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

Background and Purpose: There is a lack of potent, selective antagonists at most subtypes of P2Y receptor. The aims of this study were to characterise the pharmacological properties of the proposed P2Y ₂ receptor antagonist, AR-C118925XX, and then to use it to determine the role of P2Y ₂ receptors in the action of the P2Y ₂ agonist, UTP, in human vascular endothelial cells. Experimental Approach: Cell lines expressing native or recombinant P2Y receptors were superfused constantly, and agonist-induced changes in intracellular Ca ²⁺ levels monitored using the Ca ²⁺-sensitive fluorescent indicator, Cal-520. This set-up enabled full agonist concentration–response curves to be constructed on a single population of cells. Key Results: UTP evoked a concentration-dependent rise in intracellular Ca ²⁺ in 1321N1-hP2Y ₂ cells. AR-C118925XX (10 nM to 1 μM) had no effect per se on intracellular Ca ²⁺ but shifted the UTP concentration–response curve progressively rightwards, with no change in maximum. The inhibition was fully reversible on washout. AR-C118925XX (1 μM) had no effect at native or recombinant hP2Y ₁, hP2Y ₄, rP2Y ₆, or hP2Y ₁₁ receptors. Finally, in EAhy926 immortalised human vascular endothelial cells, AR-C118925XX (30 nM) shifted the UTP concentration–response curve rightwards, with no decrease in maximum. Conclusions and Implications: AR-C118925XX is a potent, selective and reversible, competitive P2Y ₂ receptor antagonist, which inhibited responses mediated by endogenous P2Y ₂ receptors in human vascular endothelial cells. As the only P2Y ₂-selective antagonist currently available, it will greatly enhance our ability to identify the functions of native P2Y ₂ receptors and their contribution to disease and dysfunction.