Abstract
The endocannabinoid system plays an important role in numerous physiological processes including mood, appetite, and pain sensation. A critical compound in maintaining cannabinoid tone is the endocannabinoid anandamide (AEA). We have recently shown that AEA is metabolized by several different human cytochromes P450 (P450) to form a number of metabolites, one of which exhibits increased biological activity. CYP3A4, one of the major P450s involved in the metabolism of AEA, produces four major metabolites. One of these metabolites, 5,6-epoxyeicosatrienoic acid ethanolamide (5,6-EET-EA), exhibits a much higher affinity than AEA for the cannabinoid 2 receptor (CB-2), which leads to a marked decrease in intracellular cAMP levels in cells expressing CB-2. There are multiple human alleles of CYP3A4, and the CYP3A4.4 allele has been shown to exhibit a significant decrease in activity. Recombinant CYP3A4*4 was expressed in Escherichia coli and was demonstrated to produce 60% less 6-hydroxytestosterone than the wild-type (WT) 3A4 in a reconstituted system. The metabolism of AEA by the WT and the CYP3A4.4 variant was investigated. The mutant produced 60% less of the four EET-EA metabolites than the WT. The mutant also produced a new peak on liquid chromatography-mass spectrometry not seen with the WT, which corresponded to 19-hydroxyeicosatetraenoic acid-ethanolamide. In addition, the mutant produces four novel peaks at m/z 380, which correspond to the addition of two oxygen atoms, possibly to form a peroxide bond. These data indicate that individuals expressing the CYP3A4.4 allele may exhibit significant variations in the metabolism of AEA as well as any other compounds resembling AEA.
Footnotes
This work was supported in part by the National Institutes of Health National Institute on Drug Abuse [Grant DA007268-18] (Biology of Drug Abuse Postdoctoral Training Fellowship; to M.P.-H.); and the National Institutes of Health National Cancer Institute [Grant CA016954] (to P.F.H.). N.T.S. was supported by the Michigan Institute for Clinical and Health Research Postdoctoral Translational Scholars Program [Award UL1-RR024986].
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.110.033712.
↵ The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- CB
- cannabinoid receptor
- AEA
- anandamide
- P450
- cytochrome P450
- EET
- epoxyeicosatrienoic acid
- EA
- ethanolamide
- HETE
- hydroxyeicosatetraenoic acid
- OH
- hydroxy
- ESI
- electrospray ionization
- LC/MS
- liquid chromatography/mass spectrometry
- EH
- epoxide hydrolase
- CPR
- cytochrome P450 reductase.
- Received April 1, 2010.
- Accepted August 11, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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