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Substance P (SP) is released from sensory nerves in the arteries and heart. It activates neurokinin-1 receptors (NK1Rs) causing vasodilation, immune modulation, and adverse cardiac remodeling. The hypothesis was tested: SP and SP metabolites activate different second messenger signaling pathways. Macrophages, endothelial cells, and fibroblasts metabolized SP to N- and C-terminal metabolites to varying extents. SP 5–11 was the most abundant metabolite followed by SP 1–4, SP 7–11, SP 6–11, SP 3–11, and SP 8–11. In NK1R-expressing human embryonic kidney 293 (HEK293) cells, SP and some C-terminal SP metabolites stimulate the NK1R, promoting the dissociation of several Gα proteins, including Gαs and Gαq from their βγ subunits. SP increases intracellular calcium concentrations ([Ca]i) and cyclic 3′,5′-adenosine monophosphate (cAMP) accumulation with similar −log EC50 values of 8.5 ± 0.3 and 7.8 ± 0.1 M, respectively. N-terminal metabolism of SP by up to five amino acids and C-terminal deamidation of SP produce peptides that retain activity to increase [Ca]i but not to increase cAMP. C-terminal metabolism results in the loss of both activities. Thus, [Ca]i and cAMP signaling are differentially affected by SP metabolism. To assess the role of N-terminal metabolism, SP and SP 6–11 were compared with cAMP-mediated activities in NK1R-expressing 3T3 fibroblasts. SP inhibits nuclear factor κB (NF-κB) activity, cell proliferation, and wound healing and stimulates collagen production. SP 6–11 had little or no activity. Cyclooxygenase-2 (COX-2) expression is increased by SP but not by SP 6–11. Thus, metabolism may select the cellular response to SP by inhibiting or redirecting the second messenger signaling pathway activated by the NK1R. NEW & NOTEWORTHY: Endothelial cells, macrophages, and fibroblasts metabolize substance P (SP) to N- and C-terminal metabolites with SP 5–11 as the most abundant metabolite. SP activates neurokinin-1 receptors to increase intracellular calcium and cyclic AMP. In contrast, SP metabolites of N-terminal metabolism and C-terminal deamidation retain the ability to increase calcium but lose the ability to increase cyclic AMP. These new insights indicate that the metabolism of SP directs cellular functions by regulating specific signaling pathways. [ABSTRACT FROM AUTHOR]

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

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

15-Lipoxygenase (15-LO-1) metabolizes arachidonic acid (AA) to l l,12,15-trihydroxyeicosatrienoic acids (THETAs) and 15-hydroxy-11,12-epoxyeicosatrienoic acids (HEETA) that dilate rabbit arteries. Increased endothelial 15-LO-1 expression enhances arterial relaxations to agonists. We tested the effect of hypoxia on 15-LO-1 expression, THETA and HEETA synthesis, and relaxations in rabbit arteries. The incubation of rabbit aortic endothelial cells and isolated aortas in 0.7% [O.sub.2] increased 15-LO-1 expression. Rabbits were housed in a hypoxic atmosphere of 12% [O.sub.2] for 5 days. 15-LO-1 expression increased in the endothelium of the arteries of rabbits in 12% [O.sub.2] compared with room air. THETA and HEETA synthesis was also enhanced in aortas and mesenteric arteries. AA hyperpolarized the smooth muscle cells in indomethacinand phenylephrine-treated mesenteric arteries of hypoxic rabbits from -29.4 [+ or -] 1 to -50.1 [+ or -] 3 mV. The hyperpolarization to AA was less in arteries of normoxic rabbits (from -26.0 [+ or -] 2 to -37 [+ or -] 2 mV). This AA-induced hyperpolarization was inhibited by the 15-LO inhibitor BW-755C. Nitric oxide and prostaglandin-independent maximum relaxations to acetylcholine (79.7 [+ or -] 2%) and AA (38.3 [+ or -] 4%) were enhanced in mesenteric arteries from hypoxic rabbits compared with the normoxic rabbits (49.7 [+ or -] 6% and 19.9 [+ or -] 2%, respectively). These relaxations were inhibited by BW-755C and nordihydroguaiaretic acid. Therefore, hypoxia increased the relaxations to agonists in the rabbit mesenteric arteries by enhancing endothelial 15-LO-1 expression and synthesis of the hyperpolarizing factors THETA and HEETA. endothelium; vasodilation; endothelium-derived hyperpolarizing factor

