8-9-epoxyeicosatrienoic-acid and 11-12-epoxy-5-8-14-eicosatrienoic-acid

Cytochrome P450 epoxygenases metabolize arachidonic acid to biologically active eicosanoids. Primary epoxidation products are four regioisomers of cis-epoxyeicosatrienoic acid (EET), 5,6-, 8,9-, 11,12-, and 14,15-EET. One of the predominant epoxygenase isoforms involved in EET formation belongs to the CYP2 gene family. In humans, the P450 epoxygenase, CYP2J2, is expressed in the cardiovascular system, namely the endothelium, vascular smooth muscle, and cardiomyocyte. CYP2J2 possesses vascular protective effects, which include but are not limited to, protection against ischemia-reperfusion injury, suppression of reactive oxygen species following hypoxia-reoxygenation, inhibition of the pro-inflammatory transcription factor, nuclear factor-kappaB (NF-kappaB), attenuation of vascular smooth muscle migration, and enhancement of a fibrinolytic pathway. Although regioisomers of EET elicit these effects to varying degrees, 11,12-EET appears to be the most potent with respect to anti-inflammatory, anti-migratory, and pro-fibrinolytic effects. Thus, CYP2J2 and its derived arachidonic acid metabolites may play important roles in regulating vascular function under normal and pathophysiological conditions.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Cell Movement; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Eicosanoids; Fibrinolysis; Humans; Inflammation; Isoenzymes; Models, Biological; Muscle, Smooth, Vascular; Myocytes, Cardiac; NF-kappa B; Oxygenases; Protective Agents; Stereoisomerism; Vasodilation

Epoxyeicosatrienoic acids (EETs) are lipid metabolites that are synthesized in vascular endothelial cells. They are released by stimulation of their muscarinic receptors, and induce vaso-relaxation of cerebral blood vessels. In addition, cytochrome P450 epoxygenase enzymes, which catalyze the formation of epoxyeicosatrienoic acids, especially after stimulation by the excitatory neurotransmitter glutamate, are present in astrocytes, an abundant cell type in the brain that extends foot processes onto the cerebral microvessels. Using a modification of an efficient, recently developed, fluorescent assay, we have detected the presence of EETs in endothelial cells cultured from the cortex of rat brains as well as in neonatal astrocytes. We propose that both these cell types provide a dual supply of EETs to increase cerebral blood flow in order to meet systemic as well as localized nutrient demands of cells in the brain.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Astrocytes; Brain Chemistry; Cerebrovascular Circulation; Endothelium, Vascular; Humans; Microcirculation; Muscle, Smooth, Vascular; Vasodilation

Several mediators contribute to postocclusive reactive hyperemia (PORH) of the skin, including sensory nerves and endothelium-derived hyperpolarizing factors. The main objective of our study was to investigate the specific contribution of epoxyeicosatrienoic acids in human skin PORH. Eight healthy volunteers were enrolled in two placebo-controlled experiments. In the first experiment we studied the separate and combined effects of 6.5 mM fluconazole, infused through microdialysis fibers, and lidocaine/prilocaine cream on skin PORH following 5 min arterial occlusion. In the second experiment we studied the separate and combined effects of 6.5 mM fluconazole and 10 mM N(G)-monomethyl-l-arginine (l-NMMA). Skin blood flux was recorded using two-dimensional laser speckle contrast imaging. Maximal cutaneous vascular conductance (CVC(max)) was obtained following 29 mM sodium nitroprusside perfusion. The PORH peak at the placebo site averaged 66 ± 11%CVC(max). Compared with the placebo site, the peak was significantly lower at the fluconazole (47 ± 10%CVC(max); P < 0.001), lidocaine (29 ± 10%CVC(max); P < 0.001), and fluconazole + lidocaine (30 ± 10%CVC(max); P < 0.001) sites. The effect of fluconazole on the area under the curve was more pronounced. In the second experiment, the PORH peak was significantly lower at the fluconazole site, but not at the l-NMMA or combination site, compared with the placebo site. In addition to sensory nerves cytochrome epoxygenase metabolites, putatively epoxyeicosatrienoic acids, play a major role in healthy skin PORH, their role being more important in the time course rather than the peak.

