MECHANISM OF CANALICULAR BILE FORMATION AND CHOLESTASIS

胆管形成和胆汁淤积的机制

• 批准号: 2139047
• 负责人: MOHAMMED SAWKAT ANWER
• 金额: $ 23.78万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-07-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2139047
• 关键词: bile; calcium flux; cholates; cholestasis; cyclic AMP; fluorimetry; hormone regulation /control mechanism; intracellular transport; ion transport; laboratory rat; liver cells; liver metabolism; membrane potentials; membrane transport proteins; phosphorylation; protein kinase; sodium potassium exchanging ATPase; western blottings

The long-term objective of the present proposal is to more clearly define cellular mechanisms involved in hepatic solute transport and bile formation. Solute transport from blood to bile is an important function of the liver, with accumulation of (otherwise excreted) endogenous and exogenous solutes in blood being a common feature of cholestasis. Specific aims of this proposal are to better define cellular mechanisms involved in the endocrine regulation of two hepatic solute transporters; Na+/H+ exchange being integrally involved with the regulation of intracellular pH, and Na+/taurocholate (TC) cotransport being involved in hepatic uptake of bile acids, which play an important role in file formation. The following hypotheses will be tested: 1) hormonal regulation of Na+/H+ exchange may involve kinase-mediated phosphorylation of the transporter, and 2) hormones may stimulate Na+/TC cotransport directly by translocating the transporter from intracellular organelles to the plasma membrane, and/or indirectly by changing membrane potential. Effects of hormones and kinases on Na+/TC cotransport and Na+/H+ will be studied in isolated rat hepatocytes and plasma membrane vesicles using established techniques. Intracellular [Ca++] pH and [Na+] will be quantitated using fluorometric methods. Suggestive evidence for the involvement of kinase will be obtained through transport studies in hepatocytes using known inhibitors and activators of specific kinases. Direct modification of the transporter will be assessed by studying transport in membrane vesicles treated with specific kinases. Transport studies in membrane vesicles prepared from hormone-treated hepatocytes would indicate if hormone action involves stable alteration (phosphorylation/translocation) of transport proteins. Regulation of phosphorylation of the Na+/H+ exchanger or translocation of the Na+/TC cotransporter will be evaluated by protein phosphorylation and immunoblotting studies, respectively. Subcellular fractions (microsomes and golgi complex) isolated from hormone-treated hepatocytes will be immunoblotted to determine the source(s) of the translocated transporter. Whether a hormone action is mediated indirectly via changes in membrane potential will be evaluated by correlating effects of the hormone on Na+/TC cotransport and membrane potential under different experimental conditions. Studies will also be conducted to determine the role of Na+,K+ -ATPase in hormone-induced changes in membrane potential. Collectively, proposed studies should provide further insight into cellular mechanisms involved in the hormonal regulation of hepatic Na+/H+ exchange and Na+/TC cotransport.

本提案的长期目标是更清楚地定义 肝溶质传输和胆汁涉及的细胞机制 形成。 从血到胆汁的溶质传输是重要的功能 肝脏的积累（其他排泄）内源性和 血液中的外源溶质是胆汁淤积的共同特征。 该建议的具体目的是更好地定义细胞机制 参与了两个肝溶质转运蛋白的内分泌调节； Na+/H+交换与调节的调节完全涉及 涉及细胞内pH和Na+/taurochalice（TC）共转运 在肝脏摄取中，胆汁酸在文件中起重要作用 形成。 将测试以下假设：1）激素 Na+/H+交换的调节可能涉及激酶介导的磷酸化 转运蛋白和2）激素可能会刺激Na+/Tc共转运 直接通过从细胞内细胞器转运转运蛋白 通过改变膜电位而间接地到质膜和/或间接。 激素和激酶对Na+/TC共晶和Na+/H+的影响 使用分离的大鼠肝细胞和质膜囊泡进行研究 建立的技术。 细胞内[Ca ++] pH和[Na+]将为 使用荧光测定法定量。 暗示性证据 激酶的参与将通过运输研究获得 肝细胞使用特定激酶的已知抑制剂和活化剂。 将通过研究来评估转运蛋白的直接修改 用特定激酶处理的膜囊泡的转运。 运输 由激素处理的肝细胞制备的膜囊泡研究 会表明激素作用是否涉及稳定的改变 转运蛋白的（磷酸化/易位）。 调节 Na+/H+交换器的磷酸化或Na+/Tc的易位 共转运蛋白将通过蛋白质磷酸化和 免疫印迹研究。 亚细胞级分（微粒体 从激素处理的肝细胞中分离出的高尔基体配合物将是 免疫印迹以确定易位转运蛋白的来源。 激素作用是否通过膜的变化间接介导 电位将通过将激素对对激素的影响进行评估 Na+/TC共转移和膜电位在不同的实验下 状况。 还将进行研究以确定 激素诱导的膜电位变化中的Na+，K+ -ATPase。 拟议的研究总的来说，应进一步了解 肝Na+/H+的激素调节涉及的细胞机制 Exchange和Na+/TC共晶。