Carroll P T, Badamchian M, Craig P, Lyness W H
Brain Res. 1986 Sep 24;383(1-2):83-99. doi: 10.1016/0006-8993(86)90010-7.
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 (P3) by a Ca2+-dependent process, while simultaneously accelerating the breakdown of cytosolic (S3) [3H]ACh. A portion of the [3H]choline derived from the hydrolyzed S3 [3H]ACh was donated to the P3 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 Ca2+-dependent release of [3H]ACh or the hydrolysis of cytosolic [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 Ca2+-dependent release of [3H]ACh from the P3 fraction. Instead, it then stimulated the Ca2+-independent release of [3H]ACh from the S3 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 Ca2+-dependent release of labelled ACh either. Instead, veratridine stimulated the Ca2+-independent release of labelled ACh from the S3 fraction. NVP also abolished the veratridine-induced, Ca2+-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 S3 [3H]ACh during mince incubation. Veratridine depolarization of hippocampal minces stimulated the activity of a membrane-bound fraction of ChAT associated with the P3 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 S3 [3H]ACh. The veratridine-induced increase in membrane-bound ChAT activity was dependent on the presence of extracellular Ca2+ in the incubation medium.(ABSTRACT TRUNCATED AT 400 WORDS)
在“穿透性差”的乙酰胆碱酯酶（EC 3.1.1.7，AChE）抑制剂碘磷定存在的情况下，将大鼠海马匀浆用N-甲基-[3H]乙酰胆碱（[3H]ACh）进行装载，并测定去极化剂藜芦定对[3H]ACh和[3H]胆碱亚细胞储存及释放的影响。结果表明，藜芦定通过Ca2+依赖的过程刺激了粗囊泡部分（P3）中[3H]ACh的释放，同时加速了胞质（S3）中[3H]ACh的分解。水解后的S3 [3H]ACh衍生出的一部分[3H]胆碱被提供给P3部分用于[3H]ACh的形成和释放。当使用来自隔区损伤大鼠的海马匀浆进行相同实验时，藜芦定既不刺激[3H]ACh的Ca2+依赖释放，也不刺激胞质[3H]ACh的水解。用对氧磷（一种比碘磷定能更快穿透胆碱能神经末梢的AChE抑制剂）孵育对照海马匀浆，可防止藜芦定刺激P3部分中[3H]ACh的Ca2+依赖释放。相反，它随后刺激了S3部分中[3H]ACh的Ca2+非依赖释放。当匀浆与胆碱O-乙酰转移酶（EC 2.3.1.6，ChAT）抑制剂4-(1-萘基)乙烯基吡啶（NVP）孵育时，藜芦定也不再能够刺激标记ACh的Ca2+依赖释放。相反，藜芦定刺激了S3部分中标记ACh的Ca2+非依赖释放。NVP也消除了藜芦定诱导的总ACh的Ca2+依赖释放。在体外测试时，对氧磷和NVP都抑制了从大鼠脑制备的离子结合型ChAT的可逆反应，但对氧磷的效力远低于NVP，并且在低浓度下无法抑制该反应，而该低浓度在匀浆孵育期间可防止藜芦定诱导的S3 [3H]ACh分解。海马匀浆的藜芦定去极化刺激了与P3部分相关的膜结合型ChAT的活性，但在组织孵育期间，这部分ChAT对氧磷抑制的敏感性并未增加。匀浆的藜芦定去极化也增加了膜结合型AChE的活性，但该酶不受低浓度NVP的抑制，而低浓度NVP可防止藜芦定诱导的S3 [3H]ACh分解。藜芦定诱导的膜结合型ChAT活性增加依赖于孵育培养基中细胞外Ca2+的存在。（摘要截短至400字）
