ALCOHOLIC LIVER INJURY--IMPAIRED VESICULAR TRANSPORT

酒精性肝损伤——水泡运输受损

• 批准号: 2000280
• 负责人: MARK A. MC NIVEN
• 金额: $ 18.57万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-04-01 至 1999-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2000280
• 关键词: acetaldehyde; adduct; adenosinetriphosphatase; cell free system; cellular pathology; confocal scanning microscopy; electron microscopy; ethanol; fluorescent dye /probe; guanosinetriphosphatases; immunoelectron microscopy; intracellular membranes; intracellular transport; laboratory rat; liver cells; liver disorder; membrane activity; microinjections; microtubules; protein transport; tissue /cell culture; vesicle /vacuole

The goal of this proposed study is to define the molecular mechanisms by which alcohol, and its oxidized metabolite, acetaldehyde, impairs secretory and endocytic vesicular protein transport in hepatocytes. Specifically, we will test the hypothesis that alcohol disrupts the microtubule cytoskeleton and associated ATPases, or motor enzymes, which play a major role in the organization, transport, and targeting of different vesicle populations within the hepatocyte. Numerous reports by others have suggested that alcohol and its oxidized metabolites inhibit protein trafficking indirectly through a disruption of the microtubule cytoskeleton upon which most of these transport events depend. Despite these extensive in vitro studies, it has not been determined if alcohol alters microtubule organization within the hepatocyte, how this disruption occurs, and if other microtubule associated enzymes (motors) are also perturbed. Our goal is to utilize state of the art cell biological techniques to conduct a definitive and novel study on the mechanism by which alcohol interferes with microtubule- based vesicular trafficking within the hepatocyte. The proposed study has three specific aims. First, we will examine and compare how microtubules are organized and polarized within healthy control vs. ethanol-treated hepatocytes by: a) confocal-immunofluorescent and electron microscopic examination in conjunction with computer-aided reconstruction morphometry; b) polarity assays of microtubules in cultured hepatocyte couplets. Second, through the application of cell microinjection with computer- enhanced, fluorescent, video microscopy we will determine if secretory and endocytotic vesicular transport along microtubules in cultured hepatocyte couplets is altered by alcohol treatment. Third, we will test for disruptive effects of alcohol on vesicle transport and fusion by measuring alterations in the activity of microtubule-associated motor enzymes (kinesin, dynein, dynamin) through morphological and biochemical manipulation of permeabilized and homogenized hepatocytes, and cell-free systems using purified vesicular and cytoskeletal components. The technology and experiments described in this proposal are novel to the study of alcohol-induced cell-damage and to vesicular transport in hepatocytes. They will not only greatly expand our understanding of how alcohol impairs hepatocyte function but lend insight into the basic mechanisms of secretion and endocytosis in these cells.

这项研究的目标是通过以下方式定义分子机制： 哪种酒精及其氧化代谢物乙醛会损害分泌 和肝细胞内吞的囊泡蛋白转运。 具体来说，我们 将检验酒精破坏微管细胞骨架的假设 以及相关的 ATP 酶或运动酶，它们在 不同囊泡群体的组织、运输和靶向 肝细胞内。 其他人的许多报告都表明 酒精及其氧化代谢物间接抑制蛋白质运输 通过破坏微管细胞骨架，大多数细胞都依赖于微管细胞骨架 这些运输事件取决于。 尽管进行了这些广泛的体外研究， 尚未确定酒精是否会改变微管组织 在肝细胞内，这种破坏是如何发生的，以及其他微管是否 相关的酶（马达）也受到干扰。 我们的目标是利用 最先进的细胞生物学技术进行明确和 关于酒精干扰微管机制的新研究 基于肝细胞内的囊泡运输。 拟议的研究有 三个具体目标。 首先，我们将检查并比较微管如何 在健康对照与乙醇处理之间有组织和两极分化 肝细胞：a) 共聚焦免疫荧光和电子显微镜 与计算机辅助重建形态测定相结合的检查； b) 培养的肝细胞对中微管的极性测定。 其次，通过计算机显微注射技术的应用 增强荧光视频显微镜我们将确定是否分泌和 培养肝细胞中沿着微管的内吞性囊泡运输 对联因酒精处理而改变。 第三，我们将测试 通过测量酒精对囊泡运输和融合的破坏性影响 微管相关运动酶活性的改变 （驱动蛋白、动力蛋白、动力蛋白）通过形态学和生物化学 透化和均质化肝细胞以及无细胞肝细胞的操作 使用纯化的囊泡和细胞骨架成分的系统。 这 本提案中描述的技术和实验对于 酒精诱导的细胞损伤和囊泡运输的研究 肝细胞。 它们不仅将极大地扩展我们对如何 酒精会损害肝细胞功能，但有助于深入了解肝细胞的基本功能 这些细胞的分泌和内吞作用的机制。