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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.sup.+] 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.sup.+] channel activation. Thus formation of vasodilator eicosanoids derived from LO pathways contributes to the regulation of vascular tone, local blood flow, and blood pressure. endothelium-derived hyperpolarizing factors; vascular relaxation; 15-1ipoxygenase; 12-lipoxygenase; trihydroxyeicosatrienoic acids; hydroxyepoxyeicosatrienoic acids

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce marked reductions in infarct size (IS) in canine myocardium either given before an ischemic insult or at reperfusion similar to that produced in ischemic preconditioning (IPC) and postconditioning (POC) protocols. However, no studies have addressed the possibility that EETs serve a beneficial role in IPC or POC. We tested the hypothesis that EETs may play a role in these two phenomena by preconditioning dog hearts with one 5-min period of total coronary occlusion followed by 10 rain of reperfusion before 60 rain of occlusion and 3 h of reperfusion or by postconditioning with three 30-s periods of reperfusion interspersed with three 30-s periods of occlusion. To test for a role of EETs in IPC and POC, the selective EET antagonists 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) or its derivative, 14,15-epoxyeicosa-5(Z)-enoic acid 2-[2-(3-hydroxypropoxy)-ethoxy]-ethyl ester (14,15-EEZE-PEG), were administered 10 min before IPC, 5 min after IPC, or 5 min before POC. In a separate series, the selective EET synthesis inhibitor N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide (MS-PPOH) was administered 10 min before IPC. Infarct size was determined by tetrazolium staining and coronary collateral blood flow at 30 min of occlusion and reperfusion flow at 3 h by radioactive microspheres. Both IPC and POC produced nearly equivalent reductions in IS expressed as a percentage of the area at risk (AAR) [Control 21.2 [+ or -] 1.2%, IPC 8.3 [+ or -] 2.2%, POC 10.1 [+ or -] 1.8% (P < 0.001)]. 14,15-EEZE, 14,15-EEZE-PEG, and MS-PPOH markedly attenuated the cardioprotective effects of IPC and POC (14,15-EEZE and 14,15-EEZE-PEG) at doses that had no effect on IS/AAR when given alone. These results suggest a unique role for endogenous EETs in both IPC and POC. cytochrome P-450 epoxygenases; epoxyeicosatrienoic acid antagonists; ischemia-reperfusion

15-Lipoxygenase (15-LO-1) metabolizes arachidonic acid (AA) to l l,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% [O.sub.2] increased 15-LO-1 expression. Rabbits were housed in a hypoxic atmosphere of 12% [O.sub.2] for 5 days. 15-LO-1 expression increased in the endothelium of the arteries of rabbits in 12% [O.sub.2] 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 [+ or -] 1 to -50.1 [+ or -] 3 mV. The hyperpolarization to AA was less in arteries of normoxic rabbits (from -26.0 [+ or -] 2 to -37 [+ or -] 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 [+ or -] 2%) and AA (38.3 [+ or -] 4%) were enhanced in mesenteric arteries from hypoxic rabbits compared with the normoxic rabbits (49.7 [+ or -] 6% and 19.9 [+ or -] 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. endothelium; vasodilation; endothelium-derived hyperpolarizing factor

Arachidonic acid (AA) metabolites from the 15-1ipoxygenase-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 acetylcholine (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 [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) and indomethacin (Indo), maximum responses in 1 (-54.7 [+ or -] 7.4 and -37.9 [+ or -] 3.9%)- and 4 (-48.8 [+ or -] 2.4 and -35.5 [+ or -] 7.8%)-wk-old rabbits were higher than 8 (-30.0 [+ or -] 2.8 and -26.6 [+ or -] 4.4%)-and 16 (-36.7 [+ or -] 3.5 and -27.3 [+ or -] 10%)-wk-old rabbits. A lipoxygenase inhibitor, BW755C, reduced THETA and HEETA synthesis in mesenteric arteries. In the presence of Indo and [N.sup.[omega]]-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 [+ or -] 0.9%, by apamin alone to -19.5 [+ or -] 1.4%, and by BW755C to -18.8 [+ or -] 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. endothelium-derived hyperpolarizing factor; arachidonic acid; vasodilation; mean arterial pressure

