去甲肾上腺素通过下调Bcl-2以及激活β-肾上腺素能和半胱天冬酶-2途径诱导新生大鼠内皮细胞凋亡。

Norepinephrine induces apoptosis in neonatal rat endothelial cells via down-regulation of Bcl-2 and activation of beta-adrenergic and caspase-2 pathways.

作者信息

Fu Yun-Ching, Chi Ching-Shiang, Yin Sui-Chu, Hwang Betau, Chiu Yung-Tsung, Hsu Shih-Lan

机构信息

Department of Pediatrics, Taichung Veterans General Hospital, Taichung, Taiwan, ROC.

出版信息

Cardiovasc Res. 2004 Jan 1;61(1):143-51. doi: 10.1016/j.cardiores.2003.10.014.

DOI:10.1016/j.cardiores.2003.10.014

PMID:14732211

Abstract

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 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).

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 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.

摘要

目的

心力衰竭与血浆去甲肾上腺素（NE）水平升高及内皮细胞凋亡增加有关。最近的报告表明，内皮功能障碍是未来心力衰竭治疗的一个重要靶点。然而，NE是否能诱导内皮细胞凋亡及其机制仍不清楚。

方法

用不同浓度的NE处理新生大鼠心脏来源的内皮细胞不同时间。通过末端脱氧核苷酸转移酶介导的缺口末端标记法（TUNEL）和DNA片段化分析评估细胞凋亡。使用特异性荧光底物测量半胱天冬酶活性。通过蛋白质印迹法检测Bcl-2家族蛋白和细胞色素c。

结果

NE以剂量和时间依赖性方式诱导内皮细胞凋亡。处理48小时后，将NE浓度从5、10、50、100增加到200微摩尔/升，分别导致6±3%、14±5%、43±4%、66±5%和89±6%的凋亡细胞。细胞凋亡伴随着Bcl-2蛋白合成的下调，但不伴有细胞色素c从胞质转位。凋亡过程中半胱天冬酶-2、-3、-6和-9被激活，半胱天冬酶-2抑制剂（Z-VDVAD-FMK）和半胱天冬酶-3抑制剂（Z-DEVD-FMK）显著减轻细胞凋亡。Bcl-2的过表达抑制半胱天冬酶活性并减少细胞凋亡。此外，NE介导的凋亡作用被β-（β2>β>β1）肾上腺素能拮抗剂（ICI-118,551>普萘洛尔>阿替洛尔）减弱，但不受α1或α2肾上腺素能拮抗剂（哌唑嗪或育亨宾）影响。