Translocation of PKC, protein phosphatase inhibition and preconditioning of rabbit cardiomyocytes
Translocation of PKC, protein phosphatase inhibition and preconditioning of rabbit cardiomyocytes
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DOI：
10.1006/jmcc.1996.0138
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发表时间：
1996-07-01
影响因子：
5
通讯作者：
Ganote, CE
中科院分区：
文献类型：
--
作者：
Armstrong, SC;Hoover, DB;Ganote, CE
本研究旨在验证预适应状态诱导导致蛋白激酶C（PKC）持续移位的假说，该蛋白激酶C解释了与预适应相关的记忆。在诱导预处理（PC）、孵育后或最终持续缺血期间的定时间隔，收获细胞，进行毛地黄皂苷裂解并分离成胞质和颗粒部分。通过Western印迹分析用α、β、ζ和γ PKC同工酶的单克隆抗体评价样品，并通过光密度法定量条带。每个实验的内部对照包括氧合的心肌细胞和具有通过用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）处理诱发的PKC易位的细胞。用于控制含氧细胞。颗粒部分含有约30%的PKC β、5-10%的PKC α和60-70%的PKC ζ。（100 μ M）或10分钟缺血对这些百分比没有显著影响。与对照和预处理细胞的颗粒部分相关的PKC同工酶的相对量在后处理后没有差异，在含氧缓冲液中孵育或在最终缺血孵育期间，PMA和巨大戟二萜醇完全易位PKC α和α亚型，而胸腺毒素完全易位PKC α但仅部分（50%）易位PKC β。组分之间PKC ζ的分布不受任何药物的影响，蛋白磷酸酶抑制剂calyculin A保护细胞模仿预处理。这种保护被阻断预孵育与选择性PKC抑制剂calphostin C，但在很大程度上保留，如果calphostin C仅在肛门缺血期间加入。它的结论是，PKC活性所需的预处理，但持续易位的PKC高于基础水平是不必要的保护兔心肌细胞在体外。(C)出版社：Academic Press Limited。
This study was designed to test the hypothesis that induction of the preconditioned state results in a sustained translocation of protein kinase C (PKC) which accounts for the memory associated with preconditioning, Isolated rabbit cardiomyocytes were subjected to established preconditioning protocols using either adenosine or transient ischemia. At timed intervals during induction of preconditioning (PC), post-incubation or final sustained ischemia, cells were harvested, subjected to digitonin lysis and separated into cytosolic and particulate fractions, Samples were evaluated by Western blot analysis with monoclonal antibodies to alpha, epsilon, zeta and gamma PKC isozymes, and bands were quantified by densitometry. Internal controls for each experiment included oxygenated cardiomyocytes and cells with PKC translocation evoked by treatment with phorbol 12-myristate 13-acetate (PMA). For control oxygenated cells. the particulate fraction contained about 30% of PKC epsilon, 5-10% of PKC alpha and 60-70% of PKC zeta, Preconditioning with adenosine (100 mu M) or 10 min ischemia had no significant effect on these percentages, Furthermore, the relative amounts of the PKC isozymes associated with the particulate fraction of control and preconditioned cells did not differ after a post-incubation in oxygenated buffer or during a final ischemic incubation, PMA and ingenol completely translocated the epsilon and alpha isoforms, while thymeleatoxin totally translocated PKC alpha but only partially (50%) translocated PKC epsilon, The distribution of PKC zeta between fractions was not affected by any drug, The protein phosphatase inhibitor calyculin A protected cells mimicking preconditioning. This protection was blocked by preincubation with the selective PKC inhibitor calphostin C but was largely retained if calphostin C was added only during the anal ischemic period. It is concluded that PKC activity is required for preconditioning, but a sustained translocation of PKC above basal levels is not necessary for protection of rabbit cardiomyocytes in vitro. (C) 1996 Academic Press Limited.