The Role of Trp Proteins in Myometrial Calcium Dynamics

描述(由申请人提供)：了解调节子宫肌层细胞内游离钙(CaI)动态的机制对于理解子宫肌层活动的控制至关重要。信号调节的钙离子内流(也称为容量性钙离子内流)在这里被广泛定义为既包括受体操作的钙离子内流，也包括存储操作的钙离子内流。该通路参与了对子宫肌膜电位和张力的调节，并可能在激素、生长因子和细胞因子对子宫肌层收缩活动的调节中发挥作用。因此，调节钙离子内流可能参与控制妊娠和分娩期间的子宫收缩活动。我们发现，大鼠和人类子宫肌层细胞对多种信号调节的钙离子刺激作出反应，并表达与这种类型的钙离子进入有关的许多TrpC mRNAs和蛋白。这项研究的总体目标是了解TrpC蛋白在调节子宫肌层CaI动态和控制收缩活动中的作用。 目的1确定信号调节的钙内流在控制子宫肌层钙离子动力学(荧光成像测量)和子宫肌条收缩活动中的相对重要性。 目的2将描述特定的TrpC蛋白在人子宫肌层细胞信号调节的钙内流和钙调素动力学中的作用。过表达和抑制TrpC表达对子宫肌层细胞CAI动态的影响将被确定，并与特定的反应相联系。 目的3研究其他信号通路对子宫肌层TrpC表达和信号调节的钙内流的调节作用。这些研究将利用Q-RT-PCR、免疫印迹和钙成像方法在人子宫肌层细胞和怀孕大鼠模型中进行。 目的4研究TrpC亚型表达对TrpC与子宫肌质膜结合的影响，以及信号调节的钙内流对其他膜蛋白活性的可能影响。这些研究将使用扫描共聚焦显微镜和亚细胞分离方法。这些研究有望对这种严格调控的钙离子进入机制在控制子宫肌层CaI动态和肌层收缩中的作用提供新的见解，并可能导致设计新的方法来控制怀孕和分娩期间的子宫肌层活动。

DESCRIPTION (provided by applicant): Understanding the mechanisms regulating myometrial intracellular free Ca (Cai) dynamics is critical to understanding the control of myometrial activity. Signal-regulated calcium entry (also called capacitative entry) is broadly defined here to include both receptor- and store-operated entry. This pathway has been implicated in regulation of membrane potential and tone in smooth muscle and may play a role in the regulation of myometrial contractile activity by hormones, growth factors and cytokines. Hence regulated calcium entry may participate in control of uterine contractile activity during pregnancy and parturition. We found that rat and human myometrial cells respond to more than one signal-regulated calcium stimulus and express a number of TrpC mRNAs and proteins implicated in this type of calcium entry. The overall goal of this study is to understand the role of TrpC proteins in the regulation of myometrial Cai dynamics and control of contractile activity. Aim 1 will determine the relative importance of signal-regulated Ca entry in the control of myometrial Cai dynamics, measured by fluorescence imaging, and to contractile activity in uterine strips. Aim 2 will delineate the contribution of specific TrpC proteins to signal-regulated calcium entry and Cai dynamics in human myometrial cells. The effects of both overexpression and suppression of TrpC expression on myometrial cell Cai dynamics will be determined and linked to specific responses. Aim 3 will examine the regulation of TrpC expression and of signal-regulated Ca entry by other signaling pathways in myometrium. These studies will utilize Q-RT-PCR, immunoblot and Ca imaging approaches in human myometrial cells and the pregnant rat model. Aim 4 will examine the influence of TrpC subtype expression on TrpC association with myometrial plasma membrane and possible effects of signal-regulated Ca entry on activity of other membrane proteins. These studies will use scanning confocal microscopy and subcellular fractionation approaches. These studies promise new insights into the role of this tightly regulated calcium entry mechanism in the control of myometrial Cai dynamics and myometrial contraction and may lead to design of new approaches for controlling myometrial activity during pregnancy and labor.