GLUTATHIONE REGULATES NITRIC-OXIDE SYNTHASE IN CULTURED-HEPATOCYTES

Objective The authors determine the relationship between glutathione a nd nitric oxide (NO) synthesis in cultured hepatocytes. Summary Backgr ound Data Glutathione is a cofactor for a number of enzymes, and its p resence is essential for maximal enzyme activity by the inducible macr ophage nitric oxide synthase (iNOS), which produces the reactive nitri c oxide radical. Hepatocytes contain substantial quantities of glutath ione, and this important tripeptide is decreased in hepatocytes stress ed by ischemia/reperfusion or endotoxemia. Endotoxemia also induces th e synthesis of inflammatory cytokines that result in the production of nitric oxide from hepatocytes by iNOS, suggesting that hepatocytes ma y be attempting to synthesize nitric oxide at times when intracellular glutathione is reduced. Methods Hepatocytes were cultured with buthio nine sulfoximine and 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) to inhi bit glutathione. After exposure to cytokines, NO synthesis was assesse d by supernatant nitrite levels, cytosolic INOS enzyme activity, and i NOS mRNA levels. Results Inhibition of glutathione synthesis with buth ionine sulfoximine or inhibition of glutathione reductase activity wit h BCNU inhibited nitrite synthesis. Both buthionine sulfoximine and BC NU inhibited the induction of iNOS mRNA, as detected by Northern blot analysis. Exogenous glutathione increased cytokine-stimulated INOS ind uction, overcame the inhibitory effects of BCNU, and increased nitrite production by intact hepatocytes, induced hepatocyte cytosol, and par tially purified hepatocyte iNOS. Conclusions In cultured hepatocytes, adequate glutathione levels are required for optimal nitric oxide synt hesis. This finding is predominantly due to an effect on INOS mRNA lev els, although glutathione also participates in the regulation of iNOS enzyme activity.