Calcium signaling in hypoxic pulmonary vasoconstriction

缺氧肺血管收缩中的钙信号传导

• 批准号: 6819645
• 负责人: J T SYLVESTER
• 金额: $ 36.43万
• 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/6819645
• 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）：肺血管收缩功能减退（HPV）在正常和病变肺中起重要作用。肺动脉平滑肌细胞（PASMCs）包含HPV的所有基本机制，这取决于钙内流和细胞内钙浓度的继发性增加。一般认为，这种钙内流是通过肌膜电压依赖性钙通道（VDCCs）发生的，由于缺氧抑制电压依赖性钾（Kv）通道，去极化激活了VDCCs。然而，观察到HPV是通过抑制肌浆网（SR）的钙释放而不是去极化或抑制Kv和VDCC来预防的，这表明HPV的机制更为复杂。我们的初步数据表明，缺氧增强通过SR钙耗竭激活的PASMC肌膜通道的容量性钙内流（CCE）。抑制CCE阻断缺氧诱导的PASMCs和离体肺中HPV细胞内钙浓度的增加。远端肺动脉表达TRPC mRNA和蛋白质，它们是果蝇中被认为形成CCE通道的“瞬时受体电位”蛋白质的同源物。在此基础上，我们假设HPV是由SR钙释放和二次激活CCE通过TRPC蛋白组成的通道，引起局部细胞内钙浓度的增加。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。由此产生的钙内流通过VDCCs增加CCE，提高整体细胞内钙浓度足以触发PASMC收缩。为了验证这一假设，我们将测量离体肺中的血管反应，并使用荧光显微镜、膜片钳和PASMCs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通道的激活是否引起缺氧去极化，缺氧对CCE的影响如何转导，以及负责PASMCs缺氧反应的CCE通道是否由TRPC蛋白组成。我们希望这些实验将提高对HPV的理解，并最终导致低氧性肺动脉高压患者的发病率和死亡率降低。