Two Distinct Mechanisms Underlie the Stimulation of Neurotransmitter Release by Phorbol Esters in Clonal Rat Pheochromocytoma PC 12 Cells

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Phorbol ester treatment induces the phosphorylation of SNAP-25 at Ser¹⁸⁷ and the potentiation of Ca²⁺-induced dopamine (DA) and acetylcholine (Ach) release from PC 12 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 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, 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 1min after washing PDA in the presence of okadaic acid, conditions under which the phosphorylation of SNAP-25 persisted for at least 15min. 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 PC 12 cells.