Mechanisms for hypoxic Ca2+ release in pulmonary artery myocytes

描述（由申请人提供）：低氧性肺血管收缩（HPV）是一种重要的调节机制，可在缺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，肺动脉平滑肌细胞（PASMCs）胞内Ca ~（2+）浓度（[Ca ~（2+）]i）升高在HPV的发生发展中起着至关重要的作用。[Ca 2 +]i的缺氧升高可由于电压依赖性K+通道抑制和钙库操纵的Ca 2+通道激活后肌浆网（SR）的Ca 2+释放和细胞外Ca 2+内流而发生。缺氧对电压依赖性钾通道的抑制和对钙库操纵性钾通道的激活可能与肌浆网Ca ~（2+）释放有关。然而，细胞和分子过程耦合缺氧Ca 2+释放，是不完全理解。我们最近的研究结果，加上以前的出版物，表明线粒体电子传递链可能作为一个主要的缺氧传感器，通过增加活性氧（ROS）的产生，激活磷脂酶C（PLC）和蛋白激酶C（PKC）。ROS和PKC可能对Ca ~（2+）释放通道产生协同作用。此外，PKC还可刺激NADPH氧化酶产生更多的ROS，提供正反馈机制介导缺氧反应。为了验证这一假设，本申请将解决以下问题（具体目的）：1）PLC-PKC-NADPH氧化酶信号转导是否参与缺氧Ca 2+释放？2)PLC-PKC-NADPH氧化酶信号通路是否在缺氧刺激时线粒体ROS过度产生后被激活？低氧产生的ROS如何激活ryanodine受体引起PASMCs内Ca ~（2+）释放。这些目标将通过测量小鼠PASMCs中低氧ROS产生和Ca 2+释放、单个PLC和PKC亚型以及NADPH氧化酶亚基的mRNA和蛋白质表达、单个PLC和PKC亚型以及NADPH氧化酶的活性来实现。药理学抑制剂、小干扰RNA和转基因小鼠也将用于确定缺氧与Ca 2+释放的偶联。该研究结果将增强我们对PASMCs和相关HPV中缺氧[Ca 2 +]i升高的细胞分子机制的理解，并可能导致确定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.