THE ROLE OF TRP PROTEINS IN MYOMETRIAL CALCIUM DYNAMICS

为了理解和调节子宫收缩/舒张，了解控制细胞内钙的机制是很重要的。Trp家族中的蛋白质被认为可以形成储存操作和非电容性钙通道。本研究的总体目标是验证这样一种假设，即通过形成响应与细胞内钙储存消耗相关的收缩刺激信号的通道复合物，色氨酸蛋白在肌层钙进入的重要方式中起作用。我们的初步数据表明，多种色氨酸蛋白在子宫肌瘤细胞中表达。目的1将检查人类肌层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，检查可选择的剪接模式，并通过Q-RT-PCR定量这些mRNA的相对表达。目的2将研究色氨酸蛋白在肌层钙进入的功能。在G蛋白偶联受体激活或胞内钙泵抑制刺激下，人肌层细胞中过表达选定的Trp蛋白对细胞内钙瞬态和钙进入的影响将通过以Fura-2为指示剂的双波长荧光成像来测量。将评估Trp反义mRNA抑制特定Trp蛋白表达对钙动力学的影响。电压操作与容性钙进入大鼠肌组织的相对重要性将通过同时测量力和细胞内钙变化的许多方案进行评估。目的3将检查大鼠和人类怀孕期间Trp mrna和蛋白质表达的调节以及培养的PHM1-41细胞中的激素。这里提出的研究将为这些蛋白质在肌层中的潜在重要作用提供重要的新见解。这些蛋白活性的调节可能为调节收缩活动的药物提供新的靶点，因此可能在控制早产和功能障碍分娩中有用。

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.