Regulation of Secretion by Bile Duct Epithelial Cells

本提案中描述的研究通过以下方式解决了负责分泌的细胞机制： 胆管细胞，排列在肝内胆管内腔的上皮细胞，占胆汁的约40%。 先前的研究表明，胆管细胞的分泌能力经历了动态的变化， 响应不同生理需求的变化;并已确定了多个细胞内 囊泡可以迅速动员，大小足以取代高达40%的整个 胆管细胞质膜内。此外，最好的证据支持一个模型，其中一个 这些囊泡池中富含ATP，胞吐作用导致细胞外ATP的局部增加。 核苷酸和嘌呤能信号级联的启动。这些不同的囊泡池在功能上 与细胞ATP释放、嘌呤能信号的激活和分离的C/” 通道对cAMP、胞质Ca 2+和细胞体积的响应。因此，设计了具体目标 为了进一步解决工作假设，这是不变的，即运输（内吞和 胞吐作用）的膜囊泡含有离子通道提供了早期和必要的机制， 改变胆管细胞质膜的组成和电导， 对cAMP、Ca 2+和细胞体积的分泌反应。具体目标继续侧重于1） 表征负责调节内吞作用和胞吐作用的细胞信号; 2） 小电导K ~+（SK ~ 2）通道在胆管细胞调节中的功能作用 体积和导管分泌;和3）维持多蛋白的细胞策略的定义 信号传导复合物负责顶端ATP释放、P2受体分布以及K+和Cl-分泌。到 为此，我们开发了结合成像和生物物理方法来测量膜 动力学和真实的时间中的离子运输;并且涉及宏观和微观两方面的细胞ATP释放的检测。 个体细胞模型。长期的目标是确定参与小管形成的细胞机制。 分泌，并确定通过对导管的影响促进胆汁形成的生理因素 细胞因此，这些发现仍然直接关系到诊断和管理的广泛的 以受损的胆管细胞功能为特征的胆汁淤积性疾病;以及 通过影响胆管细胞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