LIVER PROTON AND SOLUTE TRANSPORT AND ENDOCYTOSIS

肝脏质子和溶质的转运和胞吞作用

• 批准号: 2140478
• 负责人: REBECCA W. VAN DYKE
• 金额: $ 19.46万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-08-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2140478
• 关键词: G protein; acidity /alkalinity; adenosinetriphosphatase; cholera toxin; cyclic AMP; endocytosis; enzyme inhibitors; hormone regulation /control mechanism; human subject; hydrogen transport; immunofluorescence technique; intracellular transport; laboratory rat; liver cells; liver function; low density lipoprotein; membrane potentials; membrane transport proteins; pertussis toxin; protein kinase A; receptor coupling; receptor mediated endocytosis; tissue /cell culture; vesicle /vacuole; western blottings

Hepatocytes use intracellular vesicles to transport proteins and macromolecules into, out of and across cells as well as into or out of plasma membranes, processes termed endocytosis and exocytosis. Most of these vesicles are acidified by a unique electrogenic proton pump that usually functions in parallel with a chloride conductance to maximally acidify vesicle interior and limit membrane potential. Vesicle acidification is essential for efficient sorting and delivery of proteins, for degradation of macromolecules, for transport of small solutes into or out of acidified vesicles and for post-translational processing, delivery and function of lysosomal enzymes. Viruses and toxins also exploit the acidic environment of endocytic vesicles to trigger infection. Although vesicular transport and acidification have been identified in many cells, little is known regarding regulation of these important cell functions. My own work has shown that cholera and pertussis toxins, cAMP and protein kinase A all increase endosome acidification, findings that have implications for overall vesicular transport. In addition, work from others suggests that vesicle movement itself may be regulated, by a variety of agents, including cAMP, perhaps related, in part, to changes in vesicle acidification. Furthermore, endocytosis is of increasing clinical importance as a number of diseases affecting hepatic endocytosis have been identified, including familial hypercholesterolemia, alcoholic liver disease, diabetes, copper overload and lysosomal storage disorders. Finally, endocytosis is being employed for gene therapy. The long-term objectives of my research are to characterize, quantitate and compare mechanisms of vesicular acidification, to identify regulatory processes for acidification and for vesicular transport and to determine the role(s) of these regulatory processes in overall hepatic function. Based on work by myself and others, I have formulated a series of hypotheses within the context of these objectives that will be tested in the studies proposed in this application. I propose that receptor-coupled G proteins, located on intracellular vesicles, regulate endosome acidification and function with Gs proteins stimulating and Gi proteins, perhaps coupled to somatostatic, effecting tonic inhibition of vesicle acidification. I propose that these effects are mediated by cAMP, protein kinase A and, possibly, protein kinase C. I propose that these agents change vesicle acidification through effects on the proton pump, associated ion transporters, and/or changes in vesicle size, and that changes in acidification are associated with changes in rates of endocytosis and vesicular transport. I will test these hypotheses using isolated endocytic vesicles as well as the isolated perfused rat liver and cultured rat hepatocytes in conjunction with a variety of probes including toxins (cholera and pertussis toxins) and hormones known to affect G proteins as well as specific protein kinase inhibitors and agonists. The findings are expected to shed light on normal hepatic physiology and on mechanisms of those diseases affecting hepatic vesicular transport.

肝细胞使用细胞内囊泡运输蛋白质和 大分子进入，跨越细胞，进出 质膜，称为内吞作用和胞吞作用的过程。 大多数 这些囊泡是通过独特的电质子泵酸性的 通常与最大的氯化物电导并联起作用 酸化囊泡内部并限制膜电位。 囊泡 酸化对于有效的分类和蛋白质递送至关重要， 为了降解大分子，用于将小溶质传输到或 从酸化的囊泡和翻译后加工中分娩 溶酶体酶的功能。 病毒和毒素还利用 内吞囊泡的酸性环境引发感染。 虽然 在许多细胞中都发现了囊泡转运和酸化 关于这些重要细胞功能的调节，知之甚少。 我的 自己的工作表明霍乱和百日咳毒素，营地和蛋白质 激酶A全部增加内体酸化，结果 对整体囊泡运输的影响。 此外，从 其他人则建议可以通过一种多样性来调节囊泡运动本身 特工，包括营地，可能部分与囊泡变化有关 酸化。 此外，内吞作用是增加临床 重要的是影响肝内吞作用的多种疾病已经存在 确定，包括家族性高胆固醇血症，酒精性肝脏 疾病，糖尿病，铜超负荷和溶酶体储存障碍。 最后，正在用于基因疗法的内吞作用。 长期 我的研究目标是表征，定量和比较 囊泡酸化的机制，以确定调节过程 酸化和囊泡转运，并确定 这些总体肝功能中的调节过程。 基于工作 我本人和他人，我在其中提出了一系列假设 这些目标的背景将在提出的研究中进行测试 此应用程序。 我建议该受体偶联的G蛋白，位于 细胞内囊泡，调节内体酸化并与 刺激的GS蛋白和胃肠道蛋白，也许耦合到体状， 有效滋补囊泡酸化。 我建议这些 作用是由CAMP，蛋白激酶A和蛋白质介导的 激酶C.我建议这些药物通过 对质子泵，相关离子转运蛋白和/或变化的影响 囊泡大小，酸化的变化与变化有关 内吞作用和囊泡转运率。 我将测试这些 假设使用孤立的内吞囊泡以及分离的 灌注大鼠肝脏和培养的大鼠肝细胞与 包括毒素（霍乱和百日咳毒素）和 已知会影响G蛋白以及特定蛋白激酶的激素 抑制剂和激动剂。 这些发现有望散发出正常 肝生理学以及影响肝的疾病的机制 囊泡运输。