Effect of Protein Kinase C Activation on the Release of [³H]Acetylcholine in the Presence of Vesamicol

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.

The Vesicular Acetylcholine Transporter Is Required for Neuromuscular Development and Function

“…3 H]choline and monitored the release of labeled neurotransmitter as previously described (2,26,27,32). VAChT del/del mice are capable of producing ACh (see Fig.…”

“…A number of previously reported studies suggested that distinct pathways of ACh secretion might exist at cholinergic synapses (56,60,61,64). Moreover, vesamicol-independent ACh release, presumably from synaptic vesicles, can be detected in response to pharmacological treatments (2,7,10,11,46). Hence, if VAChT-independent mechanisms of ACh release have functional significance, they might partially compensate for the lack of the transporter in at least some of its physiological roles.…”

Elimination of the Vesicular Acetylcholine Transporter in the Striatum Reveals Regulation of Behaviour by Cholinergic-Glutamatergic Co-Transmission

“…Slices were obtained from the striatum and hippocampus of control and test mice, labelled with [ 3 H]methyl-choline, and the release of labelled ACh was determined essentially as described [73] except that 33 mM KCl was used as a depolarizing stimuli.…”

Trafficking of the vesicular acetylcholine transporter in SN56 cells: a dynamin‐sensitive step and interaction with the AP‐2 adaptor complex

“…Afterwards, beads were precipitated and washed 5 times with homogenization buffer and after the last wash beads were resuspended in SDS‐sample buffer. Proteins were resolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred to nitrocellulose membrane as described (Barbosa et al . 1997).…”