A new pathway of nitric oxide cyclic GMP signaling involving S-nitrosoglutathione

Nitric oxide (NO), a physiologically important activator of soluble guanylyl cyclase (sGC), is synthesized from L-arginine and O-2 in a reaction catalyzed by NO synthases (NOS), Previous studies with purified NOS failed to detect formation of free NO, presumably due to a fast inactivation of NO by simultaneously produced superoxide (O-2(radical anion)). To characterize the products involved in NOS-induced sGC activation, we measured the formation of cyclic 3',5'-guanosine monophosphate (cGMP) by purified sGC incubated in the absence and presence of GSH (1 mM) with drugs releasing different NO-related species or with purified neuronal NOS. Basal sGC activity was 0.04 +/- 0.01 and 0.19 +/- 0.06 mu mol of cGMP x mg(-1) x min(-1) without and with 1 mM GSH, respectively. The NO donor DEA/NO activated sGC in a GSH-independent manner, Peroxynitrite had no effect in the absence of GSH but significantly stimulated the enzyme in the presence of the thiol (3.45 a 0.60 mu mol of cGMP x mg(-1) x min(-1)). The NO/O-2(radical anion) donor SIN-1 caused only a slight accumulation of cGMP in the absence of GSH but was almost as effective as DEA/NO in the presence of the thiol, The profile of sGC activation by Ca2+/calmodulin-activated NOS resembled that of SIN-1; at a maximally active concentration of 200 ng/0.1 ml, NOS increased sGC activity to 1.22 +/- 0.12 and 8.51 +/- 0.88 mu mol of cGMP x mg(-1) x min(-1) in the absence and presence of GSH, respectively, The product of NOS and GSH was identified as the thionitrite GSNO, which activated sGC through Cu+-catalyzed release of free NO. In contrast to S-nitrosation by peroxynitrite, the novel NO/O-2(radical anion)-triggered pathway was very efficient (25-45% GSNO) and insensitive to CO2. Cu+-specific chelators inhibited bradykinin-induced cGMP release from rat isolated hearts but did not interfere with the direct activation of cardiac sGC, suggesting that thionitrites may occur as intermediates of NO/cGMP signaling in mammalian tissues.