Nitric oxide inhibits ischemia/reperfusion-induced myocardial apoptosis by modulating cyclin A-associated kinase activity
Nitric oxide inhibits ischemia/reperfusion-induced myocardial apoptosis by modulating cyclin A-associated kinase activity
复制标题
DOI：
10.1016/s0008-6363(03)00425-5
复制
发表时间：
2003-08-01
影响因子：
10.8
通讯作者：
Isobe, M
中科院分区：
文献类型：
--
作者：
Maejima, Y;Adachi, S;Isobe, M
目的：心脏缺血再灌注可引起心肌细胞凋亡和一氧化氮（NO）生成增加。我们已经报道了心肌细胞凋亡与细胞周期调控蛋白的激活有关。然而，一氧化氮（NO）在缺血/再灌注诱导的细胞凋亡中的作用尚不清楚。本研究旨在阐明缺血/再灌注诱导细胞凋亡的新机制，特别是NO与细胞周期调控因子的相互作用。方法和结果：1或2日龄Wistar大鼠的新生心肌细胞进行1小时的缺血，然后再灌注。TUNEL法检测心肌细胞凋亡率在再灌注24 h后明显增加。再灌注15 min，心肌细胞NO增加1.8倍。再灌注36 h后，NO合成酶抑制剂硝基-L-精氨酸甲酯（Nitro-L-arginine methyl ester，L-NAME）可显著增加细胞凋亡率，而NO供体S-亚硝基-N-乙酰青霉胺（S-nitroso-N-acetylpenicillamine，SNAP）可显著降低细胞凋亡率。免疫印迹分析表明，细胞周期蛋白A的蛋白水平积累在一个时间依赖性的方式响应缺血/再灌注，和L-NAME抑制这种反应。缺血/再灌注后细胞周期蛋白A相关激酶（cyclinA-associatedkinase，cyclinA-associatedkinase，cyclinA-associatedkinase，cyclinA-associatedkinase）活性明显升高，CDK 2腺病毒感染可抑制细胞凋亡。为了阐明参与p21（cip 1/waf 1）蛋白，这是细胞周期蛋白A相关激酶的抑制剂，我们进行免疫印迹分析，并检查其激酶活性。用L-NAME处理心肌细胞可抑制p21（cip 1/waf 1）蛋白水平，并增加细胞周期蛋白A相关激酶活性。添加SNAP显示出相反的结果。结论：我们的数据表明，NO释放的心肌细胞在缺血/再灌注条件下发挥抗凋亡作用，通过调节细胞周期蛋白A相关激酶活性，通过p21（cip 1/waf 1）积累。(C)2003年欧洲心脏病学会。Elsevier B. V.出版，保留所有权利。
Objective: Ischemia/reperfusion in the heart causes myocardial apoptosis and increase nitric oxide (NO) production. We have reported that myocardial apoptosis is related to activation of cell cycle regulatory proteins. However, the role of nitric oxide (NO) in ischemia/reperfusion-induced apoptosis is still unclear. This study was designated to elucidate novel apoptosis mechanisms induced by ischemia/reperfusion, especially the interaction between NO and cell cycle regulators. Methods and results: Neonatal cardiomyocytes from 1- or 2-day-old Wistar rats were subjected to 1-h ischemia and then to reperfusion. The rate of cardiomyocyte apoptosis increased significantly after 24 h of reperfusion as evaluated by TUNEL analysis. NO increased 1.8-fold after 15 min of reperfusion in cardiomyocytes. After 36 h of reperfusion, the apoptosis rate was greatly increased by the NO synthetase inhibitor, Nitro-L-arginine methyl ester (L-NAME), and decreased by the NO donor of S-nitroso-N-acetylpenicillamine (SNAP). Immunoblot analysis showed that the protein levels of cyclin A accumulated in a time-dependent manner in response to ischemia/reperfusion, and L-NAME inhibited this response. Ischemia/reperfusion also increased the activity of cyclin A-associated kinase, and the apoptosis was inhibited by infection of dominant-negative cdk2 adenovirus. To clarify the involvement of p21(cip1/waf1) protein, which is the suppressor of cyclin A-associated kinase, we performed immunoblot analysis and examined its kinase activity. Treatment of cardiomyocytes with L-NAME suppressed the p21(cip1/waf1) protein level and increased the cyclin A-associated kinase activity. The addition of SNAP showed inverse results. Conclusion: Our data indicates that NO released from cardiomyocytes under condition of ischemia/reperfusion exerts an antiapoptotic effect by modulating cyclin A-associated kinase activity via p21(cip1/waf1) accumulation. (C) 2003 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.