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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 It is generally accepted that the protein kinase C (PKC) signaling pathway is mediated by alpha adrenergic receptors, but recent studies have shown that cyclic AMP (cAMP) activates Epac (cAMP-activated guanine exchange factor) and thus Activation of phospholipase Cε and PKC suggests that there may be a signaling pathway mediated by Epac and phospholipase C between β adrenergic receptor (βAR) and PKC. Objective To investigate the changes (activation and translocation) of PKCε after isoprenaline (Iso) stimulation in cardiomyocytes and to explore the role of Epac in them. Methods Primary cultured Wistar rat cardiomyocytes were treated with βAR agonist Iso (1μmol / L, 1min), Epac agonist 8CPT (1μmol / L, 10min), phospholipase C inhibitor U73122 , 30 min), and Epac R279K (dominant negative, DN) virus. Western blot and confocal laser microscopy were used to detect the activation and translocation of PKCε. After transfection with specific PKCε translocation inhibitory peptide, the protein content of cardiomyocytes and the surface area of cardiomyocytes were measured 48h after Iso treatment. Results The activation of PKCε was induced by βAR stimulation. Part of PKCε of cellular granules began to increase after 1 min incubation with Iso, and continued until 15 min, restored to 30 min, and PKCε translocated around the nucleus. After incubation with Iso and 8CPT, the expression of PKCε in granule part increased (P <0.05). The EpacR279K (DN) virus infected cells, and the expression of Epac was decreased or down-regulated. The cells were treated with Iso, and the fraction of PKCε particles did not increase compared with GFP (green fluorescent protein) control group. After U73122 (2μmol / L, 30min) incubated with cardiomyocytes, the effect of Iso stimulation on activation of PKCε disappeared, and the expression of PKCε in granule part had no significant increase (P> 0.05). PKCε did not translocate. Iso-induced PKCε-induced cardiomyocyte hypertrophy. The cell surface area was (1319.8 ± 460.0) and (1874.4 ± 479.5) μm2 in the control group and the Iso group, respectively. The relative protein / DNA ratios were 0.64 ± 0.05 and 0.98 ± 0.13 (P <0.05). After transfection with PKCε inhibitor of PKCε, the myocardial cell surface area and protein / DNA content in Iso group showed no significant increase compared with the control group (all P> 0.05). CONCLUSION: βAR stimulation in cardiomyocytes causes activation of PKCε. Epac and phospholipase C mediate the activation of PKCε. Cardiomyocyte hypertrophy is one of the effects of Iso activation on PKCε signaling pathway.