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. doi: 10.1074/jbc.273.47.30879.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 8,11,14-Eicosatrienoic Acid / analogs & derivatives*
  • 8,11,14-Eicosatrienoic Acid / isolation & purification
  • 8,11,14-Eicosatrienoic Acid / pharmacology
  • Animals
  • Aorta / physiology*
  • Arachidonate 15-Lipoxygenase / metabolism
  • Arachidonic Acid / metabolism*
  • Cytochrome P-450 Enzyme Inhibitors
  • Endothelium, Vascular / physiology*
  • Gas Chromatography-Mass Spectrometry
  • Leukotrienes / metabolism
  • Lipid Peroxides / metabolism
  • Miconazole / pharmacology
  • Microsomes / metabolism
  • Models, Biological
  • Rabbits
  • Vasodilator Agents / isolation & purification*
  • Vasodilator Agents / pharmacology


  • Cytochrome P-450 Enzyme Inhibitors
  • Leukotrienes
  • Lipid Peroxides
  • Vasodilator Agents
  • Arachidonic Acid
  • 15-hydroperoxy-5,8,11,13-eicosatetraenoic acid
  • 11,14,15-trihydroxyeicosa-5,8,12-trienoic acid
  • Miconazole
  • 11,12,15-trihydroxyeicosatrienoic acid
  • Arachidonate 15-Lipoxygenase
  • 8,11,14-Eicosatrienoic Acid