P2Y Receptor Agonist Research Articles

Purinergic Receptor P2Y <sub>6</sub> Inhibits the Maturation and Function of NK Cells by Suppressing mTOR Signaling Pathway

Natural killer (NK) cells are a critical immune cells of innate immunity that can kill tumor cells, cancer-initiating cells and clear virus infections by producing cytokines and cytotoxic granules. However, there are few reports that elucidate the role of purinergic receptor in regulating NK cells. In this study, we discovered the expression of purinergic receptor P2Y6 was decreased after the activation of NK cells. In P2Y6-deficient mice, we found that P2Y6 deficiency promoted the development of NKp cells into iNK cells and the maturation of NK cells. We also found P2Y6 deficiency increased the production of IFN-γ in NK cells after stimulated with Poly (I:C), IL-15, the combination of IL-12 and IL-15 or PMA. P2Y6 receptor agonist UDP or UDP analogue 5-OMe-UDP inhibited the production of IFN-γ in NK cells after stimulation. In addition, we demonstrated P2Y6 deficiency increased the cytotoxicty function of NK cells in vitro and anti-metastatic activity in vivo. Mechanistically, we found P2Y6 deletion increased the expression of T-bet with or without IL-15 stimulation. Additionally, P2Y6 deficiency increased the level of p-AKT and pS6 after IL-15 stimulation, respectively. Collectively, we identify P2Y6 receptor, as a negative regulation of of NK cells function, inhibits the expression of T-bet by suppressing mTOR signaling pathway activation, eventually prevents NK cells maturation and effector function.

Long-Term Safety and Effectiveness of Diquafosol for the Treatment of Dry Eye in a Real-World Setting: A Prospective Observational Study.

IntroductionDiquafosol is a P2Y2 receptor agonist that has been shown to be effective in the treatment of dry eye disease (DED) in short-term studies; however, its long-term safety and effectiveness have not been evaluated in a real-world setting.MethodsThis prospective, multicentre, open-label observational study was conducted in patients with DED over 12 months. Safety endpoints included the incidence of adverse drug reactions (ADRs) and serious ADRs. Effectiveness endpoints included change from baseline in keratoconjunctival staining score, tear film break-up time (BUT) and Dry Eye-related Quality of Life Score (DEQS).ResultsA total of 580 patients were included, most of whom were female (82.9%). The proportion of patients who completed 12 months of observation was 55.0%, the most common reason for discontinuation was patient decision (54.6%). The incidence of ADRs was 10.7% and was highest during the first month of treatment (5.5%); no serious ADRs were reported. Compared with baseline, significant improvements in all effectiveness outcomes, including keratoconjunctival fluorescein staining score, BUT and DEQS summary score, were observed at each evaluation during the treatment period (p < 0.001).ConclusionThe present, real-world study showed that diquafosol 3.0% ophthalmic solution was well tolerated and effective in the long-term treatment of DED.Electronic supplementary materialThe online version of this article (10.1007/s12325-019-01188-x) contains supplementary material, which is available to authorized users.

Synthesis and preclinical validation of novel P2Y1 receptor ligands as a potent anti-prostate cancer agent

Purinergic receptor is a potential drug target for neuropathic pain, Alzheimer disease, and prostate cancer. Focusing on the structure-based ligand discovery, docking analysis on the crystal structure of P2Y1 receptor (P2Y1R) with 923 derivatives of 1-indolinoalkyl 2-phenolic compound is performed to understand the molecular insights of the receptor. The structural model identified the top novel ligands, 426 (compound 1) and 636 (compound 2) having highest binding affinity with the docking score of −7.38 and −6.92. We have reported the interaction efficacy and the dynamics of P2Y1R protein with the ligands. The best hits synthesized were experimentally optimized as a potent P2Y1 agonists. These ligands exhibits anti-proliferative effect against the PC-3 and DU-145 cells (IC50 = 15 µM – 33 µM) with significant increase in the calcium level in dose- and time-dependent manner. Moreover, the activation of P2Y1R induced the apoptosis via Capase3/7 and ROS signaling pathway. Thus it is evidenced that the newly synthesized ligands, as a P2Y1R agonists could potentially act as a therapeutic drug for treating prostate cancer.

