THE ROLE OF TRP PROTEINS IN MYOMETRIAL CALCIUM DYNAMICS

TRP 蛋白在子宫肌层钙动态中的作用

• 批准号: 6130081
• 负责人: BARBARA M SANBORN
• 金额: $ 22.89万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6130081
• 关键词: antisense nucleic acid; calcium channel; calcium flux; calcium indicator; calcium transporting ATPase; estrogens; female; fluorimetry; hormone regulation /control mechanism; human tissue; laboratory rat; muscle contraction; myometrium; pregnancy; protein protein interaction; tissue /cell culture; voltage gated channel

To understand and regulate uterine contraction/relaxation, it is important to understand the mechanisms that control intracellular Ca. Proteins in the Trp family are postulated to form store operated and non-capacitative calcium channels. The overall goal of this study is to test the hypothesis that, by forming channel complexes that respond to contractant-stimulated signals related to intracellular Ca store depletion, Trp proteins contribute in a significant way to Ca entry in myometrium. Our preliminary data indicate that multiple Trp proteins are expressed in myometrial cells. Aim 1 will examine the expression of specific Trp mRNA forms and Trp proteins in human myometrial PHM1-41 cells and will include completion of Trp cDNA sequences, examination of alternative splicing patterns, and quantitation of the relative expression of these mRNAs by Q-RT-PCR. Aim 2 will examine the function of Trp proteins in relation to capacitative calcium entry in myometrium. The effects of overexpressing selected Trp proteins in human myometrial cells on intracellular Ca transients and Ca entry in depletion/readdition protocols stimulated by G- protein coupled receptor activation or intracellutar Ca pump inhibition will be measured by dual wavelength fluorescence imaging with Fura-2 as indicator. The consequences of inhibition of specific Trp protein expression by Trp antisense mRNA on calcium dynamics will be assessed. The relative importance of voltage-operated versus capacitative calcium entry in rat myometrial tissue will be assessed in a number of protocols employing simultaneous measurement of changes in force and intracellular Ca. Aim 3 will examine the regulation of Trp mRNAs and protein expression during pregnancy in the rat and human and by hormones in PHM1-41 cells in culture. The studies proposed here will provide important new insights into the potentially important role of these proteins in the myometrium. The regulation of the activity of these proteins may provide a new target for drugs regulating contractile activity and hence may prove useful in the control of premature labor and dysfunctional labor.

要了解和调节子宫收缩/放松，了解控制细胞内Ca的机制很重要。 假定TRP家族中的蛋白质会形成储存的钙通道和非适应性钙通道。 这项研究的总体目的是检验以下假设：通过形成对与细胞内Ca储存量相关的违约信号响应的通道复合物，TRP蛋白会以重要的方式促进CA进入子宫肌层。 我们的初步数据表明，多种TRP蛋白在肌层细胞中表达。 AIM 1将检查人肌层PHM1-41细胞中特定的TRP mRNA形式和TRP蛋白的表达，并将包括完成TRP cDNA序列的完成，替代剪接模式的检查以及Q-RT-PCR对这些mRNA的相对表达的定量。 AIM 2将检查TRP蛋白与肌层中电容性钙进入有关的功能。 过度表达人肌层细胞中所选的TRP蛋白对细胞内Ca瞬变的影响以及通过双波长荧光成像以Fura-2作为指示灯来衡量的G蛋白偶联受体激活或细胞内CA泵抑制刺激的耗竭/读取方案的Ca进入。 将评估TRP反义mRNA对钙动力学抑制特异性TRP蛋白表达的后果。 在大鼠肌层组织中，电压训练与电容性钙进入的相对重要性将在许多方案中评估，这些方案同时测量了力和细胞内CA的变化。 AIM 3将检查培养物中PHM1-41细胞中大鼠和人类妊娠期间TRP mRNA和蛋白质表达的调节。 此处提出的研究将为这些蛋白质在子宫肌层中的潜在重要作用提供重要的新见解。 这些蛋白质活性的调节可能会为调节收缩活性的药物提供一个新的靶标，因此可能对控制早产和功能失调的劳动有用。