Nitric oxide inhibits ischemia/reperfusion-induced myocardial apoptosis by modulating cyclin A-associated kinase activity.
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.


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.


CONCLUSIONS
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.
Maejima
Yasuhiro
Y
Department of Cardiovascular Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8519, Japan.
Adachi
Susumu
S
Ito
Hiroshi
H
Nobori
Kiyoshi
K
Tamamori-Adachi
Mimi
M
Isobe
Mitsuaki
M
D000818:Animals; D017209:Apoptosis; D016203:CDC2 Protein Kinase / Q000378:metabolism; D002478:Cells, Cultured; D019925:Cyclin A / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D015428:Myocardial Reperfusion Injury / Q000378:metabolism* / Q000473:pathology*; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology*; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D051381:Rats; D017208:Rats, Wistar; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D013997:Time Factors
C493958:Cdkn1a protein, rat; D019925:Cyclin A; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D020030:Nitric Oxide Donors; D009569:Nitric Oxide; D026423:S-Nitroso-N-Acetylpenicillamine; D019001:Nitric Oxide Synthase; D016203:CDC2 Protein Kinase; D019331:NG-Nitroarginine Methyl Ester