Topics: 8,11,14-Eicosatrienoic Acid; Adult; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Female; Fluconazole; Humans; Hyperemia; Lidocaine; Male; NG-Nitroarginine Methyl Ester; Nitroprusside; Regional Blood Flow; Sensory Receptor Cells; Skin; Skin Diseases

Epoxyeicosatrienoic acids (EETs) are epoxy fatty acids that have biological actions that are essential for maintaining water and electrolyte homeostasis. An inability to increase EETs in response to a high-salt diet results in salt-sensitive hypertension. Vasodilation, inhibition of epithelial sodium channel, and inhibition of inflammation are the major EET actions that are beneficial to the heart, resistance arteries, and kidneys. Genetic and pharmacological means to elevate EETs demonstrated antihypertensive, anti-inflammatory, and organ protective actions. Therapeutic approaches to increase EETs were then developed for cardiovascular diseases. sEH (soluble epoxide hydrolase) inhibitors were developed and progressed to clinical trials for hypertension, diabetes mellitus, and other diseases. EET analogs were another therapeutic approach taken and these drugs are entering the early phases of clinical development. Even with the promise for these therapeutic approaches, there are still several challenges, unexplored areas, and opportunities for epoxy fatty acids.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Cardiovascular Diseases; Cytochrome P-450 Enzyme System; Disease Models, Animal; Epoxide Hydrolases; Forecasting; Humans; Hypertension; Kidney; Kidney Diseases; Mice; Natriuresis; Potassium; Rats; Rats, Inbred Dahl; Sodium Chloride; Sodium Chloride, Dietary; Vasodilation; Water-Electrolyte Balance; Water-Electrolyte Imbalance

Dronedarone and amiodarone are structurally similar antiarrhythmic drugs. Dronedarone worsens cardiac adverse effects with unknown causes while amiodarone has no cardiac adversity. Dronedarone induces preclinical mitochondrial toxicity in rat liver and exhibits clinical hepatotoxicity. Here, we further investigated the relative potential of the antiarrhythmic drugs in causing mitochondrial injury in cardiomyocytes. Differentiated rat H9c2 cardiomyocytes were treated with dronedarone, amiodarone, and their respective metabolites namely N-desbutyldronedarone (NDBD) and N-desethylamiodarone (NDEA). Intracellular ATP content, mitochondrial membrane potential (Δψm), and inhibition of carnitine palmitoyltransferase I (CPT1) activity and arachidonic acid (AA) metabolism were measured in H9c2 cells. Inhibition of electron transport chain (ETC) activities and uncoupling of ETC were further studied in isolated rat heart mitochondria. Dronedarone, amiodarone, NDBD and NDEA decreased intracellular ATP content significantly (IC50 = 0.49, 1.84, 1.07, and 0.63 µM, respectively) and dissipated Δψm potently (IC50 = 0.5, 2.94, 12.8, and 7.38 µM, respectively). Dronedarone, NDBD, and NDEA weakly inhibited CPT1 activity while amiodarone (IC50 > 100 µM) yielded negligible inhibition. Only dronedarone inhibited AA metabolism to its regioisomeric epoxyeicosatrienoic acids (EETs) consistently and potently. NADH-supplemented ETC activity was inhibited by dronedarone, amiodarone, NDBD and NDEA (IC50 = 3.07, 5.24, 11.94, and 16.16 µM, respectively). Cytotoxicity, ATP decrease and Δψm disruption were ameliorated via exogenous pre-treatment of H9c2 cells with 11, 12-EET and 14, 15-EET. Our study confirmed that dronedarone causes mitochondrial injury in cardiomyocytes by perturbing Δψm, inhibiting mitochondrial complex I, uncoupling ETC and dysregulating AA-EET metabolism. We postulate that cardiac mitochondrial injury is one potential contributing factor to dronedarone-induced cardiac failure exacerbation.

Topics: 8,11,14-Eicosatrienoic Acid; Adenosine Triphosphate; Anti-Arrhythmia Agents; Cardiotonic Agents; Cell Line; Cell Survival; Dronedarone; Humans; Membrane Potential, Mitochondrial; Mitochondria, Heart; Myocytes, Cardiac

Cytochrome P450 (CYP) 2C9, CYP2C8 and CYP2J2 enzymes, which metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids, have cardioprotective effects including anti-inflammation and vasodilation. We have recently shown that some angiotensin II receptor blockers (ARBs) may inhibit AA metabolism via CYP2C8. Using recombinant CYP2C9, CYP2J2 and human liver microsomes (HLMs), the aim was now to compare the ability of six different clinically used ARBs to inhibit AA metabolism in vitro. The rank order of the ARBs for the 50% inhibitory concentration (IC

Topics: 8,11,14-Eicosatrienoic Acid; Angiotensin II Type 1 Receptor Blockers; Arachidonic Acid; Cytochrome P-450 CYP2C9; Cytochrome P-450 CYP2C9 Inhibitors; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Drug Interactions; Humans; Kinetics; Liver; Microsomes, Liver; Recombinant Proteins

