Ca2+ Waves in Hepatocytes: Mechanisms and Effects

描述（由申请人提供）：细胞质Ca 2+是几乎所有类型细胞中的关键第二信使。相对于其他信使分子，Ca 2+是新颖的，因为它同时调节单个细胞内的多个过程。该研究项目的持续主题是研究Ca 2+如何调节特别是肝细胞中的胆汁分泌，作为Ca 2+如何调节极化上皮内局部过程的模型。在肝细胞中，Ca 2+信号完全由肌醇1，4，5-三磷酸（InsPS）介导。InsPS通过与InsPS受体（InsPSR）结合而起作用，InsPSR是内质网（ER）膜中的四聚体InsPS门控Ca 2+释放通道。InsPS因此通过InsPSR从ER释放Ca 2+来增加细胞溶质Ca 2+。InsPSR的三种已知同种型中的两种（InsP 3R-1和InsP 3R-2）在肝细胞中表达，每种都具有不同的生物物理特性。这两种亚型中的每一种都以独特的亚细胞模式分布在肝细胞中，因此可以在细胞内建立信号微区。该建议的假设是，Ca 2+信号模式及其对肝细胞胆汁分泌的影响取决于表达的InsPSR的类型及其亚细胞分布。本研究将通过以下三个具体目标来验证这一假设：（1）在分子水平和单通道水平上确定InsP 3R-1和InsP 3R-2对Ca 2+信号传导的相对作用;（2）在完整细胞中确定InsP 3R-1和InsP 3R-2对Ca 2+信号传导的相对作用;（3）将在模型细胞系统和完整的灌注肝脏中确定InsP 3R-1和InsP 3R-2对Ca 2+介导的分泌的相对作用。总之，这些研究将为胆汁淤积的分子和细胞基础提供新的见解，胆汁淤积是肝脏疾病的主要表现之一。此外，这项工作应该提供一个一般的范例，其中Ca 2+信号产生的方式，以调节特定的亚细胞区域内的事件。

DESCRIPTION (provided by the applicant): Cytosolic Ca2+ is a critical second messenger in virtually every type of cell. Ca2+ is novel relative to other messenger molecules because it regulates multiple processes simultaneously within an individual cell. The ongoing theme of this research project is to investigate how Ca2+ regulates bile secretion in hepatocytes in particular, as a model for how Ca2+ can regulate a localized process within a polarized epithelium. In the hepatocyte, Ca2+ signals are mediated entirely by inositol 1,4,5-trisphosphate (InsPS). InsPS acts by binding to the InsPS receptor (InsPSR), which is a tetrameric InsPS-gated Ca2+ release channel in the endoplasmic reticulum (ER) membrane. InsPS thus increases cytosolic Ca2+ by releasing Ca2+ from the ER via the InsPSR. Two of the three known isoforms of the InsPSR (lnsP3R-1 and lnsP3R-2) are expressed in hepatocytes, each with distinct biophysical properties. Each of these two isoforms is distributed in a distinctive subcellular pattern in the hepatocyte, which thus may establish signaling microdomains within the cell. The hypothesis of this proposal is that Ca2+ signaling patterns and their effects on hepatocyte bile secretion depend upon the types of InsPSRs that are expressed and their subcellular distributions. This hypothesis will be tested through three specific aims: (1) The relative effects of lnsP3R-1 and lnsP3R-2 on Ca2+ signaling will be determined at the molecular and single-channel level; (2) The relative effects of lnsP3R-1 and lnsP3R-2 on Ca2+ signaling will be determined in intact cells; (3) The relative effects of lnsP3R-1 and lnsP3R-2 on Ca2+-mediated secretion will be determined in model cell systems and in the intact, perfused liver. Together, these studies will provide new insights to the molecular and cellular basis for cholestasis, one of the cardinal manifestations of liver disease. Moreover, this work should provide a general paradigm for the way in which Ca2+ signals are generated in order to regulate events within specific subcellular regions.