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生长激素诱导心肌细胞外信号调节酶激活及其上游调控 |
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袁国祥 顾亚平 小室一成 矢崎义雄
摘 要 目的和方法：用生长激素(GH)刺激培养的新生大鼠心肌细胞，用含MBP凝胶分离法测定细胞外信号调节酶(ERKs)活性，用Whatman Paper Filter法测定Raf-1活性，观察GH是否激活心肌细胞Raf-1-ERK级联反应。观察负显突变Ras质粒(D.N.Ras)与HA-ERK2质粒复合转染或有关抑制剂对GH诱导ERK激活的影响。结果：GH以时间和浓度依赖性方式激活心肌细胞ERK1和ERK2。ERK上游调节酶Raf-1活性也升高。过度表达D.N.Ras明显抑制GH诱导的心肌细胞HA-ERK2激活。Ras特导性抑制剂manumycin也显著阻断GH诱导的心肌细胞ERK激活。而分别用TPA和Calphostin C耗竭和抑制心肌细胞PKC，均未阻断GH对ERK的激活作用。结论：GH激活心肌细胞的Raf-1-ERK级联反应，这种激活依赖上游Ras，而不受PKC活性和含量变化的影响。
主题词 激素类，生长；信号传递；心肌
GH-induced ERK activation and upstream regulation in cardiac myocytes
YUAN Guo-Xiang, GU Ya-Ping, Issei KOMURO, Yoshio YAZAKI
Department of Pathophysiology, Nantong Medical College, Nantong(226001)
Abstract AIM and METHODS: Cultured neonatal cardiac myocytes were stimulated by growth hormone(GH), and then the activities of extracellular signal-regulated kinases(ERKs) were assayed with the method of MBP-containing gel and the activity of Raf-1 kinase was examined with the method of Whatman Paper Filter to examine whether GH activates Raf-1-ERK cascade in cardiac myocytes. Furthermore, the effects of Dominant-negative mutant Ras plasmids( D N. Ras) and HA-ERK2 plasmids cotransfection as well as relative inhibitors on GH-induced ERK activation were observed to explore the upstream pathway leading to ERK activation stimulated by GH. RESULTS: GH activated ERK1(42 kDa) and ERK2(44 kDa) in cardiac myocytes in a time-and a dose-dependent manners. The activity of Raf-1, an upstream regulating enzyme of ERKs, was also increased after GH stimulation. Overexpression of D.N.Ras significantly inhibited GH-induced HA-ERK2 activation in cardiac myocytes. Manumycin, a specific inhibitor of Ras, also strongly blocked GH-induced ERK activation in cardiac myocytes. The depletion and inhibition of PKC by long time exposure to PTA or pretreatment with calphostin C respectively had no effects on GH-induced ERK activation in cardiac myocytes.CONCLUSION: GH activated Raf-1-ERK [1] [2] [3] 下一页
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