Arachidonic acid (ARA) is metabolized by cyclooxygenase (COX) and cytochrome P450 to produce proangiogenic metabolites. Specifically, epoxyeicosatrienoic acids (EETs) produced from the P450 pathway are angiogenic, inducing cancer tumor growth. A previous study showed that inhibiting soluble epoxide hydrolase (sEH) increased EET concentration and mildly promoted tumor growth. However, inhibiting both sEH and COX led to a dramatic decrease in tumor growth, suggesting that the contribution of EETs to angiogenesis and subsequent tumor growth may be attributed to downstream metabolites formed by COX. This study explores the fate of EETs with COX, the angiogenic activity of the primary metabolites formed, and their subsequent hydrolysis by sEH and microsomal EH. Three EET regioisomers were found to be substrates for COX, based on oxygen consumption and product formation. EET substrate preference for both COX-1 and COX-2 were estimated as 8,9-EET > 5,6-EET > 11,12-EET, whereas 14,15-EET was inactive. The structure of two major products formed from 8,9-EET in this COX pathway were confirmed by chemical synthesis:

Topics: 8,11,14-Eicosatrienoic Acid; Angiogenesis Inducing Agents; Arachidonic Acid; Cyclooxygenase 1; Cyclooxygenase 2; Humans

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid (AA) oxidation that have important cardioprotective and signaling properties. AA is an ω-6 polyunsaturated fatty acid (PUFA) that is prone to autoxidation. Although hydroperoxides and isoprostanes are major autoxidation products of AA, EETs are also formed from the largely overlooked peroxyl radical addition mechanism. While autoxidation yields both cis- and trans-EETs, cytochrome P450 (CYP) epoxygenases have been shown to exclusively catalyze the formation of all regioisomer cis-EETs, on each of the double bonds. In plasma and red blood cell (RBC) membranes, cis- and trans-EETs have been observed, and both have multiple physiological functions. We developed a sensitive ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay that separates cis- and trans- isomers of EETs and applied it to determine the relative distribution of cis- vs. trans-EETs in reaction mixtures of AA subjected to free radical oxidation in benzene and liposomes in vitro. We also determined the in vivo distribution of EETs in several tissues, including human and mouse heart, and RBC membranes. We then measured EET levels in heart and RBC of young mice compared to old. Formation of EETs in free radical reactions of AA in benzene and in liposomes exhibited time- and AA concentration-dependent increase and trans-EET levels were higher than cis-EETs under both conditions. In contrast, cis-EET levels were overall higher in biological samples. In general, trans-EETs increased with mouse age more than cis-EETs. We propose a mechanism for the non-enzymatic formation of cis- and trans-EETs involving addition of the peroxyl radical to one of AA's double bonds followed by bond rotation and intramolecular homolytic substitution (S

Topics: 8,11,14-Eicosatrienoic Acid; Aging; Animals; Arachidonic Acid; Benzene; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Erythrocyte Membrane; Female; Humans; Liposomes; Male; Mice; Mice, Inbred C57BL; Myocardium; Oxidation-Reduction; Peroxides; Stereoisomerism; Tandem Mass Spectrometry

The biological role of epoxyeicosatrienoic acids (EETs) in the regulation of pulmonary circulation is currently under debate. We hypothesized that EETs initiate increases in right ventricular systolic pressure (RVSP) via perhaps, pulmonary vasoconstriction.. Mice were anesthetized with isoflurane. Three catheters, inserted into the left jugular vein, the left carotid artery, and the right jugular vein, were used for infusing EETs, monitoring blood pressure (BP), and RVSP respectively. BP and RVSP were continuously recorded at basal conditions, in response to administration of 4 regioisomeric EETs (5,6-EET; 8,9-EET; 11,12-EET, and 14,15-EET; 1, 2, 5 and 10 ng/g body weight (BW) for each EET), and during exposure of mice to hypoxia.. All 4 EETs initiated dose-dependent increases in RVSP, though reduced BP. 11,12-EET elicited the greatest increment in RVSP among all EET isoforms. To clarify the direct elevation of RVSP in a systemic BP-independent manner, equivalent amounts of 14,15-EET were injected over 1 and 2 minutes respectively. One-minute injection of 14,15-EET elicited significantly faster and greater increases in RVSP than the 2-minute injection, whereas their BP changes were comparable. Additionally, direct injection of low doses of 14,15-EET (0.1, 0.2, 0.5, and 1 ng/g BW) into the right ventricle caused significant increases in RVSP without effects on BP, confirming that systemic vasodilation-induced increases in venous return are not the main cause for the increased RVSP. Acute exposure of mice to hypoxia significantly elevated RVSP, as well as 14,15-EET-induced increases in RVSP.. EETs directly elevate RVSP, a response that may play an important role in the development of hypoxia-induced pulmonary hypertension (PH).

