Endocannabinoids Anandamide and 2-Arachidonoylglycerol Are Substrates for Human CYP2J2 Epoxygenase

Abstract

The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are arachidonic acid (AA) derivatives that are known to regulate human cardiovascular functions. CYP2J2 is the primary cytochrome P450 in the human heart and is most well known for the metabolism of AA to the biologically active epoxyeicosatrienoic acids. In this study, we demonstrate that both 2-AG and AEA are substrates for metabolism by CYP2J2 epoxygenase in the model membrane bilayers of nanodiscs. Reactions of CYP2J2 with AEA formed four AEAepoxyeicosatrienoic acids, whereas incubations with 2-AG yielded detectable levels of only two 2-AG epoxides. Notably, 2-AG was shown to undergo enzymatic oxidative cleavage to form AA through a NADPH-dependent reaction with CYP2J2 and cytochrome P450 reductase. The formation of the predominant AEA and 2-AG epoxides was confirmed using microsomes prepared from the left myocardium of porcine and bovine heart tissues. The nuances of the ligand-protein interactions were further characterized using spectral titrations, stoppedflow small-molecule ligand egress, and molecular modeling. The experimental and theoretical data were in agreement, which showed that substitution of the AA carboxylic acid with the 2-AG ester-glycerol changes the binding interaction of these lipids within the CYP2J2 active site, leading to different product distributions. In summary, we present data for the functional metabolomics of AEA and 2-AG by a membrane-bound cardiovascular epoxygenase.

Anti-inflammatory ω-3 endocannabinoid epoxides

“…Because the rat CYP epoxygenases are enzymatically different from human CYP epoxygenases, we also demonstrated a similar metabolism profile using porcine brain microsomes (SI Appendix, Fig. S1), which contain a CYP2J isozyme with homology similar to human CYP2J2 (28). Taken together, these findings suggest that similar CYP epoxygenases are implicated in the metabolism of both DHEA and EPEA and that there is competition for the substrates among the various CYP isoforms in rat brain microsomes.…”

“…3D). The total turnover rates of AEA, EPEA, and DHEA are significantly greater than those of AA, EPA, and DHA (28,32,56). Specifically, maximal CYP2J2 conversion of DHA to 19,20-EDP was calculated at 22.5 pmol·min −1 ·pmol −1 protein (57), whereas the CYP2J2 conversion of DHEA to 19,20-EDP-EA was 542.3 ± 44.6 5 pmol·min −1 ·pmol −1 protein, thus demonstrating that the preference for the DHEA substrate is an order of magnitude greater than the preference for DHA.…”

“…CYP2J2 was recombinantly expressed and was incorporated with its redox partner into nanodiscs (28). The first indication that the CYP2J2-CPR nanodiscs could use EPEA and DHEA as substrates was found using LC-electrospray ionization (ESI)-MS (SI Appendix, Fig.…”

“…Human CYP2J2 epoxygenates AEA and 2-arachidonoylglycerol (2-AG) (28) and is physiologically relevant, because it is the second most highly expressed P450 in the human brain (29) and is the most highly expressed P450 in human cardiomyocytes (30). However, the cross-species studies are not completely translatable, because BV-2 microglial cells are derived from mice, which have 10 subfamily CYP2J isozymes (CYP2J-5, -6, -7, -8, -9, -11, -12, -13, -14, -15) but lack a comparable CYP2J2 isozyme, making comparative species studies more difficult (31).…”

The Endocannabinoid System and its Modulation by Phytocannabinoids

“…Therefore, the overlap between the "endocannabinoidome" and the "phytocannabinoidome" is only partial. Further interactions can be predicted on the basis of the capability of both polyunsaturated endocannabinoid-like mediators and plant cannabinoids to inhibit, or to be oxidized by, cytochrome p450 oxygenases [55,56], which, in theory, allows the latter compounds, when administered systemically, to modulate the levels of the former. Finally, CBD acid, and much less so THC acid, were reported to inhibit cycloxygenase-2 [57], thus potentially inhibiting the formation not only of prostanoids, but also of prostamides and prostaglandin-glycerol esters.…”