MECHANISMS OF CANALICULAR BILE FORMATION AND CHOLESTASIS

胆管形成和胆汁淤积的机制

• 批准号: 3080404
• 负责人: REBECCA W. VAN DYKE
• 金额: $ 7.56万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1989-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3080404
• 关键词: adenosinetriphosphatase; alanine; bicarbonates; bile; biliary tract disorder; cell membrane; chlorpromazine; estrogens; fluorescent dye /probe; liver; liver cells; membrane potentials; perfusion; secretion; tissue /cell culture

Recent work by the applicant and others has identified an electrogenic proton pump (H+-ATPase) in rat liver endocytic vesicles and in organelles in other tissues. This H+-ATPase plays an important role in receptor-mediated endocytosis and urinary acidification, yet little is known regarding its regulation or role in other cell functions. The proposed studies will further characterize the H+-ATPase in rat liver endocytic vesicles and determine its role in other liver processes. The specific aims of this proposal are (1) to study in detail the function of H+-ATPase and its possible regulation by anions and organic nitrates; (2) to characterize transport of C1- (essential for vesicle acidification) in the same vesicles; (3) to quantitate pH and membrane potential gradients established by H+-ATPase and to examine these aspects of H+-ATPase function in vesicles derived from four different steps along the endocytic pathway; (4) to explore a possible plasma membrane location for H+-ATPase and to determine its role in hepatocyte pHi regulation, H+ efflux, biliary HCO3- secretion, and bile acid-independent bile formation (BAIBF); and (5) to explore the hypothesis that hepatocyte H+/HCO3- transport and BAIBF may be regulated, in part, by exocytotic insertion/endocytic removal of H+-ATPase units into/from the plasma membrane. These specific aims will be addressed using a variety of experimental models and techniques. Fluorescent probes and radiolabeled tracers will be used to examine and quantitate H+-ATPase function in intracellular and plasma membrane vesicles. pHi regulation and H+ efflux will be studied in isolated hepatocytes using fluorescent and pH-STAT techniques. Bile formation and endocytosis will be studied in the isolated perfused rat liver and in cultured rat hepatocytes. All of these methods are currently in use in the applicant's laboratory, and the ability to use a variety of models and techniques and to integrate the findings from studies at the subcellular, cellular, and intact organ level will facilitate attainment of these specific aims. The H+-ATPase appears to be widely distributed in nature, yet these issues have not been systematically addressed in any cell type. Thus, the findings of the proposed studies are expected to shed new light not only on liver endocytosis, pHi regulation, bile formation, and cholestasis, but also on pHi regulation, endocytosis, lysosomal degradation, cholesterol metabolism, storage and secretion of neurotransmitters, hormones and proteins, viral infection and epithelial H+ transport in many cell types.

申请人和其他人的最新工作已经确定了电源 大鼠肝内吞囊泡和细胞器中的质子泵（H+-ATPase） 在其他组织中。 此H+-ATPase在 受体介导的内吞作用和尿酸化，但几乎没有 有关其调节或在其他细胞功能中的作用的已知。 这 拟议的研究将进一步表征大鼠肝脏中的H+-ATPase 内吞囊泡并确定其在其他肝脏过程中的作用。 这 该提案的具体目的是（1）详细研究 H+-ATPase及其可能由阴离子和有机硝酸盐调节； （2） 表征C1-（囊泡酸化必不可少的） 相同的囊泡； （3）定量pH和膜电位梯度 由H+-ATPase建立并检查H+-ATPase函数的这些方面 在沿着内吞途径的四个不同步骤中得出的囊泡中； （4）探索H+-ATPase的可能的质膜位置 确定其在肝细胞PHI调节中的作用，H+外排，胆道HCO3- 分泌和胆汁酸独立的胆汁形成（BAIBF）； （5）到 探索肝细胞H+/HCO3-运输和BAIBF的假设可能是 部分受H+-ATPase的胞吐插入/内吞去的调节 从质膜进入/从质膜进入/单位。 这些具体目标将被解决 使用多种实验模型和技术。 荧光探针 和放射标记的示踪剂将用于检查和定量H+-ATPase 在细胞内和质膜囊泡中的功能。 PHI调节和 将使用荧光和 pH-stat技术。 将研究胆汁形成和内吞作用 孤立的灌注大鼠肝脏和培养的大鼠肝细胞。 所有这些 方法目前正在申请人的实验室中使用，并且能力 使用各种模型和技术并整合发现 从亚细胞，细胞和完整器官水平的研究将 促进达到这些特定目标。 H+-ATPase似乎在本质上分布广泛，但是这些问题 在任何细胞类型中都没有系统地解决。 因此， 拟议研究的发现预计将不仅为 肝内吞作用，PHI调节，胆汁形成和胆汁淤积，但 同样关于PHI调节，内吞作用，溶酶体降解，胆固醇 神经递质，激素和激素的代谢，储存和分泌 许多细胞类型的蛋白质，病毒感染和上皮H+转运。