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Epoxyeicosatrienoic acids (EETs) contribute to haemodynamics, electrolyte homoeostasis and blood pressure regulation, leading to the concept that EETs can be therapeutically targeted for hypertension. In the present study, multiple structural EET analogues were synthesized based on the EET pharmacophore and vasodilator structure-activity studies. Four EET analogues with 91-119% vasodilatory activity in the isolated bovine coronary artery (EC50: 0.18-1.6 μM) were identified and studied for blood-pressure-lowering in hypertension. Two EET analogues in which the COOH group at carbon 1 of the EET pharmacophore was replaced with either an aspartic acid (EET-A) or a heterocyclic surrogate (EET-X) were administered for 14 days [10 mg/kg per day intraperitoneally (i.p.)]. Both EET-A and EET-X lowered blood pressure in spontaneously hypertensive rats (SHRs) and in angiotensin II (AngII) hypertension. On day 14, the mean arterial pressures in EET analogue-treated AngII-hypertensive and SHRs were 30-50 mmHg (EET-A) and 15-20 mmHg (EET-X) lower than those in vehicle-treated controls. These EET analogues (10 mg/kg per day) were further tested in AngII hypertension by administering orally in drinking water for 14 days and EET-A lowered blood pressure. Additional experiments demonstrated that EET-A inhibits epithelial sodium channel (ENaC) activity in cultured cortical collecting duct cells and reduced renal expression of ENaC subunits in AngII hypertension. In conclusion, we have characterized EET-A as an orally active antihypertensive EET analogue that protects vascular endothelial function and has ENaC inhibitory activity in AngII hypertension.

Cytochrome P-450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H(2)O(2)), are important signaling molecules in the kidney. In renal arteries, EETs cause vasodilation whereas H(2)O(2) causes vasoconstriction. To determine the physiological contribution of H(2)O(2), catalase is used to inactivate H(2)O(2). However, the consequence of catalase action on EET vascular activity has not been determined. In rat renal afferent arterioles, 14,15-EET caused concentration-related dilations that were inhibited by Sigma bovine liver (SBL) catalase (1,000 U/ml) but not Calbiochem bovine liver (CBL) catalase (1,000 U/ml). SBL catalase inhibition was reversed by the soluble epoxide hydrolase (sEH) inhibitor tAUCB (1 μM). In 14,15-EET incubations, SBL catalase caused a concentration-related increase in a polar metabolite. Using mass spectrometry, the metabolite was identified as 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), the inactive sEH metabolite. 14,15-EET hydrolysis was not altered by the catalase inhibitor 3-amino-1,2,4-triazole (3-ATZ; 10-50 mM), but was abolished by the sEH inhibitor BIRD-0826 (1-10 μM). SBL catalase EET hydrolysis showed a regioisomer preference with greatest hydrolysis of 14,15-EET followed by 11,12-, 8,9- and 5,6-EET (V(max) = 0.54 ± 0.07, 0.23 ± 0.06, 0.18 ± 0.01 and 0.08 ± 0.02 ng DHET·U catalase(-1)·min(-1), respectively). Of five different catalase preparations assayed, EET hydrolysis was observed with two Sigma liver catalases. These preparations had low specific catalase activity and positive sEH expression. Mass spectrometric analysis of the SBL catalase identified peptide fragments matching bovine sEH. Collectively, these data indicate that catalase does not affect EET-mediated dilation of renal arterioles. However, some commercial catalase preparations are contaminated with sEH, and these contaminated preparations diminish the biological activity of H(2)O(2) and EETs.

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.

