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. 2008 Jan 4;102(1):59-67.
doi: 10.1161/CIRCRESAHA.107.159129. Epub 2007 Nov 1.

Hydrogen peroxide inhibits cytochrome p450 epoxygenases: interaction between two endothelium-derived hyperpolarizing factors

Brandon T Larsen  1 , David D Gutterman, Atsushi Sato, Kazuyoshi Toyama, William B Campbell, Darryl C Zeldin, Vijay L Manthati, John R Falck, Hiroto Miura
Affiliations

Hydrogen peroxide inhibits cytochrome p450 epoxygenases: interaction between two endothelium-derived hyperpolarizing factors

Brandon T Larsen et al. Circ Res. .

Abstract

The cytochrome P450 epoxygenase (CYP)-derived metabolites of arachidonic acid the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H2O2) 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 H2O2 in human coronary arterioles, where EDHF-mediated vasodilatory mechanisms are prominent. Bradykinin induced vascular superoxide and H2O2 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 H2O2 in vitro, as observed by high-performance liquid chromatography (HPLC); however, EETs were not directly sensitive to oxidative modification. H2O2 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 H2O2, and this interaction may modulate vascular EET bioavailability.

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Figures

Figure 1
Figure 1
BK-induced dilation of HCAs in the presence of CYP/EET inhibitors or catalase. A, BK induces dilation of HCAs that is not inhibited by sulfaphenazole, EEZE, iberiotoxin, or 6-(2-propargyloxyphenyl)hexanoic acid (PPOH) (n=31, 6, 5, 6, and 7, respectively), indicating little contribution of EETs to this response. B, BK-induced dilation is reduced by catalase (n=31 and 21, respectively; *P<0.05 vs vehicle), indicating that H2O2 partially mediates this response. n indicates number of patients.
Figure 2
Figure 2
BK-induced ROS production in isolated HCAs. A, Representative histofluorescence images of HCAs incubated with DHE and DCFH to visualize superoxide and H2O2 production, respectively, in response to BK. Some vessels were denuded of endothelium or treated with catalase. Bar=100 μm. B, BK induces superoxide and H2O2 production in HCAs, as evidenced by the increased ratio of DHE and DCFH fluorescence intensity (I) vs baseline (I0), respectively (n=7). BK-induced ROS production is endothelium dependent (n=4). Catalase inhibits the increase in BK-induced DCFH fluorescence, indicating specificity for H2O2 (n=4). n indicates number of patients. *P<0.05 vs vehicle, †P<0.05 vs BK+vehicle.
Figure 3
Figure 3
Detection of an EET-mediated component of BK-induced dilation of HCAs in the presence of catalase. A, BK-induced dilation of HCAs in the presence of catalase is inhibited by sulfaphenazole, EEZE, and iberiotoxin (n=21, 5, 5, and 4, respectively; *P<0.05 vs catalase+vehicle), suggesting that EETs mediate BK-induced dilation when H2O2 is reduced. B, Diagram of the dual-cannulated, tandem-perfused HCA bioassay preparation. Endothelium-denuded detector vessels were perfused downstream of endothelium-intact donor vessels. Dilation of detectors was monitored by videomicroscopy. C, Bioassay detection of transferable vasodilators released from HCA endothelium. Donor-applied BK induces dilation of detectors (n=5) in a manner that is partially inhibited by donor-applied catalase (n=5) but not by detector-applied EEZE (n=5). However, an inhibitory effect of detector-applied EEZE is unmasked in the presence of donor-applied catalase (n=5), suggesting that EETs represent transferable vasodilators, the release of which is enhanced when H2O2 is reduced. n indicates number of patients. *P<0.05 vs BK+vehicles, †P<0.05 vs BK+donor catalase+detector vehicle.
Figure 4
Figure 4
Effect of H2O2 on 14C-AA metabolism by microsomes containing recombinant human CYP2C9 or CYP2J2. A and C, Representative chromatograms following separation of metabolites by reverse-phase HPLC. Migration times of authentic standards are noted on each chromatogram. CPM indicates counts per minute. B and D, EET formation by CYP2C9 and CYP2J2 is inhibited in a concentration-dependent fashion by H2O2 (n=3 to 8 at each concentration; *P<0.05 vs respective vehicle). n indicates number of experiments.
Figure 5
Figure 5
Direct effect of ROS on 3H-14,15-EET in vitro. A through D, Representative HPLC chromatograms following incubation of 3H-14,15-EET with H2O2 or the superoxide-generating system xanthine plus xanthine oxidase (X+XO). 14,15-EET is not directly oxidized by H2O2 or superoxide (n=3). CPM indicates counts per minute. n indicates number of experiments.
Figure 6
Figure 6
Effect of H2O2 on AA- or 11,12-EET–induced dilation of HCAs. A, AA-induced dilation of endothelium-intact HCAs is reduced by transient exposure to 10−5 mol/L H2O2 (n=7, *P<0.05 vs vehicle), suggesting that H2O2 interferes with the synthesis and/or action of EETs. B, 11,12-EET–induced dilation of endothelium-denuded HCAs is not inhibited by H2O2 (n=6), suggesting that H2O2 does not interfere with the downstream action of EETs on VSMCs. n indicates number of vessels.
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