Elsevier

Brain Research

Volume 915, Issue 1, 5 October 2001, Pages 70-78
Brain Research

Research report
Interaction of anandamide with the M1 and M4 muscarinic acetylcholine receptors

https://doi.org/10.1016/S0006-8993(01)02825-6Get rights and content

Abstract

The M1 and M4 muscarinic acetylcholine receptors are the most abundant muscarinic receptor subtypes in the brain, and are involved in learning and memory. Because cannabinoid receptors are also abundantly expressed in similar brain regions and mediate opposite effects to acetylcholine on cognition, the present study investigated whether the endocannabinoid agonist, anandamide, and its metabolically stable derivative, methanandamide, directly modified the binding properties of the human M1 and M4 receptors individually expressed in CHO cell membranes. Experiments utilized the antagonists, [3H]N-methylscopolamine and [3H]quinuclidinyl benzilate. When acetylcholine was used as the inhibiting ligand, shallow, biphasic isotherms were observed at both receptors, characterised by similar apparent dissociation constants for high and low affinity binding at each receptor but with a greater proportion of high affinity sites at the M4 (40–45%) than at the M1 receptor (17–20%). In contrast, anandamide and methanandamide inhibited the binding of both radioligands over a narrow (low micromolar) concentration range, with monophasic isotherms characterized by Hill coefficients significantly greater than 1 at both receptors. These effects were not due to the vehicle used. Further saturation binding analyses found anandamide able to significantly reduce the apparent affinity and maximal density of binding sites labeled by [3H]quinuclidinyl benzilate. Interestingly, no significant inhibition of radioligand binding was noted using the synthetic cannabinoid agonist, WIN55212-2, or the cannabinoid CB1 receptor antagonist, SR141716A. These data thus provide evidence for a direct role of anandamides in modulating muscarinic receptor binding properties through a non-competitive mechanism that is unrelated to their actions on cannabinoid receptors.

Introduction

Both acetylcholine and cannabinoid ligands have long been known to exert profound effects on a variety of processes associated with cognition, with the former agent facilitating and the latter substances causing disruptions in learning and memory [15], [16], [17], [21], [29]. At the molecular level, the majority of the effects of acetylcholine and cannabinoid ligands are mediated by specific receptors belonging to the G protein-coupled receptor superfamily [4], [5], [26], [27].

In terms of effects on memory, much attention has focused on muscarinic and cannabinoid-induced changes in the release or actions of glutamate, especially due to the known localization of many hippocampal receptors for each of these substances on or near glutamatergic neurones [15], [17]. However, cannabinoids are also able to interfere with the synthesis [13] and/or release of acetylcholine [9], and this provides another potential mechanism by which cannabinoids can exert negative modulatory effects on cognition. Indeed, more recent studies have illustrated a physiological antagonism between central cholinergic and cannabinoid mechanisms in their effects on working memory [3], [30].

With the relatively recent identification of an endogenous cannabinoid signaling system, exemplified by the arachidonic acid derivative, anandamide (Fig. 1), attention has now been focused on the possibility of more direct effects of the endocannabinoids on other receptor systems. For example, anandamide, as well as binding with nanomolar affinity to the cannabinoid receptors and acting as an agonist [26], has also been shown to inhibit ligand binding to central 5-HT receptors [19], although no effects were noted on ionotropic GABAA receptors in the same study. More recently, Frey et al. demonstrated that anandamide was also able to inhibit ligand binding to muscarinic acetylcholine receptors in homogenates derived from human brain [22]. That finding suggests that anandamide may play a more direct role on central cholinergic processes beyond its effects on cannabinoid receptors. However, the human brain is known to contain a mixture of all five subtypes of muscarinic receptors, particularly the M1 and M4[23], [31], and the effects of anandamide on the binding properties of the individual human muscarinic receptors are currently unknown. Because M1 and M4 receptors, as well as the cannabinoid CB1 receptors, are richly expressed in brain regions associated with learning and memory, the present study was undertaken in order to elucidate the effects of anandamide, and its metabolically stable derivative methanandamide (Fig. 1), on the individual binding properties of these two muscarinic receptor subtypes using CHO cell membranes stably expressing the human M1 or M4 receptor.

Section snippets

Materials

[3H]N-methylscopolamine (70 Ci/mmol) and [3H]quinuclidinyl benzilate (43 Ci/mmol) were purchased from NEN Dupont (Wilmington, DE); Dulbecco’s modified Eagle’s medium was purchased from Gibco BRL (Gaithersburg, MD); geneticin was obtained from Calbiochem (La Jolla, CA); bovine calf serum was supplied by Hyclone (Logan, UT); WIN 55,212-2 (R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]-pyrrolo[1,2,3,-de]-1,4-benzoxazin-yl]-(1-napthalenyl)-methanone mesylate) was purchased from Research

Binding of acetylcholine to the M1 and M4 receptors

Table 1 shows the saturation binding parameters for both radioligands at the M1 and M4 muscarinic acetylcholine receptors. Although there was a trend towards a higher affinity for [3H]quinuclidinyl benzilate relative to [3H]N-methylscopolamine, a one-way ANOVA found no significant difference between any of the parameters at the M1 and M4 receptors.

