated after CCT injury. In vitro, Piezo2 expression in TG neurons was significantly increased by exogenous adenosine 5'-triphosphate (ATP) and Ca2+ ionophore ionomycin. ATP pre-treated TG neurons displayed elevated [Ca2+ ]i and faster increase in responding to blockage of Na+ /Ca2+ exchanger by KB-R7943. Furthermore, mechanical stimulation of cultured TG neurons led to sustained elevation in [Ca2+ ]i in ATP pre-treated TG neurons, which is much less in naïve TG neurons, or is significantly reduced by Piezo2 inhibitor GsMTx4. These results indicated a pivotal role of Piezo2 in peripheral mechanical allodynia in the rat CCT model. Extracellular ATP, Ca2+ influx, and the cAMP-to-Epac1 signaling pathway synergistically contribute to the pathogenesis and the persistence of mechanical allodynia.

Redox regulation of hemodynamics response to diadenosine tetraphosphate an agonist of P2 receptors and renal function in diet-induced hypercholesterolemic rats.

Hypercholesterolemia and oxidative stress may lead to disturbances in the renal microvasculature in response to vasoactive agents, including P2 receptors (P2R) agonists. We investigated the renal microvascular response to diadenosine tetraphosphate (Ap4 A), an agonist of P2R, in diet-induced hypercholesteremic rats over 28 days, supplemented in the last 10 days with tempol (2 mM) or DL-buthionine-(S,R)-sulfoximine (BSO, 20 mM) in the drinking water. Using laser Doppler flowmetry, renal blood perfusion in the cortex and medulla (CBP, MBP) was measured during the infusion of Ap4 A. This induced a biphasic response in the CBP: a phase of rapid decrease was followed by one of rapid increase extended for 30 min in both the normocholesterolemic and hypercholesterolemic rats. The phase of decreased CBP was not affected by tempol or BSO in either group. Early and extended increases in CBP were prevented by tempol in the hypercholesterolemia rats, while, in the normocholesterolemic rats, only the extended increase in CBP was affected by tempol; BSO prevented extended increase in CBP in normocholesterolemic rats. MBP response is not affected by hypercholesterolemia. The hypercholesterolemic rats were characterized by increased urinary albumin and 8-isoPGF2α excretion. Moreover, BSO increased the urinary excretion of nephrin in the hypercholesterolemic rats but, similar to tempol, did not affect the excretion of albumin in their urine. The results suggest the important role of redox balance in the extracellular nucleotide regulation of the renal vasculature and glomerular injury in hypercholesterolemia.

Targeting P2 receptors in purinergic signaling: a new strategy of active ingredients in traditional Chinese herbals for diseases treatment.

Adenosine triphosphate (ATP) and its metabolites adenosine diphosphate, adenosine monophosphate, and adenosine in purinergic signaling pathway play important roles in many diseases. Activation of P2 receptors (P2R) channels and subsequent membrane depolarization can induce accumulation of extracellular ATP, and furtherly cause kinds of diseases, such as pain- and immune-related diseases, cardiac dysfunction, and tumorigenesis. Active ingredients of traditional Chinese herbals which exhibit superior pharmacological activities on diversified P2R channels have been considered as an alternative strategy of disease treatment. Experimental evidence of potential ingredients in Chinese herbs targeting P2R and their pharmacological activities were outlined in the study.

P2Y14 Receptor as a Target for Neutrophilia Attenuation in Severe COVID-19 Cases: From Hematopoietic Stem Cell Recruitment and Chemotaxis to Thrombo-inflammation.

The global SARS-CoV-2 pandemic starting in 2019 has already reached more than 2.3 million deaths. Despite the scientific community's efforts to investigate the COVID-19 disease, a drug for effectively treating or curing patients yet needs to be discovered. Hematopoietic stem cells (HSC) differentiating into immune cells for defense express COVID-19 entry receptors, and COVID-19 infection hinders their differentiation. The importance of purinergic signaling in HSC differentiation and innate immunity has been recognized. The metabotropic P2Y14 receptor subtype, activated by UDP-glucose, controls HSC differentiation and mobilization. Thereon, the exacerbated activation of blood immune cells amplifies the inflammatory state observed in COVID-19 patients, specially through the continuous release of reactive oxygen species and extracellular neutrophil traps (NETs). Further, the P2Y14 subtype, robustly inhibits the infiltration of neutrophils into various epithelial tissues, including lungs and kidneys. Here we discuss findings suggesting that antagonism of the P2Y14 receptor could prevent the progression of COVID-19-induced systemic inflammation, which often leads to severe illness and death cases. Considering the modulation of neutrophil recruitment of extreme relevance for respiratory distress and lung failure prevention, we propose that P2Y14 receptor inhibition by its selective antagonist PPTN could limit neutrophil recruitment and NETosis, hence limiting excessive formation of oxygen reactive species and proteolytic activation of the kallikrein-kinin system and subsequent bradykinin storm in the alveolar septa of COVID-19 patients.

