Role of the Purinergic P2Y2 Receptor in Pulmonary Hypertension

Pulmonary arterial hypertension (PAH), group 1 pulmonary hypertension (PH), is a fatal disease that is characterized by vasoconstriction, increased pressure in the pulmonary arteries, and right heart failure. PAH can be described by abnormal vascular remodeling, hyperproliferation in the vasculature, endothelial cell dysfunction, and vascular tone dysregulation. The disease pathomechanisms, however, are as yet not fully understood at the molecular level. Purinergic receptors P2Y within the G-protein-coupled receptor family play a major role in fluid shear stress transduction, proliferation, migration, and vascular tone regulation in systemic circulation, but less is known about their contribution in PAH. Hence, studies that focus on purinergic signaling are of great importance for the identification of new therapeutic targets in PAH. Interestingly, the role of P2Y2 receptors has not yet been sufficiently studied in PAH, whereas the relevance of other P2Ys as drug targets for PAH was shown using specific agonists or antagonists. In this review, we will shed light on P2Y receptors and focus more on the P2Y2 receptor as a potential novel player in PAH and as a new therapeutic target for disease management.

P2Y receptors in GtoPdb v.2021.3

P2Y receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on P2Y Receptors [3, 5, 192]) are activated by the endogenous ligands ATP, ADP, uridine triphosphate, uridine diphosphate and UDP-glucose. The relationship of many of the cloned receptors to endogenously expressed receptors is not yet established and so it might be appropriate to use wording such as 'uridine triphosphate-preferring (or ATP-, etc.) P2Y receptor' or 'P2Y1-like', etc., until further, as yet undefined, corroborative criteria can be applied [47, 110, 190, 383, 396]. Clinically used drugs acting on these receptors include the dinucleoside polyphosphate diquafosol, agonist of the P2Y2 receptor subtype, approved in Japan for the management of dry eye disease [241], and the P2Y12 receptor antagonists prasugrel, ticagrelor and cangrelor, all approved as antiplatelet drugs [53, 323].

P2Y2 and P2X4 Receptors Mediate Ca2+ Mobilization in DH82 Canine Macrophage Cells

Purinergic receptors of the P2 subclass are commonly found in human and rodent macrophages where they can be activated by adenosine 5′-triphosphate (ATP) or uridine 5′-triphosphate (UTP) to mediate Ca2+ mobilization, resulting in downstream signalling to promote inflammation and pain. However, little is understood regarding these receptors in canine macrophages. To establish a macrophage model of canine P2 receptor signalling, the expression of these receptors in the DH82 canine macrophage cell line was determined by reverse transcription polymerase chain reaction (RT-PCR) and immunocytochemistry. P2 receptor function in DH82 cells was pharmacologically characterised using nucleotide-induced measurements of Fura-2 AM-bound intracellular Ca2+. RT-PCR revealed predominant expression of P2X4 receptors, while immunocytochemistry confirmed predominant expression of P2Y2 receptors, with low levels of P2X4 receptor expression. ATP and UTP induced robust Ca2+ responses in the absence or presence of extracellular Ca2+. ATP-induced responses were only partially inhibited by the P2X4 receptor antagonists, 2′,3′-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), paroxetine and 5-BDBD, but were strongly potentiated by ivermectin. UTP-induced responses were near completely inhibited by the P2Y2 receptor antagonists, suramin and AR-C118925. P2Y2 receptor-mediated Ca2+ mobilization was inhibited by U-73122 and 2-aminoethoxydiphenyl borate (2-APB), indicating P2Y2 receptor coupling to the phospholipase C and inositol triphosphate signal transduction pathway. Together this data demonstrates, for the first time, the expression of functional P2 receptors in DH82 canine macrophage cells and identifies a potential cell model for studying macrophage-mediated purinergic signalling in inflammation and pain in dogs.

P2Y2 Receptor Induces L. amazonensis Infection Control in a Mechanism Dependent on Caspase-1 Activation and IL-1β 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-1β release resulting from P2X7 activation are important for outcomes of L. amazonensis infection. The cytokine IL-1β 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-1β 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-1β 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-1β 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-1β secretion during L. amazonensis infection. IL-1β/IL-1R signaling is crucial for P2Y2R-mediated protective immune response in an experimental model of cutaneous leishmaniasis.

Exercise training ameliorates cerebrovascular dysfunction in a murine model of Alzheimer’s disease: role of the P2Y2 receptor and endoplasmic reticulum stress

A limited study has investigated whether exercise training can improve cerebrovascular function in Alzheimer's disease. The novel findings of the study are that exercise training improves cerebrovascular dysfunction through enhancing P2Y2 receptor-mediated eNOS signaling and reducing ER stress-associated pathways in AD. These data suggest that exercise training, which regulates P2Y2 receptor and ER stress in AD brain, is a potential therapeutic strategy for Alzheimer's disease.