Multisite interactions between Pb2+ and protein kinase C and its role in norepinephrine release from bovine adrenal...

We investigated the interaction between Pb2+ and protein kinase C (PKC) in the Pb(2+)-induced release of norepinephrine (NE) from permeabilized adrenal chromaffin cells. Our analysis of endogenous PKC activity in permeabilized cells suggests that Pb2+ interacts with the adrenal enzyme at multiple sites. Pb2+ activates the enzyme through high-affinity (KA(Pb) = 2.4 x 10(-12) M) interactions and inhibits the enzyme by competitive and noncompetitive interactions with nanomolar-(Ki = 7.1 x 10(-9) M) and micromolar-(Ki = 2.8 x 10(-7) M) affinity sites, respectively. Activation of PKC by 12-O-tetradecanoyl-phorbol 13-acetate (TPA) in Ca(2+)-deficient, Pb(2+)-containing medium, enhances the Pb(2+)-induced NE release from permeabilized chromaffin cells by lowering the concentration of Pb2+ required for half-maximal activation of the secretory response from 7.5 x 10(-10) to 5.7 x 10(-11) M. The PKC inhibitors staurosporine and pseudosubstrate PKC (19-36) abolish the effect of TPA without affecting the Pb(2+)-induced secretion in the absence of TPA. These results indicate that (a) Pb2+ is a partial agonist of PKC, capable of both activating and inhibiting the enzyme and (b) synergistic activation of PKC by TPA and Pb2+ results in increased sensitivity of exocytosis to Pb2+ but is not obligatory for Pb(2+)-triggered secretion.