Biol. Pharm. Bull. 28(5) 779—785 (2005)

most cells at concentrations of 5 to 10 mM and has a variety of physiological functions ranging from protection of cells by serving as an antioxidant to modulation of signal transduction by controlling cellular redox state. GSH exerts its antioxidant function as a free radical scavenger and as a cofactor for antioxidant enzymes, glutathione peroxidase (GPx) and glutathione S-transferase, providing the first line of cellular defense against oxidative stress. Cells subjected to oxidative stress respond not only by eliminating and detoxifying reactive oxygen species (ROS) with such pre-existing defense system, but by enhancing the defense through newly synthesizing antioxidants such as superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase and thioredoxin as well as glutathione. Such adjustment of the cells is known as an adaptive response of the cell. Induction of GSH synthesis has been reported to be one of a common responses of the cells against a variety of stimuli, especially oxidative stress including nitric oxide (NO), and which is achieved through elevated expression of the rate limiting enzyme of glutathione biosynthesis, g-glutamylcysteine synthetase (g-GCS). NO becomes cytotoxic at higher concentrations by generating ROS including peroxynitrite and hydrogen peroxide, by directly modifying the enzymes such as GPx and catalase or by inhibiting mitochondrial respiratory chain. At lower concentrations, on the other hand, NO has been reported to exert a protective effect by interfering with lipid peroxidation process and apoptotic pathway or by inducing an adaptive defense response as mentioned above. Since induction of GSH by NO has been reported in different cell types at low, sometimes physiologically relevant concentrations, it has become recognized to be a novel function of NO. Such effect of NO may be important in modulating physiological processes where NO is involved. Peroxynitrite, which is physiologically formed in the cell through the reaction of NO with superoxide, is suggested to be generated whenever NO is available within the cell, because superoxide is constitutively produced in mitochondria under a normal condition. Because of its oxidizing reactivity against a wide range of cell components such as proteins, nucleotides, lipids, peroxynitrite has been believed to be one putative species responsible for many biological effects of NO, including deleterious one. To have a better view on the physiological role of NO, elucidation of the key species and the signaling mechanisms involved in enhancement of antioxidant defense by NO is of particular importance. In our previous study, 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 g-GCS, the rate-limiting enzyme of de novo GSH synthesis, and the GSH content. As a result, a significant elevation of expression of mRNA for g-GCS was observed at 3 h after exposure of the cells to SNP at a concentration of 0.25 mM. Carboxy-2(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3oxide (PTIO) (c-PTIO), N-acetylcysteine (NAC), or ebselen (Eb) 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 gGCS mRNA and glutathione. Chemiluminescenses induced by SIN-1, a chemical ONOO donor, and ONOO itself were completely blocked by Eb. SIN-1 also significantly eleMay 2005 Biol. Pharm. Bull. 28(5) 779—785 (2005) 779