Arachidonic acid (AA) metabolites from the 15-1ipoxygenase-1 (15-LO-1) pathway, trihydroxyeicosatrienoic acids (THETAs) and hydroxy-epoxyeicosatrienoic acids (HEETAs), are endothelium-derived hyperpolarizing factors (EDHFs) and relax rabbit arteries. Rabbit vascular 15-LO-1 expression, THETA and HEETA synthesis, and nitric oxide and prostaglandin-independent relaxations to acetylcholine (ACh) and AA decreased with age (neonates to 16-wk-old). We characterized age-dependent ACh-hypotensive responses in vivo in 1-, 4-, 8-, and 16-wk-old rabbits and the contribution of THETAs and HEETAs to these responses. In anesthetized rabbits, blood pressure responses to ACh (4-4,000 ng/kg) were determined in the presence of vehicle or various inhibitors. ACh responses decreased with age (P > 0.001). In the absence or presence of [N.sup.[omega]]-nitro-L-arginine methyl ester (L-NAME) and indomethacin (Indo), maximum responses in 1 (-54.7 [+ or -] 7.4 and -37.9 [+ or -] 3.9%)- and 4 (-48.8 [+ or -] 2.4 and -35.5 [+ or -] 7.8%)-wk-old rabbits were higher than 8 (-30.0 [+ or -] 2.8 and -26.6 [+ or -] 4.4%)-and 16 (-36.7 [+ or -] 3.5 and -27.3 [+ or -] 10%)-wk-old rabbits. A lipoxygenase inhibitor, BW755C, reduced THETA and HEETA synthesis in mesenteric arteries. In the presence of Indo and [N.sup.[omega]]-nitro-L-arginine, ACh relaxations were reduced by BW755C to a greater extent in the mesenteric arteries from the younger rabbits. In 4-wk-old rabbits treated with L-NAME and Indo, the maximum ACh hypotension was reduced by the potassium channel inhibitors apamin and charybdotoxin to -6.9 [+ or -] 0.9%, by apamin alone to -19.5 [+ or -] 1.4%, and by BW755C to -18.8 [+ or -] 3.5%. The present study indicates that the age-related decrease in ACh-induced hypotension is mediated by the decreased synthesis of the 15-LO-1 metabolites THETAs and HEETAs. endothelium-derived hyperpolarizing factor; arachidonic acid; vasodilation; mean arterial pressure

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

Previous studies indicate that 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), an endothelium-derived hyperpolarizing factor in the rabbit aorta, mediates a portion of the relaxation response to acetylcholine by sequential metabolism of arachidonic acid by 15-lipoxygenase, hydroperoxide isomerase, and epoxide hydrolase. To determine the stereochemical configuration of the endothelial 11,12,15-THETA, its activity and chromatographic migration were compared with activity and migration of eight chemically synthesized stereoisomers of 11, 12, 15(S)-THETA. Of the eight isomers, only 11(R), 12(S), 15(S)-trihydroxyeicosa-5(Z), 8(Z), 13(E)-trienoic acid comigrated with the biological 11, 12, 15-THETA on reverse- and normal-phase HPLC and gas chromatography. The same THETA isomer ([10.sup.-7] - [10.sup.-4] M) relaxed the rabbit aorta in a concentration-related manner (maximum relaxation = 69 [+ or -] 5%). These relaxations were blocked by apamin ([10.sup.-7] M), an inhibitor of small-conductance [Ca.sup.2+]-activated [K.sup.+] channels. In comparison, 11 (S), 12(R), 15(S), 5(Z), 8(Z), 13(E)-THETA ([10.sup.-4] M) relaxed the aorta by 22%. The other six stereoisomers were inactive in this assay. With use of the whole cell patch-clamp technique, it was shown that [10.sup.-4] M 11(R), 12(S), 15(S), 5(Z), 8(Z), 13(E)-THETA increased outward [K.sup.+] current in isolated aortic smooth muscle cells by 119 [+ or -] 36% at +60 mV, whereas [10.sup.-4] M 11(R), 12(R), 15(S), 5(Z), 8(Z), 13(E)-THETA increased outward [K.sup.+] current by only 20 [+ or -] 2%. The 11(R), 12(S), 15(S), 5(Z), 8(Z), 13(E)-THETA-stimulated increase in [K.sup.+] current was blocked by pretreatment with apamin. These studies suggest that 11(R), 12(S), 15(S)-trihydroxyeicosa-5(Z), 8(Z), 13(E)-trienoic acid is the active stereoisomer produced by the rabbit aorta. It relaxes smooth muscle by activating [K.sup.+] channels. The specific structural and stereochemical requirements for [K.sup.+] channel activation suggest that a specific binding site or receptor of 11, 12, 15-THETA is involved in these actions. endothelium-derived hyperpolarizing factor; smooth muscle cells; potassium channels; arachidonic acid

