title: Release of acetylcholine from murine embryonic stem cells: effect of nicotinic and muscarinic receptors and blockade of organic cation transporter.
abstract: OBJECTIVE
  The non-neuronal cholinergic system is widely expressed in nature. The present experiments were performed to characterize the non-neuronal cholinergic system in murine embryonic stem cells (CGR8 cell line).
  
  
  METHODS
  CGR8 cells were cultured in gelatinized flasks with Glasgow's buffered minimal essential medium (Gibco, Germany). Acetylcholine was measured by HPLC combined with bioreactor and electrochemical detection.
  
  
  RESULTS
  CGR8 cells contained 1.08±0.12 pmol acetylcholine/10(6) cells (n=7) which was reduced to 0.50±0.06 pmol/10(6) cells (n=6; p<0.05) in the presence (4h) of 30μM bromoacetylcholine to block choline acetyltransferase. A time-dependent release of acetylcholine into the incubation medium was demonstrated, when cholinesterase activity was blocked by 10 μM physostigmine, with 97±13, 180±15 and 216±14 pmol being released from 65×10(6) cells after incubation periods of 2, 4 and 6h, respectively. The cumulative release corresponds to a fractional release rate of 2%/min. Blockade of nicotine or muscarine receptors did not significantly modulate the release of acetylcholine which was substantially reduced by 300 μM quinine (inhibitor of organic cation transporters). This inhibition showed considerable fading over the incubation period, indicating additional release mechanisms activated upon inhibition of organic cation transporters.
  
  
  CONCLUSIONS
  Murine embryonic stem cells contain and release significant amounts of acetylcholine. The high fractional release rate and the compensation for blocked organic cation transporters indicate that non-neuronal acetylcholine may play a functional role in the homeostasis of murine embryonic stem cells.
authors.affiliation: Institute of Pathology, University Medical Center, Johannes Gutenberg University Mainz, D-55101 Mainz, Germany. wessler@uni-mainz.de
authors.forename: Ignaz
authors.initials: I
authors.lastname: Wessler
authors.forename: Rosmarie
authors.initials: R
authors.lastname: Michel-Schmidt
authors.forename: Eva
authors.initials: E
authors.lastname: Dohle
authors.forename: Charles James
authors.initials: CJ
authors.lastname: Kirkpatrick
mesh_terms: D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D027682:Cation Transport Proteins / Q000037:antagonists & inhibitors*; D002460:Cell Line; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D002802:Cholinesterases / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000378:metabolism*; D051379:Mice; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D018733:Nicotinic Antagonists / Q000494:pharmacology; D010095:Oxotremorine / Q000494:pharmacology; D010830:Physostigmine / Q000494:pharmacology; D011803:Quinine / Q000494:pharmacology*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000378:metabolism*
chemical_list: D027682:Cation Transport Proteins; D002800:Cholinesterase Inhibitors; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D018733:Nicotinic Antagonists; D011976:Receptors, Muscarinic; D011978:Receptors, Nicotinic; D010095:Oxotremorine; D010830:Physostigmine; D011803:Quinine; D002802:Cholinesterases; D000109:Acetylcholine