Converging purinergic and immune signaling pathways drive IL-6 secretion by Fragile X cortical astrocytes via STAT3.

The symptoms of Fragile X syndrome (FXS) are driven in part by abnormal glial-mediated function. FXS astrocytes release elevated levels of immune-related factors interleukin-6 (IL-6) and tenascin C (TNC), and also demonstrate increased purinergic signaling, a pathway linked to signaling factor release. Here, in cortical astrocytes from the Fmr1 knockout (KO) FXS mouse model, purinergic agonism enhanced TNC secretion and STAT3 phosphorylation, two processes linked to elevated IL-6 secretion in FXS, while STAT3 knockdown and TLR4 antagonism normalized Fmr1 KO IL-6 release. We therefore suggest that purinergic signaling and immune regulatory pathways converge to drive FXS cortical pro-inflammatory responses.

Molecular interaction of HIC, an agonist of P2Y1 receptor, and its role in prostate cancer apoptosis.

Prostate cancer is a heterogeneous, slow growing asymptomatic cancer that predominantly affects man. A purinergic G-protein coupled receptor, P2Y1R, is targeted for its therapeutic value since it plays a crucial role in many key molecular events of cancer progression and invasion. Our previous study demonstrated that indoline derivative, 1 ((1-(2-Hydroxy-5-nitrophenyl) (4-hydroxyphenyl) methyl)indoline-4­carbonitrile; HIC), stimulates prostate cancer cell (PCa) growth inhibition via P2Y1R. However, the mode of interaction of P2Y1R with HIC involved in this process remains unclear. Here, we have reported the molecular interactions of HIC with P2Y1R. Molecular dynamics simulation was performed that revealed the stable specific binding of the protein-ligand complex. In vitro analysis has shown increased apoptosis of PCa-cells, PC3, and DU145, upon specific interaction of P2Y1R-HIC. This was further validated using siRNA analysis that showed a higher percentage of apoptotic cells in PCa-cells transfected with P2Y-siRNA-MRS2365 than P2Y-siRNA-HIC treatment. Decreased mitochondrial membrane potential (MMP) activity and reduced glutathione (GSH) level show their role in P2Y1R-HIC mediated apoptosis. These in silico and in vitro results confirmed that HIC could induce mitochondrial apoptotic signaling through the P2Y1R activation. Thus, HIC being a potential ligand upon interaction with P2Y1R might have therapeutic value for the treatment of prostate cancer.

G protein-coupled purinergic P2Y receptor oligomerization: Pharmacological changes and dynamic regulation.

P2Y receptors (P2YRs) are a δ group of rhodopsin-like G protein-coupled receptors (GPCRs) with many essential functions in physiology and pathology, such as platelet aggregation, immune responses, neuroprotective effects, inflammation, and cellular proliferation. Thus, they are among the most researched therapeutic targets used for the clinical treatment of diseases (e.g., the antithrombotic drug clopidogrel and the dry eye treatment drug diquafosol). GPCRs transmit signals as dimers to increase the diversity of signalling pathways and pharmacological activities. Many studies have frequently confirmed dimerization between P2YRs and other GPCRs due to their functions in cardiovascular and cerebrovascular processes in vivo and in vitro. Recently, some P2YR dimers that dynamically balance physiological functions in the body were shown to be involved in effective signal transduction and exert pathological responses. In this review, we summarize the types, pharmacological changes, and active regulators of P2YR-related dimerization, and delineate new functions and pharmacological activities of P2YR-related dimers, which may be a novel direction to improve the effectiveness of medications.

Control of Macrophage Inflammation by P2Y Purinergic Receptors.

