iNOS Posttranslational Regulation in Cardiac Rejection

描述（由申请人提供）：诱导型一氧化氮合酶（iNOS）产生的一氧化氮（NO）在心脏移植排斥的炎症反应中起重要作用。虽然iNOS是转录调节，但iNOS活性的翻译后调节在急性排斥反应中的作用尚不清楚。假设：翻译后因子改变iNOS产生NO，并决定NO在急性心脏排斥反应中的下游作用。方法：将刘易斯（同种）或Wistar-Furth（同种）供体心脏移植到刘易斯受体大鼠体内。还将使用分离的精氨酸刺激的心肌细胞（CM）。将通过蛋白质印迹和RT-PCR测定基因表达。BH 4和NO合成将通过HPLC、NO分析仪或电子顺磁共振（EPR）光谱测定。将通过最先进的生物物理和生物化学技术，包括：用蛋白质印迹、免疫组织化学、ELISA、EPR和荧光光谱法、化学发光进行过滤，检查通过不同辅因子合成（包括GTP环化水解酶I过表达）、iNOS二聚化和硝基酪氨酸形成的NO与超氧化物产生的解偶联。目的#1：BH 4调节NO的生产从iNOS在精氨酸激活的CM和CM后同种免疫激活。目的#2：6 H4调节在马槟榔碱激活的CM和同种免疫激活后的CM中从iNOS产生超氧化物。目标3：NO和超氧化物产生的改变在精氨酸激活的CM和同种免疫激活后的CM中对脂质过氧化、蛋白质氧化和蛋白质硝化的生物标志物具有下游效应。本研究的发现可能为急性心脏排斥反应中抗氧化和硝化损伤提供独特的策略。公共卫生声明：心肌细胞的损失是人类心脏移植中的一个重要问题，可能导致心脏功能不良。我们的研究确定了一个潜在的分子问题，内在的心脏细胞，易于细胞死亡和损伤。更好地了解这一分子过程可能会导致更好的策略，以防止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.