Regulation of Hepatic Nitric Oxide Synthase by Reactive Oxygen Intermediates and Glutathione

Regulation of induced nitric oxide synthase in rat hepatocyte primary cultures was explored. Nitric oxide synthase (NOS) induction by tumor necrosis factor-α (TNFα) is synergized by interferon-γ, and both NOS activity and gene expression are maximal by 10 h and maintained through 24 h. Glutathione depletion by diethylmaleate, which conjugates reduced glutathione, 1,3-bis(chloroethyl)-1-nitrosourea (BCNU), a glutathione reductase inhibitor, or buthionine sulfoxamine, a glutathione synthesis inhibitor, abolishes or reduces NOS induction in TNF α-treated hepatocytes, whereas N-acetylcysteine has little effect. Thus, reduced glutathione is critical to NOS mRNA induction and activity in TNFα-treated hepatocytes. NOS induction in TNFα-treated cells is reduced by rotenone, a mitochondrial complex 1 inhibitor. Concurrent treatment with TNFα and the antioxidant, Trolox, or the iron-chelating agent, desferrioxamine, also reduces NOS activity. Dithiothreitol, a thiol antioxidant, reduced TNFα induction of NOS. Trolox and BCNU, combined, blocked TNFα stimulation of NOS greater than either agent alone. These results suggest that TNFα increases mitochondrial production of reactive oxygen intermediates (ROI), which contributes to NOS induction. Hepatocytes exposed to extracellular ROI generation through a xanthine/xanthine oxidase superoxide-generating system expressed increased NOS activity and mRNA levels. NOS induction by superoxide also requires reduced glutathione since diethylmaleate blocks induction by xanthine/xanthine oxidase while N-acetylcysteine elevates NOS expression. Thus, the generation of ROI by cytokines or other physiological processes stimulates the induction of NOS and this process is regulated by cellular levels of reduced glutathione.