This project is part of an IRPG application that represents a continuation of previous studies carried out under a program project grant of the same title that in turn continued studies on a longstanding RO1 grant (also of the same title) originally funded in 1966. This program project included several projects focused on the interactions of ethanol with G protein-coupled phospholipase C (PLC) signalling system in the liver. We identified multiple sites of interaction of ethanol with the control of this signalling process in liver cells resulting in the suppression by ethanol of the response to hormones. This ethanol inhibition originated in at least two mechanisms, one resulting from the direct interaction of ethanol with the receptor-G protein-PLC complex, the other resulting from the potentiation by ethanol of the feedback inhibition mediated by protein kinase C (PKC). In this project, we will pursue the identification of the molecular mechanisms underlying these effects of ethanol and characterize possible physiological consequences for liver function. First, we will further characterize the role of PKC in the inhibitory effects of ethanol. We will interfere in multiple ways with PKC activity in intact cells, by the use of multiple inhibitors, enzyme downregulation and microinjection of specific peptides that interfere with its normal function. In addition, we will use longer-term hepatocyte cultures to suppress specific PKC isoforms using antisense strategies. The phosphorylation of potential protein kinase C target proteins of receptor-Gq/G11 protein-PLCbeta complex in intact hepatocytes will also be studied.
The second aim i s to obtain molecular information on the direct interactions of ethanol with this signaling complex. For this purpose, we will obtain highly purified vasopressin V1A receptor, Gq protein and phospholipase Cbeta1 protein and reconstitute these proteins in artificial phospholipid vesicle systems, both as individual or paired functional units and as a functional complex. This reconstitution will enable us to focus on individual protein-protein interactions in the complex and investigate the effects of ethanol on these interactions and the effect of treatment with purified PKC isoforms.
The third aim i s concerned with the functional implications of the effects of ethanol on Ca2+ -mediated signalling in the liver. We will utilize newly developed fluorescence imaging techniques that allow dynamic measurements of intracellular Ca2+ concentrations in hepatocytes in the intact perfused liver. The effects of ethanol and hormones on spatial and temporal organization of intracellular Ca2+ changes in individual cells across the functional hepatic lobule will be examined. We will also investigate the ethanol-induced disruption of hepatic Ca2+ signalling on bile flow and secretion and to study the metabolic interactions between ethanol and hormonal stimulation of mitochondrial Ca2+ metabolism in the perfused liver. We expect that an understanding of the molecular mechanisms by which ethanol affects signaling systems in the liver will help elucidate the pathogenesis of alcoholic liver disease. REVIEW A
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