The objectives of the proposed studies are to investigate the mechanisms of hormonal influences on alcohol dehydrogenase, of changes in total pyridine nucleotides during ethanol elimination, and the pathway of rapid initial rates of ethanol metabolism in primary hepatocyte culture. The studies will determine the possible role in mediating increases in alcohol dehydrogenase of insulin-growth factors in the case of growth hormone, and of adrenergic receptors and second messenger pathways for the case of epinephrine. For all the hormones that affect alcohol dehydrogenase activity, it will be determined whether it is the result of changes in synthesis or degradation and whether it involves messenger RNA formation and regulation of gene transcription. A cDNA encoding rat liver alcohol dehydrogenase provided by Dr. David W. Crabb from Indiana University School of Medicine will be used in the determination of messenger RNA formation. Investigation of the effect of ethanol on total pyridine nucleotides will concentrate on the mechanism for the observed decrease in NAD + NADH with corresponding increase in NADP + NADPH during ethanol metabolism. Studied will be the time course and reversibility of the changes, their reproducibility in isolated non-cultured hepatocytes, whether the changes are produced by ethanol itself or require its metabolism, and most importantly the enzymatic pathway for the changes. For this last purpose, the effects of ethanol will be determined on the specific phosphorylation of NAD to NADP catalyzed by NAD kinase, and on more generalized phosphorylation of purines and pyrimidines, and also on salvage and biosynthetic pathways of the pyridine nucleotides. Measurements of pyridine nucleotides will be carried out by reverse phase high performance liquid chromatography. To investigate the observed rapid initial rate of ethanol elimination, the formation of acetaldehyde, changes in free NADH/NAD ratio and changes in cell respiration will be determined together with examination of the possible relative roles of alcohol dehydrogenase, the microsomal ethanol oxidizing system, and fatty acid ethyl ester formation in the ethanol elimination.
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