Abstract
Stimuli such as inflammation or hypoxia induce cytochrome P450 epoxygenase-mediated production of arachidonic acid-derived epoxyeicosatrienoic acids (EETs). EETs have cardioprotective, vasodilatory, angiogenic, anti-inflammatory, and analgesic effects, which are diminished by EET hydrolysis yielding biologically less active dihydroxyeicosatrienoic acids (DHETs). Previous in vitro assays have suggested that epoxide hydrolase 2 (EPHX2) is responsible for nearly all EET hydrolysis. EPHX1, which exhibits slow EET hydrolysis in vitro, is thought to contribute only marginally to EET hydrolysis. Using Ephx1-/-, Ephx2-/-, and Ephx1-/-Ephx2-/- mice, we show here that EPHX1 significantly contributes to EET hydrolysis in vivo Disruption of Ephx1 and/or Ephx2 genes did not induce compensatory changes in expression of other Ephx genes or CYP2 family epoxygenases. Plasma levels of 8,9-, 11,12-, and 14,15-DHET were reduced by 38, 44, and 67% in Ephx2-/- mice compared with wildtype (WT) mice, respectively; however, plasma from Ephx1-/-Ephx2-/- mice exhibited significantly greater reduction (100, 99, and 96%) of those respective DHETs. Kinetic assays and FRET experiments indicated that EPHX1 is a slow EET scavenger, but hydrolyzes EETs in a coupled reaction with cytochrome P450 to limit basal EET levels. Moreover, we also found that EPHX1 activities are biologically relevant, as Ephx1-/-Ephx2-/- hearts had significantly better postischemic functional recovery (71%) than both WT (31%) and Ephx2-/- (51%) hearts. These findings indicate that Ephx1-/-Ephx2-/- mice are a valuable model for assessing EET-mediated effects, uncover a new paradigm for EET metabolism, and suggest that dual EPHX1 and EPHX2 inhibition may represent a therapeutic approach to manage human pathologies such as myocardial infarction.
Highlights
We examined fatty acid epoxide levels and metabolism in tissues and body fluids from WT, Ephx1Ϫ/Ϫ, Ephx2Ϫ/Ϫ, and
This manuscript contains several important and novel findings regarding the functional role of epoxide hydrolases: 1) disruption of both EPHX1 and EPHX2 almost completely abolishes epoxyeicosatrienoic acids (EETs) hydrolysis in mouse plasma; 2) in vitro kinetic assays demonstrate that, relative to EPHX2, EPHX1 is a poor EET scavenge; 3) EPHX1 plays a prominent role in EET hydrolysis when EET formation rates are low; 4) disruption of both EPHX1 and EPHX2 suppresses EET hydrolysis in mouse heart; and 5) Ephx1Ϫ/ϪEphx2Ϫ/Ϫ hearts have significantly improved recovery of cardiac function after ischemia compared with both WT or Ephx2Ϫ/Ϫ hearts
The contribution of EPHX1 to EET hydrolysis is surprising in several respects
Summary
We examined fatty acid epoxide levels and metabolism in tissues and body fluids from WT, Ephx1Ϫ/Ϫ, Ephx2Ϫ/Ϫ, and. Compared with WT, Ephx1Ϫ/ϪEphx2Ϫ/Ϫ mice had significantly higher levels of every CYP-derived epoxide measured except 5,6-EET. Compared with Ephx2Ϫ/Ϫ mice, plasma from Ephx1Ϫ/ϪEphx2Ϫ/Ϫ mice had significantly higher levels of 11,12-EET, 17,18-EpETE, and 19,20-EpDPE, and significantly lower levels of all diols measured except 12,13-DiHOME. These data suggest functional compensation of EPHX1 and EPHX2 with regard to fatty acid epoxide hydrolysis in vivo. Ephx1Ϫ/ϪEphx2Ϫ/Ϫ lysates had a small additional decrease in 11,12-EET hydrolysis compared with Ephx2Ϫ/Ϫ lysates These data suggest that EPHX1 is responsible for only a small fraction (1– 4%) of 11,12-EET hydrolysis in the liver under these experimental conditions. The role of EPHX1 with regard to EET hydrolysis is unmasked in the absence of EPHX2
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