N-Ethylmaleimide differentiates between the M 2 - and M 4 -autoreceptor-mediated inhibition of acetylcholine release in the mouse brain.

Muscarinic M ₂ and M ₄ receptors resemble each other in brain distribution, function, and G _i/o protein signaling. However, there is evidence from human recombinant receptors that the M ₄ receptor also couples to G _s protein whereas such an alternative signaling is of minor importance for its M ₂ counterpart. The question arises whether this property is shared by native receptors, e.g., the murine hippocampal M ₂ - and the striatal M ₄ -autoreceptor. To this end, the electrically evoked tritium overflow was studied in mouse hippocampal and striatal slices pre-incubated with ³ H-choline. ³ H-Acetylcholine release in either region was inhibited by the potent muscarinic receptor agonist iperoxo (pIC ₅₀ 8.6-8.8) in an atropine-sensitive manner (apparent pA ₂ 8.6-8.8); iperoxo was much more potent than oxotremorine (pIC ₅₀ 6.5-6.6). In hippocampal slices, N-ethylmaleimide (NEM) 32 μM, which inactivates G _i/o proteins, tended to shift the concentration-response curve of iperoxo (pIC ₅₀ 8.8) to the right (pIC ₅₀ 8.5) and depressed its maximum from 85 to 69%. In striatal slices, the inhibitory effect of iperoxo declined at concentrations higher than 0.1 μM, yielding a biphasic curve with a pIC ₅₀ of 8.6 for the falling part and a pEC ₅₀ of 6.4 for the rising part of the curve. The inhibitory effect of iperoxo 10 μM (47%) after NEM pre-treatment was lower by about 35% compared to the maximum (74%) obtained without NEM. In conclusion, our data, which need to be confirmed by pertussis toxin, might suggest that in the striatum, unlike the hippocampus, stimulatory G _s protein comes into play at high concentrations of a muscarinic receptor agonist.