Norepinephrine induces apoptosis in neonatal rat endothelial cells via down-regulation of Bcl-2 and activation of beta-adrenergic and caspase-2 pathways.
OBJECTIVE
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 microM 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 beta- (beta2>beta>beta1) adrenergic antagonists (ICI-118,551>propranolol>atenolol) but was not affected by alpha1- or alpha2-adrenergic antagonists (prazosin or yohimbine).


CONCLUSIONS
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 beta-adrenergic (beta2>beta1) and caspase-2 pathways. beta-Adrenergic antagonists and caspases inhibitors may be useful in the prevention and management of NE-mediated endothelial apoptosis during heart failure.
Fu
Yun-Ching
YC
Department of Pediatrics, Taichung Veterans General Hospital, Taichung, Taiwan, ROC.
Chi
Ching-Shiang
CS
Yin
Sui-Chu
SC
Hwang
Betau
B
Chiu
Yung-Tsung
YT
Hsu
Shih-Lan
SL
D000319:Adrenergic beta-Antagonists / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects; D001262:Atenolol / Q000494:pharmacology; D015153:Blotting, Western / Q000379:methods; D053143:Caspase 2; D053148:Caspase 3; D053178:Caspase 6; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000378:metabolism* / Q000473:pathology; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019254:Genes, bcl-2; D009206:Myocardium / Q000378:metabolism*; D009638:Norepinephrine / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology; D011412:Propanolamines; D011433:Propranolol / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
D000319:Adrenergic beta-Antagonists; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D009842:Oligopeptides; D011412:Propanolamines; C110772:benzoylcarbonyl-aspartyl-glutamyl-valyl-aspartyl-fluoromethyl ketone; C427308:benzoylcarbonyl-valyl-aspartyl-valyl-alanyl-aspartyl-fluoromethyl ketone; C026777:ICI 118551; D001262:Atenolol; D045304:Cytochromes c; D011433:Propranolol; C505410:Casp3 protein, rat; C505518:Casp6 protein, rat; C506435:Casp9 protein, rat; D053143:Caspase 2; D053148:Caspase 3; D053178:Caspase 6; D053453:Caspase 9; D020169:Caspases; D009638:Norepinephrine