NO-Mediated Pulmonary Vasodilation After Chronic Hypoxia

描述(申请人提供)：慢性低氧(CH)与各种肺部疾病相关，可导致肺动脉高压。适应性反应，包括增强的一氧化氮(NO)介导的血管扩张，可能会减轻缺氧性肺动脉高压的严重程度。然而，无论是CH后NO依赖反应性改变的机制，还是导致NO介导的肺血管扩张的信号通路，都不是很清楚。蛋白激酶G(PKG)是NO信号转导的重要靶点，通过降低细胞内游离钙离子浓度([Ca2]i)而引起血管平滑肌(VSM)的松弛，使血管收缩机构对钙离子失去敏感性。PKG导致钙脱敏的后一种作用在很大程度上是未知的，尽管研究表明RhoA-Rho激酶信号级联的失活在这一反应中起到了作用。有趣的是，初步实验表明，CH减弱了依赖PKG的VSM[Ca2]i的下降，同时反常地增强了PKG介导的血管扩张。因此，本研究将探讨这一中心假设，即CH损害肺VSM中PKG依赖的[Ca~(2+)]i调节，并介导PKG信号的代偿性转变，从而促进收缩机构对[Ca~(2+)]的脱敏。这一假说将通过以下具体目标来解决：具体目标1：建立CH损害依赖PKG的肺VSM[Ca~(2+)]i降低的机制。此目的的工作假说是CH通过干扰钙内流和隔离机制来减少PKG介导的VSM[Ca~(2+)]i的降低。具体目标#2：确定CH增强PKG介导的肺VSM钙脱敏的机制。我们推测，CH后PKG介导的肺血管扩张增强是VSM通过抑制RhoA-Rho激酶途径的钙脱敏作用的结果。这项拟议的研究将采用一种创新的方法来研究CH改变肺VSM中PKG信号的机制，包括同时测量对照组和CH大鼠分离的加压小肺动脉的血管反应性和VSM[Ca~(2+)]i。这些研究结果有望从根本上促进我们对PKG调节肺血管张力的信号转导机制的理解，以及肺循环对CH的适应性反应。

DESCRIPTION (provided by applicant): Chronic hypoxia (CH) associated with various lung diseases leads to pulmonary hypertension. Adaptive responses, including enhanced nitric oxide (NO)-mediated vasodilation, may diminish the severity of hypoxic pulmonary hypertension. However, neither the mechanism of altered NO-dependent reactivity following CH, nor the signaling pathways leading to NO-mediated pulmonary vasodilation are well understood. Protein kinase G (PKG) is a prominent target of NO signaling that elicits relaxation of vascular smooth muscle (VSM) by decreasing the concentration of intracellular free calcium ([Ca 2+]i)as well as desensitization of the contractile apparatus to Ca 2+ . This latter effect of PKG to cause Ca2+-desensitization is largely undefined, although studies have implicated a role for inactivation of the RhoA-Rho kinase-signaling cascade in this response. Interestingly, pilot experiments suggest that CH attenuates PKG-dependent decreases in VSM [Ca 2+]i while paradoxically augmenting PKG-mediated vasodilation. Therefore, the proposed studies will investigate the central hypothesis that CH impairs PKG-dependent regulation of [Ca2+]i in pulmonary VSM and mediates a compensatory shift in PKG signaling to promote desensitization of the contractile apparatus to [Ca 2+]. This hypothesis will be addressed by the following specific aims: Specific Aim #1: Establish the mechanism by which CH impairs PKG-dependent reduction of pulmonary VSM [Ca2+]i. The working hypothesis for this aim is that CH diminishes PKG-mediated decreases in VSM [Ca2+]i by interfering with regulation of Ca 2+ influx and sequestration mechanisms. Specific Aim #2: Identify the mechanism by which CH augments PKG-mediated pulmonary VSM Ca2+-desensitization. We hypothesize that enhanced PKG-mediated pulmonary vasodilation following CH is a function of VSM Ca2+-desensitization through inhibition of the RhoA-Rho kinase pathway. The proposed studies will employ an innovative approach to address mechanisms by which CH alters PKG-signaling in pulmonary VSM, including simultaneous measurement of vasoreactivity and VSM [Ca2+]i in isolated, pressurized small pulmonary arteries from control and CH rats. Findings from studies are expected to fundamentally advance our understanding of signal transduction mechanisms by which PKG regulates pulmonary vascular tone, as well as adaptive responses of the pulmonary circulation to CH.