EFFECTS OF ETHANOL ON HEPATIC HOMEOSTASIS

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The overall purpose of this proposal is to better understand the molecular mechanisms by which ethanol alters the homeostasis of hepatic GSH and its localization in the liver acinus. The specific aims are: 1) Effect of ethanol on the GSH homeostasis in periportal (PP) and perivenous (PV) hepatocytes. We will determine the zonation f the changes in the homeostasis of GSH by chronic ethanol feeding previously observed in mixed population of isolated hepatocytes as to whether these are predominant in a specific cell population of the acinus. With enriched population of PP and PV cells we will determine the kinetics of GSH efflux and the compartmentation of GSH in pair and ethanol-fed PP and PV cells. Alternatively, we will study the effect of ethanol feeding on the GSH content in different mitochondrial populations fractionated by flow cytometry. 2) Effect of lipid composition and fluidity on the transport of GSH in sinusoidal membrane vesicles (bLPM). Chronic ethanol feeding increases the fluidity of the sinusoidal plasma membrane which may subsequently affect the hepatic transport of GSH. Therefore, we will determine the effect of changing the lipid composition of the bLPM lipids on the transport of GSH by fusing liposomes of defined lipid composition and cholesterol content to pair- and ethanol- bLPM. Also, we will determine the changing of fluidity independent of lipid composition on the transport of GSH by using agents that intercalate into the plasma membrane increasing its fluidity. 3) Characterization of the kinetics, driving forces of GSH transport in mitochondria from chronic ethanol-fed rats. We will determine in permeabilized rat hepatocytes the kinetics and driving forces of GSH transport in mitochondria and the effects of ethanol feeding on the features of the mitochondrial transport of GSH by reconstituting the extracted mitochondrial inner membrane proteins in artificial or native lipids from both groups.