Abstract

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-α (TNF-α)- 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-α/LPS/ethanol. Furthermore, the capability of these agents to induce iNOS expression is primarily determined by the age of the animals. Interestingly, antioxidants such asN-acetylcysteine (NAC), ascorbic acid, or α-tocopherol fail to inhibit TNF-α/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-α/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-α/LPS/ethanol.