Calcium signaling in hypoxic pulmonary vasoconstriction

描述（由申请人提供）：缺氧肺血管收缩（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，其依赖于钙的流入和细胞内钙浓度的继发性增加。人们普遍认为，由于电压依赖性钾通道（Kv）的缺氧抑制，去极化激活了肌层电压依赖性钙通道（VDCCs），从而导致钙内流发生。然而，观察到HPV通过抑制肌浆网（SR）钙释放而不是去极化或抑制Kv和VDCCs来预防，这表明HPV的机制更为复杂。我们的初步数据表明，缺氧增强了由SR钙耗尽激活的PASMC肌层通道的容性钙进入（CCE）。CCE抑制阻断缺氧诱导的PASMCs和离体肺HPV细胞内钙浓度升高。远端肺动脉表达TRPC mRNA和蛋白，这是果蝇中“瞬时受体电位”蛋白的同源物，被认为可以形成CCE通道。在此基础上，我们假设HPV是通过TRPC蛋白组成的通道SR钙释放和CCE的二次激活引发的，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通道的打开或间接由于局部钙依赖性的肌层K或CI通道活性的改变。由此产生的钙通过VDCCs内流增强CCE，提高细胞内钙浓度，足以触发PASMC收缩。为了验证这一假设，我们将在离体肺中测量血管舒张反应，并使用荧光显微镜、膜片夹持和PASMCs中的小干扰rna来确定缺氧如何释放SR钙并改变CCE通道的电生理特性，CCE通道的激活是否导致缺氧去极化，缺氧对CCE的影响如何被转导，以及PASMCs中负责缺氧反应的CCE通道是否由TRPC蛋白组成。我们希望这些实验将提高对HPV的了解，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。

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.