MECHANISMS OF CANALICULAR BILE FORMATION

胆管形成机制

• 批准号: 3227808
• 负责人: BRUCE F SCHARSCHMIDT
• 金额: $ 25.35万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3227808
• 关键词: G protein; Xenopus; acidity /alkalinity; animal tissue; apical membrane; bile; bilirubin; biological fluid transport; calcium; cyclic AMP; electrophysiology; fatty acid transport; fluorescent dye /probe; genetic translation; human tissue; ion transport; laboratory rat; liver cells; liver metabolism; long chain fatty acid; membrane potentials; membrane proteins; messenger RNA; molecular cloning; northern blottings; oleate; protein kinase; radiotracer; sodium; stoichiometry; transfection; transport proteins; voltage /patch clamp

The metabolic, digestive, and excretory functions of the liver are dependent on the uptake from blood and/or secretion into bile of circulating amphipathic organic solutes, including bile acids, nonbile acid organic anions such as bilirubin, and fatty acids. Accumulation of solutes such as bilirubin or bile acids in the bloodstream largely account for the biochemical or clinical manifestations of certain hereditary disorders of hepatic organic anion transport, as well as of cholestasis, a common manifestation of acquired liver disease. We and others have characterized the hepatocyte transport mechanisms for bile acids, nonbile acid organic anions such as bilirubin, and long-chain fatty acids. Progress toward the isolation of certain transport proteins has been reported; however, attempts at purification and antibody production have been frustrated by the low abundance of such proteins and/or the presence of multiple membrane proteins of similar molecular weight. Moreover, important aspects of the function of these transport proteins remain undefined. For example, our preliminary observations suggest that the uptake of taurocholate and of the long-chain fatty acid oleate is electrogenic, yet little is known regarding the relationship between transport of bile acids or fatty acids and transmembrane electrical potential difference, which we have shown to vary in response to physiologic stimuli and intracellular pH. Certain hormones alter transport of bile acids or fatty acids, but it is not known whether these changes reflect a direct effect on transport, mediated possibly by Ca2+ or cAMP, or hormonal effects on transmembrane electrical potential difference. The role of transmembrane ion gradients and ATP in the transport of certain organic anions is also unclear. The proposed studies will define the function and regulation of several liver-specific transport proteins which mediate the basolateral uptake or canalicular secretion of certain bile acids and nonbile acid organic anions. We will utilize a variety of approaches including electrophysiological and fluorescence techniques which have been developed over the previous grant period, are ideally suited to address the questions posed, and have had limited application to liver. These techniques will be applied to hepatocytes, as well as to expression systems including Xenopus laevis oocytes and transfected mammalian cells, in which the function of a single transport protein can be studied in isolation. To accomplish this objective, we and our collaborators will utilize expression cloning in Xenopus laevis oocytes, a strategy which does not depend on antibodies or synthetic nucleotides, has been successfully used to clone ion channels, receptors, and transporters, and, as demonstrated by our collaborators and our own preliminary studies, can be applied to liver specific membrane transport proteins. The proposed studies will provide new insights into mechanisms of canalicular bile formation and will advance our understanding of the pathophysiology of certain hereditary hepatic disorders and of cholestatic liver disease.

肝脏的代谢，消化和排泄功能是 依赖于血液和/或分泌到胆汁的吸收 循环两亲的有机溶质，包括胆汁酸，非胆汁酸 有机阴离子，例如胆红素和脂肪酸。 溶质的积累 例如胆红素或血液中的胆汁酸在很大程度上解释 某些遗传疾病的生化或临床表现 肝脏有机阴离子运输以及胆汁淤积 获得的肝病的表现。 我们和其他人的特征 胆汁酸，非玻璃酸有机的肝细胞转运机制 胆红素和长链脂肪酸等阴离子。 朝着 已经报道了某些转运蛋白的分离。然而， 纯化和抗体产生的尝试受到了挫败 这种蛋白质的低丰度和/或存在多个膜的存在 相似分子量的蛋白质。 而且， 这些转运蛋白的功能仍然不确定。 例如，我们的 初步观察表明，牛角胆酸盐的摄取和 长链脂肪酸是电的，但关于 胆汁酸或脂肪酸的转运与 跨膜电势差，我们已证明这有所不同 响应生理刺激和细胞内pH。 某些激素 改变胆汁酸或脂肪酸的转运，但尚不清楚是否知道 这些变化反映了对运输的直接影响，可能是由 CA2+或CAMP，或对跨膜电势的荷尔蒙影响 不同之处。 跨膜离子梯度和ATP在 某些有机阴离子的运输也不清楚。 拟议的研究将定义几个的功能和调节 介导基底外侧摄取或 某些胆汁酸和非胆汁酸有机物的口腔分泌 阴离子。 我们将采用各种方法 已经开发的电生理和荧光技术 在上一个赠款期间，非常适合解决问题 摆姿势，对肝脏的应用有限。 这些技术将是 应用于肝细胞以及包括Xenopus的表达系统 Laevis卵母细胞和转染的哺乳动物细胞，其中A的功能 单个转运蛋白可以分离研究。 实现这一目标 目的，我们和我们的合作者将使用表达克隆 Xenopus laevis卵母细胞，一种不依赖抗体或 合成核苷酸已成功用于克隆离子通道， 受体和转运蛋白，以及我们的合作者和 我们自己的初步研究可以应用于肝脏特定的膜 转运蛋白。 拟议的研究将为您提供新的见解 大口径胆汁形成的机制，并将提高我们的理解 某些遗传性肝疾病的病理生理学和 胆固性肝病。