ESTIMATION OF EDRF AND NITRIC-OXIDE RELEASE USING [H-3] GTP-LABELED HUMAN PLATELETS

We have developed a new bioassay for endothelium-derived relaxing fact or (EDRF) or nitric oxide (NO) using human [H-3]guanosine triphosphate (GTP)-labeled platelets. The labeled platelets were preincubated with isobutyl-methylxanthine and co-cultured with endothelial cells and th e [H-3]cyclic guanosine monophosphate (cGMP) formed was isolated by io n-exchange chromatography. Endothelial cells, either in monolayer cell s or in suspension, increased platelet cGMP accumulation dose-dependen tly, a significant increase being detected with 5,000 endothelial cell s or more/assay when suspended cells were used. Co-culturing with the same number of skin fibroblasts failed to elevate platelet cGMP. Prein cubation of endothelial cells with bradykinin and superoxide dismutase (SOD) synergistically potentiated the increase in platelet cGMP, but was attenuated by N-omega-nitro-L-arginine, with partial restoration b y L-arginine but not by D-arginine. These compounds, however, did not affect cGMP accumulation by sodium nitroprusside. Moreover, preincubat ion of the labeled platelets with the NO synthase inhibitor prior to E DRF assay had no effect. We conclude that [H-3]GTP-labeled platelets c ould provide a simple, sensitive and specific bioassay for estimating EDRF or NO release.