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 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.
Tomsig
J L
JL
Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, OH 45267-0576, USA.
Suszkiw
J B
JB
D000311:Adrenal Glands / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects; D002838:Chromaffin System / Q000166:cytology / Q000378:metabolism*; D007854:Lead / Q000502:physiology*; D009638:Norepinephrine / Q000378:metabolism*; D011493:Protein Kinase C / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D010738:Type C Phospholipases / Q000494:pharmacology
D007854:Lead; D011493:Protein Kinase C; D010738:Type C Phospholipases; D013755:Tetradecanoylphorbol Acetate; D009638:Norepinephrine