Regulation of secretion by bile duct epithelial cells

描述(由申请人提供)：本建议中描述的研究涉及负责胆管细胞分泌的细胞机制，胆管细胞是排列在肝内胆管管腔内的上皮细胞，约占人体胆汁容量的40%。以往的研究表明，胆管细胞的分泌能力随着生理需求的变化而发生动态变化，并依赖于对cAMP、细胞内钙离子和细胞体积的选择性地改变单独的氯离子通道。值得注意的是，这些通道存在于高度受调控的细胞内小泡中，并且有足够的容量在几分钟内取代整个胆管细胞质膜的40%。因此，特定的目的是为了解决一个工作假说，即含有离子通道的膜小泡的运输(内吞和胞吐)为改变胆管细胞膜的组成和电导提供了一个早期和必要的机制，并且对于cAMP、Ca~(2+)和细胞体积的分泌反应是必不可少的。具体目标包括：1)表征负责调节胞吞和胞吐作用的细胞信号；2)评估囊泡运输和PKCA直接调节对调节胆管细胞体积和胆小管分泌至关重要的SKCa通道的磷酸化的相对作用；3)确定维持负责顶端ATP释放、P2受体分布以及K+和Cl-分泌的多蛋白信号复合体的细胞策略。长期目标是确定胆管分泌所涉及的细胞机制，并确定通过作用于胆管细胞而促成胆汁形成的生理因素。因此，这些发现直接关系到以胆管细胞功能受损为特征的广泛胆汁淤积性疾病的诊断和治疗，以及通过对胆管细胞的影响来改变胆汁体积和成分的药理学方法的发展。

DESCRIPTION (provided by applicant): The studies described in this proposal address the cellular mechanisms responsible for secretion by cholangiocytes, the epithelial cells that line the lumen of intrahepatic bile ducts and account for approximately 40 percent) of bile volume in man. Previous studies indicate that the secretory capacity of cholangiocytes undergoes dynamic changes in response to varying physiologic demands; and depends on selective) penning of separate Cl- channels in response to cAMP, cytosolic Ca2+, and cell volume. Notably, these channels are present in intracellular vesicles that are highly regulated and are of sufficient capacity to replace 40 percent of the entire cholangiocyte plasma membrane within minutes. Accordingly, the Specific Aims are designed to address the working hypothesis that trafficking (endocytosis and exocytosis) of membrane vesicles containing ion channels provides an early and essential mechanism for modifying the composition and conductance of the cholangiocyte plasma membrane and is essential for the secretory responses to cAMP, Ca2+, and cell volume. The Specific Aims occurs on 1) characterization of the cellular signals responsible for regulation of endocytosis and exocytosis, 2) evaluation, of the relative roles of vesicular trafficking and direct regulatory phosphorylation by PKCa of SKCa channels essential for modulation of cholangiocyte volume and Juctular secretion; and 3) definition of the cellular strategies for maintaining the multiprotein signaling complex responsible for apical ATP release, P2 receptor distribution and K+ and Cl- secretion. The long-term goal is to define the cellular mechanisms involved in ductular secretion, and to identify the) physiologic factors which contribute to bile formation through effects on duct cells. Thus, the findings ire directly relevant to diagnosis and management of a broad range of cholestatic disorders characterized by impaired cholangiocyte function; and to development of pharmacologic approaches o modify the volume and composition of bile through effects on duct cells.