Identification of the 11,14,15- and 11,12,15-trihydroxyeicosatrienoic acids as endothelium-derived relaxing factors of rabbit aorta

Sandra L. Pfister, Nancy Spitzbarth, Kasem Nithipatikom, William S. Edgemond, J R Falck, William B. Campbell

Research output: Contribution to journalArticle

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Abstract

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 cytochrome 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.

Original languageEnglish (US)
Pages (from-to)30879-30887
Number of pages9
JournalJournal of Biological Chemistry
Volume273
Issue number47
DOIs
StatePublished - Nov 20 1998

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Endothelium-Dependent Relaxing Factors
Arachidonate 15-Lipoxygenase
Metabolites
Arachidonic Acid
Aorta
Rabbits
Acids
Lipoxygenase
Epoprostenol
Gas chromatography
Gas Chromatography-Mass Spectrometry
Mass spectrometry
Nitric Oxide
High Pressure Liquid Chromatography
High pressure liquid chromatography
Miconazole
Hydroxyeicosatetraenoic Acids
Eicosanoids
Endothelial cells
Chemical modification

ASJC Scopus subject areas

  • Biochemistry

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Identification of the 11,14,15- and 11,12,15-trihydroxyeicosatrienoic acids as endothelium-derived relaxing factors of rabbit aorta. / Pfister, Sandra L.; Spitzbarth, Nancy; Nithipatikom, Kasem; Edgemond, William S.; Falck, J R; Campbell, William B.

In: Journal of Biological Chemistry, Vol. 273, No. 47, 20.11.1998, p. 30879-30887.

Research output: Contribution to journalArticle

Pfister, Sandra L. ; Spitzbarth, Nancy ; Nithipatikom, Kasem ; Edgemond, William S. ; Falck, J R ; Campbell, William B. / Identification of the 11,14,15- and 11,12,15-trihydroxyeicosatrienoic acids as endothelium-derived relaxing factors of rabbit aorta. In: Journal of Biological Chemistry. 1998 ; Vol. 273, No. 47. pp. 30879-30887.
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T1 - Identification of the 11,14,15- and 11,12,15-trihydroxyeicosatrienoic acids as endothelium-derived relaxing factors of rabbit aorta

AU - Pfister, Sandra L.

AU - Spitzbarth, Nancy

AU - Nithipatikom, Kasem

AU - Edgemond, William S.

AU - Falck, J R

AU - Campbell, William B.

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N2 - 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 cytochrome 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.

AB - 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 cytochrome 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.

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