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，不仅肝细胞必须分泌胆汁，而且还必须由胆管上皮细胞或胆管细胞进一步修饰和调节。胆管细胞功能异常导致胆汁淤积，这是肝脏疾病的主要表现。胆汁淤积性肝病占美国肝脏移植的20%，是儿童移植患者中最常见的肝病原因。此外，胆管细胞功能异常是最常见的遗传性疾病之一囊性纤维化肝脏表现的原因。胆管细胞的胆汁分泌部分受胞浆Ca2+调节。一般来说，细胞受Ca2+信号随时间的模式和Ca2+信号发生的细胞区域的调节。然而，人们对胆管细胞中Ca2+信号的时间或空间方面知之甚少，也不知道这些Ca2+信号是如何调节的。肌醇1,4,5-三磷酸受体（InsP3R）在上皮细胞中介导Ca2+信号，胆管细胞表达该受体的所有三种亚型。该建议的假设是胆管细胞中的Ca2+信号受到InsP3R亚型的亚细胞分布的调节。这一假设将通过以下具体目的进行研究：将在单通道水平上比较insp3r的功能和调控。每个受体在胆管细胞中对Ca2+信号的作用将在胆管细胞系中进行检查，以表达这些受体中的一个或两个。这些发现将与Ca2+信号的组织和天然胆管细胞的分泌功能有关，如在分离的微灌注胆管段中确定的。这项工作不仅应该确定胆管细胞中Ca2+信号传导的分子机制，而且可以作为信号传导途径的分子组织如何负责调节小管分泌的模型。

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.