Protein S-nitrosothiols (PrSNOs) have been implicated in the pathophysiology of neuroinflammatory disorders characterized by extensive nitrosative stress. Using rat spinal cord slices we have previously established that S-nitrosoglutathione (GSNO) is a viable intercellular S-nitrosating agent. Moreover, generation of PrSNOs with GSNO occurs exclusively via a S-transnitrosation mechanism. Although the metabolically instability of PrSNOs is well known, there is little understanding of the factors involved in the cleavage of S-NO linkage in intact cells.To address this issue, we conducted chase experiments in spinal cord slices incubated with GSNO. The results show that removal of GSNO leads to a rapid decreasing of PrSNOs (t1/2 ~ 2h), which is greatly accelerated when glutathione (GSH) levels are raised with the permeable analogue GSH ethyl ester suggesting that GSH plays a key role in the denitrosylation process. Inhibition of both GSH-dependent enzymes and enzymes that could mediate denitrosylation do not alter the rate of PrSNO decomposition. The lack of protein glutathionylation during the chase suggest that most proteins are denitrosylated via rapid transnitrosation with GSH. The differences in the denitrosylation rate of individual proteins would indicate the existence of additional structural factors in this process. Supported by NIH grant NS 47448.