Arachidonic acid (AA) causes endothelium-dependent smooth muscle hyperpolarizations and relaxations that are mediated by a 15-lipoxygenase-I (15-LO-I) metabolite, 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA). We propose that AA is metabolized sequentially by 15-LO-I and hydroperoxide isomerase to an unidentified hydroxyepoxyeicosatrienoic acid (HEETA), which is hydrolyzed by a soluble epoxide hydrolase (sEH) to 11,12,15-THETA. After incubation of aorta with [sup.4]C-labeled AA, metabolites were extracted and the HEETAs were resolved by performing HPLC. Mass spectrometric analyses identified 15-Hydroxy-11,12-epoxyeicosatrienoic acid (15-H-11,12-EETA). Incubation of aortic incubates with methanol and acetic acid trapped the acidsensitive 15-H-11,12-EETA as methoxydihydroxyeicosatrienoic acids (MDHEs) (367 m/z, M-H). Pretreatment of the aortic tissue with the sEH inhibitor 12-(3-adamantan-l-yl-ureido)-dodecanoic acid (AUDA; [10.sup.-6] M) increased the formation of 15-H-11,12-EETA, measured as MDHEs. Thus 15-H-11,12-EETA is an acid- and sEH-sensitive precursor of 11,12,15-THETA. Aortic homogenates and endothelial cells contain a 57-kDa protein corresponding to the rabbit sEH. In preconstricted aortic rings, AA ([10.sup.-7]-[10.sup.-4] M) and acetylcholine ([10.sup.-9]-[10.sup.-6] M) caused concentration-related relaxations that were enhanced by pretreatment with AUDA. These enhanced relaxations were inhibited by increasing extracellular [[K.sup.+]] from 4.8 to 20 mM. AA (3 x [10.sup.-6] M) induced cell membrane hyperpolarization (from -31.0 [+ or -] 1 to -46.8 [+ or -] 2 mV) in aortic strips with an intact endotfielium, which was enhanced by AUDA. These results indicate that 15-H-11,12-EETA is produced by the aorta, hydrolyzed by sEH to 11,12,15-THETA, and mediates relaxations by membrane hyperpolarization. 15-H-11,12-EETA represents an endothelium-derived hyperpolarizing factor. arachidonic acid; endothelial cells; endothelium-derived hyperpolarizing factor; 15-lipoxygenase

Rabbit 15-1ipoxygenase- 1 (15-LO-1) oxygenates arachidonic acid (AA) into 15-hydroperoxyeicosatetraenoic acid, which is then converted to the vasodilatory 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) and 11,12,15-trihydroxyeicosatrienoic acid (THETA). We studied the age-dependent expression of the 15-LO-1 in rabbit aorta and its effects on the synthesis of THETA, HEETA, and vasoactivity. Aortas of 1-wk-old rabbits express greater amounts of 15-LO-1 mRNA and protein compared with aortas of 4-, 8-, or 16-wk-old rabbits. The synthesis of THETA and HEETA in the rabbit aorta was also reduced with age. THETA synthesis was maximal in 1-wk-old aortas but decreased in aortas of 4- (42%), 8- (4%), and 16-wk-old (1%) rabbits. Similarly, THETA and HEETA synthesis decreased with age in mesenteric arteries from 1-, 4-, 8-, and 16-wk-old rabbits. The maximum vasorelaxation response to acetylcholine ([10.sup.-6] M) in the presence of indomethacin and nitro-L-arginine decreased in the order of 1 wk (64.5 [+ or -] 6.9%), 4 wk (52.6 [+ or -] 8.9%), 8 wk (53.0 [+ or -] 9.4%), and 16 wk (33.3 [+ or -] 6.6%). Similarly, the maximum relaxation to AA (3 x [10.sup.-4] M) decreased with age in the order of 1 wk (60.4 [+ or -] 8.9%), 4 wk (56.3 [+ or -] 5.8%), 8 wk (41.8 [+ or -] 12.3%), and 16 wk (28.9 [+ or -] 1.6%). In contrast, the vasorelaxation to sodium nitroprusside was not significantly altered by age. These data indicate that aortic 15-LO-1 expression and activity are downregulated with aging in rabbits. This decrease is paralleled by the reduced synthesis of vasoactive THETA and HEETA and aortic relaxations to acetylcholine and AA. endothelium-derived hyperpolarizing factor; arachidonic acid; acetylcholine; vasodilation

