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Arachidonic acid is metabolized to four regioisomeric epoxyeicosatrienoic acids (EETs) by cytochrome P-450. 5,6-, 8,9-, 11,12-, and 14,15-EET are equipotent in relaxing bovine coronary arteries (BCAs). Vasorelaxant effects of EETs are nonselectively antagonized by 14,15-epoxyeicosa-5(Z)-enoic acid. The 11,12-EET analogs, 20-hydroxy-11,12-epoxyeicosa- 8(Z)-enoic acid (20-H-11,12-EE8ZE) and 11,12,20-trihydroxyeicosa- 8(Z)-enoic acid (11,12,20-THE8ZE) were synthesized and tested for antagonist activity against EET-induced relaxations in BCAs. In U-46619-preconstricted arterial rings, 5,6-, 8,9-, 11,12-, and 14,15- EET caused concentration-dependent relaxations with maximal relaxations ranging from 80 to 96%. Preincubation of arteries with 20-H- 11,12-EE8ZE (10-5 M) inhibited relaxations to 14,15- and 11,12- EET, but not 5,6- and 8,9-EET; however, greatest inhibitory effect was against 11,12-EET (maximal relaxation = 80.6 ± 4.6 vs. 26.7 ± 7.4% without and with 20-H-11,12-EE8ZE, respectively). Preincubation with the soluble epoxide hydrolase inhibitor (tAUCB, 10-6 M) significantly enhanced the antagonist effect of 20-H-11,12-EE8ZE against 14,15-EET-induced relaxations (maximal relaxation = 86.6 ± 4.4 vs. 27.8 ± 3.3%, without and with 20-H-11,12-EE8ZE and tAUCB) without any change in its effect against 11,12-EET-induced relaxations. In contrast to the parent compound, the metabolite, 11,12,20-THE8ZE (10-5 M), significantly inhibited relaxations to 11,12-EET and was without effect on other EET regioisomers. Mass spectrometric analysis revealed conversion of 20-H-11,12-EE8ZE to 11,12,20-THE8ZE by incubation with BCA. The conversion was blocked by tAUCB. 14,15-Dihydroxy-eicosa-5Z-enoic acid (a 14,15-EET antagonist), but not 11,12,20-THE8ZE (an 11,12-EET antagonist), inhibited BCA relaxations to arachidonic acid and flow-induced dilation in rat mesenteric arteries. These results indicate that 11,12,20-THE8ZE is a selective antagonist of 11,12-EET relaxations and a useful pharmacological tool to elucidate the function of 11,12-EET in the cardiovascular system. [ABSTRACT FROM AUTHOR]

Adrenic acid (docosatetraenoic acid), an abundant fatty acid in the adrenal gland, is identical to arachidonic acid except for 2 additional carbons on the carboxyl end. Adrenic acid is metabolized by cyclooxygenases, cytochrome P450s, and lipoxygenases; however, little is known regarding the role of adrenic acid and its metabolites in vascular tone. Because of its abundance in the adrenal gland, we investigated the role of adrenic acid in vascular tone of bovine adrenal cortical arteries and its metabolism by bovine adrenal zona glomerulosa cells. In adrenal cortical arteries, adrenic acid caused concentration-dependent relaxations, which were inhibited by the epoxyeicosatrienoic acid antagonist 14,15-epoxyeicosa-5(Z)-enoic acid and the cytochrome P450 inhibitor SKF-525A. The large-conductance calcium-activated potassium channel blocker iberiotoxin or removal of the endothelium abolished these relaxations. Reverse-phase high-pressure liquid chromatography and liquid chromatography/mass spectrometry isolated and identified numerous adrenic acid metabolites from zona glomerulosa cells, including dihomo-epoxyeicosatrienoic acids and dihomo-prostaglandins. In denuded adrenal cortical arteries, adrenic acid caused concentration-dependent relaxations in the presence of zona glomerulosa cells but not in their absence. These relaxations were inhibited by SKF-525A, 14,15-epoxyeicosa-5(Z)-enoic acid, and iberiotoxin. Dihomo-16,17-epoxyeicosatrienoic acid caused concentration-dependent relaxations of adrenal cortical arteries, which were inhibited by 14,15-epoxyeicosa-5(Z)-enoic acid and high potassium. Our results suggest that adrenic acid relaxations of bovine adrenal cortical arteries are mediated by endothelial and zona glomerulosa cell cytochrome P450 metabolites. Thus, adrenic acid metabolites could function as endogenous endothelium-derived and zona glomerulosa-derived hyperpolarizing factors in the adrenal cortex and contribute to the regulation of adrenal blood flow. [ABSTRACT FROM AUTHOR]