Macrophages comprise a phenotypically and functionally diverse group of hematopoietic cells. Versatile macrophage subsets engage to ensure maintenance of tissue integrity. To perform tissue stress surveillance, macrophages express many different stress-sensing receptors, including purinergic P2X and P2Y receptors that respond to extracellular nucleotides and their sugar derivatives. Activation of G protein-coupled P2Y receptors can be both pro- and anti-inflammatory. Current examples include the observation that P2Y14 receptor promotes STAT1-mediated inflammation in pro-inflammatory M1 macrophages as well as the demonstration that P2Y11 receptor suppresses the secretion of tumor necrosis factor (TNF)-α and concomitantly promotes the release of soluble TNF receptors from anti-inflammatory M2 macrophages. Here, we review macrophage regulation by P2Y purinergic receptors, both in physiological and disease-associated inflammation. Therapeutic targeting of anti-inflammatory P2Y receptor signaling is desirable to attenuate excessive inflammation in infectious diseases such as COVID-19. Conversely, anti-inflammatory P2Y receptor signaling must be suppressed during cancer therapy to preserve its efficacy.

Adenosine Diphosphate Improves Wound Healing in Diabetic Mice Through P2Y12 Receptor Activation.

Chronic wounds are a public health problem worldwide, especially those related to diabetes. Besides being an enormous burden to patients, it challenges wound care professionals and causes a great financial cost to health system. Considering the absence of effective treatments for chronic wounds, our aim was to better understand the pathophysiology of tissue repair in diabetes in order to find alternative strategies to accelerate wound healing. Nucleotides have been described as extracellular signaling molecules in different inflammatory processes, including tissue repair. Adenosine-5'-diphosphate (ADP) plays important roles in vascular and cellular response and is immediately released after tissue injury, mainly from platelets. However, despite the well described effect on platelet aggregation during inflammation and injury, little is known about the role of ADP on the multiple steps of tissue repair, particularly in skin wounds. Therefore, we used the full-thickness excisional wound model to evaluate the effect of local ADP application in wounds of diabetic mice. ADP accelerated cutaneous wound healing, improved new tissue formation, and increased both collagen deposition and transforming growth factor-ß (TGF-ß) production in the wound. These effects were mediated by P2Y12 receptor activation since they were inhibited by Clopidogrel (Clop) treatment, a P2Y12 receptor antagonist. Furthermore, P2Y1 receptor antagonist also blocked ADP-induced wound closure until day 7, suggesting its involvement early in repair process. Interestingly, ADP treatment increased the expression of P2Y12 and P2Y1 receptors in the wound. In parallel, ADP reduced reactive oxygen species (ROS) formation and tumor necrosis factor-α (TNF-α) levels, while increased IL-13 levels in the skin. Also, ADP increased the counts of neutrophils, eosinophils, mast cells, and gamma delta (γδ) T cells (Vγ4+ and Vγ5+ cells subtypes of γδ+ T cells), although reduced regulatory T (Tregs) cells in the lesion. In accordance, ADP increased fibroblast proliferation and migration, myofibroblast differentiation, and keratinocyte proliferation. In conclusion, we provide strong evidence that ADP acts as a pro-resolution mediator in diabetes-associated skin wounds and is a promising intervention target for this worldwide problem.

Diquafosol tetrasodium elicits total cholesterol release from rabbit meibomian gland cells via P2Y2 purinergic receptor signalling.

Diquafosol tetrasodium (DQS), a purinergic P2Y2 receptor agonist, stimulates secretion of both water and mucins from the conjunctiva into tears. Hence, DQS-containing eye drops have been approved as a therapeutic option for dry eye disease in some Asian countries, including Japan. Recent clinical reports state that instilling DQS-containing eye drops significantly increases the lipid layer thickness in tears. Therefore, we examined this compound's direct actions on holocrine lipid-secreting meibomian gland cells and their function. Isolated meibomian gland cells (meibocytes) were procured from rabbits and cultivated in serum-free culture medium. Differentiated meibocytes with pioglitazone were used for the subsequent experiments. Intracellular Ca2+ signalling of the cells was dramatically elevated with DQS addition in a dose-dependent manner. This DQS-induced elevation was almost completely cancelled by the coexistence of the selective P2Y2 receptor antagonist AR-C118925XX. DQS treatment also facilitated total cholesterol (TC) release from cells into the medium. This effect of DQS on TC was suppressed significantly by the intracellular Ca2+ chelator BAPTA-AM as well as by AR-C118925XX. DNA fragmentation analysis revealed that DQS may have enhanced the apoptotic DNA fragmentation caused spontaneously by cells. Thus, DQS could stimulate meibocytes to release lipids through the P2Y2 receptor and possibly facilitate holocrine cell maturation.