Cytochrome P-450 epoxygenases metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs). EETs relax vascular smooth muscle by membrane hyperpolarization. 14,15-Epoxyeicosa-5(Z)-enoic acid (14,15-EE5ZE) antagonizes many vascular actions of EETs. EETs are converted to the corresponding dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH). sEH activity in the bovine arterial endothelium and smooth muscle regulates endogenous EETs. This study examined sEH metabolism of 14,15-EE5ZE to 14,15-dihydroxy-eicosa-5(Z)-enoic acid (14,15-DHE5ZE) and the resultant consequences on EET relaxations of bovine coronary arteries (BCAs). BCAs converted 14,15-EE5ZE to 14,15-DHE5ZE. This conversion was blocked by the sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA). 14,15-EET relaxations (maximal relaxation, 83.4 ± 4.5%) were inhibited by 14,15-DHE5ZE (10 μM; maximal relaxation, 36.1 ± 9.0%; p < 0.001). In sharp contrast with 14,15-EE5ZE, 14,15-DHE5ZE is a 14,15-EET-selective inhibitor and did not inhibit 5,6-, 8,9-, or 11,12-EET relaxations. 14,15-EET and 11,12-EET relaxations were similar in the presence and absence of AUDA (1 μM). 14,15-EE5ZE inhibited 14,15-EET relaxations to a similar extent with and without AUDA pretreatment. However, 14,15-EE5ZE inhibited 11,12-EET relaxations to a greater extent with than without AUDA pretreatment. These observations indicate that sEH converts 14,15-EE5ZE to 14,15-DHE5ZE, and this alteration influences antagonist selectivity against EET-regioisomers. 14,15-DHE5ZE inhibited endothelium-dependent relaxations to AA but not endothelium-independent relaxations to sodium nitroprusside. A series of sEH-resistant ether analogs of 14,15-EE5ZE was developed, and analogs with agonist and antagonist properties were identified. The present study indicates that conversion of 14,15-EE5ZE to 14,15-DHE5ZE produces a 14,15-EET-selective antagonist that will be a useful pharmacological tool to identify EET receptor(s) and EET function in the cardiovascular system.

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.

15-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% O(2) increased 15-LO-1 expression. Rabbits were housed in a hypoxic atmosphere of 12% O(2) for 5 days. 15-LO-1 expression increased in the endothelium of the arteries of rabbits in 12% O(2) compared with room air. THETA and HEETA synthesis was also enhanced in aortas and mesenteric arteries. AA hyperpolarized the smooth muscle cells in indomethacin- and phenylephrine-treated mesenteric arteries of hypoxic rabbits from -29.4 +/- 1 to -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 mesenteric 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.

Arachidonic acid (AA) metabolites 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 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(omega)-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 N(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 +/- 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.

Angiotensin (Ang) II regulates adrenal steroidogenesis and adrenal cortical arterial tone. Vascular metabolism could decrease Ang II concentrations and produce metabolites with vascular activity. Our goals were to study adrenal artery Ang II metabolism and to characterize metabolite vascular activity. Bovine adrenal cortical arteries were incubated with Ang II (100 nmol/L) for 10 and 30 minutes. Metabolites were analyzed by mass spectrometry. Ang (1-7), Ang III, and Ang IV concentrations were 146+/-21, 173+/-42 and 58+/-11 pg/mg at 10 minutes and 845+/-163, 70+/-14, and 31+/-3 pg/mg at 30 minutes, respectively. Concentration-related relaxations of U46619-preconstricted cortical arteries to Ang II (maximum relaxation=29+/-3%; EC(50)=3.4 pmol/L) were eliminated by endothelium removal and inhibited by the NO synthase inhibitor, nitro-L-arginine (30 micromol/L; maximum relaxation=14+/-7%). Ang II relaxations were enhanced by the angiotensin type-1 receptor antagonist losartan (1 micromol/L; maximum relaxation=41+/-3%; EC(50)=11 pmol/L). Losartan-enhanced Ang II relaxations were inhibited by nitro-L-arginine (maximum relaxation=18+/-5%) and the angiotensin type-2 receptor antagonist PD123319 (10 micromol/L; maximum relaxation=27+/-5%). Ang (1-7) and Ang III caused concentration-related relaxations with less potency (EC(50)=43 and 24 nmol/L, respectively) but similar efficacy (maximum relaxations=39+/-3% and 48+/-5%, respectively) as losartan-enhanced Ang II relaxations. Ang (1-7) relaxations were inhibited by nitro-L-arginine (maximum relaxation=16+/-4%) and the Ang (1-7) receptor antagonist 7(D)-Ala-Ang (1-7) (1 micromol/L; maximum relaxation=10+/-3%) and eliminated by endothelium removal. Thus, Ang II metabolism by adrenal cortical arteries to metabolites with decreased vascular activity represents an inactivation pathway possibly decreasing Ang II presentation to adrenal steroidogenic cells and limits Ang II vascular effects.