Role of opioid receptors in modulation of P2X receptor-mediated cardiac sympathoexcitatory reflex response

Myocardial ischemia evokes powerful reflex responses through activation of vagal and sympathetic afferents in the heart through the release of ischemic metabolites. We have demonstrated that extracellular ATP stimulates cardiac sympathetic afferents through P2 receptor-mediated mechanism, and that opioid peptides suppress these afferents’ activity. However, the roles of both P2 receptor and endogenous opioids in cardiac sympathoexcitatory reflex (CSR) responses remain unclear. We therefore hypothesized that activation of cardiac P2 receptor evokes CSR responses by stimulating cardiac sympathetic afferents and these CSR responses are modulated by endogenous opioids. We observed that intrapericardial injection of α,β-methylene ATP (α,β-meATP, P2X receptor agonist), but not ADP (P2Y receptor agonist), caused a graded increase in mean arterial pressure in rats with sinoaortic denervation and vagotomy. This effect of α,β-meATP was abolished by blockade of cardiac neural transmission with intrapericardial procaine treatment and eliminated by intrapericardial A-317491, a selective P2X2/3 and P2X3 receptor antagonist. Intrapericardial α,β-meATP also evoked CSR response in vagus-intact rats. Furthermore, the P2X receptor-mediated CSR responses were enhanced by intrapericardial naloxone, a specific opioid receptor antagonist. These data suggest that stimulation of cardiac P2X2/3 and P2X3, but not P2Y receptors, powerfully evokes CSR responses through activation of cardiac spinal afferents, and that endogenous opioids suppress the P2X receptor-mediated CSR responses.

A role for PKD1 in insulin secretion downstream of P2Y1 receptor activation in mouse and human islets.

Along with insulin, β‐cells co‐secrete the neurotransmitter ATP which acts as a positive autocrine signal via P2Y1 receptors to activate phospholipase C and increase the production of diacylglycerol (DAG). However, the downstream signaling that couples P2Y1 activation to insulin secretion remains to be fully elucidated. Since DAG activates protein kinase D1 (PKD1) to potentiate glucose‐stimulated insulin release, we hypothesized that autocrine ATP signaling activates downstream PKD1 to regulate insulin secretion. Indeed, we find that the P2Y1 receptor agonists, MRS2365 and ATP induce, PKD1 phosphorylation at serine 916 in mouse islets. Similarly, direct depolarization of islets by KCl caused PKD1 activation, which is reduced upon P2Y1 antagonism. Potentiation of insulin secretion by P2Y1 activation was lost from PKD1−/− mouse islets, and knockdown of PKD1 reduced the ability of P2Y1 activation to facilitate exocytosis in single mouse β‐cells. Finally, qPCR analysis confirmed PKD1 transcript (PRKD1) expression in human islets, and insulin secretion assays showed that inhibition of either P2Y1 or PKD1 signaling impaired glucose‐stimulated insulin secretion. Human islets showed donor‐to‐donor variation in their responses to both P2Y1 and PKD1 inhibition, however, and we find that the P2Y1‐PKD1 pathway contributes a substantially greater proportion of insulin secretion from islets of overweight and obese donors. Thus, PKD1 promotes increased insulin secretion, likely mediating an autocrine ATP effect via P2Y1 receptor activation which may be more important in islets of donors who are overweight or obese.

Role of Carbon Monoxide in the Mechanisms of Action of Extracellular ATP on Contractile Activity of Vascular Smooth Muscle Cells.

We studied the role of carbon monoxide (CO) in the effect of P2X and P2Y receptor agonist ATP on the tone of rat aorta segments with intact endothelium. ATP (1-1000 μM) and P2X receptor agonist α,β-MeATP (100 μM) relaxed segments precontracted with phenylephrine (10 μM), while UTP (100-1000 μM) increased the amplitude of phenylephrine-induced contraction. The relaxing effect of ATP was enhanced by CORM II (100 μM), NO synthase inhibitor L-NAME, and guanylate cyclase inhibitor ODQ and attenuated by ZnPP IX (100 μM). The constrictive effect of UTP was weakened by CORM II (100 μM), but was not changed by ZnPP IX (100 μM). ZnPP IX (100 μM) weakened the relaxation response to α,β-MeATP. Thus, ATP involves the CO-dependent signaling cascade through P2X receptors.

