ELECTROPHYSIOLOGY OF ORGANIC ION TRANSPORT IN HEPATOCYTE

肝细胞中有机离子转运的电生理学

• 批准号: 3464233
• 负责人: STEVEN A WEINMAN
• 金额: $ 9.52万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3464233
• 关键词: binding proteins; cholanate compound; electrophysiology; fluorescence; ion transport; ligands; liver cells; microelectrodes; secretion; voltage /patch clamp

The goals of this project are to understand the mechanisms by which hepatocytes transport organic ions. These transport processes result in large concentration gradients, are impaired in cholestatic diseases, and are important considerations in the development of therapeutic and diagnostic agents. The specific aims are: 1) to determine the electrical and chemical driving forces for uptake of organic ions. 2) To measure the contribution of electrogenic and ATP-dependent mechanisms to the secretion of organic ions into the canaliculus. 3) To determine the role of the intracellular binding capacity in organic solute uptake. The experimental design will use direct electrophysiological measurements in individual hepatocytes and secreting isolated hepatocyte couplets to measure uptake and secretion. Electrogenic transport currents will be measured with microelectrodes after blocking other membrane conductances and by tight-seal whole-cell patch clamp. Uptake of fluorescent organic ions such s bile salts and fluorescein derivatives into single cells will be directly determined in impaled cells. Secretion into the biliary space will be studied in couplets by determining the rate of fluorescence appearance in the canalicular space as a function of membrane voltage and cellular ATP. ATP-dependent organic ion efflux has previously been observed in liver and may be an important component of canalicular secretion. This hypothesis will be tested by maneuvers to deplete ATP in couplets and by observing secretion in whole-cell-path- clamped couplets in which the intracellular ATP concentration is varied directly. The role of intracellular binding in transport will be evaluated by observing the effect of varying the concentration of intracellular ligands and binding proteins on apparent unidirectional influx rate. These studies will provide much needed information on the driving forces for organic ion accumulation and secretion in the liver. Ultimately, this information could be used to limit hepatocellular damage from transported toxins, to improve targeting of therapeutic agents to hepatocytes and hepatocellular carcinomas, and to assist in the development of diagnostic studies that can be applied to extremely small samples of liver, such as that from a liver biopsy.

该项目的目标是了解其机制 肝细胞运输有机离子。 这些运输过程导致 大的浓度梯度，在胆汁淤积性疾病中受损，并且 是开发治疗和治疗的重要考虑因素 诊断剂。 具体目标是： 1) 确定电 和吸收有机离子的化学驱动力。 2) 测量 生电和 ATP 依赖性机制对分泌的贡献 有机离子进入泪小管。 3）确定角色 有机溶质摄取的细胞内结合能力。 实验设计将使用直接电生理测量 在单个肝细胞中并分泌分离的肝细胞对 测量摄取和分泌。 生电传输电流将是 阻断其他膜电导后用微电极测量 以及通过紧密密封的全细胞膜片钳。荧光有机物的吸收 胆汁盐和荧光素衍生物等离子进入单细胞将 可以直接在刺穿的细胞中测定。 将通过确定对联来研究胆道空间的分泌 泪小管间隙中荧光出现率的函数 膜电压和细胞 ATP 的关系。 ATP依赖性有机离子流出 先前在肝脏中观察到，可能是 泪小管分泌。 这一假设将通过演习来检验 通过观察全细胞路径中的分泌来消耗对联中的 ATP 细胞内 ATP 浓度变化的夹紧对 直接地。 细胞内结合在运输中的作用将通过以下方式评估 观察改变细胞内配体浓度的影响 和结合蛋白对表观单向流入速率的影响。 这些研究将提供有关驱动力的急需信息 用于肝脏中有机离子的积累和分泌。最终，这 信息可用于限制运输造成的肝细胞损伤 毒素，以改善治疗剂对肝细胞的靶向性 肝细胞癌，并协助开发诊断方法 可应用于极小肝脏样本的研究，例如 来自肝活检。