Previously, we demonstrated (17) that 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) produce marked reductions in myocardial infarct size. Although it is assumed that this cardioprotective effect of the EETs is due to a specific interaction with a membrane-bound receptor, no evidence has indicated that novel EET antagonists selectively block the EET actions in dogs. Our goals were to investigate the effects of 11,12- and 14,15-EET, the soluble epoxide hydrolase inhibitor, 12-(3-adamantan-l-ylureido)-dodecanoic acid (AUDA), and the putative selective EET antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), on infarct size of barbital anesthetized dogs subjected to 60 min of coronary artery occlusion and 3 h of reperfusion. Furthermore, the effect of 14,15-EEZE on the cardioprotective actions of the selective mitochondrial ATP-sensitive potassium channel opener diazoxide was investigated. Both 11,12- and 14,15-EET markedly reduced infarct size [expressed as a percentage of the area at risk (IS/AAR)] from 21.8 [+ or -] 1.6% (vehicle) to 8.7 [+ or -] 2.2 and 9.4 [+ or -] 1.3%, respectively. Similarly, AUDA significantly reduced IS/AAR from 21.8 [+ or -] 1.6 to 14.4 [+ or -] 1.2% (low dose) and 9.4 [+ or -] 1.8% (high dose), respectively. Interestingly, the combination of the low dose of AUDA with 14,15-EET reduced IS/AAR to 5.8 [+ or -] 1.6% (P < 0.05), further than either drug alone. Diazoxide also reduced IS/AAR significantly (10.2 [+ or -] 1.9%). In contrast, 14,15-EEZE had no effect on IS/AAR by itself (21.0 [+ or -] 3.6%), but completely abolished the effect of II,12-EET (17.8 [+ or -] 1.4%) and 14,15-EET (19.2 [+ or -] 2.4%) and AUDA (19.3 [+ or -] 1.6%), but not that of diazoxide (10.4 [+ or -] 1.4%). These results suggest that activation of the EET pathway, acting on a putative receptor, by exogenous EETs or indirectly by blocking EET metabolism, produced marked cardioprotection, and the combination of these two approaches resulted in a synergistic effect. These data also suggest that 14,15-EEZE is not blocking the mitochondrial ATP-sensitive potassium channel as a mechanism for antagonizing the cardioprotective effects of the EETs. cytochrome P-450 epoxygenase; epoxyeicosatrienoic acids; soluble epoxide hydrolase; ATP-sensitive potassium channel opener

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.sup.-7] - [10.sup.-4] M) relaxed the rabbit aorta in a concentration-related manner (maximum relaxation = 69 [+ or -] 5%). These relaxations were blocked by apamin ([10.sup.-7] M), an inhibitor of small-conductance [Ca.sup.2+]-activated [K.sup.+] channels. In comparison, 11 (S), 12(R), 15(S), 5(Z), 8(Z), 13(E)-THETA ([10.sup.-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.sup.-4] M 11(R), 12(S), 15(S), 5(Z), 8(Z), 13(E)-THETA increased outward [K.sup.+] current in isolated aortic smooth muscle cells by 119 [+ or -] 36% at +60 mV, whereas [10.sup.-4] M 11(R), 12(R), 15(S), 5(Z), 8(Z), 13(E)-THETA increased outward [K.sup.+] current by only 20 [+ or -] 2%. The 11(R), 12(S), 15(S), 5(Z), 8(Z), 13(E)-THETA-stimulated increase in [K.sup.+] 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.sup.+] channels. The specific structural and stereochemical requirements for [K.sup.+] channel activation suggest that a specific binding site or receptor of 11, 12, 15-THETA is involved in these actions. endothelium-derived hyperpolarizing factor; smooth muscle cells; potassium channels; arachidonic acid