Targeting the purinergic pathway in breast cancer and its therapeutic applications.

Breast cancer (BC) is the most frequent cause of death among women, representing a global public health problem. Here, we aimed to discuss the correlation between the purinergic system and BC, recognizing therapeutic targets. For this, we analyzed the interaction of extracellular nucleotides and nucleosides with the purinergic receptors P1 and P2, as well as the influence of ectonucleotidase enzymes (CD39 and CD73) on tumor progression. A comprehensive bibliographic search was carried out. The relevant articles for this review were found in the PubMed, Scielo, Lilacs, and ScienceDirect databases. It was observed that among the P1 receptors, the A1, A2A, and A2B receptors are involved in the proliferation and invasion of BC, while the A3 receptor is related to the inhibition of tumor growth. Among the P2 receptors, the P2X7 has a dual function. When activated for a short time, it promotes metastasis, but when activated for long periods, it is related to BC cell death. P2Y2 and P2Y6 receptors are related to BC proliferation and invasiveness. Also, the high expression of CD39 and CD73 in BC is strongly related to a worse prognosis. The receptors and ectonucleotidases involved with BC become possible therapeutic targets. Several purinergic pathways have been found to be involved in BC cell survival and progression. In this review, in addition to analyzing the pathways involved, we reviewed the therapeutic interventions already studied for BC related to the purinergic system, as well as to other possible therapeutic targets.

Rhizoma Bletillae polysaccharide elicits hemostatic effects in platelet-rich plasma by activating adenosine diphosphate receptor signaling pathway.

Rhizoma Bletillae, the tubes of Bletilla striata, has been traditionally used in China as a hemostatic agent. In preliminary studies, the major active fraction responsible for its hemostatic effect have been confirmed to be Rhizoma Bletillae polysaccharide (RBp), but the hemostatic mechanism of action of RBp is still unknown.The main aim of this study was to clarify its mechanism of hemostatic effect. RBp was prepared by 80 % ethanol precipitation of the water extract of Rhizoma Bletillae followed by the Sevag method to remove proteins. The average molecular weight (Mw) of the crude RBp maintained at a range of 30.06-200 KDa. The hemostatic effects of RBp were evaluated by testing its effect on the platelet aggregation of rat platelet-rich plasma (PRP). PRP was dealt with different concentrations of RBp and platelet aggregation was measured by the turbidimetric method. The hemostatic mechanism of RBp was investigated by examining its effect on platelet shape, platelet secretion, and activation of related receptors (P2Y1, P2Y12 and TXA2) by electron microscopy and the turbidimetric method. RBp significantly enhanced the platelet aggregations at concentrations of 50-200 mg/L in a concentration-dependent manner. The inhibitory rate of platelet aggregation was significantly increased by apyrase and Ro31-8220 in a concentration-dependent manner, while RBp-induced platelet aggregation was completely inhibited by P2Y1, P2Y12 and the PKC receptor antagonists. However, the aggregation was not sensitive to TXA2. RBp, the active ingredients of Rhizoma Bletillae responsible for its hemostatic effect, could significantly accelerate the platelet aggregation and shape change. The hemostatic mechanism may involve activation of the P2Y1, P2Y12, and PKC receptors in the adenosine diphosphate (ADP) receptor signaling pathway.

Transient Receptor Potential Vanilloid 4 Regulation of Adenosine Triphosphate Release by the Adenosine Triphosphate Transporter Vesicular Nucleotide Transporter, a Novel Therapeutic Target for Gastrointestinal Baroreception and Chronic Inflammation.

