Vesamicol, an inhibitor of acetylcholine vesicle packaging, increases synaptophysin phosphorylation in rat cortical synaptosomes

Abstract Vesamicol (AH5183) is an inhibitor (IC 50 , 50 nM) of acetylcholine (ACh) vesicle packaging. Vesamicol increases the phosphorylation pattern of synaptophysin (p38), identified as a vesicle-specific phosphoprotein involved in vesicle-mediated neurotransmitter release. Percoll fractionation of the rat cortex yielded a cholinergic-enriched synaptosomal Fraction 4. Fraction 4 contained the highest enrichment of cholineacetyl-transferase activity (86 ± 4,6 μmole AcCh/g protein/hr.) in the Percoll gradient. Fraction 4 demonstrated oxygen consumption (108 ± 23.4 nmole/mg protein), levels of adenosine triphosphate, ATP, (10.29 ± 0.45 nmole/mg protein) and adenosine diphosphate, ADP, (10.54 ± 2.72 nmole/mg protein), energy potential (ATP/[ADP][Pi], (0.49) phosphate uptake (65–80 nmoles phosphate/mg tissue), 32 pi labelling (130 ± 12 × 10 5 DPM/mg tissue; 74 ± 9.8 × 10 2 nmoles phosphate/mg tissue). Synaptophysin was identified by Western blotting and confirmed by qualitative immunoprecipitation. Synaptophysin phosphorylation was confirmed by autoradiograph. Synaptophysin phosphorylation increased (225%) in the presence of vesamicol (ED50, 1 nM) in Fraction 4. Vesamicol (50 nM) and vanadate (54 μM) were compared for their effects on synaptophysin. This study suggests that during the inhibition of acetylcholine packaging by vesamicol that synaptophysin is phosphorylated. Therefore, the phosphorylation and dephosphorylation of synaptophysin may be involved in the transport of acetylcholine in or out of the synaptic vesicle.