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Endothelium-derived epoxyeicosatrienoic acids (EETs) have numerous vascular activities mediated by G protein-coupled receptors. Long-chain free fatty acids and EETs activate GPR40, prompting us to investigate the role of GPR40 in some vascular EET activities. 14,15-EET, 11,12-EET, arachidonic acid, and the GPR40 agonist GW9508 increase intracellular calcium concentrations in human GPR40-overexpressing HEK293 cells (EC 50 = 0.58 ± 0.08 μm, 0.91 ± 0.08 μm, 3.9 ± 0.06 μm, and 19 ± 0.37 nm, respectively). EETs with cis - and trans -epoxides had similar activities, whereas substitution of a thiirane sulfur for the epoxide oxygen decreased the activities. 8,9-EET, 5,6-EET, and the epoxide hydrolysis products 11,12- and 14,15-dihydroxyeicosatrienoic acids were less active than 11,12-EET. The GPR40 antagonist GW1100 and siRNA-mediated GPR40 silencing blocked the EET- and GW9508-induced calcium increases. EETs are weak GPR120 agonists. GPR40 expression was detected in human and bovine endothelial cells (ECs), smooth muscle cells, and arteries. 11,12-EET concentration-dependently relaxed preconstricted coronary arteries; however, these relaxations were not altered by GW1100. In human ECs, 11,12-EET increased MAP kinase (MAPK)-mediated ERK phosphorylation, phosphorylation and levels of connexin-43 (Cx43), and expression of cyclooxygenase-2 (COX-2), all of which were inhibited by GW1100 and the MAPK inhibitor U0126. Moreover, siRNA-mediated GPR40 silencing decreased 11,12-EET-induced ERK phosphorylation. These results indicated that GPR40 is a low-affinity EET receptor in vascular cells and arteries. We conclude that epoxidation of arachidonic acid to EETs enhances GPR40 agonist activity and that 11,12-EET stimulation of GPR40 increases Cx43 and COX-2 expression in ECs via ERK phosphorylation., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Angiotensin II (Ang II) and adrenocorticotropic hormone (ACTH) regulate adrenal vascular tone in vitro through endothelial and zona glomerulosa cell-derived mediators. The role of these mediators in regulating adrenal blood flow (ABF) and mean arterial pressure (MAP) was examined in anesthetized rats. Ang II (0.01 to 100 ng/kg) increased ABF [maximal increase of 97.2 ± 6.9 perfusion units (PUs) at 100 ng/kg] and MAP (basal, 115 ± 7 mm Hg; Ang II, 163 ± 5 mm Hg). ACTH (0.1 to 1000 ng/kg) also increased ABF (maximum increase of 91.4 ± 10.7 PU) without changing MAP. ABF increase by Ang II was partially inhibited by the nitric oxide (NO) synthase inhibitor N-nitro-l-arginine methyl ester (L-NAME) (maximum increase of 72.9 ± 4.2 PU), the cytochrome P450 inhibitor miconazole (maximum increase of 39.1 ± 6.8 PU) and the epoxyeicosatrienoic acid (EET) antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) (maximum increase of 56.0 ± 13.7 PU) alone, whereas combined administration of miconazole and L-NAME (maximum increase of 16.40 ± 8.98 PU) ablated it. These treatments had no effect on MAP. Indomethacin did not affect the increase in ABF or MAP induced by Ang II. The ABF increase by ACTH was partially ablated by miconazole and 14,15-EEZE but not by L-NAME. Steroidogenic stimuli such as Ang II and ACTH increase ABF to promote oxygen and cholesterol delivery for steroidogenesis and aldosterone transport to its target tissues. The increases in ABF induced by Ang II are mediated by release of NO and EETs, whereas ABF increases with ACTH are mediated by EETs only., (Copyright © 2018 Endocrine Society.)

Aim: 12/15-lipoxygenase (12/15-LO) metabolizes arachidonic acid (AA) into several vasoactive eicosanoids. In mouse arteries, we previously characterized the enzyme's 15-LO metabolites 12(S)-hydroxyeicosatetraenoic acid (HETE), 15-HETE, hydroxyepoxyeicosatrienoic acids (HEETAs) and 11,12,15-trihydroxyeicosatrienoic acids (11,12,15-THETAs) as endothelium-derived relaxing factors. However, the observed 12-LO metabolites remained uncharacterized. The purpose of this study was to determine the structure and biological functions of eicosanoids generated by the enzyme's 12-LO activity., Methods: Metabolites extracted from aortas of C57BL/6 male mice were separated using a series of reverse and normal phase chromatographic steps and identified as hepoxilin A 3 , trioxilin A 3 and trioxilin C 3 by mass spectrometry. Activities of these natural compounds were tested on isometric tension and intracellular calcium release. The role of thromboxane (TP) receptor was determined in HEK293 cells overexpressing TPα receptor (TPα -HEK)., Results: All identified vascular 12-LO metabolites were biologically active. In mouse mesenteric arteries, trioxilin A 3 , C 3 and hepoxilin A 3 (3 μm) relaxed arteries constricted with the thromboxane mimetic, U46619-constricted arteries (maximum relaxations of 78.9 ± 3.2, 29.7 ± 4.6, 82.2 ± 5.0 and 88.0 ± 2.4% respectively), but not phenylephrine-constricted arteries. In TPα-HEK cells, trioxilin A 3 , C 3 and hepoxilin A 3 (10 μm) inhibited U46619 (10 nM)-induced increases in intracellular calcium by 53.0 ± 7.2%, 32.8 ± 5.0% and 37.9 ± 13.5% respectively. In contrast, trioxilin B 3 and hepoxilin B 3 were not synthesized in arteries and exhibited little biological activity., Conclusion: Trioxilin A 3 and C 3 and hepoxilin A 3 are endogenous vascular relaxing factors. They are not endothelium-derived hyperpolarizing factors but mediate vascular relaxation by inhibiting TP agonist-induced increases in intracellular calcium. Thus, they regulate vascular homeostasis by acting as endogenous TP antagonists., Competing Interests: The authors declared no conflicts of interest, financial or otherwise., (© 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

