Peroxynitrite-induced Accumulation of Cyclic GMP in Endothelial Cells and Stimulation of Purified Soluble Guanylyl Cyclase

Abstract

Peroxynitrite (ONOO-) is widely recognized as mediator of NO toxicity, but recent studies have indicated that this compound may also have physiological activity and induce vascular relaxation as well as inhibition of platelet aggregation. We found that ONOO- induced a pronounced increase in endothelial cyclic GMP levels, and that this effect was significantly attenuated by pretreatment of the cells with GSH-depleting agents. In the presence of 2 mM GSH, ONOO- stimulated purified soluble guanylyl cyclase with a half-maximally effective concentration of about 20 microM. In contrast to the NO donor 2,2-Diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO), ONOO- was completely inactive in the absence of GSH, indicating that thiol-mediated bioactivation of ONOO- is involved in enzyme stimulation. Studies on the reaction between ONOO- and GSH revealed that about 1% of ONOO- was non-enzymatically converted to S-nitrosoglutathione. The authentic nitrosothiol was found to be stable in solution, but slowly decomposed in the presence of GSH. GSH-induced decomposition of S-nitrosoglutathione was apparently catalyzed by trace metals and was accompanied by a sustained release of NO and a 40-100-fold increase in its potency to stimulate purified soluble guanylyl cyclase. Our data suggest that the biologic activity of ONOO- involves S-nitrosation of cellular thiols resulting in NO-mediated cyclic GMP accumulation.