THE ROLE OF TRP PROTEINS IN MYOMETRIAL CALCIUM DYNAMICS

为了了解和调节子宫收缩/舒张，重要的是要了解控制细胞内Ca的机制。 Trp家族中的蛋白质被假定形成储存操作的和非容量性的钙通道。 本研究的总体目标是检验以下假设：通过形成响应于与细胞内钙库耗竭相关的收缩刺激信号的通道复合物，Trp蛋白以显著的方式促进子宫肌层中的钙进入。 我们的初步数据表明，多种色氨酸蛋白表达在子宫肌层细胞。 目的1将检查人子宫肌层PHM 1 -41细胞中特定Trp mRNA形式和Trp蛋白的表达，并将包括完成Trp cDNA序列，检查选择性剪接模式，并通过Q-RT-PCR定量这些mRNA的相对表达。 目的2将检查Trp蛋白在子宫肌层中与容量性钙内流相关的功能。 在人子宫肌层细胞中过表达选定的Trp蛋白对细胞内Ca瞬变和在G-蛋白偶联受体激活或细胞内Ca泵抑制刺激的耗竭/再灌注方案中Ca进入的影响将通过双波长荧光成像以Fura-2作为指示剂来测量。 将评估Trp反义mRNA抑制特异性Trp蛋白表达对钙动力学的影响。 电压操作与容量钙离子进入大鼠子宫肌层组织中的相对重要性将在一些协议中进行评估，同时测量力和细胞内Ca的变化。 目的3将研究大鼠和人类妊娠期间Trp mRNA和蛋白表达的调节以及培养的PHM 1 -41细胞中激素的调节。 这里提出的研究将提供重要的新的见解，这些蛋白质在子宫肌层的潜在重要作用。 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，因此可能证明在控制早产和功能障碍性分娩中是有用的。

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.