Activation of protein kinase C is not required for exocytosis from bovine adrenal chromaffin cells. The effects of protein kinase C(19-31), Ca/CaM kinase II(291-317), and staurosporine.
We examined whether protein kinase C activation plays a modulatory or an obligatory role in exocytosis of catecholamines from chromaffin cells by using PKC(19-31) (a protein kinase C pseudosubstrate inhibitory peptide), Ca/CaM kinase II(291-317) (a calmodulin-binding peptide), and staurosporine. In permeabilized cells, PKC (19-31) inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion as much as 90% but had no effect on Ca2(+)-dependent secretion in the absence of phorbol ester. The inhibition of the phorbol ester-induced enhancement of secretion by PKC (19-31) was correlated closely with the ability of the peptide to inhibit in situ phorbol ester-stimulated protein kinase C activity. PKC(19-31) also blocked 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced phosphorylation of numerous endogenous proteins in permeabilized cells but had no effect on Ca2(+)-stimulated phosphorylation of tyrosine hydroxylase. Ca/CaM kinase II(291-317), derived from the calmodulin binding region of Ca/calmodulin kinase II, had no effect on Ca2(+)-dependent secretion in the presence or absence of phorbol ester. The peptide completely blocked the Ca2(+)-dependent increase in tyrosine hydroxylase phosphorylation but had no effect on TPA-induced phosphorylation of endogenous proteins in permeabilized cells. To determine whether a long-lived protein kinase C substrate might be required for secretion, the lipophilic protein kinase inhibitor, staurosporine, was added to intact cells for 30 min before permeabilizing and measuring secretion. Staurosporine strongly inhibited the phorbol ester-mediated enhancement of Ca2(+)-dependent secretion. It caused a small inhibition of Ca2(+)-dependent secretion in the absence of phorbol ester which could not be readily attributed to inhibition of protein kinase C. Staurosporine also inhibited the phorbol ester-mediated enhancement of elevated K(+)-induced secretion from intact cells while it enhanced 45Ca2+ uptake. Staurosporine inhibited to a small extent secretion stimulated by elevated K+ in the absence of TPA. The data indicate that activation of protein kinase C is modulatory but not obligatory in the exocytotoxic pathway.
Terbush
D R
DR
Department of Pharmacology, University of Michigan, Ann Arbor 48109-0626.
Holz
R W
RW
D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism*; D000470:Alkaloids / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002417:Cattle; D002478:Cells, Cultured; D004789:Enzyme Activation; D005089:Exocytosis / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000470:Alkaloids; D010455:Peptides; D011494:Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium