In the current study we present evidence that the endocannabinoid system plays a role in the regulation of human endometrial cell migration and that the effect of endogenous and phytocannabinoids suggests a signalling mechanism mediated through CB2 receptors and to a greater extent, GPR18. The most effective activator of endometrial cell migration was the AEA metabolite, NAGly, which was also a potent ligand at GPR18. We further characterized GPR18 pharmacology via p44/42 MAPK activation, demonstrating agonist profile that did not include the majority of the known CB1 and CB2 receptor agonists. However, our results did reveal a surprising agonist activity for Δ9-THC at GPR18, suggesting that this orphan receptor represents an additional route for phytocannabinoid pharmacology.
Cannabinoids and endometrial migration
Recent work by Gentilini et al. (2010) reported that primary human endometrial stromal cells migrated towards R1-methAEA (10 µM) and that this was blocked by AM251 (10 µM), suggesting a CB1 receptor-mediated signalling mechanism. This is in contrast to our findings with HEC-1B endometrial epithelial cells, where the data point towards migration occurring through CB2 and GPR18 receptors, and independently of CB1 receptors. Our HEK293-GPR18 MAPK studies show that while AM251 was a mixed CB1/GPR18 antagonist, R1-methAEA remained a selective agonist for CB1 receptors, supporting the conclusions of Gentilini et al. (2010). Stromal cells are part of the support structure of the endometrium, forming thick connective tissue, as distinct from endometrial epithelium, which is parenchymal. Therefore, differences in phenotype are likely to underlie this apparent discrepancy in regulation of migration by the endocannabinoid system. Like the endometrial epithelium, stroma also undergoes many changes to mediate the dynamic proliferation, secretion and regression events associated with the various phases of the menstrual cycle (Chard and Grudzinskas, 1994). However, Taylor et al. (2010) have observed notable dissimilarities in both CB1 and CB2 receptor immunoreactivity between stroma and epithelium in human endometrial biopsies. Maccarrone et al. (2000) found FAAH localized in endometrial epithelium and this is compatible with our HPLC/MS/MS data, which indicated the functional presence of a potential FAAH-dependent ‘AEA-to-NAGly’ conversion in HEC-1B endometrial epithelial cells.
Differences in the activity curves of AEA and NAGly-induced HEC-1B migration suggest that the increased potency of AEA is due in part to AEA potentially activating CB2 receptors (as demonstrated by the attenuated response with pre-incubation of SR144528 and no effect of SR141716A) as well as GPR18 through the conversion of AEA to NAGly. In addition, AEA and NAGly each demonstrate the same bell-shape response with a maximization or ‘ceiling effect’ of migration, which does not appear to be additive as neither compound caused migration levels above ∼275% of the maximum effect of oestradiol at amounts above 1 µM. Therefore, additional activation by NAGly (probably acting on GPR18) in the presence of AEA (acting on CB2 receptors) does not increase this ceiling effect. Taken together, these results demonstrate a sophisticated role for CB1, CB2 receptors and GPR18 in endometrial signalling.
Novel cannabinoid pharmacology
The most current understanding of the endocannabinoid system holds that the pharmacology of endogenous and phytocannabinoids is complex. For several years, well-documented evidence has supported the existence of additional cannabinoid receptors, other than CB1 and CB2 receptors, contributing to the system (Begg et al., 2005; Mackie and Stella, 2006; Brown, 2007; McHugh et al., 2008). Our recent publication was the first to demonstrate that the Gi/o-coupled GPCR, GPR18, is one of these unknown receptors in that AEA and its metabolite NAGly exert potent control of CNS microglia (McHugh et al., 2010). Moreover, in that same publication we proposed the working hypothesis that GPR18 is the molecular identity of the Abn-CBD receptor, the most prominent of the non-CB1, non-CB2 receptors. A portion of the data reported showed that NAGly drove MAPK (p44/42; ERK1/2) activation in GPR18-transfected HEK293 cells. Using this same expression system we screened the specificity of cannabinoid ligands including traditional CB1 and CB2 receptor agonists and antagonists at GPR18. In order of potency, NAGly, O-1602, Abn-CBD, Δ9-THC, AEA and ACPA are full agonists at GPR18; CBD was a weak GPR18 partial agonist. WIN55212-2, CP55940, R1-methAEA, JWH-133 and JWH-015 had no effect. The ACPA data in Figure 4A suggested that there may be a two-site curve in the GPR18 cells, possibly reflecting two populations of GPR18 receptors (coupled and non-coupled) that have different affinity for the agonist. In order to test this, using GraphPad Prism, the fit of the ACPA data was compared between one-site curve versus a two-site curve. The F-test resulted in a P-value of <0.05, indicating that the one-site model is preferred.