ACh-induced endothelium-dependent relaxation in rabbit small mesenteric arteries is resistant to N-nitro-L-arginine (L-NA) and indomethacin but sensitive to high [K.sup.+], indicating the relaxations are mediated by endothelium-derived hyperpolarizing factors (EDHFs). The identity of the EDHFs in this vascular bed remains undefined. Small mesenteric arteries pretreated with L-NA and indomethacin were contracted with phenylephrine. ACh ([10.sup.-10] to [10.sup.-6] M) caused concentration-dependent relaxations that were shifted to the right by lipoxygenase inhibition and the [Ca.sup.2+]-activated [K.sup.+] channel inhibitors apamin (100 nM) or charybdotoxin (100 nM) and eliminated by the combination of apamin plus charybdotoxin. Relaxations to ACh were also blocked by a combination of barium (200 [micro]M) and apamin but not barium plus charybdotoxin. Addition of [K.sup.+] (10.9 mM final concentration) to the preconstricted arteries elicited small relaxations. [K.sup.+] addition before ACh restored the charybdotoxin-sensitive component of relaxations to ACh. [K.sup.+] (10.9 mM) also relaxed endothelium-denuded arteries, and the relaxations were inhibited by barium but not by charybdotoxin and apamin. With the use of whole cell patch-clamp analysis, ACh ([10.sup.-7] M) stimulated voltage-dependent outward [K.sup.+] current from endothelial cells, which was inhibited by charybdotoxin, indicating [K.sup.+] efflux. Arachidonic acid ([10.sup.-7] to [10.sup.-4] M) induced concentration-related relaxations that were inhibited by apamin but not by charybdotoxin and barium. Addition of arachidonic acid after [K.sup.+] (10.9 mM) resulted in more potent relaxations to arachidonic acid compared with control without [K.sup.+] (5.9 mM). These findings suggest that, in rabbit mesenteric arteries, ACh-induced, L-NA- and indomethacin-resistant relaxation is mediated by endothelial cell [K.sup.+] efflux and arachidonic acid metabolites, and a synergism exists between these two separate mechanisms. acetylcholine; potassium channels; lipoxygenase; endothelium-derived hyperpolarizing factor doi:10.1152/ajpheart.00268.2006

Adrenic acid (docosatetraenoic acid), an abundant fatty acid in the vasculature, is produced by a two-carbon chain elongation of arachidonic acid. Despite its abundance and similarity to arachidonic acid, little is known about its role in the regulation of vascular tone. Gas chromatography/mass spectrometric analysis of bovine coronary artery and endothelial cell lysates revealed arachidonic acid concentrations of 2.06 [+ or -] 0.01 and 6.18 [+ or -] 0.60 [micro]g/mg protein and adrenic acid concentrations of 0.29 [+ or -] 0.01 and 1.56 [+ or -] 0.16 [micro]g/mg protein, respectively. In bovine coronary arterial rings preconstricted with the thromboxane mimetic U-46619, adrenic acid ([10.sup.-9]-[10.sup.-5] M) induced concentration-related relaxations (maximal relaxation = 83 [+ or -] 4%) that were similar to arachidonic acid relaxations. Adrenic acid relaxations were blocked by endothelium removal and the [K.sup.+] channel inhibitor, iberiotoxin (100 nM), and inhibited by the cyclooxygenase inhibitor, indomethacin (10 [micro]M, maximal relaxation = 53 [+ or -] 4%), and the cytochrome P-450 inhibitor, miconazole (10 [micro]M, maximal relaxation = 52 [+ or -] 5%). Reverse-phase HPLC and liquid chromatography/ mass spectrometry isolated and identified numerous adrenic acid metabolites from coronary arteries including dihomo (DH)-epoxy-eicosatrienoic acids (EETs) and DH-prostaglandins. DH-EET [16,17-, 13,14-, 10,11-, and 7,8- ([10.sup.-9]-[10.sup.-5] M)] induced similar concentration-related relaxations (maximal relaxations averaged 83 [+ or -] 3%). Adrenic acid ([10.sup.-6] M) and DH-16,17-EET ([10.sup.-6] M) hyperpolarized coronary arterial smooth muscle. DH-16,17-EET ([10.sup.-8]-[10.sup.-6] M) activated iberiotoxin-sensitive, whole cell [K.sup.+] currents of isolated smooth muscle cells. Thus, in bovine coronary arteries, adrenic acid causes endothelium-dependent relaxations that are mediated by cyclooxygenase and cytochrome P-450 metabolites. The adrenic acid metabolite, DH-16,17-EET, activates smooth muscle [K.sup.+] channels to cause hyperpolarization and relaxation. Our results suggest a role of adrenic acid metabolites, specifically, DH-EETs as endothelium-derived hyperpolarizing factors in the coronary circulation. endothelium-dependent relaxation; cytochrome P-450; cyclooxygenase; potassium channels; endothelium-derived hyperpolarizing factor doi:10.1152/ajpheart.00947.2006

