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.

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中起重要作用，炎症过程中产生的高水平NO或与超氧化物结合形成过氧亚硝酸盐，可损伤内皮屏障。虽然iNOS被认为主要是 转录调控，我们的证据表明，iNOS的活性和NO的生产在尼古丁刺激的人肺微血管内皮细胞（HLMVECs）是在更精细调谐的翻译后调节。在项目2中，我们将解决新的和重要的信号转导途径，高输出NO是在姜黄素刺激HLMVECs的调节。在具体目标#1中，我们将阐明通过iNOS产生NO的机制，通过膜羧肽酶（CP）M和CPD从含Arg的肽中释放Arg有效地诱导NO的产生。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，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 激肽B1受体通过其刺激iNOS活性和高输出NO产生的信号通路，并决定其对肺内皮屏障功能的影响。我们的假设是，B1受体的刺激，在苦参碱处理的HLMVECs激活iNOS通过偶联通过异源三聚体G蛋白，Galpha-i，Src激酶导致的变化，磷酸化和/或亚细胞定位的iNOS上调NO的生产。在具体目标#3中，我们将确定缓激肽B2受体刺激精氨酸激活的HLMVEC中延长的高输出NO的途径，以及CPM和CPD通过将激肽B2激动剂转化为脱精氨酸-激肽B1激动剂在放大NO输出中的作用，以及对内皮通透性的结果。我们的假设是B2受体通过Galpha-i偶联在苦参碱刺激的HLMVECs中激活 Src激酶和Akt，导致eNOS的磷酸化和延长的激活。这与羧肽酶介导的iNOS生成或B1激动剂和B1受体活化相结合，导致NO产生的进一步放大和内皮屏障的破坏。这些研究将确定新的机制，肺微血管内皮细胞在炎症条件下可以产生高输出NO作为自分泌和旁分泌信号，以增加内皮通透性，从而将允许识别潜在的治疗干预，以改善内皮屏障功能的目标。