Lipoxygenase (15-LO-1) metabolizes arachidonic acid (AA) to 11,12,15-trihydroxyeicosatrienoic acids (THETAs) and 15-hydroxy-11,12-epoxyeicosatrienoic acids (HEETA) that dilate rabbit arteries. Increased endothelial 15-LO-1 expression enhances arterial relaxations to agonists. We tested the effect of hypoxia on 15-LO-1 expression, THETA and HEETA synthesis, and relaxations in rabbit arteries. The incubation of rabbit aortic endothelial cells and isolated aortas in 0.7% 02 increased 15-LO-1 expression. Rabbits were housed in a hypoxic atmosphere of 12% 02 for 5 days. 15-LO-1 expression increased in the endothelium of the arteries of rabbits in 12% O2 compared with room air. THETA and HEETA synthesis was also enhanced in aortas and mesenteric arteries. AA hyperpolarized the smooth muscle cells in indomethacinand phenylephrine-treated mesenteric arteries of hypoxic rabbits from -29.4 ± Ito -50.1 ± 3 mV. The hyperpolarization to AA was less in arteries of normoxic rabbits (from -26.0 ± 2 to -37 ± 2 mV). This AA-induced hyperpolarization was inhibited by the 15-LO inhibitor BW-755C. Nitric oxide and prostaglandin-independent maximum relaxations to acetylcholine (79.7 ± 2%) and AA (38.3 ± 4%) were enhanced in mesentenc arteries from hypoxic rabbits compared with the normoxic rabbits (49.7 ± 6% and 19.9 ± 2%, respectively). These relaxations were inhibited by BW-755C and nordihydroguaiaretic acid. Therefore, hypoxia increased the relaxations to agonists in the rabbit mesenteric arteries by enhancing endothelial 15-LO-1 expression and synthesis of the hyperpolarizing factors THETA and HEETA. [ABSTRACT FROM AUTHOR]

Arachidonic acid (AA) from the 15-lipoxygenase-1 (15-LO-1) pathway, trihydroxyeicosatrienoic acids (THETAs) and hydroxy-epoxyeicosatrienoic acids (HEETAs), are endothelium-derived hyperpolarizing factors (EDHFs) and relax rabbit arteries. Rabbit vascular 15-LO-1 expression, THETA and HEETA synthesis, and nitric oxide and prostaglandin-independent relaxations to acetyicholine (ACh) and AA decreased with age (neonates to 16-wk-old). We characterized age-dependent ACh-hypotensive responses in vivo in 1-, 4-, 8-, and 16-wk-old rabbits and the contribution of THETAs and HEETAs to these responses. In anesthetized rabbits, blood pressure responses to ACh (4-4,000 ng/kg) were determined in the presence of vehicle or various inhibitors. ACh responses decreased with age (P > 0.001). In the absence or presence of Nω-nitro-L-arginine methyl ester (L- NAME) and indomethacin (Indo), maximum responses in 1 (-54.7 ± 7.4 and -37.9 ± 3.9%)- and 4 (-48.8 ± 2.4 and -35.5 ± 7.8%)- wk-old rabbits were higher than 8 (-30.0 ± 2.8 and -26.6 ± 4.4%)- and 16 (-36.7 ± 3.5 and -27.3 ± 10%)-wk-old rabbits. A lipoxygenase inhibitor, BW755C, reduced THETA and HEETA synthesis in mesenteric arteries. In the presence of Indo and Nω-nitro-L-arginine, ACh relaxations were reduced by BW755C to a greater extent in the mesenteric arteries from the younger rabbits. In 4-wk-old rabbits treated with L-NAME and Indo, the maximum ACh hypotension was reduced by the potassium channel inhibitors apamin and charybdotoxin to -6.9 ± 0.9%, by apamin alone to -19.5 ± 1.4%, and by BW755C to -18.8 ± 3.5%. The present study indicates that the age-related decrease in ACh-induced hypotension is mediated by the decreased synthesis of the 15-LO-1 metabolites THETAs and HEETAs. [ABSTRACT FROM AUTHOR]