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.

P2Y6 Receptor-Mediated Spinal Microglial Activation in Neuropathic Pain

Objective To explore the role of purine family member P2Y6 receptors in regulating neuropathic pain (NP) via neuroinflammation in the spinal cord. Methods Chronic constriction injury of the sciatic nerve (CCI) of NP was classic in setting up models on Sprague-Dawley (SD) rats. Experiments were performed on rats with sham surgery, CCI, CCI + MRS2578 (a P2Y6 receptor antagonist), and UDP (a P2Y6 receptor agonist). The hyperalgesia intensity was mirrored by paw withdrawal threshold (PWT) and thermal withdrawal latency (TWL). Immunofluorescence staining and western blot were used to evaluate activated microglial marker Iba-1. Enzyme-linked immunosorbent assay (ELISA) was used to access levels of IL-6. Conventional reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis were used to detect the expression of P2Y6 mRNA and activation of JAK/STAT signaling. Results Among all groups, CCI caused decreased PWT and TWL compared to sham surgery, meaning a successful establishment of the NP model. These decreased values of PWT and TWL tests could be prevented by intraperitoneally injected MRS2578 and enhanced by UDP administration. Similarly, CCI induced increase of Iba-1 protein, P2Y6 mRNA expression, and circulating IL-6 secretion, as well as increased JAK2/STAT3 mRNA expression and phosphorylating modification in spinal cord tissues could also be diminished by MRS2578 treatment and exacerbated by UDP. Conclusions These findings indicated the crucial role of the P2Y6 receptor in modulating the microglial and inflammatory responses in the process of NP in vivo. Results from this study would provide insights into targeting the P2Y6 receptor to treat NP in the near future.

Alterations in Nitric Oxide‐Mediated Signals in Coronary Arteries Following Spaceflight

Proportional cardiovascular disease mortality rates are not different between US astronauts that have flown in low Earth orbit (LEO) and those that have never flown orbital missions in space. While these data suggest that spaceflight (SF) in LEO holds nominal long‐term cardiovascular health consequences, to date the acute effect of SF on coronary artery function has not been explored. The purpose of this study was to examine murine coronary artery contractile, dilatory, and mechanical properties following SF in LEO aboard the International Space Station (ISS). Twenty C57BL/6J mice were housed in a specially designed rodent habitat and were launched to the ISS aboard a Falcon 9 rocket (Space‐X CRS‐12). Following ~33 days of SF in LEO, the Dragon capsule containing the mice splashed down in the Pacific Ocean. Within 33–41 hours of returning to Earth, the left anterior descending (LAD) coronary arteries were cleared of surrounding cardiac tissue and mounted in a Danish Myotech wire myograph. LADs were then exposed to agonists that allowed for the examination of endothelial (EC) and smooth muscle cell (SMC) function. Vivarium controls (VC), consisting of mice housed in conventional vivarium cages, and habitat controls (HC), which were housed in the same type cages as those flown on orbit and maintained under similar pCO2, temperature, humidity, and light conditions, were used for comparison. The passive pressure‐diameter relationship was not different between SF and either control. SMC‐dependent vasoconstriction to U46619, a thromboxane receptor agonist, was significantly greater in HC versus SF at only a single dose, while vasoconstriction to isosmotic KCl was unaltered. LADs from SF mice preconstricted with U46619 exhibited similar EC‐dependent vasodilation to the muscarinic receptor agonist acetylcholine when compared to VC and HC. Acetylcholine and ADPβS (a P2Y1 receptor agonist)‐mediated vasodilation were completely absent in all LADs preconstricted with KCl in the presence of the nitric oxide synthase inhibitor, L‐NAME. In vessels preconstricted with U46619 vasodilation to the EC‐dependent vasodilator ADPβS was similar between SF and both controls, with SF LADs exhibiting less dependence on nitric oxide (NO)‐mediated mechanisms as observed in the presence of L‐NAME. LADs from SF mice exhibited a right‐ward shift to the NO donor and SMC‐dependent vasodilator, DEA NONOate. Collectively, these data suggest that subtle changes in LAD function occur following spaceflight in LEO. In particular, NO‐mediated signals were the most affected, as indicated by decreased SMC sensitivity to exogenous NO. In addition, while the magnitude of vasodilation to ADPβS was unchanged, SF animals exhibited less dependence on NO‐mediated signals. These ADPβS responses suggest a shift away from the canonical NO‐mediated pathway of vasodilation. These data represent the first examination of coronary artery function following SF and suggest that function is well maintained due to subtle mechanistic adaptations.Support or Funding InformationThis work was supported by NASA Space Biology grant NNX16AC28G.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Purinergic modulation of neuronal activity in the rat prepositus hypoglossi nucleus.

