2,2$prime;-Pyridylisatogen tosylate antagonizes P2Y1 receptor signaling without affecting nucleotide binding

Abstract

The effect of 2,2′-pyridylisatogen tosylate (PIT) on the human P2Y 1 receptor and on other recombinant P2Y receptors has been studied. We first examined the modulation by PIT of the agonist-induced accumulation of inositol phosphates. PIT blocked 2-methylthio-ADP (2-MeSADP)-induced accumulation of inositol phosphates in 1321N1 astrocytoma cells stably expressing human P2Y 1 receptors in a non-competitive and concentration-dependent manner. The IC 50 for reduction of the maximal agonist effect was 0.14 μM. In contrast, MRS2179, a competitive P2Y 1 receptor antagonist, parallel-shifted the agonist concentration–response curve to the right. PIT also concentration-dependently blocked the P2Y 1 receptor signaling induced by the endogenous agonists, ADP and ATP. A simple structural analogue of PIT was synthesized and found to be inactive as a P2Y 1 receptor antagonist, suggesting that the nitroxyl group of PIT is a necessary structural component for P2Y 1 receptor antagonism. We next examined the possible modulation of the binding of the newly available antagonist radioligand for the P2Y 1 receptor, [ 3 H ] MRS2279. It was found that PIT (0.01–10 μM) did not inhibit [ 3 H ] MRS2279 binding to the human P2Y 1 receptor. PIT (10 μM) had no effect on the competition for [ 3 H ] MRS2279 binding by agonists, ADP and ATP, suggesting that its antagonism of the P2Y 1 receptor may be allosteric. PIT had no significant effect on agonist activation of other P2Y receptors, including P2Y 2 , P2Y 4 , P2Y 6 , P2Y 11 and P2Y 12 receptors. Thus, PIT selectively and non-competitively blocked P2Y 1 receptor signaling without affecting nucleotide binding.