Novel synthesis of S-nitrosoglutathione and degradation by human neutrophils

S-nitrosoglutathione (SNO-GSH), a stable derivative of nitric oxide, is an endothelium-derived relaxation factor, which provokes vasodilation, inhibits platelet aggregation, and inhibits neutrophil (PMN) superoxide anion (O2+) generation. We have established a novel method for synthesis of S-nitrosoglutathione using a column containing S-nitrosothiol covalently attached to agarose. S-nitrosoglutathione was a product as assessed after separation using C-18 reverse-phase HPLC and absorption spectroscopy. We examined the stability of SNO-GSH in the presence or absence of PMN. The half-life (mercuric acid diazotization) of SNO-GSH in Hepes was >60 min. The addition of resting PMN did not affect the T12 of SNO-GSH. PMN exposed to N-fMet-Leu-Phe (FMLP, 10−7m) reduced measurable SNO-GSH (15 μm) at 5 min (48 ± 5.0% control, P < 0.05). Incubation (5 min, 37°C) of PMN with 10 μm tenidap (an anti-inflammatory drug which inhibits PMN activation) before addition of FMLP blocked the PMN-dependent degradation of SNO-GSH (42 ± 3 vs 78 ± 1.3% control, P = 0.01). We confirmed the recovery of SNO-GSH through measurements by bioassay (platelet aggregation) and HPLC analysis. The degradation of S-nitrosothiols by activated neutrophils may reverse the inhibitory effect of S-nitrosothiols on PMN functions and contribute to tissue injury at sites of inflammation.