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）是维持缺氧时动脉氧合充足的重要调节机制，但也可能导致肺动脉高压。越来越多的证据表明，肺动脉平滑肌细胞（PASMCs）细胞内Ca2+浓度（[Ca2+]i）的升高在HPV的发展中起着至关重要的作用。低氧升高[Ca2+]i可发生由于Ca2+从肌浆网（SR）释放和细胞外Ca2+内流后抑制电压依赖性K+通道和激活储存操作的Ca2+通道。低氧对电压依赖性K+通道的抑制和存储操作通道的激活可能与SR Ca2+释放有关。然而，细胞和分子过程耦合缺氧Ca2+释放不完全了解。我们最近的研究结果以及之前的出版物表明，线粒体电子传递链可能通过增加活性氧（ROS）的产生而发挥主要的缺氧传感器的作用，活性氧可以激活磷脂酶C （PLC）和蛋白激酶C （PKC）。ROS和PKC都可能对Ca2+释放通道产生协同作用。此外，PKC可能刺激NADPH氧化酶，从而产生更多的ROS，为介导缺氧反应提供了正反馈机制。为了验证这一假设，本应用程序将解决以下问题（具体目标）：1)PLC-PKC-NADPH氧化酶信号是否参与缺氧Ca2+释放？2) PLC-PKC-NADPH氧化酶信号是否在缺氧刺激时线粒体ROS过量产生后被激活？3)缺氧产生的ROS如何激活ryanodine受体导致PASMCs中Ca2+释放。这些目标将通过测量小鼠pasmc中缺氧ROS生成和Ca2+释放，单个PLC和PKC异构体的mRNA和蛋白质表达，以及NADPH氧化酶亚基，单个PLC和PKC异构体的活性以及NADPH氧化酶来实现。药理抑制剂、小干扰rna和转基因小鼠也将被用来定义缺氧与Ca2+释放的耦合。这一发现将增强我们对PASMCs和相关HPV中缺氧[Ca2+]i升高的细胞分子机制的理解，并可能导致发现治疗肺动脉高压的潜在新靶点。

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.