血管紧张素Ⅱ经AT1受体和ERK1/2上调心肌细胞Cx43间隙连接

目的：探讨血管紧张素Ⅱ对培养新生鼠心室肌细胞Cx43间隙连接的影响及其机制。 方法： AngⅡ处理培养心肌细胞24 h。缬沙坦、PD98059在AngⅡ刺激细胞前1 h加到培养基中，对照组加等体积药物溶剂DMSO。用Western blotting分析、代谢标记和免疫沉淀测定、电镜观察心肌细胞Cx43表达、合成和间隙连接。 结果： Western blotting分析显示用10-9-10-6 mol/L AngⅡ刺激细胞24 h，Cx43的表达与对照组相比呈浓度依赖性增加；用AngⅡ 0.1 μmol/L刺激心肌细胞24 h，磷酸化ERK1/2(P-ERK1/2)活性高于对照组（P<0.01），AT1受体拮抗剂缬沙坦（1 μmol/L）能完全阻断AngⅡ增加P-ERK1/2活性；用AngⅡ 0.1 μmol/L刺激心肌细胞24 h，Cx43表达及P-ERK1/2活性均高于对照组(P<0.01)，ERK1/2激酶特异性抑制剂PD98059(1 μmol/L) 能阻断AngⅡ上调Cx43表达和增加P-ERK1/2活性。代谢标记和免疫沉淀测定显示AngⅡ处理组放射渗入Cx43的量明显高于对照组（P<0.01）,缬沙坦(1 μmol/L)能完全阻断AngⅡ增加放射渗入Cx43的量。电镜观察表明用AngⅡ 0.1 μmol/L刺激心肌细胞24 h，AngⅡ处理组细胞间隙连接数目和大小大于对照组（P<0.05）。 结论： AngⅡ通过AT1受体和ERK1/2促进培养心肌细胞合成Cx43，上调Cx43表达和增加间隙连接数目及大小。

Angiotensin Ⅱ up- regulates cardiomyocyte Cx43 gap expression via AT1 receptor and ERK1/2

AIM: To investigate the effect of angiotensinⅡon Cx43 gap junction in cultured neonatal rat cardiac myocytes and its mechanism. METHODS: The cardiomyocytes were treated with AngⅡ for 24 h, which were pretreated with valsartan or PD98059 for 60 min before AngⅡ treatment. The controls were treated with equal amount of DMSO. The Cx43 expression, synthesis and gap junction in cardiomyocytes were characterized by Western blotting, metabolic labeling and immunoprecipitation assay, and electron microscope. RESULTS: Western blotting analysis revealed that Cx43 content concentration-dependently increased in cells treated with 10-9-10-6 mol/L AngⅡfor 24 h. Phosphorylated extracellular signal regulated kinase (P-ERK) 1/2 activity increased in cells treated with 0.1 μmol/L AngⅡ for 24 h (P<0.01 vs control), which was inhibited by the presence of 1 μmol/L valsartan, an AT1-receptor blocker. Cx43 expression and P-ERK1/2 activity increased in cells treated with 0.1 μmol/L AngⅡ for 24 h (P<0.01 vs control), which was inhibited by the presence of 1 μmol/L PD98059, a specific inhibitor of ERK1/2 kinase. Metabolic labeling and immunoprecipitation assay revealed that AngⅡ increased the amount of radioactivity incorporated into Cx43, which was blocked by valsartan. Electron microscopy demonstrated increases in the number and size of gap junction profiles in cells exposed to 0.1 μmol/L AngⅡ for 24 h (P<0.05 vs control). CONCLUSIONS: AngⅡ facilitates Cx43 synthesis , up-regulates Cx43 expression and increases the number and size of gap junction profiles in cultured neonatal rat cardiac myocytes via AT1 receptor and ERK1/2.