A New Pathway of Nitric Oxide/Cyclic GMP Signaling InvolvingS-Nitrosoglutathione*

Nitric oxide (NO), a physiologically important activator of soluble guanylyl cyclase (sGC), is synthesized from l-arginine and O2 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). 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 μmol of cGMP × mg−1 × 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 ± 0.60 μmol of cGMP × mg−1 × min−1). The NO/O·̄2 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 μmol of cGMP × mg−1 × 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-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.