Estimation of EDRF and nitric oxide release using [3H]GTP-labeled human platelets

We have developed a new bioassay for endothelium-derived relaxing factor (EDRF) or nitric oxide (NO) using human [3H]guanosine triphosphate (GTP)-labeled platelets. The labeled platelets were preincubated with isobutyl-methylxanthine and co-cultured with endothelial cells and the [3H]cyclic guanosine monophosphate (cGMP) formed was isolated by ion-exchange chromatography. Endothelial cells, either in monolayer cells or in suspension, increased platelet cGMP accumulation dose-dependently, a significant increase being detected with 5000 endothelial cells or more/assay when suspended cells were used. Co-culturing with the same number of skin fibroblasts failed to elevate platelet cGMP. Preincubation of endothelial cells with bradykinin and superoxide dismutase (SOD) synergistically potentiated the increase in platelet cGMP, but was attenuated by Nomega-nitro-L-arginine, with partial restoration by L-arginine but not by D-arginine. These compounds, however, did not affect cGMP accumulation by sodium nitroprusside. Moreover, preincubation of the labeled platelets with the NO synthase inhibitor prior to EDRF assay had no effect. We conclude that [3H]GTP-labeled platelets could provide a simple, sensitive and specific bioassay for estimating EDRF or NO release.