This component will examine the role of protein kinases in the initial sensitivity to ethanol. We propose to study interactions between ethanol and protein kinase C and between ethanol and a ganglioside dependent protein kinase. The contribution made by protein kinases in the differential sensitivity to acute ethanol actions or development of functional tolerance will be addressed. The relationship between acute sensitivity and protein kinases will be addressed in long sleep and short sleep mice. The role protein kinases play in the acquisition of alcohol functional tolerance will be evaluated at each generation of the HAFT and LAFT selection. We propose to study the effect of ethanol on production and cellular concentration of protein kinase lipid activators, substrates and inhibitors of protein kinase C. The interaction between ethanol and the lipid activator of protein kinase C, 1,2-diacyl-sn-glycerol emphasizes the phospholipase D dependent production of diacylglycerol and synthesis of phosphatidylethanol via a transphosphatidylation. Studies are also proposed to evaluate the direct effect of phosphatidylethanol on protein kinase C activity as well as explore the function of phosphatidic acid. The studies will utilize both subcellular fraction (microsacs) isolated from the experimental models and primary cell cultures (cerebellar granule cells) obtained from LS, SS, HAFT and LAFT mice. The experiments are intended to address the effect of ethanol in functionally intact preparations. The in vitro treatment of LS and SS mice with ethanol elicits increases in protein kinase C activity measured in vitro. The relationship will be further evaluated by determining the effect of ethanol on the phosphorylation of specific substrates including, neuromodulin, neurogranin, myristoylated alanine-rich C kinase substrate (MARKS), the gama aminobutric acid receptor and N-methyl-D-aspartate receptor in the LS, SS the selected mice lines. A difference in CNS ganglioside concentration and topography has been established in the LS and SS mice and we have shown that chronic infusion of mixed gangliosides into brain ventricles can alter acute ethanol sensitivity in the LS and SS mice. The possibility that ganglioside modulated protein kinases may play a role in mediating the anesthetic effects of ethanol will be investigated.
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