Identification of the 11,14,15- and 11,12,15-Trihydroxyeicosatrienoic Acids as Endothelium-derived Relaxing Factors of Rabbit Aorta

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-14 C]arachidonic acid, [U-14 C]arachidonic acid plus 15-lipoxygenase (soybean lipoxidase), or [U-14 C]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.The vascular endothelium synthesizes and releases compounds that are involved in the regulation of vascular tone (1). These endothelial-derived vasoactive compounds include prostacyclin, endothelium-derived relaxing factor or nitric oxide, endothelium-derived hyperpolarizing factor, endothelium-derived contracting factor, and endothelin. These endothelial factors mediate the vasoactive effects of a number of hormones including acetylcholine, bradykinin, and ATP (1). Alterations in the production of these compounds may be associated with cardiovascular diseases, including atherosclerosis, coronary vasospasm, and hypertension.Arachidonic acid is metabolized by the vascular endothelium to a variety of cyclooxygenase, lipoxygenase, and cytochrome P450 epoxygenase products (2). The iden...

Role of phospholipase C and diacylglyceride lipase pathway in arachidonic acid release and acetylcholine-induced vascular relaxation in rabbit aorta

“…In rabbit aortas, ACh elicits synthesis of prostaglandins (26) and other AA metabolites (40) by activating muscarinic receptors (25). We reported that AA metabolites of 15-LO pathways, specifically 11,12,15-THETA and 15-H-11,12-EETA, mediate these L-NNA-and indomethacin-resistant vasorelaxations through opening apamin-sensitive K ϩ channels (11,23,42). The studies suggest that THETA and HEETA serve as EDHFs in rabbit aorta.…”

“…Using specific inhibitors in rabbit aorta, Singer and Peach (51) first concluded that a LO metabolite mediates the NO-and PG-resistant relaxation to ACh (51). The LO inhibitors cinnamyl-3,4-dihydroxy-␣-cyanocinnamate, nordihydroguaiaretic acid, and ebselen block ACh and AA-induced vasodilation in rabbit aorta (11,39,42). Previous studies in our lab identified the 15-LO metabolites of AA in rabbit aorta.…”

“…The buffer was acidified to pH 3.5 with glacial acetic acid and extracted using Bond Elute octadecylsilyl columns (Varian, Palo Alto). The extracted lipid metabolites were analyzed by reverse-phase high-pressure liquid chromatography (HPLC) using solvent system III and a Nucleosil C 18 (5 m, 4.6 ϫ 250 mm) column (42). A 40-min linear gradient from 50% solvent B (acetonitrile with 0.1% glacial acetic acid) in solvent A (deionized water) to 100% solvent B was used.…”

“…AA is oxidized by 15-LO to 15-hydroperoxyeicosatetraenoic acid (15-HPETE), which is converted to hydroxyepoxyeicosatrienoic acid (HEETA) and trihydroxyeicosatrienoic acid (THETA) (11,42). 15-Hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA) and 11,12,15-THETA relax the aorta, an effect blocked by high potassium, or by apamin, an inhibitor of small-conductance, calcium-activated potassium channels (11,23,42). Therefore, in rabbit aorta, 15-LO metabolites of AA represent a new group of endothelium-dependent hyperpolarizing factors (EDHF) that are involved in regulation of vascular tone.…”

11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid: an endothelium-derived 15-lipoxygenase metabolite that relaxes rabbit aorta

“…Several substances, including epoxyeicosatrienoic acids, K ϩ , H 2 O 2 , and C-type natriuretic peptide, exhibit EDHF activity (2,4,7,16). In rabbit arteries, we found that EDHF activity is mediated by a 15-lipoxygenase (15-LO) metabolite of arachidonic acid (AA) (3,23). The rabbit endothelium metabolizes AA to 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] through 15-LO (23,24,26).…”