Previous studies indicate that 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), an endothelium-derived hyperpolarizing factor in the rabbit aorta, mediates a portion of the relaxation response to acetylcholine by sequential metabolism of arachidonic acid by 15-lipoxygenase, hydroperoxide isomerase, and epoxide hydrolase. To determine the stereochemical configuration of the endothelial 11,12,15-THETA, its activity and chromatographic migration were compared with activity and migration of eight chemically synthesized stereoisomers of 11,12,15(S)-THETA. Of the eight isomers, only 11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z), 13(E)-trienoic acid comigrated with the biological 11,12,15-THETA on reverse- and normal-phase HPLC and gas chromatography. The same THETA isomer (10-7-10-4 M) relaxed the rabbit aorta in a concentration-related manner (maximum relaxation = 69 ± 5%). These relaxations were blocked by apamin (10-7 M), an inhibitor of small-conductance Ca2+-activated K+ channels. In comparison, 11(S), 12(R), 15(S),5 (Z) ,8 (Z) .1 3(E)-THETA (10-4 M) relaxed the aorta by 22%. The other six stereoisomers were inactive in this assay. With use of the whole cell patch-clamp technique, it was shown that 10-4 M 11(R),12(S),15(S),5(Z), 8(Z),13(E)-THETA increased outward K+ current in isolated aortic smooth muscle cells by 119 ± 36% at +60 mV, whereas 10-4 M 11(R), 2(R),15(S),5(Z),8(Z), 13(E)-THETA increased outward K+ current by only 20 ± 2%. The 11(R),12(S),15(S),5(Z),8(Z),13(E)-THETA-stimulated increase in K+ current was blocked by pretreatment with apamin. These studies suggest that 11(R),12(S),15(S)- trihydroxyeicosa-5(Z),8(Z), 13(E)-trienoic acid is the active stereoisomer produced by the rabbit aorta. It relaxes smooth muscle by activating K+ channels. The specific structural and stereochemical requirements for K+ channel activation suggest that a specific binding site or receptor of 11,12,15-THETA is involved in these actions. [ABSTRACT FROM AUTHOR]

Arachidonic acid (AA) causes endothelium-dependent smooth muscle hyperpolarizations and relaxations that are mediated by a 15-lipoxygenase-1 (15-LO-1) metabolite, 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA). We propose that AA is metabolized sequentially by 15-LO-1 and hydroperoxide isomerase to an unidentified hydroxyepoxyeicosatrienoic acid (HEETA), which is hydrolyzed by a soluble epoxide hydrolase (sEH) to 11,12,15-THETA. After incubation of aorta with 14C-labeled AA, metabolites were extracted and the HEETAs were resolved by per- forming HPLC. Mass spectrometric analyses identified 15-Hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Incubation of aortic incubates with methanol and acetic acid trapped the acid- sensitive 15-H-11,12-EETA as methoxydihydroxyeicosatrienoic acids (MDHEs) (367 m/z, M-H). Pretreatment of the aortic tissue with the sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA; 10-6 M) increased the formation of 15-H-11,12-EETA, measured as MDHEs. Thus 15-H-11,12-EETA is an acid- and sEH-sensitive precursor of 11,12,15-THETA. Aortic homogenates and endothelial cells contain a 57-kDa protein corresponding to the rabbit sEH. In preconstricted aortic rings, AA (10-7-10-4 M) and acetylcholine (10-9- 10-6 M) caused concentration-related relaxations that were enhanced by pretreatment with AUDA. These enhanced relaxations were inhibited by increasing extracellular [K+] from 4.8 to 20 mM. AA (3 × 10-6 M) induced cell membrane hyperpolarization (from -31.0 ± 1 to -46.8 ± 2 mV) in aortic strips with an intact endothelium, which was enhanced by AUDA. These results indicate that 15-H-11,12-EETA is produced by the aorta, hydrolyzed by sEH to 11,12,15-THETA, and mediates relaxations by membrane hyperpolarization. 15-H-11,12-EETA represents an endothelium-derived hyperpolarizing factor. [ABSTRACT FROM AUTHOR]

