S-Transnitrosation Reactions Are Involved in the Metabolic Fate and Biological Actions of Nitric Oxide
AbstractAbstract S-Nitrosothiols are a group of potent, bioactive compounds that form through the reaction of nitric oxide (NO) with thiols in the presence of oxygen. These compounds are naturally occurring in vivo , stabilize NO and potentiate its biological effects. S-Nitrosoglutathione is the most abundant intracellular S-nitrosothiol, and the kinetics for its formation favors de novo synthesis. In this analysis, we studied the formation of S-nitrosothiols by S-transnitrosation, or exchange of -NO for -H between sulfur atoms; we synthesized S-nitroso-glutathionyl-Sepharose 4B beads (SNO-4B) as a reagent with which to measure S-transnitrosation reactions. We detected a maximum of 1.57 ± 0.24 pmol NO/bead ( n = 5) after S-nitrosation of the beads with acidified nitrite. The stability of the S-NO bond was dependent on temperature, but not pH over the 5 to 9 range (except at pH 9 at 37°), with an estimated t 1/2 of 30 hr at 22°C and of approximately 2 wk at 4°C. We demonstrated that SNO-4B transfers -NO to glutathione and to cysteine rapidly and in a pH-dependent manner. The initial rate of transfer of -NO from SNO-4B to glutathione at room temperature was 0.53, 3.03 and 5.14 μM/min at pH 5.0, 7.4 and 9.0, respectively (P < .05). Under the same conditions, the initial rate of -NO transfer to cysteine was 0.72, 3.71 and 4.69 μM/min at pH 5.0, 7.4 and 9.0, respectively (P < .05). There was no appreciable S-transnitrosation between SNO-4B and bovine serum albumin. We further demonstrated that SNO-4B evokes significant vasodilator and platelet inhibitory responses in plasma-free systems and activates platelet soluble guanylyl cyclase. These data suggest a mechanism by which to explain the metabolic fate and distribution of NO among thiol pools in the vasculature, and implicate S-transnitrosation at the cell surface in NO signal transduction.