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.

Background and Purpose. In rat middle cerebral arteries, endothelium-dependent hyperpolarization (EDH) is mediated by activation of calcium-activated potassium (KCa) channels specifically KCa2.3 and KCa3.1. Lipoxygenase (LOX) products function as endothelium-derived hyperpolarizing factors (EDHFs) in rabbit arteries by stimulating KCa2.3. We investigated if LOX products contribute to EDH in rat cerebral arteries. Methods. Arachidonic acid (AA) metabolites produced in middle cerebral arteries were measured using HPLC and LC/MS. Vascular tension and membrane potential responses to SLIGRL were simultaneously recorded using wire myography and intracellular microelectrodes. Results. SLIGRL, an agonist at PAR2 receptors, caused EDH that was inhibited by a combination of KCa2.3 and KCa3.1 blockade. Non-selective LOX-inhibition reduced EDH, whereas inhibition of 12-LOX had no effect. Soluble epoxide hydrolase (sEH) inhibition enhanced the KCa2.3 component of EDH. Following NO synthase (NOS) inhibition, the KCa2.3 component of EDH was absent. Using HPLC, middle cerebral arteries metabolized (14)C-AA to 15- and 12-LOX products under control conditions. With NOS inhibition, there was little change in LOX metabolites, but increased F-type isoprostanes. 8-iso-PGF2α inhibited the KCa2.3 component of EDH. Conclusions. LOX metabolites mediate EDH in rat middle cerebral arteries. Inhibition of sEH increases the KCa2.3 component of EDH. Following NOS inhibition, loss of KCa2.3 function is independent of changes in LOX production or sEH inhibition but due to increased isoprostane production and subsequent stimulation of TP receptors. These findings have important implications in diseases associated with loss of NO signaling such as stroke; where inhibition of sEH and/or isoprostane formation may of benefit.

Targeted disruption of the Alox15 gene makes mice resistant to angiotensin II-, DOCA/salt-, and N(ω)-nitro-L-arginine methyl ester (L-NAME)-induced experimental hypertension. Macrophages, a primary source of Alox15, are facilitating this resistance, but the underlying mechanism is not known. Because Alox15 metabolites are peroxisome proliferator-activated receptor (PPAR)γ agonists, we hypothesized that activation of macrophage PPARγ is the key step in Alox15 mediation of hypertension. Thioglycollate, used for macrophage elicitation, selectively upregulated PPARγ and its target gene CD36 in peritoneal macrophages of both wild-type (WT) and Alox15(-/-) mice. Moreover, thioglycollate-injected Alox15(-/-) mice became hypertensive upon L-NAME treatment. A similar hypertensive effect was observed with adoptive transfer of thioglycollate-elicited Alox15(-/-) macrophages into Alox15(-/-) recipient mice. The role of PPARγ was further specified by using the selective PPARγ antagonist GW9662. WT mice treated with 50 μg/kg daily dose of GW9662 for 12 days became resistant to L-NAME-induced hypertension. The PPARγ antagonist treatment also prevented L-NAME-induced hypertension in thioglycollate-injected Alox15(-/-) mice, indicating a PPARγ-mediated effect in macrophage elicitation and the resultant hypertension. These results indicate a regulatory role for macrophage-localized PPARγ in L-NAME-induced experimental hypertension.

Adrenal blood flow (ABF) is closely coupled to steroid hormone release. ACTH and angiotensin (Ang) II stimulate cortisol and aldosterone secretion; however, their effects on ABF remain poorly defined. We used the laser-Doppler technique to measure rat ABF. Anesthetized male Sprague-Dawley rats were cannulated for mean arterial pressure (MAP) measurement and drug infusion. The left adrenal gland was exposed for ABF measurement. ABF and MAP changes to ACTH and Ang II were determined. Bolus injections of Ang II (0.01-1000 ng/kg) increased ABF (maximal increase = 110 ± 18 perfusion units at 1000 ng/kg) and increased MAP at doses greater than 10 ng/kg (basal, 99.2 ± 1.4 mm Hg; 1000 ng/kg Ang II, 149.7 ± 3.9 mm Hg). ACTH (0.1-1000 ng/kg) increased ABF (maximum increase = 158 ± 33 perfusion units) without increasing MAP. ABF increases induced by Ang II and ACTH were ablated by the cytochrome 450 inhibitor miconazole (2 mg/kg). Bolus injections of endothelin-1 (1-1000 ng/kg) increased ABF only at 1 ng/kg and increased MAP at 1000 ng/kg. Bolus injections of sodium nitroprusside increased ABF at 1 and 10 μg/kg and decreased MAP at 10 μg/kg. Thus, laser-Doppler flowmetry is a useful tool for understanding ABF regulation by peptides that stimulate steroid hormone release. Our results demonstrate that Ang II and ACTH increases in ABF are mediated by a cytochrome P450 metabolite.

