Identification of the 11,14,15- and 11,12, 15-trihydroxyeicosatrienoic acids as endothelium-derived relaxing factors of rabbit aorta. J Biol Chem 1998 Nov 20;273(47):30879-87
Date
11/13/1998Pubmed ID
9812980DOI
10.1074/jbc.273.47.30879Scopus ID
2-s2.0-0032553415 (requires institutional sign-in at Scopus site) 75 CitationsAbstract
A number of endothelium-derived relaxing factors have been identified including nitric oxide, prostacyclin, and the epoxyeicosatrienoic acids. Previous work showed that in rabbit aortic endothelial cells, arachidonic acid was metabolized by a lipoxygenase to vasodilatory eicosanoids. The identity was determined by the present study. Aortic homogenates were incubated in the presence of [U-14C]arachidonic acid, [U-14C]arachidonic acid plus 15-lipoxygenase (soybean lipoxidase), or [U-14C]15-hydroxyeicosatetraenoic acid (15-HPETE) and analyzed by reverse phase high pressure liquid chromatography (RP-HPLC). Under both experimental conditions, there was a radioactive metabolite that migrated at 17.5-18.5 min on RP-HPLC. When the metabolite was isolated from aortic homogenates, it relaxed precontracted aortas in a concentration-dependent manner. Gas chromatography/mass spectrometry (GC/MS) of the derivatized metabolite indicated the presence of two products; 11,12,15-trihydroxyeicosatrienoic acid (THETA) and 11,14,15-THETA. A variety of chemical modifications of the metabolite supported these structures and confirmed the presence of a carboxyl group, double bonds, and hydroxyl groups. With the combination of 15-lipoxygenase, arachidonic acid, and aortic homogenate, an additional major radioactive peak was observed. This fraction was analyzed by GC/MS. The mass spectrum was consistent with this peak, containing both the 11-hydroxy-14, 15-epoxyeicosatrienoic acid (11-H-14,15-EETA) and 15-H-11,12-EETA. The hydroxyepoxyeicosatrienoic acid (HEETA) fraction also relaxed precontracted rabbit aorta. Microsomes derived from rabbit aortas also synthesized 11,12,15- and 11,14,15-THETAs from 15-HPETE, and pretreatment with the cyctochrome P450 inhibitor, miconazole, blocked the formation of these products. The present studies suggest that arachidonic acid is metabolized by 15-lipoxygenase to 15-HPETE, which undergoes an enzymatic rearrangement to 11-H-14,15-EETA and 15-H-11,12-EETA. Hydrolysis of the epoxy group results in the formation of 11,14,15- and 11,12,15-THETA, which relaxed rabbit aorta. Thus, the 15-series THETAs join prostacyclin, nitric oxide, and epoxyeicosatrienoic acids as new members of the family of endothelium-derived relaxing factors.
Author List
Pfister SL, Spitzbarth N, Nithipatikom K, Edgemond WS, Falck JR, Campbell WBAuthors
William B. Campbell PhD Professor in the Pharmacology and Toxicology department at Medical College of WisconsinSandra L. Pfister PhD Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
8,11,14-Eicosatrienoic AcidAnimals
Aorta
Arachidonate 15-Lipoxygenase
Arachidonic Acid
Cytochrome P-450 Enzyme Inhibitors
Endothelium, Vascular
Gas Chromatography-Mass Spectrometry
Leukotrienes
Lipid Peroxides
Miconazole
Microsomes
Models, Biological
Rabbits
Vasodilator Agents