Marais E, Genade S, Huisamen B, Strijdom J G, Moolman J A, Lochner A
Department of Medical Physiology and Biochemistry, University of Stellenbosch Faculty of Medicine, Tygerberg, Republic of South Africa.
J Mol Cell Cardiol. 2001 Apr;33(4):769-78. doi: 10.1006/jmcc.2001.1347.
The role of p38 mitogen-activated protein kinase (MAPK) in ischaemic preconditioning remains controversial. Since most previous studies focussed on events only during sustained ischaemia, the aim of this study was to establish the activation pattern of p38 MAPK during a multicycle preconditioning protocol, sustained ischaemia as well as reperfusion and to correlate these events with functional recovery of the isolated perfused rat heart. Isolated perfused rat hearts were preconditioned by 3x5 min global ischaemia followed by 25 min global ischaemia and 30 min reperfusion. Non-preconditioned hearts were subjected to 25 min global ischaemia and 30 min reperfusion. Hearts were freeze-clamped and p38 MAPK activation in tissue lysates was assessed by standard Western blotting techniques, using a dual phospho-p38 MAPK antibody as well as a non-radioactive IP-kinase assay. The results showed that transient dual phosphorylation and activation of p38 MAPK occurs during a 3x5 min preconditioning protocol: the activation was maximal during the first episode, becoming progressively lower during the second and third episodes. p38 MAPK activation was significantly less during both sustained ischaemia and reperfusion in preconditioned hearts, when compared with non-preconditioned hearts. Attenuation of p38 MAPK activity during sustained ischaemia and reperfusion was associated with improved functional recovery. The effect of inhibition of p38 MAPK activation on cardioprotection was further evaluated in adult, isolated cardiomyocytes. Administration of SB 203580 (1-10 microM) before and during the preconditioning protocol, had no effect on cell morphology and viability after 2 h hypoxia, compared to untreated preconditioned cardiomyocytes. When administered to non-preconditioned cells before the onset of 2 h hypoxia, it caused a significant improvement in both morphology and viability. In summary, the results suggest that attenuation of the kinase activity during sustained ischaemia and reperfusion may be an essential element of the preconditioning process.
p38丝裂原活化蛋白激酶（MAPK）在缺血预处理中的作用仍存在争议。由于此前大多数研究仅关注持续性缺血期间的事件，本研究的目的是确定多周期预处理方案、持续性缺血以及再灌注期间p38 MAPK的激活模式，并将这些事件与离体灌注大鼠心脏的功能恢复相关联。离体灌注大鼠心脏先经3次5分钟全心缺血预处理，随后进行25分钟全心缺血和30分钟再灌注。未预处理的心脏进行25分钟全心缺血和30分钟再灌注。心脏经冷冻钳夹处理，使用双磷酸化p38 MAPK抗体以及非放射性IP激酶测定法，通过标准蛋白质印迹技术评估组织裂解物中p38 MAPK的激活情况。结果显示，在3次5分钟预处理方案期间，p38 MAPK会发生短暂的双磷酸化和激活：在第一次发作时激活程度最高，在第二次和第三次发作时逐渐降低。与未预处理的心脏相比，预处理心脏在持续性缺血和再灌注期间p38 MAPK的激活明显较少。持续性缺血和再灌注期间p38 MAPK活性的减弱与功能恢复改善相关。在成年离体心肌细胞中进一步评估了抑制p38 MAPK激活对心脏保护的作用。与未处理的预处理心肌细胞相比，在预处理方案之前和期间给予SB 203580（1 - 10 microM），对缺氧2小时后的细胞形态和活力没有影响。当在2小时缺氧开始前给予未预处理的细胞时，它能显著改善细胞形态和活力。总之，结果表明持续性缺血和再灌注期间激酶活性的减弱可能是预处理过程的一个关键要素。
