Skip to main content
SpringerLink
Log in

N-Ethylmaleimide differentiates between the M2- and M4-autoreceptor-mediated inhibition of acetylcholine release in the mouse brain

  • Brief Communication
  • Published:
Naunyn-Schmiedeberg's Archives of Pharmacology Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

References

  • Alexander SPH, Christopoulos A, Davenport AP, Kelly E, Marrison NV, Peters JA, Faccenda E, Harding SD, Pawson AJ, Sharman JL, Southan C, Davies JA, collaborators CGTP (2017) The concise guide to pharmacology 2017/18: G protein-coupled receptors. Br J Pharmacol 174:S17–S129

    Article  CAS  Google Scholar 

  • Allgaier C, Choi BK, Hertting G (1993) Muscarine receptors regulating electrically evoked release of acetylcholine in hippocampus are linked to pertussis toxin-sensitive G proteins but not to adenylate cyclase. J Neurochem 61:1043–1049

    Article  CAS  Google Scholar 

  • Bymaster FP, Calligaro DO, Falcone JF (1999) Arachidonic acid release in cell lines transfected with muscarinic receptors: a simple functional assay to determine response of agonists. Cell Signal 11:405–413

    Article  CAS  Google Scholar 

  • Caulfield MP, Birdsall NJ (1998) International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol Rev 50:279–290

    CAS  PubMed  Google Scholar 

  • Croy CH, Schober DA, Xiao H, Quets A, Christopoulos A, Felder CC (2014) Characterization of the novel positive allosteric modulator, LY2119620, at the muscarinic M2 and M4 receptors. Mol Pharmacol 86:106–115

    Article  Google Scholar 

  • Dallanoce C, Conti P, De Amici M, De Micheli C, Barocelli E, Chiavarini M, Ballabeni V, Bertoni S, Impicciatore M (1999) Synthesis and functional characterization of novel derivatives related to oxotremorine and oxotremorine-M. Bioorg Med Chem 7:1539–1547

    Article  CAS  Google Scholar 

  • Huang F, Buchwald P, Browne CE, Farag HH, Wu WM, Ji F, Hochhaus G, Bodor N (2001) Receptor binding studies of soft anticholinergic agents. AAPS Pharm Sci 3(4):E30

    Article  CAS  Google Scholar 

  • Jackisch R, Hotz H, Allgaier C, Hertting G (1994) Presynaptic opioid receptors on dopaminergic nerves in the rabbit caudate nucleus: coupling to pertussis toxin-sensitive G-proteins and interaction with D2 auto receptors? Naunyn Schmiedeberg’s Arch Pharmacol 349:250–258

    Article  CAS  Google Scholar 

  • Janßen N (2011) Zelluläre dynamische Massenumverteilung zur Erfassung der Signalwegsaktivierung 7-transmembranärer Rezeptoren unter besonderer Berücksichtigung des M4-Acetylcholinrezeptors. Dissertation, University of Bonn

  • Janßen N, Kebig A, Mohr K (2011) Differential sensitivity of the muscarinic M2 and M4 receptor for biased signaling in response to dualsteric agonists. Naunyn Schmiedeberg’s Arch Pharmacol 383(Suppl 1):54

    Google Scholar 

  • Lebois EP, Thorn C, Edgerton JR, Popiolek M, Xi S (2017) Muscarinic receptor subtype distribution in the central nervous system and relevance to aging and Alzheimer’s disease. Neuropharmacology 136:362–373. https://doi.org/10.1016/j.neuropharm.2017.11.018

    Article  CAS  PubMed  Google Scholar 

  • Michal P, El-Fakahany EE, Doležal V (2007) Muscarinic M2 receptors directly activate Gq/11 and Gs G-proteins. J Pharmacol Exp Ther 320:607–614

    Article  CAS  Google Scholar 

  • Richards MH (1990) Relative potencies of agonists and differential sensitivity to N-ethylmaleimide of muscarinic autoreceptors and postsynaptic receptors in rat hippocampus. J Pharmacol Exp Ther 255:83–89

    CAS  PubMed  Google Scholar 

  • Schlicker E, Malinowska B, Kathmann M, Göthert M (1994) Modulation of neurotransmitter release via histamine H3 heteroreceptors. Fundam Clin Pharmacol 8:128–137

    Article  CAS  Google Scholar 

  • Schulte K, Steingrüber N, Jergas B, Redmer A, Kurz CM, Buchalla R, Lutz B, Zimmer A, Schlicker E (2012) Cannabinoid CB1 receptor activation, pharmacological blockade, or genetic ablation affects the function of the muscarinic auto- and heteroreceptor. Naunyn Schmiedeberg’s Arch Pharmacol 385:385–396

    Article  CAS  Google Scholar 

  • Scimeni A, Beato M (2009) Determining the neurotransmitter concentration profile at active synapses. Mol Neurobiol 40:289–306

    Article  Google Scholar 

  • Shinoda M, Katada T, Ui M (1990) Selective coupling of purified alpha-subunits of pertussis toxin-substrate GTP-binding proteins to endogenous receptors in rat brain membranes treated with N-ethylmaleimide. Cell Signal 2:403–414

    Article  CAS  Google Scholar 

  • Valuskova P, Farar V, Forczek S, Krizova I, Myslivecek J (2018) Autoradiography of 3H-pirenzepine and 3H-AFDX-384 in mouse brain regions: possible insights in M1, M2 and M4 muscarinic receptors distribution. Front Pharmacol 9:124

    Article  Google Scholar 

  • Wess J, Eglen RM, Gautam D (2007) Muscarinic acetylcholine receptors: mutant mice provide new insights for drug development. Nat Rev Drug Discov 6:721–733

    Article  CAS  Google Scholar 

  • Zhang W, Basile AS, Gomeza J, Volpicelli LA, Levey AI, Wess J (2002) Characterization of central inhibitory muscarinic autoreceptors by the use of muscarinic acetylcholine receptor knock-out mice. J Neurosci 22:1709–1717

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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 information

Authors and Affiliations

  1. Pharmacology and Toxicology Section, Institute of Pharmacy, University of Bonn, Bonn, Germany

    Justine Etscheid & Klaus Mohr

  2. Institute of Pharmacology and Toxicology, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn, Germany

    Justine Etscheid & Eberhard Schlicker

Authors
  1. Justine Etscheid
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Klaus Mohr
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eberhard Schlicker
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Eberhard Schlicker.

Ethics declarations

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.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Etscheid, J., Mohr, K. & Schlicker, E. N-Ethylmaleimide differentiates between the M2- and M4-autoreceptor-mediated inhibition of acetylcholine release in the mouse brain. Naunyn-Schmiedeberg's Arch Pharmacol 391, 1295–1299 (2018). https://doi.org/10.1007/s00210-018-1539-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00210-018-1539-8

Keywords

Search

Navigation