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...

. Pfister. 11,12,15-Trihydroxyeicosatrienoic acid mediates ACh-induced relaxations in rabbit aorta. Am J Physiol Heart Circ Physiol 285: H2648-H2656, 2003. First published August 7, 2003 10.1152/ajpheart.00412.2003.-Rabbit aortic endothelium metabolizes arachidonic acid (AA) by the 15-lipoxygenase pathway to vasodilatory eicosanoids, hydroxyepoxyeicosatrienoic acids (HEETAs), and trihydroxyeicosatrienoic acids (THETAs). The present study determined the chemical identity of the vasoactive THETA and investigated its role in ACh-induced relaxation in the rabbit aorta. AA caused endothelium-dependent, concentration-related relaxations of the rabbit aorta. Increasing the extracellular KCl concentration from 4.8 to 20 mM inhibited the relaxations to AA by ϳ60%, thereby implicating K ϩ -channel activation in the relaxations. In addition, AA caused an endothelium-dependent hyperpolarization of aortic smooth muscle from Ϫ39.6 Ϯ 2.7 to Ϫ56.1 Ϯ 3.4 mV. In rabbit aortic rings, [ 14 C]AA was metabolized to prostaglandins, HEETAs, THETAs, and 15-hydroxyeicosatetraenoic acid. Additional purification of the THETAs by HPLC resolved the mixture into its 14 C-labeled products. Gas chromatography/ mass spectrometry identified the metabolites as isomers of 11,12,15-THETA and 11,14,15-THETA. The 11,12,15-THETA relaxed and hyperpolarized the rabbit aorta, whereas 11,14,15-THETA had no vasoactive effect. The relaxations to 11,12,15-THETA were blocked by 20 mM KCl. In aortic rings pretreated with inhibitors of nitric oxide and prostaglandin synthesis, ACh caused a concentration-related relaxation that was completely blocked by 20 mM KCl. Pretreatment with the phospholipase A 2 inhibitors mepacrine and 7,7-dimethyl-5,8-eicosadienoic acid, the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-␣-cyanocinnamate, nordihydroguaiaretic acid, and ebselen, or the hydroperoxide isomerase inhibitors miconazole and clotrimazole also blocked ACh-induced relaxations. ACh caused a threefold increase in THETA release. These studies indicate that AA is metabolized by endothelial cells to 11,12,15-THETA, which activates K ϩ channels to hyperpolarize the aortic smooth muscle membrane and induce relaxation. Additionally, this lipoxygenase pathway mediates the nonnitric oxide, nonprostaglandin relaxations to ACh in the rabbit aorta by acting as a source of an endothelium-derived hyperpolarizing factor. trihydroxyeicosatrienoic acid; arachidonic acid; endotheliumderived hyperpolarizing factor; potassium channels; membrane potential; lipoxygenase ACETYLCHOLINE AND BRADYKININ STIMULATE the release of soluble mediators from the vascular endothelium that act on the adjacent vascular smooth muscle to cause vasodilation (3,11,15,19,20). These mediators include prostacyclin and nitric oxide (NO). However, when the synthesis of NO and prostacyclin are inhibited by the combination of nitro-L-arginine (L-NNA) and indomethacin, a portion of the relaxation response to ACh persists (2-4, 6, 9, 21). These endothelium-dependent, L-NNA-and indomethacin-resistant relaxations a...

Abstract-Hypoxia causes localized pulmonary arterial (PA) constriction to divert blood flow to optimally ventilated regions of the lung. is an essential mechanism to balance perfusion with ventilation and is unique to the pulmonary circulation. This action regulates one of the most important physiological parameters in mammals, arterial oxygen tension. The acute response is biphasic in isolated perfused vessels, whereas sustained hypoxia in vivo can lead to structural remodeling and matrix deposition in pulmonary arteries, which results in increased arterial tone. 1 The biochemical mechanisms that have been hypothesized to underlie this response are varied, 1-3 except for the consensus that HPV is a multifactorial manifestation, with "elements of energy, oxygen and lipid metabolism" (page 1192). 3 Recently, specific candidates that mediate HPV have been carefully examined (eg, Archer et al 2 ). Nitric oxide (NO), cytochrome P450 metabolites, leukotrienes, or direct effects of oxygen on ion channels in vascular smooth muscle cells have been proposed as modulators/mediators of acute hypoxic responses of the pulmonary vasculature, but none appears to account for all the features of hypoxic vasoconstriction. 2 Our overriding goal was to understand adaptation of pulmonary arteries (PAs) to chronic hypoxia. Arachidonic acid (AA) metabolites modulate vasoactivity, and oxygen is a substrate in eicosanoid synthesis, which makes these products ideal candidates to contribute to HPV. 5-hydroxyeicosatetraenoic acid (HETE), the major metabolites of the 5-lipoxygenase pathway, is increased in macrophages exposed to hypoxia. 4 However, hypoxic pulmonary vasoconstriction is not affected by the 5-lipoxygenase inhibitor MK886 in perfused rabbit lungs. 5 To examine the effects of subacute hypoxia on pulmonary metabolism of AA and the reactivity of PAs to these metabolites, we used an animal model in which rabbit kits Original

Arachidonic acid elicited relaxation responses in normal rabbit aorta precontracted with norepinephrine. The relaxation response was enhanced by the cyclooxygenase inhibitor indomethacin and inhibited by lipoxygenase inhibitors, including nordihydroguaiaretic acid and cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate. The cytochrome P-450 epoxygenase inhibitor metyrapone had no effect on arachidonic acid-induced relaxations. The present study hypothesized that a lipoxygenase metabolite of arachidonic acid mediated the response. Incubation of rabbit aorta with [14C]arachidonic acid resulted in the synthesis of a previously unidentified 14C-labeled metabolite and was called the unknown factor. Production of the unknown factor was not inhibited by indomethacin and decreased by lipoxygenase inhibitors. Production of the unknown factor and arachidonic acid-induced relaxations were dependent on an intact endothelium, indicating that the cellular source of the unknown relaxant factor was the endothelial cell. This was confirmed by demonstrating the ability of cultured rabbit aortic endothelial cells to produce the unknown factor from [14C]arachidonic acid. Feeding rabbits a 2% cholesterol diet for 2 wk induced hypercholesterolemia without causing atherosclerosis. In the cholesterol-fed rabbits, indomethacin enhanced arachidonic acid-induced relaxations in norepinephrine-precontracted aortas (maximal relaxation 49.0 +/- 2.5 vs. 35.5 +/- 1.7%, cholesterol-fed vs. normal) and increased production of the unknown factor compared with normal rabbits. The partially purified unknown factor elicited an approximately 26% inhibition of the vasoconstrictor response to norepinephrine in intact rabbit aorta. Further purification of the unknown factor by reverse-phase high-pressure liquid chromatography system resulted in isolation of a radioactive product that relaxed precontracted rabbit aorta. Therefore these data suggest that in normal and hypercholesterolemic rabbit aorta the endothelium produces an unknown metabolite of arachidonic acid that causes vasorelaxation and may regulate vascular tone.