Endothelium-dependent vasorelaxation of the rabbit aorta is mediated by either nitric oxide (NO) or arachidonic acid (AA) metabolites from cyclooxygenase (COX) and 15-lipoxygenase (15-LO) pathways. 15-LO-1 metabolites of AA, 11,12,15-trihydroxyeicosatrienoic acid (THETA), and 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) cause concentration-dependent relaxation. We tested the hypothesis that in the 15-LO pathway of AA metabolism, 15-LO-1 is sufficient and is the rate-limiting step in inducing relaxations in rabbit aorta. Aorta and rabbit aortic endothelial cells were treated with adenoviruses containing human 15-LO-1 cDNA (Ad-15-LO-1) or [beta]-galactosidase (Ad-[beta]-Gal). Ad-15-LO-l-transduction increased the expression of a 75-kDa protein corresponding to 15-LO-1, detected by immunoblotting with an anti-human15-LO-1 antibody, and increased the production of HEETA and THETA from [[sup.14]C]AA. Immunohistochemical studies on Ad- 15-LO-1-transduced rabbit aorta showed the presence of 15-LO-1 in endothelial cells. Ad-15-LO-1-treated aortic rings showed enhanced relaxation to AA (max 31.7 [+ or -] 3.2%) compared with Ad-[beta]-Gal-treated (max 12.7 [+ or -] 3.2%) or control nontreated rings (max 13.1 [+ or -] 1.6%) (P < 0.01). The relaxations in Ad-15-LO-1-treated aorta were blocked by the 15-LO inhibitor cinnamyl-3,4-dihydroxy-a-cyanocinnamate. Overexpression of 15-LO-1 in the rabbit aortic endothelium is sufficient to increase the production of the vasodilatory HEETA and THETA and enhance the relaxations to AA. This confirms the role of HEETA and THETA as endothelium-derived relaxing factors. vasodilation; trihydroxyeicosatrienoic acid

Epoxyeicosatrienoic acids (EETs), derived from arachidonic acid by cytochrome P450 epoxygenases, are potent vasodilators that function as endothelium-derived hyperpolarizing factors in some vascular beds. EETs are rapidly metabolized by soluble epoxide hydrolase to form dihydroxyeicosatrienoic acids (DHETs). Recent reports indicate that EETs have several important non-vasomotor regulatory roles in the cardiovascular system. EETs are potent anti-inflammatory agents and might function as endogenous anti-atherogenic compounds. In addition, EETs and DHETs might stimulate lipid metabolism and regulate insulin sensitivity. Thus, pharmacological inhibition of soluble epoxide hydrolase might be useful not only for hypertension but also for abating atherosclerosis, diabetes mellitus and the metabolic syndrome. Finally, although usually protective in the systemic circulation, EETs might adversely affect the pulmonary circulation.

Epoxyeicosatrienoic acids (EETs) are metabolized by soluble epoxide hydrolase (sEH) to form dihydroxyeicosatrienoic acids (DHETs) and are putative endothelium-derived hyperpolarizing factors (EDHFs). EDHFs modulate microvascular tone; however, the chemical identity of EDHF in the human coronary microcirculation is not known. We examined the capacity of EETs, DHETs, and sEH inhibition to affect vasomotor tone in isolated human coronary arterioles (HCAs). HCAs from right atrial appendages were prepared for videomicroscopy and immunohistochemistry. In vessels preconstricted with endothelin-1, three EET regioisomers (8,9-, 11,12-, and 14,15-EET) each induced a concentration-dependent dilation that was sensitive to blockade of large-conductance Ca2+-activated K| (BKca) channels by iberiotoxin. EET-induced dilation was not altered by endothelial denudation. 8,9-, 11,12-, and 14,15-DHET also dilated HCA via activation of BKca channels. Dilation was less with 8,9- and 14,15-DHET but was similar with 11,12-DHET, compared with the corresponding EETs. Immunohistochemistry revealed prominent expression of cytochrome P-450 (CYP450) 2C8, 2C9, and 2J2, enzymes that may produce EETs, as well as sEH, in HCA. Inhibition of sEH by 1-cyclohexyl-3-dodecylurea (CDU) enhanced dilation caused by 14,15-EET but reduced dilation observed with 11, 12-EET. DHET production from exogenous EETs was reduced in vessels pretreated with CDU compared with control, as measured by liquid chromatography electrospray-ionization mass spectrometry. In conclusion, EETs and DHETs dilate HCA by activating BKca channels, supporting a role for EETs/DHETs as EDHFs in the human heart. CYP450s and sEH may be endogenous sources of these compounds, and sEH inhibition has the potential to alter myocardial perfusion, depending on which EETs are produced endogenously. endothelium-derived hyperpolafizing factor; endothelium; cytochrome P-450 epoxygenase; soluble epoxide hydrolase

