Research reportInteraction of anandamide with the M1 and M4 muscarinic acetylcholine 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
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