The endocannabinoid 2-arachidonylglycerol is a negative allosteric modulator of the human A3 adenosine receptor
Graphical abstract
Introduction
The cannabinoid and adenosine pathways have overlapping roles in both the modulation of neurotransmitter release in the central nervous system and modulation of the immune response [1], [2]. The endogenous nucleoside adenosine activates four distinct adenosine receptors, which all belong to the family of G protein coupled receptors (GPCR): the adenosine A1, A2A, A2B and A3 receptor [1]. Of these the adenosine A3 receptor is the most recently discovered and has a wide distribution, with its mRNA being present in testis, lung, heart, placenta, brain, spleen, liver, uterus, proximal colon and bladder [3], [4], [5]. There is now extensive evidence for the involvement of the A3 adenosine receptor in many disease pathways [6]. The A3 receptor has been implicated in the modulation of inflammatory effects, for example stimulation of the A3 receptor expressed in RBL-2H3 rat mast cells with adenosine leads to degranulation and may protect mast cells from apoptosis [7]. The A3 receptor also plays an important role in both neuroprotective and neurodegenerative effects in the brain [6].
Of several endogenous agonists for cannabinoid receptors identified thus far the most notable are anandamide and 2-arachidonylglycerol (2-AG) [2]. Their physiological effects are mainly mediated via the cannabinoid receptors CB1 and CB2[2]. These endocannabinoids have been implicated in both neuromodulatory roles and immunomodulatory functions (e.g. through the regulation of cytokine release and immune cell migration) [8]. CB1 receptor expression levels are highest in the CNS, whereas CB2 receptors have a close association with the immune system and mRNA has been detected in a wide variety of immune cells and tissues such as mast cells and microglia [2]. Interestingly, the endocannabinoid 2-AG has been shown to decrease the immunological activation of guinea pig mast cells in a mechanism mediated by CB2 receptors and opposite to the effect mediated by adenosine described above [9]. Consequently, it was of interest to investigate the potential for cannabinoid ligands to modulate the action of the adenosine A3 receptor.
It should be noted that a number of studies have implicated other sites of action of these endocannabinoids in addition to their ability to activate cannabinoid CB1 and CB2 receptors [10]. For example, neurobehavioural studies carried out in cannabinoid CB1 knockout mice showed that regardless of the lack of CB1 receptors, anandamide still exerted cannabimimetic-like activity, even though the CB1 receptor is believed to be the only receptor for cannabinoids in the brain [11]. 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], [15], [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. This is particularly relevant for disease states such as ischemia and schizophrenia where the local concentration of the endocannabinoid can be elevated [17], [18], [19], [20]. Given that cannabinoid receptors and adenosine A3 receptors are co-localised, for example in glia and immune cells such as mast cells [2], [6], [21], [22], it is possible that elevated levels of endocannabinoids may exert a ‘spill-over’ effect on adenosine receptor binding and/or functional properties. In the present study we focus on the hA3 receptor and demonstrate that the endocannabinoid ligands do indeed have a direct effect on the ligand binding of hA3 receptors. Furthermore, this study provides evidence that this interaction is non-competitive and independent of an interaction with either CB1 or CB2 receptors. The inhibition of both agonist and antagonist binding is characterised by steep Hill slopes over a narrow concentration range. This effect is specific to the A3 receptor, with no effect of cannabinoid ligands being observed on the ligand binding ability of A1 or A2A adenosine receptors. Moreover, measuring intracellular cAMP levels, we demonstrate that 2-AG decreases both the potency of an agonist at the hA3R and the basal signalling of this receptor. An investigation of ligand dissociation kinetics demonstrated that eicosanoid ligands, including the endogenous ligands anandamide and 2-AG, are negative allosteric modulators of ligand binding at the adenosine A3 receptor.
Section snippets
Materials
[125I] AB MECA was purchased from PerkinElmer BV, Groningen, NL. The following cannabinoid ligands were obtained from Tocris Ltd., Avonmouth, UK: ACEA, N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide; ACPA, N-(cyclopropyl)-5Z,8Z,11Z,14Z-eicosatetraenamide; AM 251, N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide; AM630, 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone; CP55940,
Saturation and inhibition binding assays in membranes of CHO cells stably expressing the human hA3 adenosine receptor
Saturation binding experiments performed for [125I] AB MECA at the hA3R expressed in CHO membranes yielded a Kd of 1.86 ± 0.69 nM and a Bmax of 1.16 ± 0.28 pmol/mg (Table 2). At the working concentration of 0.1 nM [125I] AB MECA, binding to the hA3R was completely abolished with the addition of 100 μM GTP indicating that only the high affinity (G protein-coupled) state of the receptor was occupied at this concentration (data not shown). To examine the effects of various cannabinoid ligands (Fig. 1) on
Discussion
This study demonstrates a direct interaction of endocannabinoid ligands with the adenosine A3 receptor which leads to the inhibition of ligand binding. Our study suggests that this is an allosteric effect rather than a direct competition with the orthosteric binding site of the A3 receptor. Accordingly kinetic studies and functional studies demonstrate that eicosanoids act as allosteric inhibitors at the A3 receptor.
The direct effects of endocannabinoid ligands tested at the A3 receptor
Acknowledgment
We thank Prof C.E. Müller (University of Bonn, Germany) for providing us with [3H] PSB-11.
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