Arachidonic acid is metabolized by the 15-lipoxygenase-1 pathway to the vasodilatory eicosanoids hydroxy-epoxyeicosatrienoic acid and trihydroxyeicosatrienoic acid. We determined the in vitro and in vivo effects of the 15-lipoxygenase-1 metabolites in rabbits infected with adenovirus containing cDNA for human 15-lipoxygenase-1 (Ad-15-LO-1). Forty hours after intravenous adenoviral injection, 15-lipoxygenase-1 expression increased in liver and mesenteric arteries 10-fold and 3-fold, respectively. Expression of 15-LO-1 was limited to the endothelium of mesenteric arteries. Overexpression did not occur in tissues from rabbits infected with a beta-galactosidase containing adenovirus. Trihydroxyeicosatrienoic acid and hydroxy-epoxyeicosatrienoic acid synthesis per milligram of tissue increased by 2.1- and 1.5-fold, respectively, in mesenteric arteries from Ad-15-LO-1-infected rabbits compared with normal rabbits. Pretreatment with a 15-lipoxygenase inhibitor BW755C inhibited the synthesis. NO and prostaglandin-independent, maximal relaxations to acetylcholine were greater in mesenteric arteries from Ad-15-LO-1-infected rabbits (98.3+/-1.7%) compared with normal (60.93+/-10.5%) and beta-galactosidase containing adenovirus-infected rabbits (68.3+/-7.7%). Pretreatment with BW755C decreased these relaxations. Mean arterial pressure and heart rate did not differ in Ad-15-LO-1-infected rabbits compared with normal or beta-galactosidase containing adenovirus-infected rabbits. The hypotensive responses to acetylcholine in the presence and absence of inhibition of NO and prostaglandins were greater in Ad-15-LO-1-infected rabbits (-52+/-2% and -47+/-2%) compared with normal (-31+/-3% and -25+/-5%) or beta-galactosidase containing adenovirus-infected rabbits (-38+/-2% and -30+/-3%). Therefore, increased expression of 15-LO-1 increases acetylcholine relaxation in arteries and hypotensive responses in rabbits because of increased hydroxy-epoxyeicosatrienoic acid and trihydroxyeicosatrienoic acid synthesis.

Adrenal blood flow is coupled to adrenal hormone secretion. ACTH increases adrenal blood flow and stimulates the secretion of aldosterone and cortisol in vivo. However, ACTH does not alter vascular tone of isolated adrenal cortical arteries. Mechanisms underlying this discrepancy remain unsolved. The present study examined the effect of zona glomerulosa (ZG) cells on cortical arterial tone. ZG cells (10(5) to 10(7) cells) and ZG cell-conditioned medium relaxed preconstricted adrenal arteries (maximal relaxations = 79 +/- 4 and 66 +/- 4%, respectively). In adrenal arteries coincubated with a small number of ZG cells (0.5-1 x 10(6)), ACTH (10(-12) to 10(-8) m) induced concentration-dependent relaxations (maximal relaxation = 67 +/- 4%). Similarly, ACTH (10(-8) m) dilated (55 +/- 10%) perfused arteries embedded in adrenal cortical slices. ZG cell-dependent relaxations to ACTH were endothelium-independent and inhibited by high extracellular K(+) (60 mm); the K(+) channel blocker, iberiotoxin (100 nm); the cytochrome P450 inhibitors SKF 525A (10 microm) and miconazole (10 microm); and the epoxyeicosatrienoic acid (EET) antagonist 14,15-EEZE (2 microm). Four EET regioisomers were identified in ZG cell-conditioned media. EET production was stimulated by ACTH. We conclude that ZG cells release EETs and this release is stimulated by ACTH. Interaction of endocrine and vascular cells represents a mechanism for regulating adrenal blood flow and couples steroidogenesis to increased blood flow.

