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CARDIOVASCULAR

Hydrolysis of cis- and trans-Epoxyeicosatrienoic Acids by Rat Red Blood Cells

Department of Pharmacology, New York Medical College, Valhalla, New York (H.J., A.G.Z., M.M., J.C.M.); Entomology and Cancer Center, University of California Davis, Davis, California (C.M., B.D.H.); and Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas (J.R.F.)

Erythrocytes serve as reservoirs for cis- and trans-epoxyeicosatrienoic acids (EETs). Incubation of rat red blood cells (RBCs) with cis- and trans-EETs produces threo- and erythro-dihydroxyeicosatrienoic acids, respectively. The V_max of EET hydrolysis by rat intact RBCs (2.35 ± 0.24 pmol/min/10⁸ RBCs for 14,15-trans-EET) decreased by approximately 2 to 3-fold sequentially from 14,15-, 11,12- to 8,9-EETs for both cis- and trans-isomers. The V_max of trans-EET hydrolysis by RBCs is approximately 2 to 3 times that of the corresponding cis-EETs. Incubation of EETs with recombinant murine soluble epoxide hydrolase (sEH) yielded the same geometric and regio preferences of EET hydrolysis as with rat intact RBCs. The principal epoxide hydrolase activity for EET hydrolysis (approximately 90%) is present in the erythrocyte cytosol. Western blots of sEH suggested a concentration of sEH protein to be approximately 2 µg/mg protein or 0.4 µg/10⁹ RBCs. The apparent K_m values of EETs were between 1 and 2 µM, close to the K_m for purified sEH as reported. Erythrocyte hydration of cis- and trans-EETs was blocked by sEH inhibitors, 1,3-dicyclohexylurea and 4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid. Erythrocyte sEH activity was inhibited more than 80% by 0.2% bovine serum albumin in the buffer. Preferred hydrolysis of 14,15-EETs and trans-epoxides characterizes sEH activity in RBCs that regulates the hydrolysis and release of cis- and trans-EETs in the circulation. Inhibition of sEH has produced antihypertensive and antiinflammatory effects. Because plasma trans-EETs would increase more than cis-EETs with sEH inhibition, the potential roles of trans-EETs and erythrocyte sEH in terms of circulatory regulation deserve attention.