BACKGROUND: Transient receptor potential vanilloid 4 (TRPV4) is activated by stretch (mechanical), warm temperature, some epoxyeicosatrienoic acids, and lipopolysaccharide. TRPV4 is expressed throughout the gastrointestinal epithelia and its activation induces adenosine triphosphate (ATP) exocytosis that is involved in visceral hypersensitivity. As an ATP transporter, vesicular nucleotide transporter (VNUT) mediates ATP storage in secretory vesicles and ATP release via exocytosis upon stimulation. SUMMARY: TRPV4 is sensitized under inflammatory conditions by a variety of factors, including proteases and serotonin, whereas methylation-dependent silencing of TRPV4 expression is associated with various pathophysiological conditions. Gastrointestinal epithelia also release ATP in response to hypo-osmolality or acid through molecular mechanisms that remain unclear. These synergistically released ATP could be involved in visceral hypersensitivity. Low concentrations of the first generation bisphosphate, clodronate, were recently reported to inhibit VNUT activity and thus clodronate may be a safe and potent therapeutic option to treat visceral pain. Key Messages: This review focuses on: (1) ATP and TRPV4 activities in gastrointestinal epithelia; (2) factors that could modulate TRPV4 activity in gastrointestinal epithelia; and (3) the inhibition of VNUT as a potential novel therapeutic strategy for functional gastrointestinal disorders.

Stimulation of P2Y11 receptor protects human cardiomyocytes against Hypoxia/Reoxygenation injury and involves PKCε signaling pathway.

Sterile inflammation is a key determinant of myocardial reperfusion injuries. It participates in infarct size determination in acute myocardial infarction and graft rejection following heart transplantation. We previously showed that P2Y11 exerted an immunosuppressive role in human dendritic cells, modulated cardiofibroblasts' response to ischemia/reperfusion in vitro and delayed graft rejection in an allogeneic heterotopic heart transplantation model. We sought to investigate a possible role of P2Y11 in the cellular response of cardiomyocytes to ischemia/reperfusion. We subjected human AC16 cardiomyocytes to 5 h hypoxia/1 h reoxygenation (H/R). P2Y11R (P2Y11 receptor) selective agonist NF546 and/or antagonist NF340 were added at the onset of reoxygenation. Cellular damages were assessed by LDH release, MTT assay and intracellular ATP level; intracellular signaling pathways were explored. The role of P2Y11R in mitochondria-derived ROS production and mitochondrial respiration was investigated. In vitro H/R injuries were significantly reduced by P2Y11R stimulation at reoxygenation. This protection was suppressed with P2Y11R antagonism. P2Y11R stimulation following H2O2-induced oxidative stress reduced mitochondria-derived ROS production and damages through PKCε signaling pathway activation. Our results suggest a novel protective role of P2Y11 in cardiomyocytes against reperfusion injuries. Pharmacological post-conditioning targeting P2Y11R could therefore contribute to improve myocardial ischemia/reperfusion outcomes in acute myocardial infarction and cardiac transplantation.

P2Y11 receptor antagonist NF340 ameliorates inflammation in human fibroblast-like synoviocytes: An implication in rheumatoid arthritis.

Rheumatoid arthritis is a common chronic inflammatory joint disease. Fibroblast-like synoviocytes-mediated inflammation is closely associated with the development of rheumatoid arthritis. In this study, we report that P2Y11 receptor activity is required for cytokine-induced inflammation in primary fibroblast-like synoviocytes (FLS). P2Y11R is fairly expressed in primary FLS isolated from healthy subjects and is elevated to around three- to four-fold in rheumatoid arthritis-derived FLS. The expression of P2Y11R is inducible upon IL-1ß treatment. Blockage of P2Y11R by its antagonist suppresses IL-1ß-induced TNF-α and IL-6 induction and ameliorates oxidative stress as determined by levels of cellular ROS and the oxidative byproduct 4-HNE. Moreover, blockage of P2Y11R by NF340 inhibits IL-1ß-induced matrix metalloproteinase protein expression as indicated by the levels of MMP-1, MMP-3, and MMP-13. Mechanistically, blockage of P2Y11R mitigates IL-1ß-activated NFκB signaling, which was revealed by reduced IκBα phosphorylation, nuclear p65 accumulation, and NFκB promoter activity. Our study provides evidence of a protective mechanism of P2Y11R antagonist NF340 against cytokine-induced inflammation. Therefore, targeting P2Y11R could have potential therapeutic implication in the treatment of RA.

