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This Article Full Text Full Text (PDF) All Versions of this Article: 294/3/H1348    most recent 01326.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Chawengsub, Y. Articles by Campbell, W. B. PubMed PubMed Citation Articles by Chawengsub, Y. Articles by Campbell, W. B.

Identification of 15-hydroxy-11,12-epoxyeicosatrienoic acid as a vasoactive 15-lipoxygenase metabolite in rabbit aorta

¹Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin; ²Department of Biochemistry, University of Texas Southwestern Medical School, Dallas, Texas; and ³Department of Entomology and Cancer Research Center, University of California, Davis, California

Submitted 12 November 2007 ; accepted in final form 11 January 2008

Arachidonic acid (AA) causes endothelium-dependent smooth muscle hyperpolarizations and relaxations that are mediated by a 15-lipoxygenase-I (15-LO-I) metabolite, 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA). We propose that AA is metabolized sequentially by 15-LO-I and hydroperoxide isomerase to an unidentified hydroxyepoxyeicosatrienoic acid (HEETA), which is hydrolyzed by a soluble epoxide hydrolase (sEH) to 11,12,15-THETA. After incubation of aorta with ¹⁴C-labeled AA, metabolites were extracted and the HEETAs were resolved by performing HPLC. Mass spectrometric analyses identified 15-Hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Incubation of aortic incubates with methanol and acetic acid trapped the acid-sensitive 15-H-11,12-EETA as methoxydihydroxyeicosatrienoic acids (MDHEs) (367 m/z, M-H). Pretreatment of the aortic tissue with the sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA; 10^–6 M) increased the formation of 15-H-11,12-EETA, measured as MDHEs. Thus 15-H-11,12-EETA is an acid- and sEH-sensitive precursor of 11,12,15-THETA. Aortic homogenates and endothelial cells contain a 57-kDa protein corresponding to the rabbit sEH. In preconstricted aortic rings, AA (10^–7–10^–4 M) and acetylcholine (10^–9–10^–6 M) caused concentration-related relaxations that were enhanced by pretreatment with AUDA. These enhanced relaxations were inhibited by increasing extracellular [K⁺] from 4.8 to 20 mM. AA (3 x 10^–6 M) induced cell membrane hyperpolarization (from –31.0 ± 1 to –46.8 ± 2 mV) in aortic strips with an intact endothelium, which was enhanced by AUDA. These results indicate that 15-H-11,12-EETA is produced by the aorta, hydrolyzed by sEH to 11,12,15-THETA, and mediates relaxations by membrane hyperpolarization. 15-H-11,12-EETA represents an endothelium-derived hyperpolarizing factor.

arachidonic acid; endothelial cells; endothelium-derived hyperpolarizing factor; 15-lipoxygenase