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MOLECULAR PHARMACOLOGY 52:535-541 (1997).

The Role of Src Kinase in the Potentiation by Ethanol of Cytokine- and Endotoxin-Mediated Nitric Oxide Synthase Expression in Rat Hepatocytes

Min-Liang Kuo, Yat-Pang Chau, Jyh-Horng Wang, and Pei-Jung Lin

Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan (M.-L.K., P.-J.L.), Institute of Anatomy, School of Life Science, National Yang-Ming University, Shih-Pai, Taipei, Taiwan (Y.-P.C.), and Department of Orthopaedics, National Taiwan University Hospital, Taipei, Taiwan (J.-H.W.)

This study demonstrates that exposure of primary rat hepatocytes or mouse BNL Cl.2 liver cell line to ethanol causes potentiation of tumor necrosis factor- (TNF-)- and lipopolysaccharide (LPS)-stimulated nitrite accumulation. The potentiating effect of ethanol (0.02-2 mM ) appears to be time and concentration dependent. Consistent with nitrite production, the amount of inducible nitric oxide synthase (iNOS) mRNA and protein is initially detected at 4 hr after treatment with TNF-/LPS/ethanol. Furthermore, the capability of these agents to induce iNOS expression is primarily determined by the age of the animals. Interestingly, antioxidants such as N-acetylcysteine (NAC), ascorbic acid, or -tocopherol fail to inhibit TNF-/LPS/ethanol-induced increase in iNOS protein. In addition, several kinase inhibitors, including staurosporine, genistein, curcumin, and herbimycin A, were used to examine their effects on this induction. Among them, only herbimycin A potently inhibits the accumulation of nitrite and iNOS expression. In vitro kinase assay verifies that Src tyrosine kinase is rapidly activated with a peak at 1 hr after treatment with TNF-/LPS/ethanol but is not activated by these agents singly or doubly. As expected, herbimycin A can block Src kinase activity under circumstances in which iNOS expression is also inhibited. However, our results do not indicate that the mitogen-activated protein kinase is activated after treatment with these agents. The study results suggest that Src tyrosine kinase plays a prominent role in transducing the signal to induce iNOS expression in hepatocytes treated with TNF-/LPS/ethanol.