EFFECTS OF ETHANOL ON HEPATIC HOMEOSTASIS

这项建议的总体目的是为了更好地了解 乙醇改变肝脏动态平衡的分子机制 谷胱甘肽及其在肝腺泡内的定位。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 静脉周围(PV)肝细胞。我们将确定该区域的分区 慢性酒精喂养对谷胱甘肽稳态的影响 在分离的肝细胞混合种群中观察到这些 在腺泡的特定细胞群中占主导地位。使用 PP和PV细胞的富集群我们将确定其动力学 GSH外流和GSH在乙醇喂养PP和PP中的区划 光伏细胞。或者，我们将研究乙醇饲喂对 不同线粒体群体中的GSH含量由 流式细胞术。 2)脂质组成和流动性对谷胱甘肽转运的影响 正弦状膜小泡(BLPM)。慢性乙醇摄入量增加 正弦质膜的流动性，随后可能 影响谷胱甘肽的肝脏转运。因此，我们将确定 改变bLPM脂类的脂组成对大鼠血脂的影响 确定脂组成的脂质体与谷胱甘肽的融合转运GSH 胆固醇含量对-和乙醇-bLPM。此外，我们还将确定 不依赖于脂质成分的流动性的变化 谷胱甘肽的血浆插入剂转运 膜的流动性增加。 3)GSH转运动力学、驱动力的表征 慢性酒精喂养大鼠的线粒体。我们将在年确定 大鼠肝细胞通透性GSH的动力学和驱动力 线粒体转运及乙醇摄取对线粒体功能的影响 重组谷胱甘肽对线粒体转运的影响 人工或天然提取的线粒体内膜蛋白 两组的脂类。

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.