In the nervous system, adenosine 5'-trisphosphate (ATP) functions as a neurotransmitter and binds to ionotropic P2X receptors and metabotropic P2Y receptors. Although ATP receptors are expressed in the prepositus hypoglossi nucleus (PHN), which is a brainstem structure involved in controlling horizontal gaze, it is unclear whether ATP indeed affects the activity of PHN neurons. In this study, we investigated the effects of ATP on spontaneous firing of PHN neurons using whole-cell recordings in rat brainstem slices. Bath application of ATP increased or decreased the spontaneous firing rate of the neurons in a dose-dependent manner, indicating that ATP indeed affects PHN neuronal activity. To clarify the mechanisms of the ATP effects, we investigated the current responses of PHN neurons to a local application of ATP. The ATP application induced a fast inward (FI) current, a slow inward (SI) current, and/or a slow outward (SO) current in the neurons. The agonists of P2X and P2Y receptors induced FI and SI currents, respectively. The SO currents were not induced by the ATP agonists but were induced by adenosine, which may be extracellularly converted from ATP by ectonucleotidases. An antagonist of adenosine P1 (A1 ) receptors abolished the adenosine-induced SO currents and bath application of adenosine decreased the spontaneous firing rate of all PHN neurons tested. These results indicate that PHN neurons express functional purinoceptors and show that the FI, SI, and SO currents were mediated via P2X, P2Y, and A1 receptors, respectively.

Comparison of inhibitory neuromuscular transmission in the Cynomolgus monkey IAS and rectum: special emphasis on differences in purinergic transmission.

Inhibitory neuromuscular transmission (NMT) was compared in the internal anal sphincter (IAS) and rectum of the Cynomolgus monkey, an animal with high gene sequence identity to humans. Nitrergic NMT was present in both muscles while purinergic NMT was limited to the rectum and VIPergic NMT to the IAS. The profile for monkey IAS more closely resembles humans than rodents. In both muscles, SK3 channels were localized to PDGFRα+ cells that were closely associated with nNOS+ /VIP+ nerves. Gene expression levels of P2RY subtypes were the same in IAS and rectum while KCNN expression levels were very similar. SK3 channel activation and inhibition caused faster/greater changes in contractile activity in rectum than IAS. P2Y1 receptor activation inhibited contraction in rectum while increasing contraction in IAS. The absence of purinergic NMT in the IAS may be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα+ cells. Inhibitory neuromuscular transmission (NMT) was compared in the internal anal sphincter (IAS) and rectum of the Cynomolgus monkey, an animal with a high gene sequence identity to humans. Electrical field stimulation produced nitric oxide synthase (NOS)-dependent contractile inhibition in both muscles whereas P2Y1-dependent purinergic NMT was restricted to rectum. An additional NOS-independent, α-chymotrypsin-sensitive component was identified in the IAS consistent with vasoactive intestinal peptide-ergic (VIPergic) NMT. Microelectrode recordings revealed slow NOS-dependent inhibitory junction potentials (IJPs) in both muscles and fast P2Y1-dependent IJPs in rectum. The basis for the difference in purinergic NMT was investigated. PDGFRα+ /SK3+ cells were closely aligned with nNOS+ /VIP+ neurons in both muscles. Gene expression of P2RY was the same in IAS and rectum (P2RY1>>P2RY2-14) while KCNN3 expression was 32% greater in rectum. The SK channel inhibitor apamin doubled contractile activity in rectum while having minimal effect in the IAS. Contractile inhibition elicited with the SK channel agonist CyPPA was five times faster in rectum than in the IAS. The P2Y1 receptor agonist MRS2365 inhibited contraction in rectum but increased contraction in the IAS. In conclusion, both the IAS and the rectum have nitrergic NMT whereas purinergic NMT is limited to rectum and VIPergic NMT to the IAS. The profile in monkey IAS more closely resembles that of humans than rodents. The lack of purinergic NMT in the IAS cannot be attributed to the absence of PDGFRα+ cells, P2Y1 receptors or SK3 channels. Rather, it appears to be due to poor coupling between P2Y1 receptors and SK3 channels on PDGFRα+ cells.