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

OBJECTIVE: Myocardial 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. METHODS: We 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. RESULTS: P2Y11R 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. CONCLUSIONS: Our 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.

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.

Microvesicles generated following traumatic brain injury induce platelet dysfunction via adenosine diphosphate receptor.

BACKGROUND: Traumatic brain injury (TBI) can result in an acute coagulopathy including platelet dysfunction that can contribute to ongoing intracranial hemorrhage. Previous studies have shown adenosine diphosphate (ADP)-induced platelet aggregation to be reduced after TBI. In addition, circulating microvesicles (MVs) are increased following TBI and have been shown to play a role in post-TBI coagulopathy and platelet function. We hypothesized that post-TBI MVs would affect platelet aggregation in a murine head injury model. METHODS: Moderate TBI was performed using a weight-drop method in male C57BL6 mice. Whole blood, plasma, MVs, and MV-poor plasma were isolated from blood collected 10 minutes following TBI and were mixed separately with whole blood from uninjured mice. Platelet aggregation was measured with Multiplate impedance platelet aggregometry in response to ADP. The ADP P2Y12 receptor inhibitor, R-138727, was incubated with plasma and MVs from TBI mice, and platelet inhibition was again measured. RESULTS: Whole blood taken from 10-minute post-TBI mice demonstrated diminished ADP-induced platelet aggregation compared with sham mice. When mixed with normal donor blood, post-TBI plasma and MVs induced diminished ADP-induced platelet aggregation compared with sham plasma and sham MVs. By contrast, the addition of post-TBI MV-poor plasma to normal blood did not change ADP-induced platelet aggregation. The observed dysfunction in post-TBI ADP platelet aggregation was prevented by the pretreatment of post-TBI plasma with R-138727. Treatment of post-TBI MVs with R-138727 resulted in similar findings of improved ADP-induced platelet aggregation compared with nontreated post-TBI MVs. CONCLUSION: Adenosine diphosphate-induced platelet aggregation is inhibited acutely following TBI in a murine model. This platelet inhibition is reproduced in normal blood by the introduction of post-TBI plasma and MVs. Furthermore, observed platelet dysfunction is prevented when post-TBI plasma and MVs are treated with an inhibitor of the P2Y12 ADP receptor. Clinically observed post-TBI platelet dysfunction may therefore be partially explained by the presence of the ADP P2Y12 receptor within post-TBI MVs. LEVEL OF EVIDENCE: Level III.

I-Tiao-Gung extract through its active component daidzin improves cyclophosphamide-induced bladder dysfunction in rat model.

AIMS: We explored the therapeutic potential of intragastric administration traditional Chinese medicine Glycine tomentella Hayata (I-Tiao-Gung, ITG) extract and its active component Daidzin on cyclophosphamide (CYP)-induced cystitis and bladder hyperactivity in rats. METHODS: Female Wistar rats were divided into control, CYP (200 mg/kg), CYP + ITG (1.17 g/kg/day), CYP + Daidzin (12.5 mg/kg/day), and 1 week of ITG preconditioning with CYP (ITG + CYP) groups. We determined the trans cystometrogram associated with external urethral sphincter electromyogram, and the expression of M2 and M3 muscarinic and P2 × 2 and P2 × 3 purinergic receptors by Western blot in these animals. RESULTS: ITG extract contains 1.07% of Daidzin and 0.77% of Daidzein by high-performance liquid chromatography. Daidzin was more efficient than Daidzein in scavenging H2 O2 activity by a chemiluminescence analyzer. CYP induced higher frequency, shorter intercontraction interval, lower maximal voiding pressure, lower threshold pressure, and Phase-2 emptying contraction with a depressed external urethral sphincter electromyogram activity, and hemorrhagic cystitis in the bladders. The altered parameters by CYP were significantly improved in CYP + ITG, CYP + Daidzin, and ITG + CYP groups. The P2 × 2 and P2 × 3 expressions were significantly upregulated in CYP group, but were depressed in CYP + ITG, CYP + Daidzin, and ITG + CYP groups. The M2 expression was not significantly different among these five groups. The M3 expression was significantly upregulated in CYP group, but was significantly depressed in CYP + ITG, CYP + Daidzin, and ITG + CYP groups. CONCLUSIONS: These data suggest that ITG extract through its active component Daidzin effectively improved CYP-induced cystitis by the action of restoring Phase 2 activity and inhibiting the expressions of P2 × 2, P2 × 3, and M3 receptors.

