ATP, an agonist at the rat P2Y4 receptor, is an antagonist at the human P2Y4 receptor

The nucleotide selectivities of the human P2Y(4) (hP2Y(4)) and rat P2Y(4) (rP2Y(4)) receptor stably expressed in 1321N1 human astrocytoma cells were determined by measuring increases in intracellular [Ca2+] under conditions that minimized metabolism, bioconversion, and endogenous nucleotide release. In cells expressing the hP2Y4 receptor, UTP, GTP, and ITP all increased intracellular [Ca2+] with a rank order of potency of UTP (0.55) > GTP (6.59) = ITP (7.38), (EC50, mu M). ATP, CTP, xanthine 5'-triphosphate (XTP), and diadenosine 5',5'''-P-1,P-4-tetraphosphate (Ap(4)A), all at 100 mu M, were inactive at the hP2Y4 receptor. In cells expressing the rP2Y4 receptor, all seven nucleotides increased intracellular [Ca2+] with similar maximal effects and a rank order of potency of UTP (0.20) > ATP (0.51) > Ap4(A) (1.24) approximate to ITP (1.82) approximate to GTP (2.28) > CTP (7.24). XTP (22.9). Because ATP is inactive at the hP2Y(4) receptor, we assessed whether ATP displayed antagonist activity. When coapplied, ATP shifted the concentration-response curve to UTP rightward in a concentration-dependent manner, with no change in the maximal response. A Schild plot derived from these data gave a pA(2) value of 6.15 (K-B = 708 nM) and a slope near unity. Additionally, CTP and Ap(4)A (each at 100 mu M) inhibited the response to an EC50 concentration of UTP by similar to 40 and similar to 50%, respectively, whereas XTP had no effect. The inhibitory effects of ATP, CTP, and Ap(4)A were reversible on washout. Thus, ATP is a potent agonist at the rP2Y(4) receptor but is a competitive antagonist with moderate potency at the hP2Y(4) receptor.