The chemistry of the S -nitrosoglutathione/glutathione system

Abstract

S-Nitrosothiols have generated considerable interest due to their ability to act as nitric oxide (NO) donors and due to their possible involvement in bioregulatory systems-e.g., NO transfer reactions. Elucidation of the reaction pathways involved in the modification of the thiol group by S-nitrosothiols is important for understanding the role of S-nitroso compounds in vivo. The modification of glutathione (GSH) in the presence of S-nitrosoglutathione (GSNO) was examined as a model reaction. Incubation of GSNO (1 mM) with GSH at various concentrations (1-10 mM) in phosphate buffer (pH 7.4) yielded oxidized glutathione, nitrite, nitrous oxide, and ammonia as end products. The product yields were dependent on the concentrations of GSH and oxygen. Transient signals corresponding to GSH conjugates, which increased by one mass unit when the reaction was carried out with 15 N-labeled GSNO, were identified by electrospray ionization mass spectrometry. When morpholine was present in the reaction system, N-nitrosomorpholine was formed. Increasing concentrations of either phosphate or GSH led to lower yields of N-nitrosomorpholine. The inhibitory effect of phosphate may be due to reaction with the nitrosating agent, nitrous anhydride (N 2 O 3 ), formed by oxidation of NO. This supports the release of NO during the reaction of GSNO with GSH. The products noted above account quantitatively for virtually all of the GSNO nitrogen consumed during the reaction, and it is now possible to construct a complete set of pathways for the complex transformations arising from GSNO ؉ GSH.S-Nitrosothiols, RSNO, with certain exceptions, are unstable in aqueous solution. For example, S-nitrosoglutathione (GSNO) undergoes decomposition over hours, whereas Snitrosocysteine has a half-life of less than 2 min. The initial step in the decomposition of RSNO is believed to be homolytic cleavage of the SON bond to give nitric oxide (NO) and a thiyl radical (1, 2). These compounds are involved in many bioregulatory functions, including vasodilation and inhibition of platelet aggregation. The existence of more stable transport forms of NO has been postulated in view of the short half-life of authentic NO in vivo (3). Low molecular weight thiols such as cysteine, glutathione (GSH), and penicillamine are prime candidates for such carrier molecules, and they can form S-nitrosothiols on reaction with oxides of nitrogen (4). It has been assumed that the biological effects of these compounds are due to the spontaneous release of NO; however, this hypothesis is not supported by currently available data (5-7).Although a few studies have been carried out in an attempt to determine the reaction products and to deduce the mechanism of the modification of the thiol group by S-nitrosothiols, the experiments were purely qualitative and no clear mechanistic picture has emerged (8,9). In this report, we describe the reaction of GSNO with GSH, a tripeptide with intracelluclar concentrations as high as 10 mM (10). It is involved in the cell's antioxidant defens...

Formation of S-Nitrosothiols from Regiospecific Reaction of Thiols with N-Nitrosotryptophan Derivatives

“…When equimolar concentrations of NANT and GSH were used, the UV-visible maximum at 545 nm did not increase continuously (Fig. 3B), probably because of a GS Ϫ -induced depletion of S-nitrosoglutathione as reported by Singh et al (17).…”

Nitric Oxide Inhibits c-Jun DNA Binding by Specifically TargetedS-Glutathionylation

“…Dependent on the protein examined half-maximal glutathionylation was observed at GSH/GSSG ratios ranging from 27 to 10 Ϫ5 (34). NO was reported to oxidize GSH to GSSG under anaerobic conditions, at low GSH/NO ratios, or via a secondary reaction of GSNO with GSH (22,(35)(36)(37). In our experimental system, GSSG concentrations were 15 Ϯ 4 M (n ϭ 6) under control conditions, i.e.…”

“…In our in vitro system, special emphasis was given to the role of the reduced sulfhydryl compound GSH, which is present in concentrations of 1-10 mM in mammalian cells (18). GSH not only protects oxidant-sensitive protein thiols against oxidative damage (19,20) but also critically determines the biological activity of NO (21)(22)(23). We show here that in the presence of physiologically relevant concentrations of GSH, NO inhibits c-Jun DNA binding in vitro by specifically targeting the formation of a mixed disulfide with GSH to a conserved cysteine residue in the DNA-binding site of the transcription factor.…”

S-Nitrosoglutathione Reduces Inflammation and Protects Brain against Focal Cerebral Ischemia in a Rat Model of Experimental Stroke

“…Metabolism of GSNO has been described as a complex phenomenon especially in vivo as it is dependent on several factors, both enzymatic and nonenzymatic (Chiueh, 2002;Eiserich et al, 1998;Singh et al, 1996;Zeng et al, 2001). We used a cell culture model of BBB consisting of a coculture of mouse brain capillary endothelial cells (Omidi et al, 2003) and rat astrocytes in vitro (Dehouck et al, 1990;Deplanque et al, 2003) to assess the membrane permeability of GSNO.…”