Abstract
The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB2 receptors (CB2Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB2R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB2R. Using membrane preparations from CB2R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB2R where the synthetic cannabinoid, [3H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB2R-selective compound, CM-157. The effect on binding to CB2R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the KD. CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB2R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.
Highlights
The endocannabinoid system consists of two compounds with amphipathic structure, 2-arachidonyl glycerol (2-AG) and anandamide (AEA), the enzymes that produce and degrade them, and two cannabinoid, CB1 and CB2, receptors
Cannabinoid receptors belong to the superfamily of G-protein-coupled receptors (GPCRs) and are reportedly coupled to the heterotrimeric Gi protein1, i.e., their activation leads to a decrease in the intracellular level of a second messenger, cAMP
Some authors open new avenues in the therapeutic use of this phytocannabinoid. They seem to demonstrate that CBD was able to reduce the effect of 2-AG and 9-THC on cannabinoid receptor subtype 1 (CB1R) internalization and PLCβ3 and ERK1/2 phosphorylation, proposing that CBD may act as a negative allosteric modulator of these receptors (Laprairie et al, 2015)
Summary
The endocannabinoid system consists of two compounds with amphipathic structure, 2-arachidonyl glycerol (2-AG) and anandamide (AEA), the enzymes that produce and degrade them, and two cannabinoid, CB1 and CB2, receptors (see Lu and MacKie, 2016 and references therein). Further behavioral studies suggest that CBD may act via 5-HT1A receptors (Russo et al, 2005; Alves et al, 2010; Magen et al, 2010; Rock et al, 2012; Espejo-Porras et al, 2013; Sartim et al, 2016) Such assumption is intriguing as the reported affinity of CBD binding to these serotonin receptors does not lie within the nanomolar high affinity range (Russo et al, 2005). Some authors open new avenues in the therapeutic use of this phytocannabinoid They seem to demonstrate that CBD was able to reduce the effect of 2-AG and 9-THC on CB1R internalization and PLCβ3 and ERK1/2 phosphorylation, proposing that CBD may act as a negative allosteric modulator of these receptors (Laprairie et al, 2015). The aim of this work was to search for the possibility that CBD acts as an allosteric ligand of CB2R by checking whether it is able to modulate the binding and functional effect of CB2R agonists
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