Effect of protein kinase C activation on the release of [3H]acetylcholine in the presence of vesamicol.
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 alpha-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.
Barbosa
J
J
Departamento de Farmacologia, Universidade Federal de Minas Gerais, Belo Horizonte, Brasil.
Clarizia
A D
AD
Gomez
M V
MV
Romano-Silva
M A
MA
Prado
V F
VF
Prado
M A
MA
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D004558:Electric Stimulation; D004789:Enzyme Activation / Q000502:physiology; D005260:Female; D006624:Hippocampus / Q000187:drug effects / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D026901:Membrane Transport Proteins; D010766:Phosphorylation; D010880:Piperidines / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014316:Tritium; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; D026901:Membrane Transport Proteins; D010880:Piperidines; C493461:Slc18a3 protein, rat; D050494:Vesicular Acetylcholine Transport Proteins; D033921:Vesicular Transport Proteins; D014316:Tritium; C006189:vesamicol; D011493:Protein Kinase C; D000109:Acetylcholine; D013755:Tetradecanoylphorbol Acetate