Effect of 2‐(4‐Phenylpiperidino)cyclohexanol (AH 5183) on the Veratridine‐Induced Increase in Acetylcholine Synthesis by Rat Hippocampal Tissue

Abstract

The intent of this study was to determine whether the drug 2-(4-phenylpiperidino)cyclohexanol (AH 5183 or vesamicol) might inhibit the veratridine-induced increase in acetylcholine (ACh) synthesis by reducing the veratridine-induced activation of a detergent-soluble choline-O-acetyltransferase (EC 2.3.1.6; ChAT) fraction associated with a vesicle-bound store of ACh. When minces of rat hippocampal tissue were loaded with [14C]choline and subsequently depolarized with veratridine, an increase in the synthesis of [14C]ACh occurred that could be abolished by L-AH 5183 (75 nM). When minces were depolarized with veratridine in the presence of L-AH 5183 (75 nM), the depolarization-induced activation of a detergent-soluble ChAT fraction associated with a vesicle-bound store of ACh was blocked. Conversely, the veratridine-induced activation of a water-soluble ChAT fraction believed to be cytosolic was not. AH 5183 also blocked the repletion of the vesicle-bound store with newly synthesized ACh following veratridine-induced depletion of ACh, a result that appeared to be mediated by an effect on the synthesis of ACh at the vesicular surface. These results suggest that veratridine depolarization of rat hippocampal nerve terminals stimulates the synthesis of ACh by activating a detergent-soluble fraction of ChAT closely associated with synaptic vesicle release sites. ACh synthesis and transport at the vesicular surface may be influenced by a common AH 5183-sensitive regulatory protein.

Suppression of In Vivo Neostriatal Acetylcholine Release by Vesamicol: Evidence for a Functional Role of Vesamicol Receptors in Brain

“…In the present study, vesamicol maximally suppressed the release of ACh by 52-55%, an effect that saturated at 3-10 HMofdrug. Comparably, in vitro studies have shown that similar or higher concentrations of vesamicol reduce the K+ or veratridine evoked release of ACh by 44-75% in cortical synaptosomes and slices (10 pM, Otero et al, 1985;Jope and Johnson, 1985), by up to 79% in mouse forebrain minces (17 pM, Carroll, 1985), by 77% in rat hippocampal slices (75 nM, Carroll and Ivy, 1988), and by as much as 90% in rat striatal slices (10 pM, R%n$ and Collier, 1986). Because the anticholinergic effect of vesamicol is stimulus dependent (Marshall, 1970;Whitton et al, 1986;Van der Kloot, 1986), the slightly greater efficacy of vesamicol observed in vitro may be related to the high depolarizing concentrations of K+ (30-60 mM) routinely applied in these studies.…”

Effect of Phospholipase C from Bacillus cereus on the Release of Membrane‐Bound Choline‐O‐Acetyltransferase from Rat Hippocampal Tissue

“…Male Sprague-Dawley rats were decapitated, and the brains were quickly removed and placed into an ice-cold normal Krebs solution (pH 7.4) vigorously bubbled with 95% 0 2 / 5% COz as described previously (Carroll and Ivy, 1988). In the case of the zinc experiments, the brains were placed into a 15 mM Tris buffer of the following composition: NaCI, 157 mM, KCl, 5.7 mM; MgCl2.6HzO, 1.5 mM; CaC12, 2.5 d, glucose, 1 1 .…”

“…Hippocampal minces (each incubated sample contained two hippocampi of -160-200 mg) were incubated in a normal Krebs solution (pH 7.4) for 30 rnin at 30°C in the absence or presence of varying concentrations of PLC from B. cereus (0.125, 1.25, and 12.5 U/ml). The activities ofChAT, AChE, and LDH and the amount of protein in the release media were determined as previously described (Benishin and Carroll, 1983;Carroll and Ivy, 1988).…”

“…The effect of PLC on the formation of [I4C]ACh was determined by incubating hippocampal minces in a normal Krebs solution for 30 rnin at 30°C in the absence or presence of varying concentrations of PLC (0.125, 1.25, and 12.5 U/ ml). Following the incubation, the minces were washed twice with 5 ml of 0.32 Msucrose and crude synaptosomal fractions (P2) prepared as previously described (Carroll and Ivy, 1988).…”

“…Although most of the choline-0-acetyltransferase (EC 2.3.1.6; ChAT) in central cholinergic nerve terminals is considered to be soluble (Fonnum, 1968), some also appears to be nonionically associated with membranes (Smith and Carroll, 1980;Benishin and Carroll, 1983;Peng et al, 1986;Rylett, 1989). Some investigators have suggested that particulate ChAT may play a critical role in the activation of acetylcholine (ACh) synthesis during depolarization (Carroll and Ivy, 1988;Myluta and Collier, 1989). Others have suggested that membrane-bound ChAT may serve as a cell-surface marker (Docherty and Bradford, 1986; Barochovsky et al, 1988).…”