CAJ | 학술논문

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,本实验在大鼠心室肌源细胞和大鼠心脏中观察了α1-AR对AMPK的激活作用,利用Westernblot检测了AMPK-α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-α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β-AR的拮抗剂普萘洛尔(propranolol)不敏感,但是可以被α1-AR拮抗剂哌唑嗪(prazosin)所阻断.结果提示,α1-AR介导AMPK的磷酸化,但β-AR无此作用.AR激动剂持续灌注7 d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(P＜0.05,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.本文证明α1-AR激动剂可以增强AMPK的活性,揭示了心脏中α1-AR激动在调控AMPK活性方面的重要作用.深入了解α1-AR介导的AMPK激活可能在心衰治疗中具有重要的临床意义.
위료험증심장선감산활화단백격매(AMP-activated protein kinase,AMPK)시부위신상선소수체(adrenergic receptor,AR)적하유신호분자,본실험재대서심실기원세포화대서심장중관찰료α1-AR대AMPK적격활작용,이용Westernblot검측료AMPK-α총단백표체량급기172위소안산린산화수평.웅성Sprague-Dawley대서피하식입거갑신상선소(norepinephrine,NE),분신상선소(phenylephrine,PE)혹자용제재체[0.01%(W/V)유생소C]적완석미빙(osmotic minipump).NE혹PE이매소시0.2 mg/kg적속솔지속수주,7 d후용AMPK-α항체면역침정처리양본병측정AMPK적활성.결과현시,재세포수평,NE인기적AMPK린산화수평증고구유시간의뢰화제량의뢰특점,NE처리세포10 min후AMPK린산화수평체도최고봉;NE인기적저충효응대β-AR적길항제보내락이(propranolol)불민감,단시가이피α1-AR길항제고서진(prazosin)소조단.결과제시,α1-AR개도AMPK적린산화,단β-AR무차작용.AR격동제지속관주7 d후,AMPK적활성재NE(7.4배)화PE(6.0배)관주조교대조조현저증고(P＜0.05,n=6).PE지속관주조대서여대조조상비무명현적심기비후화조직섬유화변화.본문증명α1-AR격동제가이증강AMPK적활성,게시료심장중α1-AR격동재조공AMPK활성방면적중요작용.심입료해α1-AR개도적AMPK격활가능재심쇠치료중구유중요적림상의의.
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-α subunit and the phosphorylation level of Thr172-AMPK-αt 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-α 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 μmol/L NE after 10-minute treatment. This effect was insensitive to propranolol, a specific β-AR antagonist, but abolished by prazosin, an α1-AR antagonist, suggesting that α1-AR but not β-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 α1-AR stimulation enhances the activity of AMPK, indicating an important role of α1-AR stimulation in the regulation of AMPK in the heart.Understanding the activation of AMPK mediated by α1-AR might have clinical implications in the therapy of heart failure.