N-Ethylmaleimide differentiates between the M2- and M4-autoreceptor-mediated inhibition of acetylcholine release in the mouse brain
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Muscarinic M2 and M4 receptors resemble each other in brain distribution, function, and Gi/o protein signaling. However, there is evidence from human recombinant receptors that the M4 receptor also couples to Gs protein whereas such an alternative signaling is of minor importance for its M2 counterpart. The question arises whether this property is shared by native receptors, e.g., the murine hippocampal M2- and the striatal M4-autoreceptor. To this end, the electrically evoked tritium overflow was studied in mouse hippocampal and striatal slices pre-incubated with 3H-choline. 3H-Acetylcholine release in either region was inhibited by the potent muscarinic receptor agonist iperoxo (pIC50 8.6–8.8) in an atropine-sensitive manner (apparent pA2 8.6–8.8); iperoxo was much more potent than oxotremorine (pIC50 6.5–6.6). In hippocampal slices, N-ethylmaleimide (NEM) 32 μM, which inactivates Gi/o proteins, tended to shift the concentration-response curve of iperoxo (pIC50 8.8) to the right (pIC50 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 pIC50 of 8.6 for the falling part and a pEC50 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 Gs protein comes into play at high concentrations of a muscarinic receptor agonist.
KeywordsAcetylcholine release N-Ethylmaleimide Iperoxo M2 receptor M4 receptor
The authors would like to thank the NRW International Graduate Research School Biotech-Pharma for a scholarship to J.E., Mrs. D. Petri † for her skilled technical assistance, and Prof. U. Holzgrabe for a gift of iperoxo.
Author contribution statement
KM and ES conceived and designed the research. JE conducted experiments. JE and ES analyzed the data. ES wrote the manuscript. All authors read and approved the manuscript.
Compliance with ethical standards
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
Conflict of interest
The authors declare that they have no conflict of interest.
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