Background: Ethanol abuse has been shown to up-regulate muscarinic acetylcholine receptors (mAChRs) in the central nervous system, both in vivo and in vitro, but the detailed mechanisms for this action are not known. Recent studies suggest that the actions of ethanol in several biological systems involve nitric oxide (NO) pathways. The aim of this work was therefore to determine whether the effects of long-term ethanol treatment on mAChRs involved actions upon NO pathways in a well defined cell culture system.
Methods: Human neuroblastoma SH-SY5Y cells were used as an in vitro model system. The time- and dose-dependent effects of ethanol on endogenous NO production, as well as the effects of the NO donor 3-morpholino-sydnonimine-chloride and of the neuronal NO synthase (nNOS) inhibitor N(omega)-propyl-L-arginine on mAChR number and on ethanol effects upon these receptors, were studied.
Results: Ethanol time- and dose-dependently decreased the production of NO in the cells. Exogenous NO decreased the number of mAChRs and totally blocked the effects of ethanol upon these receptors. Inhibition of nNOS up-regulated the number of mAChRs, but this effect was not additive to the effects of ethanol.
Conclusions: The results of this study suggest that the number of cell-surface mAChRs in SH-SY5Y cells may be correlated with changes in NO levels. The number of cell surface mAChRs decreased with NO-elevating treatment and increased with NO-lowering treatment. Because ethanol, which is known to up-regulate mAChRs in SH-SY5Y cells, also decreased NO levels and because nNOS inhibition and ethanol effects on mAChRs were not additive, it is conceivable that ethanol-induced up-regulation of mAChRs involves inhibition of nNOS.