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Ca（Cai）动力学的机制对于了解子宫肌层活性的控制至关重要。信号调节的钙进入（也称为容量进入）在这里被广泛定义为包括受体和储存操作的进入。该途径与平滑肌中膜电位和张力的调节有关，并可能在激素、生长因子和细胞因子调节子宫肌层收缩活性中发挥作用。因此，调节钙进入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响应于一个以上的信号调节的钙刺激，并表达了一些TrpC mRNA和蛋白质参与这种类型的钙进入。本研究的总体目标是了解TrpC蛋白在子宫肌层Cai动力学调节和收缩活动控制中的作用。 目的1将确定信号调节的Ca进入子宫肌层Cai动力学控制中的相对重要性，通过荧光成像测量，以及子宫条的收缩活动。 目的2将描绘特定的TrpC蛋白的信号调节的钙离子内流和蔡动力学在人类子宫肌层细胞的贡献。将确定TrpC表达的过表达和抑制对子宫肌层细胞Cai动力学的影响，并将其与特异性反应联系起来。 目的3将检查TrpC表达的调节和信号调节的Ca内流的其他信号通路在子宫肌层。这些研究将利用人子宫肌层细胞和妊娠大鼠模型中的Q-RT-PCR、免疫印迹和Ca成像方法。 目的4将检查TrpC亚型表达对TrpC与子宫肌层质膜的结合的影响，以及信号调节的Ca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节的钙离子进入机制在控制子宫肌层Cai动力学和子宫肌层收缩中的作用有新的认识，并可能导致设计新的方法来控制妊娠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.