Expression of inducible nitric oxide synthase in endotoxemic rat hepatocytes is dependent on the cellular glutathione status

The inducible nitric oxide synthase (iNOS) promoter contains nuclear factor kappa B (NF-kappa B) binding sites. NF-kappa B activation is determined, i n part, by the intracellular redox status, The aim of this study was to det ermine the importance of the cellular glutathione status in relation to NF- kappa B activation and iNOS expression in hepatocytes in vivo and in vitro, For in vivo experiments, rats were injected with endotoxin and sacrificed 6 hours later. Glutathione was depleted by diethylmaleate. For in vitro exp eriments, cultured hepatocytes from untreated rats were exposed to a cytoki ne mixture, Glutathione levels were depleted by diethylmaleate and restored by N-acetylcysteine. iNOS expression was assessed by Western blot, reverse transcription polymerase chain reaction, nitric oxide (NO) metabolites, an d immunohistochemistry. NF-kappa B binding was assessed by electrophoretic mobility shift assay. Endotoxin-induced iNOS expression in rat liver was pr ominent in hepatocytes, Kupffer cells, and inflammatory cells, in particula r neutrophils. Glutathione depletion prevented iNOS induction in hepatocyte s, but not in inflammatory cells. iNOS protein levels were in accordance wi th iNOS messenger RNA and NO metabolites in plasma. Glutathione depletion d id not affect neutrophil infiltration. Cytokines strongly induced iNOS in c ultured hepatocytes, Induction was prevented by glutathione depletion and c ould be restored by addition of N-acetylcysteine, NF-kappa B binding correl ated with iNOS induction. In conclusion, in this study we show that iNOS in duction in hepatocytes in vivo and in vitro is dependent on the intracellul ar glutathione status and correlates with NF-kappa B binding. Glutathione-d epletion has no effect on the expression of iNOS in inflammatory cells, nor on neutrophil infiltration.