Intracellular Ca²⁺ and phorbol esters synergistically inhibit internalization of epidermal growth factor in pancreatic acini

The association of ¹²⁵I-labelled epidermal growth factor (¹²⁵I-EGF) with mouse pancreatic acinar cells was inhibited by secretagogues which increase intracellular free Ca²⁺ concentrations. These agents included cholecystokinin-octapeptide (CCK₈) and the Ca²⁺ ionophore A23187. Inhibition by CCK₈ was blocked by lowering the incubation temperature from 37°C to 15° C. Moreover, in contrast with studies of intact acini, the binding of ¹²⁵I-EGF to isolated acinar membrane particles was not affected either by CCK₈, or by varying the level of Ca²⁺ in the incubation medium. These results indicated, therefore, that the inhibition of ¹²⁵I-EGF association with acinar cells required intact cells that are metabolically active. Since intact cells at 37°C are known to internalize bound EGF rapidly, acid washing was used to distinguish membrane-associated hormone from internalized hormone. Under steady-state conditions 86% of the ¹²⁵I-EGF associated with the acini was found to be internalized by this technique. When agents that increased intracellular Ca²⁺ were tested they all markedly reduced the amount of internalized hormone, whereas surface binding was only minimally affected. The phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), which is known to activate protein kinase C, a Ca²⁺-regulated enzyme, also inhibited the association of EGF with acini. This inhibition was similar to that induced by elevated intracellular Ca²⁺. To test whether these two inhibitory phenomena were related, the effects of TPA in combination with the Ca²⁺ ionophore A 23187 were examined. At low concentrations the effects were synergistic, whereas at high concentrations the maximal level of inhibition was not changed. We suggest therefore that elevated intracellular Ca²⁺ and phorbol esters may inhibit EGF internalization by a mechanism involving activation of protein kinase C.