title: The role of Src kinase in the potentiation by ethanol of cytokine- and endotoxin-mediated nitric oxide synthase expression in rat hepatocytes.
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-alpha (TNF-alpha)- 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-alpha/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 alpha-tocopherol fail to inhibit TNF-alpha/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-alpha/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-alpha/LPS/ethanol.
authors.affiliation: Institute of Toxicology, College of Medicine, National Taiwan University, Taipei, Taiwan.
authors.forename: M L
authors.initials: ML
authors.lastname: Kuo
authors.forename: Y P
authors.initials: YP
authors.lastname: Chau
authors.forename: J H
authors.initials: JH
authors.lastname: Wang
authors.forename: P J
authors.initials: PJ
authors.lastname: Lin
mesh_terms: D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D004357:Drug Synergism; D004789:Enzyme Activation; D004790:Enzyme Induction; D000431:Ethanol / Q000494:pharmacology*; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008099:Liver / Q000187:drug effects* / Q000201:enzymology*; D008297:Male; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000378:metabolism; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015398:Signal Transduction / Q000187:drug effects; D013268:Stimulation, Chemical; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D019061:src-Family Kinases / Q000502:physiology*
chemical_list: D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D000431:Ethanol; D019001:Nitric Oxide Synthase; D016392:Proto-Oncogene Proteins pp60(c-src); D019061:src-Family Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases