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We previously demonstrated that several epoxyeicosatrienoic acids (EETs) produce reductions in myocardial infarct size in rats and dogs. Since a recent study demonstrated the release of opioids in mediating the antinociceptive effect of 14,15-EET, we hypothesized that endogenous opioids may also be involved in mediating the cardioprotective effect of the EETs. To test this hypothesis, we used an in vivo rat model of infarction and a rat Langendorff model. In the infarct model, hearts were subjected to 30 min occlusion of the left coronary artery and 2 h reperfusion. Animals were treated with 11,12-EET or 14,15-EET (2.5 mg/kg) alone 15 min before occlusion or with opioid antagonists [naloxone, naltrindole, nor-binaltorphimine (nor-BNI), and D-Phe-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-N[H.sub.2] (CTOP), a nonselective, a selective [delta], a selective [kappa], and a selective [mu] receptor antagonist, respectively] 10 min before EET administration. In four separate groups, antiserum to Met- and Leuenkephalin and dynorphin-A-(1-17) was administered 50 min before the 11,12-EET administration. Infarct size expressed as a percent of the area at risk (IS/AAR) was 63.5 [+ or -] 1.2, 45.3 [+ or -] 1.0, and 40.9 [+ or -] 1.2% for control, 11,12-EET, and 14,15-EET, respectively. The protective effects of 11,12-EET were abolished by pretreatment with either naloxone (60.5 [+ or -] 1.8%), naltrindole (60.8 [+ or -] 1.0%), nor-BNI (62.3 [+ or -] 2.8%), or Met-enkephalin antiserum (63.2 [+ or -] 1.7%) but not CTOP (42.0 [+ or -] 3.0%). In isolated heart experiments, 11,12-EET was administered to the perfusate 15 min before 20 min global ischemia followed by 45 min reperfusion in control hearts or in those pretreated with pertussis toxin (48 h). 11,12-EET increased the recovery of left ventricular developed pressure from 33 [+ or -] 1 to 45 [+ or -] 6% (P < 0.05) and reduced ISIAAR from 37 [+ or -] 4 to 20 [+ or -] 3% (P < 0.05). Both pertussis toxin and naloxone abolished these beneficial effects of 11,12-EET. Taken together, these results suggest that the major cardioprotective effects of the EETs depend on activation of a [G.sub.i/o] protein-coupled [delta]- and/or [kappa]-opioid receptor. nor-binaltorphimine; infarct size; area at risk doi: 10.1152/ajpheart.00815.2009.

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

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

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

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

Cytochrome P-450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce increases in postischemic function via ATP-sensitive potassium channels ([K.sub.ATP]); however, the direct effects of EETs on infarct size (IS) have not been investigated. We demonstrate that two major regioisomers of CYP epoxygenases, 11,12-EET and 14,15-EET, significantly reduced IS in dogs compared to control (22.1 [+ or -] 1.8%), whether administered 15 min before 60 min of coronary occlusion (6.4 [+ or -] 1.9%, 11,12-EET; and 8.4 [+ or -] 2.4%, 14.15-EET) or 5 min before 3 h of reperfusion (8.8 [+ or -] 2.1%, 11,12-EET; and 9.7 [+ or -] 1.4%, 14,15-EET). Pretreatment with the epoxide hydrolase metabolite of 14,15-EET, 14,15-dihydroxyeicosatrienoic acid, had no effect. The protective effect of 11,12-EET was abolished (24.3 [+ or -] 4.6%) by the [K.sub.ATP] channel antagonist glibenclamide. Furthermore, one 5-min period of ischemic preconditioning (IPC) reduced IS to a similar extent (8.7 [+ or -] 2.8%) to that observed with the EETs. The selective CYP epoxygenase inhibitor, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), did not block the effect of IPC. However, administration of MS-PPOH concomitantly with N-methylsulfonyl-12, 12-dibromododec-11-enanide (DDMS), a selective inhibitor of endogenous CYP [omega]-hydroxylases, abolished the reduction in myocardial IS expressed as a percentage of area at risk (IS/AAR) produced by DDMS (4.6 [+ or -] 1.2%, DDMS; and 22.2 [+ or -] 3.4%, MS-PPOH + DDMS). These data suggest that 11,12-EET and 14,15-EET produce reductions in IS/AAR primarily at reperfusion. Conversely, inhibition of CYP epoxygenases and endogenous EET formation by MS-PPOH, in the presence of the CYP [omega]-hydroxylase inhibitor DDMS blocked cardioprotection, which suggests that endogenous EETs are important for the beneficial effects observed when CYP to-hydroxylases are inhibited. Finally, the protective effects of EETs are mediated by cardiac [K.sub.ATP] channels. cytochrome P-450 epoxygenase; 11,12-epoxyeicosatrienoic acid; 14,15-epoxyeicosatrienoic acid; 20-hydroxyeicosatetraenoic acid; ischemic preconditioning; adenosine 5'-triphosphate-sensitive potassium channels doi:10.1152/ajpheart.00071.2006

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

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

Overexpression of human cyclooxygenase 2 (COX-2) in the mammary glands of transgenic mice induces tissue-specific tumorigenic transformation. However, the molecular mechanisms involved are not yet defined. Here we show that COX-2 expressed in the epithelial cell compartment regulates angiogenesis in the stromal tissues of the mammary gland. Microvessel density increased before visible tumor growth and exponentially during tumor progression. Inhibition of prostanoid synthesis with indomethacin strongly decreased microvessel density and inhibited tumor progression. Up-regulation of angiogenic regulatory genes in COX-2 transgenic mammary tissue was also potently inhibited by indomethacin treatment, suggesting that prostanoids released from COX-2-expressing mammary epithelial cells induce angiogenesis. G protein-coupled receptors for the major product, prostaglandin [E.sub.2] (PG[E.sub.2]) E[P.sub.1-4], are expressed during mammary gland development, and E[P.sub.1,2,4] receptors were up-regulated in tumor tissue. PG[E.sub.2] stimulated the expression angiogenic regulatory genes in mammary tumor cells isolated from COX-2 transgenic mice. Such cells are tumorigenic in nude mice; however, treatment with Celecoxib, a COX-2-specific inhibitor, reduced tumor growth and microvessel density. These results define COX-2-derived PG[E.sub.2] as a potent inducer of angiogenic switch during mammary cancer progression. tumor angiogenesis | prostagiandin [E.sub.2] receptors | mammary cancer

Levels of adenosine in the renal medullary interstitium were determined using microdialysis and fluorescence HPLC methods. The role of endogenous adenosine in the control of blood medullary flow and sodium excretion was then examined with the infusion of specific adenosine receptor antagonists or agonists into the renal medulla of anaesthesized Sprague-Dawley rats. Results suggest that endogenous medullary adenosine at physiological concentrations serves to dilate medullary vessels via A2 receptors resulting in natriuretic response that overrides the tubular antinatriuteric effects.

Nithipatikom, Kasem, Phillip F. Pratt, and William B. Campbell. Determination of EETs using microbore liquid chromatography with fluorescence detection. Am J Physiol Heart Circ Physiol 279: H857-H862, 2000.--Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 metabolites of arachidonic acid involved in the regulation of vascular tone. The method of microbore column high-performance liquid chromatography with fluorescence detection was developed to determine 14,15-EET, 11,12-EET, and the mixture of 8,9-EET and 5,6-EET. Tridecanoic acid (TA) was used as an internal standard. EETs were reacted with 2-(2,3-naphthalimino) ethyl trifluoromethanesulfonate (NT) to form highly fluorescent derivatives. A [C.sub.18] microbore column and a water-acetonitrile mobile phase were used for separation. Samples were excited at 259 nm, and the fluorescence was detected at 395 nm. The overall recoveries were 88% for EETs and 40% for TA. EETs were detected in concentrations as low as 2 pg (signal-to-noise ratio = 3). The method was used to determine the EET production from endothelial cells (ECs). Bradykinin and methacholine ([10.sup.-6] M) stimulated an increase in the production of EETs by ECs two- and fivefold, respectively. This sensitive method may be used for determination of EETs at low concentrations normally detected in complex biological samples. cytochrome P-450; endothelial cells; epoxyeicosatrienoic acids; high-performance liquid chromatography