Reticulocyte 15-Lipoxygenase-I Is Important in Acetylcholine-Induced Endothelium-Dependent Vasorelaxation in Rabbit Aorta

Abstract

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...

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

“…The prostaglandin-and nitric oxide-independent relaxations to acetylcholine are inhibited by the SK Ca channel blocker apamin, high extracellular K ϩ , phospholipase inhibitors, and lipoxygenase inhibitors (3,12,20,27). They are also inhibited by antisense oligonucleotides against 15-LO, but not by scrambled oligonucleotides (26). These studies indicate that the EDHF is an AA metabolite of 15-LO.…”

“…The exact structure of the biologically active 11,12,15-THETA was not previously known. Since rabbit aorta contains 15-LO and synthesizes 15(S)-HPETE at 94% optical purity (21,26), the THETA must contain a 15(S)-hydroxyl. However, configurations of the 11-and 12-hydroxyls were not known.…”

“…Since rabbit endothelium contains 15-LO and synthesizes 15(S)-HPETE at 96% optical purity, only isomers of 11,12,15(S)-THETA were studied (21,26). The hydroxyl groups at carbons 11 and 12 can have the R or S configuration, and the ⌬ 13,14 -olefin can have the cis (Z) or trans (E) configuration.…”

“…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). 15-HPETE is rearranged to 15-hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA) by a hydroperoxide isomerase, possibly a cytochrome P-450.…”

“…The rabbit endothelium contains 15-LO and produces the S isomer of 15-hydroxyeicosatetraenoic acid at 96% optical purity (21,26). Thus the 15-hydroxy group has the S configuration.…”