A purinergic P2Y6 receptor agonist prodrug modulates airway inflammation, remodeling, and hyperreactivity in a mouse model of asthma.

BackgroundPurinergic receptors control cell proliferation, apoptosis, migration, inflammation, and cytokine secretion. Increased expression of specific purinergic receptors is reported in asthma. The role of purinergic P2Y6 receptors (P2Y6R) in asthma is controversial.HypothesisP2Y6R activation in asthma improves pulmonary function and reduces inflammation and smooth muscle amount.MethodsFemale mice (C57/BL6, age 30 days) were randomly assigned to receive intranasal house dust mite (HDM) antigen (40 or 80 µg) or saline, 5 days/week, for 6 weeks. Randomly selected subgroups received intraperitoneal P2Y6R agonist prodrug (GC021109; 10 or 100 µg/kg weight/dose) simultaneously with HDM. After 6 weeks, lung function was measured. Lung lavage fluid (LLF) was used to measure total cell count, total protein, and cytokines. Immunohistochemistry for alpha smooth muscle actin (α-SMA) was done. Airway wall thickness was measured on micro-computed tomography (micro-CT) images.ResultsPulmonary function testing revealed a HDM dose-dependent airway hyperresponsiveness. Airway resistance was increased 2-fold while compliance was decreased by 50% at the higher HDM dose (P<0.05). GC021109 prevented these changes. HDM-exposed mice had elevated inflammatory cell and total protein levels in LLF which were prevented by GC021109 (P<0.05). HDM mice also had elevated LLF levels of interleukin (IL)-4, IL-5, IL-12, granulocyte colony stimulating factor, chemokine (C-X-C) motif ligand 1, and leukemia inhibitory factor that were reduced by GC021109 with a dose-dependent pattern. HDM mice had increased peribronchial and perivascular inflammatory cell infiltration and increased α-SMA; these changes were absent with GC021109. Airway wall thickness measured on micro-CT images was increased after HDM exposure and significantly reduced by GC021109 treatment.ConclusionThe P2Y6R prodrug GC021109 inhibited allergen-induced changes in pulmonary function, inflammatory responses, and airway and vascular smooth muscle mass. P2Y6R activation may be an effective therapeutic maintenance strategy in asthma.

The inhibitory role of purinergic P2Y receptor on Mg2+ transport across intestinal epithelium-like Caco-2 monolayer.

The mechanism of proton pump inhibitors (PPIs) suppressing intestinal Mg2+ uptake is unknown. The present study aimed to investigate the role of purinergic P2Y receptors in the regulation of Mg2+ absorption in normal and omeprazole-treated intestinal epithelium-like Caco-2 monolayers. Omeprazole suppressed Mg2+ transport across Caco-2 monolayers. An agonist of the P2Y2 receptor, but not the P2Y4 or P2Y6 receptor, suppressed Mg2+ transport across control and omeprazole-treated monolayers. Omeprazole enhanced P2Y2 receptor expression in Caco-2 cells. Forskolin and P2Y2 receptor agonist markedly enhanced apical HCO3- secretion by control and omeprazole-treated monolayers. The P2Y2 receptor agonist suppressed Mg2+ transport and stimulated apical HCO3- secretion through the Gq-protein coupled-phospholipase C (PLC) dependent pathway. Antagonists of cystic fibrosis transmembrane conductance regulator (CFTR) and Na+-HCO3- cotransporter-1 (NBCe1) could nullify the inhibitory effect of P2Y2 receptor agonist on Mg2+ transport across control and omeprazole-treated Caco-2 monolayers. Our results propose an inhibitory role of P2Y2 on intestinal Mg2+ absorption.

