OXYGEN RADICALS, NEUTROPHILS AND MYOCARDIAL INJURY

缺血/再灌注(I/R)损伤已被公认为是一种重要的 病理过程；这在临床上表现为再灌注 心律失常、心肌顿抑现象和 细胞坏死率。与氧衍生的自由基有牵连 作为这场伤害的重要调解人。当前的总体目标 建议(A)调查是否在下列情况下产生单线态氧 I/R，(B)制定新的战略，以减少这种伤害 单线态氧清除和(C)研究潜在的生物化学， 损伤的生理和形态机制。因此， 该项目的具体目标是检验以下假设。这个 第一种假设是单线态氧的产生是I/R的结果 在隔离灌流的大鼠心脏中。使用电子顺磁 我们将测量共振(EPR)、化学发光和高效液相技术 单次产氧。特异单重态氧自旋陷阱，2，2，6，6- 四甲基哌啶(TEMP)将用于电子顺磁共振和高效液相色谱测量。 单线态氧的特异性将被清道夫证实，例如 组氨酸、色氨酸和B-胡萝卜素。我们还将监控 收缩、心律失常、心肌代谢、细胞膜 高通透性肌膜和肌浆网功能及 超微结构形态。第二个假设是单线态氧 聚集失活肌浆网钙-三磷酸腺苷酶 和碎裂或通过氨基酸残基的修饰。我们会 染尘后细胞内钙离子-三磷酸腺苷酶分子结构的研究 单线态氧。使用十二烷基硫酸钠-PAGE和高效液相技术，我们将评估 Ca~(2+)-ATPase对97K道尔顿单体蛋白的损伤及与 O2、H_2O_2和OH自由基。组氨酸的保护作用，α- 生育酚、B-胡萝卜素和抗坏血酸也将被评估。这个 第三种假设是单线态氧和激活的中性粒细胞产生 严重的机械和血流动力学功能障碍，类似于I/R 离体心准备。我们将灌流辐射过的玫瑰孟加拉和 离体灌流心脏和监护仪中激活的中性粒细胞 等容左室压、+和-dp/dt最大值、心电图和 冠状动脉流量和测定肌浆网功能、肌膜 Na+K+-ATPase活性、硫醇基团和脂质过氧化。药效 组氨酸-β-胡萝卜素、抗坏血酸、α-生育酚(单发) 氧)，超氧化物歧化酶，过氧化氢酶，脱铁胺(O2，H_2O_2，OH自由基人 血小板(具有抗氧化活性)将在此进行测试。 模特。第四种假设是单线态氧本身是一种直接的， 负性变力物质并通过以下方式发挥其负性变力作用 扰乱心肌兴奋收缩偶联系统。 利用离体乳头肌等长收缩模型， 单线态氧损伤相关机械功能障碍的研究 通过监测乳头肌收缩的变化。这个 潜在清道夫的影响(保护、改善效果和 回收)也将进行研究。这项工作将启动首批工作之一 旨在(1)测量以下单线态氧的产生的研究 I/R(2)显示生理、生化和形态 自由基/中性粒细胞介导的损伤的特征。我们 预计我们的发现将有助于我们对 单线态氧介导生理和免疫调节的作用和机制(S) 生化事件。

Ischemia/reperfusion (I/R) injury has been recognized to be an important pathologic process; this is manifest clinically as reperfusion induced arrhythmias, the phenomenon of myocardial stunning and an increase in the rate of cell necrosis. Oxygen-derived free radicals have been implicated as important mediators of this injury. The overall goals of the present proposal are (a) to investigate if singlet oxygen is generated following I/R, (b) to develop novel strategies to reduce this injury based on singlet oxygen scavenging and (c) to study the underlying biochemical, physiological and morphological mechanisms of injury. Accordingly, the specific aims of the project are to test the following hypotheses. The first hypothesis is that singlet oxygen is generated as a result of I/R in the isolated perfused rat heart. Using electron paramagnetic resonance (EPR), chemiluminescence and HPLC techniques we will measure single oxygen generation. Specific singlet oxygen spin trap, 2,2,6,6- tetramethylpiperidine (TEMP) will be used in EPR and HPLC measurements. Specificity of singlet oxygen will be confirmed by scavengers such as histidine, tryptophan and B-carotene. We will also monitor contractility, arrhythmia, myocardial metabolism, cell membrane hyperpermeability sarcolemmal and sarcoplasmic reticulum functions and ultrastructural morphology. The second hypothesis is that singlet oxygen inactivates Ca2+-ATPase enzyme of sarcoplasmic reticulum by aggregation and fragmentation or by modification of amino acid residues. We shall study the molecular structure of Ca2+-ATPase following exposure to singlet oxygen. Using SDS-PAGE and HPLC techniques, we shall assess the damage to the 97 K dalton monomer protein of Ca2+-ATPase and compare with O2, H2O2 and OH radical. Protective effects of histidine, alpha- tocopherol B-carotene and ascorbic acid will also be evaluated. The third hypothesis is that singlet oxygen and activate neutrophils produce significant mechanical and hemodynamic dysfunction, similar to I/R in the isolated heart preparation. We will perfuse irradiated rose bengal and activated neutrophils in the isolated perfused heart and monitor isovolumetric left ventricular pressure, + and - dP/dt max, ECG and coronary flow and measure sarcoplasmic reticulum function, sarcolemmal Na+K+-ATPase activity, thiol groups and lipid peroxidation. The efficacy of histidine beta-carotene, ascorbic acid, alpha-tocopherol (for singlet oxygen), SOD, catalase, deferoxamine (O2, H2O2, OH radical human platelets (which possess antioxidative activity) will be tested in this model. the fourth hypothesis is that singlet oxygen itself is a direct, negative inotropic species and exerts its negative inotropic action by disrupting the excitation-contraction coupling system of cardiac muscle. Using isolated papillary muscle isometric contraction model we shall study the mechanical dysfunction associated with singlet oxygen injury by monitoring alterations in contraction of the papillary muscle. The effects of potential scavengers (protective, ameliorating effect and recovery) will also be studied. This work will initiate one of the first studies aimed at (1) measuring the generation of singlet oxygen following I/R (2) demonstrating the physiological, biochemical and morphological characterization of free radicals/neutrophils mediated injury. We anticipate that our findings will foster a precise understanding of the role and mechanism(s) of singlet oxygen mediated physiological and biochemical events.