Abstract

The nucleotide selectivities of the human P2Y₄(hP2Y₄) and rat P2Y₄ (rP2Y₄) 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, μM). ATP, CTP, xanthine 5′-triphosphate (XTP), and diadenosine 5′,5‴-P¹,P⁴-tetraphosphate (Ap₄A), all at 100 μM, were inactive at the hP2Y₄ receptor. In cells expressing the rP2Y₄receptor, all seven nucleotides increased intracellular [Ca²⁺] with similar maximal effects and a rank order of potency of UTP (0.20) > ATP (0.51) > Ap₄A (1.24) ≈ ITP (1.82) ≈ GTP (2.28) > CTP (7.24) > XTP (22.9). Because ATP is inactive at the hP2Y₄ 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 pA₂ value of 6.15 (K_B = 708 nM) and a slope near unity. Additionally, CTP and Ap₄A (each at 100 μM) inhibited the response to an EC₅₀ concentration of UTP by ∼40 and ∼50%, respectively, whereas XTP had no effect. The inhibitory effects of ATP, CTP, and Ap₄A were reversible on washout. Thus, ATP is a potent agonist at the rP2Y₄receptor but is a competitive antagonist with moderate potency at the hP2Y₄ receptor.