It is becomingly increasingly clear that a proper understanding of GPR18 pharmacology and its physiological role is essential firstly, for an adequate understanding of the complexity of the endocannabinoid system. Secondly, to avoid misinterpretation of effects observed with ligands previously regarded as CB1 or CB2 receptor selective, which are also pharmacologically active at GPR18. For instance, R1-methAEA is the most potent agonist in the methanandamide series. It is approximately fourfold more potent at CB1 receptors and more resistant to hydrolytic inactivation by FAAH, than AEA (Abadji et al., 1994). These characteristics have made this ligand a convenient substitute for AEA in various tissues. However, now on the basis of our results, caution should be exercised when interpreting the outcome. Our data indicate activity for AEA but not R1-methAEA at GPR18 and that FAAH-dependent hydrolysis of AEA is likely to be an integral biosynthetic step towards the production of NAGly, which is a more potent agonist at GPR18. An appreciation of this will avoid misconstruing effects as solely CB1 receptor- versus GPR18-mediated, especially given the antagonism of GPR18 by AM251 we also report here. It will also allow for a more nuanced use of R1-methAEA to selectively target CB1 receptor signalling over that of GPR18 and CB2 receptors. Thirdly, a more complete picture of GPR18 pharmacology will help to generate future hypotheses in cannabinoid research.
There are many reports of AEA and Abn-CBD activity associated with Abn-CBD receptors (also known as the ‘endothelial anandamide receptor’), that are either insensitive to SR141617A antagonism (Mukhopadhyay et al., 2002; Walter et al., 2003; Milman et al., 2005; O'Sullivan et al., 2005; McHugh et al., 2010) or require substantially greater concentrations than those required to block CB1 receptors (White and Hiley, 1998; Járai et al., 1999; Wagner et al., 1999; McHugh et al., 2008). Our data showed that SR141716A had no effect on migration of HEC-1B cells (Figure 1C).
The lack of consistency for SR141716A antagonism at Abn-CBD receptors may be explained as a product of its affinity for Abn-CBD receptors and the number that need to be blocked in order to observe a reduction in signalling, which will in turn vary according to the degree of receptor reserve in any given tissue. AM251 is structurally very similar to SR141716A. In light of this, we hypothesized that AM251 may be able to antagonize GPR18 receptors, where SR141716A failed to do so. SR141617A and AM251 are both biarylpyrazole antagonists. In AM251, the p-chloro group attached to the phenyl substituent at C-5 of the pyrazole ring of SR141716A is replaced with a p-iodo group – essentially a halogen substitution (Lan et al., 1999). Structure–activity studies where comparable halogen substitutions were made on the vanillyl moieties of the TRPV1 agonists resiniferatoxin and nordihydrocapsaicin, have yielded potent TRPV1 antagonists (Wahl et al., 2001; Appendino et al., 2003). In addition, Liu and Simon (1997) found that the inhibitory potency of TRPV1 antagonists correlated directly with the size of the halogen substituent (I > Br > Cl) and inversely with its electronegativity. Based on these data, and in order to further test our hypothesis that GPR18 is the molecular identity of the Abn-CBD receptor, we predicted that AM251 would be a more potent antagonist at GPR18 than SR141716A. Our results are in support of this. AM251 antagonized NAGly and Δ9-THC activation of p44/42 MAPK in HEK293-GPR18 cells with IC50 values of ∼144 nM and ∼47 nM respectively (Figure 5D). AM251 also antagonized NAGly and Δ9-THC-induced migration of HEC-1B cells, where SR141716A had no effect (Figures 1C and 6B,C); AM251's IC50 values here were ∼ 112 nM and ∼ 80 nM respectively. Furthermore, the IC50 values for AM251 antagonism of GPR18 (Abn-CBD receptors) are lower than the reported IC50 for SR141716A, of ∼ 600 nM at Abn-CBD receptors (Jung et al., 1997; Bukoski et al., 2002).
In conclusion, the discovery of the endocannabinoid system began with the identification of a single GPCR, the CB1 receptor and the first endogenous ligand, anandamide. However, a very few additional components have accrued over the last two decades to produce a small group of endocannabinoid receptors and ligands. Recent discussions have centred on what it means to be a member of the ‘cannabinoid’ family and whether or not the levels of inclusion and exclusion are beneficial in terms of getting to a better understanding of the science related to how the phytocannabinoids either mimic or interfere with endogenous mammalian signalling systems (see Pertwee et al., 2010). Here, we provide data that the Gi/o-coupled GPCR, GPR18, was activated by Δ9-THC and additional data showing that CBD acted as an antagonist at this same receptor. These results demonstrate that greater concentrations of Δ9-THC are required to activate GPR18 receptors, than of CBD to produce GPR18 antagonism, a difference that is likely to affect the therapeutic outcome of existing pharmacotherapies that combine both Δ9-THC and CBD.
We also provide evidence that an endogenous metabolite of anandamide, NAGly, mimicked the effects of Δ9-THC in driving migration of the human endometrial cell line, HEC-1B, which was also blocked by <100 nM of CBD. These data potentially explain the ‘idiosyncratic’ outcomes from some previous studies in the field in which the effects of CB1 and CB2 receptors were unable to account for the observed phenomena.
The endocannabinoid system branch of neuroscience research is still evolving; however, it is already clear that it powerfully regulates neuronal, vascular, immune and reproductive functions. An understanding of the expression, function and regulation of the hitherto unidentified cannabinoid receptors such as GPR18, their molecular interactions with endogenous ligands, and how phytocannabinoids play a role with their signalling is vital if we are to comprehensively assess the function of the cannabinoid signalling system in human health and disease.