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

Abstract

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 (O2/(·-). 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 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 ± 0.60 μmol of cGMP x mg-1 x min-1). The NO/O2/(·-) 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 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/O2/(·- )-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.