Lipoxygenases regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids. Previously, we showed that endothelium-targeted adenoviral vector-mediated gene transfer of the human 15-lipoxygenase-1 (h15-LO-1) enhances arterial relaxation through the production of vasodilatory hydroxyepoxyeicosatrienoic acid (HEETA) and trihydroxyeicosatrienoic acid (THETA) metabolites. To further define this function, a transgenic (Tg) mouse line that overexpresses h15-LO-1 was studied. Western blot, immunohistochemistry and RT-PCR results confirmed expression of 15-LO-1 transgene in tissues, especially high quantity in coronary arterial wall, of Tg mice. Reverse-phase HPLC analysis of [(14)C]-AA metabolites in heart tissues revealed enhanced 15-HETE synthesis in Tg vs. WT mice. Among the 15-LO-1 metabolites, 15-HETE, erythro-13-H-14,15-EETA, and 11(R),12(S),15(S)-THETA relaxed the mouse mesenteric arteries to the greatest extent. The presence of h15-LO-1 increased acetylcholine- and AA-mediated relaxation in mesenteric arteries of Tg mice compared to WT mice. 15-LO-1 was most abundant in the heart; therefore, we used the Langendorff heart model to test the hypothesis that elevated 15-LO-1 levels would increase coronary flow following a short ischemia episode. Both peak flow and excess flow of reperfused hearts were significantly elevated in hearts from Tg compared to WT mice being 2.03 and 3.22 times greater, respectively. These results indicate that h15-LO-1-derived metabolites are highly vasoactive and may play a critical role in regulating coronary blood flow., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Bradykinin causes vascular relaxations through release of endothelial relaxing factors including prostacyclin, nitric oxide (NO) and epoxyeicosatrienoic acids (EETs). Bradykinin is metabolized by angiotensin converting enzyme (ACE) and ACE inhibition enhances bradykinin relaxations. Our goal was to characterize the role of bradykinin receptors and endothelial factors in ACE inhibitor-enhanced relaxations in bovine coronary arteries. In U46619 preconstricted arteries, bradykinin (10-11-10-8m) caused concentration-dependent relaxations (maximal relaxation ≥100%, log EC50=-9.8±0.1). In the presence of the NO synthase inhibitor, N-nitro-L-arginine (L-NA, 30 μm) and the cyclooxygenase inhibitor, indomethacin (10 μm), relaxations were reduced by an inhibitor of EET synthesis, miconazole (10 μm) (maximal relaxation=55±10%). Bradykinin relaxations were inhibited by the bradykinin 2 (B2) receptor antagonist, D-Arg0-Hyp3-Thi5,8-D-Phe7-bradykinin (1 μm) (log EC50=-8.5±0.1) but not altered by the B1 receptor antagonist, des-Arg9[Leu8]bradykinin (1 μm). Mass spectrometric analysis of bovine coronary artery bradykinin metabolites revealed a time-dependent increase in bradykinin (1-5) and (1-7) suggesting metabolism by ACE. ACE inhibition with captopril (50 μm) enhanced bradykinin relaxations (log EC50=-10.3±0.1). The enhanced relaxations were eliminated by L-NA or the B1 receptor antagonist but not the B2 receptor antagonist. Our results demonstrate that ACE inhibitor-enhanced bradykinin relaxations of bovine coronary arteries occur through endothelial cell B1 receptor activation and NO.

Background: Arachidonic acid (AA) and/or its enzymatic metabolites are important lipid mediators contributing to endothelium-derived hyperpolarizing factor (EDHF)-mediated dilation in multiple vascular beds, including human coronary arterioles (HCAs). However, the mechanisms of action of these lipid mediators in endothelial cells (ECs) remain incompletely defined. In this study, we investigated the role of the transient receptor potential vanilloid 4 (TRPV4) channel in AA-induced endothelial Ca(2+) response and dilation of HCAs., Methods and Results: AA induced concentration-dependent dilation in isolated HCAs. The dilation was largely abolished by the TRPV4 antagonist RN-1734 and by inhibition of endothelial Ca(2+)-activated K(+) channels. In native and TRPV4-overexpressing human coronary artery ECs (HCAECs), AA increased intracellular Ca(2+) concentration ([Ca(2+)]i), which was mediated by TRPV4-dependent Ca(2+) entry. The AA-induced [Ca(2+)]i increase was inhibited by cytochrome P450 (CYP) inhibitors. Surprisingly, the CYP metabolites of AA, epoxyeicosatrienoic acids (EETs), were much less potent activators of TRPV4, and CYP inhibitors did not affect EET production in HCAECs. Apart from its effect on [Ca(2+)]i, AA induced endothelial hyperpolarization, and this effect was required for Ca(2+) entry through TRPV4. AA-induced and TRPV4-mediated Ca(2+) entry was also inhibited by the protein kinase A inhibitor PKI. TRPV4 exhibited a basal level of phosphorylation, which was inhibited by PKI. Patch-clamp studies indicated that AA activated TRPV4 single-channel currents in cell-attached and inside-out patches of HCAECs., Conclusions: AA dilates HCAs through a novel mechanism involving endothelial TRPV4 channel-dependent Ca(2+) entry that requires endothelial hyperpolarization, PKA-mediated basal phosphorylation of TRPV4, and direct activation of TRPV4 channels by AA.

