iNOS Posttranslational Regulation in Cardiac Rejection

心脏排斥反应中 iNOS 的翻译后调节

• 批准号: 7088167
• 负责人: GALEN M PIEPER
• 金额: $ 37.88万
• 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/7088167
• 关键词: cardiac myocytes; cytokine; electron spin resonance spectroscopy; gene expression; genetic transcription; heart transplantation; high performance liquid chromatography; homologous transplantation; laboratory rat; lipid peroxides; nitration; nitric oxide; nitric oxide synthase; oxidation; oxidative stress; peroxidation; polymerase chain reaction; posttranslational modifications; superoxides; tetrahydrobiopterin; transplant rejection; western blottings

DESCRIPTION (provided by applicant): 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 spectroscopy, 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.

描述（由申请人提供）：诱导型NO合酶（INOS）的一氧化氮（NO）产生在心脏移植排斥反应的炎症反应中起重要作用。虽然iNOS受到转录的调节，但iNOS活性在急性排斥反应中的翻译后调节的作用尚不清楚。假设：翻译后因素改变了INOS的NO产生，并确定急性心脏排斥反应中NO的下游作用。方法：刘易斯（等距移植）或wistar-furth（同种异体移植）供体心脏将被移植到刘易斯接受者大鼠中。还将使用分离的细胞因子刺激的心肌细胞（CM）。基因表达将由Western印迹和RT-PCR确定。 BH4和无合成将由HPLC，无分析仪或电子顺磁共振（EPR）光谱确定。 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 spectroscopy,化学发光。 AIM＃1：BH4在AlloMune激活后，在细胞因子激活的CM和CM中没有调节INOS的产生。 AIM＃2：6H4调节来自细胞因子活化的CM中的iNOS和AlloMune激活后CM中的超氧化物产生。 AIM＃3：在AlloMune激活后，NO的改变和细胞因子激活CM和CM中的超氧化物的改变对脂质过氧化，蛋白质氧化和蛋白质硝化的生物学标志物具有下游影响。这项研究的发现可能提供了独特的策略，以防止急性心脏排斥反应中的牛和硝化损伤。公共卫生陈述：心脏肌肉细胞的丧失是人类心脏移植的重大问题，可能导致心脏功能不佳。我们的研究确定了易于细胞死亡和损伤的心脏细胞固有的潜在分子问题。更好地了解这种分子过程可能会导致更好的策略来预防这些患者的损伤心脏细胞。