EFFECTS OF ETHANOL ON HEPATIC HOMEOSTASIS

本提案的总体目的是为了更好地了解 乙醇改变肝细胞内稳态的分子机制 谷胱甘肽及其在肝腺泡中的定位。 具体目标是： 1)乙醇对门静脉周围（PP）和门静脉周围（PP）GSH稳态的影响 静脉周（PV）肝细胞。 我们将确定 先前慢性乙醇喂养引起的GSH稳态变化 在分离的肝细胞的混合群体中观察到， 在腺泡的特定细胞群中占主导地位。 与 富集的PP和PV电池群，我们将确定 GSH流出和GSH在成对和乙醇喂养的PP中的区室化， 光伏电池。 或者，我们将研究乙醇喂养对 不同线粒体群体的GSH含量通过 流式细胞仪 2)脂质组成和流动性对谷胱甘肽转运的影响 窦状膜囊泡（bLPM）。 慢性乙醇喂养增加 窦状隙质膜的流动性， 影响GSH的肝脏转运。 因此，我们将确定 改变bLPM脂质的脂质组成对 通过融合确定脂质组成的脂质体转运GSH， 胆固醇含量对-和乙醇- bLPM。 同时，我们将确定 流动性的变化不依赖于脂质成分， 通过使用插入血浆的试剂转运GSH 膜增加其流动性。 3)谷胱甘肽转运的动力学，驱动力， 慢性乙醇喂养大鼠的线粒体。 我们将决定在 透化大鼠肝细胞GSH的动力学和驱动力 线粒体的转运和乙醇喂养对线粒体的影响 还原型谷胱甘肽的线粒体转运的特点， 提取的线粒体内膜蛋白在人工或天然 两组的脂质。

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.