Background: Microglia provide continuous immune surveillance of the CNS and upon activation rapidly change phenotype to express receptors that respond to chemoattractants during CNS damage or infection. These activated microglia undergo directed migration towards affected tissue. Importantly, the molecular species of chemoattractant encountered determines if microglia respond with pro- or anti-inflammatory behaviour, yet the signaling molecules that trigger migration remain poorly understood. The endogenous cannabinoid system regulates microglial migration via CB₂receptors and an as yet unidentified GPCR termed the 'abnormal cannabidiol' (Abn-CBD) receptor. Abn-CBD is a synthetic isomer of the phytocannabinoid cannabidiol (CBD) and is inactive at CB₁or CB₂receptors, but functions as a selective agonist at this G_i/o-coupled GPCR.N-arachidonoyl glycine (NAGly) is an endogenous metabolite of the endocannabinoid anandamide and acts as an efficacious agonist at GPR18. Here, we investigate the relationship between NAGly, Abn-CBD, the unidentified 'Abn-CBD' receptor, GPR18, and BV-2 microglial migration.Results: Using Boyden chamber migration experiments, yellow tetrazolium (MTT) conversion, In-cell Western, qPCR and immunocytochemistry we show that NAGly, at sub-nanomolar concentrations, and Abn-CBD potently drive cellular migration in both BV-2 microglia and HEK293-GPR18 transfected cells, but neither induce migration in HEK-GPR55 or non-transfected HEK293 wildtype cells. Migration effects are blocked or attenuated in both systems by the 'Abn-CBD' receptor antagonist O-1918, and low efficacy agonistsN-arachidonoyl-serine and cannabidiol. NAGly promotes proliferation and activation of MAP kinases in BV-2 microglia and HEK293-GPR18 cells at low nanomolar concentrations - cellular responses correlated with microglial migration. Additionally, BV-2 cells show GPR18 immunocytochemical staining and abundant GPR18 mRNA. qPCR demonstrates that primary microglia, likewise, express abundant amounts of GPR18 mRNA.Conclusions: NAGly is the most effective lipid recruiter of BV-2 microglia currently reported and its effects mimic those of Abn-CBD. The data generated from this study supports the hypothesis that GPR18 is the previously unidentified 'Abn-CBD' receptor. The marked potency of NAGly acting on GPR18 to elicit directed migration, proliferation and perhaps other MAPK-dependent phenomena advances our understanding of the lipid-based signaling mechanisms employed by the CNS to actively recruit microglia to sites of interest. It offers a novel research avenue for developing therapeutics to elicit a self-renewing population of neuroregenerative microglia, or alternatively, to prevent the accumulation of misdirected, pro-inflammatory microglia which contribute to and exacerbate neurodegenerative disease.

BACKGROUND AND PURPOSEEndometriosis is a disorder in which the endometrium forms growths outside the uterus and is associated with chronic pain. Recent evidence suggests that endometrial motility plays a role in the aetiology of endometriosis. The endocannabinoid system regulates cellular migration. Given the growing involvement of the endocannabinoids in reproduction, we investigated the role of the endocannabinoid system in migration of endometrial cells.EXPERIMENTAL APPROACHMigration of the human endometrial HEC‐1B cells was assayed. Standard PCR techniques were used to determine the presence of the GPCR, GPR18, in HEC‐1B cells, and p44/42 MAPK was assayed in stably transfected HEK293‐GPR18 cells to determine receptor specificity for known cannabinoid agonists and antagonists.N‐arachidonoyl ethanolamine (AEA) metabolism was measured, using HPLC/MS/MS for lipid analysis.KEY RESULTSAEA, Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC) andN‐arachidonoyl glycine (NAGly) induce migration of HEC‐1B cells through cannabinoid CB₁receptor‐independent mechanisms. MAPK activation in HEK293‐GPR18 cells revealed novel pharmacology for known CB₁and CB₂receptor ligands at GPR18 receptors, including Δ⁹‐THC, which activates MAPK at nanomolar concentrations, whereas WIN 55212‐2, CP55940, JWH‐133 and JWH‐015, and arachidonyl‐1‐hydroxy‐2‐propylamide (R1‐methanandamide) had no effect. Moreover, HEC‐1B migration and MAPK activation by NAGly and Δ⁹‐THC were antagonized byPertussistoxin, AM251 and cannabidiol.CONCLUSIONS AND IMPLICATIONSAn understanding of the function and regulation of GPR18 and its molecular interactions with endogenous ligands, and how phytocannabinoids play a role with GPR18 signalling is vital if we are to comprehensively assess the function of the cannabinoid signalling system in human health and disease.LINKED ARTICLESThis article is commented on by Alexander, pp. 2411–2413 of this issue and is part of a themed section on Cannabinoids in Biology and Medicine. To view Alexander visithttp://dx.doi.org/10.1111/j.1476‐5381.2011.01731.x. To view the other articles in this section visithttp://dx.doi.org/10.1111/bph.2012.165.issue‐8. To view Part I of Cannabinoids in Biology and Medicine visithttp://dx.doi.org/10.1111/bph.2011.163.issue‐7