P2Y2 receptor activation promotes esophageal cancer cells proliferation via ERK1/2 pathway.

Esophageal cancer is a prominent worldwide illness that is divided into two main subtypes: esophageal squamous cell carcinoma and esophageal adenocarcinoma. Mortality rates are alarming, and the understanding of the mechanisms involved in esophageal cancer development, becomes essential. Purinergic signaling is related to many diseases and among these various types of tumors. Here we studied the effects of the P2Y2 receptor activation in different types of esophageal cancer. Esophageal tissue samples of healthy controls were used for P2Y2R expression quantification. Two human esophageal cancer cell lines Kyse-450 (squamous cell carcinoma) and OE-33 (adenocarcinoma) were used to perform in vitro analysis of cell proliferation, migration, adhesion, and the signaling pathways involved in P2Y2R activation. Data showed that P2Y2R was expressed in biopsies of patients with ESCC and adenocarcinoma, as well as in the two human esophageal cancer cell lines studied. The RT-qPCR analysis demonstrated that OE-33 cells have higher P2RY2 expression than Kyse-450 squamous cell line. Results showed that P2Y2R activation, induced by ATP or UTP, promoted esophageal cancer cells proliferation and colony formation. P2Y2R blockage with the selective antagonist, AR-C 118925XX, led to decreased proliferation, colony formation and adhesion. Treatments with ATP or UTP activated ERK 1/2 pathway in ESCC and ECA cells. The P2Y2R antagonism did not alter the migration of esophageal cancer cells. Interestingly, the esophageal cancer cell lines presented a distinct profile of nucleotide hydrolysis activity. The modulation of P2Y2 receptors may be a promising target for esophageal cancer treatment.

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

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

P2Y2 Receptor Induces L. amazonensis Infection Control in a Mechanism Dependent on Caspase-1 Activation and IL-1ß Secretion.

Leishmaniasis is a neglected tropical disease caused by an intracellular parasite of the genus Leishmania. Damage-associated molecular patterns (DAMPs) such as UTP and ATP are released from infected cells and, once in the extracellular medium, activate P2 purinergic receptors. P2Y2 and P2X7 receptors cooperate to control Leishmania amazonensis infection. NLRP3 inflammasome activation and IL-1ß release resulting from P2X7 activation are important for outcomes of L. amazonensis infection. The cytokine IL-1ß is required for the control of intracellular parasites. In the present study, we investigated the involvement of the P2Y2 receptor in the activation of NLRP3 inflammasome elements (caspase-1 and 11) and IL-1ß secretion during L. amazonensis infection in peritoneal macrophages as well as in a murine model of cutaneous leishmaniasis. We found that 2-thio-UTP (a selective P2Y2 agonist) reduced parasite load in L. amazonensis-infected murine macrophages and in the footpads and lymph nodes of infected mice. The antiparasitic effects triggered by P2Y2 activation were not observed when cells were pretreated with a caspase-1 inhibitor (Z-YVAD-FMK) or in macrophages from caspase-1/11 knockout mice (CASP-1,11-/-). We also found that UTP treatment induced IL-1ß secretion in wild-type (WT) infected macrophages but not in cells from CASP-1,11-/- mice, suggesting that caspase-1 activation by UTP triggers IL-1ß secretion in L. amazonensis-infected macrophages. Infected cells pretreated with IL-1R antagonist did not show reduced parasitic load after UTP and ATP treatment. Our in vivo experiments also showed that intralesional UTP treatment reduced both parasite load (in the footpads and popliteal lymph nodes) and lesion size in wild-type (WT) and CASP-11-/- but not in CASP-1,11-/- mice. Taken together, our findings suggest that P2Y2R activation induces CASP-1 activation and IL-1ß secretion during L. amazonensis infection. IL-1ß/IL-1R signaling is crucial for P2Y2R-mediated protective immune response in an experimental model of cutaneous leishmaniasis.

