Multisite Interactions Between Pb2+ and Protein Kinase C and Its Role in Norepinephrine Release from Bovine Adrenal Chromaffin Cells

: We investigated the interaction between Pb2+ and protein kinase C (PKC) in the Pb2+‐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 × 10−12M) interactions and inhibits the enzyme by competitive and noncompetitive interactions with nanomolar‐(Ki = 7.1 × 10−9M) and micromolar‐ (K′i = 2.8 × 10−7M) affinity sites, respectively. Activation of PKC by 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) in Ca2+‐deficient, Pb2+‐containing medium, enhances the Pb2+‐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 × 10−10 to 5.7 × 10−11M. The PKC inhibitors staurosporine and pseudosubstrate PKC (19–36) abolish the effect of TPA without affecting the Pb2+‐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 Pb2+‐triggered secretion.