Here, we show a novel pharmacology for inhibition of human neutrophil migration by endocannabinoids, phytocannabinoids, and related compounds. The endocannabinoids virodhamine and N-arachidonoyl dopamine are potent inhibitors of N-formyl-l-methionyl-l-leucyl-l-phenylalanine-induced migration of human neutrophils, with IC₅₀ values of 0.2 and 8.80 nM, respectively. The endocannabinoid anandamide inhibits human neutrophil migration at nanomolar concentrations in a biphasic manner. The phytocannabinoid (-)-cannabidiol is a partial agonist, being ∼40 fold more potent than (+)-cannabidiol; abnormal-cannabidiol is a full agonist. Furthermore, the abnormal-cannabidiol (CBD) analog trans-4-[3-methyl-6-(1-methylethenyl)-2-cyclohexen-1-yl]-5-methyl-1,3-benzenediol (O-1602) inhibits migration, with an IC₅₀ value of 33 nM. This reported profile of agonist efficacy and potency parallels with the pharmacology of the novel “abnormal-cannabidiol” receptor or a related orphan G protein-coupled receptor, which are already known to modulate cell migration. Although having no effect alone, N-arachidonoyl l-serine attenuated inhibition of human neutrophil migration induced by anandamide, virodhamine, and abnormal-CBD. Our data also suggest that there is cross-talk/negative co-operativity between the cannabinoid CB₂ receptor and this novel target: CB₂ receptor antagonists significantly enhance the inhibition observed with anandamide and virodhamine. This study reveals that certain endogenous lipids, phytocannabinoids, and related ligands are potent inhibitors of human neutrophil migration, and it implicates a novel pharmacological target distinct from cannabinoid CB₁ and CB₂ receptors; this target is antagonized by the endogenous compound N-arachidonoyl l-serine. Furthermore, our findings have implications for the potential pharmacological manipulation of elements of the endocannabinoid system for the treatment of various inflammatory conditions.

A family of endogenous lipids, structurally analogous to the endogenous cannabinoid, N-arachidonoyl ethanolamine (Anandamide), and called N-acyl amides have emerged as a family of biologically active compounds at TRP receptors. N-acyl amides are constructed from an acyl group and an amine via an amide bond. This same structure can be modified by changing either the fatty acid or the amide to form potentially hundreds of lipids. More than 70 N-acyl amides have been identified in nature. We have ongoing studies aimed at isolating and characterizing additional members of the family of N-acyl amides in both central and peripheral tissues in mammalian systems. Here, using a unique in-house library of over 70 N-acyl amides we tested the following three hypotheses: 1) Additional N-acyl amides will have activity at TRPV1-4, 2) Acute peripheral injury will drive changes in CNS levels of N-acyl amides, and 3) N-acyl amides will regulate calcium in CNS-derived microglia. Through these studies, we have identified 20 novel N-acyl amides that collectively activate (stimulating or inhibiting) TRPV1-4. Using lipid extraction and HPLC coupled to tandem mass spectrometry we showed that levels of at least 10 of these N-acyl amides that activate TRPVs are regulated in brain after intraplantar carrageenan injection. We then screened the BV2 microglial cell line for activity with this N-acyl amide library and found overlap with TRPV receptor activity as well as additional activators of calcium mobilization from these lipids. Together these data provide new insight into the family of N-acyl amides and their roles as signaling molecules at ion channels, in microglia, and in the brain in the context of inflammation.

Background. Cannabinoids are known to reduce intestinal inflammation. Atypical cannabinoids produce pharmacological effects via unidentified targets. We

N-arachidonoyl glycine is an endogenous arachidonoyl amide that activates the orphan G protein-coupled receptor (GPCR) GPR18 in a pertussis toxin (PTX)-sensitive manner and produces antinociceptive and antiinflammatory effects. It is produced by direct conjugation of arachidonic acid to glycine and by oxidative metabolism of the endocannabinoid anandamide. Based on the presence of enzymes that conjugate fatty acids with glycine and the high abundance of palmitic acid in the brain, we hypothesized the endogenous formation of the saturated N-acyl amide N-palmitoyl glycine (PalGly). PalGly was partially purified from rat lipid extracts and identified using nano-high-performance liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry. Here, we show that PalGly is produced after cellular stimulation and that it occurs in high levels in rat skin and spinal cord. PalGly was up-regulated in fatty acid amide hydrolase knockout mice, suggesting a pathway for enzymatic regulation. PalGly potently inhibited heat-evoked firing of nociceptive neurons in rat dorsal horn. In addition, PalGly induced transient calcium influx in native adult dorsal root ganglion (DRG) cells and a DRG-like cell line (F-11). The effect of PalGly on the latter cells was characterized by strict structural requirements, PTX sensitivity, and dependence on the presence of extracellular calcium. PalGly-induced calcium influx was blocked by the nonselective calcium channel blockers ruthenium red, 1-(β-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SK&F96365), and La³⁺. Furthermore, PalGly contributed to the production of NO through calcium-sensitive nitric-oxide synthase enzymes present in F-11 cells and was inhibited by the nitric-oxide synthase inhibitor 7-nitroindazole.