iNOS Posttranslational Regulation in Cardiac Rejection

心脏排斥反应中 iNOS 的翻译后调节

• 批准号: 7373617
• 负责人: GALEN M PIEPER
• 金额: $ 36.78万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-15 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7373617
• 关键词: 3-nitrotyrosine; Acute; Allografting; Arts; Biochemical; Biological Markers; Cardiac; Cardiac Myocytes; Cell Death; Cells; Dimerization; Electron Spin Resonance Spectroscopy; Enzyme-Linked Immunosorbent Assay; Filtration; Fluorescence; GTP Cyclohydrolase I; Gene Expression; Goals; Graft Rejection; Heart; High Pressure Liquid Chromatography; Human; Immunohistochemistry; In Vitro; Inflammatory Response; Injury; Isogenic transplantation; Lead; Lipid Peroxidation; Modeling; Molecular; Nitric Oxide; Nitric Oxide Synthase; Organ Transplantation; Oxidative Stress; Patients; Play; Post-Translational Regulation; Process; Production; Protein Overexpression; Proteins; Public Health; Rattus; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Role; Solid; Superoxides; Techniques; Transplantation; Up-Regulation; Western Blotting; cytokine; heart allograft; heart function; human NOS2A protein; in vivo; nitration; oxidation; prevent; protein expression; tetrahydrobiopterin

Nitric oxide (NO) production from inducible NO synthase (iNOS) plays an important role in the inflammatory response of cardiac transplant rejection. While iNOS is regulated transcriptionally, the role of posttranslational regulation of iNOS activity in acute rejection is unknown. HYPOTHESIS: Posttranslational factors alter the production of NO from iNOS and determine the downstream actions of NO in acute cardiac rejection. METHODS: Lewis (isograft) or Wistar-Furth (allograft) donor hearts will be transplanted into Lewis recipient rats. Isolated cytokine-stimulated cardiac myocytes(CM) will also be used. Gene expression will be determined by Western blot and RT-PCR. BH4 and NO synthesis will be determined by HPLC, NO analyzer or electron paramagnetic resonance (EPR) spectroscopy. Uncoupling of NO vs. superoxide production by varying co-factor synthesis (including GTP cyclohydrolase I overexpression), iNOS dimerization and nitrotyrosine formation will be examined by state-of-the-art biophysical and biochemical techniques including: get filtration with Western blot, immunohistochemistry, ELISA, EPR and fluorescence spectrosocpy, chemiluminescence. AIM#1: BH4 regulates NO production from iNOS in cytokine-activated CM and in CM after alloimmune activation. AIM #2: 6H4 regulates superoxide production from iNOS in cytokine-actived CM and in CM after alloimmune activation. AIM #3: Alteration in NO and superoxide production in cytokine-activated CM and in CM after alloimmune activation has downstream effects on biological markers of lipid peroxidation, protein oxidation and protein nitration. Findings from this study may provide unique strategies to protect against oxidate and nitrative injury in acute cardiac rejection. Public Health Statement: The loss of cardiac muscle cells is a significant problem in human cardiac transplants that may contribute to poor heart function. Our studies identify a potential molecular problem intrinsic to cardiac cells that predisposes to cell death and injury. A better understanding of this molecular process may lead to better strategies to prevent injury cardiac cells in these patients.

诱导型一氧化氮合酶（inducible NO synthase，iNOS）产生的一氧化氮（Nitric oxide，NO）在炎症反应中起重要作用。 心脏移植排斥反应。虽然iNOS是转录调节的，但 在急性排斥反应中iNOS活性的翻译后调节是未知的。假设：翻译后 这些因素改变了iNOS产生NO的过程，并决定了NO在急性心肌梗死中的下游作用。 排斥反应方法：将刘易斯（同种异体）或Wistar-Furth（同种异体）供体心脏移植到刘易斯体内 受体大鼠还将使用分离的精氨酸刺激的心肌细胞（CM）。基因表达将 通过蛋白质印迹和RT-PCR进行测定。BH 4和NO的合成将通过HPLC测定，NO 分析仪或电子顺磁共振（EPR）光谱。NO与超氧化物的解偶联 通过改变辅因子合成（包括GTP环化水解酶I过表达）产生，iNOS 二聚化和硝基酪氨酸形成将通过最先进的生物物理和生物化学方法进行检查。 方法：采用Western blot、免疫组化、ELISA、EPR和荧光等方法进行筛选 光谱学化学发光目的#1：BH 4调节精氨酸激活的细胞中iNOS的NO产生 CM和同种免疫激活后的CM。目的#2：6 H4调节iNOS的超氧化物产生， 在同种异体免疫激活后的CM中。目的#3：NO和超氧化物的改变 在精氨酸激活的CM中和同种免疫激活后的CM中的产生具有下游效应， 脂质过氧化、蛋白质氧化和蛋白质硝化的生物标志物。这项研究的结果可能 提供了独特的策略，以防止氧化和硝化损伤的急性心脏排斥反应。 公共卫生声明：心肌细胞的损失是人类心脏病的一个重要问题。 可能导致心脏功能不佳的移植手术。我们的研究发现了一个潜在的分子问题 心脏细胞固有的，易导致细胞死亡和损伤。更好地理解这种分子 这一过程可能会导致更好的策略，以防止这些患者的心肌细胞损伤。