Endothelium-derived hyperpolarizing factors (EDHFs) regulate vascular tone by contributing to the vasorelaxations to shear stress and endothelial agonists such as bradykinin and acetylcholine. 15(S)-Hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA) and 11(R),12(S),15(S)-trihydroxyeicosatrienoic acid (11,12,15-THETA) are endothelial metabolites of the 15-lipoxygenase (15-LO) pathway of arachidonic acid metabolism and are EDHFs. 11,12,15-THETA activates small conductance, calcium-activated potassium channels on smooth muscle cells causing membrane hyperpolarization, and relaxation. Expression levels of 15-LO in the endothelium regulate the activity of the 15-LO/15-H-11,12-EETA/11,12,15-THETA pathway and its contribution to vascular tone. Regulation of its expression is by transcriptional, translational, and epigenetic mechanisms. Hypoxia, hypercholesterolemia, atherosclerosis, anemia, estrogen, interleukins, and possibly other hormones increase 15-LO expression. An increase in 15-LO results in increased synthesis of 15-H-11,12-EETA and 11,12,15-THETA, increased membrane hyperpolarization, and enhanced contribution to relaxation by endothelial agonists. Thus, the 15-LO pathway represents the first example of an inducible EDHF. In addition to 15-LO metabolites, a number of chemicals have been identified as EDHFs and their contributions to vascular tone vary with species and vascular bed. The reason for multiple EDHFs has evaded explanation. However, EDHF functioning as constitutive EDHFs or inducible EDHFs may explain the need for chemically and biochemically distinct pathways for EDHF activity and the variation in EDHFs between species and vascular beds. This new EDHF classification provides a framework for understanding EDHF activity in physiological and pathological conditions.

Arachidonic acid (AA) metabolites mediate endothelium-dependent relaxation in many vascular beds. Previously, we identified the major AA 12/15-lipoxygenase (12/15-LO) metabolite of mouse arteries as 12-hydroxyeicosatetraenoic acid (12-HETE). The goal was to determine the stereospecific configuration of mouse vascular 12-HETE and characterize the role of 12-HETE stereoisomers in the regulation of vascular tone. Using normal, reverse phase, and chiral HPLC, the stereospecific configuration was identified as 12(S)-HETE. 12(S)-HETE relaxed U46619-, carbocyclic thromboxane A(2)-, PGF(2α)-, and 8-iso PGF(2α)-preconstricted mesenteric arteries, but not phenylephrine-preconstricted arteries. 12(R)-HETE was more potent than 12(S)-HETE in relaxing U46619-preconstricted mouse arteries (maximum relaxations = 91.4 ± 2.7% and 71.8 ± 5.9%, respectively). Neither 12-HETE isomer caused constriction. Pretreatment with 12(S)- or 12(R)-HETE (1 μM) inhibited constrictions to U46619 but not phenylephrine. To investigate the role of thromboxane A(2) (TP) receptors in 12-HETE vascular actions, [(3)H]SQ29548 radioligand binding studies were performed in mouse platelets. U46619, 12(R)-HETE, and 12(S)-HETE displaced [(3)H]SQ29548 binding with IC(50)s of 0.07, 0.32, and 1.73 μM, respectively. Both 12(S)- and 12(R)-HETE inhibited intracellular calcium increases induced by U46619 (10 nM) in HEK293 cells overexpressing TP(α) receptor (65.5% and 45.1%, respectively) and coexpressing prostacyclin (IP) and TP(α) receptors (58.0% and 27.1%, respectively). The LO inhibitor NDGA (10 μM) reduced AA relaxations in arteries preconstricted with U46619 but not phenylephrine. These results indicate that exogenous and endogenous 12(S)-HETE relax mouse mesenteric arteries that are preconstricted with thromboxane agonists. These 12(S)-HETE relaxations are mediated by TP receptor competitive inhibition and inhibition of TP agonist-induced increases in intracellular calcium.