ATP and ADP enhance DNA damage repair in γ-irradiated BEAS-2B human bronchial epithelial cells through activation of P2X7 and P2Y12 receptors.

Agents that promote DNA repair may be useful as radioprotectants to minimize side effects such as radiation pneumonia caused by damage to normal cells during radiation therapy to treat lung cancer. We have reported that extracellular nucleotides and nucleosides are involved in the P2 or P1 receptor-mediated DNA damage response (DDR) after γ-irradiation. Here, we investigated the effects of ATP, UTP, GTP, ITP and their metabolites on the γH2AX/53BP1 focus formation in nuclei (a measure of γ-irradiation-induced DDR) and the survival of γ-irradiated immortalized human bronchial epithelial (BEAS-2B) cells. Fluorescence immunostaining showed that ATP and ADP increase DDR and DNA repair, and exhibit radioprotective effects as evaluated by colony formation assay. These effects of ATP or ADP were blocked by inhibitors of P2X7 or P2Y12 receptor, respectively, and by ERK1/2 inhibitor. ATP and ADP enhanced phosphorylation of ERK1/2 by suppressing MKP-1 and MKP-3 expression after γ-irradiation. These results indicate that ATP and ADP exhibit radioprotective effects by phosphorylation of ERK1/2 via activation of P2X7 and P2Y12 receptors, respectively, to promote γ-irradiation-induced DDR and DNA repair. ATP and ADP appear to be candidates for radioprotectants to reduce damage to non-cancerous cells during lung cancer radiotherapy by promoting DDR and DNA repair.

Expanding Role of Dopaminergic Inhibition in Hypercapnic Responses of Cultured Rat Carotid Body Cells: Involvement of Type II Glial Cells.

Dopamine (DA) is a well-studied neurochemical in the mammalian carotid body (CB), a chemosensory organ involved in O2 and CO2/H+ homeostasis. DA released from receptor (type I) cells during chemostimulation is predominantly inhibitory, acting via pre- and post-synaptic dopamine D2 receptors (D2R) on type I cells and afferent (petrosal) terminals respectively. By contrast, co-released ATP is excitatory at postsynaptic P2X2/3R, though paracrine P2Y2R activation of neighboring glial-like type II cells may boost further ATP release. Here, we tested the hypothesis that DA may also inhibit type II cell function. When applied alone, DA (10 µM) had negligible effects on basal [Ca2+]i in isolated rat type II cells. However, DA strongly inhibited [Ca2+]i elevations (Δ[Ca2+]i) evoked by the P2Y2R agonist UTP (100 µM), an effect opposed by the D2/3R antagonist, sulpiride (1-10 µM). As expected, acute hypercapnia (10% CO2; pH 7.4), or high K+ (30 mM) caused Δ[Ca2+]i in type I cells. However, these stimuli sometimes triggered a secondary, delayed Δ[Ca2+]i in nearby type II cells, attributable to crosstalk involving ATP-P2Y2R interactions. Interestingly sulpiride, or DA store-depletion using reserpine, potentiated both the frequency and magnitude of the secondary Δ[Ca2+]i in type II cells. In functional CB-petrosal neuron cocultures, sulpiride potentiated hypercapnia-induced Δ[Ca2+]i in type I cells, type II cells, and petrosal neurons. Moreover, stimulation of type II cells with UTP could directly evoke Δ[Ca2+]i in nearby petrosal neurons. Thus, dopaminergic inhibition of purinergic signalling in type II cells may help control the integrated sensory output of the CB during hypercapnia.

