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.