Subsequent competition experiments were undertaken to characterize the binding of the endogenous muscarinic receptor agonist, acetylcholine, to the

Discussion

In the present study, we have identified a direct effect of the endocannabinoid, anandamide, on the binding properties of the human M1 and M4 muscarinic acetylcholine receptors. Importantly, these proteins are the most abundantly expressed subtypes of muscarinic acetylcholine receptors in the hippocampus [23], [31], a region that has a well-established role in memory processing. Our findings suggest a potentially novel mode of regulation of central cholinergic processes by endogenous lipid

Acknowledgements

The authors would like to acknowledge the generous gift of SR141716A by Drs. Murielle Rinaldi-Carmona and Mandelainne Mosse, Sanofi-Synthélabo Recherché, France. We are also grateful to Dr. Fred Mitchelson, for critical review of the manuscript, and to AMRAD, Australia, for financial support of the AMRAD Drug Discovery Laboratory in the Department of Pharmacology, University of Melbourne. Arthur Christopoulos is a C.R. Roper Research Fellow of the Faculty of Medicine, Dentistry and Health

References (33)

  • M. Segal et al.

    Muscarinic receptors involved in hippocampal plasticity

    Life Sci.

    (1997)
  • P.M. Zygmunt et al.

    Anandamide — the other side of the coin

    Trends Pharmacol. Sci.

    (2000)
  • D. Braida et al.

    Cannabinoid-induced working memory impairment is reversed by a second generation cholinesterase inhibitor in rats

    Neuroreport

    (2000)
  • M.P. Caulfield et al.

    International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors

    Pharmacol. Rev.

    (1998)
  • Y.-C. Cheng et al.

    Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction

    Biochem. Pharmacol.

    (1973)
  • A. Christopoulos

    Overview of receptor allosterism

  • Cited by (36)

    • Memory Consolidation Depends on Endogenous Hippocampal Levels of Anandamide: CB1 and M4, but Possibly not TRPV1 Receptors Mediate AM404 effects

      2022, Neuroscience
      Citation Excerpt :

      Synthetic and natural agonists acutely infused, however, tend to produce diverging results (Terranova et al., 1995; de Oliveira Alvares et al., 2006; Yim et al., 2008), some of which can be explained by the administration route – systemic vs. intra-cerebral – or by the poor selectivity/specificity of ligands, as is the case of AEA itself (Morena and Campolongo, 2014; Quillfeldt and de Oliveira Alvares, 2015). Anandamide is known to be a promiscuous ligand (Di Marzo and Piscitelli, 2015) with several non-cannabinoid targets, and besides TRPV1, has an interesting relation with the G protein coupled receptor (GPCR) Muscarinic acetylcholine (MAChR) M4 inhibitory receptors (Gessa et al., 1997; Christopoulos and Wilson, 2001; McPartland et al., 2008), which possibly involve mutual interactions though allosteric modulators (Turu et al., 2009; Bock et al., 2018). All these receptors have in common the fact that they are involved both in synaptic plasticity and memory processing (Diehl et al., 2007; Genro et al., 2012; Gobira et al., 2017; Ratano et al., 2017; Cui et al., 2018).

    • Crosstalk between the M<inf>1</inf> muscarinic acetylcholine receptor and the endocannabinoid system: A relevance for Alzheimer's disease?

      2020, Cellular Signalling
      Citation Excerpt :

      Indeed, as discussed above, the activation of GPR55 leads to the release of glutamate and thus supports LTP formation [111,112]. Furthermore, anandamide has been shown to bind to M1 and M4 mAChRs, where it is thought to bind to allosteric sites [137,138]. It is therefore possible that the CB1-independent properties of genetic FAAH ablation could, in part, be explained by activation of GPR55, CB2 or even binding to non-CB receptors such as the mAChRs.

    • The endocannabinoid 2-arachidonylglycerol is a negative allosteric modulator of the human A<inf>3</inf> adenosine receptor

      2010, Biochemical Pharmacology
      Citation Excerpt :

      Ligands with an eicosanoid structure, including the endocannabinoids 2-AG and anandamide act as negative allosteric modulators and increase the rate of dissociation of the agonist [125I] AB MECA. This is in contrast to the previous findings of Christopoulos and Wilson who demonstrated that anandamide, whilst inhibiting radioligand binding at the muscarinic M1 and M4 receptors with high Hill coefficients, did not affect the rate of ligand dissociation [15]. We extended the study to monitor the effect of 2-AG on the rate of dissociation of the selective hA3 receptor antagonist [3H] PSB-11.

    • An introduction to the endocannabinoid system: from the early to the latest concepts

      2009, Best Practice and Research: Clinical Endocrinology and Metabolism
    View all citing articles on Scopus
    View full text