Arachidonic acid (AA) causes endothelium-dependent smooth muscle hyperpolarizations and relaxations that are mediated by a 15-lipoxygenase-I (15-LO-I) metabolite, 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA). We propose that AA is metabolized sequentially by 15-LO-I 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 [sup.4]C-labeled AA, metabolites were extracted and the HEETAs were resolved by performing 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 acidsensitive 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-l-yl-ureido)-dodecanoic acid (AUDA; [10.sup.-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.sup.-7]-[10.sup.-4] M) and acetylcholine ([10.sup.-9]-[10.sup.-6] M) caused concentration-related relaxations that were enhanced by pretreatment with AUDA. These enhanced relaxations were inhibited by increasing extracellular [[K.sup.+]] from 4.8 to 20 mM. AA (3 x [10.sup.-6] M) induced cell membrane hyperpolarization (from -31.0 [+ or -] 1 to -46.8 [+ or -] 2 mV) in aortic strips with an intact endotfielium, 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. arachidonic acid; endothelial cells; endothelium-derived hyperpolarizing factor; 15-lipoxygenase

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.sup.+], 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.sup.-10] to [10.sup.-6] M) caused concentration-dependent relaxations that were shifted to the right by lipoxygenase inhibition and the [Ca.sup.2+]-activated [K.sup.+] 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 [micro]M) and apamin but not barium plus charybdotoxin. Addition of [K.sup.+] (10.9 mM final concentration) to the preconstricted arteries elicited small relaxations. [K.sup.+] addition before ACh restored the charybdotoxin-sensitive component of relaxations to ACh. [K.sup.+] (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.sup.-7] M) stimulated voltage-dependent outward [K.sup.+] current from endothelial cells, which was inhibited by charybdotoxin, indicating [K.sup.+] efflux. Arachidonic acid ([10.sup.-7] to [10.sup.-4] M) induced concentration-related relaxations that were inhibited by apamin but not by charybdotoxin and barium. Addition of arachidonic acid after [K.sup.+] (10.9 mM) resulted in more potent relaxations to arachidonic acid compared with control without [K.sup.+] (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.sup.+] efflux and arachidonic acid metabolites, and a synergism exists between these two separate mechanisms. acetylcholine; potassium channels; lipoxygenase; endothelium-derived hyperpolarizing factor doi:10.1152/ajpheart.00268.2006

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 [+ or -] 0.01 and 6.18 [+ or -] 0.60 [micro]g/mg protein and adrenic acid concentrations of 0.29 [+ or -] 0.01 and 1.56 [+ or -] 0.16 [micro]g/mg protein, respectively. In bovine coronary arterial rings preconstricted with the thromboxane mimetic U-46619, adrenic acid ([10.sup.-9]-[10.sup.-5] M) induced concentration-related relaxations (maximal relaxation = 83 [+ or -] 4%) that were similar to arachidonic acid relaxations. Adrenic acid relaxations were blocked by endothelium removal and the [K.sup.+] channel inhibitor, iberiotoxin (100 nM), and inhibited by the cyclooxygenase inhibitor, indomethacin (10 [micro]M, maximal relaxation = 53 [+ or -] 4%), and the cytochrome P-450 inhibitor, miconazole (10 [micro]M, maximal relaxation = 52 [+ or -] 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.sup.-9]-[10.sup.-5] M)] induced similar concentration-related relaxations (maximal relaxations averaged 83 [+ or -] 3%). Adrenic acid ([10.sup.-6] M) and DH-16,17-EET ([10.sup.-6] M) hyperpolarized coronary arterial smooth muscle. DH-16,17-EET ([10.sup.-8]-[10.sup.-6] M) activated iberiotoxin-sensitive, whole cell [K.sup.+] 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.sup.+] 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. endothelium-dependent relaxation; cytochrome P-450; cyclooxygenase; potassium channels; endothelium-derived hyperpolarizing factor doi:10.1152/ajpheart.00947.2006