Cytochrome P-450 (CYP) [omega]-hydroxylases and their arachidonic acid (AA) metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), produce a detrimental effect on ischemia-reperfusion injury in canine hearts, and the inhibition of CYP [omega]-hydroxylases markedly reduces myocardial infarct size expressed as a percentage of the area at risk (IS/AAR, %). In this study, we demonstrated that a specific CYP -hydroxylase inhibitor, N-methylsultbnyl-12,12-dibromododec-11-enamide (DDMS), markedly reduced 20-HETE production during ischemia-reperfusion and reduced myocardial infarct size compared with control [19.5 [+ or -] 1.0% (control), 9.6 | 1.5% (0.40 mg/kg DDMS), 4.0 _+ 2.0% (0.81 mg/kg DDMS), P < 0.01]. In addition, 20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid (20-HEDE, a putative 20-HETE antagonist) significantly reduced myocardial infarct size from control [10.3 [+ or -] 1.3% (0.032 mg/kg 20-HEDE) and 5.9 [+ or -] 1.9% (0.064 mg/kg 20-HEDE), P < 0.05]. We further demonstrated that one 5-min period of ischemic preconditioning (IPC) reduced infarct size to a similar extent as that observed with the high doses of DDMS and 20-HEDE, and the higher dose of DDMS given simultaneously with IPC augmented the infarct size reduction [9.9 [+ or -] 2.8% (IPC) to 2.5 [+ or -] 1.4% (0.81 mg/kg DDMS), P < 0.05] to a greater degree than that observed with either treatment alone. These results suggest an important negative role for endogenous CYP [omega]-hydroxylases and their product, 20-HETE, to exacerbate myocardial injury in canine myocardium. Furthermore, for the first time, this study demonstrates that the effect of IPC and the inhibition of CYP [omega]-hydroxylase synthesis (DDMS) or its actions (20-HEDE) may have additive effects in protecting the canine heart from ischemia-reperfusion injury. 20-hydroxyeicosa-6(Z), 15(Z)-dienoic acid; 20-hydroxyeicosatetraenoic acid; ischemia; ischemic preconditioning; reperfusion

Epoxyeicosatrienoic acids (EETs) are endothelium-derived cytochrome P-450 (CYP) metabolites of arachidonic acid that relax vascular smooth muscle by large-conductance calcium-activated potassium ([BK.sub.Ca]) channel activation and membrane hyperpolarization. We hypothesized that if smooth muscle cells (SMCs) had the capacity to synthesize EETs, endogenous EET production would increase [BK.sub.Ca] channel activity. Bovine coronary SMCs were transduced with adenovirus coding the CYP Bacillus megaterium -3 (F87V) (CYP BM-3) epoxygenase that metabolizes arachidonic acid exclusively to 14(S),15(R)-EET. Adenovirus containing the cytomegalovirus promoter-Escherichia coli [beta]-galactosidase was used as a control. With the use of an anti-CYP BM-3 (F87V) antibody, a 124-kDa immunoreactive protein was detected only in CYP BM-3-transduced cells. Protein expression increased with increasing amounts of virus. When CYP BM-3-transduced cells were incubated with [[sup.14]C]arachidonic acid, HPLC analysis detected 14,15-dihydroxyeicosatrienoic acid (14,15-DHET) and 14,15-EET. The identity of 14,15-EET and 14,15-DHET was confirmed by mass spectrometry. In CYP BM-3-transduced cells, methacholine ([10.sup.-5] M) increased 14,15-EET release twofold and [BK.sub.Ca] channel activity fourfold in cell-attached patches. Methacholine-induced increases in BKca channel activity were blocked by the CYP inhibitor 17-octadecynoic acid ([10.sup.-5] M). 14(S),15(R)-EET was more potent than 14(R), 15(S)-EET in relaxing bovine coronary arteries and activating [BK.sub.Ca] channels. Thus CYP BM-3 adenoviral transduction confers SMCs with epoxygenase activity. These cells acquire the capacity to respond to the vasodilator agonist by synthesizing 14(S), 15(R)-EET from endogenous arachidonic acid to activate [BK.sup.Ca] channels. These studies indicate that 14(S),15(R)-EET is a sufficient endogenous activator of [BK.sub.Ca], channels in coronary SMCs. endothelium-derived hyperpolarizing factor; arachidonic acid; vascular relaxation

