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 Ki 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 μM 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 (IC50 = 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-γ 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.
Footnotes
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This work was supported in part by the National Institutes of Health National Cancer Institute [Grant CA16954]; the National Institutes of Health National Institute of General Medical Sciences [Grant T32-GM007767]; a predoctoral fellowship support from Merck and Co., Inc.; and a Howard Hughes Medical Institute through an Undergraduate Science Education Program to Kalamazoo College [Award 52005128].
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ABBREVIATIONS: CNS, central nervous system; AM1241, (2-iodo-5-nitrophenyl)-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl]-methanone; AUDA, 2-(3-adamantan-1-yl-ureido)-dodecanoic acid; CB, cannabinoid receptor; CHO, Chinese hamster ovary; CP-55940, 5-(1,1-dimethylheptyl)-2-[(1R,2R,5R)-5-hydroxy-2-(3-hydroxy-propyl)-cyclohexyl]-phenol; DHET, dihydroxyeicosatrienoic acid; EA, ethanolamide; EET, epoxyeicosatrienoic acid; ESI-LC/MS, electrospray ionization-liquid chromatography/mass spectrometry; FAAH, fatty acid amide hydrolase; HETE, hydroxyeicosatetraenoic acid; IBMX, 3-isobutyl-1-methylxanthine; IFNγ, interferon γ; LPS, lipopolysaccharide; P450, cytochrome P450; WIN-55212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-napthalenylmethanone; TME, Tris/MgCl2/EDTA; TMEB, Tris/MgCl2/EDTA/bovine serum albumin; 5,6-EET-EA, 5,6-epoxyeicosatrienoic acid ethanolamide; 5,6-DHET-EA, 5,6-dihydroxyeicosatrienoic acid ethanolamide.
- Received November 13, 2008.
- Accepted January 26, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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