Astrocytes Modulate Baroreflex Sensitivity at the Level of the Nucleus of the Solitary Tract.

Maintenance of cardiorespiratory homeostasis depends on autonomic reflexes controlled by neuronal circuits of the brainstem. The neurophysiology and neuroanatomy of these reflex pathways are well understood, however, the mechanisms and functional significance of autonomic circuit modulation by glial cells remain largely unknown. In the experiments conducted in male laboratory rats we show that astrocytes of the nucleus of the solitary tract (NTS), the brain area that receives and integrates sensory information from the heart and blood vessels, respond to incoming afferent inputs with [Ca2+]i elevations. Astroglial [Ca2+]i responses are triggered by transmitters released by vagal afferents, glutamate acting at AMPA receptors and 5-HT acting at 5-HT2A receptors. In conscious freely behaving animals blockade of Ca2+-dependent vesicular release mechanisms in NTS astrocytes by virally driven expression of a dominant-negative SNARE protein (dnSNARE) increased baroreflex sensitivity by 70% (p < 0.001). This effect of compromised astroglial function was specific to the NTS as expression of dnSNARE in astrocytes of the ventrolateral brainstem had no effect. ATP is considered the principle gliotransmitter and is released by vesicular mechanisms blocked by dnSNARE expression. Consistent with this hypothesis, in anesthetized rats, pharmacological activation of P2Y1 purinoceptors in the NTS decreased baroreflex gain by 40% (p = 0.031), whereas blockade of P2Y1 receptors increased baroreflex gain by 57% (p = 0.018). These results suggest that glutamate and 5-HT, released by NTS afferent terminals, trigger Ca2+-dependent astroglial release of ATP to modulate baroreflex sensitivity via P2Y1 receptors. These data add to the growing body of evidence supporting an active role of astrocytes in brain information processing.SIGNIFICANCE STATEMENT Cardiorespiratory reflexes maintain autonomic balance and ensure cardiovascular health. Impaired baroreflex may contribute to the development of cardiovascular disease and serves as a robust predictor of cardiovascular and all-cause mortality. The data obtained in this study suggest that astrocytes are integral components of the brainstem mechanisms that process afferent information and modulate baroreflex sensitivity via the release of ATP. Any condition associated with higher levels of "ambient" ATP in the NTS would be expected to decrease baroreflex gain by the mechanism described here. As ATP is the primary signaling molecule of glial cells (astrocytes, microglia), responding to metabolic stress and inflammatory stimuli, our study suggests a plausible mechanism of how the central component of the baroreflex is affected in pathological conditions.

Cardioprotective properties of the platelet P2Y12 receptor inhibitor prasugrel on cardiac ischemia/reperfusion injury.

OBJECTIVE: The effects of prasugrel, a third-generation thienopyridine, on myocardial infarction, and ischemia-induced ventricular arrhythmias was evaluated in open-chest anesthetized rats. The role of protein kinase C and phosphoinositide 3-kinase pathways in these effects was also examined. METHODS: The effect of P2Y12 receptor inhibition by prasugrel (3-10 mg/kg, po) on infarct size after 30-min coronary artery occlusion and 120-min reperfusion or on arrhythmias after 7-min coronary occlusion and 7-min reperfusion was evaluated. RESULTS: In the control group, 31.25 ± 3.01% of the risk zone infarcted. At both prasugrel doses, infarct size was significantly smaller than that in the control group: 5.03 ± 0.81% for 3 mg/kg (p < 0.0001), and 8.78 ± 2.04% for 10 mg/kg (p < 0.0001). The protein kinase C antagonist chelerythrine abolished the anti-infarct effect of prasugrel at 24.77 ± 1.73% as did the phosphoinositide 3-kinase antagonist wortmannin abolished the anti-infarct effect of prasugrel at 27.45 ± 2.74%. Ten mg/kg prasugrel reduced the duration of VT (p = 0.0152 vs control), and wortmannin, but not chelerythrine, reversed the effect of prasugrel on arrhythmias (p = 0.0295). CONCLUSION: The selective P2Y12 inhibitor prasugrel provides effective protection against myocardial infarction and ischemia-induced ventricular arrhythmias in rats. As in ischemic postconditioning, protein kinase C and phosphoinositide 3-kinase signaling pathways play a role in this protection.

