Stimulation of vascular endothelial cells with agonists such as acetylcholine (ACh) or bradykinin or with shear stress activates phospholipases and releases arachidonic acid (AA). AA is metabolized by cyclooxygenases, cytochrome P-450s, and lipoxygenases (LOs) to vasoactive products. In some arteries, a substantial component of the vasodilator response is dependent on LO metabolites of AA. Nitric oxide (NO)- and prostaglandin (PG)-independent vasodilatory responses to ACh and AA are reduced by inhibitors of LO and by antisense oligonucleotides specifically against 15-LO-1. Vasoactive 15-LO metabolites derived from the vascular endothelium include 15-hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-HEETA) that is hydrolyzed by soluble epoxide hydrolase to 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA). HEETA and THETA are endothelium-derived hyperpolarizing factors that induce vascular relaxations by activation of smooth muscle apamin-sensitive, calcium-activated, small-conductance K(+) channels causing hyperpolarization. In other arteries, the 12-LO metabolite 12-hydroxyeicosatetraenoic acid is synthesized by the vascular endothelium and relaxes smooth muscle by large-conductance, calcium-activated K(+) channel activation. Thus formation of vasodilator eicosanoids derived from LO pathways contributes to the regulation of vascular tone, local blood flow, and blood pressure.

Arachidonic acid (AA) metabolites function as EDHFs in arteries of many species. They mediate cyclooxygenase (COX)- and nitric oxide (NO)-independent relaxations to acetylcholine (ACh). However, the role of AA metabolites as relaxing factors in mouse arteries remains incompletely defined. ACh caused concentration-dependent relaxations of the mouse thoracic and abdominal aorta and carotid, femoral, and mesentery arteries (maximal relaxation: 57 ± 4%, 72 ± 4%, 82 ± 3%, 80 ± 3%, and 85 ± 3%, respectively). The NO synthase inhibitor nitro-L-arginine (L-NA; 30 μM) blocked relaxations in the thoracic aorta, and L-NA plus the COX inhibitor indomethacin (10 μM) inhibited relaxations in the abdominal aorta and carotid, femoral, and mesenteric arteries (maximal relaxation: 31 ± 10%, 33 ± 5%, 41 ± 8%, and 73 ± 3%, respectively). In mesenteric arteries, NO- and COX-independent relaxations to ACh were inhibited by the lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA; 10 μM) and BW-755C (200 μM), the K(+) channel inhibitor apamin (1 μM), and 60 mM KCl and eliminated by endothelium removal. They were not altered by the cytochrome P-450 inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (20 μM) or the epoxyeicosatrienoic acid antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 μM). AA relaxations were attenuated by NDGA or apamin and eliminated by 60 mM KCl. Reverse-phase HPLC analysis revealed arterial [(14)C]AA metabolites that comigrated with prostaglandins, trihydroxyeicosatrienoic acids (THETAs), hydroxyepoxyeicosatrienoic acids (HEETAs), and hydroxyeicosatetraenoic acids (HETEs). Epoxyeicosatrienoic acids were not observed. Mass spectrometry confirmed the identity of 6-keto-PGF(1α), PGE(2), 12-HETE, 15-HETE, HEETAs, 11,12,15-THETA, and 11,14,15-THETA. AA metabolism was blocked by NDGA and endothelium removal. 11(R),12(S),15(S)-THETA relaxations (maximal relaxation: 73 ± 3%) were endothelium independent and blocked by 60 mM KCl. Western immunoblot analysis and RT-PCR of the aorta and mesenteric arteries demonstrated protein and mRNA expression of leukocyte-type 12/15-LO. Thus, in mouse resistance arteries, 12/15-LO AA metabolites mediate endothelium-dependent relaxations to ACh and AA.

Chawengsub Y, Aggarwal NT, Nithipatikom K, Gauthier KM, Anjaiah S, Hammock BD, Falck JR, Campbell WB. Identification of 15-hydroxy-11,12-epoxyeicosatrienoic acid as a vasoactive 15-lipoxygenase metabolite in rabbit aorta.

