Calcium signaling in hypoxic pulmonary vasoconstriction

描述(申请人提供)：缺氧性肺血管收缩(HPV)在正常肺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，依赖于钙离子内流和继发性细胞内钙离子浓度的升高。人们普遍认为，这种钙内流是通过肌膜电压依赖性钙通道(VDCC)发生的，由于低氧对电压依赖性钾通道(Kv)的抑制，去极化激活了VDCC。然而，观察到HPV是通过抑制肌浆网(SR)的钙释放来预防的，而不是通过去极化或抑制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，低氧通过肌浆网钙耗竭激活的PASMC肌膜通道促进了电容性钙内流(CCE)。抑制CCE可阻断低氧引起的PASMCs内钙离子浓度升高和HPV的升高。远端肺动脉表达TRPC mRNA和蛋白质，它们是果蝇“瞬时受体潜力”蛋白质的同源物，被认为形成CCE通道。在此基础上，我们假设HPV是由SR钙释放和通过TRPC蛋白组成的通道二次激活CCE，导致局部细胞内钙浓度升高而启动的。接下来发生去极化，要么直接由于CCE通道的开放，要么间接由于局部钙依赖的肌膜K或CI通道活性的改变。由此产生的钙内流通过VDCC增加CCE，充分提高全球细胞内钙浓度，从而触发PASMC收缩。为了验证这一假说，我们将测量离体肺的血管舒缩反应，并使用荧光显微镜、膜片钳和PASMCs上的小干扰RNA来确定低氧如何释放SR钙并改变CCE通道的电生理特性，CCE通道的激活是否导致缺氧性去极化，低氧对CCE的影响是如何转导的，以及PASMCs中负责低氧反应的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。

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.