In mouse arteries, Alox15 [leukocyte-type 12/15-lipoxygenase (LO)] is assumed to regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids that mediate the endothelium-dependent relaxations to AA and acetylcholine (ACh). We used Alox15(-/-) mice, made by targeted disruption of the Alox15 gene, to characterize its role in the regulation of blood pressure and vascular tone. Systolic blood pressures did not differ between wild-type (WT) and Alox15(-/-) mice between 8-12 wk of age, but Alox15(-/-) mice exhibited resistance toward both N(G)-nitro-L-arginine-methyl ester (L-NAME)- and deoxycorticosterone acetate (DOCA)/high-salt-induced hypertension. ACh relaxed mesenteric arteries and abdominal aortas of WT and Alox15(-/-) mice to an identical extent. The LO inhibitor nordihydroguaiaretic acid attenuated the ACh relaxations by 35% in arteries from both WT and Alox15(-/-) mice. Reverse-phase HPLC analysis of [(14)C]AA metabolites in aorta and peritoneal macrophages (PM) revealed differences. Unlike PM, aorta tissue did not produce detectable amounts of 15-hydroxyeicosatetraenoic acid. Although Alox15 mRNA was detected in aorta, high-resolution gel electrophoresis with immunodetection revealed no Alox15 protein expression. Unlike aorta, Alox15 protein was detected in PM, intestine, fat, lung, spleen, and skin from WT, but not Alox15(-/-), mice. Injection of WT PM, a primary source of Alox15 protein, into Alox15(-/-) mice abolished their resistance toward L-NAME-induced hypertension. On the other hand, WT mice acquired resistance to L-NAME-induced hypertension after depletion of macrophages by clodronate injection. These studies indicate that Alox15 is involved in development of experimental hypertension by altering macrophage functions but not via synthesis of the vasoactive LO metabolites in mouse arteries.

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.

Endothelial 15-lipoxygenase-1 (15-LO-1) metabolites of arachidonic acid (AA), 11,12,15-trihydroxyeicosatrienoic acid (THETA) and 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) and nitric oxide (NO) mediate relaxations to acetylcholine (ACH). However, interactions between NO and the 15-LO-1 pathway have not been explored. Therefore, the effect of physiological and pharmacological concentrations of NO on 15-LO activity and relaxation was studied in rabbit aorta. In indomethacin-treated aortic rings, maximal ACH relaxations of 91.3±4.0%, decreased to 54.5±3.0% by the NO synthase inhibitor, nitro-l-arginine (LNA), to 49.8±3% by the guanylate cyclase (GC) inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, to 63.7±4.9% by the lipoxygenase (LO) inhibitor, nordihydroguaiaretic acid (NDGA) and were completely inhibited by the combination of LNA and NDGA. AA relaxations were not affected by GC inhibition but were reduced by LO inhibition. The NO donor, dipropylenetriamine-NONOate (DPTA) caused concentration-related relaxations (EC(50)=4.7×10(-6)M). Aortic metabolism of (14)C-AA to THETA and HEETA was not altered by EC(50) concentrations of DPTA but were reduced 10-fold by 10(-3)M DPTA. In LNA-treated aorta, DPTA (3×10(-6)M) caused relaxations of 38.2.5±4%. Maximum relaxations to ACH did not differ in the presence and absence 3×10(-6)M DPTA (49.5±5% and 44.2±4%, respectively). These results indicate that NO and 15-LO-1 act in parallel to mediate ACH relaxations and NO does not alter 15-LO-1 activity., (Copyright © 2011 Elsevier Inc. All rights reserved.)

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.

Our goal was to quantify mitochondrial and plasma potential (Δψ(m) and Δψ(p)) based on the disposition of rhodamine 123 (R123) or tetramethylrhodamine ethyl ester (TMRE) in the medium surrounding pulmonary endothelial cells. Dyes were added to the medium, and their concentrations in extracellular medium ([R(e)]) were measured over time. R123 [R(e)] fell from 10 nM to 6.6 ± 0.1 (SE) nM over 120 min. TMRE [R(e)] fell from 20 nM to a steady state of 4.9 ± 0.4 nM after ∼30 min. Protonophore or high K(+) concentration ([K(+)]), used to manipulate contributions of membrane potentials, attenuated decreases in [R(e)], and P-glycoprotein (Pgp) inhibition had the opposite effect, demonstrating the qualitative impact of these processes on [R(e)]. A kinetic model incorporating a modified Goldman-Hodgkin-Katz model was fit to [R(e)] vs. time data for R123 and TMRE, respectively, under various conditions to obtain (means ± 95% confidence intervals) Δψ(m) (-130 ± 7 and -133 ± 4 mV), Δψ(p) (-36 ± 4 and -49 ± 4 mV), and a Pgp activity parameter (K(Pgp), 25 ± 5 and 51 ± 11 μl/min). The higher membrane permeability of TMRE also allowed application of steady-state analysis to obtain Δψ(m) (-124 ± 6 mV). The consistency of kinetic parameter values obtained from R123 and TMRE data demonstrates the utility of this experimental and theoretical approach for quantifying intact cell Δψ(m) and Δψ(p.) Finally, steady-state analysis revealed that although room air- and hyperoxia-exposed (95% O(2) for 48 h) cells have equivalent resting Δψ(m), hyperoxic cell Δψ(m) was more sensitive to depolarization with protonophore, consistent with previous observations of pulmonary endothelial hyperoxia-induced mitochondrial dysfunction.