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

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 microg/mg protein and adrenic acid concentrations of 0.29 +/- 0.01 and 1.56 +/- 0.16 microg/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 microM, maximal relaxation = 53 +/- 4%), and the cytochrome P-450 inhibitor, miconazole (10 microM, 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)-epoxyeicosatrienoic 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.

Objective: Endothelium-dependent dilation to acetylcholine (Ach) is reduced in mucosal arterioles from patients with inflammatory bowel disease (IBD). The contributions of both nitric oxide (NO) and endothelial-derived hyperpolarizing factor (EDHF) are decreased. We hypothesized that the remaining dilation results from products of cyclooxygenase., Methods and Results: High-performance liquid chromatography (HPLC) was used to isolate eicosanoid vasodilator products and videomicroscopy was used to examine vasomotor responses in human mucosal arterioles from subjects with or without IBD undergoing bowel resection surgeries. In subjects without IBD, Ach constricted (-52%+/-10%) arterioles devoid of endothelium. Indomethacin (INDO) (cyclooxygenase inhibitor) had no effect. In contrast, Ach dose-dependently dilated both intact and endothelial denuded arterioles from patients with IBD. The dilation was converted to constriction by INDO (-54%+/-9%; P<0.05 versus non-IBD) or by BWA868C (PGD2 receptor antagonist). Only in arterioles from subjects with IBD did Ach produce an arachidonic acid metabolite that comigrated on HPLC with PG D2 (PGD2). Exogenous PGD2 dilated (max=66%+/-4%) IBD arterioles., Conclusions: In arterioles from IBD patients, Ach-mediated dilation shifts from endothelial production of NO and EDHF to nonendothelial generation of a PG, likely PGD2. This is a novel dilator mechanism arising from nonendothelial vascular tissue that compensates for loss of endothelium-dependent dilation. PGD2 appears to be important in regulating mucosal blood flow in patients with IBD, implicating potentially detrimental effects from nonsteroidal antiinflammatory drugs.

5,6-epoxyeicosatrienoic acid (5,6-EET) is a cytochrome P450 epoxygenase metabolite of arachidonic acid that causes vasorelaxation. However, investigations of its role in biological systems have been limited by its chemical instability. We developed a stable agonist of 5,6-EET, 5-(pentadeca-3(Z),6(Z),9(Z)-trienyloxy)pentanoic acid (PTPA), in which the 5,6-epoxide was replaced with a 5-ether. PTPA obviates chemical and enzymatic hydrolysis. In bovine coronary artery rings precontracted with U46619, PTPA (1 nmol/L to 10 micromol/L) induced concentration-dependent relaxations, with maximal relaxation of 86+/-5% and EC50 of 1 micromol/L. The relaxations were inhibited by the cyclooxygenase inhibitor indomethacin (10 micromol/L; max relaxation 43+/-9%); the ATP-sensitive K+ channel inhibitor glybenclamide (10 micromol/L; max relaxation 49+/-6%); and the large conductance calcium-activated K+ channel inhibitor iberiotoxin (100 nmol/L; max relaxation 38+/-6%) and abolished by the combination of iberiotoxin with indomethacin or glybenclamide or increasing extracellular K+ to 20 mmol/L. Whole-cell outward K+ current was increased nearly 6-fold by PTPA (10 micromol/L), which was also blocked by iberiotoxin. Additionally, we synthesized 5-(pentadeca-6(Z),9(Z)-dienyloxy)pentanoic acid and 5-(pentadeca-3(Z),9(Z)-dienyloxy)pentanoic acid (PDPA), PTPA analogs that lack the 8,9 or 11,12 double bonds of arachidonic acid and therefore are not substrates for cyclooxygenase. The PDPAs caused concentration-dependent relaxations (max relaxations 46+/-13% and 52+/-7%, respectively; EC50 1micromol/L), which were not altered by glybenclamide but blocked by iberiotoxin. These studies suggested that PTPA induces relaxation through 2 mechanisms: (1) cyclooxygenase-dependent metabolism to 5-ether-containing prostaglandins that activate ATP-sensitive K+ channels and (2) activation of smooth muscle large conductance calcium-activated K+ channels. PDPAs only activate large conductance calcium-activated K+ channels.

