Regulation of Secretion by Bile Duct Epithelial Cells

这项建议中描述的研究解决了负责分泌的细胞机制。 胆管细胞是排列在肝内胆管管腔内的上皮细胞，占胆汁的40%左右 人类的音量。以往的研究表明，胆管细胞的分泌能力是动态的。 对不同生理需求的反应变化；并确定了细胞内的多个池 可快速动员的囊泡，其大小足以取代多达40%的整个 胆管细胞质膜在几分钟内。此外，最好的证据支持这样一种模型，即 这些囊泡池中的大多数富含三磷酸腺苷，胞吐作用导致细胞外局部增加。 核苷酸与嘌呤能信号级联反应的启动。这些不同的水泡池在功能上 与细胞内ATP的释放、嘌呤能信号的激活和选择性开放分离的C/ 对cAMP、细胞内钙离子和细胞体积的反应。相应地，设计了具体目标 为了进一步解决工作假说，这一假说没有改变，即贩运(内吞和 含离子通道的膜小泡的胞吐作用)提供了一种早期和必要的机制 改变胆管细胞质膜的组成和电导是必要的 对cAMP、钙离子和细胞体积的分泌反应。具体目标继续集中在1) 负责调节胞吞和胞吐的细胞信号的特征；2) 小电导K~+(SK2)通道在胆管细胞调节中的功能评价 体积和导管分泌物；以及3)维持多蛋白的细胞策略的定义 负责顶端ATP释放、P2受体分布以及K+和CI“分泌的信号复合体。 为此，我们开发了成像和生物物理相结合的方法来测量膜 动力学和离子传输的实时；以及检测细胞ATP释放的宏观和 单个细胞模型。长期目标是确定导管相关的细胞机制。 分泌物，并确定通过影响胆管形成胆汁的生理因素 细胞。因此，这些发现继续直接与诊断和治疗广泛的 以胆管细胞功能受损为特征的胆汁淤积性疾病； 通过对胆管细胞的作用改变胆汁体积和成分的药理学方法

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 ~40% of bile volume in man. Previous studies indicate that the secretory capacity of cholangiocytes undergoes dynamic changes in response to varying physiologic demands; and have identified multiple pools of intracellular vesicles that can be mobilized rapidly and are sufficient in size to replace up to 40% of the entire cholangiocyte plasma membrane within minutes. Further, the best evidence supports a model wherein one of these vesicular pools is enriched in ATP, and exocytosis leads to a local increase in extracellular nucleotides and initiation of a purinergic signaling cascade. These different vesicular pools are functionally linked to cellular ATP release, activation of purinergic signaling and selective opening of separateC/" channels in response to cAMP, cytosolic Ca2+, and cell volume. Accordingly, the Specific Aims are designed to further address the working hypothesis, which is unchanged, 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 continue to focus on 1) characterization of the cellular signals responsible for regulation of endocytosis and exocytosis; 2) assessment of the functional roles of small conductance K+ (SK2) channels in regulation of cholangiocyte volume and ductular 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 CI"secretion. To that end, we have developed combined imaging and biophysical approaches to measurement membrane dynamics and ion transport in real time; and to detection of cellular ATP release in both macroscopic and individual cell models. 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 continue to be directly relevant to diagnosis and management of a broad range of cholestatic disorders characterized by impaired cholangiocyte function; and to development of pharmacologic approaches to modify the volume and composition of bile through effects on duct cells