Hypoxic Pulmonary Hypertension:Contractile Mechanisms

描述(由申请人提供)： 长时间暴露在氧分压降低的环境中，就像许多肺病患者一样 疾病，导致肺动脉高压(CHPH)。新出现的证据 提示持续的肺动脉平滑肌细胞收缩 慢性缺氧(CH)可能与K通道的改变有关， 膜电位和细胞内钙离子浓度([Ca2+]i)；然而， 这一过程背后的确切机制和中介因素仍然存在 未知。低氧诱导因子1(HIF-1)是一种转录因子，介导 许多对低氧的适应性反应。HIF-1控制低氧诱导 许多可能在CHPH的发生发展中起重要作用的基因，包括内皮素- I(ET-12)。ET-1受体拮抗剂预防CHPH，ET-1抑制CH。 此外，在慢性低氧动物的PASMC中，ET-1信号 信号转导通路发生改变，使ET-1诱导的收缩 增强，似乎主要是通过激活非钙依赖的 收缩通路，可能是由于蛋白激酶C(PKC)和 丝裂原活化蛋白激酶(MAPK)。增强的收缩与 ET-1水平升高可能导致正反馈机制，通过 维持持续的血管收缩。我们建立了CHPH的小鼠模型 再加上部分缺陷转基因小鼠的产生 对于作为HIF-1亚单位的Varies，创建了一个独特的动物模型系统， 使我们能够特别针对HIF-1在这一疾病过程中的作用。 我们将使用这个模型来检验假设，在CH期间，诱导 HIF-1是CHPH发生发展的关键启动步骤， 缺氧诱导的HIF-1导致ET-1水平升高，从而激活 PASMQ收缩机制的组合，包括：a)去极化 由于K+通道减少；b)去极化驱动的升高 静息[Ca~(2+)]i和c)收缩肌对Ca~(2+)敏感性的变化 这是由于依赖于PKC和MAPK的通路被激活所致。为了测试 在这个假设中，我们将使用组合技术，包括等距技术 动脉节段张力记录和Western印迹分析，以及 PASMCs的全细胞膜片钳和显微荧光测量 完成以下具体目标：1)确定HIF-1是否调节 在PASM观察到ET-1水平及其介导的CH诱导的改变；2)确定 ET-1是否介导了CH诱导的PASM改变；3)确定 CH是否降低K+通道蛋白表达 受HIF-1和ET-1的调节；4)确定CH诱导的高血压是否 静息[Ca~(2+)]i是去极化引起的钙内流增强所致 通过Na~+/Ca~(2+)交换和5)确定PKC和MAPK在ET-1中的作用 -在暴露于CH后，PASM对钙的敏感性增加。(完) 的摘要)

DESCRIPTION (provided by applicant): Prolonged exposure to decreased oxygen tension, as occurs in many pulmonary diseases, results in pulmonary hypertension (CHPH). Emerging evidence indicates sustained pulmonary arterial smooth muscle cell (PASMC) contraction associated with chronic hypoxia (CH) may be related to changes in K channels, membrane potential and intracellular Ca2+ concentration ([Ca2+]i); however, the exact mechanisms underlying, and factors mediating, this process remain unknown. Hypoxia-inducible factor 1 (HIF-1), a transcription factor, mediates numerous adaptive responses to hypoxia. HIF-1 controls hypoxic induction of many genes that may be important in development of CHPH, including endothelin- I (ET-12). ET-1 receptor antagonists prevent CHPH and ET-1 inhibits CH. Furthermore, in PASMC from chronically hypoxic animals, ET-1 signal transduction pathways are altered such that ET-1 -induced contraction is enhanced and appears to occur primarily via activation of Ca2+ -independent contractile pathways, possibly due to activation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Enhanced contraction coupled with elevated ET-1 levels could result in a positive feedback mechanism by which sustained vasoconstriction is maintained. We developed a murine model of CHPH that, together with the generation of transgenic mice with partial deficiency for the varies as subunit of HIF-1, creates a unique animal model system that allows us to specifically target the role of HIF-1 in this disease process. We will use this model to test the hypothesis that during CH, induction of HIF-1 is a critical initiating step in the development of CHPH, and that hypoxic induction of HIF-1 results in elevated ET-1 levels which activate a combination of contractile mechanisms in PASMQs, including: a) depolarization due to reduction of K+ channels; b) depolarization-driven elevation of resting [Ca2+]i and c) changes in Ca2+ -sensitivity of the contractile apparatus due to activation of PKC- and MAPK- dependent pathways. To test this hypothesis, we will use a combination of techniques, including isometric tension recording and Western blot analysis in arterial segments, and whole-cell patch-clamp and microfluorescence measurement in PASMCs, to accomplish the following Specific Aims: 1) determine whether HIF-1 regulates ET-1 levels and mediates CH-induced alterations observed in PASM; 2) determine whether ET-1 mediates CH-induced alterations observed in PASM; 3) determine whether CH decreases K+ channel protein expression and whether this is regulated by HIF-1 and ET-1; 4) determine whether the CH-induced elevation in resting [Ca 2+]i is due to depolarization-driven enhancement of Ca2+ influx through Na+/Ca2+ exchange and 5) determine the role of PKC and MAPK in ET-1 -induced increase in Ca2+ sensitivity in PASM following exposure to CH. (End of Abstract)