This proposal will test the HYPOTHESIS that ethanol desensitizes hepatocytes to the trophic effects of growth factors by altering the expression and/or function of certain guanine nucleotide binding proteins (G proteins) which transduce growth regulatory signals. Since cAMP- dependent signals modulate growth and differentiation and our preliminary data suggest that ethanol alters receptor-G protein coupled activation of adenylyl cyclase, this proposal will focus on defining the effects of ethanol on G proteins coupled to adenylyl cyclase. 1) The effect of ethanol on hepatic EXPRESSION of G proteins will be determined. Immunoblot, immunocytochemistry, and quantitative immunoprecipitation of G proteins will be used to identify differences in membrane levels of G proteins. Northern analysis and nuclear run-on studies will be performed to determine if differences in gene transcription or mRNA stability explain differences in expression. 2) The effect of ethanol on G protein FUNCTION will be assessed by determining basal and stimulated adenylyl cyclase activity; evaluating restoration of adenylyl cyclase activity in cyc(-) membranes; and quantitating hepatic cAMP. 3) If altered G protein expression and function cannot be correlated, then perturbations of receptor kinetics, adenylyl cyclase expression or post-translational modification of G proteins will be sought. Post-translational modification will be assessed by 2-D gel and quantitative determination of phosphorylation and GTPase activity. 4) To determine the relevance of altered adenylyl cyclase signalling to growth regulation, G protein function will be selectively manipulated with pertussis and/or cholera toxin. Differences in growth factor induction of pre-replicative enzymes, growth-related and differentiated gene expression, and DNA synthesis will be noted. These experiments will use tissues from non-regenerating and regenerating (post-partial hepatectomy) livers from rats fed either ethanol-containing diets, isocaloric non-ethanol diets, or ad libitum non- ethanol diets, as well as cultured hepatocytes obtained from these 3 feeding groups and grown in the presence of growth factors +/- ethanol. Information obtained will advance the development of rational therapies for alcoholic liver disease and provide novel insights into regulation of cellular proliferation and organ regeneration.

National Institute of Health (NIH)
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
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Special Emphasis Panel (SRCA (40))
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Johns Hopkins University
Internal Medicine/Medicine
Schools of Medicine
United States
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