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: 6536138
• 负责人: BARBARA M SANBORN
• 金额: $ 23.49万
• 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 2003-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6536138
• 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.

为了了解和调节子宫收缩/舒张，了解控制细胞内 Ca 的机制非常重要。 假定色氨酸家族中的蛋白质形成钙池操作的非电容性钙通道。 本研究的总体目标是检验以下假设：通过形成对与细胞内 Ca 储存耗尽相关的收缩刺激信号做出反应的通道复合物，Trp 蛋白对 Ca 进入子宫肌层发挥重要作用。 我们的初步数据表明多种色氨酸蛋白在子宫肌细胞中表达。 目标 1 将检查人子宫 PHM1-41 细胞中特定 Trp mRNA 形式和 Trp 蛋白的表达，包括完成 Trp cDNA 序列、检查可变剪接模式以及通过 Q-RT-PCR 对这些 mRNA 的相对表达进行定量。 目标 2 将检查色氨酸蛋白与子宫肌层中钙的电容性进入相关的功能。 通过以 Fura-2 作为指示剂的双波长荧光成像，测量人子宫肌细胞中过表达选定的 Trp 蛋白对 G 蛋白偶联受体激活或细胞内 Ca 泵抑制刺激的消耗/再添加方案中的细胞内 Ca 瞬变和 Ca 进入的影响。 将评估 Trp 反义 mRNA 抑制特定 Trp 蛋白表达对钙动态的影响。 电压操作与电容性钙进入大鼠子宫肌层组织的相对重要性将通过同时测量力和细胞内 Ca 变化的多种方案进行评估。 目标 3 将检查大鼠和人类妊娠期间 Trp mRNA 和蛋白质表达的调节以及培养的 PHM1-41 细胞中激素的调节。 这里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为调节收缩活性的药物提供新的靶点，因此可能有助于控制早产和功能障碍性分娩。