Release of acetylcholine from murine embryonic stem cells: Effect of nicotinic and muscarinic receptors and blockade of organic cation transporter

AimsThe 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).Main methodsCGR8 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.Key findingsCGR8 cells contained 1.08 ± 0.12 pmol acetylcholine/106 cells (n = 7) which was reduced to 0.50 ± 0.06 pmol/106 cells (n = 6; p < 0.05) in the presence (4 h) of 30 μM 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 × 106 cells after incubation periods of 2, 4 and 6 h, 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.SignificanceMurine 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.