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

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 P2Y2 receptor antagonist, AR-C118925XX, and then to use it to determine the role of P2Y2 receptors in the action of the P2Y2 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 Ca2+ levels monitored using the Ca2+ -sensitive fluorescent indicator, Cal-520. This set-up enabled full agonist concentration-response curves to be constructed on a single population of cells. Key

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 P2Y2 receptor antagonist, AR-C118925XX, and then to use it to determine the role of P2Y2 receptors in the action of the P2Y2 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 Ca2+ levels monitored using the Ca2+ -sensitive fluorescent indicator, Cal-520. This set-up enabled full agonist concentration-response curves to be constructed on a single population of cells. Key

UTP evoked a concentration-dependent rise in intracellular Ca2+ in 1321N1-hP2Y2 cells. AR-C118925XX (10 nM to 1 μM) had no effect per se on intracellular Ca2+ 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 hP2Y1 , hP2Y4 , rP2Y6 , or hP2Y11 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 P2Y2 receptor antagonist, which inhibited responses mediated by endogenous P2Y2 receptors in human vascular endothelial cells. As the only P2Y2 -selective antagonist currently available, it will greatly enhance our ability to identify the functions of native P2Y2 receptors and their contribution to disease and dysfunction.