生长激素诱导心肌细胞外信号调节酶激活及其上游调控

GH-induced ERK activation and upstream regulation in cardiac myocytes

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ＷｈａｔｍａｎＰａｐｅｒＦｉｌｔｅｒ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ＣａｌｐｈｏｓｔｉｎＣ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。

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-ERK 2 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 ERK 1(42 kDa) and ERK 2(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-ERK 2 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 cascade in cardiac myocytes was dependent on upstream Ras, but not affected by the changes of PKC activity and PKC quantity in cardiomyocytes.