β肾上腺素能受体活化蛋白激酶C诱导心肌细胞肥大  

β-Adrenergic Stimulation Activate Protein Kinase Cε Mediating Cardiohypertrophy

作　　者：李琳[1] 蔡红雁[1] 郭涛[1] 

机构地区：[1]昆明医学院第一附属医院心内科,云南昆明650032

出　　处：《中华高血压杂志》2010年第2期165-170,共6页Chinese Journal of Hypertension

摘　　要：背景通常认为蛋白激酶C(PKC)信号转导通路是由α肾上腺素受体介导的,但最近研究表明,环磷酸腺苷(cAMP)可活化Epac(cAMP活化的鸟嘌呤交换因子),从而激活磷脂酶Cε和PKC,提示β肾上腺素能受体(βAR)与PKC之间可能存在由Epac和磷脂酶C介导的信号传导通路。目的探讨心肌细胞中对异丙肾上腺素(Iso)刺激后PKCε的变化(活化和移位),探讨Epac在其中的作用。方法以原代培养Wistar乳鼠心肌细胞为实验模型,分别与βAR激动剂Iso(1μmol/L,1min)、Epac激动剂8CPT(1μmol/L,10min)、磷脂酶C抑制剂U73122(2μmol/L,30min)处理细胞,及Epac R279K(dominant negative,DN)病毒感染细胞,采用Western blot和共聚焦激光显微镜检测PKCε的活化转位情况。予特异性PKCε转位抑制肽转染细胞后,测定Iso处理48h后心肌细胞蛋白质含量和心肌细胞表面积。结果βAR刺激引起PKCε活化移位,细胞颗粒部分PKCε在与Iso孵育1min后开始增加,一直持续到15min,到30min时恢复,PKCε转位于细胞核周围。Iso和8CPT与细胞孵育后,颗粒部分PKCε表达增加(P<0.05)。EpacR279K(dominant negative,DN)病毒感染细胞,减少或下调Epac的表达后再予Iso处理,颗粒部分PKCε与GFP(绿色荧光蛋白)对照组相比没有增加。U73122(2μmol/L,30min)与心肌细胞孵育后,Iso刺激引起PKCε活化移位的作用消失,颗粒部分PKCε表达无明显增加(P>0.05),PKCε未发生核周转位。Iso引起PKCε活化导致心肌细胞肥大,对照组和Iso组细胞表面积分别为(1319.8±460.0)和(1874.4±479.5)μm2(P<0.05),蛋白质/DNA相对比值分别为0.64±0.05和0.98±0.13(P<0.05);予PKCε特异性抑制剂PKCε转位抑制肽转染细胞后,Iso组心肌细胞表面积和蛋白质/DNA含量与对照组比较无明显增加(均P>0.05)。结论心肌细胞中βAR刺激引起PKCε活化移位,Epac、磷脂酶C介导了PKCε的激活,心肌细胞肥大是Iso活化PKCε信号通路的效应之一。Background The protein kinase C （PKC） pathway is classically considered as independent of the β-adrenergic receptor （βAR） pathway.However, it was recently shown that the exchange factor Epac, exchange protein directly activated by cAMP, activates phospholipase Cε, suggesting that β AR stimulation may activate PKC. ObjectiveTo evaluate PKCε translocation after βAR stimulation in isolated cardiomyocytes, and its physiological effects. Methods Rat neonatal cardiomyocytes were cultured and treated with isoproterenol （Iso, 1 μmol/L for 1 min）, Epac activator 8CPT （1 μmol/L for 10 min）, and PLC inhibitor U73122 （2 μmol/L for 30 min）.After infected with a virus coding for the green fluorescent protein （GFP） and dominant negative （DN） form of Epac, cells were subjected to Iso.PKCε content was measured in the particulate fraction of cell lysates obtained by differential centrifugation.The localization of translocation of PKCε was studied by confocal microscopy.The physiological role of translocated PKCε was shown by the use of a specific PKCε inhibitor peptide and a scramble peptide （negative control） treated with Iso （10 μmol/L for 48 h）.ResultsIn response to isoproterenol, PKCε content was increased in particulate fractions （PF） of cell lysates and, PKCε was translocated to the perinuclear area determined by confcocal microscopy.The PKCε content in particulate fractions increased as early as 1 min after stimulation with isoproterenol.Compared with control, this change persisted for 15 min and return to control level at 30 min.After incubation with 8-CPT, an activator of Epac, both particulate PKCε content and PKCε perinulear accumulation increased. Epac R279K （dominant negative）, down-regulating of Epac expression, blocked isoproterenol-induced PKCε activation.Isoproterenol-inducedPKCεactivationwasblockedafterincubatingwithPLCinhibitorU73122 （2 μmol/L, 30 min）.The functional role of isoproterenol-induced PKCε activation was evidenced by the effect of

关 键 词：Β肾上腺素能受体 信号转导 蛋白激酶CΕ 磷脂酶C 心肌细胞肥大 

分 类 号：R96[医药卫生—药理学]