THE ROLE OF TRP PROTEINS IN MYOMETRIAL CALCIUM DYNAMICS

要了解和调节子宫收缩/松弛，重要的是要了解细胞内钙的控制机制。色氨酸家族中的蛋白质被认为可以形成储存操作和非容量性的钙通道。本研究的总体目标是验证这一假说，即Trp蛋白通过形成通道复合体来响应与细胞内钙库耗竭相关的收缩刺激信号，从而对子宫肌层的钙进入做出重要贡献。我们的初步数据表明，在子宫肌层细胞中表达多种Trp蛋白。目的1检测人子宫肌层PHM1-41细胞中特定Trp mRNA形式和Trp蛋白的表达，包括Trp基因序列的完成，不同剪接模式的检测，以及通过Q-RT-PCR定量这些mRNAs的相对表达。目的2将研究色氨酸蛋白与子宫肌层容量性钙内流有关的功能。以Fura-2为指示剂，用双波长荧光成像法检测在G蛋白偶联受体激活或细胞内钙泵抑制刺激下，人子宫肌层细胞过度表达Trp蛋白对细胞内钙瞬变和细胞内钙内流的影响。我们将评估Trp反义mRNA抑制特定Trp蛋白表达对钙动力学的影响。在大鼠子宫肌层组织中，电压操作和容积性钙离子进入的相对重要性将通过同时测量力量和细胞内钙离子变化的多个方案来评估。目的3研究妊娠期大鼠和人的色氨酸蛋白mRNAs和蛋白表达的调节，以及激素对PHM1-41细胞培养的调节。这里提出的研究将为这些蛋白在子宫肌层中潜在的重要作用提供重要的新见解。对这些蛋白活性的调节可能为调节收缩活动的药物提供一个新的靶点，因此可能被证明在控制早产和功能障碍分娩方面是有用的。

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.