A cytochrome P450-derived epoxygenated metabolite of anandamide is a potent cannabinoid receptor 2-selective agonist

Abstract

Oxidation of the endocannabinoid anandamide by cytochrome P450 (P450) enzymes has the potential to affect signaling pathways within the endocannabinoid system and pharmacological responses to novel drug candidates targeting this system. We previously reported that the human cytochromes P450 2D6, 3A4, and 4F2 are high-affinity, high-turnover anandamide oxygenases in vitro, forming the novel metabolites hydroxyeicosatetraenoic acid ethanolamides and epoxyeicosatrienoic acid ethanolamides. The objective of this study was to investigate the possible biological significance of these metabolic pathways. We report that the 5,6-epoxide of anandamide, 5,6-epoxyeicosatrienoic acid ethanolamide (5,6-EET-EA), is a potent and selective cannabinoid receptor 2 (CB2) agonist. The K(i) values for the binding of 5,6-EET-EA to membranes from Chinese hamster ovary (CHO) cells expressing either recombinant human CB1 or CB2 receptor were 11.4 microM and 8.9 nM, respectively. In addition, 5,6-EET-EA inhibited the forskolin-stimulated accumulation of cAMP in CHO cells stably expressing the CB2 receptor (IC(50) = 9.8 +/- 1.3 nM). Within the central nervous system, the CB2 receptor is expressed on activated microglia and is a potential therapeutic target for neuroinflammation. BV-2 microglial cells stimulated with low doses of interferon-gamma exhibited an increased capacity for converting anandamide to 5,6-EET-EA, which correlated with increased protein expression of microglial P450 4F and 3A isoforms. Finally, we demonstrate that 5,6-EET-EA is more stable than anandamide in mouse brain homogenates and is primarily metabolized by epoxide hydrolase. Combined, our results suggest that epoxidation of anandamide by P450s to form 5,6-EET-EA represents an endocannabinoid bioactivation pathway in the context of immune cell function.

Fig. 1.

Binding of anandamide and 5,6-EET-EA to human CB1 and CB2 receptors. Membrane protein from CHO cells stably expressing either the human CB1 (A) or the human CB2 receptor (B) were incubated with radiolabeled CP-55940 at its K_d value (5.1 nM for CB1 and 1.4 nM for CB2) in the presence of vehicle (ethanol) or various concentrations of anandamide or 5,6-EET-EA (0.3 nM to 100 μM), and the reactions were allowed to reach equilibrium. The binding of CP-55940 in the presence of a saturating concentration (10 μM) of the cannabinoid agonist WIN-55212-2 was considered to be due to nonspecific binding. The specific binding in the presence of the various concentrations of competitor was expressed as a percentage of the specific binding in the presence of vehicle.

Fig. 2.

Inhibition of cAMP accumulation in CHO-CB2 cells by AM1241 and 5,6-EET-EA. cAMP levels were measured in CHO-CB2 cells that were either untreated or treated with 10 μM forskolin and 100 μM IBMX in the presence or absence of AM1241 (A) or 5,6-EET-EA (B) for 10 min. Control cells (0, -) received medium alone. The results are the mean ± S.E. of triplicate cultures. ^*, p < 0.05; ^**, p < 0.01; ^***, p < 0.001, compared with vehicle (0,+) group.

Fig. 3.

Effect of 5,6-EET-EA on intracellular cAMP levels in CHO-K1 and CHO-CB2 cells. cAMP levels were measured in untreated or CHO-K1 (A) or CHO-CB2 (B) cells treated with 10 μM forskolin and 100 μM IBMX in the presence or absence of various doses of 5,6-EET-EA for 10 min. Control cells (-) received medium alone. The results are the mean ± S.E. of triplicate cultures. ^***, p < 0.001 compared with vehicle (+) group.

Fig. 4.

Metabolism of anandamide by microglial cells in culture. Unstimulated or BV-2 microglial cells stimulated with IFNγ (10 ng/ml) for 24 h were incubated in serum-free medium containing anandamide (20 μM) for 45 min. Metabolites were extracted and analyzed by ESI-LC/MS as described under Materials and Methods. A, selected ion chromatogram showing monooxygenated anandamide metabolites with mass to charge (m/z) ratios of 364. B, quantitation of 5,6-EET-EA based on a standard curve generated using an authentic standard. The results are the mean ± S.E. from triplicate cultures. ^**, p < 0.01.

Fig. 5.

Induction of anandamide-metabolizing cytochrome P450 enzymes in activated microglial cells. Mouse microglial BV-2 cells were stimulated for 24 h with the treatments indicated. Whole-cell lysates (60 μg protein/lane) from the BV-2 cells were separated on a 4 to 20% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were probed with antibodies against either P450 4F (A), P450 3A (B), or β-actin (A and B) followed by horseradish peroxidase-conjugated secondary antibodies. The signals were detected using the enhanced chemiluminescence system, and band densities were quantitated as described under Materials and Methods. Shown are data obtained from individual experiments with a single replicate per treatment and are representative of at least four independently performed experiments.

Fig. 6.

The effect of ketoconazole upon 5,6-EET-EA and 20-HETE-EA formation by IFNγ-stimulated BV-2 microglia. BV-2 cells stimulated with IFNγ (10 ng/ml) for 24 h were incubated in serum-free medium containing anandamide (10 μM) and either DMSO vehicle (0.01%) or 0.5 μM ketoconazole for 45 min. Metabolites were extracted and analyzed by ESI-LC/MS as described under Materials and Methods. Quantitation of 5,6-EET-EA and 20-HETE-EA was performed based on standard curves generated by using authentic standards. The results are the mean ± S.E. from triplicate cultures. ^**, p < 0.01.

Fig. 7.

Degradation of anandamide and 5,6-EET-EA by mouse brain proteins. Anandamide or 5,6-EET-EA (5 μM) were incubated in the presence of mouse brain homogenate (0.5 mg protein/reaction) in phosphate buffer, pH 7.4, at 37°C for 0 to 180 min. At the designated time points and after the addition of internal standard, the reaction mixtures were extracted with 3 volumes of ethyl acetate and analyzed by ESI-LC/MS as described under Materials and Methods. The amounts of 5,6-EET-EA or anandamide remaining at each time point were plotted as a percentage of the starting amount (at time 0). Control samples contained 0.5 mg of heat-inactivated mouse brain homogenate. ^**, p < 0.01; ^***, p < 0.001 compared with the anandamide group at the same time point.

Fig. 8.

Mouse brain epoxide hydrolase converts 5,6-EET-EA to 5,6-DHET-EA. 5,6-EET-EA (5 μM) was incubated in the presence of mouse brain homogenate (0.5 mg protein/reaction) in phosphate buffer, pH 7.4, at 37°C for 0 to 180 min. Reaction mixtures were extracted with 3 volumes of ethyl acetate and analyzed by ESI-LC/MS as described under Materials and Methods. A, selected ion chromatograms of 5,6-EET-EA and its epoxide hydrolase-derived metabolite 5,6-DHET-EA at times 0 and 120 min. A, inset, 5,6-DHET-EA formation in the presence of various concentrations (1-100 nM) of the soluble epoxide hydrolase inhibitor AUDA. B, time course for the formation of 5,6-DHET-EA from 5,6-EET-EA.