The endocannabinoid 2-arachidonylglycerol is a negative allosteric modulator of the human A₃ adenosine receptor

Abstract

Studies of endogenous cannabinoid agonists, such as 2-arachidonylglycerol (2-AG), have revealed their potential to exert modulatory actions on other receptor systems in addition to their ability to activate cannabinoid receptors. This study investigated the effect of cannabinoid ligands on the human adenosine A₃ (hA₃R) receptor. The endocannabinoid 2-AG was able to inhibit agonist ([¹²⁵I]N⁶-(4-amino-3-iodobenzyl) adenosine-5′-(N-methyluronamide) – [¹²⁵I] AB MECA) binding at the hA₃R. This inhibition occurred over a narrow range of ligand concentration and was characterized by high Hill coefficients suggesting a non-competitive interaction. Furthermore, in the presence of 2-AG, the rate of [¹²⁵I] AB MECA dissociation was increased, consistent with an action as a negative allosteric modulator of the hA₃R. Moreover, by 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. Since the hA₃R has been shown to be expressed in astrocytes and microglia, these findings may be particularly relevant in certain pathological states such as cerebral ischemia where levels of 2-AG and anandamide are raised.

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, A_2A, A_2B 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 A_2A 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.