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 micromol/l), methacholine (10 micromol/l)-stimulated 2-AG release was blocked by the phospholipase C inhibitor U-73122 (10 micromol/l) or the diacylglycerol lipase inhibitor RHC-80267 (40 micromol/l). In U-46619-preconstricted bovine coronary arterial rings, 2-AG relaxations averaging 100% at 10 micromol/l were inhibited by endothelium removal, by DAK, by the hydrolase inhibitor methyl arachidonylfluorophosphate (10 micromol/l), by the cyclooxygenase inhibitor indomethacin (10 micromol/l), but not by the CB1 cannabinoid receptor antagonist SR-141716 (1 micromol/l). The cytochrome P-450 inhibitor SKF-525a (10 micromol/l) and the 14,15-epoxyeicosa-5Z-enoic acid EET antagonist (14,15-EEZE; 10 micromol/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.

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 +/- 0.5% and EC(50) of 50 nM. The cyclooxygenase (COX) inhibitors indomethacin (10 microM), NS-398 (10 microM, 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 muM), nordihydroguaiaretic acid (NDGA; 1 microM), and ebselen (1 microM). HPLC analysis showed that [(14)C]AA was metabolized by mesenteric arteries to PGI(2), PGE(2), THETAs, hydroxyepoxyeicosatrienoic acids (HEETAs), and 15-hydroxyeicosatetraenoic acid (15-HETE). The production of PGI(2) and PGE(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. PGI(2), PGE(2), 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.

The present study characterizes the vascular responses of isolated small bovine adrenal cortical arteries to acetylcholine, an endogenous neurotransmitter in the adrenal gland. Acetylcholine (10(-10) to 10(-6) m) elicited a concentration-dependent relaxation, with a maximal relaxation of 96 +/- 1% and EC50 of 4.2 nm. The relaxation was abolished by endothelial removal and attenuated by the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NA, 30 microm) but not by the cyclooxygenase inhibitor indomethacin (10 microm). The maximal relaxation and EC50 of acetylcholine in the presence of L-NA were 87 +/- 4% and 22 nm, respectively. The acetylcholine-induced, indomethacin- and L-NA-resistant relaxation was eliminated by high K+ and markedly inhibited by the cytochrome P450 inhibitors SKF 525A (10 microm) and miconazole (10 microm). The maximal relaxations and EC50s with SKF 525A and miconazole were 56 +/- 8 and 72 +/- 2% and 0.8 and 0.5 microm, respectively. In indomethacin- and L-NA-treated arteries, acetylcholine induced a smooth muscle hyperpolarization, which was blocked by SKF 525A (3 +/- 1 mV vs. 15 +/- 2 mV of control). Arachidonic acid (10(-9) to 10(-5) m) and 14,15-epoxyeicosatrienoic acid (14,15-EET, 10(-9) to 10(-5) m), a cytochrome P450 metabolite of arachidonic acid, also evoked relaxations in small adrenal arteries, with maximal relaxations of 56 +/- 4 and 90 +/- 5%, respectively. The arachidonic acid-induced relaxation was blocked by SKF 525A. Using high-pressure liquid chromatography and gas chromatography/mass spectrometry analysis, EETs were identified in small adrenal arteries. These results demonstrate that acetylcholine is a potent vasodilator of small adrenal cortical arteries. The acetylcholine-induced relaxation is largely mediated by an endothelium-dependent hyperpolarization mechanism, presumably through cytochrome P450 metabolites of arachidonic acid.

