摘要
目的研究异丙肾上腺素(Iso)刺激心肌细胞后是否激活PKCε,探讨直接被cAMP激活的交换蛋白(Epac)在其中的作用及其与ERK信号通路的关系。方法以原代培养的Wistar乳鼠心肌细胞为实验细胞,用β肾上腺素能受体(β-AR)激动剂Iso、Epac激动剂8-CPT、Epac抑制剂Epac R279K(DN)及腺病毒编码兔肌肉cAMP依赖的蛋白激酶抑制剂(Ad.PKI)和PKCε转位抑制肽处理细胞,Western blot检测细胞颗粒部分PKCε及pERK1/2,激光共聚焦显微镜观察PKCε转位情况。结果 Iso引起心肌细胞颗粒部分PKCε增加,PKCε转位于胞核周围。8-CPT增加细胞颗粒部分PKCε(P<0.05)。Epac R279K感染细胞后再予Iso处理,颗粒部分PKCε与对照组相比无增加。Ad.PKI未抑制Iso引起的颗粒部分PKCε增加(P<0.01)。PKCε转位抑制肽阻断Iso诱导的ERK1/2磷酸化。结论心肌细胞中β-AR通过Epac介导以不依赖于PKA的方式活化PKCε并诱导ERK1/2磷酸化。
Objective To evaluate PKCe translocation after β-adrenergic stimulation in isolated cardiomyocytes and the cross-talk with Epac and ERK phosphorylation. Methods Rat neonatal cardiomyocytes were cultured and treated with isoproterenol （Iso） and Epac activator 8-CPT. After infection with adenovirus coding for dominant negative （DN） form of Epac （ Epac R279K） and adenovirus coding for rabbit muscle cAMP-dependent protein kinase inhibitor （Ad. PKI）, cells were subjected to Iso. PKCe content was examined in the particulate fractions of cell lysates by Western blot. The localization of translocation of PKCe was examined by confocal microscope. After using of a specific PKCε inhibitor peptide, cells were treated with Iso, and pERK1/2 expression was assessed by Western blot. Results In cultured rat neonatal cardiomyoeytes it was shown that, in response to Iso, PKCε content was increased in particulate fractions of cell lysates, and PKCe was transloeated to the perinuelear area. After incubation with 8-CPT, PKCe content in particulate fractions was increased. Epac R279K blocked Iso-induced PKCe activation. After infected with Ad. PKI, PKCε content was not decreased in particulate fractions by Iso stimulation. Isoinduced ERK phosphorylation was blocked by the specific PKCε inhibitor peptide. Conclusions β-adrenergic stimulation activates PKCε in an Epac-dependent and PKA-independent fashion inducing ERK phosphorylation incardiomyocytes.
出处
《基础医学与临床》
CSCD
北大核心
2012年第8期948-952,共5页
Basic and Clinical Medicine
