Infarct size-limiting effect of epoxyeicosatrienoic acid analog EET-B is mediated by hypoxia-inducible factor-1α via downregulation of prolyl hydroxylase 3.

Epoxyeicosatrienoic acids (EETs) decrease cardiac ischemia-reperfusion injury; however, the mechanism of their protective effect remains elusive. Here, we investigated the cardioprotective action of a novel EET analog, EET-B, in reperfusion and the role of hypoxia-inducible factor (HIF)-1α in such action of EET-B. Adult male rats were subjected to 30 min of left coronary artery occlusion followed by 2 h of reperfusion. Administration of 14,15-EET (2.5 mg/kg) or EET-B (2.5 mg/kg) 5 min before reperfusion reduced infarct size expressed as a percentage of the area at risk from 64.3 ± 1.3% in control to 42.6 ± 1.9% and 46.0 ± 1.6%, respectively, and their coadministration did not provide any stronger effect. The 14,15-EET antagonist 14,15-epoxyeicosa-5( Z)-enoic acid (2.5 mg/kg) inhibited the infarct size-limiting effect of EET-B (62.5 ± 1.1%). Similarly, the HIF-1α inhibitors 2-methoxyestradiol (2.5 mg/kg) and acriflavine (2 mg/kg) completely abolished the cardioprotective effect of EET-B. In a separate set of experiments, the immunoreactivity of HIF-1α and its degrading enzyme prolyl hydroxylase domain protein 3 (PHD3) were analyzed in the ischemic areas and nonischemic septa. At the end of ischemia, the HIF-1α immunogenic signal markedly increased in the ischemic area compared with the septum (10.31 ± 0.78% vs. 0.34 ± 0.08%). After 20 min and 2 h of reperfusion, HIF-1α immunoreactivity decreased to 2.40 ± 0.48% and 1.85 ± 0.43%, respectively, in the controls. EET-B blunted the decrease of HIF-1α immunoreactivity (7.80 ± 0.69% and 6.44 ± 1.37%, respectively) and significantly reduced PHD3 immunogenic signal in ischemic tissue after reperfusion. In conclusion, EET-B provides an infarct size-limiting effect at reperfusion that is mediated by HIF-1α and downregulation of its degrading enzyme PHD3. NEW & NOTEWORTHY The present study shows that EET-B is an effective agonistic 14,15-epoxyeicosatrienoic acid analog, and its administration before reperfusion markedly reduced myocardial infarction in rats. Most importantly, we demonstrate that increased hypoxia-inducible factor-1α levels play a role in cardioprotection mediated by EET-B in reperfusion likely by mechanisms including downregulation of the hypoxia-inducible factor -1α-degrading enzyme prolyl hydroxylase domain protein 3.

A novel activity of microsomal epoxide hydrolase: metabolism of the endocannabinoid 2-arachidonoylglycerol.

Microsomal epoxide hydrolase (EPHX1, EC 3.3.2.9) is a highly abundant α/ß-hydrolase enzyme that is known for its catalytical epoxide hydrolase activity. A wide range of EPHX1 functions have been demonstrated including xenobiotic metabolism; however, characterization of its endogenous substrates is limited. In this study, we present evidence that EPHX1 metabolizes the abundant endocannabinoid 2-arachidonoylglycerol (2-AG) to free arachidonic acid (AA) and glycerol. The EPHX1 metabolism of 2-AG was demonstrated using commercially available EPHX1 microsomes as well as PC-3 cells overexpressing EPHX1. Conversely, EPHX1 siRNA markedly reduced the EPHX1 expression and 2-AG metabolism in HepG2 cells and LNCaP cells. A selective EPHX1 inhibitor, 10-hydroxystearamide, inhibited 2-AG metabolism and hydrolysis of a well-known EPHX1 substrate, cis-stilbene oxide. Among the inhibitors studied, a serine hydrolase inhibitor, methoxy-arachidonyl fluorophosphate, was the most potent inhibitor of 2-AG metabolism by EPHX1 microsomes. These results demonstrate that 2-AG is an endogenous substrate for EPHX1, a potential role of EPHX1 in the endocannabinoid signaling and a new AA biosynthetic pathway.

Roles of endothelial nitric oxide synthase (eNOS) and mitochondrial permeability transition pore (MPTP) in epoxyeicosatrienoic acid (EET)-induced cardioprotection against infarction in intact rat hearts.