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.

Elevated concentrations of aldosterone are associated with several cardiovascular diseases. Angiotensin II (Ang II) increases aldosterone secretion and adrenal blood flow. This concurrent increase in steroidogenesis and adrenal blood flow is not understood. We investigated the role of zona glomerulosa (ZG) cells in the regulation of vascular tone of bovine adrenal cortical arteries by Ang II. ZG cells enhanced endothelium-dependent relaxations to Ang II. The ZG cell-dependent relaxations to Ang II were unchanged by removing the endothelium-dependent response to Ang II. These ZG cell-mediated relaxations were ablated by cytochrome P450 inhibition, epoxyeicosatrienoic acid (EET) antagonism, and potassium channel blockade. Analysis of ZG cell EET production by liquid chromatography/mass spectrometry demonstrated an increase in EETs and dihydroxyeicosatrienoic acids with Ang II stimulation. These EETs and dihydroxyeicosatrienoic acids produced similar concentration-dependent relaxations of adrenal arteries, which were attenuated by EET antagonism. Whole-cell potassium currents of adrenal artery smooth muscle cells were increased by Ang II stimulation in the presence of ZG cells but decreased in the absence of ZG cells. This increase in potassium current was abolished by iberiotoxin. Similarly, 14,15-EET induced concentration-dependent increases in potassium current, which was abolished by iberiotoxin. ZG cell aldosterone release was not directly altered by EETs. These data suggest that Ang II stimulates ZG cells to release EETs and dihydroxyeicosatrienoic acids, resulting in potassium channel activation and relaxation of adrenal arteries. This provides a mechanism by which Ang II concurrently increases adrenal blood flow and steroidogenesis.

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.

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.

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid that are produced by the vascular endothelium in responses to various stimuli such as the agonists acetylcholine (ACH) or bradykinin or by shear stress which activates phospholipase A(2) to release arachidonic acid. EETs are important regulators of vascular tone and homeostasis. In the modulation of vascular tone, EETs function as endothelium-derived hyperpolarizing factors (EDHFs). In models of vascular inflammation, EETs attenuate inflammatory signaling pathways in both the endothelium and vascular smooth muscle. Likewise, EETs regulate blood vessel formation or angiogenesis by mechanisms that are still not completely understood. Soluble epoxide hydrolase (sEH) converts EETs to dihydroxyeicosatrienoic acids (DHETs) and this metabolism limits many of the biological actions of EETs. The recent development of inhibitors of sEH provides an emerging target for pharmacological manipulation of EETs. Additionally, EETs may initiate their biological effects by interacting with a cell surface protein that is a G protein-coupled receptor (GPCR). Since GPCRs represent a common target of most drugs, further characterization of the EET receptor and synthesis of specific EET agonists and antagonist can be used to exploit many of the beneficial effects of EETs in vascular diseases, such as hypertension and atherosclerosis. This review will focus on the current understanding of the contribution of EETs to the regulation of vascular tone, inflammation, and angiogenesis. Furthermore, the therapeutic potential of targeting the EET pathway in vascular disease will be highlighted., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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. Mass spectrometric analyses and HPLC comigration identified the HEETAs as threo- and erythro-diastereomers of 13-H-trans-14,15-EETA. Erythro- and threo-diastereomers of 13-H-trans-14,15-EETA relaxed endothelium-denuded rabbit small mesenteric arteries with maximum relaxations of 22.6 +/- 6.0% and 8.6 +/- 4.3%, respectively. Apamin (10(-7) m) inhibited the relaxations to the erythro-isomer (maximum relaxation = 1.2 +/- 5.6%) and increasing [K(+)](o) from 4.6 to 30 mm blocked relaxations to both isomers. In cell-attached patches of mesenteric arterial smooth muscle cells (SMCs), erythro-13-H-trans-14,15-EETA (1-3 x 10(-6) m) increased mean open time of small conductance K(+) channels (13-14 pS) from 0.0007 +/- 0.0007 to 0.0053 +/- 0.0042. This activation was inhibited by apamin. The erythro, but not the threo, isomer blocked angiotensin II-stimulated aortic SMC migration. These studies demonstrate that 13-H-14,15-EETAs induces vascular relaxation via K(+) channel activation to cause SMC hyperpolarization. Thus, 13-H-14,15-EETA represents a new endothelial factor.

We investigated the hypothesis that oxidative stress in persistent pulmonary hypertension of the newborn (PPHN) impairs voltage gated potassium (Kv) channel function. We induced PPHN in fetal lambs by prenatal ligation of ductus arteriosus; controls had sham ligation. We studied changes in the tone of pulmonary artery (PA) rings and Kv channel current of freshly isolated PA smooth muscle cells (PASMC) using standard techniques. 4-Aminopyridine (4-AP), a Kv channel antagonist, induced dose-dependent constriction of control PA rings; this response was attenuated in PPHN pulmonary arteries. Exogenous superoxide and peroxynitrite inhibited the response to 4-AP in control rings. Tiron, a superoxide scavenger, improved the response to 4-AP in PPHN rings. 4-AP inhibited the NOS-independent relaxation response to ATP in control PA rings. Relaxation response to ATP was blunted in PPHN rings and was improved by NOS antagonist, N-nitro-L-arginine methyl ester (L-NAME). 4-AP attenuated this response in L-NAME-treated PPHN rings. Exogenous superoxide suppressed 4-AP sensitive Kv current in control PASMC. Kv channel current was attenuated in cells from PPHN lambs and was restored by tiron. Oxidative stress impairs Kv channel function in PPHN. Superoxide scavengers may improve pulmonary vasodilation in PPHN in part by restoring Kv channel function.

