Uterine Relaxing Factors: Molecular Aspects of Action

描述（由申请人提供）：这项工作的长期目标是了解激素影响子宫平滑肌收缩和松弛的分子机制。本研究的重点是确定肌层主要收缩和松弛信号通路之间串扰的细胞和亚细胞机制，特别是支架蛋白和信号单元的区隔化在这些机制中的作用。目的1将确定怀孕大鼠分娩前子宫肌质膜相关PKA下降的机制，以及这种下降与cAMP调节子宫收缩活动的关系。我们将采用肌条、细胞培养和全动物模型相结合的方法来研究子宫收缩的基本机制，并将生化变化与子宫收缩活性的影响联系起来。目的2将确定是否类似的变化在子宫肌质膜PKA发生在灵长类动物妊娠与劳动。我们将检查怀孕狒狒在从挛缩到收缩的不同阶段的眼底和下子宫段组织的变化，以及在分娩前或分娩后剖宫产时获得的妇女子宫肌组织的变化。将在体内和怀孕的人子宫肌瘤细胞系中探索调节机制。目的3将确定PKA和PP2B与AKAP79/150的关联在控制肌层plcbet3磷酸化中的重要性。在我们知道Galpha(q)偶联受到影响的情况下，我们将确定PLCbeta3在细胞和组织中响应PKA激活的磷酸化状态，并将探索PKA在细胞培养和妊娠大鼠肌层模型中影响自身、PP2B亚基和AKAP79/150的活性和膜关联的机制。Aim 4将确定OTR和cAMP调控通路以及K（Ca）通道的成分如何在质膜微域之间分布，并评估分布是否受到收缩或松弛信号的影响，从而影响信号串扰。在这些研究中，我们将采用免疫印迹、共聚焦显微免疫定位和膜片钳方法。了解cAMP通路和信号串扰平衡影响变化的机制，将增强对妊娠和分娩期间子宫肌活动的理解，并可能导致设计改进的方法来管理子宫收缩活动和预防早产。

DESCRIPTION (provided by applicant): The long-term objective of this work is to understand the molecular mechanisms by which hormones influence uterine smooth muscle contraction and relaxation. This proposal focuses on defining cellular and subcellular mechanisms involved in crosstalk between major contractant and relaxant signaling pathways in the myometrium, in particular the role of scaffolding proteins and compartmentalization of signaling units in these mechanisms. Aim 1 will determine the mechanism responsible for the decline in myometrial plasma membrane-associated PKA prior to delivery in the pregnant rat and the relationship of this decline to the regulation of uterine contractile activity by cAMP. We will use a combination of muscle strip, cell culture and whole animal models to study basic mechanism and relate biochemical changes to effects on uterine contractile activity. Aim 2 will determine if similar changes in myometrial plasma membrane PKA occur in primate pregnancy in relation to labor. We will examine changes in tissue from both fundus and lower uterine segment in pregnant baboons at different stages in the transition from contractures to contractions and in myometrial tissues from women obtained at the time of cesarean section before or after the onset of labor. Mechanisms of regulation will be explored in vivo and in a pregnant human myometrial cell line. Aim 3 will determine the importance of the association of PKA and PP2B with AKAP79/150 in the control of myometrial PLCbeta3 phosphorylation. We will determine the phosphorylation status of PLCbeta3 in response to PKA activation in cells and tissue under conditions where we know Galpha(q) coupling is affected and will explore mechanisms by which PKA can influence activity and membrane association of itself, PP2B subunits and AKAP79/150 in cell culture and pregnant rat myometrial strip models. Aim 4 will determine how the constituents of OTR and cAMP regulatory pathways and K(Ca) channels are distributed between plasma membrane microdomains and assess whether distribution is affected by contractant or relaxant signals to influence signaling crosstalk. For these studies we will immunoblot, confocal microscopic immunolocalization and patch clamp methodology. Understanding the mechanisms for the changes in the influence of the cAMP pathway and signaling crosstalk balance will enhance understanding of myometrial activity during pregnancy and labor and may lead to design of improved methods for management of uterine contractile activity and the prevention of premature labor.