We previously demonstrated that 11,12 and 14,15-epoxeicosatrienoic acids (EETs) produce cardioprotection against ischemia-reperfusion injury in dogs and rats. Several signaling mechanisms have been implicated in the cardioprotective actions of the EETs; however, their mechanisms remain largely elusive. Since nitric oxide (NO) plays a significant role in cardioprotection and EETs have been demonstrated to induce NO production in various tissues, we hypothesized that NO is involved in mediating the EET actions in cardioprotection. To test this hypothesis, we used an in vivo rat model of infarction in which intact rat hearts were subjected to 30-min occlusion of the left coronary artery and 2-hr reperfusion. 11,12-EET or 14,15-EET (2.5mg/kg) administered 10min prior to the occlusion reduced infarct size, expressed as a percentage of the AAR (IS/AAR), from 63.9±0.8% (control) to 45.3±1.2% and 45.5±1.7%, respectively. A nonselective nitric oxide synthase (NOS) inhibitor, L-NAME (1.0mg/kg) or a selective endothelial NOS inhibitor, L-NIO (0.30mg/kg) alone did not affect IS/AAR but they completely abolished the cardioprotective effects of the EETs. On the other hand, a selective neuronal NOS inhibitor, nNOS I (0.03mg/kg) and a selective inducible NOS inhibitor, 1400W (0.10mg/kg) did not affect IS/AAR or block the cardioprotective effects of the EETs. Administration of 11,12-EET (2.5mg/kg) to the rats also transiently increased the plasma NO concentration. 14,15-EET (10µM) induced the phosphorylation of eNOS (Ser(1177)) as well as a transient increase of NO production in rat cardiomyoblast cell line (H9c2 cells). When 11,12-EET or 14,15-EET was administered at 5min prior to reperfusion, infarct size was also reduced to 42.8±2.2% and 42.6±1.9%, respectively. Interestingly, L-NAME (1.0mg/kg) and a mitochondrial KATP channel blocker, 5-HD (10mg/kg) did not abolish while a sarcolemmal KATP channel blocker, HMR 1098 (6.0mg/kg) and a mitochondrial permeability transition pore (MPTP) opener, atractyloside (5.0mg/kg) completely abolished the cardioprotection produced by the EETs. 14,15-EET (1.5mg/kg) with an inhibitor of MPTP opening, cyclosporin A (CsA, 1.0mg/kg) produced a greater reduction of infarct size than their individual administration. Conversely, an EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 2.5mg/kg) completely abolished the cardioprotective effects of CsA, suggesting a role of MPTP in mediating the EET actions. Taken together, these results suggest that the cardioprotective effects of the EETs in an acute ischemia-reperfusion model are mediated by distinct mediators depending on the time of EET administration. The cardioprotective effects of EETs administered prior to ischemia were regulated by the activation of eNOS and increased NO production, while sarcKATP channels and MPTP were involved in the beneficial effects of the EETs when administered just prior to reperfusion.

Protection from myocardial ischemia/reperfusion injury by a positive allosteric modulator of the A3 adenosine receptor.

Adenosine is increased in ischemic tissues where it serves a protective role by activating adenosine receptors (ARs), including the A3 AR subtype. We investigated the effect of N-{2-[(3,4-dichlorophenyl)amino]quinolin-4-yl}cyclohexanecarboxamide (LUF6096), a positive allosteric modulator of the A3 AR, on infarct size in a barbital-anesthetized dog model of myocardial ischemia/reperfusion injury. Dogs were subjected to 60 min of coronary artery occlusion and 3 h of reperfusion. Infarct size was assessed by macrohistochemical staining. Three experimental groups were included in the study. Groups I and II received two doses of vehicle or LUF6096 (0.5 mg/kg i.v. bolus), one administered before ischemia and the other immediately before reperfusion. Group III received a single dose of LUF6096 (1 mg/kg i.v. bolus) immediately before reperfusion. In preliminary in vitro studies, LUF6096 was found to exert potent enhancing activity (EC50 114.3 ± 15.9 nM) with the canine A3 AR in a guanosine 5'-[γ-[³5S]thio]triphosphate binding assay. LUF6096 increased the maximal efficacy of the partial A3 AR agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide and the native agonist adenosine more than 2-fold while producing a slight decrease in potency. In the dog studies, administration of LUF6096 had no effect on any hemodynamic parameter measured. Pretreatment with LUF6096 before coronary occlusion and during reperfusion in group II dogs produced a marked reduction in infarct size (∼50% reduction) compared with group I vehicle-treated dogs. An equivalent reduction in infarct size was observed when LUF6096 was administered immediately before reperfusion in group III dogs. This is the first study to demonstrate efficacy of an A3 AR allosteric enhancer in an in vivo model of infarction.

