REGULATION OF SECRETION BY BILE DUCT EPITHELIAL CELLS

这项建议中描述的研究检查了负责的机制 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% 胆汁成分的体积和修饰，但对其知之甚少 负责的细胞机制。膜片钳技术的最新观察 记录技术表明，顶膜中的C1-通道可能 在调节分泌方面起着关键作用。使用膜片钳和其他 技术，第一个工作假说的长期目标是 识别和表征细胞内信号转导的作用 BDE细胞膜C1-(和其他)通道的调节途径。 具体目标是：i)评估 Ca~(2+)和cAMP依赖的C_1电流并确定其作用 钙和cAMP依赖的蛋白激酶对磷酸化的调节 在当前的激活中；ii)识别对 对这些电流，重点是cAMP激活的~8pS c1-通道 Iii)确定cAMP依赖的电流是否 囊性纤维化跨膜的内源性表达 电导调节蛋白(CFTR)，一种可能的C1通道蛋白；以及iv)到 评价GTP结合(G)蛋白在抑制HIGH中的作用 电导阴离子通道，并在激活整个细胞的C1-电流。 第二个工作假设的长期目标是确定 以及有助于独立调节的生理因素 导管细胞分泌物。具体目标是：1)开发模型系统 受体介导的跨上皮细胞调节的研究 运输；二)界定依赖营地和不依赖营地的机制 负责由促胰液素激活C1-通道；以及iii) 评价细胞外腺苷分泌的调节作用 和ATP，它通过嘌呤能受体，增加腺酰环化酶 活性和细胞内钙离子。膜的研究 C1-和其他通道为生理学和药理学提供了焦点 胆管细胞分泌的调节直接促进胆汁淤积。 胆汁的体积和成分，以及异常的调节也可能是 对于胆汁淤积表现的囊性纤维化和其他原发疾病 影响胆管上皮细胞的疾病。

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.