title: Identification of 15-hydroxy-11,12-epoxyeicosatrienoic acid as a vasoactive 15-lipoxygenase metabolite in rabbit aorta.
abstract: 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.
authors.affiliation: Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
authors.forename: Yuttana
authors.initials: Y
authors.lastname: Chawengsub
authors.forename: Nitin T
authors.initials: NT
authors.lastname: Aggarwal
authors.forename: Kasem
authors.initials: K
authors.lastname: Nithipatikom
authors.forename: Kathryn M
authors.initials: KM
authors.lastname: Gauthier
authors.forename: Siddam
authors.initials: S
authors.lastname: Anjaiah
authors.forename: Bruce D
authors.initials: BD
authors.lastname: Hammock
authors.forename: John R
authors.initials: JR
authors.lastname: Falck
authors.forename: William B
authors.initials: WB
authors.lastname: Campbell
mesh_terms: D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D001013:Aorta, Thoracic / Q000378:metabolism*; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism*; D001095:Arachidonic Acids / Q000378:metabolism; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D004851:Epoxide Hydrolases / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D008564:Membrane Potentials / Q000502:physiology; D011817:Rabbits
chemical_list: C528813:15-hydroxy-11,12-epoxyeicosatrienoic acid; D001095:Arachidonic Acids; D001093:Arachidonate 15-Lipoxygenase; D004851:Epoxide Hydrolases; D015126:8,11,14-Eicosatrienoic Acid; D000109:Acetylcholine