The Role of Trp Proteins in Myometrial Calcium Dynamics

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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.