Depolarization of mouse forebrain minces with veratridine and high K+: failure to stimulate the Ca2+ independent, spontaneous release of acetylcholine from the cytoplasm due to hydrolysis of the acetylcholine stored there.

Both high K+ and veratridine, depolarizing agents with different mechanisms of action, lowered the ACh content of the cytoplasmic (S3) fraction of mouse forebrain minces incubated in a Ca2+-free Krebs solution, without stimulating ACh release or altering the level of ACh in the vesicle-bound (P3) fraction. Veratridine increased the level of choline in the P3 fraction by the same amount as it reduced the level of ACh in the S3 fraction, and these changes did not occur in the presence of tetrodotoxin (TTX). Pretreatment of minces in normal Krebs increased the ACh but not the choline content of the S3 fraction. Following this expansion of the S3 ACh content, veratridine caused an even greater loss of S3 ACh, and increased the Ca2+-independent release of ACh slightly. Under these conditions, veratridine also stimulated the Ca2+ independent release of choline, and this increase exceeded that obtained for the Ca2+-independent release of ACh. Preincubation in normal Krebs with paraoxon did not alter the S3 ACh content after 5 min, but raised it by 78% after 30 min. Under the latter conditions of pretreatment, veratridine then stimulated the Ca2+-independent release of ACh even more, but did not stimulate the release of choline. These results suggest that depolarization of brain tissue does not facilitate the Ca2+-independent release of ACh from the cytoplasm because a portion of ACh stored there is hydrolyzed. When the cytoplasmic level of ACh is sufficiently elevated prior to depolarization, then some ACh escapes hydrolysis and is released independently of Ca2+. It is suggested that the depolarization-induced hydrolysis of cytoplasmic ACh may be mediated by an intraterminal form of AChE and may, in addition to the hydrolysis of extracellular ACh, provide substrate for the formation and release of ACh by the vesicle-bound fraction.