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.