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.

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 min of reperfusion before 60 min 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-hydroxy-propoxy)-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 +/- 1.2%, IPC 8.3 +/- 2.2%, POC 10.1 +/- 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.

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.

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-1-yl-ureido)-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 +/- 1.6% (vehicle) to 8.7 +/- 2.2 and 9.4 +/- 1.3%, respectively. Similarly, AUDA significantly reduced IS/AAR from 21.8 +/- 1.6 to 14.4 +/- 1.2% (low dose) and 9.4 +/- 1.8% (high dose), respectively. Interestingly, the combination of the low dose of AUDA with 14,15-EET reduced IS/AAR to 5.8 +/- 1.6% (P < 0.05), further than either drug alone. Diazoxide also reduced IS/AAR significantly (10.2 +/- 1.9%). In contrast, 14,15-EEZE had no effect on IS/AAR by itself (21.0 +/- 3.6%), but completely abolished the effect of 11,12-EET (17.8 +/- 1.4%) and 14,15-EET (19.2 +/- 2.4%) and AUDA (19.3 +/- 1.6%), but not that of diazoxide (10.4 +/- 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.

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),13(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),12(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.

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 14C-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 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 x 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.

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.

Continuing investigations of the roles of cytochrome P450 (CYP) arachidonic acid epoxygenase metabolites in the regulation of cardiovascular physiology and pathophysiology have revealed their complex and diverse biological effects. Often these metabolites demonstrate protective properties that are revealed during cardiovascular disease. In this regard, the epoxyeicosatrienoic acids (EETs) are an emerging target for pharmacological manipulation aimed at enhancing their cardiac and vascular protective mechanisms. This review will focus on the role of EETs in the regulation of vascular tone, with emphasis on the coronary circulation, their role in limiting platelet aggregation, vascular inflammation and EET contribution to preconditioning of the ischemic myocardium. Production and metabolism of EETs as well as their specific cellular signaling mechanisms are discussed.

Background and Purpose: Cerebrovascular smooth muscle cells express the CB1 cannabinoid receptor and CB1 agonists produce vasodilatation of the middle cerebral artery (MCA). The thromboxane A2 mimetic, U-46619, increased the content of the endocannabinoid, 2-arachidonoylglycerol (2-AG) in the MCA and 2-AG moderated the vasoconstriction produced by U46619 in this tissue. The purposes of this study were to examine the extent to which 2-AG is catabolized by cerebral arteries and to determine whether blockade of 2-AG inactivation potentiates its feedback inhibition of U-44619-mediated vasoconstriction., Experimental Approach: The diameters of isolated, perfused MCA from male rats were measured using videomicroscopy., Key Results: Exogenous 2-AG produces a CB1 receptor-dependent and concentration-related increase in the diameter of MCA constricted with 5-HT. The E (max) for 2-AG dilation is increased 4-fold in the presence of the metabolic inhibitors 3-(decylthio)-1,1,1-trifluropropan-2-one (DETFP), URB754 and URB597. To examine the role of catabolism in the effects of endogenous 2-AG, vasoconstriction induced by U-46619 was studied. DETFP and URB754, but not the fatty acid amide hydrolase inhibitor, URB597, significantly increased the EC(50) for U-46619. These data support a physiological role for endocannabinoid feedback inhibition in the effects of U-46619 and indicate that endogenously produced 2-AG is also efficiently catabolized within the MCA., Conclusions and Implications: MCA express mechanisms for the efficient inactivation of 2-AG, providing further support for an endocannabinoid feedback mechanism that opposes thromboxane-mediated vasoconstriction. These data suggest that potentiation of endogenously produced 2-AG could be a novel therapeutic approach to the treatment of thrombotic stroke.

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.

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(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(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 [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 BK(Ca) channel activity fourfold in cell-attached patches. Methacholine-induced increases in BK(Ca) 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 BK(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(Ca) channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of BK(Ca) channels in coronary SMCs.

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(-9)-5 x 10(-6) M) elicited concentration-dependent relaxations. The relaxations were blocked by the H(1) receptor antagonists diphenhydramine (10 microM) or mepyramine (1 microM) (maximal relaxations of 18 +/- 6 and 22 +/- 6%, respectively, vs. 55 +/- 5% of control) but only partially inhibited by the H(2) receptor antagonist cimetidine (10 microM) and the H(3) receptor antagonist thioperamide (1 microM). Histamine-induced relaxations were also blocked by the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NA, 30 microM; maximal relaxation of 13 +/- 7%) and eliminated by endothelial removal or L-NA combined with the cyclooxgenase inhibitor indomethacin (10 microM). In the presence of adrenal zona glomerulosa (ZG) cells, histamine did not induce further relaxations compared with histamine alone. Histamine (10(-7)-10(-5) M) concentration-dependently increased aldosterone production by adrenal ZG cells. Compound 48/80 (10 microg/ml), a mast cell degranulator, induced significant relaxations (93 +/- 0.6%), which were blocked by L-NA plus indomethacin or endothelium removal, partially inhibited by the combination of the H(1), H(2), and H(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(1) receptors. The potential role of histamine in linking adrenal vascular events and steroid secretion requires further investigation.

