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, 165 (8), 2414-24

Δ(9) -Tetrahydrocannabinol and N-arachidonyl Glycine Are Full Agonists at GPR18 Receptors and Induce Migration in Human Endometrial HEC-1B Cells

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Δ(9) -Tetrahydrocannabinol and N-arachidonyl Glycine Are Full Agonists at GPR18 Receptors and Induce Migration in Human Endometrial HEC-1B Cells

Douglas McHugh et al. Br J Pharmacol.

Abstract

Background and purpose: Endometriosis 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 approach: Migration 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 results: AEA, Δ(9) -tetrahydrocannabinol (Δ(9) -THC) and N-arachidonoyl glycine (NAGly) induce migration of HEC-1B cells through cannabinoid CB(1) receptor-independent mechanisms. MAPK activation in HEK293-GPR18 cells revealed novel pharmacology for known CB(1) and CB(2) receptor ligands at GPR18 receptors, including Δ(9) -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 Δ(9) -THC were antagonized by Pertussis toxin, AM251 and cannabidiol.

Conclusions and implications: An 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 articles: This 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 visit http://dx.doi.org/10.1111/j.1476-5381.2011.01731.x. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Figures

Figure 1
Figure 1
Oestradiol, AEA and NAGly induced HEC-1B endometrial cell migration. (A) HEC-1B cell migration in response to basal conditions; vehicle (vh; 0.1% DMSO); 0.1 nM–10 µM concentrations of oestradiol, AEA and NAGly; n= 3. (B) Filter photographs of one random field of view at ×40 magnification indicating the migration produced by the vehicle; 1 nM oestradiol, 1 nM AEA and 1 nM NAGly. The 10 µm diameter pores can be discerned as the clear unstained circles. (C) HEC-1B cell migration in response to 100 nM oestradiol + vehicle (0.1% DMSO), 100 nM oestradiol + 100 nM SR141716A (rimonabant), 100 nM oestradiol + 100 nM SR144528, 100 nM oestradiol + 1 µM CBD, 100 nM oestradiol + 1 µg·mL−1 PTX; 100 nM AEA + vehicle, 100 nM AEA + 100 nM SR141716A (rimonabant), 100 nM AEA + 100 nM SR144528, 100 nM AEA + 1 µM CBD, 100 nM AEA + 1 µg·mL−1 PTX; 100 nM NAGly + vehicle, 100 nM NAGly + 100 nM SR141716A (rimonabant),100 nM NAGly + 100 nM SR144528, 100 nM NAGly + 1 µM CBD, 100 nM NAGly + 1 µg·mL−1 PTX. ##P < 0.01, significantly different from 100 nM oestradiol; **P < 0.01, significantly different from 100 nM AEA; ††P < 0.01, significantly different from 100 nM NAGly; one-way anova; n= 3–6.
Figure 2
Figure 2
Deuterium-labelled anandamide is a precursor for NAGly in HEC-1B through two pathways. (A) Molecular structures of d4-anandamide, d0-NAGly and d2-NAGly are illustrated. The NAGly products were formed after d4-AEA was incubated with HEC-1B cells. The chromatogram shows the relative peaks of the d0-NAGly and the d2-NAGly products from a 90 min incubation of d4-AEA. (B) The left hand axis represents the percentage of metabolism (the inverse of d4-AEA recovery measured over time), whereas the right hand axis represents the level of two different NAGly products (d0-NAGly and d2-NAGly) measured at the same time points in the same experiments.
Figure 3
Figure 3
HEC-1B endometrial cells express GPR18 mRNA. Gel electrophoresis of HEC-1B endometrial cell RT-qPCR products. RT-qPCR products were collected from the RT-qPCR run; loading buffer was added to the samples, which were run on a 2% agarose gel.
Figure 4
Figure 4
Concentration–response curves indicating activation of p44/42 MAPK in HEK293-GPR18 and HEK293-CB1 cells. (A) p44/42 MAPK activation in HEK293-GPR18 cells in response to 0.1 nM–100 µM concentrations of NAGly, O-1602, Abn-CBD, Δ9-THC, AEA and ACPA; n= 3. (B) p44/42 MAPK activation in HEK293-GPR18 cells in response to 0.1 nM–100 µM concentrations of O-1602, Abn-CBD and CBD; n= 3. (C) p44/42 MAPK activation in HEK293-GPR18 cells in response to 0.1 nM–100 µM concentrations of WIN55212-2, CP55940, R1-methAEA, JWH-133 and JWH-015; n= 3. (D) p44/42 MAPK activation in HEK293-CB1 cells in response to 0.1 nM–100 µM concentrations of WIN55212-2, CP55940, R1-methAEA and ACPA; n= 3.
Figure 5
Figure 5
Concentration–response curves indicating activation of p44/42 MAPK in HEK293-GPR18 and HEK293 wildtype cells. (A) p44/42 MAPK activation in HEK293-GPR18 cells in response to 0.1 nM–100 µM concentrations of Δ9-THC alone and 0.1 nM–1 mM Δ9-THC + 20 µM CBD; n= 3. (B) HEK293 wildtype cell p44/42 MAPK activation in response to 0.1 nM–100 µM concentrations of Δ9-THC; n= 3. (C) p44/42 MAPK activation in HEK293-GPR18 cells in response to 0.1 nM–100 µM concentrations of AM251; n= 3. (D) p44/42 MAPK activation in HEK293-GPR18 cells in response to 100 nM NAGly + 0.1 nM–100 µM AM251 and 100 nM Δ9-THC + 0.1 nM–100 µM AM251; n= 3.
Figure 6
Figure 6
Δ9-THC and NAGly-induced HEC-1B endometrial cell migration is antagonized by AM251 and CBD. (A) HEC-1B cell migration in response to basal conditions; vh (0.1% DMSO); 0.1 nM–10 µM concentrations of NAGly, Δ9-THC, CBD and AM251; n= 3. (B) HEC-1B cell migration in response to basal conditions; 100 nM NAGly + 0.1 nM–10 µM CBD; 100 nM NAGly + 0.1 nM–10 µM AM251; n= 3. (C) HEC-1B cell migration in response to basal conditions; 100 nM Δ9-THC + 0.1 nM–10 µM CBD; 100 nM Δ9-THC + 0.1 nM–10 µM AM251; n= 3.

Comment in

  • So What Do We Call GPR18 Now?
    SP Alexander. Br J Pharmacol 165 (8), 2411-3. PMID 22014123.
    The further characterization of the orphan GPCR GPR18 conducted by McHugh et al. in this issue of the British Journal of Pharmacology has generated a pharmacological prof …

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