Translocation of cytosolic acetylcholine into synaptic vesicles and demonstration of vesicular release.
The rate of translocation of newly synthesized acetylcholine (ACh) from the presynaptic cytosol of Torpedo electric organ nerve terminals into synaptic vesicles and the extent to which ACh release from these neurons is mediated by a vesicular mechanism were investigated. For this purpose the compound 2(4-phenylpiperidino)cyclohexanol (AH5183), which inhibits the active transport of ACh into isolated cholinergic synaptic vesicles, was employed. Preincubation of purified Torpedo nerve terminals (synaptosomes) with AH5183 does not affect the intraterminal synthesis of [3H]ACh but results in a marked inhibition (85%) of its Ca2+-dependent K+-evoked release. By contrast, the evoked release of the endogenous nonlabeled ACh is not affected by this compound. When AH5183 is added during radiolabeling, it causes a progressively smaller inhibition of [3H]ACh release which is completely abolished if the drug is added after the preparation has been labeled. These findings suggest that most of the newly synthesized synaptosomal [3H]ACh (85%) is released by a vesicular mechanism and that some [3H]ACh (15%) may be released by a different process. The translocation of cytosolic [3H]ACh into the synaptic vesicles was monitored by determining the time course of the loss of susceptibility of [3H]ACh release to AH5183. It was found not to be coupled kinetically to [3H]ACh synthesis and to lag behind it. The nature of the intraterminal processes underlying this lag is discussed.
Michaelson
D M
DM
Burstein
M
M
Licht
R
R
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D003600:Cytosol / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism*; D007700:Kinetics; D009467:Neuromuscular Depolarizing Agents / Q000494:pharmacology; D010622:Phencyclidine / Q000031:analogs & derivatives / Q000494:pharmacology; D010880:Piperidines; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism*; D014101:Torpedo
D009467:Neuromuscular Depolarizing Agents; D010880:Piperidines; C006189:vesamicol; D010622:Phencyclidine; D000109:Acetylcholine