Protein kinase C controls the priming step of regulated exocytosis in adrenal chromaffin cells.
1. To investigate the mechanism whereby protein kinase C enhances secretory function in adrenal chromaffin cells, we examined the effects of 12-O-tetradecanoylphorbor-13-acetate (TPA) on Ca(2+)-induced catecholamine release from digitonin-permeabilized cells, resolving the release into a MgATP-dependent priming step and a MgATP-independent Ca(2+)-triggered step. Treatment with TPA selectively potentiated the priming activity of MgATP, with little increase in the MgATP-independent release. The potentiation by TPA of the MgATP-dependent priming was blocked by [Ser25]protein kinase C(19-31), a specific substrate of protein kinase C. Gö 6976, an inhibitor selective for protein kinase C alpha and beta isoforms, also blocked the potentiation by TPA. These results suggest that activation of protein kinase C, probably the alpha isoform, potentiates the MgATP-dependent priming step. 2. The antibody raised against GAP-43, a known substrate of protein kinase C, also potentiated the MgATP-dependent priming. The effect of TPA and that of the anti-GAP-43 antibody were not additive. Calmodulin, which binds to GAP-43 and inhibits its phosphorylation by protein kinase C, abolished the effect of TPA. Thus, the present results suggest that protein kinase C potentiates MgATP-dependent priming, at least in part, through phosphorylation of GAP-43.
Misonou
H
H
Life Science Institute, Sophia University, Tokyo, Japan.
Ohara-Imaizumi
M
M
Murakami
T
T
Kawasaki
M
M
Ikeda
K
K
Wakai
T
T
Kumakura
K
K
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000313:Adrenal Medulla / Q000166:cytology / Q000502:physiology*; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis; D019922:GAP-43 Protein / Q000276:immunology / Q000502:physiology; D007211:Indoles / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D000906:Antibodies; D002227:Carbazoles; D002395:Catecholamines; D004791:Enzyme Inhibitors; D019922:GAP-43 Protein; D007211:Indoles; D010446:Peptide Fragments; C081021:Go 6976; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium