Regulation of cholangiocytes by InsP3 receptor isoforms

描述(申请人提供)：胆汁分泌是肝脏的主要功能之一。为了维持胆汁流动，肝细胞不仅必须分泌胆汁，而且还必须由胆管上皮细胞或胆管细胞进一步修改和调节胆汁。胆管细胞功能异常导致胆汁淤积，是肝病的主要表现。胆汁淤积性肝病是美国20%的肝移植手术的原因，也是儿童肝移植患者中最常见的肝病原因。此外，囊性纤维化的肝脏表现与胆管细胞功能异常有关，囊性纤维化是最常见的遗传性疾病之一。胆管细胞的胆汁分泌在一定程度上受细胞内钙离子的调节。一般说来，细胞既受钙信号随时间变化的模式的调节，也受细胞内出现钙信号的区域的调节。然而，对胆管细胞中钙信号的时间或空间方面知之甚少，也不知道这些钙信号是如何调节的。1，4，5-三磷酸肌醇受体(InsP3R)介导上皮细胞的钙信号转导，胆管细胞表达该受体的三种亚型。这一假设认为，胆管细胞内的钙信号受InsP3R亚型亚细胞分布的调控。这一假说将通过以下具体目标进行研究：在单通道水平上比较InsP3Rs的功能和调节。每种受体在胆管细胞中对钙信号的作用将在胆管细胞系中进行检测，该细胞系被改造为表达这些受体中的一种或一种。这些发现将与天然胆管细胞中钙信号的组织和分泌功能有关，正如在分离的微灌流胆管节段中所确定的那样。这项工作不仅应该确定胆管细胞中负责钙信号转导的分子机制，而且应该作为信号通路的分子组织如何负责调节胆管分泌的模型。

DESCRIPTION (provided by applicant): Bile secretion is one of the principal functions of the liver. In order to maintain bile flow, not only must hepatocytes secrete bile, but this must then be modified and conditioned further by bile duct epithelial cells, or cholangiocytes. Abnormal cholangiocytes function results in cholestasis, which is a cardinal manifestation of liver disease. Cholestatic liver diseases are responsible for 20% of liver transplants in the US, and are the most common cause of liver disease among pediatric transplant patients. In addition, abnormal cholangiocyte function is responsible for the hepatic manifestations of cystic fibrosis, one of the most common inherited diseases. Bile secretion in cholangiocytes is regulated in part by cytosolic Ca2+. In general, cells are regulated both by the pattern of Ca2+ signals over time and by the regions of the cells in which Ca2+ signals occur. However, little is known about temporal or spatial aspects of Ca2+ signaling in cholangiocytes, and nothing is known about how these Ca2+ signals are regulated. Inositol 1,4,5-trisphosphate receptors (InsP3R) mediate Ca2+ signaling in epithelia, and cholangiocytes express all three isoforms of this receptor. The hypothesis of this proposal is that Ca2+ signals in the cholangiocyte are regulated by the subcellular distribution of the InsP3R isoforms. This hypothesis will be investigated through the following specific aims: The function and regulation of the InsP3Rs will be compared at the single channel level. The contribution that each of the receptors plays to Ca2+ signaling in cholangiocytes will be examined in a bile duct cell line modified to express either one or a combination of these receptors. These findings will be related to the organization of Ca2+ signals and secretory function in native cholangiocytes, as determined in isolated microperfused bile duct segments. This work should not only identify the molecular mechanisms responsible for Ca2+ signaling in cholangiocytes, but serve as a model for how the molecular organization of signaling pathways is responsible for regulation of ductular secretion.