Epoxyeicosatrienoic acids as antagonists of prostanoid receptor dependend contraction

The endothelium is important for vascular homeostasis as it tightly regulates the production of vasoactive metabolites that control vascular pressure and blood flow. Nitric oxide, prostacyclin and endothelium-derived hyperpolarizing factors are important vasodilating principles that compete with potent vasoconstrictors such as thromboxane or endothelin-1 to determine vascular tone. Arachidonic acid (AA, 20 carbons Ѡ-6 PUFA) is a precursor of several vasoactive lipids. When metabolized by cyclooxygenases, AA generates prostaglandins and thromboxane. The cytochrome P450 monooxygenases (CYP450) metabolize AA to epoxyeicosatrienoic acids (EETs) through epoxidation of any of its four double bounds. This process generates four isomers: 5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET. The soluble epoxide hydrolase can inactivate these isomers by forming the corresponding dihydroxyeicosatrienoic acids. The aim of this study was to characterize EETs as antagonists of prostanoid receptors and elucidate associated changes in intracellular signaling. For this we generated an endothelial cell-specific, tamoxifen-inducible knockout mouse model of the CYP450 reductase (POR, ecPOR-/-). The rational of this was that POR is the only enzyme to catalyze electron transfer to all CYP450 enzymes. It is therefore vital for their function. Thus, this model is a novel approach to study EETs through inhibiting the function of all endothelial CYP450 enzymes. Organ chamber experiments in isolated vessels showed that ecPOR-/- mice have a lower constriction threshold to U46619, a thromboxane receptor agonist, and prostaglandin E2 (PGE2). This effect could be abolished by incubation of ecPOR-/- with 14,15-EE-8(Z)-E (EEZE), a stable EET analog. While EETs were unable to induce relaxation in phenylephrine pre-constricted vessels, U46619 and PGE2 induced constrictions were reversed. Incubation of aortic rings with Rho-kinase inhibitor SAR407899 showed a significant decrease in constriction response to U46619. RTqPCR experiments showed a compensatory upregulation of Rho-Kinase after 24h incubation with EEZE in CTR mice, while prostanoid receptor levels remained unchanged. In conclusion, we show that EETs antagonize prostanoid receptor-dependent contraction. Additionally, we demonstrate that aortae from endothelial cell-specific POR knockout mice have a lower constriction threshold for the thromboxane receptor agonist U46619 and PGE2, which is likely mediated through changes in the Rho signaling system. This effect was abolished after incubation with 14,15-EE-8(Z)-E, suggesting that EETs and prostanoids crosstalk at receptor and intracellular signaling levels.