Veratridine-induced breakdown of cytosolic acetylcholine in rat hippocampal minces: An intraterminal form of acetylcholinesterase or choline O-acetyltransferase?

Rat hippocampal minces were loaded with N-methyl-[ 3H]acetylcholine ([ 3H]ACh) in the presence of the ‘poorly penetrating’ acetylcholinesterase (EC 3.1.1.7, AChE) inhibitor echothiophate and the effect of the depolarizing agent veratridine determined on the subcellular storage and release of [ 3H]ACh and [ 3H]choline. Results indicated that veratridine stimulated the release of [ 3H]ACh from a crude vesicular fraction (P 3) by a Ca 2+-dependent process, while simultaneously accelerating the breakdown of cytosolic (S 3) [ 3H]ACh. A portion of the [ 3H]choline derived from the hydrolyzed S 3[ 3H]ACh was donated to the P 3 fraction for [ 3H]ACh formation and release. When the identical experiment was done using hippocampal minces from septal lesioned rats, veratridine did not stimulate either the Ca 2+-dependent release of [ 3H]ACh or the hydrolysis of cystolic [ 3H]ACh. Incubation of control hippocampal minces with paraoxon, an AChE inhibitor which can penetrate cholinergic nerve terminals more rapidly than echothiophate, prevented veratridine from stimulating the Ca 2+-dependent release of [ 3H]ACh from the P 3 fraction. Instead, it then stimulated the Ca 2+-independent release of [ 3H]ACh from the S 3 fraction. When minces were incubated with the choline O-acetyltransferase (EC 2.3.1.6, ChAT) inhibitor 4-(1-naphthyl)vinyl pyridine (NVP), veratridine was no longer able to stimulate the Ca 2+-dependent release of labelled ACh either. Instead, veratridine stimulated the Ca 2+-independent release of labelled ACh from the S 3 fraction. NVP also abolished the veratridine-induced, Ca 2+-dependent release of total ACh. Both paraoxon and NVP inhibited the reversible reaction of ionically bound ChAT prepared from rat brain when tested in vitro, yet paraoxon was much less potent than NVP, and was unable to inhibit this reaction at the low concentration which prevented the veratridine induced breakdown of S 3[ 3H]ACh during mince incubation. Veratridine depolarization of hippocampal minces stimulated the activity of a membrane-bound fraction of ChAT associated with the P 3 fraction, but this fraction of ChAT did not become more sensitive to inhibition by paraoxon during tissue incubation. Veratridine depolarization of minces also increased the activity of membrane-bound AChE, but this enzyme was not inhibited by the low NVP concentration which prevented the veratridine-induced breakdown of S 3[ 3H]ACh. The veratridine-induced increase in membrane-bound ChAT activity w