Two distinct mechanisms underlie the stimulation of neurotransmitter release by phorbol esters in clonal rat pheochromocytoma PC12 cells.
Phorbol ester treatment induces the phosphorylation of SNAP-25 at Ser(187) and the potentiation of Ca(2+)-induced dopamine (DA) and acetylcholine (Ach) release from PC12 cells. In order to evaluate the functional consequences of phosphorylation, quantitative analysis was carried out using an anti-phosphopeptide antibody that specifically recognizes SNAP-25 phosphorylated at Ser(187). DA and ACh release, assayed in low-K(+) as well as high-K(+) solution, increased by treating the cells with phorbol-12-myristate-13-acetate (PMA); however, the stimulation of high-K(+)-dependent release occurred at lower concentrations and with shorter exposures to PMA than that of the basal release in low-K(+)-solution. The PMA-induced phosphorylation of SNAP-25 did not correlate with the potentiation of high-K(+)-dependent neurotransmitter release. The potentiation of high-K(+)-dependent DA release by phorbol 12,13-diacetate (PDA), a water soluble phorbol ester, almost completely disappeared within 1 min after washing PDA in the presence of okadaic acid, conditions under which the phosphorylation of SNAP-25 persisted for at least 15 min. PMA-induced phosphorylation of SNAP-25 was inhibited by staurosporine, however, the potentiation of high-K(+)-dependent DA release was suppressed only partially. These results indicate that protein kinase activation does not account for a large fraction of the phorbol ester-induced potentiation of depolarization-dependent neurotransmitter release from PC12 cells.
Iwasaki
S
S
Department of Life Sciences (Biology), Graduate School of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153-8902, Japan.
Kataoka
M
M
Sekiguchi
M
M
Shimazaki
Y
Y
Sato
K
K
Takahashi
M
M
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004298:Dopamine / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008565:Membrane Proteins; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D016716:PC12 Cells / Q000187:drug effects* / Q000378:metabolism; D010703:Phorbol Esters / Q000494:pharmacology*; D010748:Phosphopeptides / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D051381:Rats; D019311:Staurosporine / Q000494:pharmacology; D050825:Synaptosomal-Associated Protein 25
D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010703:Phorbol Esters; D010748:Phosphopeptides; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; D019319:Okadaic Acid; C008874:phorbol-12,13-diacetate; D011493:Protein Kinase C; D019311:Staurosporine; D000109:Acetylcholine; D011188:Potassium; D004298:Dopamine