Maejima Yasuhiro, Adachi Susumu, Ito Hiroshi, Nobori Kiyoshi, Tamamori-Adachi Mimi, Isobe Mitsuaki
Department of Cardiovascular Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8519, Japan.
Cardiovasc Res. 2003 Aug 1;59(2):308-20. doi: 10.1016/s0008-6363(03)00425-5.
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.
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.
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.
心脏缺血/再灌注会导致心肌细胞凋亡并增加一氧化氮（NO）的生成。我们曾报道心肌细胞凋亡与细胞周期调节蛋白的激活有关。然而，一氧化氮（NO）在缺血/再灌注诱导的凋亡中的作用仍不清楚。本研究旨在阐明缺血/再灌注诱导的新型凋亡机制，尤其是NO与细胞周期调节因子之间的相互作用。
对1日龄或2日龄Wistar大鼠的新生心肌细胞进行1小时缺血，然后再灌注。通过TUNEL分析评估，再灌注24小时后心肌细胞凋亡率显著增加。心肌细胞再灌注15分钟后，NO增加了1.8倍。再灌注36小时后，一氧化氮合酶抑制剂L-硝基精氨酸甲酯（L-NAME）使凋亡率大幅增加，而NO供体S-亚硝基-N-乙酰青霉胺（SNAP）则使其降低。免疫印迹分析表明，细胞周期蛋白A的蛋白水平在缺血/再灌注后呈时间依赖性积累，L-NAME抑制了这种反应。缺血/再灌注还增加了细胞周期蛋白A相关激酶的活性，显性负性cdk2腺病毒感染可抑制凋亡。为了阐明细胞周期蛋白A相关激酶的抑制剂p21（cip1/waf1）蛋白的作用，我们进行了免疫印迹分析并检测了其激酶活性。用L-NAME处理心肌细胞可抑制p21（cip1/waf1）蛋白水平并增加细胞周期蛋白A相关激酶活性。添加SNAP则显示出相反的结果。
我们的数据表明，在缺血/再灌注条件下心肌细胞释放的NO通过p21（cip1/waf1）积累调节细胞周期蛋白A相关激酶活性，从而发挥抗凋亡作用。