Eribis peptide 94 reduces infarct size in rat hearts via activation of centrally located µ opioid receptors.

Eribis peptide 94 (EP 94) is a novel enkephalin derivative that binds with high potency to µ and δ opioid receptors with less affinity for the κ opioid receptor. This compound has recently been shown to produce an acute reduction in myocardial infarct size in the anesthetized pig and rat partially via an endothelial nitric oxide synthase and KATP channel-dependent mechanism. EP 94 also was found to produce a chronic reduction in infarct size 24 hours postdrug administration via the upregulation of inducible nitric oxide synthase in rats. Despite these findings, no data have emerged in which the opioid receptor subtype responsible for cardioprotection has been identified and the site of action, heart, other peripheral organs, or the central nervous system, has not been addressed. In the current study, EP 94, was administered in 2 divided doses (0.5 µg/kg, intravenously) at 5 and 10 minutes into the ischemic period, and the opioid antagonists were administered 10 minutes before the onset of the 30-minute ischemic period. The selective antagonists used were the µ receptor antagonist CTOP (D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2), the δ receptor antagonists naltrindole and BNTX (7-benzylidenenaltrexone), and the κ receptor antagonist nor-BNI (norbinaltorphimine). Surprisingly, only CTOP completely blocked the cardioprotective effect of EP 94, whereas naltrindole, BNTX, and nor-BNI had modest but nonsignificant effects. Because there is controversial evidence suggesting that µ receptors may be absent in the adult rat myocardium, it was hypothesized that the protective effect of EP 94 may be mediated by an action outside the heart, perhaps in the central nervous system. To test this hypothesis, rats were pretreated with the nonselective opioid antagonist, naloxone hydrochloride, which penetrates the blood-brain barrier or naloxone methiodide, the quaternary salt of naloxone hydrochloride, which does not penetrate the blood-brain barrier before EP 94 administration. In support of a central nervous system site of action for EP 94, naloxone hydrochloride completely blocked its cardioprotective effect, whereas naloxone methiodide had no effect. These results suggest that EP 94 reduces infarct size (expressed as a percent of the area at risk) in the rat primarily via activation of central µ opioid receptors.

Acute and chronic cardioprotection by the enkephalin analogue, Eribis peptide 94, is mediated via activation of nitric oxide synthase and adenosine triphosphate-regulated potassium channels.

BACKGROUND/AIMS: Eribis peptide 94 (EP 94) is a new enkephalin derivative which potently binds to the µ- and δ-opioid receptor. In this study, we determined the effects of EP 94 and potential mechanism(s) involved in cardioprotection of the rat heart. METHODS AND RESULTS: An acute (5 and10 min into ischemia) and a chronic (24 h prior to ischemia) EP 94 administration produced a similar 30-40% reduction in infarct size/area at risk and the effects were blocked by the K(ATP) channel antagonists, HMR 1098 and 5-HD. The cardioprotective effects were blocked by a nonselective nitric oxide synthase (NOS) inhibitor (L-NAME) following acute administration and by a selective iNOS inhibitor (1400W) following chronic administration. CONCLUSION: These results suggest that EP 94 may have potential for the treatment of ischemic heart disease via a nitric oxide (NO)-K(ATP)-mediated mechanism.

Role of superoxide and thromboxane receptors in acute angiotensin II-induced vasoconstriction of rabbit vessels.

This study explored the hypothesis that a portion of angiotensin II-induced contractions is dependent on superoxide generation and release of a previously unidentified arachidonic acid metabolite that activates vascular smooth muscle thromboxane receptors. Treatment of rabbit aorta or mesentery artery with the thromboxane receptor antagonist SQ29548 (10 µM) reduced angiotensin II-induced contractions (maximal contraction in aorta; control vs. SQ29548: 134 ± 16 vs. 93 ± 10%). A subset of rabbits deficient in vascular thromboxane receptors also displayed decreased contractions to angiotensin II. The superoxide dismutase mimetic Tiron (30 mM) attenuated angiotensin II-induced contractions only in rabbits with functional vascular thromboxane receptors (maximal contraction in aorta; control vs. Tiron: 105 ± 5 vs. 69 ± 11%). Removal of the endothelium or treatment with a nitric oxide synthase inhibitor, nitro-l-arginine (30 µM) did not alter angiotensin II-induced contractions. Tiron and SQ29548 decreased angiotensin II-induced contractions in the denuded aortas by a similar percentage as that observed in intact vessels. The cyclooxygenase inhibitor indomethacin (10 µM) or thromboxane synthase inhibitor dazoxiben (10 µM) had no effect on angiotensin II-induced contractions indicating that the vasoconstrictor was not thromboxane. Angiotensin II increased the formation of a 15-series isoprostane. Isoprostanes are free radical-derived products of arachidonic acid. The unidentified isoprostane increased when vessels were incubated with the superoxide-generating system xanthine/xanthine oxidase. Pretreatment of rabbit aorta with the isoprostane isolated from aortic incubations enhanced angiotensin II-induced contractions. Results suggest the factor activating thromboxane receptors and contributing to angiotensin II vasoconstriction involves the superoxide-mediated generation of a 15-series isoprostane.

Anti-proliferative effect of a putative endocannabinoid, 2-arachidonylglyceryl ether in prostate carcinoma cells.

Endocannabinoids (ECs), anandamide (AEA) and 2-arachidonoylglycerol (2-AG), inhibit proliferation of carcinoma cells. Several enzymes hydrolyze ECs to reduce endogenous EC concentrations and produce eicosanoids that promote cell growth. In this study, we determined the effects of EC hydrolysis inhibitors and a putative EC, 2-arachidonylglyceryl ether (noladin ether, NE) on proliferation of prostate carcinoma (PC-3, DU-145, and LNCaP) cells. PC-3 cells had the least specific hydrolysis activity for AEA and administration of AEA effectively inhibited cell proliferation. The proliferation inhibition was blocked by SR141716A (a selective CB1R antagonist) but not SR144528 (a selective CB2R antagonist), suggesting a CB1R-mediated inhibition mechanism. On the other hand, specific hydrolysis activity for 2-AG was high and 2-AG inhibited proliferation only in the presence of EC hydrolysis inhibitors. NE inhibited proliferation in a concentration-dependent manner; however, SR141716A, SR144528 and pertussis toxin did not block the NE-inhibited proliferation, suggesting a CBR-independent mechanism of NE. A peroxisome proliferator-activated receptor gamma (PPARγ) antagonist GW9662 did not block the NE-inhibited proliferation, suggesting that PPARγ was not involved. NE also induced cell cycle arrest in G(0)/G(1) phase in PC-3 cells. NE inhibited the nuclear translocation of nuclear factor-kappa B (NF-κB p65) and down-regulated the expression of cyclin D1 and cyclin E in PC-3 cells, suggesting the NF-κB/cyclin D and cyclin E pathways are involved in the arrest of G1 cell cycle and inhibition of cell growth. These results indicate therapeutic potentials of EC hydrolysis inhibitors and the enzymatically stable NE in prostate cancer.

Abdominal surgical incision induces remote preconditioning of trauma (RPCT) via activation of bradykinin receptors (BK2R) and the cytochrome P450 epoxygenase pathway in canine hearts.

OBJECTIVE: Recently, a novel observation was made in which nonischemic trauma at a site remote from the heart produced by a transverse abdominal incision resulted in a marked reduction of infarct size (IS) in the mouse heart via activation of sensory nerve fibers in the skin and subsequent activation of bradykinin 2 receptors (BK2R). This phenomenon was termed remote preconditioning of trauma (RPCT). Since RPCT may have potential clinical implications we attempted to confirm these findings in a large animal model, the dog. The epoxyeicosatrienoic acids (EETs) have also recently been shown to be antinociceptive and have been shown to mimic ischemic preconditioning (IPC) and postconditioning (POC) in dogs, therefore, we tested the role of the EETs in RPCT. METHODS: Anesthetized adult mongrel dogs of either sex were subjected to 60 min of left anterior descending (LAD) coronary artery occlusion followed by 3 h of reperfusion. In all groups except the controls (no slit), a transverse slit (9 cm) was applied to the abdominal wall of the dog being careful to only slit the skin. Subsequently, 15 min after the slit the heart was subjected to the ischemia/reperfusion protocol. RESULTS: In the control dogs, the IS as a percent of the area at risk (AAR) was 22.5 ± 2.4%, whereas in the dogs subjected to the slit alone the IS/AAR was reduced to 9.2 ± 1.2% (*P < 0.01). The BR2R blocker, HOE 140 (50 ug/kg, iv) given 10 min prior to the slit, completely abolished the protective effects of RCPT as did pretreatment with 14,15-EEZE, a putative EET receptor blocker or pretreatment with the selective EET synthesis inhibitor, MSPPOH. CONCLUSIONS: These results suggest that BK and the EETs share cardioprotective properties in a large animal model of RPCT.

Identification of 13-hydroxy-14,15-epoxyeicosatrienoic acid as an acid-stable endothelium-derived hyperpolarizing factor in rabbit arteries.

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.

Inhibition of carcinoma cell motility by epoxyeicosatrienoic acid (EET) antagonists.

Cytochrome P450 (CYP) epoxygenases, CYP2C8, 2C9 and 2J2 mRNA and proteins, were expressed in prostate carcinoma (PC-3, DU-145 and LNCaP) cells. 11,12-Epoxyeicosatrienoic acid (11,12-EET) was the major arachidonic acid metabolite in these cells. Blocking EET synthesis by a selective CYP epoxygenase inhibitor (N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide [MS-PPOH]) inhibited tonic (basal) invasion and migration (motility) while exogenously added EET induced cell motility in a concentration-dependent manner. An epidermal growth factor receptor (EGFR) kinase inhibitor (AG494) or a PI3 kinase inhibitor (LY294002) inhibited cell migration and reduced 11,12-EET-induced cell migration. Importantly, synthetic EET antagonists (14,15-epoxyeicosa-5(Z)-enoic acid [14,15-EEZE], 14,15-epoxyeicosa-5(Z)-enoic acid 2-[2-(3-hydroxy-propoxy)-ethoxy]-ethyl ester [14,15-EEZE-PEG] and 14,15-epoxyeicosa-5(Z)-enoic-methylsulfonylimide [14,15-EEZE-mSI]) inhibited EET-induced cell invasion and migration. 11,12-EET induced cell stretching and myosin-actin microfilament formation as well as increased phosphorylation of EGFR and Akt (Ser473), while 14,15-EEZE inhibited these effects. These results suggest that EET induce and EET antagonists inhibit cell motility, possibly by putative EET receptor-mediated EGFR and PI3K/Akt pathways, and suggest that EET antagonists are potential therapeutic agents for prostate cancer.

Evidence for a role of opioids in epoxyeicosatrienoic acid-induced cardioprotection in rat hearts.

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-NH(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 Leu-enkephalin 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 IS/AAR 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(i/o) protein-coupled delta- and/or kappa-opioid receptor.

Evidence for role of epoxyeicosatrienoic acids in mediating ischemic preconditioning and postconditioning in dog.

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.

Expression and function of fatty acid amide hydrolase in prostate cancer.

The hydrolysis of endocannabinoids has profound effects on the function of the endocannabinoid signaling system in the regulation of prostate carcinoma cells. Prostate carcinoma cells exhibit a wide range of hydrolysis activity for 2-arachidonoylglycerol (2-AG), the major endocannabinoid. However, enzyme(s) responsible for 2-AG hydrolysis and their functions in prostate cancer have not been characterized. In this study, we demonstrated that fatty acid amide hydrolase (FAAH) was differentially expressed in normal and prostate carcinoma cells. In PC-3 cells, overexpression of FAAH resulted in increased FAAH protein, 2-AG hydrolysis, cell invasion and cell migration. Conversely, small-interfering RNA (siRNA) knockdown of FAAH in LNCaP cells decreased FAAH protein, 2-AG hydrolysis and cell invasion. Furthermore, CAY10401, a FAAH inhibitor, decreased cell invasion and it enhanced the reduction of invasion in FAAH siRNA-transfected LNCaP cells. Immunohistochemistry staining of commercial tissue microarrays (TMAs) demonstrated FAAH staining in 109 of 157 cores of prostate adenocarcinomas but weak staining in 1 of 8 cores of normal prostate tissues. These results suggest that FAAH regulates 2-AG hydrolysis and invasion of prostate carcinoma cells and is potentially involved in prostate tumorigenesis.

Structural characterization of monohydroxyeicosatetraenoic acids and dihydroxy- and trihydroxyeicosatrienoic acids by ESI-FTICR.

The fragmentation characteristics of monohydroxyeicosatetraenoic acids and dihydroxy- and trihydroxyeicosatrienoic acids were investigated by electrospray ionization Fourier transform ion cyclotron resonance (FTICR) mass spectrometry using sustained off-resonance irradiation collision-induced dissociation (SORI-CID) and infrared multiphoton dissociation (IRMPD). The fragmentation patterns of these compounds were associated with the number and positions of the hydroxyl substituents. The fragmentation is more complicated with increasing number of the hydroxyl groups of the compounds. In general, the major carbon-carbon cleavage of [M - H](-) ions occurred at the alpha-position to the hydroxyl group, and the carbon-carbon cleavage occurred when there was a double-bond at the beta-position to the hydroxyl group. SORI-CID and IRMPD produced some common fragmentation patterns; however, each technique provided some unique patterns that are useful for structural identification of these compounds. This study demonstrated the application of FTICR via the identification of regioisomers of trihydroxyeicosatrienoic acids in rabbit aorta samples.

Endothelium-derived steroidogenic factor enhances angiotensin II-stimulated aldosterone release by bovine zona glomerulosa cells.

Endothelium-derived steroidogenic factor (EDSF) is an endothelial peptide that stimulates aldosterone release from bovine adrenal zona glomerulosa (ZG) cells. The regulation of aldosterone release by combinations of EDSF and angiotensin II (AII) or EDSF and ACTH was investigated. Endothelial cells (ECs) and EC-conditioned media (ECCM) increased aldosterone release from ZG cells, an activity attributed to EDSF. AII (10(-12) to 10(-8) M) and ACTH (10(-12) to 10(-9) M) also stimulated the release of aldosterone from ZG cells. The stimulation by AII, but not ACTH, was greatly enhanced when ZG cells were coincubated with ECs. AII was metabolized by ECs to peptides identified by mass spectrometry as angiotensin (1-7) and angiotensin IV. There was very little metabolism of AII by ZG cells. Neither of these two AII metabolites altered aldosterone release from ZG cells, so they could not account for the enhanced response with ECs. AII-induced aldosterone release from ZG cells was enhanced by ECCM but not cell-free conditioned medium. This enhanced response was not due to increased EDSF release from ECs by AII. The synergistic effect of EDSF and AII was apparent when AII was added during or after the generation of ECCM and not observed when the AII component of the enhancement was blocked by the AII antagonist, losartan. These studies indicate that EDSF enhances the steroidogenic effect of AII. In the adrenal gland, ECs are in close anatomical relationship with ZG cells and may sensitize ZG cells to the steroidogenic action of AII by releasing EDSF.

Reticulocyte 15-lipoxygenase-I is important in acetylcholine-induced endothelium-dependent vasorelaxation in rabbit aorta.

OBJECTIVE: Aortic 15-lipoxygenase (15-LO) metabolizes arachidonic acid (AA) to 15-hydroperoxyeicosatetraenoic acid, which is then converted to the vasodilators 15-hydroxy-11,12-epoxyeicosatrienoic acid and 11,12,15-trihydroxyeicosatrienoic acid. These metabolites contribute to endothelium-dependent relaxations of rabbit aorta to AA and acetylcholine. We investigated the identity of rabbit aortic 15-LO and studied its importance in the regulation of vascular tone. METHODS AND RESULTS: RT-PCR using 12-lipoxygenase/15-LO specific primers resulted in a 572-bp product with a sequence identical to 15-LO-I from rabbit aorta. A RT-PCR/restriction digest strategy excluded expression of 12-lipoxygenase. Immunoblotting revealed 15-LO-I expression in rabbit endothelial and smooth muscle cells. Aortic homogenates and cytosolic fractions metabolize AA to 15(S)-hydroxyeicosatetraenoic acid and linoleic acid to 13(S)-hydroxyoctadecadienoic acid. This activity was blocked by LO inhibitors. The kinetic characteristics (Michaelis constant of aortic 15-LO is 2.2+/-0.3 micromol/L for AA and 23.5+/-3.3 micromol/L for linoleic acid) of aortic 15-LO were similar to those of the purified 15-LO-I. An antisense oligonucleotide inhibited 15-LO-I expression in rabbit aorta. Indomethacin and nitro-L-arginine-resistant relaxations to acetylcholine were inhibited by 15-LO-I antisense oligonucleotide but not by the scrambled oligonucleotide. CONCLUSIONS: 15-LO-I is expressed in rabbit aortic endothelium and is important in endothelium-dependent regulation of vascular tone.

