OXYGEN RADICALS, NEUTROPHILS, AND MYOCARDIAL INJURY

氧自由基、中性粒细胞和心肌损伤

• 批准号: 2223190
• 负责人: Rakesh C Kukreja
• 金额: $ 17.95万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2223190
• 关键词: calcium transporting ATPase; electron microscopy; electron spin resonance spectroscopy; electrophysiology; free radical oxygen; heart contraction; high performance liquid chromatography; isolation perfusion; laboratory rat; myocardial ischemia /hypoxia; neutrophil; reperfusion; sarcolemma; sarcoplasmic reticulum; singlet oxygen

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.

缺血/再灌注（I/R）受伤已被认为是重要的 病理过程；随着再灌注引起的，这在临床上表现出来 心律不齐，心肌惊人的现象和增加 细胞坏死率。 氧衍生的自由基已与 作为这种伤害的重要调解人。 当前的总体目标 提案是（a）调查是否生成单线氧 I/R，（b）制定新的策略来减少基于 单线氧气清除和（c）研究潜在的生化， 损伤的生理和形态学机制。 因此， 该项目的具体目的是检验以下假设。 这 第一个假设是单重氧是由于I/R产生的 在孤立的灌注大鼠心脏中。 使用电子顺磁性 共振（EPR），化学发光和HPLC技术我们将测量 单一氧气产生。 特定的单线氧自旋陷阱，2,2,6,6-- 四甲基磷酸（TEMP）将用于EPR和HPLC测量。 单线氧的特异性将由清道夫（例如 组氨酸，色氨酸和B-胡萝卜素。 我们还将监视 收缩力，心律不齐，心肌代谢，细胞膜 过度过敏性肌膜和核质网函数以及 超微结构形态。 第二个假设是单线氧 通过聚集使肌质网的Ca2+-ATPase酶失活 和碎裂或通过修饰氨基酸残基。 我们将 研究暴露后Ca2+-ATPase的分子结构 单线氧。 使用SDS-PAGE和HPLC技术，我们将评估 损坏Ca2+-ATPase的97 K道尔顿单体蛋白质，并与 O2，H2O2和OH激进。 组氨酸，α-的保护作用 还将评估生育酚B-羟基烯和抗坏血酸。 这 第三个假设是单线氧和激活中性粒细胞会产生 显着的机械和血液动力学功能障碍，类似于I/R 孤立的心脏准备。 我们将灌注辐照的玫瑰孟加拉和 孤立的灌注心脏中激活的中性粒细胞和监测 等光量表左心压， +和-DP/DT Max，ECG和 冠状动脉流量和测量肌质网，肌膜片 Na+K+-ATPase活性，硫醇基和脂质过氧化。 功效 组氨酸β-胡萝卜素，抗坏血酸，α-生育酚 氧），SOD，过氧化氢酶，脱氧胺（O2，H2O2，OH激进的人类 血小板（具有抗氧化活性）将在此测试 模型。 第四个假设是单线氧本身是直接的， 负肌部阴性物种，并通过 破坏心肌的激发反应耦合系统。 使用孤立的乳头肌肉等距收缩模型，我们将 研究与单线氧损伤相关的机械功能障碍 通过监测乳头肌收缩的改变。 这 潜在的清除剂的影响（保护性，改善效果和 还将研究恢复）。 这项工作将启动第一个 旨在（1）测量单线氧的产生的研究 I/R（2）证明生理，生化和形态学 自由基/中性粒细胞介导的损伤的表征。 我们 预计我们的发现将促进对 单线氧介导的生理和机制 生化事件。