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.

这项拟议的研究的目的是通过 哪种酒精及其氧化的代谢物乙醛会损害分泌物 和肝细胞中的内吞囊泡蛋白转运。 具体来说，我们 将测试酒精破坏微管细胞骨架的假设 以及相关的ATPases或运动酶，它们在 不同囊泡群体的组织，运输和靶向 在肝细胞中。 其他人的大量报告表明 酒精及其氧化代谢产物间接抑制蛋白质运输 通过微管细胞骨架的破坏。 这些运输事件取决于。 尽管经过广泛的体外研究， 尚未确定酒精是否改变微管组织 在肝细胞中，这种破坏是如何发生的，以及其他微管 相关的酶（电动机）也受到干扰。 我们的目标是利用 采用最终细胞生物学技术来进行确定的和 关于酒精干扰微管的机制的新研究 基于肝细胞内的囊泡贩运。 拟议的研究有 三个具体目标。 首先，我们将检查和比较微管的方式 在健康对照中组织和两极分化与乙醇处理 肝细胞通过：a）共聚焦免疫荧光和电子显微镜 结合计算机辅助重建形态测定法； b）培养的肝细胞对联中微管的极性测定。 其次，通过将细胞对计算机的微注射应用 增强，荧光，视频显微镜我们将确定分泌和分泌率是否 培养的肝细胞中沿微管的内吞囊泡转运 对联通过酒精处理改变。 第三，我们将测试 通过测量酒精对囊泡传输和融合的破坏性影响 微管相关运动酶的活性改变 （动力学，动力蛋白，动力学）通过形态学和生化 操纵透化和均质的肝细胞和无细胞 使用纯化的水泡和细胞骨架成分的系统。 这 该提案中描述的技术和实验对 研究酒精引起的细胞破坏和囊泡转运 肝细胞。 他们不仅会大大扩展我们对如何的理解 酒精会损害肝细胞功能，但可以深入了解基本 这些细胞中分泌和内吞作用的机制。