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

M. O. Muoboghare, R. Drummond, C. Kennedy. SIPBS, University of strathclyde, Glasgow, United Kingdom.

Introduction: P2Y receptors are a family of eight G protein-coupled receptors that mediate the actions of endogenous nucleotides, such as uridine 5'-triphosphate (UTP) (1,2). The physiological functions of many of the subtypes are unclear due to the limited selectivity and low potency of most currently available antagonists. A putative P2Y₂ antagonist, AR-C118925XX, has recently become available, so the aims here were to quantify the action of AR-C118925XX at recombinant P2Y₂ receptors and then to determine the role of native P2Y₂ receptors in the actions of UTP in human vascular endothelial cells.

Method: EAhy926 cells, immortalised human umbilical vein endothelial cells (3), and 1321N1 cells stably expressing recombinant human P2Y₁, P2Y_2, P2Y₄, P2Y₁₁ or rat P2Y₆ receptors, were grown on glass coverslips. Following incubation with the Ca²⁺-sensitive dye, Cal-520AM (5 μM), they were placed in a fluorimeter and intracellular Ca²⁺ measured. Cells were superfused continuously and agonists were applied in the superfusate (ADP-P2Y₁; UTP-P2Y₂, EAhy926; ATP-P2Y₄, P2Y₁₁; UDP-P2Y₆), before and after 5 min superfusion with AR-C118925XX. Where appropriate the Hill equation was fitted to the data, and antagonist potency calculated using the Gaddum-Schild equation or a Schild plot.

Results: UTP (10nM-3μM) evoked a concentration-dependent rise in intracellular Ca²⁺ in 1321N1 cells expressing recombinant P2Y₂ receptors (EC₅₀=54nM, 95% cl=43-67nM, n=5). AR-C118925XX (10nM-1μM), produced a progressive rightward shift in the UTP concentration-response curve, with no effect on maximum response (n=6 each). Schild analysis gave a pA₂=8.30 and slope=0.985. In contrast, AR-C118925XX (1μM), a concentration 200x greater than its K_B at P2Y₂ receptors, had no effect at recombinant P2Y_1, P2Y₄, P2Y₆ and P2Y₁₁ receptors (n=5 each). UTP (100nM-30μM) also increased intracellular Ca²⁺ in EAhy926 endothelial cells in a concentration-dependent manner (EC₅₀=680nM, 95% cl=506-912nM, n=5). AR-C118925XX (30nM), shifted the UTP curve rightwards (EC₅₀=7.6μM, 95% cl. 4.3-13.2μM, n=5), with no decrease in maximum response. Gaddum-Schild analysis gave a K_B=3.0nM (95% cl=1.3-4.6nM).

Conclusion: These data show that AR-C118925XX is a potent and selective P2Y₂ antagonist, which enabled us to identify P2Y₂ receptors as the P2Y subtype that mediates UTP-evoked increases in intracellular Ca²⁺ in human endothelial cells. Currently, AR-C118925XX is the only selective P2Y₂ antagonist available and so will be invaluable in identifying the physiological functions of other native P2Y₂ receptors.

References:

(1) Abbracchio MP et al. (2006). Pharmacol Rev 58: 281-341.

(2) Kennedy C et al. (2013). Fut Med Chem 5: 355-357.

(3) Graham A et al. (1996). Br J Pharmacol 117: 1341-1347.