Arachidonic acid induces an endothelium-dependent relaxation of the rabbit aorta that is blocked by lipoxygenase inhibitors. The cellular vasodilatory mechanisms activated by arachidonic acid metabolites remain undefined. In rabbit thoracic aortic rings pretreated with indomethacin (10 micromol/L) and contracted with phenylephrine, arachidonic acid (0.1 to 100 micromol/L) induced concentration-dependent relaxations. Maximal relaxations averaged 45+/-3% and were inhibited by increasing extracellular K+ (30 mmol/L, 15+/-5%; P<0.001) or incubation with apamin (100 nmol/L, 26+/-7%; P<0.05) but not incubation with charybdotoxin (100 nmol/L, 41+/-5%). In aortic strips with an intact endothelium that were treated with phenylephrine, arachidonic acid (10 micromol/L) increased the membrane potential from -28.7+/-1.3 to -37.8+/-3.0 mV (P<0.01). Preincubation with apamin did not alter basal membrane potential but inhibited arachidonic acid-induced hyperpolarization (-31.5+/-1.5 mV). Incubation of rabbit aortic segments with apamin or charybdotoxin did not alter [14C]arachidonic acid metabolism. Whole-cell outward K+ currents from isolated rabbit aortic smooth muscle cells averaged 43.0+/-4.8 pA/pF at 60 mV and were significantly decreased to 35.7+/-4.2 pA/pF by apamin (P<0.001). Subsequent addition of charybdotoxin further decreased maximal currents to 14.4+/-2.3 pA/pF. Addition of 11,12,15-trihydroxyeicosatrienoic acid increased the outward whole-cell K+ current. In inside-out patches of aortic smooth muscle, apamin inhibited the calcium activation (100 to 300 nmol/L; P<0.001) of a small-conductance K+ channel (approximately 24 pS). These results suggest that arachidonic acid induces endothelium-dependent hyperpolarization and relaxation of rabbit aorta through activation of smooth muscle, apamin-sensitive K+ currents.

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 approximately 60%, thereby implicating K+-channel activation in the relaxations. In addition, AA caused an endothelium-dependent hyperpolarization of aortic smooth muscle from -39.6 +/- 2.7 to -56.1 +/- 3.4 mV. In rabbit aortic rings, [14C]AA was metabolized to prostaglandins, HEETAs, THETAs, and 15-hydroxyeicosatetraenoic acid. Additional purification of the THETAs by HPLC resolved the mixture into its 14C-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 A2 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+ 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.

Endothelium-dependent hyperpolarization and relaxation of vascular smooth muscle are mediated by endothelium-derived hyperpolarizing factors (EDHFs). EDHF candidates include cytochrome P-450 metabolites of arachidonic acid, K(+), hydrogen peroxide, or electrical coupling through gap junctions. In bovine coronary arteries, epoxyeicosatrienoic acids (EETs) appear to function as EDHFs. A 14,15-EET analogue, 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) was synthesized and identified as an EET-specific antagonist. In bovine coronary arterial rings preconstricted with U46619, 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET induced concentration-related relaxations. Preincubation of the arterial rings with 14,15-EEZE (10 micromol/L) inhibited the relaxations to 14,15-EET, 11,12-EET, 8,9-EET, and 5,6-EET but was most effective in inhibiting 14,15-EET-induced relaxations. 14,15-EEZE also inhibited indomethacin-resistant relaxations to methacholine and arachidonic acid and indomethacin-resistant and L-nitroarginine-resistant relaxations to bradykinin. It did not alter relaxation responses to sodium nitroprusside, iloprost, or the K(+) channel activators (NS1619 and bimakalim). Additionally, in small bovine coronary arteries pretreated with indomethacin and L-nitroarginine and preconstricted with U46619, 14,15-EEZE (3 micromol/L) inhibited bradykinin (10 nmol/L)-induced smooth muscle hyperpolarizations and relaxations. In rat renal microsomes, 14,15-EEZE (10 micromol/L) did not decrease EET synthesis and did not alter 20-hydroxyeicosatetraenoic acid synthesis. This analogue acts as an EET antagonist by inhibiting the following: (1) EET-induced relaxations, (2) the EDHF component of methacholine-induced, bradykinin-induced, and arachidonic acid-induced relaxations, and (3) the smooth muscle hyperpolarization response to bradykinin. Thus, a distinct molecular structure is required for EET activity, and alteration of this structure modifies agonist and antagonist activity. These findings support a role of EETs as EDHFs.

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 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 (ED[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 Δ8 double bond had full activity and potency. However, the analogs with only a Δ5 or All 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 Δ8 double bond, and a 14(S),15(R)-(cis)-epoxy group. [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]