Protein kinase C and clostridial neurotoxins affect discrete and related steps in the secretory pathway

1. The effects on catecholamine secretion of activation of protein kinase C and clostridial neurotoxins were examined in digitonin-permeabilized bovine adrenal chromaffin cells. 2. The enhancement by phorbol esters increased only the initial rate of secretion; later rates were unaffected. This enhancement was present over a wide range of Ca2+ concentrations and was elicited at 18 as well as at 27 degrees C. 3. Tetanus toxin inhibited both ATP-dependent and ATP-independent secretion, indicating that the tetanus toxin target is important during the final steps in the pathway. 4. Prior activation of protein kinase C by the phorbol ester 12-O-tetradecanoyl phorbol acetate rendered the primed state more sensitive to inhibition by tetanus toxin. The data indicate that a phosphorylated protein kinase C substrate is either identical to or closely associated with the tetanus toxin target protein at the final steps in the pathway. 5. The interaction between the effect of protein kinase activation and that of tetanus toxin suggests that protein kinase C activation does not stimulate a separate pathway of secretion but, rather, modulates the activity of the ongoing pathway. 6. The enhancement of secretion by protein kinase C is caused, at least in part, by a qualitative change in the characteristics of the primed state. This is indicated by the increased sensitivity of primed secretion to inhibition by tetanus toxin and a threefold increase in sensitivity of primed secretion to Ca2+. 7. Because activation of protein kinase C does not increase the later rates of secretion that are limited by ATP-dependent priming reactions, it is unlikely that enhancement of the maximal rate of secretion by TPA is due to an increased amount of the primed state. Instead, protein kinase C activation may increase the efficacy with which Ca2+ stimulates secretion at all Ca2+ concentrations.