ACh stimulates arachidonic acid (AA) release from membrane phospholipids of vascular endothelial cells (ECs). In rabbit aorta, AA is metabolized through the 15-lipoxygenase pathway to form vasodilatory eicosanoids 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) and 11,12,15-trihydroxyeicosatrienoic acid (THETA). AA is released from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by phospholipase [A.sub.2] ([PLA.sub.2]), or from phosphatidylinositol (PI) by phospholipase C (PLC) pathway. The diacylglycerol (DAG) lipase can convert DAG into 2-arachidonoylglycerol from which free AA can be released by monoacylglycerol (MAG) lipase or fatty acid amidohydrolase (FAAH). We used specific inhibitors to determine the involvement of the PLC pathway in ACh-induced AA release. In rabbit aortic rings precontracted by phenylephrine, ACh induced relaxation in the presence of indomethacin and N[omega]-nitro-L-arginine (L-NNA). These relaxations were blocked by the PLC inhibitor U-73122, DAG lipase inhibitor RHC-80267, and MAG lipase/FAAH inhibitor URB-532. Cultured rabbit aortic ECs were labeled with [[sup.14]C]AA and stimulated with methacholine ([10.sup.-5] M). Free [[sup.14]C]AA was released by methacholine. Methacholine decreased the [[sup.14]C]AA content of PI, DAG, and MAG fractions but not PC or PE fractions. Methacholine-induced release of [[sup.14]C]AA was blocked by U-73122, RHC-80267, and URB-532 but not by U-73343, an inactive analog of U-73122. The data suggested that ACh activates PLC, DAG lipase, and MAG lipase pathway to release AA from membrane lipids. This pathway is important in regulating vasodilatory eicosanoid synthesis and vascular relaxation in rabbit aorta. endothelium-dependent hyperpolarizing factor; phospholipid; monoacylglycerol lipase; trihydroxyeicosatrienoic acid; hydroxyepoxyeicosatrienoic acid

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

The present study tested the hypothesis that endostatin stimulates superoxide ([O.sup.-.sub.2]*) production through a ceramide-mediating signaling pathway and thereby results in an uncoupling of bradykinin (BK)-induced increases in intracellular [Ca.sup.2+] concentration ([[[Ca.sup.2+]].sub.i]) from nitric oxide (NO) production in coronary endothelial cells. With the use of high-speed, wavelength-switching, fluorescence-imaging techniques, the [[[Ca.sup.2+]].sub.i] and NO levels were simultaneously monitored in the intact endothelium of freshly isolated bovine coronary arteries. Under control conditions, BK was found to increase NO production and [[[Ca.sup.2+]].sub.i] in parallel. When the arteries were pretreated with 100 nM human recombinant endostatin for 1 h, this BK-induced NO production was reduced by 89%, whereas [[[Ca.sup.2+]].sub.i] was unchanged. With the conversion rate of L-[[sup.3.H]]arginine to L-[[sup.3.H]citrulline measured, endostatin had no effect on endothelial NO synthase (NOS) activity, but it stimulated ceramide by activation of sphingomyelinase (SMase), whereby [O.sup.-.sub.2]* production was enhanced in endothelial cells. [O.sup.-.sub.2]*. scavenging by tiron and inhibition of NAD(P)H oxidase by apocynin markedly reversed the effect of endostatin on the NO response to BK. These results indicate that endostatin increases intracellular ceramide levels, which enhances [O.sup.-.sub.2]* production through activation of NAD(P)H oxidase. This ceramide-[O.sup.-.sub.2]* signaling pathway may contribute importantly to endostatin-induced endothelial dysfunction. collagen; sphingolipid; free radicals; signal transduction; coronary artery

We recently reported that the lipoxygenase product 11,12,15-trihydroxyeicosatrienoic acid (THETA) mediates arachidonic acid (AA)-induced relaxation in the rabbit aorta. This study was designed to determine whether this lipoxygenase metabolite is involved in relaxation responses to AA in rabbit small mesenteric arteries. AA ([10.sup.-9]-[10.sup.-4] M) produced potent relaxations in isolated phenylephrine-preconstricted arteries, with a maximal relaxation of 99 [+ or -] 0.5% and ECso of 50 nM. The cyclooxygenase (COX) inhibitors indomethacin (10 txM), NS-398 (10 [micro]M, selective for COX-2), and SC-560 (100 nM, selective for COX-1) caused a marked rightward shift of concentration responses to AA. With the use of immunohistochemical analysis, both COX-1 and COX-2 were detected in endothelium and smooth muscle of small mesenteric arteries. Indomethacin-resistant relaxations were further reduced by the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC; 1 [micro]M), nordihydroguaiaretic acid (NDGA; 1 [micro]M), and ebselen (1 [micro]M). HPLC analysis showed that [[sup.14]C]AA was metabolized by mesenteric arteries to PG[I.sub.2], PG[E.sub.2], THETAs, hydroxyepoxyeicosatrienoic acids (HEETAs), and 15-hydroxyeicosatetraenoic acid (15-HETE). The production of PG[I.sub.2] and PG[E.sub.2] was blocked by indomethacin, and the production of THETAs, HEETAs, and 15-HETE was inhibited by CDC and NDGA. Column fractions corresponding to THETAs were further purified, analyzed by gas chromatography/mass spectrometry, and identified as 11,12,15- and 11,14,15-THETA. PG[I.sub.2], PG[E.sub.2], and purified THETA fractions relaxed mesenteric arteries precontracted with phenylephrine. The AA- and THETA-induced relaxations were blocked by high [K.sup.+] (60 mM). These findings provide functional and biochemical evidence that AA-induced relaxation in rabbit small mesenteric arteries is mediated through both COX and lipoxygenase pathways. endothelium-derived factors; trihydroxyeicosatrienoic acid; prostaglandin [E.sub.2]; prostaglandin [I.sub.2]; endothelium-derived hyperpolarizing factor

