Effect of epidermal growth factor on sodium-dependent L-alanine transport in LLC-PK1 cells

We evaluated the role of epidermal growth factor (EGF) in the regulation of L-alanine transport in LLC-PK1 renal epithelia. After 2 h of incubation, EGF had no significant effect on L-alanine uptake by LLC-PK1 cells. However, prolonged (16 h) incubation with 2 and 20 ng/ml of EGF resulted in significant increases in sodium-dependent L-alanine uptake as compared with controls. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA; 20 ng/ ml) caused a marked increase in sodium-dependent L-alanine uptake after both 2 and 16 h of incubation, and the treatment with TPA (20 ng/ml) EGF (20 ng/ml) for 16 h resulted in significant acceleration of the TPA-stimulated increase in L-alanine uptake by LLC-PK1 cells. Coincubation with H-7 (20 microM) inhibited both EGF- and TPA-stimulated increases in L-alanine uptake, and genistein (20 microg/ml) blocked the stimulatory effect of EGF in L-alanine transport to the control level. Furthermore, coincubation with cycloheximide (20 microg/ml) for 16 h inhibited both EGF- and TPA-stimulated increases in L-alanine transport to a great extent. The sodium- independent L-alanine uptake was not affected by treatment with either EGF or TPA. These results suggest that the activation of protein kinase C through tyrosine kinase activation plays a role in the EGF effect of stimulating L-alanine transport in LLC-PK1 cells and that the effect is mainly due to increased protein de novo synthesis which occurs after protein kinase C activation.