P2Y12 antagonists and cardiac repair post-myocardial infarction: global and regional heart function analysis and molecular assessments in pigs.

Aims: P2Y12 antagonists are the standard in antiplatelet therapy but their potential effects on functional myocardial recovery and cardioprotection post-myocardial infarction (MI) are unknown. We investigated in a preclinical model of MI whether ticagrelor and clopidogrel differently affect cardiac repair post-MI. Methods and results: Pigs either received: (i) clopidogrel (600 mg; 75 mg/qd); (ii) ticagrelor (180 mg; 90 mg/bid); and (iii) placebo control. MI was induced by mid-left anterior descending coronary artery balloon occlusion (60 min) and animals received the maintenance doses for the following 42 days. Serial cardiac magnetic resonance was performed at Day 3 and Day 42 for the assessment of global and regional cardiac parameters. We determined cardiac AMP-activated protein kinase (AMPK), Akt/PKB, aquaporin-4, vascular density, and fibrosis. In comparison to controls, both P2Y12 antagonists limited infarct expansion at Day 3, although ticagrelor induced a further 5% reduction (P < 0.05 vs. clopidogrel) whereas oedema was only reduced by ticagrelor (≈23% P < 0.05). Scar size decreased at Day 42 in ticagrelor-treated pigs vs. controls but not in clopidogrel-treated pigs. Left ventricular ejection fraction was higher 3 days post-MI in ticagrelor-treated pigs and persisted up to Day 42 (P < 0.05 vs. post-MI). Regional analysis revealed that control and clopidogrel-treated pigs had severe and extensive wall motion abnormalities in the jeopardized myocardium and a reduced myocardial viability that was not as evident in ticagrelor-treated pigs (χ2P < 0.05 vs. ticagrelor). Only ticagrelor enhanced myocardial AMPK and Akt/PKB activation and reduced aquaporin-4 levels (P < 0.05 vs. control and clopidogrel). No differences were observed in vessel density and fibrosis markers among groups. Conclusions: Ticagrelor is more efficient than clopidogrel in attenuating myocardial structural and functional alterations post-MI and in improving cardiac healing. These benefits are associated with persistent AMPK and Akt/PKB activation.

Activation of basal forebrain purinergic P2 receptors promotes wakefulness in mice.

The functions of purinergic P2 receptors (P2Rs) for extracellular adenosine triphosphate (ATP) are poorly understood. Here, for the first time, we show that activation of P2Rs in an important arousal region, the basal forebrain (BF), promotes wakefulness, whereas inhibition of P2Rs promotes sleep. Infusion of a non-hydrolysable P2R agonist, ATP-γ-S, into mouse BF increased wakefulness following sleep deprivation. ATP-γ-S depolarized BF cholinergic and cortically-projecting GABAergic neurons in vitro, an effect blocked by antagonists of ionotropic P2Rs (P2XRs) or glutamate receptors. In vivo, ATP-γ-S infusion increased BF glutamate release. Thus, activation of BF P2XRs promotes glutamate release and excitation of wake-active neurons. Conversely, pharmacological antagonism of BF P2XRs decreased spontaneous wakefulness during the dark (active) period. Together with previous findings, our results suggest sleep-wake regulation by BF extracellular ATP involves a balance between excitatory, wakefulness-promoting effects mediated by direct activation of P2XRs and inhibitory, sleep-promoting effects mediated by degradation to adenosine.

A promising drug candidate for the treatment of glaucoma based on a P2Y6-receptor agonist.

