Effect of Protein Kinase C Activation on the Release of [3H]Acetylcholine in the Presence of Vesamicol |
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Authors: | J. Barbosa Jr. A. D. Clarizia M. V. Gomez M. A. Romano-Silva V. F. Prado M. A. M. Prado |
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Affiliation: | Laboratório de Neurofarmacologia, Departamentos de Farmacologia,; Bioquímica-Imunologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brasil |
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Abstract: | Abstract: The present work tested whether pharmacological activation of protein kinase C (PKC) influences the release of [3H]-acetylcholine ([3H]ACh) synthesized in the presence of vesamicol, an inhibitor of the vesicular acetylcholine transporter (VAChT). Newly synthesized [3H]ACh was released from hippocampal slices by field stimulation (15 Hz) in the absence of vesamicol, but as expected [3H]ACh synthesized during exposure to vesamicol was not released significantly by stimulation. Treatment of slices with the PKC activator phorbol myristate acetate (PMA) decreased the inhibitory effect of vesamicol on [3H]ACh release. The effect of PMA was dose-dependent, was sensitive to calphostin C, a PKC-selective inhibitor, and could not be mimicked by α-PMA, an inactive phorbol ester. PMA did not alter the release of [3H]ACh in the absence of vesamicol, suggesting that the site of PKC action could be related to the VAChT. In agreement with this observation, immunoprecipitation of VAChT from 32P-labeled synaptosomes showed that phosphorylation occurs and that incorporation of 32P in the VAChT protein increases in the presence of PMA. We suggest that PKC alters the output of [3H]ACh formed in the presence of vesamicol and also provide circumstantial evidence for a role of phosphorylation of VAChT in this process. |
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Keywords: | Acetylcholine release Vesamicol Vesicular acetylcholine transporter Synaptic vesicles Protein kinase C |
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