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 100mM vs. trusopt and timolol in MTT assay in ocular and liver cells. In normotensive rabbits, 100μM 1A vs. xalatan, trusopt, and pilocarpine reduced IOP by 45 vs. 20-30%, respectively. In the phenol animal model, 1A (100μM) 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.

CB1 Receptors Mediated Inhibition of ATP-Induced [Ca2+]i Increase in Cultured Rat Spinal Dorsal Horn Neurons.

Spinal cannabinoid receptor 1 (CB1R) and purinergic P2X receptors (P2XR) play a critical role in the process of pathological pain. Both CB1R and P2XR are expressed in spinal dorsal horn (DH) neurons. It is not clear whether CB1 receptor activation modulates the function of P2X receptor channels within dorsal horn. For this reason, we observed the effect of CP55940 (cannabinoid receptor agonist) on ATP-induced Ca2+ mobilization in cultured rat DH neurons. The changes of intracellular calcium concentration ([Ca2+]i) were detected with confocal laser scanning microscopy using fluo-4/AM as a calcium fluorescent indicator. 100μM ATP caused [Ca2+]i increase in cultured DH neurons. ATP-evoked [Ca2+]i increase in DH neurons was blocked by chelating extracellular Ca2+ and P2 purinoceptor antagonist PPADS. At the same time, ATP-γ-S (a non-hydrolyzable ATP analogue) mimicked the ATP action, while P2Y receptor agonist ADP failed to evoke [Ca2+]i increase in cultured DH neurons. These data suggest that ATP-induced [Ca2+]i elevation in cultured DH neurons is mediated by P2X receptor. Subsequently, we noticed that, in cultured rat DH neurons, ATP-induced Ca2+ mobilization was inhibited after pretreated with CP55940 with a concentration-dependent manner, which implies that the opening of P2X receptor channels are down-regulated by activation of cannabinoid receptor. The inhibitory effect of CP55940 on ATP-induced Ca2+ response was mimicked by ACEA (CB1R agonist), but was not influenced by AM1241 (CB2R agonist). Moreover, the inhibitory effect of CP55940 on ATP-induced Ca2+ mobilization was blocked by AM251 (CB1 receptor antagonist), but was not influenced by AM630 (CB2 receptor antagonist). In addition, we also observed that forskolin (an activator of adenylate cyclase) and 8-Br-cAMP (a cell-permeable cAMP analog) reversed the inhibitory effect of CP55940, respectively. In a summary, our observations raise a possibility that CB1R rather than CB2R can downregulate the opening of P2X receptor channels in DH neurons. The reduction of cAMP/PKA signaling is a key element in the inhibitory effect of CB1R on P2X-channel-induced Ca2+ mobilization.

Purinergic Signalling: Therapeutic Developments.

Purinergic signalling, i.e., the role of nucleotides as extracellular signalling molecules, was proposed in 1972. However, this concept was not well accepted until the early 1990’s when receptor subtypes for purines and pyrimidines were cloned and characterised, which includes four subtypes of the P1 (adenosine) receptor, seven subtypes of P2X ion channel receptors and 8 subtypes of the P2Y G protein-coupled receptor. Early studies were largely concerned with the physiology, pharmacology and biochemistry of purinergic signalling. More recently, the focus has been on the pathophysiology and therapeutic potential. There was early recognition of the use of P1 receptor agonists for the treatment of supraventricular tachycardia and A2A receptor antagonists are promising for the treatment of Parkinson’s disease. Clopidogrel, a P2Y12 antagonist, is widely used for the treatment of thrombosis and stroke, blocking P2Y12 receptor-mediated platelet aggregation. Diquafosol, a long acting P2Y2 receptor agonist, is being used for the treatment of dry eye. P2X3 receptor antagonists have been developed that are orally bioavailable and stable in vivo and are currently in clinical trials for the treatment of chronic cough, bladder incontinence, visceral pain and hypertension. Antagonists to P2X7 receptors are being investigated for the treatment of inflammatory disorders, including neurodegenerative diseases. Other investigations are in progress for the use of purinergic agents for the treatment of osteoporosis, myocardial infarction, irritable bowel syndrome, epilepsy, atherosclerosis, depression, autism, diabetes, and cancer.

