REGULATION OF SECRETION BY BILE DUCT EPITHELIAL CELLS

本提案中所述的研究审查了 用于肝内胆管分泌液体和电解质 上皮（BDE）细胞。 胆管分泌物占胆汁的13-40% 胆汁成分的体积和改变，但对 细胞机制负责。 膜片钳的最新观察 记录技术表明，顶膜中的C1通道可能 在调节分泌方面起关键作用。 使用膜片钳和其他 技术，第一个工作假设的长期目标是 识别和表征细胞内信号传导的作用， BDE细胞膜C1-（和其他）通道的调节途径。 具体目标是：i）评价 Ca 2+和cAMP依赖的C1-电流，并确定 Ca 2+和cAMP依赖性蛋白激酶调节磷酸化 在当前激活; ii）以确定离子通道， 这些电流，重点是一个约8 pS的C1-通道激活的cAMP iii）确定cAMP依赖性电流是否在细胞内表达; 与囊性纤维化跨膜蛋白的内源性表达相关 电导调节因子（CFTR），一种推定的C1-通道蛋白;和iv） 评估GTP结合（G）蛋白在抑制高表达的 电导阴离子通道，并激活整个细胞C1电流。 第二个工作假设的长期目标是确定 和生理因素，有助于独立调节 导管细胞分泌 具体目标是：i）开发模型系统 用于研究受体介导的跨上皮调节 运输; ii）定义cAMP依赖性和非依赖性机制 负责分泌素激活C1通道;和iii） 评价在调节细胞外腺苷分泌中的作用 和ATP，通过嘌呤能受体，增加腺苷酸环化酶 活性和细胞内Ca ~（2+）。 膜的研究 C1-和其他通道为生理和药理学研究提供了焦点。 调节胆管细胞分泌，这直接有助于 胆汁的体积和成分，以及异常调节也可能导致 对于囊性纤维化和其他原发性胆汁淤积表现， 影响胆管上皮的疾病。

The studies described in this proposal examine the mechanisms responsible for secretion of fluid and electrolytes by intrahepatic bile duct epithelial (BDE) cells. Ductular secretion accounts for 13-40% of bile volume and modification of bile composition, but little is known of the cellular mechanisms responsible. Recent observations using patch clamp recording techniques suggest that C1-channels in the apical membrane may play a key role in regulation of secretion. Using patch clamp and other techniques, the long term objective of the first working hypothesis is to identify and characterize the role of intracellular signalling pathways in regulation of BDE cell membrane C1- (and other) channels. The Specific Aims are: i) to evaluate the distinctive properties of Ca2+- and cAMP-dependent C1- currents and determine the role of regulatory phosphorylation by Ca2+- and cAMP-dependent protein kinases in current activation; ii) to identify the ion channels which contribute to these currents, with emphasis on an ~8 pS C1-channel activated by cAMP in the intact cell; iii) to determine whether cAMP-dependent currents are related to endogenous expression of cystic fibrosis transmembrane conductance regulator (CFTR), a putative C1- channel protein; and iv) to evaluate the role of GTP-binding (G) proteins in inhibition of high conductance anion channels, and in activation of whole cell C1- currents. The long term objective of the second working hypothesis is to identify and physiologic factors which contribute to independent regulation of duct cell secretion. The Specific Aims are i) to develop model systems for investigation of receptor-mediated regulation of transepithelial transport; ii) to define the cAMP-dependent and -independent mechanisms responsible for activation of C1- channels by secretin; and iii) to evaluate the role in regulation of secretion of extracellular adenosine and ATP which, through purinergic receptors, increase adenylyl cyclase activity and intracellular Ca2+, respectively. Investigation of membrane C1-and other channels provides a focus for physiologic and pharmacologic regulation of bile duct cell secretion which contributes directly to the volume and composition of bile, and abnormal regulation may also account for the cholestatic manifestations of cystic fibrosis and other primary disorders affecting biliary epithelia.