© 2004 by European Society of Cardiology
Copyright © 2004, European Society of Cardiology
Norepinephrine induces apoptosis in neonatal rat endothelial cells via down-regulation of Bcl-2 and activation of β-adrenergic and caspase-2 pathways
aDepartment of Pediatrics, Taichung Veterans General Hospital, Taichung, Taiwan, ROC
bInstitute of Clinical Medicine, National Yang-Ming University, Taipei, Taiwan, ROC
cDepartment of Education and Research, Taichung Veterans General Hospital, Taichung, Taiwan, ROC
dDepartment of Pediatrics, Taipei Veterans General Hospital, Taipei, Taiwan, ROC
* Corresponding author. Department of Education and Research, Taichung Veterans General Hospital, 160, Section 3, Chung-Kang Road, Taichung 40705, Taiwan, ROC. Tel.: +886-4-23592525x4037; fax: +886-4-23592705. h2326{at}vghtc.vghtc.gov.tw
Objectives: Heart failure is associated with increased plasma norepinephrine (NE) and endothelial apoptosis. Recent reports have suggested that endothelial dysfunction is an important target for future therapies of heart failure. However, whether NE can induce endothelial apoptosis and its mechanism remains unknown. Methods: Endothelial cells from neonatal rat heart were treated with various concentrations of NE for different durations. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated nick end-labeling (TUNEL) and DNA fragmentation assays. Caspase activity was measured using specific fluorogenic substrates. Proteins of Bcl-2 family and cytochrome c were assayed by Western blotting. Results: NE induced endothelial apoptosis in a dose- and time-dependent manner. After treatment for 48 h, increasing NE concentration from 5, 10, 50, 100 to 200 µM resulted in 6±3%, 14±5%, 43±4%, 66±5%, and 89±6% apoptotic cells, respectively. The apoptosis was accompanied by down-regulation of Bcl-2 protein synthesis but not by cytosolic cytochrome c translocation. Caspase-2, -3, -6 and -9 were activated during apoptosis and caspase-2 inhibitor (Z-VDVAD-FMK) and caspase-3 inhibitor (Z-DEVD-FMK) significantly attenuated the apoptosis. Overexpression of Bcl-2 inhibited caspase activity and decreased the apoptosis. Moreover, the NE-mediated apoptotic effect was attenuated by β- (β2>β>β1) adrenergic antagonists (ICI-118,551>propranolol>atenolol) but was not affected by 
1- or 2-adrenergic antagonists (prazosin or yohimbine). Conclusion: Our study is the first report documenting that NE induces apoptosis in neonatal rat endothelial cells mainly through down-regulation of Bcl-2 protein and activation of the β-adrenergic (β2>β1) and caspase-2 pathways. β-Adrenergic antagonists and caspases inhibitors may be useful in the prevention and management of NE-mediated endothelial apoptosis during heart failure.
KEYWORDS Apoptosis; Bcl-2; Caspase; Endothelial cells; Neonatal rat; Norepinephrine