Mechanisms for hypoxic Ca2+ release in pulmonary artery myocytes

描述(申请人提供)：缺氧性肺血管收缩(HPV)是一种重要的调节机制，可在低氧时维持足够的动脉氧合，但也可导致肺动脉高压。越来越多的证据表明，PASMCs细胞内钙离子浓度([Ca~(2+)]i)升高在HPV的发生发展中起重要作用。[Ca~(2+)]i的缺氧性升高可能是由于肌浆网(SR)的钙释放和电压依赖性K~+通道的抑制和钙离子通道的激活引起的细胞外钙内流。低氧对电压依赖性K+通道的抑制和对钙通道的激活可能与肌质网钙离子释放有关。然而，低氧与钙离子释放的细胞和分子过程还不完全清楚。我们最近的发现和以前的文献一起表明，线粒体电子传递链可能通过增加激活磷脂酶C(PLC)和蛋白激酶C(PKC)的活性氧物种(ROS)的产生而起到主要的缺氧感受器的作用。ROS和PKC可能对钙释放通道产生协同作用。此外，PKC还可以刺激NADPH氧化酶，产生更多的ROS，提供一种正反馈机制来调节缺氧反应。为了验证这一假说，这一应用将解决以下问题(具体目的)：1)PLC-PKC-NADPH氧化酶信号是否参与低氧钙释放？2)PLC-PKC-NADPH氧化酶信号是否在低氧刺激期间线粒体ROS过度产生后被激活？3)低氧产生的ROS如何激活Ryanodine受体，引起PASMCs内钙释放。通过检测小鼠PASMCs中低氧ROS的产生和钙释放、PLC和PKC亚型以及NADPH氧化酶亚基的mRNA和蛋白表达，以及PLC和PKC亚型和NADPH氧化酶的活性来实现这些目标。药物抑制剂、小干扰RNA和转基因小鼠也将被用来确定低氧与钙释放的偶联。这项研究的发现将加深我们对PASMCs和相关HPV缺氧性[Ca~(2+)]i升高的细胞分子机制的理解，并可能导致发现潜在的治疗肺动脉高压的新靶点。

DESCRIPTION (provided by applicant): Hypoxic pulmonary vasoconstriction (HPV) serves as an important regulatory mechanism to maintain adequate arterial oxygenation in response to hypoxia, but can also result in pulmonary hypertension. Increasing evidence indicates that a rise in intracellular Ca2+ concentration ([Ca2+]i) in pulmonary artery smooth muscle cells (PASMCs) plays a crucial role in the development of HPV. The hypoxic rise in [Ca2+]i can occur due to Ca2+ release from the sarcoplasmic reticulum (SR) and extracellular Ca2+ influx following inhibition of voltage-dependent K+ channels and activation of store-operated Ca2+ channels. The inhibition of voltage-dependent K+ channels and activation of store-operated channels by hypoxia are possibly associated with the SR Ca2+ release. The cellular and molecular processes coupling hypoxia to Ca2+ release, however, are incompletely understood. Our recent findings, together with previous publications, suggest that mitochondrial electron transport chain may function as a primary hypoxia sensor by increasing the generation of reactive oxygen species (ROS), which activate phospholipase C (PLC) and protein kinase C (PKC). Both ROS and PKC are likely to produce a synergetic effect on Ca2+ release channels. In addition, PKC may stimulate NADPH oxidase and then generate more ROS, providing a positive feedback mechanism to mediate hypoxic responses. To test this hypothesis, this application will address the following questions (specific aims): 1) Is the PLC-PKC-NADPH oxidase signaling involved in hypoxic Ca2+ release? 2) Is the PLC-PKC-NADPH oxidase signaling activated following the excessive generation of mitochondrial ROS during hypoxic stimulation? and 3) How do ROS produced by hypoxia activate ryanodine receptors to cause Ca2+ release in PASMCs. These aims will be pursued by measuring hypoxic ROS generation and Ca2+ release, mRNA and protein expression of individual PLC and PKC isoforms, as well as NADPH oxidase subunits, and the activity of individual PLC and PKC isoforms, as well as NADPH oxidase in mouse PASMCs. Pharmacological inhibitors, small interfering RNAs and transgenic mice will also be used to define the coupling of hypoxia to Ca2+ release. The findings from this proposal will enhance our understanding of cellular molecular mechanisms for hypoxic [Ca2+]i rise in PASMCs and associated HPV, and may lead to identify potential novel targets to treat pulmonary hypertension.