P2Y12 and P2Y13 receptors involved in ADPβs induced the release of IL-1β, IL-6 and TNF-α from cultured dorsal horn microglia.

ObjectiveP2 receptors have been implicated in the release of neurotransmitter and pro-inflammatory cytokines due to their response to neuroexcitatory substances in the microglia. Dorsal horn P2Y12 and P2Y13 receptors are involved in the development of pain behavior induced by peripheral nerve injury. However, it is not known whether P2Y12 and P2Y13 receptors activation is associated with the expression and the release of interleukin-1B (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) in cultured dorsal spinal cord microglia. For this reason, we examined the effects of ADPβs (ADP analog) on the expression and the release of IL-1β, IL-6, and TNF-α.Methods and resultsIn this study, we observed the effect of P2Y receptor agonist ADPβs on the expression and release of IL-1β, IL-6 and TNF-α by using real-time fluorescence quantitative polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). ADPβs induced the increased expression of Iba-1, IL-1β, IL-6 and TNF-α at the level of messenger RNA (mRNA). ADPβs-evoked increase in Iba-1, IL-1β, IL-6 and TNF-α mRNA expression was inhibited only partially by P2Y12 receptor antagonist MRS2395 or P2Y13 receptor antagonist MRS2211, respectively. Similarly, ADPβs-evoked release of IL-1β, IL-6 and TNF-α was inhibited only partially by MRS2395 or MRS2211. Furthermore, ADPβs-evoked increased expression of Iba-1, IL-1β, IL-6 and TNF-α mRNA, and release of IL-1β, IL-6 and TNF-α were nearly all blocked after co-administration of MRS2395 plus MRS2179. Further evidence indicated that P2Y12 and P2Y13 receptor-evoked increased gene expression of IL-1β, IL-6 and TNF-α were inhibited by Y-27632 (ROCK inhibitor), SB203580 (P38MAPK inhibitor) and PDTC (NF-κb inhibitor), respectively. Subsequently, P2Y12 and P2Y13 receptor-evoked release of IL-1β, IL-6 and TNF-α, were also inhibited by Y-27632, SB203580 and PDTC, respectively.ConclusionThese observations suggest that P2Y12 and P2Y13 receptor-evoked gene expression and release of IL-1β, IL-6 and TNF-α are associated with ROCK/P38MAPK/NF-κb signaling pathway.

Adenosine triphosphate induces P2Y2 activation and interleukin-8 release in human esophageal epithelial cells.

Immune-mediated mucosal inflammation characterized by the release of interleukin (IL)-8 is associated with gastroesophageal reflux disease. ATP released by human esophageal epithelial cells (HEECs) mediates the release of cytokines through P2 nucleotide receptors that are present on various cells, including HEECs. This study characterized and identified human esophageal epithelial P2 receptors that are responsible for ATP-mediated release of IL-8 by using a human esophageal stratified squamous epithelial model. Primary HEECs were cultured with the use of an air-liquid interface (ALI) system. The ATP analogue adenosine 5'-O-3-thiotriphosphate (ATP-γ-S) was added to the basolateral compartment, and IL-8 release was measured. Involvement of the P2Y2 receptor was assessed with the use of selective and non-selective receptor antagonists and a P2Y2 receptor agonist. Expression of the P2Y2 receptor was assessed using western blotting and immunohistochemistry. Adenosine triphosphate-γ-S induced IL-8 release through the P2Y2 receptor. A P2Y2 receptor antagonist but not a P2X3 receptor antagonist or a P2Y1 receptor antagonist blocked ATP-γ-S-mediated IL-8 release. Conversely, a P2Y2 receptor agonist induced IL-8 release. Western blotting and immunohistochemistry of the P2Y2 receptor showed strong expression of the P2Y2 receptor on ALI-cultured HEECs and in human esophagus. Inhibition of extracellular signal-regulated kinase but not of protein kinase C blocked the ATP-mediated release of IL-8. ATP-γ-S induced phosphorylation of extracellular signal-regulated kinase, and a P2Y2 receptor antagonist blocked this phosphorylation. Interleukin-8 release after purinergic stimulation in ALI-cultured HEECs is mediated through P2Y2 receptor activation. ATP-induced IL-8 release maybe involved in the pathogenesis of refractory gastroesophageal reflux disease.

