Identification of 13-hydroxy-14,15-epoxyeicosatrienoic acid as an acid-stable endothelium-derived hyperpolarizing factor in rabbit arteries

Arachidonic acid (AA) is metabolized by endothelial 15-lipoxygenase (15-LO) to several vasodilatory eicosanoids such as 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA) and its proposed unstable precursor 15-hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). In the present study, the acid-stable 13-hydroxy-trans-14,15-epoxy-eicosatrienoic acid (13-H-14,15-EETA) was identified and its vascular activities characterized. Rabbit aorta, mesenteric arteries, and the combination of 15-LO and cytochrome P450 2J2 converted AAto two distinct HEETA metabolites. The HEETA metabolites were resistant to acidic hydrolysis but were hydrolyzed by recombinant sEH to a more polar metabolite identified by mass spectrometry as 13,14,15-THETA. Mass spectrometric analyses and HPLC comigration identified the HEETAs as threo- and erythro-diastereomers of 13-H-trans-14,15-EETA. Erythro- and threo-diastereomers of 13-H-trans-14,15-EETA relaxed endothelium-denuded rabbit small mesenteric arteries with maximum relaxations of 22.6±6.0% and 8.6±4.3%, respectively. Apamin (10^-7 M) inhibited the relaxations to the erythro-isomer(maximumrelaxation=1.2±5.6%) and increasing [K⁺]_o from 4.6 to 30 mM blocked relaxations to both isomers. In cell-attached patches of mesenteric arterial smooth muscle cells (SMCs), erythro-13-H-trans-14,15-EETA (1-3 × 10^-6 M) increased mean open time of small conductance K⁺ channels (13-14 pS) from 0.0007 ± 0.0007 to 0.0053 ± 0.0042. This activation was inhibited by apamin. The erythro, but not the threo, isomer blocked angiotensin II-stimulated aortic SMC migration. These studies demonstrate that 13-H-14,15-EETAs induces vascular relaxation via K⁺ channel activation to cause SMC hyperpolarization. Thus, 13-H-14,15-EETA represents a new endothelial factor.