Chronic administration of nitric oxide reduces angiotensin II receptor type 1 expression and aldosterone synthesis in zona glomerulosa cells. Am J Physiol Endocrinol Metab 287:E820-E827, 2004. First published June 16, 2004; doi:10.1152/ajpendo.00183.2004.--Acute nitric oxide (NO) inhibits angiotensin II (ANG II)-stimulated aldosterone synthesis in zona glomerulosa (ZG) cells. In this study, we investigated the effects of chronic administration of NO on the ANG II receptor type 1 (AT1) expression and aldosterone synthesis. ZG cells were treated daily with DETA NONOate ([10.sup.-4] M), an NO donor, for 0. 12, 24, 48, 72, and 96 h. Chinese hamster ovary (CHO) cells, stably transfected with the AT1B receptor, were used as a positive control. Western blot analysis indicated that AT1 receptor expression was decreased as a function of time of NO administration in both CHO and ZG cells. ANG II binding to its receptors was determined by radioligand binding. NO treatment of ZG cells for 96 h resulted in a decrease in ANG II binding compared with control. The receptor density was decreased to 1.864 [+ or -] 129 fmol/mg protein from 3,157 [+ or -] 220 fmol/mg protein (P < 0.005), but the affinity was not changed (1.95 [+ or -] 0.22 vs. 1.88 [+ or -] 0.21 nM). Confocal Raman microspectroscopy and immunocytochemistry both confirmed that the expression of AT1 receptors in ZG cells decreased with chronic NO administration. In addition, chronic NO administration also decreased the expression of cholesterol side chain cleavage enzyme in ZG cells and inhibited ANG II- and 25-hydroxycholesterol-stimulated aldosterone synthesis in ZG cells. This study demonstrates that chronic administration of NO inhibits aldosterone synthesis in ZG cells by downregulation of the expression of both AT1 receptors and cholesterol side-chain cleavage enzyme. surface-enhanced Raman scattering

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

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

12/15-LO (12/15-lipoxygenase), encoded by Alox15 gene, metabolizes arachidonic acid to 12(S)-HETE (12-hydroxyeicosatetraenoic acid). Macrophages are the major source of 12/15-LO among immune cells, and 12/15-LO plays a crucial role in development of hypertension. Global Alox15- or macrophage-deficient mice are resistant to Ang II (angiotensin II)-induced hypertension. This study tests the hypothesis that macrophage 12(S)-HETE contributes to Ang II-mediated arterial constriction and thus to development of Ang II-induced hypertension. Ang II constricted isolated abdominal aortic and mesenteric arterial rings. 12(S)-HETE (100 nmol/L) alone was without effect; however, it significantly enhanced Ang II-induced constriction. The presence of wild-type macrophages also enhanced the Ang II-induced constriction, while Alox15-/- macrophages did not. Using this model, pretreatment of aortic rings with inhibitors, receptor agonists/antagonists, or removal of the endothelium, systematically uncovered an endothelium-mediated, Ang II receptor-2-mediated and superoxide-mediated enhancing effect of 12(S)-HETE on Ang II constrictions. The role of superoxide was confirmed using aortas from p47phox-/- mice where 12(S)-HETE failed to enhance constriction to Ang II. In cultured arterial endothelial cells, 12(S)-HETE increased the production of superoxide, and 12(S)-HETE or Ang II increased the production of an isothromboxane-like metabolite. A TP (thromboxane receptor) antagonist inhibited 12(S)-HETE enhancement of Ang II constriction. Both Ang II-induced hypertension and the enhancing effect of 12(S)-HETE on Ang II contractions were eliminated by a BLT2 (leukotriene B4 receptor-2) antagonist. These results outline a mechanism where the macrophage 12/15-LO pathway enhances the action of Ang II. 12(S)-HETE, acting on the BLT2, contributes to the hypertensive action of Ang II in part by promoting endothelial synthesis of a superoxide-derived TP agonist. [ABSTRACT FROM AUTHOR]