OXYGEN RADICALS, NEUTROPHILS AND MYOCARDIAL INJURY

缺血/再灌注（I/R）损伤已被认为是重要的 病理过程;这在临床上表现为再灌注诱导 心律失常、心肌顿抑现象和 细胞坏死率。 氧衍生的自由基与 作为这种损伤的重要介质。 当前的总体目标 建议是（a）调查单线态氧是否在以下产生 I/R，（B）开发新的策略以减少这种损伤， 单线态氧清除和（c）研究潜在的生物化学， 损伤的生理和形态学机制。 因此 该项目的具体目标是检验以下假设。 的 第一种假设是单线态氧是由于I/R而产生的 离体灌流大鼠心脏。 利用电子顺磁 共振（EPR），化学发光和HPLC技术，我们将测量 单氧生成。 特定单线态氧自旋陷阱，2，2，6，6- 四甲基哌啶（TEMP）将用于EPR和HPLC测量。 单线态氧的特异性将通过清除剂如 组氨酸、色氨酸和β-胡萝卜素。 我们还将监测 收缩力，心律失常，心肌代谢，细胞膜 高渗透性肌膜和肌浆网功能， 超微结构形态学 第二个假设是单线态氧 通过聚集使肌浆网的Ca 2 +-ATP酶失活 和片段化或通过修饰氨基酸残基。 我们将 研究了暴露后Ca 2 +-ATP酶的分子结构 单线态氧 使用SDS-PAGE和HPLC技术，我们将评估 对Ca ~（2+）-ATP酶97 K道尔顿单体蛋白的损伤， O2、H2 O2和OH自由基。 组氨酸的保护作用， 还将评价生育酚、B-胡萝卜素和抗坏血酸。 的 第三种假设是单线态氧和活化的中性粒细胞产生 严重的机械和血液动力学功能障碍，类似于I/R， 离体心脏制备。 我们将灌注辐照过的玫瑰红， 离体灌流心脏中激活的中性粒细胞和监测 等容左心室压、+和- dP/dt max、ECG和 冠状动脉血流和测量肌浆网功能，肌膜 Na+K+-ATP酶活性、巯基和脂质过氧化。 疗效 组氨酸、β-胡萝卜素、抗坏血酸、α-生育酚（对于单线态 氧），SOD，过氧化氢酶，去铁胺（O2，H2 O2，OH自由基人类 血小板（具有抗氧化活性）将在本试验中进行测试。 模型 第四个假设是单线态氧本身是直接的， 负性肌力物质，并通过以下方式发挥其负性肌力作用： 破坏心肌的兴奋-收缩耦合系统。 采用离体乳头肌等长收缩模型， 研究与单线态氧损伤相关的机械功能障碍 通过监测乳头肌收缩的变化。 的 潜在清除剂的作用（保护、改善作用和 恢复）也将进行研究。 这项工作将启动第一个 研究的目的是（1）测量单线态氧的产生， I/R（2）证明了生理、生化和形态 自由基/中性粒细胞介导的损伤的表征。 我们 我希望我们的研究结果能帮助人们更准确地理解 单线态氧介导的生理和 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.