MOLECULAR TARGETS OF NITRIC OXIDE IN CARDIAC TRANSPLANTS

心脏移植中一氧化氮的分子靶标

• 批准号: 6537767
• 负责人: GALEN M PIEPER
• 金额: $ 37.5万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6537767
• 关键词: apoptosis; electron spin resonance spectroscopy; enzyme induction /repression; gel electrophoresis; gene targeting; genetic transcription; genetically modified animals; heart contraction; heart transplantation; immunoprecipitation; laboratory mouse; laboratory rat; nitric oxide; nitric oxide synthase; nitroso compounds; northern blottings; nuclear factor kappa beta; oxidative stress; superoxide dismutase; transplant rejection; western blottings

DESCRIPTION (Verbatim from the application): While nitric oxide (NO) is believed to contribute to cardiac allograft rejection, me precise mechanism is not understood. We will examine the intracellular molecular mechanisms for the actions of excess NO on cardiac allograft contractile dyskinesis relating to a paradigm which includes both oxidative and nitrosative stress. We will examine the regulation of inducible NO synthase (iNOS) gene expression by the oxidant-sensitive NF-kB transcription factor and examine key candidate cytosolic and mitochondrial proteins as molecular targets of NO. These proteins include myoglobin and aconitase. In vitro nitrosylation of these proteins are known to inhibit enzyme activity and 02 binding, thus, interfering with efficient 02 utilization by this O2-demanding organ. Hypothesis: [NO derived from iNOS targets certain cellular proteins for nitrosylation within cardiac grafts that contribute to myocardial contractile dysfunction. Furthermore, reactive oxygen plays a role in regulating iNOS gene expression at the transcriptional level via activation of the oxidant-sensitive transcription factor (NF-KB).] The applicant will use advanced, state-of-the-art molecular and biophysical techniques including: transcription factor regulation of iNOS gene (gel shift assays; Northern and Western analysis); in situ sonomicrometry; measures of nitrosyl protein and function (EPR, electron paramagnetic resonance spectroscopy and immunoprecipitation); assay for apoptosis; quantitation of reactive oxygen (EPR spin trapping; salicylate trapping); evaluation with iNOS knockout and SOD1 or SOD2 transgenic mice. The applicant will show that following allogeneic cardiac transplantation: Aim #1: [Induction of iNOS leads to iron-nitrosyl complex formation within myocardium and contractile dysfunction during allogeneic cardiac transplantation.]; Aim #2: [Certain candidate proteins are molecular targets of nitrosylation by excess NO in allogeneic transplantation.]; Aim #3: [iNOS gene deletion prevents nitrosyiprotein formation and prolongs graft survival. ]; Aim #4: [iNOS gene expression during allogeneic transplantation is regulated by activation of NF-KB.]; Aim #5: [Antioxidants inhibit activation of NF-KB, induction of iNOS, formation of nitrosyl complexes and enhance contractile function.]; Aim #6: [Antioxidant transgene overexpression inhibits activation of NF-kB, induction of iNOS, formation of nitrosyl complexes and enhances contractile function during allogeneic cardiac transplantion.] These studies will provide a novel mechanism to explain the pathogenesis of dyskinesis and graft failure during cardiac transplant rejection.

描述（来自应用程序的逐字）：虽然一氧化氮（NO）是 被认为是心脏移植排斥反应的原因，确切的机制是 不理解。我们将研究细胞内的分子机制， 过量NO对移植心脏收缩功能障碍的影响 包括氧化应激和亚硝化应激的范例。我们将研究 诱导型一氧化氮合酶（iNOS）基因表达的调控 氧化剂敏感的NF-κ B转录因子和检测关键候选物 细胞溶质和线粒体蛋白质作为NO的分子靶点。这些蛋白质 包括肌红蛋白和乌头酸酶。这些蛋白质的体外亚硝基化是 已知其抑制酶活性和O2结合，因此干扰 通过该O2需求器官的有效O2利用。 假设：[来自iNOS的NO靶向某些细胞蛋白， 在心脏移植物内的亚硝基化有助于心肌收缩 功能障碍此外，活性氧还参与了iNOS基因的调控 在转录水平上通过激活氧化剂敏感的 转录因子（NF-κ B）。申请人将使用先进的， 最先进的分子和生物物理技术，包括：转录 诱导型一氧化氮合酶基因的因子调节（凝胶迁移分析;北方和西方 亚硝酰蛋白及其功能的测定 (EPR电子顺磁共振光谱和免疫沉淀）; 细胞凋亡的测定;活性氧的定量（EPR自旋捕获; 水杨酸捕获）;用iNOS敲除和SOD 1或SOD 2转基因进行评估 小鼠申请人将证明在同种异体心脏移植后， 移植：目的#1：[诱导iNOS导致铁-亚硝酰复合物 同种异体移植过程中心肌内的形成和收缩功能障碍 心脏移植];目标#2：[某些候选蛋白质是分子 异基因移植中过量NO亚硝基化的靶点];目标3： [iNOS基因缺失阻止亚硝酰基蛋白的形成和移植 生存];目的#4：[异基因移植过程中iNOS基因的表达， 受NF-κ B活化调节];目的#5：[抗氧化剂抑制 NF-κ B、诱导iNOS、形成亚硝酰复合物和增强 收缩功能];目的#6：[抗氧化转基因过表达抑制 NF-kB的活化，iNOS的诱导，亚硝酰基复合物的形成， 在同种异体心脏移植期间增强收缩功能。这些 这些研究将提供一种新的机制来解释 在心脏移植排斥反应期间的运动障碍和移植物衰竭。