Adrenic acid (docosatetraenoic acid), an abundant fatty acid in the vasculature, is produced by a two-carbon chain elongation of arachidonic acid. Despite its abundance and similarity to arachidonic acid, little is known about its role in the regulation of vascular tone. Gas chromatography/mass spectrometric analysis of bovine coronary artery and endothelial cell lysates revealed arachidonic acid concentrations of 2.06 ± 0.01 and 6.18 ± 0.60 µg/mg protein and adrenic acid concentrations of 0.29 ± 0.01 and 1.56 ± 0.16 µg/mg protein, respectively. In bovine coronary arterial rings preconstricted with the thromboxane mimetic U-46619, adrenic acid (10-9-10-5 M) induced concentration-related relaxations (maximal relaxation 83 ± 4%) that were similar to arachidonic acid relaxations. Adrenic acid relaxations were blocked by endothelium removal and the K+ channel inhibitor, iberiotoxin (100 nM), and inhibited by the cyclooxygenase inhibitor, indomethacin (10 µM. maximal relaxation = 53 ± 4%), and the cytochrome P-450 inhibitor, miconazole (10 µM, maximal relaxation 52 ± 5%). Reverse-phase HPLC and liquid chromatography/mass spectrometry isolated and identified numerous adrenic acid metabolites from coronary arteries including dihomo (DH)-epoxy-eicosatrienoic acids (EETs) and DH-prostaglandins. DH-EET [16,17-, 13,14-, 10,11-, and 7,8- (10-9-10-5 M)] induced similar concentration-related relaxations (maximal relaxations averaged 83 ± 3%). Adrenic acid (10-6 M) and DH-16,17-EET (10-6 M) hyperpolarized coronary arterial smooth muscle. DH-16,17-EET (10-8-10-6 M) activated iberiotoxin-sensitive, whole cell K+ currents of isolated smooth muscle cells. Thus, in bovine coronary arteries, adrenic acid causes endothelium-dependent relaxations that are mediated by cyclooxygenase and cytochrome P-450 metabolites. The adrenic acid metabolite, DH-16,17-EET, activates smooth muscle K+ channels to cause hyperpolarization and relaxation. Our results suggest a role of adrenic acid metabolites, specifically, DH-EETs as endothelium- derived hyperpolarizing factors in the coronary circulation. [ABSTRACT FROM AUTHOR]

Epoxyeicosatrienoic acids (EETs) are endothelium-derived cytochrome P-450 (CYP) metabolites of arachidonic acid that relax vascular smooth muscle by large-conductance calcium-activated potassium (BKCa) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EFT production would increase BKCa channel activity. Bovine coronary SMCs were transduced with adenovirus coding the CYP Bacillus megaterium -3 (F87V) (CYP BM-3) epoxygenase that metabolizes arachidonic acid exclusively to 14(S),15(R)- EET. Adenovirus containing the cytomegalovirus promoter-Escherichia coli β-galactosidase was used as a control. With the use of an anti-CYP BM-3 (F87V) antibody, a 124-kDa immunoreactive protein was detected only in CYP BM-3-transduced cells. Protein expression increased with increasing amounts of virus. When CYP BM-3-transduced cells were incubated with [14C]arachidonic acid, HPLC analysis detected 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) and 14,15-EET. The identity of 14,15-EET and 14,15-DHET was confirmed by mass spectrometry. In CYP BM-3-transduced cells, methacholine (10-5 M) increased 14,15-EET release twofold and BKCa channel activity fourfold in cell-attached patches. Methacholine-induced increases in BKCa channel activity were blocked by the CYP inhibitor 17-octadecynoic acid (10-5 M). 14(S),15(R)-EET was more potent than 14(R),15(S)-EET in relaxing bovine coronary arteries and activating BKCa channels. Thus CYP BM-3 adenoviral transduction confers SMCs with epoxygenase activity. These cells acquire the capacity to respond to the vasodilator agonist by synthesizing 14(5),15(R)-EET from endogenous arachidonic acid to activate BKCa channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of BKCa channels in coronary SMCs. [ABSTRACT FROM AUTHOR]

