Stimuli such as inflammation or hypoxia induce cytochrome P450 epoxygenase-mediated production of arachidonic acid-derived epoxyeicosatrienoic acids (EETs). EETs have cardioprotective, vasodilatory, angiogenic, antiinflammatory, 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 herein 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 wild-type (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 cytochromes 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. ________________________________________ Cytochrome P450 epoxygenases can oxidize arachidonic acid (AA) to form epoxyeicosatrienoic acids (EETs) which have potent cardiovascular effects. The biological effects of EETs are shortlived in that they are rapidly hydrolyzed to less active dihydroxyeicosatrienoic acids (DHETs) by EPHX2, also known as soluble epoxide hydrolase (sEH) (1). Indeed, Ephx2 -/-mice have increased EETs, decreased DHETs and improved outcomes in vascular disease models, which provide the basis for development of pharmacological EPHX2 inhibitors (1,2). EPHX2 inhibition increases EET levels in vivo, and leads to cardioprotective, vasodilatory, angiogenic, anti-inflammatory and analgesic effects (1-3). EPHX1 Regulates EET Hydrolysis and Postischemic Recovery2 EPHX2 inhibitors, which have completed phase I clinical trials, are under investigation for treatment of neuropathic pain, and may hold promise for treatment of other ailments. However, genetic disruption of Ephx2 or EPHX2 pharmacological inhibition does not completely abolish EET hydrolysis in vivo. Among the EETs, EPHX2 has the biggest effect on levels of its preferred substrate, 14,15-EET, but has diminishing effects on 11,12-and 8,9-...

Recent studies suggest a role for the arachidonic acid-derived epoxyeicosatrienoic acids (EETs) in attenuating epileptic seizures. However, their effect on neurotransmission has never been investigated in detail. Here, we studied how 11,12- and 14,15 EET affect excitability and excitatory neurotransmission in mouse hippocampus. 11,12 EET (2 μM), but not 14,15 EET (2 μM), induced the opening of a hyperpolarizing K conductance in CA1 pyramidal cells. This action could be blocked by BaCl, the G protein blocker GDPβ-S and the GIRK1/4 blocker tertiapin Q and the channel was thus identified as a GIRK channel. The 11,12 EET-mediated opening of this channel significantly reduced excitability of CA1 pyramidal cells, which could not be blocked by the functional antagonist EEZE (10 μM). Furthermore, both 11,12 EET and 14,15 EET reduced glutamate release on CA1 pyramidal cells with 14,15 EET being the less potent regioisomer. In CA1 pyramidal cells, 11,12 EET reduced the amplitude of excitatory postsynaptic currents (EPSCs) by 20% and the slope of field excitatory postsynaptic potentials (fEPSPs) by 50%, presumably via a presynaptic mechanism. EEZE increased both EPSC amplitude and fEPSP slope by 40%, also via a presynaptic mechanism, but failed to block 11,12 EET-mediated reduction of EPSCs and fEPSPs. This strongly suggests the existence of distinct targets for 11,12 EET and EEZE in neurons. In summary, 11,12 EET substantially reduced excitation in CA1 pyramidal cells by inhibiting the release of glutamate and opening a GIRK channel. These findings might explain the therapeutic potential of EETs in reducing epileptiform activity.

The potential complex formation between microsomal epoxide hydrolase (mEH) and cytochrome P450-dependent monooxygenase (CYP) has been a subject of research for many decades. Such an association would enable efficient substrate channeling between CYP and mEH and as such represent an attractive strategy to prevent deleterious accumulation of harmful metabolic by-products such as CYP-generated epoxide intermediates. However, such complex formation is experimentally difficult to prove, because CYP and mEH are membrane-bound proteins that are prone to unspecific aggregation after solubilization. Here, we report the development of a FRET-based procedure to analyze the mEH–CYP interaction in living cells by fluorescence-activated cell sorting. With this non-invasive procedure, we demonstrate that CYP2J5 and mEH associate in the endoplasmic reticulum of recombinant HEK293 cells to the same extent as do CYP2J5 and its indispensible redox partner cytochrome P450 reductase. This presents final proof for a very close proximity of CYP and mEH in the endoplasmic reticulum, compatible with and indicative of their physical interaction. In addition, we provide with FAMPIR a robust and easy-to-implement general method for analyzing the interaction of ER membrane-resident proteins that share a type I topology.Electronic supplementary materialThe online version of this article (doi:10.1007/s00204-017-2072-0) contains supplementary material, which is available to authorized users.