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.
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