Capacitative Ca2+ Entry in Pulmonary Myocyte Growth

描述（由申请人提供）：肺血管收缩和血管 平滑肌增生在很大程度上有助于肺动脉高压 原发性肺动脉高压患者的血管阻力和动脉压 高血压（PPH），一种原因不明的致命疾病。一个普遍的理论是 血管收缩和细胞增殖使用重叠信号 导致平行细胞内事件的过程。细胞质中 Ca 2+（[Ca 2 +]cyt）刺激细胞收缩和增殖。因此，在本发明中， 细胞内Ca 2+可能作为一个共享的信号转导元件， PPH时肺血管收缩和血管重构。[Ca2+]cyt关于 肺动脉平滑肌细胞（PASMC）的钙释放增加， 肌浆网（SR）和Ca ~（2+）通过肌膜内流 Ca 2+渗透通道。丝裂原诱导的[Ca 2 +]cyt的变化包括： 从SR中初始释放Ca 2+，随后持续的Ca 2+内流。 SR Ca 2+的耗尽诱导电容性Ca 2+内流（CCE），其维持 持续的Ca 2+内流并将Ca 2+重新填充到SR中。TRP编码的 蛋白质可以形成负责CCE的Ca 2+渗透通道。在 在人PASMC中，TRP 1的mRNA和蛋白水平显著高于人PASMC， 增殖的细胞比生长停滞的细胞。增强的TRP 1 mRNA和 蛋白表达与[Ca 2 +]cyt增加相关， 从SR和CCE释放。这些结果表明，TRP 1的上调可能与TRP 1的表达有关。 可能导致CCE增加，[Ca 2 +]cyt升高， 细胞内储存的[Ca 2 +]在SR（[Ca 2 +]SR）中。根据这些数据，我们 假设TRP基因的上调导致了 TRP编码的Ca 2+通道的活性。该通道将作为 关键的Ca 2+进入途径，以提高[Ca 2 +]cyt并将Ca 2+重新填充到SR中， 这两者对于肺血管收缩和PASMC都是必需的 增殖上调的TRP基因，增强了Ca 2+释放激活的 （钙库耗竭介导的）Ca 2+电流（ICRAC），因此增强的CCE可能 在PPH中肺血管阻力升高中起关键作用 患者三个具体目标是解决测试的假设：1） 分析正常人TRP基因表达、ICRAC、CCE和[Ca 2 +]SR PASMC，并比较这些参数之间的生长停滞和 增殖细胞; 2）研究TRP的功能表达是否 通过增加静息[Ca 2 +]cyt、CCE和 [Ca2+]正常人PASMC中的SR;和3）研究和比较ICRAC， TRP通道的分子表达，[Ca 2 +]cyt的时空变化 正常人和肺心病患者PASMC内[Ca ~（2+）]SR 非肺动脉高压疾病、继发性肺动脉高压和PPH。

DESCRIPTION (provided by applicant): Pulmonary vasoconstriction and vascular smooth muscle proliferation greatly contribute to the elevated pulmonary vascular resistance and arterial pressure in patients with primary pulmonary hypertension (PPH), a fatal disease with unknown causes. A common theory is that vasoconstriction and cell proliferation use overlapping signaling processes that result in parallel intracellular events. A rise in cytosolic Ca2+ ([Ca2+]cyt) stimulates cell contraction and proliferation. Thus, intracellular Ca2+ may serve as a shared signal transduction element that leads to pulmonary vasoconstriction and vascular remodeling in PPH. [Ca2+]cyt about pulmonary artery smooth muscle cells (PASMC) is increased by Ca2+ release from the sarcoplasmic reticulum (SR) and Ca2+ influx through sarcolemmal Ca2+-permeable channels. The mitogen-induced changes in [Ca2+]cyt consist of an initial release of Ca2+ from the SR followed by a sustained Ca2+ influx. Depletion of the SR Ca2+ induces capacitative Ca2+ entry (CCE), which maintains the sustained Ca2+ influx and refills Ca2+ into the SR. The TRP-encoded proteins may form the Ca2+-permeable channels that are responsible for CCE. In human PASMC, the mRNA and protein levels of TRP1 were significantly higher in proliferating cells than in growth-arrested cells. The enhanced TRP1 mRNA and protein expression was associated with increases in [Ca2+]cyt due to Ca2+ release from the SR and CCE. These results imply that the up-regulation o TRP1 may contribute to the increased CCE, and elevated [Ca2+]cyt and intracellularly-stored [Ca2+] in the SR ([Ca2+]SR). Based on these data, we hypothesize that up-regulation of TRP genes leads to an increase in the activity of a TRP-encoded Ca2+ channel. This channel would then serve as a critical Ca2+ entry pathway to raise [Ca2+]cyt and refill Ca2+ into the SR, both of which are necessary for pulmonary vasoconstriction and PASMC proliferation. The up-regulated TRP genes, augmented Ca2+ release-activated (store depletion-mediated) Ca2+ currents (ICRAC), and enhanced CCE may thus play a critical role in the elevated pulmonary vascular resistance in PPH patients. Three Specific Aims are addressed to test the hypotheses: 1) to characterize the TRP gene expression, ICRAC, CCE, and [Ca2+]SR in normal human PASMC, and to compare these parameters between growth-arrested and proliferating cells; 2) to investigate whether functional expression of TRPs facilitates cell proliferation by increasing resting [Ca2+]cyt, CCE, and [Ca2+]SR in normal human PASMC; and 3) to investigate and compare ICRAC, molecular expression of TRP channels, spatial and temporal changes of [Ca2+]cyt through CCE, and [Ca2+]SR in PASMC from normal subjects and patients with non-pulmonary hypertension diseases, secondary pulmonary hypertension, and PPH.