Anales RANF

P.17 CHARACTERISATION OF P2Y 2 RECEPTORS IN HUMAN VASCULAR ENDOTHELIAL CELLS USING AR-C118925XX, A POTENT AND SELECTIVE P2Y 2 ANTAGONIST C. Kennedy, R. Drummond, M.O. Muoboghare. 1 University of Strathclyde, Glasgow, Scotland The physiological functions of many of the eight P2Y receptor subtypes are unclear due to the limited selectivity and low potency of most antagonists. A P2Y 2 antagonist, AR- C118925XX, is now available, so the aims were to quantify the action of AR- C118925XX at recombinant P2Y 2 receptors and then to determine the role of native P2Y 2 receptors in the actions of UTP in human vascular endothelial cells. Human EAhy926 umbilical vein endothelial cells, and 1321N1 cells stably expressing recombinant human P2Y 1 , P2Y 2, P2Y 4 , P2Y 11 or rat P2Y 6 receptors, were grown on glass coverslips. Following incubation with the Ca 2+ -sensitive dye, Cal-520AM, they were placed in a fluorimeter and intracellular Ca 2+ measured. Cells were superfused continuously and agonists applied in the superfusate before and after 5 min superfusion with AR-C118925XX. Data were quantified using the Hill equation, the Gaddum- Schild equation or a Schild plot, as appropriate. UTP evoked a concentration-dependent rise in intracellular Ca 2+ in 1321N1-P2Y 2 cells (EC 50 =54nM). AR-C118925XX (10nM-1µM) had no effect per se on intracellular Ca 2+ , but shifted the UTP concentration-response curve progressively rightwards, with no change in maximum. Schild analysis gave a pA 2 =8.30 and slope=0.985. In contrast, AR-C118925XX (1µM), a concentration 200x greater than its K B at P2Y 2 receptors, had no effect at recombinant P2Y 1, P2Y 4 , P2Y 6 and P2Y 11 receptors. UTP also increased intracellular Ca 2+ in EAhy926 cells in a concentration-dependent manner (EC 50 =680nM). AR-C118925XX (30nM), shifted the UTP curve rightwards (EC 50 =7.6µM), with no decrease in maximum. Gaddum-Schild analysis gave a K B =3.0nM. These data show that AR-C118925XX is a potent and selective P2Y 2 antagonist, which enabled us to identify P2Y 2 receptors as the P2Y subtype that mediates UTP-evoked increases in intracellular Ca 2+ in human endothelial cells. Currently, AR-C118925XX is the only selective P2Y 2 antagonist available and so will be invaluable in identifying the physiological functions of other native P2Y 2 receptors.