Elsevier

Biochemical Pharmacology

Volume 68, Issue 11, 1 December 2004, Pages 2207-2219
Biochemical Pharmacology

Effects of anandamide on the binding and signaling properties of M1 muscarinic acetylcholine receptors

https://doi.org/10.1016/j.bcp.2004.08.005Get rights and content

Abstract

This study investigated the effects of the endocannabinoid, anandamide, on M1 muscarinic acetylcholine receptors (mAChRs) expressed in Chinese hamster ovary (CHO) cells. In the presence of anandamide, [3H]N-methylscopolamine ([3H]NMS) inhibition binding was characterized by Hill coefficients greater than 1 while saturation binding isotherms were characterized by a reduction in radioligand Bmax. Anandamide did not affect the potency of classic agonists, antagonists or allosteric modulators to inhibit [3H]NMS binding, indicating that the site of anandamide action did not involve receptor regions recognized by these compounds. Although the mode of binding of anandamide was reversible, the order of ligand addition was important; the inhibitory effect was greatest when anandamide was equilibrated with the receptor prior to radioligand addition, and weakest in the converse situation. Interestingly, the inhibitory potency of anandamide was reduced on pre-equilibration with non-transfected CHO cell membranes, prior to addition of M1 mAChR-transfected membranes. In phosphoinositide (PI) hydrolysis assays, anandamide significantly reduced the maximal response to acetylcholine, but at higher concentrations than those needed to fully inhibit radioligand binding. Studies utilizing a range of agonists with varying intrinsic activities showed that the inhibitory effects of anandamide on agonist function were most pronounced with the lowest efficacy agonists. These findings suggest that the mechanism of action of anandamide at the M1 mAChR involves perturbation of the receptor via the membrane in a manner that is sensitive to the conformation of the receptor (occupied versus vacant).

Section snippets

Materials

Drugs and chemicals were obtained from the following sources: [3H]NMS from NEN Life Science Products, (Boston, MA, USA), [3H]myo-inositol from Amersham Pharmacia Biotech (Buckinghamshire, UK), alcuronium chloride (generous gift from F. Hoffmann-La Roche Ltd., Basle, Switzerland), DMEM and geneticin were from Life Technologies GIBCO BRL (Grand Island, NY, USA), fetal bovine serum was from ThermoTrace, (Melbourne, VIC, Australia), and DOWEX AG1-X8 ion-exchange resin was obtained from Bio-Rad

Inhibition binding assays in CHO membranes

To examine the effects of anandamide on the binding of [3H]NMS to M1 CHO membranes, inhibition binding experiments were performed. Incubation of [3H]NMS and anandamide for 1 h revealed that the endocannabinoid was able to completely inhibit radioligand binding at the M1 mAChR, yielding a log IC50 of −5.55 ± 0.04 and nH of 2.23 ± 0.42 (n = 5; Fig. 2A). The steep Hill slope for anandamide was significantly different from unity (p < 0.05) and was not due to an equilibration artifact, since extending

Discussion

ACh and the endogenous cannabinoid, anandamide, exert profound, albeit opposing, regulatory effects on learning and memory. Although ACh mediates most of its effects through M1 mAChRs and anandamide through interaction with CB1 cannabinoid receptors, studies have recently demonstrated a direct effect of anandamide on antagonist binding to the mAChRs [21], [22]. The present study confirms and extends these previous findings, and suggests that anandamide may interact with the M1 mAChR through a

Acknowledgments

The authors are grateful to Dr. Fred Mitchelson for helpful discussions. This work was funded by Project Grant No. 209083 of the National Health and Medical Research Council (NHMRC) of Australia. Arthur Christopoulos is a senior research fellow of the NHMRC.

References (32)

  • A. Christopoulos

    Assessing the distribution of parameters in models of ligand–receptor interaction: to log or not to log

    Trends Pharmacol Sci

    (1998)
  • C.J. Ladner et al.