Zhang DX, Gauthier KM, Chawengsub Y, Campbell WB. ACh-induced relaxations of rabbit small mesenteric arteries: role of arachidonic acid metabolites and K ϩ . Am J Physiol Heart Circ Physiol 293: H152-H159, 2007. First published March 2, 2007; doi:10.1152/ajpheart.00268.2006.-ACh-induced endothelium-dependent relaxation in rabbit small mesenteric arteries is resistant to N-nitro-L-arginine (L-NA) and indomethacin but sensitive to high K ϩ , indicating the relaxations are mediated by endothelium-derived hyperpolarizing factors (EDHFs). The identity of the EDHFs in this vascular bed remains undefined. Small mesenteric arteries pretreated with L-NA and indomethacin were contracted with phenylephrine. ACh (10 Ϫ10 to 10 Ϫ6 M) caused concentration-dependent relaxations that were shifted to the right by lipoxygenase inhibition and the Ca 2ϩ -activated K ϩ channel inhibitors apamin (100 nM) or charybdotoxin (100 nM) and eliminated by the combination of apamin plus charybdotoxin. Relaxations to ACh were also blocked by a combination of barium (200 M) and apamin but not barium plus charybdotoxin. Addition of K ϩ (10.9 mM final concentration) to the preconstricted arteries elicited small relaxations. K ϩ addition before ACh restored the charybdotoxin-sensitive component of relaxations to ACh. K ϩ (10.9 mM) also relaxed endothelium-denuded arteries, and the relaxations were inhibited by barium but not by charybdotoxin and apamin. With the use of whole cell patch-clamp analysis, ACh (10 Ϫ7 M) stimulated voltage-dependent outward K ϩ current from endothelial cells, which was inhibited by charybdotoxin, indicating K ϩ efflux. Arachidonic acid (10 Ϫ7 to 10 Ϫ4 M) induced concentration-related relaxations that were inhibited by apamin but not by charybdotoxin and barium. Addition of arachidonic acid after K ϩ (10.9 mM) resulted in more potent relaxations to arachidonic acid compared with control without K ϩ (5.9 mM). These findings suggest that, in rabbit mesenteric arteries, ACh-induced, L-NA-and indomethacin-resistant relaxation is mediated by endothelial cell K ϩ efflux and arachidonic acid metabolites, and a synergism exists between these two separate mechanisms.acetylcholine; potassium channels; lipoxygenase; endothelium-derived hyperpolarizing factor STIMULATION OF THE VASCULAR endothelium with ACh, bradykinin, and increases in flow induces vasodilation by releasing soluble factors such as nitric oxide (NO), prostacyclin, and endothelium-derived hyperpolarizing factors (EDHFs; see Refs.

Gauthier KM, Chawengsub Y, Goldman DH, Conrow RE, Anjaiah S, Falck JR, Campbell WB. 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.

. Cyclooxygenase-and lipoxygenase-dependent relaxation to arachidonic acid in rabbit small mesenteric arteries. Am J Physiol Heart Circ Physiol 288: H302-H309, 2005. First published September 23, 2004; doi:10.1152/ ajpheart.00661.2004We 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 Ϫ9 -10 Ϫ4 M) produced potent relaxations in isolated phenylephrine-preconstricted arteries, with a maximal relaxation of 99 Ϯ 0.5% and EC50 of 50 nM. The cyclooxygenase (COX) inhibitors indomethacin (10 M), NS-398 (10 M, 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. Indomethacin-resistant relaxations were further reduced by the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC; 1 M), nordihydroguaiaretic acid (NDGA; 1 M), and ebselen (1 M). HPLC analysis showed that [ 14 C]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 ϩ (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.endothelium-derived factors; trihydroxyeicosatrienoic acid; prostaglandin E2; prostaglandin I2; endothelium-derived hyperpolarizing factor ACETYLCHOLINE, BRADYKININ, AND OTHER VASODILATORS elicit endothelium-dependent relaxation by release of a number of soluble factors from the vascular endothelium (8,17,22,23). These relaxing factors include nitric oxide (NO), prostacyclin, and a group of compounds termed EDHFs. Although the chemical identity of EDHF remains controversial, its action has been demonstrated in a number of vascular beds, where a significant portion of endothelium-dependent relaxation persists in the presence of NO synthase (NOS) and cyclooxygenase (COX) inhibitors such as N-nitro-L-arginine (L-NNA) and indomethacin. This endothelium-dependent, non-NO, and nonprostacyclin relaxation is associated with smooth muscle hyperpolarization and is sensitive to K ϩ channel blockers such as apamin and charybdotoxin (7,9,14,16,24). The mediators of EDHF ...

Arachidonic acid (AA) is metabolized by endothelial 15-lipoxygenase (15-LO) to several vasodilatory eicosanoids such as 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA) and its proposed unstable precursor 15-hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). In the present study, the acid-stable 13-hydroxy-trans-14,15-epoxy-eicosatrienoic acid (13-H-14,15-EETA) was identified and its vascular activities characterized. Rabbit aorta, mesenteric arteries, and the combination of 15-LO and cytochrome P450 2J2 converted AA to two distinct HEETA metabolites. The HEETA metabolites were resistant to acidic hydrolysis but were hydrolyzed by recombinant sEH to a more polar metabolite identified by mass spectrometry as 13,14,15-THETA.

The fragmentation characteristics of monohydroxyeicosatetraenoic acids and dihydroxy-and trihydroxyeicosatrienoic acids were investigated by electrospray ionization Fourier transform ion cyclotron resonance (FTICR) mass spectrometry using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) and infrared multiphoton dissociation (IRMPD). The fragmentation patterns of these compounds were associated with the number and positions of the hydroxyl substituents. The fragmentation is more complicated with increasing number of the hydroxyl groups of the compounds. In general, the major carbon-carbon cleavage of [M Ϫ H] Ϫ ions occurred at the ␣-position to the hydroxyl group, and the ␣ carbon-carbon cleavage occurred when there was a double-bond at the ␤-position to the ␤ hydroxyl group. SORI-CID and IRMPD produced some common fragmentation patterns; however, each technique provided some unique patterns that are useful for structural identification of these compounds. This study demonstrated the application of FTICR via the identification of regioisomers of trihydroxyeicosatrienoic acids in rabbit aorta samples. (J Am