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 ([BK.sub.Ca]) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase [BK.sub.Ca] 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 [beta]-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 [[sup.14]C]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.sup.-5] M) increased 14,15-EET release twofold and [BK.sub.Ca] channel activity fourfold in cell-attached patches. Methacholine-induced increases in BKca channel activity were blocked by the CYP inhibitor 17-octadecynoic acid ([10.sup.-5] M). 14(S),15(R)-EET was more potent than 14(R), 15(S)-EET in relaxing bovine coronary arteries and activating [BK.sub.Ca] 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(S), 15(R)-EET from endogenous arachidonic acid to activate [BK.sup.Ca] channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of [BK.sub.Ca], channels in coronary SMCs. endothelium-derived hyperpolarizing factor; arachidonic acid; vascular relaxation

Adrenal steroidogenesis is closely correlated with increases in adrenal blood flow. Many reports have studied the regulation of adrenal blood flow in vivo and in perfused glands, but until recently few studies have been conducted on isolated adrenal arteries. The present study examined vasomotor responses of isolated bovine small adrenal cortical arteries to histamine, an endogenous vasoactive compound, and its mechanism of action. In U-46619-precontracted arteries, histamine ([10.sup.-9-5 X [10.sup.-6] M) elicited concentration-dependent relaxations. The relaxations were blocked by the [H.sub.1] receptor antagonists diphenhydramine (10 [micro]M) or mepyramine (1 [micro]M) (maximal relaxations of 18 [+ or -] 6 and 22 [+ or -] 6%, respectively, vs. 55 [+ or -] 5% of control) but only partially inhibited by the [H.sub.2] receptor antagonist cimetidine ( 10 [micro]M) and the [H.sub.3] receptor antagonist thioperamide (1 [micro]M). Histamine-induced relaxations were also blocked by the nitric oxide synthase inhibitor N-nitroL-arginine (L-NA, 30 [micro]M; maximal relaxation of 13 [+ or -] 7%) and eliminated by endothelial removal or L-NA combined with the cyclooxgenase inhibitor indomethacin (10 [micro]M). In the presence of adrenal zona glomerulosa (ZG) cells, histamine did not induce further relaxations compared with histamine alone. Histamine ([10.sup.-7]-[10.sup.-5] M) concentration-dependently increased aldosterone production by adrenal ZG cells. Compound 48/80 (10 [micro]g/ml), a mast cell degranulator, induced significant relaxations (93 [+ or -] 0.6%), which were blocked by L-NA plus indomethacin or endothelium removal, partially inhibited by the combination of the HI, [H.sub.2], and [H.sub.3] receptor antagonists, but not affected by the mast cell stabilizer sodium cromoglycate (1 mM). These results demonstrate that histamine causes direct relaxation of small adrenal cortical arteries, which is largely mediated by endothelial NO and prostaglandins via [H.sub.1] receptors. The potential role of histamine in linking adrenal vascular events and steroid secretion requires further investigation. endothelium; nitric oxide; prostacyclin; aldosterone

