Although P2 receptors mediate a myriad of physiological effects of extracellular
adenine nucleotides, study of this broad class of receptors has been compromised by a lack of P2 receptor-selective antagonist molecules. The
adenine nucleotide-promoted
inositol lipid hydrolysis response 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' -phosphosulfate (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 result of interaction with the P2Y receptor, because A3P5PS had no effect on activation of
phospholipase C through the
beta-adrenergic receptor and produced a concentration-dependent inhibition of 2MeSATP-promoted activity over the same range of A3P5PS concentrations that alone caused a small activation of
phospholipase C. Increasing concentrations of A3P5PS 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 pKB of 6.46 +/- 0.17. The presence of a
phosphate in the 2'- or 3'-position appears to be crucial for antagonist activity, because adenosine-3' -phosphate-5'-
phosphate (A3P5P) and adenosine-2'- phosphate-5'-phosphate also exhibited competitive 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 cloned human
P2Y1 receptor stably expressed in 1321N1 human
astrocytoma cells. Moreover, both A3P5PS and A3P5P were devoid of agonist activity at the human
P2Y1 receptor. The effects of these 2'- and 3'-phosphate analogues were specific for the
phospholipase C-coupled
P2Y1 receptor, because no agonistic or antagonistic effects on the
adenylyl cyclase-coupled P2Y receptor of C6
glioma cells or on P2Y2, P2Y4, or
P2Y6 receptors stably expressed in 1321N1 human
astrocytoma cells were observed. These results describe specific competitive antagonism of the
P2Y1 receptor by an
adenine nucleotide derivative and provide a potential new avenue for P2
receptor drug development.