    Reduced high-affinity agonist binding at the M1 muscarinic receptor in Alzheimer's disease brain: differential sensitivity to agonists and divalent cations

    Exp Neurol

    (1999)
  • D. Piomelli et al.

    Endogenous cannabinoid signaling

    Neurobiol Dis

    (1998)
  • D. Jerusalinsky et al.

    Cholinergic neurotransmission and synaptic plasticity concerning memory processing

    Neurochem Res

    (1997)
  • C.J. Ladner et al.

    Pharmacological drug treatment of Alzheimer disease: the cholinergic hypothesis revisited

    J. Neuropathol Exp Neurol

    (1998)
  • M.P. Caulfield et al.

    International Union of PharmacologyXVII. Classification of muscarinic acetylcholine receptors

    Pharmacol Rev

    (1998)
  • Cited by (19)

    • 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 :

      Meanwhile, the genetic deletion of CB1 has been shown to create an imbalance of excitatory/inhibitory neurotransmission in the hippocampus, drastically accelerating memory impairment and reducing survival in APP/PS1 transgenic mice. The APP/PS1 mouse model is a double transgenic mouse model, expressing a chimeric mouse/human amyloid precursor protein, and a mutant human presenilin 1(PS1-dE9), and represents a model of familial AD with cognitive impairments, Aβ plaque deposition and synaptic abnormalities from 6 months of age [138]. The effects of activating, blocking, or deleting the CB1 receptor in mouse models of AD therefore highlight the importance of CB1 in the modulation of AD-like symptoms, particularly those attributed to cholinergic dysfunction.

    • Allosteric modulators of rhodopsin-like G protein-coupled receptors: Opportunities in drug development

      2012, Pharmacology and Therapeutics
      Citation Excerpt :

      GPCR function may be allosterically modified not only by drugs but also by endogenous allosteric modulators (Christopoulos & Kenakin, 2002). These include for example ions (e.g. Na+, Ca2+, Zn2+), lipid metabolites (Lanzafame et al., 2004; Lane et al., 2010), steroids (Rossi et al., 2009), and auto-antibodies (van Koppen & Kaiser, 2003; Jensen & Spalding, 2004; May et al., 2007). Besides modulation by ions, small molecules or antibodies, GPCRs can also be modulated by neighboring or accessory proteins (Hall & Lefkowitz, 2002; Christopoulos et al., 2004; Milligan & Smith, 2007; Smith & Milligan, 2010; Kenakin, 2012).

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

      2010, Biochemical Pharmacology
      Citation Excerpt :

      Interestingly, cannabinoids inhibit the production of cytokines such as tumour necrosis factor-α from activated microglial cells but this activity does not involve any known CB receptor subtype for it occurs at high concentrations of ligands [12,13]. Furthermore anandamide has been shown to inhibit ligand binding to central 5-HT receptors and muscarinic acetylcholine receptors [14–16]. In both cases this has been shown to be a direct effect, and not mediated by the interaction of the endocannabinoid with either CB1 or CB2 receptors.

    • Endocannabinoids in the dentate gyrus

      2007, Progress in Brain Research
      Citation Excerpt :

      Interestingly, in both these cases, action of AEA has been shown to have inhibitory effects on recombinant receptors expressed in Xenopus oocytes (Oz et al., 2002, 2003), while additional lines of evidence implicating cannabinoids in serotonergic function have been previously reviewed (Morales, 2006). Further, a potential non-competitive interaction between AEA and muscarinic acetylcholine receptors has been suggested in both binding and functional assays (Christopoulos and Wilson, 2001; Lanzafame et al., 2004). The data reviewed here indicate the presence of a robust system for EC-mediated signaling in the dentate gyrus that is clearly composed of both synthetic and degradative enzymes for endocannabinoids as well as cannabinoid receptors.

    View all citing articles on Scopus
    1

    Present address: Department of Pharmacology, Monash University, Clayton 3800, Australia.

    View full text