Cerebral vascular smooth muscle cells express the C[B.sub.1] cannabinoid receptor, and C[B.sub.1] receptor agonists produce vasodilation of cerebral arteries. The purpose of this study was to determine whether vasoconstriction of rat middle cerebral artery (MCA) results in the local formation of endocannabinoids (eCBs), which, via activation of C[B.sub.1] receptors, oppose the vasoconstriction in a feedback manner. The thromboxane [A.sub.2] (TX[A.sub.2]) mimetic U-46619 significantly increased N-arachidonylethanolamine (AEA) and 2-arachidonylglycerol (2-AG) content of isolated MCA, whereas 5-hydroxytrypamine (5-HT) decreased AEA and 2-AG content. If eCBs play a feedback role in the regulation of MCA tone, then C[B.sub.1] receptor antagonists should enhance the constriction of MCA produced by U-46619 but not 5-HT. U-46619 caused concentration-dependent constrictions of endothelium-denuded MCA. Two C[B.sub.1] receptor antagonists SR-141716 and AM-251 decreased the E[C.sub.50] value for U-46619 to constrict endothelium-denuded MCA without affecting the maximal effect. A low concentration of C[B.sub.1] receptor agonist Win-55212-2 (30 nM) produced vasodilation of MCAs constricted with low but not saturating concentrations of U-46619. SR-141716 had no effect on the 5-HT concentration-contraction relationship. These data suggest that TX[A.sub.2] receptor activation increases MCA eCB content, which, via activation of C[B.sub.1] receptors, reduces the constriction produced by moderate concentrations of the TX[A.sub.2] agonist. Although 5-HT-induced vasoconstriction is reduced by exogenous C[B.sub.1] receptor agonist, activation of 5-HT receptors does not increase eCB content. These results suggest that MCA production of eCBs is not regulated by constriction per se but likely via a signaling pathway that is specific for TX[A.sub.2] receptors and not 5-HT receptors. C[B.sub.1] receptor; cannabinoids; SR-141716; AM-251; N-arachidonyleth-anolamine; 2-arachidonylglycerol; mass spectrometry

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.sup.-9]-[10.sup.-4] M) produced potent relaxations in isolated phenylephrine-preconstricted arteries, with a maximal relaxation of 99 [+ or -] 0.5% and ECso of 50 nM. The cyclooxygenase (COX) inhibitors indomethacin (10 txM), NS-398 (10 [micro]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 [micro]M), nordihydroguaiaretic acid (NDGA; 1 [micro]M), and ebselen (1 [micro]M). HPLC analysis showed that [[sup.14]C]AA was metabolized by mesenteric arteries to PG[I.sub.2], PG[E.sub.2], THETAs, hydroxyepoxyeicosatrienoic acids (HEETAs), and 15-hydroxyeicosatetraenoic acid (15-HETE). The production of PG[I.sub.2] and PG[E.sub.2] 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. PG[I.sub.2], PG[E.sub.2], and purified THETA fractions relaxed mesenteric arteries precontracted with phenylephrine. The AA- and THETA-induced relaxations were blocked by high [K.sup.+] (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 [E.sub.2]; prostaglandin [I.sub.2]; endothelium-derived hyperpolarizing factor

Rabbit aortic endothelium metabolizes arachidonic acid (AA) by the 15-lipoxygenase pathway to vasodilatory eicosanoids, hydroxyepoxyeicosatrienoic acids (HEETAs), and trihydroxyeicosatrienoic acids (THETAs). The present study determined the chemical identity of the vasoactive THETA and investigated its role in ACh-induced relaxation in the rabbit aorta. AA caused endothelium-dependent, concentration-related relaxations of the rabbit aorta. Increasing the extracellular KCl concentration from 4.8 to 20 mM inhibited the relaxations to AA by ~60%, thereby implicating [K.sup.+]-channel activation in the relaxations. In addition, AA caused an endothelium-dependent hyperpolarization of aortic smooth muscle from -39.6 [+ or -] 2.7 to -56.1 [+ or -] 3.4 mV. In rabbit aortic rings, [[sup.14]C]AA was metabolized to prostaglandins, HEETAs, THETAs, and 15-hydroxyeicosatetraenoic acid. Additional purification of the THETAs by HPLC resolved the mixture into its [sup.14]C-labeled products. Gas chromatography/ mass spectrometry identified the metabolites as isomers of 11,12,15-THETA and 11,14,15-THETA. The 11,12,15-THETA relaxed and hyperpolarized the rabbit aorta, whereas 11,14,15-THETA had no vasoactive effect. The relaxations to 11,12,15-THETA were blocked by 20 mM KCl. In aortic rings pretreated with inhibitors of nitric oxide and prostaglandin synthesis, ACh caused a concentration-related relaxation that was completely blocked by 20 mM KCl. Pretreatment with the phospholipase [A.sub.2] inhibitors mepacrine and 7,7-dimethyl-5,8-eicosadienoic acid, the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-[alpha]-cyanocinnamate, nordihydroguaiaretic acid, and ebselen, or the hydroperoxide isomerase inhibitors miconazole and clotrimazole also blocked ACh-induced relaxations. ACh caused a threefold increase in THETA release. These studies indicate that AA is metabolized by endothelial cells to 11,12,15-THETA, which activates [K.sup.+] channels to hyperpolarize the aortic smooth muscle membrane and induce relaxation. Additionally, this lipoxygenase pathway mediates the nonnitric oxide, nonprostaglandin relaxations to ACh in the rabbit aorta by acting as a source of an endothelium-derived hyperpolarizing factor. trihydroxyeicosatrienoic acid; arachidonic acid; endothelium-derived hyperpolarizing factor; potassium channels; membrane potential; lipoxygenase

