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

高输出 NO 和内皮屏障功能障碍的翻译后调节

• 批准号: 7367821
• 负责人: Randal A Skidgel
• 金额: $ 30.81万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7367821
• 关键词: Acute Lung Injury; Address; Agonist; Albumins; Arginine; Basic Amino Acid Transport Systems; Binding; Blood capillaries; Bradykinin; Bradykinin B1 Receptor; Bradykinin B2 Receptor; C-terminal; Calcium; Carboxypeptidase; Caveolae; Caveolins; Cells; Citrulline; Condition; Coupled; Coupling; Data; Disruption; Endothelial Cells; Endothelium; GTP-Binding Proteins; Generations; Heterotrimeric GTP-Binding Proteins; Human; Inflammation; Inflammatory; Kallidin; Kinins; Label; Ligation; Lipids; Liquid substance; Lung; Macromolecular Complexes; Mediating; Mediator of activation protein; Membrane; Membrane Lipids; Membrane Microdomains; Molecular Genetics; Mus; Nitric Oxide; Output; Paracrine Communication; Pathway interactions; Peptides; Permeability; Peroxonitrite; Phospholipase C; Phosphorylation; Play; Pneumonia; Post-Translational Regulation; Production; Property; Publishing; Receptor Activation; Regulation; Relative (related person); Role; Sepsis; Signal Pathway; Signal Transduction; Site; Source; Stimulus; Superoxides; System; Therapeutic Intervention; Thinking; Vascular Endothelial Cell; Vascular Permeabilities; Work; arginylarginine; autocrine; base; capillary; carboxypeptidase M; caveolin 1; cell type; cytokine; genetic regulatory protein; human NOS2A protein; improved; inhibitor/antagonist; injured; membrane activity; monolayer; novel; receptor; receptor coupling; response; src-Family Kinases

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.

败血症是急性肺损伤的主要原因，会引起肺部炎症和毛细血管内皮渗透性增加，并且是诱导型一氧化氮合酶（INOS）表达的有效刺激。一氧化氮（NO）在调节肺血管渗透性以及在炎症过程中产生的高水平或与超氧化物结合形成过氧亚硝酸盐的高水平起着重要作用，可能会伤害内皮屏障。虽然iNOS被认为主要是 在转录调节的情况下，我们的证据表明，在细胞因子刺激的人肺微血管内皮细胞（HLMVEC）中，iNOS活性和无生产在更精细的翻译后调节下。在项目2中，我们将解决新的和重要的信号通路，通过这些信号通路，高输出NO在细胞因子刺激的HLMVEC中受到调节。在特定的目标＃1中，我们将阐明通过膜羧肽酶（CP）M和CPD从ARG释放出ARG从ARG释放有效诱导的机制。我们的假设是，CPM和/或CPD与ARG转运蛋白和iNOS形成大分子复合物，从而导致从肽底物释放到INOS的ARG转移紧密耦合。在特定的目标＃2中，我们将确定 Kinin B1受体刺激iNOS活性和高输出无产生的信号通路在细胞因子刺激的HLMVEC中无产生，并确定其对肺内皮屏障功能的后果。我们的假设是，通过通过异三聚体G蛋白，Galpha-I和SRC激酶耦合，细胞因子处理的HLMVEC中的B1受体刺激激活iNOS，从而导致磷酸化和/或亚细胞定位的变化，从而导致iNOS的亚细胞定位。在特定的目标＃3中，我们将确定延长的细胞因子吸收性HLMVEC的延长，高输出NO的途径，以及CPM和CPD在扩增NO输出中的作用，通过将Kinin B2激动剂转换为Des-Arg-arg-kinginb1 agonists，以及对生成的允许，并在des-arg-kinerist上转换出来。我们的假设是，在细胞因子刺激的HLMVEC中，通过Galpha-i通过Galpha-I夫妇激活 SRC激酶和AKT，导致磷酸化和延长的eNOS激活。与羧肽酶介导的产生或B1激动剂以及iNOS的B1受体激活相结合，导致无生产和内皮屏障的破坏进一步扩增。这些研究将确定在炎症条件下肺微血管内皮细胞的新型机制，可以产生高输入NO作为自分泌和旁分泌信号，以提高内皮渗透性，因此将允许鉴定治疗性干预措施以提高内皮屏障功能。