Capacitative Ca2+ Entry in Pulmonary Myocyte Growth

描述(申请人提供)：肺血管收缩和血管 平滑肌增殖是导致肺组织肥大的重要原因。 原发性肺心病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。一种常见的理论是 血管收缩和细胞增殖使用重叠信号 导致并行细胞内事件的过程。细胞质的上升 钙离子([Ca~(2+)]Cyt)刺激细胞收缩和增殖。因此， 细胞内钙离子可能是共同的信号转导元件，导致 与PPH的肺血管收缩和血管重构有关。[Ca2+]细胞关于 大鼠肺动脉平滑肌细胞(PASMC)钙离子释放增加 肌浆网(SR)和钙离子通过肌膜内流 钙离子通透通道。丝裂原诱导的细胞内钙离子的变化包括一种 初始钙离子从SR释放，随后持续的钙离子内流。 肌质网钙离子耗竭诱导大鼠钙离子内流(CCE) 持续的Ca~(2+)内流和补充Ca~(2+)进入SR。TRP编码的 蛋白质可能形成导致CCE的钙离子通透通道。在……里面 人PASMC中，TRP1的mRNA和蛋白水平显著高于正常对照组。 与生长受阻的细胞相比，在增殖的细胞中。增强的TRP1mRNA和 蛋白表达与钙离子引起的细胞内[钙离子]细胞计数增加有关 从SR和CCE释放。这些结果表明，TRP1的上调 可能与CCE升高、[Ca~(2+)]Cyt升高和 胞内储存[Ca~(2+)]到SR([Ca~(2+)]SR)。根据这些数据，我们 假设TRP基因上调会导致 Trp编码的钙通道的活动。然后，该通道将充当 关键的Ca~(2+)进入途径使[Ca~(2+)]Cyt升高，使Ca~(2+)重新进入SR， 两者都是肺血管收缩和PASMC所必需的 扩散。上调的色氨酸蛋白基因，促进钙释放激活 (存储耗竭介导的)钙电流(ICRAC)和增强的CCE可能因此 肺血管阻力升高在PPH中的重要作用 病人。三个具体目标被用来检验这些假设：1) 正常人色氨酸蛋白基因表达、ICRAC、CCE和[Ca~(2+)]SR的研究 PASMC，并比较生长停滞和生长停滞的这些参数 2)研究TRPs的功能性表达 通过增加静息[Ca~(2+)]Cyt、CCE和 [Ca~(2+)]SR在正常人PASMC中的表达；3)调查和比较ICRAC， 色氨酸通道的分子表达与细胞内钙离子浓度的时空变化 通过CCE和[Ca~(2+)]SR检测正常人和慢性阻塞性肺疾病患者PASMC内钙离子浓度 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.