Extracellular nucleotides can regulate the production/drainage of the aqueous humor via activation of P2 receptors, thus affecting the intraocular pressure (IOP). We evaluated 5-OMe-UDP(α-B), 1A, a potent P2Y6-receptor agonist, for reducing IOP and treating glaucoma. Cell viability in the presence of 1A was measured using [3-(4, 5-dimethyl-thiazol-2-yl) 2, 5-diphenyl-tetrazolium bromide] (MTT) assay in rabbit NPE ciliary non-pigmented and corneal epithelial cells, human retinoblastoma, and liver Huh7 cells. The effect of 1A on IOP was determined in acute glaucomatous rabbit hyaluronate model and phenol-induced chronic glaucomatous rabbit model. The origin of activity of 1A was investigated by generation of a homology model of hP2Y6-R and docking studies. 1A did not exert cytotoxic effects up to 100 mM vs. trusopt and timolol in MTT assay in ocular and liver cells. In normotensive rabbits, 100 µM 1A vs. xalatan, trusopt, and pilocarpine reduced IOP by 45 vs. 20-30%, respectively. In the phenol animal model, 1A (100 µM) showed reduction of IOP by 40 and 20%, following early and late administration, respectively. Docking results suggest that the high activity and selectivity of 1A is due to intramolecular interaction between Pα-BH3 and C5-OMe which positions 1A in a most favorable site inside the receptor. P2Y6-receptor agonist 1A effectively and safely reduces IOP in normotense, acute, and chronic glaucomatous rabbits, and hence may be suggested as a novel approach for the treatment of glaucoma.

Caspase-1 inhibition by VX-765 administered at reperfusion in P2Y12 receptor antagonist-treated rats provides long-term reduction in myocardial infarct size and preservation of ventricular function.

Patients with acute myocardial infarction receive a P2Y12 receptor antagonist prior to reperfusion, a treatment that has reduced, but not eliminated, mortality, or heart failure. We tested whether the caspase-1 inhibitor VX-765 given at reperfusion (a requirement for clinical use) can provide sustained reduction of infarction and long-term preservation of ventricular function in a pre-clinical model of ischemia/reperfusion that had been treated with a P2Y12 receptor antagonist. To address, the hypothesis open-chest rats were subjected to 60-min left coronary artery branch occlusion/120-min reperfusion. Vehicle or inhibitors were administered intravenously immediately before reperfusion. With vehicle only, 60.3 ± 3.8% of the risk zone suffered infarction. Ticagrelor, a P2Y12 antagonist, and VX-765 decreased infarct size to 42.8 ± 3.3 and 29.2 ± 4.9%, respectively. Combining ticagrelor with VX-765 further decreased infarction to 17.5 ± 2.3%. Similar to recent clinical trials, combining ticagrelor and ischemic postconditioning did not result in additional cardioprotection. VX-765 plus another P2Y12 antagonist, cangrelor, also decreased infarction and preserved ventricular function when reperfusion was increased to 3 days. In addition, VX-765 reduced infarction in blood-free, isolated rat hearts indicating at least a portion of injurious caspase-1 activation originates in cardiac tissue. While the pro-drug VX-765 only protected isolated hearts when started prior to ischemia, its active derivative VRT-043198 provided the same amount of protection when started at reperfusion, indicating that even in blood-free hearts, caspase-1 appears to exert its injury only at reperfusion. Moreover, VX-765 decreased circulating IL-1ß, prevented loss of cardiac glycolytic enzymes, preserved mitochondrial complex I activity, and decreased release of lactate dehydrogenase, a marker of pyroptosis. Our results are the first demonstration of a clinical-grade drug given at reperfusion providing additional, sustained infarct size reduction when added to a P2Y12 receptor antagonist.

Identification of novel selective P2Y6 receptor antagonists by high-throughput screening assay.

AIMS: The P2Y6 nucleotide receptor is widely involved in inflammatory responses, and is a promising molecular target for the treatment of inflammatory diseases. Although several P2Y6 receptor antagonists have been developed and evaluated thus far, none has successfully been developed into a therapeutic drug. In this study, we explored new promising compounds that inhibit the human P2Y6 receptor. MAIN METHODS: High-throughput screening (HTS) was used to study the effects of various compounds on human P2Y6 receptors expressed in 1321N1 human astrocytoma cells by monitoring intracellular Ca2+ concentration ([Ca2+]i) levels using an FDSS7000 real-time fluorescence detector. IL-8 concentration was measured by enzyme-linked immunosorbent assay. KEY FINDINGS: Among structurally diverse chemical libraries totalling 141,700 compounds, 43 compounds with an inhibitory activity against the P2Y6 receptor were identified. Further studies using a dose-response assay, receptor selectivity assay, and chemokine measurement assay revealed the selective P2Y6 receptor inhibitor TIM-38, which inhibited UDP-induced [Ca2+]i elevation in a dose-dependent manner. TIM-38 had an IC50 value of 4.3µM and inhibited P2Y6 without affecting the response induced by four other human P2Y or muscarinic receptors. In addition, TIM-38 inhibited UDP-induced interleukin-8 release in a dose-dependent manner without affecting releases caused by other stimulus such as interleukin-1ß or tumour necrosis factor-α. Analyses of TIM-38 derivatives revealed that the nitro moiety is vital to P2Y6 receptor inhibition. SIGNIFICANCE: TIM-38 acts as a novel structural antagonist of P2Y6 receptor and may be a good lead compound for developing a P2Y6 receptor-targeted anti-inflammatory drug.

