PKG Signaling and Sepsis Induced ALI

血管内表面衬里的内皮细胞的一个重要功能是提供选择性的 血液和周围组织之间的屏障。脓毒症所致急性肺损伤的发生发展过程 (ALI)肺血管内皮细胞屏障被破坏，导致通透性增加和大量 富含蛋白质的液体渗入肺泡导致急性呼吸窘迫综合征(ARDS)。在阿里， 反应性氮物种(RNS)的增加与内皮屏障的破坏有关。 RNS、一氧化氮(NO)在调节内皮屏障中的作用是复杂的。NO-cGMP信号转导 在生理条件下维持内皮细胞屏障，而一氧化氮的损伤效应可以 归因于它与超氧化物反应生成过氧亚硝酸根。我们进行了开创性的研究，证明 过氧亚硝酸盐和蛋白质硝化在脓毒症介导的ALI发生发展中的重要作用 通过激活屏障破坏蛋白RhoA进行调节。我们最近还表明， 蛋白激酶G-Iα(PKG-Iα)在酪氨酸(Y)247时硝化会减弱其酶活性。PKG-Iα保护 通过激活cAMP-PKA信号和抑制RhoA对抗损伤的内皮屏障 发出信号，但通过未解决的机制。我们已经确定了一个抑制磷酸化的位点 磷酸二酯酶3A(PDE3A，肺内皮细胞中主要的cAMP降解系统)，即PKG-Iα 依附的。因此，我们的数据暗示PKG-Iα是肺内皮细胞屏障功能的关键调节因子。 基于这些发现，这项提议将检验过氧亚硝酸盐直接影响人体健康的总体假设 至少部分通过pDE3A-cAMP-pKA和pDE3A-cAMP-pKA介导的pKG-Iα活性和间接效应 RhoA和rac1信号的下游调制。我们还将确定防止PKG-Iα硝化是否 体外屏障保护和减少临床前ARDS小鼠模型的肺损伤。我们还将开展 评估PDE3A非同义编码小核苷酸影响的遗传学研究 多态(SNPs)与人类ARDS的发生发展我们期待这项提议，使用最新的- 最先进的细胞、分子、生化、生物物理、遗传学和整体动物方法将增加我们的 蛋白激酶G-Iα与蛋白激酶A相互作用调节肺功能的机制 内皮屏障在生理和病理条件下均有作用。此外，我们的学习应该 促进制定治疗ALI/ARDS的新战略和目标，这是一种仍有 死亡率为30%。

An important function of the endothelium lining the inner surface of blood vessels is to provide a selective barrier between blood and the surrounding tissues. During the development of sepsis-induced acute lung injury (ALI) the pulmonary vascular endothelial barrier is disrupted, leading to increased permeability and massive leakage of protein rich fluids into alveoli leading to acute respiratory distress syndrome (ARDS). In ALI, the increased generation of reactive nitrogen species (RNS) is involved in the disruption of the endothelial barrier. The role of the RNS, nitric oxide (NO) in regulating the endothelial barrier is complex. NO-cGMP signaling maintains the endothelial barrier under physiologic conditions, while the injurious effects of NO can be attributed to its reaction with superoxide to generate peroxynitrite. We have pioneered studies demonstrating the important role played by peroxynitrite, and protein nitration, in the development of sepsis-mediated ALI mediated via the activation of the barrier disruptive protein, RhoA. We have also recently shown that the nitration of protein kinase G-Iα (PKG-Iα) at tyrosine (Y) 247 attenuates its enzymatic activity. PKG-Iα protects the endothelial barrier against injury via the activation of cAMP-PKA signaling and the suppression of RhoA signaling but through unresolved mechanisms. We have identified an inhibitory phosphorylation site on phosphodiesterase 3A (PDE3A, a major cAMP-degrading system in the lung endothelium) that is PKG-Iα dependent. Thus, our data implicate PKG-Iα as a critical regulator of endothelial barrier function in the lung. Based on these findings this proposal will test the overall hypothesis that peroxynitrite exerts direct effects on PKG-Iα activity as well as indirect effects mediated, at least in part, via PDE3A-cAMP-PKA and the downstream modulation of RhoA and Rac1 signaling. We will also determine if preventing PKG-Iα nitration is barrier protective in vitro and reduces lung injury in pre-clinical ARDS mouse models. We will also carry out genetic investigations to evaluate the influence of PDE3A non-synonymous coding small nucleotide polymorphisms (SNPs) in the development of ARDS in humans. We anticipate this proposal, using state-of- the-art cellular, molecular, biochemical, biophysical, genetic, and whole animal approaches will increase our understanding of the mechanisms by which interactions between PKG-Iα and PKA regulate pulmonary endothelial barrier function under both physiologic and pathologic conditions. Further, our studies should facilitate the development of new strategies and targets for the treatment of ALI/ARDS, a disease that still has a mortality rate of >30%.