Background and purpose: Red blood cells (RBCs) are reservoirs of vasodilatory, antiaggregatory, and antiinflammatory lipid mediators-epoxyeicosatrienoic acids (EETs). This study addresses the formation and release of erythrocyte-derived EETs in response to ATP receptor stimulation that may represent an important mechanism regarding circulatory regulation. Experimental approach: Erythrocyte EET formation and release were investigated by incubating rat RBCs in physiological salt solution with agents that effected ATP release via P2 receptor stimulation of phospholipase A2 and epoxygenase-like activities with activation of the ATP secretory mechanism. EETs were analyzed by gas and liquid chromatography-mass spectrometry. Key results: EETs were released from rat RBCs: 14, 11, 8,6-EETs in a ratio of 1.2:1.0:0.9:0.8. EETs were produced by epoxidation of arachidonic acid catalyzed by hemoglobin. Spontaneous release of EETs, 0.6670.14 ng per 10 9 RBCs, was dose-dependently increased by an ATP analog, BzATP, and inhibited by P2X 7 receptor antagonists. 5 mM ATP increased release of EETs over 20% to 0.8370.15 ng per 10 9 RBCs; 10 mM BzATP tripled the amount of EET release to 1.8770.20 ng per 10 9 RBCs. EET release by ATP or BzATP was not associated with hemolysis. Carbenoxolone, a gap junction inhibitor that inhibits ATP release, and glibenclamide, an inhibitor of the cystic fibrosis transmembrane conductance regulator (CFTR), which is required for ATP release, inhibited the spontaneous and stimulated EET release from RBCs. Conclusions and implications: EETs are produced and released from RBCs via a mechanism that is mediated by ATP stimulation of P2X 7 receptors coupled to ATP transporters, pannexin-1 and CFTR.

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 Ϯ 0.24 pmol/min/10 8 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 g/mg protein or 0.4 g/10 9 RBCs. The apparent K m values of EETs were between 1 and 2 M, 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.

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