Ping P, Zhang J, Cao X, Li R C, Kong D, Tang X L, Qiu Y, Manchikalapudi S, Auchampach J A, Black R G, Bolli R
Experimental Research Laboratory, Division of Cardiology, University of Louisville and Jewish Hospital Heart and Lung Institute, Louisville, Kentucky 40202, USA.
Am J Physiol. 1999 May;276(5):H1468-81. doi: 10.1152/ajpheart.1999.276.5.H1468.
Using conscious rabbits, we examined the effect of ischemic preconditioning (PC) on p44 and p42 mitogen-activated protein kinases (MAPKs). We found that both isoforms contribute significantly to total MAPK activity in the heart (in-gel kinase assay: p44, 59 +/- 1%; p42, 41 +/- 1%). Ischemic PC (6 cycles of 4-min occlusion/4-min reperfusion) elicited a pronounced increase in total cellular MAPK activity (+89%). This increase, which occurred exclusively in the nuclear fraction, was contributed by both isoforms (in-gel kinase assay: p44, +97%; p42, +210%) and was accompanied by migration of the two proteins from the cytosolic to the nuclear compartment. In control rabbits, MAPK kinase (MEK)1 and MEK2, direct activators of p44 and p42 MAPKs, were located almost exclusively in the cytosolic fraction. Ischemic PC induced a marked increase in cytosolic MEK activity (+164%), whereas nuclear MEK activity did not change, indicating that MEK-induced activation of MAPKs occurred in the cytosolic compartment. Activation of MAPKs after ischemic PC was completely blocked by the protein kinase C (PKC) inhibitor chelerythrine. Selective overexpression of PKC-epsilon in adult rabbit cardiomyocytes induced activation of both p44 and p42 MAPKs and reduced lactate dehydrogenase release during simulated ischemia-reperfusion, which was abolished by the MEK inhibitor PD-98059. The results demonstrate that 1) ischemic PC induces a rapid activation of p44 and p42 MAPKs in hearts of conscious rabbits; 2) the mechanism of this phenomenon involves activation of p44 and p42 MAPKs in the cytosol and their subsequent translocation to the nucleus; and 3) it occurs via a PKC-mediated signaling pathway. The in vitro data implicate PKC-epsilon as the specific isoform responsible for PKC-induced MAPK activation and suggest that p44/p42 MAPKs contribute to PKC-epsilon-mediated protection against simulated ischemia. The results are compatible with the hypothesis that p44 and p42 MAPKs may play a role in myocardial adaptations to ischemic stress.
我们使用清醒的兔子，研究了缺血预处理（PC）对p44和p42丝裂原活化蛋白激酶（MAPK）的影响。我们发现，这两种同工型对心脏中的总MAPK活性有显著贡献（凝胶内激酶测定：p44为59±1%；p42为41±1%）。缺血预处理（6个周期的4分钟闭塞/4分钟再灌注）使总细胞MAPK活性显著增加（+89%）。这种增加仅发生在细胞核部分，由两种同工型共同促成（凝胶内激酶测定：p44为+97%；p42为+210%），并伴随着这两种蛋白质从细胞质向细胞核区室的迁移。在对照兔子中，p44和p42 MAPK的直接激活剂MAPK激酶（MEK）1和MEK2几乎完全位于细胞质部分。缺血预处理导致细胞质中MEK活性显著增加（+164%），而细胞核中MEK活性未改变，这表明MEK诱导的MAPK激活发生在细胞质区室。缺血预处理后MAPK的激活被蛋白激酶C（PKC）抑制剂白屈菜红碱完全阻断。在成年兔心肌细胞中选择性过表达PKC-ε可诱导p44和p42 MAPK的激活，并减少模拟缺血再灌注期间乳酸脱氢酶的释放，而MEK抑制剂PD-98059可消除这种作用。结果表明：1）缺血预处理可在清醒兔心脏中快速激活p44和p42 MAPK；2）这一现象的机制涉及细胞质中p44和p42 MAPK的激活及其随后向细胞核转运；3）其通过PKC介导的信号通路发生。体外数据表明PKC-ε是负责PKC诱导的MAPK激活的特定同工型，并提示p44/p42 MAPK有助于PKC-ε介导的对模拟缺血的保护作用。这些结果与p44和p42 MAPK可能在心肌对缺血应激的适应中起作用的假设相符。
