ALCOHOL EFFECTS ON RECEPTOR G PROTEIN COUPLING

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The proposed studies will examine the effects of chronic ethanol on G- protein-coupled receptor function. Neurotransmitter receptors in specific brain regions are thought to be affected by ethanol. The G- protein-coupled receptors constitute a major class of receptors in brain, and changes in G-protein levels have been detected after chronic ethanol administration. However, there is little data regarding the effects of chronic ethanol on receptor-coupled G-protein activity. The proposed studies will determine whether changes in the functional activity of G-protein-coupled receptors in specific brain regions are affected by a chronic ethanol diet. Initial studies will involve an anatomical survey of the activity of 5-HT1A and opioid receptors in brains from rats that receive ethanol and control rats. In addition, the activity of several G-protein-coupled receptors in the cerebellum will be examined. These experiments will use a novel technique that directly measures receptor-mediated G-protein activation in an anatomically and pharmacologically specific manner: in vitro autoradiography of agonist-stimulated [35S]GTPgammaS binding. Regions in which receptor-G-protein function is altered by ethanol administration will then be examined in [35S]GTPgammaS membrane binding assays. Membrane [35S]GTPgammaS binding assays will allow the determination of changes in the potency or maximal effect of agonists after ethanol administration. Saturation analysis of basal and agonist- stimulated [35S]GTPgammaS binding will be used to determine whether changes occur in the catalytic activation of G-proteins by receptors, or in the affinity of the receptor-coupled G-proteins for GTPgammaS, after chronic ethanol administration. These studies will be combined with receptor binding assays using radiolabeled receptor antagonists. Saturation analysis of receptor binding will be performed to measure any changes in receptor number in those systems that are affected by chronic ethanol administration. The purpose of this R03 application is to provide data that will be used to develop an R01 application in the future by allowing the generation of hypotheses regarding the effects of ethanol on G-protein-coupled receptor functional activity.