Inhibition of ecto-ATPase by PPADS, suramin and reactive blue in endothelial cells, C₆ 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 C₆ 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 μM, competitively inhibited the PI responses induced by 2MeSATP and UTP, with pA₂ values of 5.5 ± 0.3 and 4.4 ± 0.4, respectively. Reactive blue, at 1-3 μM, produced shifts to the right of the 2MeSATP and UTP curves, but no further right shift at 10 μM. PPADS, at 10 μM, caused a 3 fold right shift of the 2MeSATP curve, but no further shift at concentrations up to 100 μM. 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 pA₂ values of 4.8 ± 0.5 and 5.8 ± 0.7, respectively. 4. In C₆ 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, C₆ and RAW 264.7 cells, with IC₅₀ values of 4, 4.8 and 4.7 for PPADS, 4, 4.4 and >> 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.