Phorbol ester treatment induces the phosphorylation of SNAP-25 at Ser187 and the potentiation of Ca2+ induced dopamine (DA) and acetylcholine (Ach) release from PC12 cell. 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 Ser187. 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, 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
Keywords: neurotransmitter release; phorbol ester; phosphorylation; PKC; SNAP-25
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