Modulation of cyclic AMP levels by EGF and effect of cAMP on the mitogenicity of serum and EGF.

Epidermal Growth Factor (EGF) modulates cyclic AMP (cAMP) levels in non-neoplastic T51B rat liver epithelial cells induced by the $\beta$-adrenergic agonist isoproterenol (IPR) and by forskolin (FSK). In general the effect obtained depends upon whether pretreatment has taken place in the presence of serum (stimulation) or hepes (inhibition) on the duration of the pretreatment and on whether EGF was added before or after IPR or FSK. Activation of protein kinase C (PKC) by the tumor promoting phorbol ester, TPA, potentiated cAMP synthesis stimulated by FSK in both EGF pretreated cells and untreated cells although EGF pretreatment partially attenuated the effect of TPA. Cyclic AMP secretion is one method by which cells regulate the changes in intracellular cAMP. It was observed that cAMP secretion gradually increased with time when untreated and EGF pretreated cells were stimulated with FSK or IPR. The increase in cAMP secretion did not account for all the changes in intracellular cAMP. In fact, although all treatments resulted in some accumulation of external cAMP, there was no correlation between the level of internal cAMP obtained or the decrease in internal cAMP and the amount secreted. DNA synthesis was induced in serum deprived (0.2% BCS) cells by EGF or serum (10% BCS). This induction was inhibited by most cAMP elevating agents except isoproterenol and pertussis toxin. A combination of forskolin and the phosphodiesterase inhibitor, RO 201724 was most effective in inhibiting the DNA synthesis stimulated by EGF. Forskolin and RO 201724 inhibited DNA synthesis in a time dependent manner whereas maximum isoproterenol inhibition (approximately 22%) was observed regardless of time of isoproterenol addition. Genistein, a tyrosine kinase inhibitor, also inhibited EGF induced DNA synthesis. The inhibitory effect of genistein and cAMP elevating agents were not additive. These results suggest that EGF modulates cAMP levels and the mitogenic effect of serum and EGF is inhibited by cAMP elevating agents