ELECTROPHYSIOLOGY OF ORGANIC ION TRANSPORT IN HEPATOCYTE

这个项目的目标是了解 肝细胞运输有机离子。这些传输过程导致 大浓度梯度，在胆汁淤积性疾病中受损，以及 是发展治疗和治疗的重要考虑因素 诊断试剂。具体目标是：1)确定电气参数 以及吸收有机离子的化学驱动力。2)测量 电生机制和ATP依赖机制对分泌的贡献 有机离子进入小管。3)确定 细胞内有机溶质吸收的结合能力。 实验设计将使用直接电生理测量。 在单个肝细胞中和分泌分离的肝细胞偶联 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 采用全细胞膜片钳全细胞紧密封闭。有机荧光物质的摄取 S胆盐和荧光素衍生物等离子会变成单细胞 在穿孔细胞中直接测定。 进入胆管间隙的分泌物将在对联中研究，通过确定 小管间隙内的荧光出现率作为函数 膜电压和细胞内三磷酸腺苷。依赖于ATP的有机离子外流有 以前在肝脏中观察到，可能是 小管分泌物。这一假设将通过以下策略得到检验： 消耗偶联中的ATP，并通过观察全细胞路径的分泌- 细胞内三磷酸腺苷浓度变化的钳位对 直接去吧。 细胞内结合在运输中的作用将通过以下方式进行评估 改变细胞内配体浓度的效果观察 结合蛋白对表观单向内流速率的影响。 这些研究将提供有关驱动力的迫切需要的信息 用于肝脏中有机离子的积累和分泌。归根结底，这 信息可以用来限制运输对肝细胞的损害 毒素，以改善治疗剂对肝细胞的靶向性 肝细胞癌，并协助制定诊断 可以应用于极小的肝脏样本的研究，例如 从肝脏活检中得出的结论。

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.