Purinergic signaling in kidney disease.

Nucleotides are key subunits for nucleic acids and provide energy for intracellular metabolism. They can also be released from cells to act physiologically as extracellular messengers or pathologically as danger signals. Extracellular nucleotides stimulate membrane receptors in the P2 and P1 family. P2X are ATP-activated cation channels; P2Y and P1 are G-protein coupled receptors activated by ATP, ADP, UTP, and UDP in the case of P2 or adenosine for P1. Renal P2 receptors influence both vascular contractility and tubular function. Renal cells also express ectonucleotidases that rapidly hydrolyze extracellular nucleotides. These enzymes integrate this multireceptor purinergic-signaling complex by determining the nucleotide milieu to titrate receptor activation. Purinergic signaling also regulates immune cell function by modulating the synthesis and release of various cytokines such as IL1-ß and IL-18 as part of inflammasome activation. Abnormal or excessive stimulation of this intricate paracrine system can be pro- or anti-inflammatory, and is also linked to necrosis and apoptosis. Kidney tissue injury causes a localized increase in ATP concentration, and sustained activation of P2 receptors can lead to renal glomerular, tubular, and vascular cell damage. Purinergic receptors also regulate the activity and proliferation of fibroblasts, promoting both inflammation and fibrosis in chronic disease. In this short review we summarize some of the recent findings related to purinergic signaling in the kidney. We focus predominantly on the P2X7 receptor, discussing why antagonists have so far disappointed in clinical trials and how advances in our understanding of purinergic signaling might help to reposition these compounds as potential treatments for renal disease.

Central Role of P2Y6 UDP Receptor in Arteriolar Myogenic Tone.

OBJECTIVE: Myogenic tone (MT) of resistance arteries ensures autoregulation of blood flow in organs and relies on the intrinsic property of smooth muscle to contract in response to stretch. Nucleotides released by mechanical strain on cells are responsible for pleiotropic vascular effects, including vasoconstriction. Here, we evaluated the contribution of extracellular nucleotides to MT. APPROACH AND RESULTS: We measured MT and the associated pathway in mouse mesenteric resistance arteries using arteriography for small arteries and molecular biology. Of the P2 receptors in mouse mesenteric resistance arteries, mRNA expression of P2X1 and P2Y6 was dominant. P2Y6 fully sustained UDP/UTP-induced contraction (abrogated in P2ry6(-/-) arteries). Preventing nucleotide hydrolysis with the ectonucleotidase inhibitor ARL67156 enhanced pressure-induced MT by 20%, whereas P2Y6 receptor blockade blunted MT in mouse mesenteric resistance arteries and human subcutaneous arteries. Despite normal hemodynamic parameters, P2ry6(-/-) mice were protected against MT elevation in myocardial infarction-induced heart failure. Although both P2Y6 and P2Y2 receptors contributed to calcium mobilization, P2Y6 activation was mandatory for RhoA-GTP binding, myosin light chain, P42-P44, and c-Jun N-terminal kinase phosphorylation in arterial smooth muscle cells. In accordance with the opening of a nucleotide conduit in pressurized arteries, MT was altered by hemichannel pharmacological inhibitors and impaired in Cx43(+/-) and P2rx7(-/-) mesenteric resistance arteries. CONCLUSIONS: Signaling through P2 nucleotide receptors contributes to MT. This mechanism encompasses the release of nucleotides coupled to specific autocrine/paracrine activation of the uracil nucleotide P2Y6 receptor and may contribute to impaired tissue perfusion in cardiovascular diseases.