Role of epoxyeicosatrienoic acids in protecting the myocardium following ischemia/reperfusion injury.

Cardiomyocyte injury following ischemia-reperfusion can lead to cell death and result in cardiac dysfunction. A wide range of cardioprotective factors have been studied to date, but only recently has the cardioprotective role of fatty acids, specifically arachidonic acid (AA), been investigated. This fatty acid can be found in the membranes of cells in an inactive state and can be released by phospholipases in response to several stimuli, such as ischemia. The metabolism of AA involves the cycloxygenase (COX) and lipoxygenase (LOX) pathways, as well as the less well characterized cytochrome P450 (CYP) monooxygenase pathway. Current research suggests important differences with respect to the cardiovascular actions of specific CYP mediated arachidonic acid metabolites. For example, CYP mediated hydroxylation of AA produces 20-hydroxyeicosatetraenoic acid (20-HETE) which has detrimental effects in the heart during ischemia, pro-inflammatory effects during reperfusion and potent vasoconstrictor effects in the coronary circulation. Conversely, epoxidation of AA by CYP enzymes generates 5,6-, 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) that have been shown to reduce ischemia-reperfusion injury, have potent anti-inflammatory effects within the vasculature, and are potent vasodilators in the coronary circulation. This review aims to provide an overview of current data on the role of these CYP pathways in the heart with an emphasis on their involvement as mediators of ischemia-reperfusion injury. A better understanding of these relationships will facilitate identification of novel targets for the prevention and/or treatment of ischemic heart disease, a major worldwide public health problem.

Elevated 12- and 20-hydroxyeicosatetraenoic acid in urine of patients with prostatic diseases.

The role of eicosanoids (metabolites of arachidonic acid) in prostate diseases is receiving increased attention. We investigated the relationship between the concentrations of urinary free acids of 12- and 20-hydroxyeicosatetraenoic acids (12- and 20-HETE) and the benign prostatic hypertrophy (BPH) and prostate cancer (Pca). Urinary concentrations of 12-HETE and 20-HETE of BPH and Pca patients were significantly higher than normal subjects. After removal of the prostate gland, the urinary concentrations of these eicosanoids decreased to concentrations similar to the normal subjects. These results suggest that urinary free acids of 12-HETE and 20-HETE indicate an abnormality of the prostate gland.

Inhibition of cytochrome P450omega-hydroxylase: a novel endogenous cardioprotective pathway.

Cytochrome P450s (CYP) and their arachidonic acid (AA) metabolites have important roles in regulating vascular tone, but their function and specific pathways involved in modulating myocardial ischemia-reperfusion injury have not been clearly established. Thus, we characterized the effects of several selective CYPomega-hydroxylase inhibitors and a CYPomega-hydroxylase metabolite of AA, 20-hydroxyeicosatetraenoic acid (20-HETE), on the extent of ischemia-reperfusion injury in canine hearts. During 60 minutes of ischemia and particularly after 3 hours of reperfusion, 20-HETE was produced at high concentrations. A nonspecific CYP inhibitor, miconazole, and 2 specific CYPomega-hydroxylase inhibitors, 17-octadecanoic acid (17-ODYA) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), markedly inhibited 20-HETE production during ischemia-reperfusion and produced a profound reduction in myocardial infarct size (expressed as a percent of the area at risk) (19.6+/-1.7% [control], 8.4+/-2.5% [0.96 mg/kg miconazole], 5.9+/-2.2% [0.28 mg/kg 17-ODYA], and 10.8+/-1.8% [0.40 mg/kg DDMS], P<0.05, respectively). Conversely, exogenous 20-HETE administration significantly increased infarct size (26.9+/-1.9%, P<0.05). Several CYPomega-hydroxylase isoforms, which are known to produce 20-HETE such as CYP4A1, CYP4A2, and CYP4F, were demonstrated to be present in canine heart tissue and their activity was markedly inhibited by incubation with 17-ODYA. These results indicate an important endogenous role for CYPomega-hydroxylases and in particular their product, 20-HETE, in exacerbating myocardial injury in canine myocardium. The full text of this article is available online at http://circres.ahajournals.org.