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

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.

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

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

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

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

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

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.