Inhibition of ecto-ATPase by PPADS, suramin and reactive blue in endothelial cells, C-6 glioma cells and RAW 264.7 macrophages

1 Previous studies have shown that bovine pulmonary artery endothelium (CPAE) has P-2Y and P-2U purinoceptors, rat C-6 glioma cells have P-2U purinoceptors and mouse RAW 264.7 cells have pyrimidinoceptors, all of which are coupled to phosphoinositide-specific phospholipase C (PI-PLC). The dual actions of PPADS, suramin and reactive blue as antagonists of receptor subtypes and ecto-ATPase inhibitors were studied in these three cell types. 2 In CPAE, suramin, at 3-100 mu M, competitively inhibited the PI responses induced by 2MeSATP and UTP, with pA(2) values of 5.5 +/- 0.3 and 4.4 +/- 0.4, respectively. Reactive blue, at 1-3 mu M, produced shifts to the right of the 2MeSATP and UTP curves, but no further right shift at 10 mu M. PPADS, at 10 mu M, caused a 3 fold right shift of the 2MeSATP curve, but no further shift at concentrations up to 100 mu M. In contrast, a dose-dependent shift to the left of the UTP curve and a weak inhibition of the ATP response were seen with PPADS. 3 In RAW 264.7 cells, suramin and reactive blue, but not PPADS, competitively inhibited the UTP response, with pA(2) values of 4.8 +/- 0.5 and 5.8 +/- 0.7, respectively. 4 In C-6 glioma cells, although suramin and reactive blue inhibited the ATP response, a potentiation effect on ATP and UTP responses was seen with PPADS. 5 The ecto-ATPase inhibitory activity of these three receptor antagonists were determined. All three inhibited ecto-ATPase present in CPAE, C-6 and RAW 264.7 cells, with IC50 values of 4, 4.8 and 4.7 for PPADS, 4, 4.4 and much greater than 4 for suramin, and 4.5, 4.7 and 4.7 for reactive blue. 6 This study indicates that PPADS, suramin and reactive blue are ecto-ATPase inhibitors. This property, combined with their antagonistic selectivity for receptor subtypes, can result in inhibition of, potentiation of, or lack of effect on agonist-mediated PI responses. Reactive blue is a more potent antagonist than suramin on P-2Y, P-2U and pyrimidinoceptors, and PPADS is a weak antagonist for P-2Y receptors.