EFFECT OF DRUGS UPON MYOCARDIAL HYPOXIA

在长时间缺血之前，单次或多次短暂的心肌缺血已被证明能显著减少心肌梗死面积，这种现象被称为缺血预处理（IPC）。IPC发生在两个阶段，急性阶段，心脏保护作用仅持续1-2小时，延迟阶段，在12-24小时再次发生，持续48-96小时。我们的实验室首次证明了内源性阿片类药物通过δ (delta)阿片类受体起作用，并增强了腺苷-三磷酸敏感钾通道（KATP通道）的开放，这是触发和维持完整大鼠心脏急性和延迟IPC的关键因素。基于这些发现，本研究的长期目标是阐明δ 1-阿片受体激活导致大鼠心脏急性和/或延迟PC的细胞内信号通路。需要验证的主要假设是，δ 1-阿片受体的激活通过激活几种细胞内激酶途径产生急性和延迟的PC，这些途径包括酪氨酸激酶（TK）、蛋白激酶C （PKC）和丝裂原活化蛋白激酶（MAPK）级联，导致线粒体KATP通道（mito KATP）的激活和心脏保护。实验将在分离的缓冲灌注大鼠心脏和完整的血液灌注大鼠心脏中进行。通过选择性药理学探针、免疫细胞化学和Western blot分析，我们将确定TK、PKC的不同亚型和MAPK途径的3个主要成分——细胞外信号调节激酶（ERK 1/2）、Jun n -末端激酶（JNK）和p38 MAPK在阿片类药物诱导的PC中的作用。我们将使用两个指标，器官水平的梗死面积和亚细胞水平的线粒体功能，来评估离体和完整心脏长时间缺血和再灌注后的心肌损伤。线粒体KATP通道功能将通过测量ATP合成速率、线粒体膜电位和线粒体耗氧量来评估。线粒体将从对照组、阿片类药物和ipc处理的心脏中收集，在存在和不存在mito KATP激动剂或拮抗剂的情况下，以确定这种亚细胞细胞器在心脏保护中的作用。阿片类药物产生延迟PC的机制将与热休克反应作为比较标准。本研究结果新颖，具有重要的临床意义，可能为治疗急慢性缺血性心脏病患者提供一种有效的新手段。这个项目特别令人兴奋，因为许多阿片类激动剂已经可供医生使用，在将这一概念应用于患者之前，可能不需要长时间的药物开发。

Single or multiple brief periods of myocardial ischemia prior to a prolonged period of ischemia have been shown to produce a marked reduction in myocardial infarct size, a phenomenon termed ischemic preconditioning (IPC). IPC occurs in two phases, an acute phase in which the cardioprotective effects only persist for 1-2 hours and a delayed phase which reoccurs at 12-24 hours and persists for 48-96 hours. Our laboratory was the first demonstrate that endogenous opioids acting via a delta (delta) opioid receptor and enhanced opening of the adenosine-triphosphate sensitive potassium channel (KATP channel) were key factors in triggering and maintaining acute and delayed IPC in the intact rat heart. Based on these findings, the long- term goal of the present proposal is to elucidate intracellular signalling pathways by which delta1-opioid receptor activation leads to acute and/or delayed PC in the rat heart. The major hypothesis to be tested is that activation of the delta1-opioid receptor produces acute and delayed PC by activating several intracellular kinase pathways consisting of tyrosine kinase (TK), protein kinase C (PKC) and the mitogen-activated protein kinase (MAPK) cascade which leads to activation of the mitochondrial KATP channel (mito KATP) and cardioprotection. Experiments will be performed in isolated buffer-perfused rat hearts and intact blood-perfused rat hearts. By use of selective pharmacological probes, immunocytochemistry and Western blot analysis, we will determine the role of TK, different isoforms of PKC and the 3 major components of the MAPK pathway, extracellular signal- regulated kinase (ERK 1/2), Jun N-terminal kinase (JNK) and p38 MAPK in opioid-induced PC. We will use 2 indices, infarct size at the organ level and mitochondrial function at the subcellular level, to assess myocardial injury following prolonged ischemia and reperfusion in isolated and intact hearts. Mitochondrial KATP channel function will be assessed by measurements of ATP synthesis rates, mitochondrial membrane potential and mitochondrial oxygen consumption. Mitochondria will be harvested from control, opioid- and IPC-treated hearts in the presence and absence of agonists or antagonists of mito KATP to determine the role of this subcellular organelle in cardioprotection. Mechanisms of delayed PC produced by opioids will be compared to the heat shock response as a standard of comparison. The results of the proposal are novel and have great clinical significance and may result in an effective new means of treating patients with acute or chronic ischemic heart disease. This project is particularly exciting since many opioid agonists are already available to physicians and a long period of drug development may not be necessary before implementing this concept in patients.