Post-Translational Regulation of High Output NO and Endothelial Barrier Dysfuncti

脓毒症是急性肺损伤的主要原因，可引起肺部炎症和毛细血管内皮细胞通透性增加，是诱导型一氧化氮合酶(INOS)表达的有力刺激。一氧化氮(NO)在调节肺血管通透性方面起着重要作用，炎症过程中产生较高水平的NO，或与超氧化物结合形成过氧亚硝酸盐，可损伤内皮屏障。尽管iNOS被认为主要是 在转录调控方面，我们的证据表明，细胞因子刺激的人肺微血管内皮细胞(HLMVECs)的iNOS活性和NO产生受到更精细的翻译后调节。在项目2中，我们将讨论在细胞因子刺激的HLMVECs中调控高产量NO的新的和重要的信号通路。在特定的目标#1中，我们将阐明膜羧肽酶(CP)M和CPD从含Arg的多肽中释放Arg，从而有效地通过iNOS产生NO的机制。我们的假设是，CPM和/或CPD与Arg转运体和iNOS形成大分子复合体，导致从肽底物释放的Arg转移到iNOS的紧密耦合。在具体目标2中，我们将确定 激动素B1受体在细胞因子刺激的HLMVECs中刺激iNOS活性和大量产生NO的信号通路，并决定其对肺内皮细胞屏障功能的影响。我们的假设是，在细胞因子处理的HLMVECs中，B1受体的刺激通过异源三聚体G蛋白、Galpha-I和Src激酶的偶联来激活iNOS，导致iNOS的磷酸化和/或亚细胞定位的改变，从而上调NO的产生。在特定的目标#3中，我们将确定细胞因子激活的HLMVECs中缓激肽B2受体刺激延长的、高产量的NO的途径，以及CPM和CPD在将激动素B2激动剂转化为Des-Arg-激动素B1激动剂而放大NO产量中的作用，以及由此对内皮通透性的影响。我们的假设是，在细胞因子刺激的HLMVECs中，B2受体通过Galpha-I偶联激活 SRC激酶和Akt，导致eNOS的磷酸化和延长激活。这与羧肽酶介导的B1激动剂的产生和iNOS的B1受体激活相结合，导致NO产生的进一步放大和内皮屏障的破坏。这些研究将确定炎症条件下肺微血管内皮细胞产生高产量NO作为自分泌和旁分泌信号以增加内皮通透性的新机制，从而为治疗干预改善内皮屏障功能提供潜在的靶点。

Sepsis, a leading cause of acute lung injury, causes pulmonary inflammation and increased capillary endothelial permeability and is a potent stimulus for inducible nitric oxide synthase (iNOS) expression. Nitric oxide (NO) plays an important role in regulating lung vascular permeability, and high levels produced during inflammation, or combined with superoxide to form peroxynitrite, can injure the endothelial barrier. Although iNOS is thought to be primarily transcriptionally regulated, our evidence shows that iNOS activity and NO production in cytokine-stimulated human lung microvascular endothelial cells (HLMVECs) are under more finely-tuned post-translational regulation. In Project 2, we will address new and important signaling pathways by which high output NO is regulated in cytokine-stimulated HLMVECs. In Specific Aim #1, we will elucidate the mechanisms by which NO production via iNOS is efficiently induced by the release of Arg from Arg-containing peptides by the membrane carboxypeptidases (CP) M and CPD. Our hypothesis is that CPM and/or CPD form a macromolecular complex with the Arg transporter and iNOS resulting in tight coupling of the transfer of Arg released from peptide substrates to iNOS. In Specific Aim #2, we will identify the signaling pathways by which the kinin B1 receptor stimulates iNOS activity and high output NO production in cytokine-stimulated HLMVECs and determine its consequences on lung endothelial barrier function. Our hypothesis is that B1 receptor stimulation in cytokine-treated HLMVECs activates iNOS by coupling through the heterotrimeric G protein, Galpha-i, and Src kinase leading to a change in phosphorylation and/or subcellular localization of iNOS to up-regulate NO production. In Specific Aim #3, we will determine the pathway of the bradykinin B2 receptor stimulation of the prolonged, high output NO in cytokine-acfivated HLMVECs and the role of CPM and CPD in amplifying NO output by converting kinin B2 agonists to the des-Arg-kinin B1 agonists, and the resultant consequences on endothelial permeability. Our hypothesis is that the B2 receptor couples through Galpha-i in cytokine-stimulated HLMVECs to activate Src kinase and Akt, resulting in phosphorylation and the prolonged activation of eNOS. This, coupled with carboxypeptidase-mediated generation or B1 agonists and B1 receptor activation of iNOS, results in the further amplification of NO production and disruption of the endothelial barrier. These studies will identify novel mechanisms by which lung microvascular endothelial cells under inflammatory conditions can generate high-output NO as autocrine and paracrine signals to increase endothelial permeability, and thus will allow identification of potential targets for therapeutic intervention to improve endothelial barrier function.