IDENTIFICATION OF COMPETITIVE ANTAGONISTS OF THE P2Y(1) RECEPTOR

Although P2 receptors mediate a myriad of physiological effects of ext racellular adenine nucleotides, study of this broad class of receptors has been compromised by a lack of P2 receptor-selective antagonist mo lecules. The adenine nucleotide-promoted inositol lipid hydrolysis res ponse of turkey erythrocyte membranes, which has been used extensively as a model for P2Y receptors, has been applied to identify molecules that competitively block these receptors. Adenosine-3'-phosphate-5'-ph osphosulfate (A3P5PS) promoted activation of phospholipase C that was only 10-25% of that observed with the full P2Y receptor agonists ATP, ADP, and 2-methylthio-ATP (2MeSATP). The small stimulatory effects of A3P5PS were saturable. Moreover, these effects were entirely the resul t of interaction with the P2Y receptor, because A3P5PS had no effect o n activation of phospholipase C through the beta-adrenergic receptor a nd produced a concentration-dependent inhibition of 2MeSATP-promoted a ctivity over the same range of A3P5PS concentrations that alone caused a small activation of phospholipase C. Increasing concentrations of A 3P5PS produced a rightward shift of the concentration-effect curve for 2MeSATP, and Schild transformation of these data revealed that A3P5PS is a competitive P2Y receptor antagonist with a pK(B) of 6.46+/-0.17. The presence of a phosphate in the 2'- or 3'-position appears to be c rucial for antagonist activity, because adenosine-3'-phosphate-5'-phos phate (A3P5P) and adenosine-2'-phosphate-5'-phosphate also exhibited c ompetitive antagonist/partial agonist activities. Other 3'-substituted analogues, such as 3'-amino-ATP and 3'-benzoylbenzoyl-ATP, were full agonists with no antagonist activity. A3P5PS, A3P5P, and adenosine-2', 5'-diphosphate also were competitive antagonists in studies with the c loned human P2Y, receptor stably expressed in 1321N1 human astrocytoma cells. Moreover, both A3P5PS and A3P5P were devoid of agonist activit y at the human P2Y, receptor. The effects of these 2'- and 3'-phosphat e analogues were specific for the phospholipase C-coupled P2Y, recepto r, because no agonistic or antagonistic effects on the adenylyl cyclas e-coupled P2Y receptor of C-6 glioma cells or on P2Y(2), P2Y(4), or P2 Y(6) receptors stably expressed in 1321N1 human astrocytoma cells were observed. These results describe specific competitive antagonism of t he P2Y(1) receptor by an adenine nucleotide derivative and provide a p otential new avenue for P2 receptor drug development.