iNOS Posttranslational Regulation in Cardiac Rejection

诱导型一氧化氮合酶(INOS)产生的一氧化氮合酶(NO)在炎症反应中起重要作用。 心脏移植排斥反应。虽然iNOS是转录调控的，但它的作用是 急性排斥反应中iNOS活性的翻译后调节尚不清楚。假设：翻译后 影响诱导型一氧化氮合酶产生一氧化氮的因素及其在急性心脏疾病中的下游作用 拒绝。方法：将Lewis(同种异体)或Wistar-Furth(同种异体)供心移植到Lewis体内。 受体大鼠。也将使用分离的细胞因子刺激的心肌细胞(CM)。基因表达将 用Western印迹和RT-PCR方法进行检测。BH4和NO的合成将通过高效液相色谱法进行测定， 分析仪或电子顺磁共振(EPR)光谱分析。NO与超氧化物的解偶联 通过不同的辅因子合成(包括GTP环水解酶I过表达)、iNOS产生 二聚化和硝基酪氨酸的形成将通过最先进的生物物理和生物化学进行检测 方法：免疫印迹、免疫组织化学、ELISA法、EPR法、荧光法等 光谱学，化学发光。目的#1：BH4调节细胞因子激活的iNOS产生NO CM和同种异体免疫激活后的CM。目的#2：6H4调节大鼠iNOS产生超氧化物歧化酶 细胞因子激活的CM和同种异体免疫激活后的CM。目标3：一氧化氮和超氧化物歧化 细胞因子激活的CM和同种异体免疫激活后的CM中的产物对 脂质过氧化、蛋白质氧化和蛋白质硝化的生物标志物。这项研究的发现可能 提供独特的策略，防止急性心脏排斥反应中的氧化和硝化损伤。 公共卫生声明：心肌细胞的丧失是人类心脏的一个重大问题 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 心肌细胞固有的，易导致细胞死亡和损伤的。对这种分子有更好的了解 这一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。

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.