title: Acetylcholine synthesis and release in NIH3T3 cells coexpressing the high-affinity choline transporter and choline acetyltransferase.
abstract: 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 [(3)H]hemicholinium-3 (HC-3) and greater [(3)H]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.
authors.affiliation: Department of Pharmacology, Faculty of Pharmacy, Keio University, Tokyo, Japan. tfujii-dwc.doshisha.ac.jp
authors.forename: Takeshi
authors.initials: T
authors.lastname: Fujii
authors.forename: Manabu
authors.initials: M
authors.lastname: Masai
authors.forename: Hidemi
authors.initials: H
authors.lastname: Misawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Okuda
authors.forename: Yuki
authors.initials: Y
authors.lastname: Takada-Takatori
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Moriwaki
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Haga
authors.forename: Koichiro
authors.initials: K
authors.lastname: Kawashima
mesh_terms: D000109:Acetylcholine / Q000096:biosynthesis* / Q000378:metabolism; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D027682:Cation Transport Proteins / Q000235:genetics / Q000502:physiology*; D002794:Choline / Q000378:metabolism*; D002795:Choline O-Acetyltransferase / Q000378:metabolism; D006426:Hemicholinium 3 / Q000494:pharmacology; D008012:Lidocaine / Q000494:pharmacology; D008563:Membrane Lipids / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells / Q000201:enzymology / Q000378:metabolism; D027701:Organic Cation Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D011343:Procaine / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012964:Sodium / Q000378:metabolism
chemical_list: C501512:CHT1 protein, rat; D027682:Cation Transport Proteins; D008563:Membrane Lipids; D027701:Organic Cation Transport Proteins; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D006426:Hemicholinium 3; D011343:Procaine; D000584:Amiloride; D008012:Lidocaine; D012964:Sodium; D002795:Choline O-Acetyltransferase; D002794:Choline; D000109:Acetylcholine