Identification of the 11,14,15- and 11,12, 15-trihydroxyeicosatrienoic acids as endothelium-derived relaxing factors of rabbit aorta.
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.
Pfister
S L
SL
Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Spitzbarth
N
N
Nithipatikom
K
K
Edgemond
W S
WS
Falck
J R
JR
Campbell
W B
WB
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000502:physiology*; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism; D016718:Arachidonic Acid / Q000378:metabolism*; D065607:Cytochrome P-450 Enzyme Inhibitors; D004730:Endothelium, Vascular / Q000502:physiology*; D008401:Gas Chromatography-Mass Spectrometry; D015289:Leukotrienes / Q000378:metabolism; D008054:Lipid Peroxides / Q000378:metabolism; D008825:Miconazole / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism; D008954:Models, Biological; D011817:Rabbits; D014665:Vasodilator Agents / Q000302:isolation & purification* / Q000494:pharmacology
D065607:Cytochrome P-450 Enzyme Inhibitors; D015289:Leukotrienes; D008054:Lipid Peroxides; D014665:Vasodilator Agents; D016718:Arachidonic Acid; C025086:15-hydroperoxy-5,8,11,13-eicosatetraenoic acid; C051283:11,14,15-trihydroxyeicosa-5,8,12-trienoic acid; D008825:Miconazole; C066049:11,12,15-trihydroxyeicosatrienoic acid; D001093:Arachidonate 15-Lipoxygenase; D015126:8,11,14-Eicosatrienoic Acid