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#alpha#_(1)--肾上腺素受体激活大鼠心脏腺苷酸活化蛋白激酶

徐明, 赵燕婷, 宋峣, 郝天袍, 吕志珍, 韩启德, 王世强, 张幼怡

北京大学第三医院血管医学研究所,分子心血管教育部重点实验室.北京 100083；北京大学生命科学学院,生物膜与膜生物工程国家重点实验室.北京 100087

摘要

为了验证心脏腺苷酸活化蛋白激酶(AMP--activated protein kinase,AMPK)是否为肾上腺素受体(adrenergic receptor,AR)的下游信号分子,该实验在大鼠心室肌源细胞和大鼠心脏中观察了#alpha#_(1)--AR对AMPK的激活作用,利用Westernblot检测了AMPK--#alpha#总蛋白表达量及其172位苏氨酸磷酸化水平。雄性Sprague--Dawley大鼠皮下植入去甲肾上腺素(norepinephrine,NE),苯肾上腺素(phenylephrine,PE)或者溶剂载体[0.01%(W/V)维生素C]的缓释微泵(osmotic minipump)。NE或PE以每小时0.2 mg/kg的速率持续输注,7 d后用AMPK--#alpha#抗体免疫沉淀处理样本并测定AMPK的活性。结果显示,在细胞水平,NE引起的AMPK磷酸化水平增高具有时间依赖和剂量依赖特点,NE处理细胞10 min后AMPK磷酸化水平达到最高峰;NE引起的这种效应对#beta#--AR的拮抗剂普萘洛尔(propranolol)不敏感,但是可以被#alpha#_(1)--AR拮抗剂哌唑嗪(prazosin)所阻断。结果提示,#alpha#_(1)--AR介导AMPK的磷酸化,但#beta#--AR无此作用。AR激动剂持续灌注7d后,AMPK的活性在NE(7.4倍)和PE(6.0倍)灌注组较对照组显著增高({sl P}＜0.05,{sl n}=6)。PE持续灌注组大鼠与对照组相比无明显的心肌肥厚和组织纤维化变化。该文证明#alpha#_(1)--AR激动剂可以增强AMPK的活性,揭示了心脏中#alpha#_(1)--AR激动在调控AMPK活性方面的重要作用。深入了解#alpha#_(1)--AR介导的AMPK激活可能在心衰治疗中具有重要的临床意义。

关键词： 肾上腺素受体; 腺苷酸活化蛋白激酶; 心脏

#alpha#_(1)--adrenergic receptors activate AMP--activated protein kinase in rat hearts

Xu Ming, Zhao Yanting, Song Yao, Hao Tianpao, Lu Zhizhen, Han Qide, Wang Shiqiang, Zhang Youyi

Institute of Vascular Medicine, Peking University Third Hospital and Key Laboratory of Molecular Cardiovascular Sciences,Ministry of Education.Beijing 100083；China

Abstract

To test the hypothesis that AMP-activated protein kinase (AMPK) is possibly the downstream signaling molecule of certain subtypes of adrenergic receptor (AR) in the heart, we evaluated AMPK activation mediated by ARs in H9C2 cells, a rat cardiac source cell line, and rat hearts. The AMPK-#alpha# subunit and the phosphorylation level of Thr~(172)-AMPK-#alpha# subunit were subjected to Western blot analysis. Osmotic minipumps filled with norepinephrine (NE), phenylephrine (PE) or vehicle [0.01% (W/V) vitamin C solution]were implanted into male Sprague-Dawley rats subcutaneously. The pumps delivered NE or PE continuously at the rate of 0.2 mg/kg per hour. After 7-day infusion, the activity of AMPK was examined following immunoprecipitation with anti-AMPK-#alpha# antibody. At the cellular level, we found that NE elevated AMPK phosphorylation level in a dose- and time-dependent manner, with the maximal effect at 10 #mu#mol/L NE after 10-minute treatment. This effect was insensitive to propranolol, a specific #beta#-AR antagonist, but abolished by prazosin, an #alpha#_(1)-AR antagonist, suggesting that #alpha#_(1)-AR but not #beta#-AR mediated the phosphorylation of AMPK. Moreover, the results from rat models of 7-day-infusion of AR agonists demonstrated that the activity of AMPK was significantly higher in NE (7.4-fold) and PE (6.0-fold) infusion groups than that in the vehicle group (P＜0.05, n=6). On the other hand, no obvious cardiac hypertrophy and tissue fibrosis changes were observed in PE-infused rats. Taken together, our results demonstrate that #alpha#_(1)-AR stimulation enhances the activity of AMPK, indicating an important role of #alpha#_(1)-AR stimulation in the regulation of AMPK in the heart.Understanding the activation of AMPK mediated by #alpha#_(1)-AR might have clinical implications in the therapy of heart failure.

Key words: adrenergic receptor;AMP-activated protein kinase;Heart

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引用本文：

徐明, 赵燕婷, 宋峣, 郝天袍, 吕志珍, 韩启德, 王世强, 张幼怡. #alpha#_(1)--肾上腺素受体激活大鼠心脏腺苷酸活化蛋白激酶[J]. 生理学报 2007; 59 (2): 175-182.

Xu Ming, Zhao Yanting, Song Yao, Hao Tianpao, Lu Zhizhen, Han Qide, Wang Shiqiang, Zhang Youyi. #alpha#_(1)--adrenergic receptors activate AMP--activated protein kinase in rat hearts. Acta Physiol Sin 2007; 59 (2): 175-182 (in Chinese with English abstract).