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.

Adrenal steroidogenesis is modulated by humoral and neuronal factors and blood flow. Angiotensin II (AII) stimulates adrenal cortical aldosterone and cortisol production and medullary catecholamine release. However, AII regulation of adrenal vascular tone has not been characterized. We examined the effect of AII on diameters of cannulated bovine adrenal cortical arteries. Cortical arteries (average internal diameter = 230 microm) were constricted with U46619 and concentration-diameter responses to AII (10(-13) to 10(-8) mol/liter) were measured. In endothelium-intact arteries, AII induced dilations at low concentrations (maximum dilation = 25 +/- 6% at 10(-10) mol/liter) and constrictions at high concentrations (maximum constriction = 25 +/- 18% at 10(-8) mol/liter). AII constrictions were blocked by the angiotensin type 1 (AT1) receptor antagonist, losartan (10(-6) mol/liter). AII dilations were enhanced by losartan (maximal dilation = 48 +/- 8%), abolished by endothelial cell removal or N-nitro-L-arginine (L-NA, 3 x 10(-5) mol/liter) and inhibited by the angiotensin type 2 (AT2) receptor antagonist, PD123319 (10(-6) mol/liter, maximal dilation = 18 +/- 4%). In a 4,5-diaminofluorescein diacetate nitric oxide (NO) assay of isolated cortical arteries, AII stimulated NO production, which was abolished by PD123319, L-NA, or endothelial cell removal. Western immunoblot of arterial homogenates and endothelial and zona glomerulosa cell lysates revealed 48-kD and 50-kD bands corresponding to AT1 and AT2 receptors, respectively, in all three and a 140-kD band corresponding to endothelial NO synthase in endothelial cells and arteries. Our results demonstrate that AII stimulates adrenal cortical arterial dilation through endothelial cell AT2 receptor activation and NO release and AT1 receptor-dependent constriction.

Cerebral vascular smooth muscle cells express the CB(1) cannabinoid receptor, and CB(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 CB(1) receptors, oppose the vasoconstriction in a feedback manner. The thromboxane A(2) (TXA(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 CB(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 CB(1) receptor antagonists SR-141716 and AM-251 decreased the EC(50) value for U-46619 to constrict endothelium-denuded MCA without affecting the maximal effect. A low concentration of CB(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 TXA(2) receptor activation increases MCA eCB content, which, via activation of CB(1) receptors, reduces the constriction produced by moderate concentrations of the TXA(2) agonist. Although 5-HT-induced vasoconstriction is reduced by exogenous CB(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 TXA(2) receptors and not 5-HT receptors.

Bradykinin causes arterial relaxation and hyperpolarization, which is mediated by a transferable endothelium-derived hyperpolarizing factor (EDHF). In coronary arteries, epoxyeicosatrienoic acids (EETs) are involved in the EDHF response. However, the role of EETs as transferable mediators of EDHF-dependent relaxation remains poorly defined. Two small bovine coronary arteries were cannulated and perfused in tandem in the presence of the nitric oxide synthase inhibitor, nitro-L-arginine (30 micromol/L), and the cyclooxygenase inhibitor, indomethacin (10 micromol/L). Luminal perfusate from donor arteries with intact endothelium perfused endothelium-denuded detector arteries. Detector arteries were constricted with U46619 and diameters were monitored. Bradykinin (10 nmol/L) added to detector arteries did not induce dilation (5+/-2%), whereas bradykinin addition to donor arteries dilated detector arteries by 26.5+/-7% (P<0.05). These dilations were blocked by donor artery endothelium removal and detector artery treatment with the EET-selective antagonist, 14,15-epoxyeicosa-5(Z)-monoenoic acid (14,15-EEZE; 10 micromol/L, -5+/-6%) but not 14,15-EEZE treatment of donor arteries (20+/-5%). 14,15-EET (0.1 to 10 micromol/L) added to detector arteries induced maximal dilations of 82+/-5% that were inhibited 50% by detector artery treatment with 14,15-EEZE (32+/-12%) but not donor artery treatment with 14,15-EEZE. Liquid chromatography-electrospray ionization mass spectrometry analysis verified the presence of 14,15-EET in the perfusate from an endothelium-intact but not denuded artery. These results show that bradykinin stimulates donor artery 14,15-EET release that dilates detector arteries. 14,15-EEZE blocked the donor artery, endothelium-dependent, bradykinin-induced relaxations, and attenuated relaxations to 14,15-EET. These results suggest that EETs are transferable EDHFs in coronary arteries.

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.