Ray Balmiki, Bailey Jason A, Simon Jay R, Lahiri Debomoy K
Department of Psychiatry, Institute of Psychiatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA.
Curr Protoc Neurosci. 2012 Jul;Chapter 7:Unit 7.23. doi: 10.1002/0471142301.ns0723s60.
Acetylcholine (ACh) is the neurotransmitter used by cholinergic neurons at the neuromuscular junction, in parasympathetic peripheral nerve terminals, and in important memory-related circuits in the brain, and takes part in other critical functions. ACh is synthesized from choline and acetyl coenzyme A by the enzyme choline acetyltransferase (ChAT). The formation of ACh in cholinergic nerve terminals requires the transport of choline into cells from the extracellular space and the activity of ChAT. High-affinity choline uptake (HACU) represents the majority of choline uptake into the nerve terminal and is the acutely regulated, rate-limiting step in ACh synthesis. HACU can be differentiated from nonspecific choline uptake by inhibition of the choline transporter with hemicholinium. Several methods have been described previously to measure HACU and ChAT activity simultaneously in synaptosomes, but a well-documented protocol for cultured cells is lacking. We describe a procedure for simultaneous measurement of HACU and ChAT in cultured cells by simple radionuclide-based techniques. Using this procedure, we have quantitatively determined HACU and ChAT activity in cholinergically differentiated human neuroblastoma (SK-N-SH) cells. These simple methods can be used for neurochemical and drug discovery studies relevant to several disorders, including Alzheimer's disease, myasthenia gravis, and cardiovascular disease.
乙酰胆碱（ACh）是神经肌肉接头处、副交感神经外周神经末梢以及大脑中与记忆相关的重要回路中的胆碱能神经元所使用的神经递质，并参与其他关键功能。ACh由胆碱和乙酰辅酶A通过胆碱乙酰转移酶（ChAT）合成。胆碱能神经末梢中ACh的形成需要将胆碱从细胞外空间转运到细胞内以及ChAT的活性。高亲和力胆碱摄取（HACU）占神经末梢胆碱摄取的大部分，并且是ACh合成中急性调节的限速步骤。通过用半胱氨酸抑制胆碱转运体，可以将HACU与非特异性胆碱摄取区分开来。先前已经描述了几种在突触体中同时测量HACU和ChAT活性的方法，但缺乏一种针对培养细胞的详细记录的方案。我们描述了一种通过基于简单放射性核素的技术在培养细胞中同时测量HACU和ChAT的程序。使用该程序，我们已经定量测定了胆碱能分化的人神经母细胞瘤（SK-N-SH）细胞中的HACU和ChAT活性。这些简单的方法可用于与多种疾病相关的神经化学和药物发现研究，包括阿尔茨海默病、重症肌无力和心血管疾病。
