CARDIAC OXIDANTS & APOPTOSIS: LESSONS OF PRECONDITIONING

描述：这项提案代表了调查人员在 下的肺内科、重症监护和急诊内科 新成立的加州大学紧急复苏研究中心 芝加哥。有人建议进行研究，以检验这些机制和后果 缺血/再灌注诱导心肌细胞凋亡及其机制的研究 低氧预适应对细胞凋亡的保护作用。这个 这一提议的中心假设是，预适应减少 通过多种机制阻止启动细胞的凋亡 和/或细胞凋亡的传播。为了检验这些假设，我们提出了三个假设 具体目标：(1)检测哪些活性氧物种(ROS)起作用 细胞内的细胞凋亡激活剂。实验将(A)确定 包括线粒体膜破坏在内的一系列细胞凋亡事件 电位，将细胞色素c释放到胞浆中，以及激活 Caspase-9作为“启动子”的Caspase以及下游的Casase-3等； (B)使用特定的抑制治疗，评估改变的效果 在(I/R)期间超氧化物和/或H202对这些凋亡事件的影响。(二)确定 预适应是否通过Bcl2家族阻断某些细胞凋亡事件 通过PKC介导的磷酸化进行调节。实验将(A)确定 大鼠脑缺血前后Bcl2、Bclx和Bax的表达水平 预适应(PC)或非PC细胞中I/R期间的心肌细胞；并确定 PC对Bcl2蛋白丝氨酸磷酸化的影响(B) 高、低表达/阻断PKC、Bcl2和a蛋白表达的影响 氧化应激、一氧化氮合成、非磷酸化的Bcl-2蛋白 核因子-kappaB活化与细胞凋亡事件。(三)确定关系 核因子-kappaB和一氧化氮(NO)合成在防止细胞凋亡中的作用。(A) 确定PC对IkappaB及随后的磷酸化的影响 核转录因子-kappaB的活化和核转位；(B)确定其表达 即刻早期基因A1和CHAAP，并检测它们对 线粒体事件、ROS生成、氧化应激、NO生成、caspase 活性与细胞死亡的相关性和直接性 表达和反义研究。(C)确定NO的产生水平 在使用或不使用PC的再灌流过程中以及产生的NO或 特异性阻断NO生成对半胱氨酸天冬氨酸酶活性及后续细胞的影响 每种情况下都会死亡。这些研究的结果有可能 改善心脏骤停和其他缺血后的复苏后护理 心肌梗死和中风等疾病。

DESCRIPTION: This proposal represents collaboration between investigators in the Sections of Pulmonary Medicine & Critical Care and Emergency Medicine under the newly formed Emergency Resuscitation Research Center at the University of Chicago. Studies are proposed to examine the mechanisms and consequences of ischemia/reperfusion (I/R)-induced apoptosis in cardiomyocytes and to examine the protective effects of hypoxic preconditioning on this apoptosis. The central hypotheses of this proposal are that preconditioning decreases apoptotic cell death through a number of mechanisms that block the initiation and/or propagation of apoptosis. To test these hypotheses, we propose three specific aims: (1) Test which reactive oxygen species (ROS) act as intracellular activators of apoptosis. Experiments will (a) Determine the sequence of apoptotic events including disruption of mitochondria membrane potential, release of cytochrome c into the cytosol, and activation of caspase-9 as "initiator" caspase as well as downstream casases-3 and others; (b) Using specific inhibitory treatments, assess the effects of altered superoxide and/or H202 during (I/R) on these apoptotic events. (2) Determine whether preconditioning blocks certain apoptotic events via Bcl-2 family regulation through PKC-mediated phosphorylation. Experiments will (a) Determine pre- and post-ischemic levels of expression of Bcl-2, Bcl-x, and Bax in cardiomyocytes during I/R in preconditioned (PC) or non-PC cells; and determine the effects of PC on serine phopshorylation of Bcl-2 proteins (b) Assess the effects of over and underexpression/blockade of PKC, Bcl-2, and expression of a nonphosphorylatable Bcl-2 protein on oxidant stress, nitric oxide synthesis, NF-kappaB activation and apoptotic events. (3) Determine the relationship between NF-kappaB and nitric oxide (NO) synthesis in preventing apoptosis. (a) Determine the effect of PC on phosphorylation Of IkappaB and subsequent activation and nuclear translocation of NF-kappaB; (b) Determine the expression of immediate-early genes, Al and chIAP, and examine their effects on mitochondria events, ROS generation, oxidant stress, NO generation, caspase activity and apoptotic cell death through correlative and direct over expression and antisense studies. (c) Determine the levels of NO generation during reperfusion with or without PC and the direct effects of generated NO or specific blockade of NO generation on caspase activity and subsequent cell death in each condition. Results from these studies have the potential to improve post-resuscitation care following cardiac arrest and other ischemic diseases such as myocardial infarction and stroke.