Activation of extracellular-signal regulated kinase by epidermal growth factor is potentiated by cAMP-elevating agents in primary cultures of adult rat hepatocytes

Abstract

We investigated the effects of α- and β-adrenergic agonists on epidermal growth factor (EGF)-stimulated extracellular-signal regulated kinase (ERK) isoforms in primary cultures of adult rat hepatocytes. Hepatocytes were isolated and cultured with EGF (20 ng/ml) and/or α 1-, α 2- and β 2-adrenergic agonists. Phosphorylated ERK isoforms (ERK1; p44 mitogen-activated protein kinase (MAPK) and ERK2; p42 MAPK) were detected by Western blotting analysis using anti-phospho-ERK1/2 antibody. The results show that EGF induced a 2.5-fold increase in ERK2-, but not ERK1-, phosphorylation within 3 min. This EGF-induced ERK2 activation was abolished by treatment with the EGF-receptor kinase inhibitor AG1478 (10 -7 M) or the MEK (MAPK kinase) inhibitor PD98059 (10 -6 M). The α 2-adrenergic and β 2-adrenergic agonists, UK14304 (10 -6 M) and metaproterenol (10 -6 M), respectively, had no effect in the absence of EGF, but metaproterenol significantly potentiated EGF-induced ERK2 phosphorylation. Moreover, the cell-permeable cAMP analog 8-bromo cAMP (10 -7 M), also potentiated EGF-induced ERK2 phosphorylation. The effects of these analogs were antagonized by the protein kinase A (PKA) inhibitor H-89 (10 -7 M). These results suggest that direct or indirect activation of PKA represents a positive regulatory mechanism for EGF stimulation of ERK2 induction. © 2011 Pharmaceutical Society of Japan.