蛋白激酶C在心肌缺血预处理中的作用机制研究

Role of Protein kinase C in the Protection of Ischemic Preconditioning on the Myocardial Ischemia and Repurfusion Injury in Cat Heart

目的观察猫心肺转流(Card iopu lmonary bypass,CPB)中应用蛋白激酶C(PKC)抑制剂-多粘菌素B(PolyB)及PKC激动剂-佛波酯(PMA)对缺血再灌注(IR)心肌组织中PKC激活程度的影响及其与心肌组织内Ca2+含量变化的关系,评价PKC在缺血预处理(IPC)机制中的作用。方法将120只健康家猫随机分为对照组、IR组、IPC组、Poly B组和PMA组;建立CPB模型。应用底物蛋白磷酸化法和原子吸收光谱法,分别测定CPB中主动脉阻断(ACC)及再灌注期间心肌组织中胞膜和胞浆中PKC活性以及心肌组织中Ca2+含量的变化。结果IR组ACC 60 m in后心肌组织胞膜和胞浆中PKC活性均明显降低(P<0.01),并于再灌注期间进一步下降,同时伴有心肌组织中Ca2+含量迅速升高;ACC后各时间点PKC活性均较对照组明显降低(P<0.01);IPC组于ACC及再灌注后胞浆PKC活性较CPB前显著下降(P<0.01),但胞膜PKC活性却显著升高(P<0.01),而且均明显高于IR组(P<0.01);其心肌组织Ca2+含量在ACC 60 m in以及再灌注期间虽较对照组有所升高(P<0.05),但明显低于IR组(P<0.01);Poly B组心肌组织胞膜和胞浆中PKC活性的变化趋势与IR组相似,再灌注期间其Ca2+含量虽较IPC组有升高趋势,但仍明显低于IR组(P<0.05);PMA组PKC活性以及Ca2+含量变化趋势与IPC组相近。结论PKC的激活参与介导了猫IPC的心肌保护作用;PKC抑制剂Poly B不能完全阻断IPC的效应,而PKC激动剂PMA可部分模拟IPC的作用。

OBJECTIVE To investigate the role of protein kinase C （PKC） in mediation of ischemic preconditioning （IPC） against myocardial reperfusion injury by using PKC inhibitor polymyxin B （Poly B） and PKC activator 413 -phorbol - 12 - myristate - 13 - acetate （PMA） during cardiopulmonary bypass （CPB） in feline. METHODS One hundred and twenty felines were randomized into five groups ： control group （ n - 24）, in which CPB was conducted without aortic cross -clamping （ACC） ; IR group （n =24）, with 60 min cardiac arrest by ACC followed by 30 min reperfusion, and cardioplegia used during the period of ACC ; IPC group （ n = 24）, with protocol similar to that of IR group except for three -round 15 rain IPC applied before ACC; Poly B group （n =24）, with protocol similar to that of IPC group except for polymyxin B administered after starting of IPC; PMA group （n =24）, with protocol similar to that of IR group except for PMA administered before ACC. Membrane and cytosol fraction of PKC activity was assessed by biochemical assays, and myocardial Ca^2＋ content was determined simultaneously. RESULTS PKC activity in both membrane and cytosol fractions was significantly reduced after 60 min cardiac arrest with ACC and during myocardial reperfusion in IR group while the Ca^2＋ content in myocardium was significant increased. However, IPC enhanced the activation and translocation of PKC to the membrane and significantly reduced the rise in myocardial Ca^2＋ content. Although membrane and cytosol fraction of PKC activity were also both inhibited by Poly B after ACC, the increase of myocardial Ca^2＋ content was markedly attenuated which was comparable with that in IPC group, whereas the change patterns of PKC activities and myocardial Ca^2＋ content were similar to IPC in PMA group CONCLUSION Cardioprotection by IPC is mediated through enhanced translocation of PKC to the membrane. PKC inhibitor Poly B cannot completely abolish IPC - induced cardioprotection. PKC activator PMA mimics at least partially the protective effect of IPC against myocardial calcium overload.