Calcium signaling in hypoxic pulmonary vasoconstriction

缺氧肺血管收缩中的钙信号传导

• 批准号: 7076861
• 负责人: J T SYLVESTER
• 金额: $ 39.91万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7076861
• 关键词: RNA interference; biological signal transduction; calcium channel; calcium flux; calcium metabolism; electrophysiology; enzyme activity; fluorescence microscopy; laboratory rat; membrane proteins; muscle cells; myosin light chain kinase; respiratory epithelium; respiratory hypoxia; sarcoplasmic reticulum; serine threonine protein kinase; vascular smooth muscle; vasoconstriction; voltage /patch clamp

DESCRIPTION (provided by applicant): Hypoxic pulmonary vasoconstriction (HPV) plays important roles in normal and diseased lungs. Pulmonary arterial smooth muscle cells (PASMCs) contain all essential mechanisms of HPV, which depends on influx of calcium and secondary increases in intracellular calcium concentration. It is generally accepted that this calcium influx occurs through sarcolemmal voltage-dependent calcium channels (VDCCs) activated by depolarization due to hypoxic inhibition of voltage-dependent potassium (Kv) channels. However, observations that HPV was prevented by inhibition of calcium release from sarcoplasmic reticulum (SR), but not depolarization or inhibition of Kv and VDCCs, suggest that mechanisms of HPV are more complicated. Our preliminary data demonstrate that hypoxia enhanced capacitative calcium entry (CCE) through PASMC sarcolemmal channels activated by SR calcium depletion. Inhibition of CCE blocked hypoxia-induced increases in intracellular calcium concentration in PASMCs and HPV in isolated lungs. Distal pulmonary arteries expressed TRPC mRNA and proteins, homologs of "transient receptor potential" proteins in Drosophila that are thought to form CCE channels. On this basis, we hypothesize that HPV is initiated by SR calcium release and secondary activation of CCE through channels composed of TRPC proteins, causing local increases in intracellular calcium concentration. Depolarization occurs next, either directly due to opening of CCE channels or indirectly due to local calcium-dependent alteration of sarcolemmal K or CI channel-activity. The resultant calcium influx through VDCCs augments CCE, raising global intracellular calcium concentration sufficiently to trigger PASMC contraction. To test this hypothesis, we will measure vasomotor responses in isolated lungs and use fluorescent microscopy, patch clamping, and small interfering RNAs in PASMCs to determine how hypoxia releases SR calcium and alters the electro-physiologic characteristics of CCE channels, whether activation of CCE channels causes hypoxic depolarization, how effects of hypoxia on CCE are transduced, and whether CCE channels responsible for hypoxic responses in PASMCs are composed of TRPC proteins. We hope that these experiments will improve understanding of HPV and ultimately lead to decreased morbidity and mortality in patients with hypoxic pulmonary hypertension.

描述（由申请人提供）：缺氧性肺血管收缩（HPV）在正常和患病的肺部中发挥着重要作用。肺动脉平滑肌细胞 (PASMC) 包含 HPV 的所有基本机制，这取决于钙的流入和细胞内钙浓度的继发性增加。人们普遍认为，这种钙流入是通过肌膜电压依赖性钙通道（VDCC）发生的，该通道是由于电压依赖性钾（Kv）通道的缺氧抑制而被去极化激活的。然而，通过抑制肌浆网 (SR) 的钙释放来预防 HPV，而不是去极化或抑制 Kv 和 VDCC，这表明 HPV 的机制更为复杂。我们的初步数据表明，缺氧通过 SR 钙消耗激活的 PASMC 肌膜通道增强了电容性钙进入 (CCE)。抑制 CCE 可以阻止缺氧引起的离体肺 PASMC 和 HPV 细胞内钙浓度的增加。远端肺动脉表达 TRPC mRNA 和蛋白质，它们是果蝇中“瞬时受体电位”蛋白质的同源物，被认为形成 CCE 通道。在此基础上，我们推测HPV是由SR钙释放和CCE通过TRPC蛋白组成的通道二次激活引发的，导致细胞内钙浓度局部升高。接下来发生去极化，直接由于 CCE 通道的开放，或间接由于肌膜 K 或 CI 通道活性的局部钙依赖性改变。由此产生的钙通过 VDCC 流入会增强 CCE，从而提高整体细胞内钙浓度，足以触发 PASMC 收缩。为了检验这一假设，我们将测量离体肺部的血管舒缩反应，并使用荧光显微镜、膜片钳和 PASMC 中的小干扰 RNA 来确定缺氧如何释放 SR 钙并改变 CCE 通道的电生理特征、CCE 通道的激活是否会导致缺氧去极化、缺氧对 CCE 的影响如何转导以及 CCE 是否 PASMC 中负责缺氧反应的通道由 TRPC 蛋白组成。我们希望这些实验能够增进对 HPV 的了解，并最终降低缺氧性肺动脉高压患者的发病率和死亡率。