MKP-1在血管紧张素Ⅱ导致心肌肥大反应中的调控作用

MKP-1 regulates the cardiomyocyte hypertrophic responses induced by angiotensin Ⅱ 

本研究主要从丝裂原活化蛋白激酶磷酸酶 1(MKP 1)角度 ,研究丝裂原活化蛋白激酶 (MAPK)信号途径在血管紧张素Ⅱ介导的新生大鼠心肌细胞肥大反应中的作用及调控机制。实验以心肌细胞蛋白合成速率、蛋白含量及细胞表面积作为心肌肥大反应的指标 ,以凝胶内MBP原位磷酸化测定MAPK活性 ,以免疫印迹法 (Westernboltting)分别测定MKP 1及磷酸化p44MAPK、p42MAPK蛋白表达。结果发现 :(1)AngⅡ (10 -7mol/L)处理 48h ,心肌细胞 3H 亮氨酸掺入率、蛋白含量及细胞表面积明显增加 ,AngⅡ增加 3H 亮氨酸掺入的作用可被血管紧张素Ⅱ 1型受体 (AT1受体 )拮抗剂CV11974(10 -6mol/L)明显抑制 (抑制 85 % ) ,被MAPK激酶 (MEK)特异性抑制剂PD0 980 5 9(5× 10 -5mol/L)部分抑制 (抑制 32 5 % ) ;(2 )CV11974或PD0 980 5 9可明显抑制AngⅡ介导的磷酸化MAPK蛋白表达及MAPK酶活性 (以γ 32 P ATP掺入表示 ) ;(3)以磷酸化MAPK蛋白表达反映MAPK活性 ,可见AngⅡ处理心肌细胞5min ,MAPK活性即开始增加 ,30min左右达到高峰 ,2h后基本恢复正常 ;而MKP 1蛋白表达 30min即见增加 ,持续 2h以上 ;(4 )用放线菌素D (actinomycinD)处理心肌细胞 30min可明显抑制MKP 1的表达 ,同时使AngⅡ致磷酸化MAPK蛋白表达时间延长至 2h以上。

The aim of this study was to determine the regulation by MKP 1 of MAPK activity and protein expression in cardiomyocyte hypertrophic response induced by Ang Ⅱ. Neonatal rat cardiomyocyte hypertrophic response was assayed by cell surface area, protein synthesis rate and protein content. MAPK activity was determined by an in gel kinase assay. Protein expression of MAPK and MKP 1 were detected by Western blotting. The results are as follows. (1) Ang Ⅱ induced promotion of ~ 3H leucine incorporation and increase in cell protein content and cell surface area in a dose dependent manner. Pretreatment with a selective AT 1 receptor antagonist CV11974 or a specific MEK inhibitor PD098059, cardiomyocyte hypertrophic response induced by Ang Ⅱ was inhibited by 85% and 32 5%, respectively. (2) After pretreatment with PD098059 or CV11974, AngⅡ induced increases in p44MAPK and p42MAPK protein expression and enzyme activity (expressed by γ 32 P ATP incorporation) were all inhibited obviously. (3) With treatment of myocytes by Ang Ⅱ for 5 min, MAPK activity determined by p44MAPK and p42MAPK protein expression began to increase, while MKP 1 protein expression was detected within 30 min and lasted more than 2 h following treatment with Ang Ⅱ. (4) Pretreatment of cardiomyocytes with actinomycin D (3 μg/ml) for 30 min inhibited MKP 1 protein expression, while p44MAPK and p42MAPK protein expression was still detected 120 min after AngⅡ treatment. The above results demonstrate that activation of MAPK plays an important role in Ang Ⅱ induced cardiomyocyte hypertrophic response in neonatal rat cardiomyocytes through MKP 1 mediated inactivation of p44MAPK and p42MAPK.[WT5HZ]