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, 57 (46), 6489-6499

Endocannabinoid Virodhamine Is an Endogenous Inhibitor of Human Cardiovascular CYP2J2 Epoxygenase

Endocannabinoid Virodhamine Is an Endogenous Inhibitor of Human Cardiovascular CYP2J2 Epoxygenase

Lauren N Carnevale et al. Biochemistry.

Abstract

The human body contains endogenous cannabinoids (endocannabinoids) that elicit effects similar to those of Δ9-tetrahydrocanabinol, the principal bioactive component of cannabis. The endocannabinoid virodhamine (O-AEA) is the constitutional isomer of the well-characterized cardioprotective and anti-inflammatory endocannabinoid anandamide (AEA). The chemical structures of O-AEA and AEA contain arachidonic acid (AA) and ethanolamine; however, AA in O-AEA is connected to ethanolamine via an ester linkage, whereas AA in AEA is connected through an amide linkage. O-AEA is involved in regulating blood pressure and cardiovascular function. We show that O-AEA is found at levels 9.6-fold higher than that of AEA in porcine left ventricle. On a separate note, the cytochrome P450 (CYP) epoxygenase CYP2J2 is the most abundant CYP in the heart where it catalyzes the metabolism of AA and AA-derived eCBs to bioactive epoxides that are involved in diverse cardiovascular functions. Herein, using competitive binding studies, kinetic metabolism measurements, molecular dynamics, and wound healing assays, we have shown that O-AEA is an endogenous inhibitor of CYP2J2 epoxygenase. As a result, the role of O-AEA as an endogenous eCB inhibitor of CYP2J2 may provide a new mode of regulation to control the activity of cardiovascular CYP2J2 in vivo and suggests a potential cross-talk between the cardiovascular endocannabinoids and the cytochrome P450 system.

Conflict of interest statement

CONFLICTS OF INTEREST

Authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1.
Figure 1.. Metabolism of O-AEA and AEA by CYP2J2-CPR Nanodiscs
(A) Schematic of CYP2J2-CPR-nanodisc system showing metabolism of O-AEA to O-AEA epoxides (CPR, cyan; CYP2J2, ice blue) (B) Total metabolism of O-AEA by CYP2J2-CPR-Nanodiscs. (C) O-AEA structure (red, closest interactions to heme group; blue, furthest interactions from heme group) metabolism of O-AEA by CYP2J2 showing the rate of reaction for each regioisomer. The part of O-AEA containing the 5,6- and 8,9-alkenes as well as the ester bond and amine was shown to interact more closely with the heme of CYP2J2 than the 11,12-alkene to the terminal methyl. (D) Metabolism of AEA by CYP2J2-CPR nanodiscs in the absence and presence of O-AEA. (E) Plot of AEA metabolism to the EET-EAs alone (black curve) and in the presence of 10 μM (solid blue curve) and 50 μM (dotted blue curve). Total metabolism fits best to a competitive inhibition model. Rates are in units of pmol of product per minute per nmol of CYP2J2. (F) Table of kinetic parameters for AEA metabolism alone and in the presence of O-AEA.
Figure. 2.
Figure. 2.. Spectral Interactions of O-AEA at CYP2J2.
(A) Structure of Virodhamine (O-AEA) and Ebastine (EBS). (B) Binding of O-AEA to CYP2J2 Nanodiscs (0.2 μM CYP2J2). (C) Binding of EBS to CYP2J2 Nanodiscs (0.2 μM CYP2J2 and competitive binding of O-AEA (30 μM) to CYP2J2 Nanodiscs (0.2 μM CYP2J2) in the presence of EBS (0–155 μM).
Figure 3
Figure 3. Interactions of O-AEA to CYP2J2.
(A) MD simulations of O-AEA show a strong and consistent interaction between O-AEA and glutamate 314. (B) To identify the key residues, an MD contact analysis was performed where the y-axis corresponds to the normalized interaction counts throughout the trajectory. The results show E314 as the main contributor to binding for O-AEA, as well as some contributions from nearby residues, mainly F310 and T219, which likely aids in stabilizing binding. (C) Time series of the interaction energy between O-AEA and selected residues for the last 100 ns of the three molecular dynamics trajectories that see binding. The average interaction energy of E314 was calculated to be – 86.2 ± 14.8 kcal/mol, with additional interaction energies for every other residue listed in the supplemental material.
Figure 4.
Figure 4.. Inhibition of CYP2J2-mediated wound healing by O-AEA.
(A) Cell viability of HUVECs incubated alone (control) or with O-AEA (20 μM) and/or hCYP2J2 as determined by MTT assays over a 24 h period. (B) Wound healing assay shown in hCYP2J2-transfected cells at 0 h and 24 h in the absence or presence of O-AEA (20 μM). (C) Percent wound closure in wild type HUVECs in the absence (control) and presence of O-AEA (20 μM). Cellular wound healing was observed using a microscope and images were processed using the Image J plugin. (D) Wound healing in transfected HUVEC is represented as a percentage of wound closed in treatment relative to control HUVECs without hCYP2J2. (E) Dose-dependent inhibition of wound healing via O-AEA in hCYP2J2-transfected HUVEC cells. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001 ****p ≤ 0.0001 calculated using two-way ANOVA with Tukey’s multiple comparisons test (GraphPad Prism version 6.00); n (sample size) =3.

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