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: 3237686
• 负责人: REBECCA W. VAN DYKE
• 金额: $ 20.42万
• 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 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3237686
• 关键词: Golgi apparatus; acidity /alkalinity; adenosinetriphosphatase; aminoacid metabolism; biliary tract pharmacology; bioenergetics; calcium metabolism; cations; cholelithiasis; electron microscopy; exocytosis; homeostasis; liver cells; lysosomes; membrane potentials; organelles; receptor mediated endocytosis; solute

Hepatocytes contain many vesicular organelles, including lysosomes, Golgi, endocytic vesicles and protein secretory vesicles, the interior of which is acidified by an electrogenic proton pump (H+-ATPase). Such acidification appears essential for numerous cell functions including receptor-mediated endocytosis, degradation of exogenous and endogenous materials and efficient secretion of proteins. In turn, certain viruses and toxins exploit the acid pH of endocytic vesicles to gain entry to cell cytoplasm. The long-term goals of the proposed research are to characterize the function, distribution, and regulation of proton pumps in hepatic endocytic vesicles. The specific aims of this proposal will test two new hypotheses. First, it is proposed that vesicles at distinct stages of the endocytic pathway are acidified to differing degrees, that this is due principally to changes in ion transport rather than to time-dependent acidification and that it reflects insertion of functional proton pumps into endocytic vesicles. If so, differences in the internal pH (pHi) of vesicles are likely to determine the endocytic stage(s) where pH-dependent processes differing in pH optimum occur. Second, it is proposed that endocytic vesicles take up and accumulate a number of small molecular weight solutes including calcium, cationic drugs, organic amines, and basic amino acids. This hypothesis further holds that the proton pump-generated electrochemical gradient drives vesicular uptake and storage of solutes via either H+/solute antiporters or protonation and sequestration of lipophilic weak bases. Stored solutes may be released to cytoplasm or transferred into bile as the vesicles in which they are contained fuse with the bile canalicular membrane. These hypotheses will be tested principally with purified endocytic vesicles using radiolabeled probes and fluorescent dyes. In complementary studies, proton pumps will be localized by means of electron microscopy employing specific antibodies. The relationship of vesicular uptake to overall hepatic extraction and biliary secretion of solutes will be defined in the perfused rat liver. The proposed studies are expected to provide new insight into poorly understood aspects of vesicle acidification and of solute transport across vesicular membranes in hepatocytes and in other cell types. They have important implications for a wide range of processes including receptor-mediated endocytosis, calcium homeostasis and signal transduction, biliary calcium secretion and gallstone formation, hepatic uptake and biliary secretion of drugs, amino acid metabolism, and drug-induced phospholipidoses.

肝细胞包含许多囊泡细胞器，包括 溶酶体，高尔基体，内吞囊泡和蛋白质分泌 囊泡，其内部由电源酸化 质子泵（H+-ATPase）。 这种酸化似乎是必不可少的 对于包括受体介导的许多细胞功能 内吞作用，外源性和内源物质的降解 和有效的蛋白质分泌。 反过来，某些病毒和 毒素利用内吞囊泡的酸pH来进入 细胞细胞质。 拟议研究的长期目标是 表征功能，分布和调节 肝内吞囊泡中的质子泵。 具体目的 该建议将检验两个新的假设。 首先，提议 在内吞途径不同阶段处的囊泡是 在不同程度上酸性，这主要是由于 离子传输而不是时间依赖性的变化 酸化并反映了功能质子的插入 泵入内吞囊泡。 如果是这样，内部的差异 囊泡的pH（phi）可能决定了内吞作用阶段 pH最佳的pH依赖性过程发生不同的地方。 其次，有人提出，内吞囊泡占用， 积累了许多小分子量溶质，包括 钙，阳离子药物，有机胺和碱性氨基酸。 该假设进一步认为质子泵生成 电化学梯度驱动囊泡吸收和存储 通过H+/溶质抗植物或质子化溶质，并且 亲脂性弱碱基的隔离。 存储的溶质可能是 在囊泡中释放到细胞质或转移到胆汁中 它们被包含与胆汁的融合 膜。 这些假设将主要通过 使用放射性标记的探针纯化的内吞囊泡和 荧光染料。 在互补研究中，质子泵将是 通过电子显微镜局部使用特定的电子显微镜 抗体。 囊泡吸收与总体肝的关系 溶质的提取和胆道分泌将在 灌注大鼠肝脏。 预计拟议的研究将为您提供新的见解 囊泡酸化和溶质的方面知之甚少 在肝细胞中的囊泡膜和其他 细胞类型。 它们对广泛的重要含义 包括受体介导的内吞作用，钙的过程 稳态和信号转导，胆道分泌和 胆结石形成，肝摄取和胆道分泌，药物的分泌， 氨基酸代谢和药物诱导的磷脂。