RETRACTED ARTICLE: Modulation of the TGF-β1-induced epithelial to mesenchymal transition (EMT) mediated by P1 and P2 purine receptors in MDCK cells

Epithelial to mesenchymal transition (EMT) occurs during embryogenesis or under pathological conditions such as hypoxia, injury, chronic inflammation, or tissue fibrosis. In renal tubular epithelial cells (MDCK), TGF-β1 induces EMT by reducing or increasing epithelial or mesenchymal marker expression, respectively. In this study, we confirmed that the cAMP analogues, 8-CPT-cAMP or N6-Ph-cAMP, inhibited the TGF-β1-driven overexpression of the mesenchymal markers ZEB-1, Slug, Fibronectin, and α-SMA. Furthermore, we showed that A1, A2A, P2Y1, P2Y11, and P2X7 purine receptor agonists modulated the TGF-β1-induced EMT through the involvement of PKA and/or MAPK/ERK signaling. The stimulation of A2A receptor reduced the overexpression of the EMT-related markers, mainly through the cAMP-dependent PKA pathway, as confirmed by cell pre-treatment with Myr-PKI. Both A1 and P2Y1 receptor stimulation exacerbated the TGF-β1-driven effects, which were reduced by cell pre-treatment with the MAPK inhibitor PD98059, according to the increased ERK1/2 phosphorylation upon receptor activation. The effects induced by P2Y11 receptor activation were oppositely modulated by PKA or MAPK inhibition, in line with the dual nature of the Gs- and Gq-coupled receptor. Differently, P2X7 receptor induced, per se, similar and not additive effects compared to TGF-β1, after prolonged cell exposure to BzATP. These results suggest a putative role of purine receptors as target for anti-fibrotic agents.

Large‐conductance Ca2+‐activated K+ channel activation by apical P2Y receptor agonists requires hydrocortisone in differentiated airway epithelium

Hydrocortisone (HC) is required for activation of large-conductance Ca2+ -activated K+ current (BK) by purinergic receptor agonists. HC reduces insertion of the stress-regulated exon (STREX) in the KCNMA1 gene, permitting protein kinase C (PKC)-dependent channel activation. Overlapping and unique purinergic signalling regions exist at the apical border of differentiated surface cells. BK channels localize in the cilia of surface cells. In the present study we investigated the role of hydrocortisone (HC) on uridine-5'-triphosphate (UTP)-stimulated ion transport in differentiated, pseudostratified epithelia derived from normal human bronchial basal cells. The presence of a UTP-stimulated, paxilline-sensitive large-conductance Ca2+ -activated K+ (BK) current was demonstrated in control epithelia but was not stimulated in epithelia differentiated in the absence of HC (HC0). Addition of the BK channel opener NS11021 directly activated channels in control epithelia; however, under HC0 conditions, activation only occurred when UTP was added after NS11021. The PKC inhibitors GF109203x and Gö6983 blocked BK activation by UTP in control epithelia, suggesting that PKC-mediated phosphorylation plays a permissive role in purinoceptor-stimulated BK activation. Moreover, HC0 epithelia expressed significantly more KCNMA1 containing the stress-regulated exon (STREX), a splice-variant of the α-subunit that displays altered channel regulation by phosphorylation, compared to control epithelia. Furthermore, BK channels as well as purinergic receptors were shown to localize in unique and overlapping domains at the apical membrane of ciliated surface cells. These results establish a previously unrecognized role for glucocorticoids in regulation of BK channels in airway epithelial cells.