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 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,ß-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,ß-MeATP. Thus, ATP involves the CO-dependent signaling cascade through P2X receptors.

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

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

Renal Na+ excretion consequent to pharmacogenetic activation of Gq-DREADD in principal cells.

Stimulation of metabotropic Gq-coupled purinergic P2Y2 receptors decreases activity of the epithelial Na+ channel (ENaC) in renal principal cells of the distal nephron. The physiological consequences of P2Y2 receptor signaling disruption in the P2Y2 receptor knockout mouse are decreased Na+ excretion and increased arterial blood pressure. However, because of the global nature of this knockout model, the quantitative contribution of ENaC and distal nephron compared with that of upstream renal vascular and tubular elements to changes in urinary excretion and arterial blood pressure is obscure. Moreover, it is uncertain whether stimulation of P2Y2 receptor inhibition of ENaC is sufficient to drive renal (urinary) Na+ excretion (UNaV). Here, using a pharmacogenetic approach and selective agonism of the P2Y2 receptor, we test the sufficiency of targeted stimulation of Gq signaling in principal cells of the distal nephron and P2Y2 receptors to increase UNaV. Selective stimulation of the P2Y2 receptor with the ligand MRS2768 decreased ENaC activity in freshly isolated tubules (as assessed by patch-clamp electrophysiology) and increased UNaV (as assessed in metabolic cages). Similarly, selective agonism of hM3Dq-designer receptors exclusively activated by designer drugs (DREADD) restrictively expressed in principal cells of the distal nephron with clozapine- N-oxide decreased ENaC activity and, consequently, increased UNaV. Clozapine- N-oxide, when applied to control littermates, failed to affect ENaC and UNaV. This study represents the first use of pharmacogenetic (DREADD) technology in the renal tubule and demonstrated that selective activation of the P2Y2 receptor and Gq signaling in principal cells is sufficient to promote renal salt excretion.

Deciphering biased inverse agonism of cangrelor and ticagrelor at P2Y12 receptor.

P2Y12 receptor (P2Y12-R) is one of the major targets for drug inhibiting platelet aggregation in the treatment/prevention of arterial thrombosis. However, the clinical use of P2Y12-R antagonists faces some limitations, such as a delayed onset of action (clopidogrel) or adverse effect profile (ticagrelor, cangrelor), justifying the development of a new generation of P2Y12-R antagonists with a better clinical benefit-risk balance. Although the recent concept of biased agonism offers the possibility to alleviate undesirable adverse effects while preserving therapeutic outcomes, it has never been explored at P2Y12-R. For the first time, using highly sensitive BRET2-based probes, we accurately delineated biased ligand efficacy at P2Y12-R in living HEK293T cells on G protein activation and downstream effectors. We demonstrated that P2Y12-R displayed constitutive Gi/o-dependent signaling that is impaired by the R122C mutation, previously associated with a bleeding disorder. More importantly, we reported the biased inverse agonist efficacy of cangrelor and ticagrelor that could underlie their clinical efficacy. Our study points out that constitutive P2Y12-R signaling is a normal feature of the receptor that might be essential for platelets to respond faster to a vessel injury. From a therapeutic standpoint, our data suggest that the beneficial advantages of antiplatelet drugs might be more related to inverse agonism at P2Y12-R than to antagonism of ADP-mediated signaling. In the future, deciphering P2Y12-R constitutive activity should allow the discovery of more selective biased P2Y12-R blockers demonstrating therapeutic advantages over classical antiplatelet drugs by improving therapeutic outcomes and concomitantly relieving undesirable adverse effects.

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 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.