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Objective-Aortic 15-lipoxygenase (15-LO) metabolizes arachidonic acid (AA) to 15-hydroperoxyeicosatetraenoic acid, which is then converted to the vasodilators 15-hydroxy-11,12-epoxyeicosatrienoic acid and 11,12,15-trihydroxyeicosatrienoic acid. These metabolites contribute to endothelium-dependent relaxations of rabbit aorta to AA and acetylcholine. We investigated the identity of rabbit aortic 15-LO and studied its importance in the regulation of vascular tone. Methods and Results-RT-PCR using 12-lipoxygenase/15-LO specific primers resulted in a 572-bp product with a sequence identical to 15-LO-I from rabbit aorta. A RT-PCR/restriction digest strategy excluded expression of 12-lipoxygenase. Immunoblotting revealed 15-LO-I expression in rabbit endothelial and smooth muscle cells. Aortic homogenates and cytosolic fractions metabolize AA to 15(S)-hydroxyeicosatetraenoic acid and linoleic acid to 13(S)-hydroxyoctadecadienoic acid. This activity was blocked by LO inhibitors. The kinetic characteristics (Michaelis constant of aortic 15-LO is 2.2Ϯ0.3 mol/L for AA and 23.5Ϯ3.3 mol/L for linoleic acid) of aortic 15-LO were similar to those of the purified 15-LO-I. An antisense oligonucleotide inhibited 15-LO-I expression in rabbit aorta. Indomethacin and nitro-L-arginine-resistant relaxations to acetylcholine were inhibited by 15-LO-I antisense oligonucleotide but not by the scrambled oligonucleotide. Key Words: endothelium Ⅲ arachidonic acid Ⅲ 15-lipoxygenase Ⅲ endothelium-derived hyperpolarizing factor E ndothelial cells (ECs) release factors that control vascular tone in response to acetylcholine, bradykinin, and other hormones, as well as by shear stress. 1 Some of these vasoactive factors are metabolites of arachidonic acid (AA). In rabbit aortic ECs, AA is metabolized through cyclooxygenase, lipoxygenase (LO), and cytochrome P 450 pathways into bioactive eicosanoids. [2][3][4] Rabbit aorta relaxes to acetylcholine and AA in the presence of indomethacin and L-nitroarginine (L-NA), which block cyclooxygenase and nitric oxide synthase, respectively. 2,5 These relaxation responses are blocked by LO inhibitors, such as nordihydroguaiaretic acid (NDGA), cinnamyl-3,4-dihydroxy-a-cyanocinnamate (CDC), and ebselen. 4 -6 AA is oxidized via the 15-LO pathway to 15(S)-hydroperoxyeicosatetraenoic acid, which is additionally converted to hydroxy-epoxyeicosatrienoic acids (HEETAs) and trihydroxyeicosatrienoic acids (THETAs). Both the HEETA and 11,12,15-THETA relax the rabbit aorta. 7 These studies implicate the aortic 15-LO pathway in the regulation of vascular tone. Thus, the rabbit aortic 15-LO, which initiates the synthesis of these vasodilatory eicosanoids, represents an important regulatory site of vascular eicosanoid metabolism. Conclusions-15-LO-I is expressed in rabbit aortic endothelium and is important in endothelium-In humans, there are 2 15-LO isozymes (15-LO-I and 15-LO-II) originating from 2 different genes. 8,9 15-LO-I was cloned from rabbit reticulocytes 10 and human leukocytes and oxygenates AA to 15(S)-hydrope...

(ECs). In rabbit aorta, AA is metabolized through the 15-lipoxygenase pathway to form vasodilatory eicosanoids 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) and 11,12,15-trihydroxyeicosatrienoic acid (THETA). AA is released from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by phospholipase A 2 (PLA2), or from phosphatidylinositol (PI) by phospholipase C (PLC) pathway. The diacylglycerol (DAG) lipase can convert DAG into 2-arachidonoylglycerol from which free AA can be released by monoacylglycerol (MAG) lipase or fatty acid amidohydrolase (FAAH). We used specific inhibitors to determine the involvement of the PLC pathway in ACh-induced AA release. In rabbit aortic rings precontracted by phenylephrine, ACh induced relaxation in the presence of indomethacin and N -nitro-L-arginine (L-NNA). These relaxations were blocked by the PLC inhibitor U-73122, DAG lipase inhibitor RHC-80267, and MAG lipase/FAAH inhibitor URB-532. Cultured rabbit aortic ECs were labeled with [ 14 C]AA and stimulated with methacholine (10 Ϫ5 M). Free [ 14 C]AA was released by methacholine. Methacholine decreased the [ 14 C]AA content of PI, DAG, and MAG fractions but not PC or PE fractions. Methacholine-induced release of [ 14 C]AA was blocked by U-73122, RHC-80267, and URB-532 but not by U-73343, an inactive analog of U-73122. The data suggested that ACh activates PLC, DAG lipase, and MAG lipase pathway to release AA from membrane lipids. This pathway is important in regulating vasodilatory eicosanoid synthesis and vascular relaxation in rabbit aorta. endothelium-dependent hyperpolarizing factor; phospholipid; monoacylglycerol lipase; trihydroxyeicosatrienoic acid; hydroxyepoxyeicosatrienoic acid VASCULAR ENDOTHELIAL CELLS (ECs) regulate vascular tone by synthesis and release of many bioactive compounds. Ligands such as hormones, ACh, bradykinin, and cytokines stimulate release of arachidonic acid (AA) from cellular membranes (6,15,26,43,55), and AA induces endothelium-dependent vasodilation in isolated rabbit aorta preconstricted with phenylephrine, which is not blocked by nitric oxide (NO) synthase (NOS) and cyclooxygenase (COX) inhibitors (19,39,50). The phospholipase inhibitors mepacrine and dimethyl-eicosadienoic acid (DEDA) decrease relaxation to ACh but not AA, suggesting AA or a metabolite may be involved in the relaxations (11). AA is metabolized by COX, lipoxygenase (LO), and cytochrome P-450 pathways into bioactive eicosanoids (10,30,40,48). 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. AA is oxidized by 15-LO to 15-hydroperoxyeicosatetraenoic acid (15-HPETE), which is converted to hydroxyepoxyeicosatrienoic acid (HEETA) and trihydroxyeicosatrienoic acid (THET...

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid. EETs mediate numerous biological functions. In coronary arteries, they regulate vascular tone by the activation of smooth muscle large-conductance, calcium-activated potassium (BK Ca ) channels to cause hyperpolarization and relaxation. We developed a series of 14,