Endogenous nitrosothiols (RSNO), such as S-nitrosoglutathione (GSNO) and nitrosoalbumin (nALB), may act as a reservoir for the release of biologically active nitric oxide (NO). Cu⁺ ions are known to catalyse the release of NO from RSNO (Dicks et al., 1996) and vitamin C has been implicated in the release of NO from RSNO through the reduction of Cu⁺⁺ to Cu⁺. Vitamin C can, however, interact with metal ions to generate superoxide anion (O₂^-) which may inactivate NO. The aim of this study was to examine the effects of the reducing agents vitamin C and glutathione (GSH) on the release of NO from RSNO and another NO donor (NONOate). NONOates decompose spontaneously in aqueous media, mimicking authentic NO. The role of metal ions was examined using neocuproine, a specific Cu⁺ chelator, and EDTA, a non-specific metal ion chelator.

Concentrations of NO in Krebs’ solution were measured at 23 °C using an NO specific electrochemical detector (ISO-NO Mark II meter, World Precision Instruments Ltd, UK). Effects of vitamin C/GSH on NO release from GSNO, nALB, the synthetic RSNO S-nitroso-N-acetylpenicillamine (SNAP), and diethylamine NONOate (D-NONOate) were examined. nALB was prepared by exposing bovine serum albumin to acidified NaNO₂ for 30 min at room temperature, (Stamler, 1992). Release of NO was quantified in terms of the maximum concentration of NO attained after addition of vitamin C/GSH to the NO donors. Further experiments were performed in the presence of EDTA (5 mM) or neocuproine (100µM). In order to maintain a constant background concentration of copper, Cu(II)SO₄ (Cu⁺⁺, 1µM) was present in all experiments

Vitamin C (1µM-10mM) caused an increase in NO release from GSNO with a “bell-shaped” concentration-response curve. Maximal NO release was seen at physiological concentrations of vitamin C (100µM), with vitamin C increasing NO production from GSNO from 22±0.8 nM (vitamin C absent) to 2472±139 nM (vitamin C present, n=3, P<0.005). GSH (1µM-10mM) caused an increase in NO production from GSNO that was maximal at physiological concentrations (1 µM), with GSH increasing NO from 46.5±6.9 nM to 750±6.3 nM (n=3, P<0.001). A similar pattern of NO release by vitamin C and GSH was seen for both nALB and SNAP. Effects of vitamin C and GSH on NO release from GSNO, nALB, and SNAP were abolished by the presence of either EDTA or neocuproine. By contrast, vitamin C (1µM-1mM) caused a concentration dependent decrease in NO production from D-NONOate (from 882±19 nM to 102±4.9 nM for vitamin C, 100µM, n=3, P<0.001). This effect was also abolished by the addition of EDTA and neocuproine. GSH had no significant effect on NO release from D-NONOate.

These results are consistent with the reduction of Cu⁺⁺ to Cu⁺ by vitamin C and GSH leading to the release of NO from RSNO. The bell-shaped concentration-response curve for effects of vitamin C on RSNO and also the inhibitory effects of vitamin C on D-NONOate can be explained by generation of O₂^- at high concentrations of vitamin C. Maximal release of NO from the endogenous RSNOs, GSNO and nALB, occurs at physiological concentrations of vitamin C/GSH and this mechanism may be of physiological relevance.

Dicks, A.P. et al. (1996) J. Chem. Soc.2:481-7
Stamler, J.S. et al. (1992) Proc. Natl. Acad. Sci. USA. 89 :444-448