Department of Pharmacology, School of Medicine, Kanazawa Medical University, Uchinada, Ishikawa, Japan.
Division of Genomic Science and Microbiology, School of Medicine, University of Fukui, Eiheiji, Fukui, Japan.
J Neurochem. 2022 Feb;160(3):342-355. doi: 10.1111/jnc.15556. Epub 2021 Dec 17.
Cholinergic transmission underlies higher brain functions such as cognition and movement. To elucidate the process whereby acetylcholine (ACh) release is maintained and regulated in the central nervous system, uptake of [ H]choline and subsequent synthesis and release of [ H]ACh were investigated in rat striatal segments. Incubation with [ H]choline elicited efficient uptake via high-affinity choline transporter-1, resulting in accumulation of [ H]choline and [ H]ACh. However, following inhibition of ACh esterase (AChE), incubation with [ H]choline led predominantly to the accumulation of [ H]ACh. Electrical stimulation and KCl depolarization selectively released [ H]ACh but not [ H]choline. [ H]ACh release gradually declined upon repetitive stimulation, whereas the release was reproducible under inhibition of AChE. [ H]ACh release was abolished after treatment with vesamicol, an inhibitor of vesicular ACh transporter. These results suggest that releasable ACh is continually replenished from the cytosol to releasable pools of cholinergic vesicles to maintain cholinergic transmission. [ H]ACh release evoked by electrical stimulation was abolished by tetrodotoxin, but that induced by KCl was largely resistant. ACh release was Ca dependent and exhibited slightly different sensitivities to N- and P-type Ca channel toxins (ω-conotoxin GVIA and ω-agatoxin IVA, respectively) between both stimuli. [ H]ACh release was negatively regulated by M2 muscarinic and D2 dopaminergic receptors. The present results suggest that inhibition of AChE within cholinergic neurons and of presynaptic negative regulation of ACh release contributes to maintenance and facilitation of cholinergic transmission, providing a potentially useful clue for the development of therapies for cholinergic dysfunction-associated disorders, in addition to inhibition of synaptic cleft AChE.
胆碱能传递是大脑高级功能（如认知和运动）的基础。为了阐明乙酰胆碱（ACh）在中枢神经系统中释放的维持和调节过程，我们研究了大鼠纹状体段中[ H]胆碱的摄取以及随后的[ H]ACh 的合成和释放。用[ H]胆碱孵育通过高亲和力胆碱转运体-1有效摄取，导致[ H]胆碱和[ H]ACh 的积累。然而，在抑制乙酰胆碱酯酶（AChE）后，孵育[ H]胆碱主要导致[ H]ACh 的积累。电刺激和 KCl 去极化选择性释放[ H]ACh，但不释放[ H]胆碱。重复刺激会导致[ H]ACh 释放逐渐减少，而在抑制 AChE 下释放是可重复的。用囊泡 ACh 转运体抑制剂维拉米考尔处理后，[ H]ACh 释放被消除。这些结果表明，可释放的 ACh 不断从细胞质补充到可释放的囊泡胆碱能池，以维持胆碱能传递。电刺激引起的[ H]ACh 释放被河豚毒素消除，但 KCl 诱导的释放则在很大程度上不受影响。ACh 释放依赖于 Ca2+，并且对 N 型和 P 型钙通道毒素（ω-芋螺毒素 GVIA 和 ω-海葵毒素 IVA）的敏感性略有不同，这两种刺激物之间存在差异。ACh 释放受 M2 毒蕈碱和 D2 多巴胺能受体的负调控。本研究结果表明，在胆碱能神经元中抑制 AChE 以及抑制 ACh 释放的突触前负调节有助于维持和促进胆碱能传递，为开发与胆碱能功能障碍相关疾病的治疗方法提供了有用的线索，除了抑制突触间隙 AChE 之外。
