iNOS Posttranslational Regulation in Cardiac Rejection

描述（由申请人提供）：诱导NO合成酶（iNOS）产生一氧化氮（NO）在心脏移植排斥反应的炎症反应中起重要作用。虽然iNOS受转录调控，但翻译后iNOS活性调控在急性排斥反应中的作用尚不清楚。假设：翻译后因子改变一氧化氮的产生，并决定一氧化氮在急性心脏排斥反应中的下游作用。方法：Lewis（同种异体移植）或Wistar-Furth（同种异体移植）供体心脏移植到Lewis受体大鼠体内。分离的细胞因子刺激心肌细胞（CM）也将被使用。Western blot和RT-PCR检测基因表达。采用高效液相色谱法、NO分析仪或电子顺磁共振（EPR）光谱法测定BH4和NO的合成。通过不同的辅助因子合成（包括GTP环水解酶I过表达）、iNOS二聚化和硝基酪氨酸形成来解偶联NO与超氧化物的产生将通过最先进的生物物理和生化技术进行检测，包括：用Western blot、免疫组织化学、ELISA、EPR和荧光光谱、化学发光进行过滤。目的1:BH4调节细胞因子激活CM和同种免疫激活后CM中iNOS产生NO。AIM #2: 6H4调节细胞因子激活CM和同种免疫激活后CM中iNOS的超氧化物产生。AIM #3：细胞因子激活CM和同种免疫激活后CM中NO和超氧化物产生的改变对脂质过氧化、蛋白质氧化和蛋白质硝化等生物标志物有下游影响。本研究结果可能为防止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：心肌细胞的损失是人类心脏移植的一个重要问题，可能导致心脏功能低下。我们的研究确定了心脏细胞固有的潜在分子问题，容易导致细胞死亡和损伤。对这一分子过程的更好理解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.