Abstract 1136: Hydrogen Peroxide Modulates Bioavailability of Epoxyeicosatrienoic Acids in the Human Coronary Microcirculation by Directly Inhibiting Cytochrome P450 Epoxygenases
Epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP)-derived metabolites of arachi- donic acid (AA). In human coronary arterioles (HCAs), endogenous H2O2 reduces CYP-mediated dilation to bradykinin (BK) by an unknown mechanism. We investigated whether H2O2 directly inhibits CYPs to reduce EET production. In vitro metabolism of 14C-AA by human recombinant CYP2C9 and CYP2J2 was assayed by HPLC. P450 reductase activity was determined using a cytochrome c reduction assay. HCAs from atrial appendages were cannulated and diameter changes were measured by videomicroscopy in the presence of L-NAME (nitric oxide synthase inhibitor) and indomethacin (cyclooxygenase inhibitor). EET production by CYP2C9 was inhibited in a concentration-dependent manner by H2O2 (IC50 = 13±3 μm, n = 4). Interestingly, CYP2J2 displayed greater sensitivity to H2O2 (IC50 = 0.3±0.05 μm, n = 3). Similar concentrations of H2O2 did not inhibit P450 reductase. Dithiothreitol, a thiol reductant, reversed the H2O2-induced inhibition of CYP2C9 but not CYP2J2, suggesting that H2O2 oxidizes key thiol groups on CYP2C9 but does not inhibit CYP2J2 by the same mechanism. Transient exposure to H2O2 reduced dilation of endothelium-intact HCAs to AA (% max dilation 44±13% vs. 86±9%, p<0.05, n=7). However, dilation of endothelium-denuded HCAs to 11,12-EET was not inhibited by H2O2 (68±7% vs. 66±8%, n=6, p=ns). Endothelium-denuded detector HCAs were perfused downstream of endothelium-intact donors as a bioassay. BK, which stimulates endothelial H2O2 formation, produced detector dilation when added to the donor, but not when added to the detector (56±4% vs. 3±1%, respectively, n=5, p<0.05). Detector dilation to donor-applied BK was partially inhibited by donor-applied catalase (31±3%, n=5, p<0.05), and the residual dilation was abolished when 14,15-EEZE (EET antagonist) was added to the detector (7±6%, n=5, p<0.05). However, in the absence of donor-applied catalase, detector-applied 14,15-EEZE had no effect (53±8%, n=5, p=ns), suggesting that EETs represent a transferable vasodilator whose release is inhibited by endogenous H2O2. We conclude that CYPs are directly inhibited by H2O2, and that this interaction may modulate EET bioavailability in the human coronary microcirculation.