Acetylcholine Synthesis and Release in NIH3T3 Cells Coexpressing the High-Affinity Choline Transporter and Choline Acetyltransferase
Acetylcholine Synthesis and Release in NIH3T3 Cells Coexpressing the High-Affinity Choline Transporter and Choline Acetyltransferase
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DOI：
10.1002/jnr.22117
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发表时间：
2009-10-01
影响因子：
4.2
通讯作者：
Kawashima, Koichiro
中科院分区：
文献类型：
--
作者：
Fujii, Takeshi;Masai, Manabu;Kawashima, Koichiro
已知乙酰胆碱（ACh）是中枢和外周神经系统中的关键神经递质，但它也在各种非神经元组织和细胞中产生，包括淋巴细胞、胎盘、羊膜、血管内皮细胞、角质形成细胞以及消化道和呼吸道中的上皮细胞。为了研究高亲和力胆碱转运蛋白（choline transporter，CHT 1）对胆碱能神经元和非神经元细胞合成乙酰胆碱（ACh）的作用，我们将大鼠CHT 1 cDNA转染表达小鼠胆碱乙酰转移酶（choline acetyltransferase，ChAT）的小鼠成纤维细胞系NIH 3 T3 ChAT 112-1，建立了稳定表达CHT 1和ChAT的NIH 3 T3 ChAT 112-1细胞系。与NIH 3 T3 ChAT 103-1细胞（CHT 1阴性对照细胞系）相比，NIH 3 T3 ChAT 112-1细胞显示CHT 1抑制剂[H-3]半胆碱-3（HC-3）的结合增加，[H-3]胆碱摄取和ACh合成增加。HC-3显着抑制NIH 3 T3 ChAT 112-1细胞的ACh合成，但不影响NIH 3 T3 ChAT 103-1细胞的ACh合成。乙酰胆碱合成在NIH 3 T3 ChAT 112-1细胞也减少阿米洛利，有机阳离子转运蛋白（OCT）的抑制剂参与低亲和力胆碱摄取，普鲁卡因和利多卡因，两种局部麻醉剂，抑制质膜磷脂代谢。这些结果表明，CHT 1在NIH 3 T3 ChAT 112-1细胞中的乙酰胆碱合成中起着关键作用，并且在胆碱能神经元和非神经元胆碱能细胞（如淋巴细胞）中，由OCT摄取或来自质膜的胆碱也用于乙酰胆碱合成。(C)2009 wiley-Liss，Inc.
Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, but it is also produced in a variety of non-neuronal tissues and cells, including lymphocytes, placenta, amniotic membrane, vascular endothelial cells, keratinocytes, and epithelial cells in the digestive and respiratory tracts. To investigate contribution made by the high-affinity choline transporter (CHT1) to ACh synthesis in both cholinergic neurons and nonneuronal cells, we transfected rat CHT1 cDNA into NIH3T3ChAT cells, a mouse fibroblast line expressing mouse choline acetyltransferase (ChAT), to establish the NIH3T3ChAT 112-1 cell line, which stably expresses both CHT1 and ChAT. NIH3T3ChAT 112-1 cells showed increased binding of the CHT1 inhibitor [H-3]hemicholinium-3 (HC-3) and greater [H-3]choline uptake and ACh synthesis than NIH3T3ChAT 103-1 cells, a CHT1-negative control cell line. HC-3 significantly inhibited ACh synthesis in NIH3T3ChAT 112-1 cells but did not affect synthesis in NIH3T3ChAT 103-1 cells. ACh synthesis in NIH3T3ChAT 112-1 cells was also reduced by amiloride, an inhibitor of organic cation transporters (OCTs) involved in low-affinity choline uptake, and by procaine and lidocaine, two local anesthetics that inhibit plasma membrane phospholipid metabolism. These results suggest that CHT1 plays a key role in ACh synthesis in NIH3T3ChAT 112-1 cells and that choline taken up by OCTs or derived from the plasma membrane is also utilized for ACh synthesis in both cholinergic neurons and nonneuronal cholinergic cells, such as lymphocytes. (C) 2009 wiley-Liss, Inc.