Ca2+ Waves in Hepatocytes: Mechanisms and Effects

描述(由申请人提供)：胞内钙离子是几乎每种类型细胞中的关键第二信使。与其他信使分子相比，Ca2+是新的，因为它同时调节单个细胞内的多个过程。这项研究项目正在进行的主题是研究钙离子如何调节肝细胞的胆汁分泌，作为钙离子如何调节极化上皮内局部过程的模型。在肝细胞中，钙信号完全由肌醇1，4，5-三磷酸(Insps)介导。Insps通过与Insps受体(InsPSR)结合发挥作用，Insps受体是内质网(ER)膜上的一个四聚体Insps门控钙释放通道。因此，INSPS通过INsPSR从内质网释放钙离子，从而增加细胞内钙离子浓度。已知的三种InsPSR亚型中的两种(lnsP3R-1和lnsP3R-2)在肝细胞中表达，每种亚型都具有不同的生物物理特性。这两种异构体中的每一种都以独特的亚细胞模式分布在肝细胞中，从而可能在细胞内建立信号微域。这一设想的假设是，钙信号模式及其对肝细胞胆汁分泌的影响取决于表达的InsPSRs的类型及其亚细胞分布。这一假说将通过三个特定的目标来验证：(1)将在分子和单通道水平上确定lnsP3R-1和lnsP3R-2对钙信号的相对影响；(2)将在完整细胞中确定lnsP3R-1和lnsP3R-2对钙信号的相对影响；(3)将在模型细胞系统和完整的灌流肝脏中确定lnsP3R-1和lnsP3R-2对钙介导的分泌的相对影响。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，胆汁淤积是肝病的主要表现之一。此外，这项工作应该为钙信号的产生方式提供一个通用的范例，以便调节特定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.