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 (KB = 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.
Footnotes
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Send reprint requests to: Dr. Robert Nicholas, Department of Pharmacology, University of North Carolina, CB #7365 Mary Ellen Jones Bldg., Chapel Hill, NC 27599-7365. E-mail:nicholas{at}med.unc.edu
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This work was supported by United States Public Health Service Grant GM38213 (T.K.H., R.A.N.) and grants from The Wellcome Trust and The Caledonian Research Foundation (C.K.). During this study, R.A.N. was an Established Investigator of the American Heart Association.
- Abbreviations:
- hP2Y4
- human P2Y4receptor
- Ap4A
- diadenosine 5′, 5‴-P1,P4-tetraphosphate
- rP2Y4
- rat P2Y4 receptor
- XTP
- xanthine 5′-triphosphate
- NDPK
- nucleoside diphosphokinase
- Received September 20, 1999.
- Accepted January 18, 2000.
- The American Society for Pharmacology and Experimental Therapeutics
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