Inhibition of ecto-ATPase by the P-2 purinoceptor agonists, ATP gamma S, alpha,beta-methylene-ATP, and AMP-PNP, in endothelial cells

Ecto ATPase is a plasma membrane-bound enzyme that sequentially dephosphorylates extracellular nucleotides such as ATP. This breakdown of ATP and other nucleotides makes it difficult to characterize and classify P-2 purinoceptors. We have previously shown that the P-2 purinergic antagonists, PPADS, suramin and reactive blue, act as ecto-ATPase inhibitors in various cell lines. Here, we show that the P-2 purinergic agonists, ATP gamma S, alpha,beta-methylene ATP (alpha,beta-MeATP) and AMP-PNP, inhibit the ecto-ATPase of bovine pulmonary artery endothelial cells (CPAE), with pIC(50) values of 5.2, 4.5 and 4.0, respectively, In CPAE, FPL67156, a selective ecto-ATPase inhibitor, also inhibits ecto-ATPase activity, with a pIC(50) value of 4.0. In addition, alpha,beta-MeATP (3-100 mu M), which itself does not induce phosphoinositide (PI) turnover, left-shifted the agonist-concentration effect (E/[A]) curves for ATP, 2MeS-ATP and UTP by approximate 100-300 fold, while those for ATP gamma S and AMP-PNP were only shifted approximately 2-3 fold. Moreover, in the presence of alpha,beta-MeATP, not only was the potentiation effect of PPADS on the UTP response lost, but a slight inhibition of the UTP response by PPADS was also seen. Thus, we conclude that the action of ATP gamma S, alpha,beta-MeATP and AMP-PNP as ecto-ATPase inhibitors account for their high agonist potency, and also provide information for the development of ecto ATPase inhibitors of high selectivity and potency. (C) 1997 Academic Press.