Hydrogen Peroxide Inhibits Cytochrome P450 Epoxygenases

Abstract

Abstract-The cytochrome P450 epoxygenase (CYP)-derived metabolites of arachidonic acid the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H 2 O 2 ) both function as endothelium-derived hyperpolarizing factors (EDHFs) in the human coronary microcirculation. However, the relative importance of and potential interactions between these 2 vasodilators remain unexplored. We identified a novel inhibitory interaction between CYPs and H 2 O 2 in human coronary arterioles, where EDHF-mediated vasodilatory mechanisms are prominent. Bradykinin induced vascular superoxide and H 2 O 2 production in an endothelium-dependent manner and elicited a concentration-dependent dilation that was reduced by catalase but not by 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE), 6-(2-propargyloxyphenyl)hexanoic acid, sulfaphenazole, or iberiotoxin. However, in the presence of catalase, an inhibitory effect of these compounds was unmasked.In a tandem-bioassay preparation, application of bradykinin to endothelium-intact donor vessels elicited dilation of downstream endothelium-denuded detectors that was partially inhibited by donor-applied catalase but not by detector-applied EEZE; however, EEZE significantly inhibited dilation in the presence of catalase. EET production by human recombinant CYP 2C9 and 2J2, 2 major epoxygenase isozymes expressed in human coronary arterioles, was directly inhibited in a concentration-dependent fashion by H 2 O 2 in vitro, as observed by high-performance liquid chromatography (HPLC); however, EETs were not directly sensitive to oxidative modification. H 2 O 2 inhibited dilation to arachidonic acid but not to 11,12-EET. These findings suggest that an inhibitory interaction exists between 2 EDHFs in the human coronary microcirculation. CYP epoxygenases are directly inhibited by H 2 O 2 , and this interaction may modulate vascular EET bioavailability. (Circ Res. 2008;102:59-67.)Key Words: endothelium-derived hyperpolarizing factor Ⅲ hydrogen peroxide Ⅲ epoxyeicosatrienoic acid Ⅲ cytochrome P450 Ⅲ reactive oxygen species T he vascular endothelium releases numerous vasodilatory substances, including nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factor (EDHF). EDHF is an important modulator of vasomotor tone, particularly in the microcirculation. 1,2 Despite more than a decade of investigation, the chemical identity of EDHF remains controversial, and multiple EDHFs have been proposed. Among these, substantial evidence now points to epoxyeicosatrienoic acids (EETs), [3][4][5] cytochrome P450 epoxygenase (CYP)-derived metabolites of arachidonic acid (AA), as EDHFs with another prominent candidate being the reactive oxygen species (ROS) hydrogen peroxide (H 2 O 2 ). 6,7 It has been reported that a significant inhibitory interaction occurs between NO and EET, in which NO inhibits CYPmediated production of EET from AA. 8 Such an interaction presumably exists to conserve vasodilator substances, while at the same time providing a compensatory mechanism of dilation when one is impaired. It is...

Arachidonic acid cytochrome P450 epoxygenase pathway

“…EETs produce autacrine and paracrine effects, but these actions often are overlooked in experimental studies because CYP epoxygenases are labile in cultured cells and are inhibited under conditions where H 2 O 2 is formed (18). The paracrine effects are produced by the EET released into the extracellular fluid (Fig.…”

The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to atherogenesis in mice and humans

“…ECs were removed without detachment of plaques by gently rubbing the intimal surface with a moist cotton swab under a microscope, which was followed by rinsing, as previously reported (23). ECs in HCAs and mouse arteries were also mechanically denuded using a modification of a method used previously by us (53)(54)(55). Removal of ECs was confirmed by lack of immunostaining for vWF and by abolishment of EC-dependent vasodilation and is described in Supplemental Methods and Supplemental Figure 3.…”

“…HCAs (150~1,000 μm internal diameter, 1-2 mm length; n = 26) were isolated as reported previously (53)(54)(55). The schedules and procedures of cardiac surgery (coronary artery bypass graft, valve replacement, and congenital repair) were obtained from the hospitals mentioned below.…”

“…ies were examined by videomicroscopy as previously described (53)(54)(55). Vessels pressurized at 60 mmHg for HCAs and at 40 mmHg for mouse arteries were preconstricted with acetylcholine (HCAs) or U46619 (mouse arteries), and vasodilations to 1-EBIO and pimaric acid were studied.…”

“…Fluorescence microscopy was performed as previously described (55). Changes in superoxide generation in vessels and VSMCs were assessed by measuring dihydroethidine fluorescence intensity.…”