Acetylcholine Release from Isolated Synaptic Vesicles Related to Ionic Permeability Changes: Continuous Detection with a Chemiluminescent Method

Abstract

The effect of ionic permeability changes on acetylcholine (ACh) release from isolated cholinergic synaptic vesicles of Torpedo was studied using a chemiluminescent method for continuous ACh detection. Vesicles rendered freely permeable to K+ by valinomycin lost most of their ACh content in K+ media, if the accompanying anion was permeant; Ach leakage probably occurred as the result of internal osmotic changes. Upon addition of ionophores that catalyze monovalent cation/H+ exchange (gramicidin D or a mixture of valinomycin plus protonophore FCCP [carbonylcyanide-p-trifluoromethoxyphenylhydrazone]), a rapid but transient ACh release was observed. Nigericin which also catalyzes K+/H+ exchange, had no effect on ACh release. The divalent cation ionophore A23187 [calcimycin] promoted ACh release only when Ca (and not Mg) was introduced into the external medium in a millimolar concentration range. As the simultaneous addition of the protonophore FCCP and A23187 decreased this Ca-dependent ACh leakage, a releasing effect of A23187 through Ca2+/H+ exchange is suspected. The present results emphasise the role of internal protons for ACh retention inside synaptic vesicles.