Epoxyeicosatrienoic acids (EETs) are endothelium-derived eicosanoids that activate potassium channels, hyperpolarize the membrane, and cause relaxation. We tested 19 analogs of 14,15-EET on vascular tone to determine the structural features required for activity. 14,15-EET relaxed bovine coronary arterial rings in a concentration-related manner (E[D.sub.50] = [10.sup.-6] M). Changing the carboxyl to an alcohol eliminated dilator activity, whereas 14,15-EET-methyl ester and 14,15-EET-methylsulfonimide retained full activity. Shortening the distance between the carboxyl and epoxy groups reduced the agonist potency and activity. Removal of all three double bonds decreased potency. An analog with a [DELTA]8 double bond had full activity and potency. However, the analogs with only a [DELTA]5 or [DELTA]11 double bond had reduced potency. Conversion of the epoxy oxygen to a sulfur or nitrogen resulted in loss of activity. 14(S),15(R)-EET was more potent than 14(R),15(S)-EET, and 14,15-(cis)-EET was more potent than 14,15-(trans)-EET. These studies indicate that the structural features of 14,15-EET required for relaxation of the bovine coronary artery include a carbon-1 acidic group, a [DELTA]8 double bond, and a 14(S),15(R)-(cis)-epoxy group. endothelium-derived hyperpolarizing factor; cytochrome P-450; arachidonic acid; epoxyeicosatrienoic acid

Epoxyeicosatrienoic acids (EETs) cause vascular relaxation by activating smooth muscle large conductance [Ca.sup.2+]-activated [K.sup.+] ([K.sub.Ca]) channels. EETs are metabolized to dihydroxyeicosatrienoic acids (DHETs) by epoxide hydrolase. We examined the contribution of 14,15-DHET to 14,15-EET-induced relaxations and characterized its mechanism of action. 14,15-DHET relaxed U-46619-precontracted bovine coronary artery rings but was approximately fivefold less potent than 14,15-EET. The relaxations were inhibited by charybdotoxin, iberiotoxin, and increasing extracellular [K.sub.+] to 20 mM. In isolated smooth muscle cells, 14,15-DHET increased an iberiotoxin-sensitive, outward [K.sub.+] current and increased [K.sub.Ca] channel activity in cell-attached patches and inside-out patches only when GTP was present. 14,15-[[sup.14]C]EET methyl ester (Me) was converted to 14,15-[[sup.14]C]DHET-Me, 14,15-[[sup.14]C]DHET, and 14,15-[[sup.14]C]EET by coronary arterial rings and endothelial cells but not by smooth muscle cells. The metabolism to 14,15-DHET was inhibited by the epoxide hydrolase inhibitors 4-phenylchalcone oxide (4-PCO) and BIRD-0826. Neither inhibitor altered relaxations to acetylcholine, whereas relaxations to 14,15-EET-Me were increased slightly by BIRD-0826 but not by 4-PCO. 14,15-DHET relaxes coronary arteries through activation of [K.sub.Ca] channels. Endothelial cells, but not smooth muscle cells, convert EETs to DHETs, and this conversion results in a loss of vasodilator activity. endothelium-derived hyperpolarizing factor; epoxyeicosatrienoic acids; potassium channels; epoxide hydrolase