Acetylcholine stimulates the release of endothelium-derived arachidonic acid (AA) metabolites including prostacyclin and epoxyeicosatrienoic acids (EETs), which relax coronary arteries. However, mechanisms of endothelial cell (EC AA activation remain undefined. We propose that 2-arachidonylglycerol (2-AG) plays an important role in this pathway. An AA metabolite isolated from bovine coronary ECs was identified as 2-AG by mass spectrometry. In ECs pretreated with the fatty acid amidohydrolase inhibitor diazomethylarachidonyl ketone (DAK; 20 μmol/l), methacholine (10 μmol/l)-stimulated 2-AG release was blocked by the phospholipase C inhibitor U-73122 (10 μmol/l) or the diacylglycerol lipase inhibitor RHC-80267 (40 μmol/l). In U-46619-preconstricted bovine coronary arterial rings, 2-AG relaxations averaging 100% at 10 μmol/l were inhibited by endothelium removal, by DAK, by the hydrolase inhibitor methyl arachidonylfluorophosphate (10 μmol/l), by the cyclooxygenase inhibitor indomethacin (10 μmol/l), but not by the CB1 cannabinoid receptor antagonist SR-141716 (1 μmol/l). The cytochrome P-450 inhibitor SKF-525a (10 μmol/l) and the 14,15-epoxyeicosa-5Z-enoic acid EET antagonist (14,15-EEZE; 10 μmol/l) further attenuated the indomethacin-resistant relaxations. The nonhydrolyzable 2-AG analogs noladin ether, 2-AG amide, and 14,15-EET glycerol amide did not induce relaxation. N-nitro-L-arginine-resistant relaxations to methacholine were also inhibited by U-73122, RHC-80267, and DAK. 14,15-EET glycerol ester increased opening of large-conductance K+ channels 12-fold in cell-attached patches of isolated smooth muscle cells and induced relaxations averaging 95%. These results suggest that methacholine stimulates EC 2-AG production through phospholipase C and diacylglycerol lipase activation. 2-AG is further hydrolyzed to AA, which is metabolized to vasoactive eicosanoids. These studies reveal a role for 2-AG in EC AA release and the regulation of coronary tone. [ABSTRACT FROM AUTHOR]

We recently reported that the lipoxygenase product 11,12,15-trihydroxyeicosatrienoic acid (THETA) mediates arachidonic acid (AA)-induced relaxation in the rabbit aorta. This study was designed to determine whether this lipoxygenase metabolite is involved in relaxation responses to AA in rabbit small mesenteric arteries. AA (10-9-10-4 M) produced potent relaxations in isolated phenylephrine-preconstricted arteries, with a maximal relaxation of 99 &plusm; 0.5% and EC50 of 50 nM. The cyclooxygenase (COX) inhibitors indomethacin (10 μM), NS-398 (10 μM, selective for COX-2), and SC-560 (100 nM, selective for COX-1) caused a marked rightward shift of concentration responses to AA. With the use of immunohistochemical analysis, both COX-1 and COX-2 were detected in endothelium and smooth muscle of small mesenteric arteries. lndomethacin-resistant relaxations were further reduced by the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC; 1 μM). nordihydroguaiaretic acid (NDGA; 1 μM), and ebselen (1 μM). HPLC analysis showed that [14C]AA was metabolized by mesenteric arteries to PGI2, PGE2, THETAs, hydroxyepoxyeicosatrienoic acids (HEETAs), and 15-hydroxyeicosatetraenoic acid (15-HETE). The production of PGI2 and PGE2 was blocked by indomethacin, and the production of THETAs, HEETAs, and 15 HETE was inhibited by CDC and NDGA. Column fractions corresponding to THETAs were further purified, analyzed by gas chromatography/mass spectrometry, and identified as 11,12,15- and 11,14,15 THETA. PGI2, PGE2, and purified THETA fractions relaxed mesenteric arteries precontracted with phenylephrine. The AA- and THETA-induced relaxations were blocked by high K+ (60 mM). These findings provide functional and biochemical evidence that AA-induced relaxation in rabbit small mesenteric arteries is mediated through both COX and lipoxygenase pathways. [ABSTRACT FROM AUTHOR]

Highlights: [•] Arachidonic acid metabolites of human 15-LO enzyme are vasoactive. [•] Preproendothelin-1 controlled human 15-LO transgene is highly expressed in heart. [•] Human 15-LO transgene enhances coronary flow following an ischemia. [•] 15-LO metabolites contribute to coronary reactive hyperemia. [ABSTRACT FROM AUTHOR]