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

基本信息

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
项目类别：
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

项目摘要

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，会引起肺部炎症和毛细血管内皮通透性增加，并且是诱导型一氧化氮合酶（iNOS）表达的有效刺激物。一氧化氮（NO）在调节肺血管通透性方面发挥着重要作用，炎症过程中产生的高水平一氧化氮，或与超氧化物结合形成过氧亚硝酸盐，会损伤内皮屏障。虽然 iNOS 被认为主要是我们的证据表明，细胞因子刺激的人肺微血管内皮细胞 (HLMVEC) 中的 iNOS 活性和 NO 产生受到更精细的翻译后调节。在项目 2 中，我们将研究新的重要信号通路，通过这些通路在细胞因子刺激的 HLMVEC 中调节高输出 NO。在具体目标#1中，我们将阐明膜羧肽酶 (CP) M 和 CPD 从含精氨酸肽中释放精氨酸，有效诱导通过 iNOS 产生 NO 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，导致从肽底物释放的 Arg 转移到 iNOS 的转移紧密耦合。在具体目标 #2 中，我们将确定激肽 B1 受体刺激细胞因子刺激的 HLMVEC 中 iNOS 活性和高输出 NO 产生的信号通路，并确定其对肺内皮屏障功能的影响。我们的假设是，细胞因子处理的 HLMVEC 中的 B1 受体刺激通过异源三聚体 G 蛋白、Galpha-i 和 Src 激酶偶联来激活 iNOS，导致 iNOS 磷酸化和/或亚细胞定位发生变化，从而上调 NO 产生。在具体目标#3中，我们将确定细胞因子激活的 HLMVEC 中缓激肽 B2 受体刺激延长、高输出 NO 的途径，以及 CPM 和 CPD 通过将激肽 B2 激动剂转化为 des-Arg 来放大 NO 输出的作用-激肽 B1 激动剂，以及由此产生的对内皮通透性的影响。我们的假设是 B2 受体通过细胞因子刺激的 HLMVEC 中的 Galpha-i 偶联来激活 Src 激酶和 Akt，导致 eNOS 磷酸化和延长激活。这与羧肽酶介导的 B1 激动剂生成和 iNOS 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输出NO作为自分泌和旁分泌信号以增加内皮通透性的新机制，从而确定治疗干预的潜在靶点，以改善内皮屏障功能。