Role of glutathione in nitric oxide-dependent regulation of energy metabolism in rat hepatoma cells

Abstract

Previous studies in this laboratory revealed that nitric oxide (NO) reversibly inhibits the respiration of isolated mitochondria and ascites hepatoma (AH-130) cells by an oxygen concentration-dependent mechanism. The inhibitory effect of NO on the respiration of AH-130 cells was enhanced by treating with digitonin that selectively permeabilized plasma membranes and released cytosolic lowmolecular-weight compounds. Reduced glutathione (GSH) is the most abundant cytosolic thiol that easily reacts with NO. To elucidate the mechanism by which digitonin enhanced the inhibitory action of NO, the effect of GSH and related thiols was studied with AH-130 cells and their mitochondria. The inhibitory effect of NO on the respiration of digitonin-treated cells was suppressed by either GSH, L-cysteine, or N-acetylcysteine, but not by oxidized glutathione. The inhibitory effect of NO on the respiration of their mitochondria was also decreased by GSH. In contrast, the inhibitory effect of NO was markedly enhanced with AH-130 cells obtained from animals that were pretreated with L-buthionine sulfoximine (BSO), a specific inhibitor for GSH synthesis. Kinetic analysis revealed that NO dose-dependently decreased GSH levels in AH-130 cells with concomitant generation of S-nitrosothiols. Although S-nitrosoglutathione (GSNO), a slow releaser of NO, also inhibited the respiration of tumor cell mitochondria, its effect was significantly lower than that of NO. These results suggest that cellular GSH might play pivotal roles in the regulation of energy metabolism in hepatoma cells by modulating free forms of NO. (HEPATOLOGY 1998;27:422-426.)Nitric oxide (NO) interacts with various molecules, such as hemeproteins and nonhemeproteins, thereby acting as a multifunctional molecule. Through the modification of such proteins, NO reversibly inhibits the respiration of mitochondria. [1][2][3][4] Previous studies in this laboratory 5 revealed that NO reversibly binds to cytochrome c oxidase and inhibits the respiration of tumor cells. Because NO also reacts with molecular oxygen (k ϭ 6 ϫ 10 6 mol/L Ϫ2 s Ϫ1 ), mitochondrial respiration was inhibited by this gaseous radical more strongly at low oxygen tensions than at its high concentrations. 3,5 Furthermore, the inhibitory effect of NO is enhanced by selectively permeabilizing plasma membranes of tumor cells by digitonin. 5 These observations suggested the presence of some cytosolic molecule(s) that suppressed the effect of NO on mitochondrial respiration. Because NO forms nitrosoadducts with various thiols, such as reduced glutathione (GSH) and cysteine-containing proteins, 6 these thiol compounds might decrease the levels of free forms of NO, thereby affecting its fate and biological activity. Among various thiols in cells, GSH is a naturally occurring major thiol. Preliminary experiments in this laboratory revealed that permeabilization of plasma membrane by digitonin released cytosolic GSH and decreased cellular levels of it. The inhibitory effect of NO on the respiration of isolated mitochondr...

The Effects of Hyperbaric Oxygen Treatment on Oxidant and Antioxidants Levels during Liver Regeneration in Rats

“…Conversely, high levels of ROS and low levels of GSH can be deleterious by enhancing lipid peroxidation and apoptosis (Peterhans 1997;Jones et al 1998;Vos et al 1999;Rolla et al 2000;Kaplowitz 2000). By inducing GSH depletion, ROS induce oxidative stress and reduce the antioxidant capability of other antioxidants (Huang et al 1998;Nishikawa et al 1998;Hall 1999). Depletion in GSH may also be a consequence of liver damage (Loguercio et al 2002).…”

Tumoricidal Activity of Endothelial Cells

“…NO reversibly binds to cytochrome c oxidase and inhibits the respiration of tumor cells, an effect that can be suppressed by GSH (32). By interacting with their iron centers or forming RSNO, NO can affect the activity of membrane-bound, cytosolic, and nuclear proteins, including the N-methyl-D-aspartic acid receptor, hemoglobin, and transcription factors such as nuclear factor-B and SoxR; and in addition, by inducing S-nitrosylation of caspases and tissue transglutaminase, NO may regulate the balance between apoptosis and necrosis (57).…”

“…Then, 250 l of a 0.5 N HCl solution containing 0.25% N-(1-naphthyl)ethylenediamide dihydrochloride were added to the mixtures. After incubation for 30 min at 25°C, the amount of S-nitrosothiols (RSNO) was determined spectrophotometrically at 550 nm using S-nitrosoglutathione (GSNO) as a standard (32). Fresh GSNO solution was obtained, before each experiment, by incubating equimolar GSH and sodium nitrite in acidified water at 0°C as previously described (33).…”

“…GSNO, a slow releaser of NO, may also inhibit the respiration of tumor cell mitochondria, but its effect is much lower than that of NO (32). Nevertheless, NO-induced RSNO formation was not increased by H 2 O 2 (Table VI), which suggests that RSNO formation is not required in the mechanism of NO-and H 2 O 2 -induced cooperative tumor cytotoxicity (see Tables III and IV).…”

“…In addition, NO and Ϫ OONO can increase intracellular H 2 O 2 by inhibiting the mitochondrial respiratory chain (32,60) or through the inhibition of catalase (Table VI). Therefore, it is possible that NO may induce the release of catalytic amounts of free iron and increase H 2 O 2 levels in the intact tumor cells.…”