iNOS Posttranslational Regulation in Cardiac Rejection

描述(申请人提供)：诱导型一氧化氮合酶(INOS)产生的一氧化氮(NO)在心脏移植排斥反应的炎症反应中起重要作用。虽然iNOS是转录调节的，但翻译后调节iNOS活性在急性排斥反应中的作用尚不清楚。假设：翻译后因素改变iNOS产生的NO，并决定NO在急性心脏排斥反应中的下游作用。方法：将Lewis(同种异体)或Wistar-Furth(同种异体)供心移植到Lewis受体大鼠体内。也将使用分离的细胞因子刺激的心肌细胞(CM)。基因的表达将通过Western印迹和RT-PCR来确定。BH4和NO的合成将通过高效液相色谱、NO分析仪或电子顺磁共振(EPR)光谱来确定。通过不同的辅因子合成(包括GTP环水解酶I的过度表达)、iNOS二聚体和硝基酪氨酸的形成，将通过不同的辅因子合成(包括GTP环水解酶I过表达)来检测NO和超氧化物的解偶联，将通过最先进的生物物理和生化技术来检测，包括：用Western印迹、免疫组织化学、ELISA法、EPR和荧光光谱、化学发光来检测GET过滤。目的#1：BH4调节细胞因子激活的CM和同种免疫激活后的CM中iNOS产生的NO。目的#2：6H4调节细胞因子激活的CM和同种免疫激活后的CM中iNOS产生的超氧化物歧化酶。目的#3：在细胞因子激活的CM和同种异体免疫激活后的CM中，NO和超氧化物产生的变化对脂质过氧化、蛋白质氧化和蛋白质硝化等生物标志物具有下游影响。这项研究的结果可能为预防急性心脏排斥反应中的氧化和硝化损伤提供独特的策略。公共卫生声明：心肌细胞的丧失是人类心脏移植中的一个重大问题，可能会导致心脏功能不佳。我们的研究发现了心肌细胞固有的潜在分子问题，容易导致细胞死亡和损伤。更好地了解这一分子过程可能会导致更好的策略来预防这些患者的心肌细胞损伤。

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.