Does peroxynitrite involve in the elevation of cellular glutathione induced by sodium nitroprusside (SNP) in RAW 264.7 cells?

The mechanism underlying the elevation of intracellular glutathione (GSH) in RAW 264.7 cells exposed to low-level sodium nitroprusside (SNP) was investigated by measuring the expression of mRNA for gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of de novo GSH synthesis, and the GSH content. A significant elevation of expression of mRNA for gamma-GCS was observed at 3 h after exposure of the cells to SNP at a concentration of 0.25 mM. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), N-acetylcysteine (NAC), or ebselen (Ebs) significantly suppressed the elevations induced by SNP, suggesting that hydrogen peroxide or peroxynitrite (ONOO(-)) is involved in this event as a triggering molecule. Hydrogen peroxide itself, however, did not induce the elevation of gamma-GCS mRNA and glutathione. Chemiluminescenses induced by SIN-1, a chemical ONOO(-) donor, and ONOO(-) itself were completely blocked by Ebs. SIN-1 also significantly elevated the cellular glutathione level, and the elevation was absolutely blocked by Ebs. These results suggest that the elevation of intracellular GSH in RAW 264.7 cells exposed to low-level SNP occurs via the de novo GSH pathway through transcriptional up-regulation of the gamma-GCS gene induced by peroxynitrite molecule.