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arterial Pressure; Disease Models, Animal; Dose-Response Relationship, Drug; Hypertension, Pulmonary; Hypoxia; Infusions, Intravenous; Male; Mice, Inbred C57BL; Pulmonary Artery; Time Factors; Ventricular Function, Right; Ventricular Pressure

Epoxyeicosatrienoic acids (EETs) are potent lipid mediators formed by cytochrome P450 epoxygenases from arachidonic acid. They consist of four regioisomers of cis-epoxyeicosatrienoic acids: 5,6-, 8,9-, 11,12- and 14,15-EET. Here we investigated whether these triene epoxides are electrophilic enough to form covalent adducts with DNA in vitro. Using the thin-layer chromatography (TLC) (32)P-postlabelling method for adduct detection we studied the reaction of individual deoxynucleoside 3'-monophosphates and calf thymus DNA with the four racemic EETs. Under physiological conditions (pH 7.4) only ±11,12-EET11,12-EET formed adducts with DNA in a dose dependent manner detectable by the (32)P-postlabelling method. However, when pre-incubated at pH 4 all four racemic EETs were capable to bind to DNA forming several adducts. Under these conditions highest DNA adduct levels were found with ±11,12-EET followed by ±5,6-EET, ±8,9-EET, and ±14,15-EET, all of them two orders of magnitude higher (between 3 and 1 adducts per 10(5) normal nucleotides) than those obtained with ±11,12-EET at pH 7.4. Similar DNA adduct patterns consisting of up to seven spots were observed with all four racemic EETs the most abundant adducts being derived from the reaction with deoxyguanosine and deoxyadenosine. In summary, when analysed by the (32)P-postlabelling method all four racemic EETs formed multiple DNA adducts after activation by acidic pH, only ±11,12-EET produced DNA adducts in aqueous solution at neutral pH. Therefore, we conclude from our in vitro studies that EETs might be endogenous genotoxic compounds.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Cattle; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; DNA; DNA Adducts; Hydrogen-Ion Concentration; Kinetics; Phosphorus Radioisotopes; Solutions; Stereoisomerism

Prolonged endoplasmic reticulum (ER) stress causes apoptosis and is associated with heart failure. Whether CYP2J2 and its arachidonic acid metabolites [epoxyeicosatrienoic acids (EETs)] have a protective influence on ER stress and heart failure has not been studied. Assays of myocardial samples from patients with end-stage heart failure showed evidence of ER stress. Chronic infusion of isoproterenol (ISO) or angiotensin II (AngII) by osmotic mini-pump induced cardiac hypertrophy and heart failure in mice as evaluated by hemodynamic measurements and echocardiography. Interestingly, transgenic (Tr) mice with cardiomyocyte-specific CYP2J2 expression were protected against heart failure compared with wild-type mice. ISO or AngII administration induced ER stress and apoptosis, and increased levels of intracellular Ca(2+). These phenotypes were abolished by CYP2J2 overexpression in vivo or exogenous EETs treatment of cardiomyocytes in vitro. ISO or AngII reduced sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a) expression in hearts or isolated cardiomyocytes; however, loss of SERCA2a expression was prevented in CYP2J2 Tr hearts in vivo or in cardiomyocytes treated with EETs in vitro. The reduction of SERCA2a activity was concomitant with increased oxidation of SERCA2a. EETs reversed SERCA2a oxidation through increased expression of antioxidant enzymes and reduced reactive oxygen species levels. Tempol, a membrane-permeable radical scavenger, similarly decreased oxidized SERCA2a levels, restored SERCA2a activity, and markedly reduced ER stress response in the mice treated with ISO. In conclusion, CYP2J2-derived EETs suppress ER stress response in the heart and protect against cardiac failure by maintaining intracellular Ca(2+) homeostasis and SERCA2a expression and activity.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Apoptosis; Calcium; Cardiomegaly; Cell Line; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Endoplasmic Reticulum Stress; Heart Failure; Humans; In Vitro Techniques; Major Histocompatibility Complex; Mice; Mice, Transgenic; Myocardium; Myocytes, Cardiac; Rats; Reactive Oxygen Species; Sarcoplasmic Reticulum Calcium-Transporting ATPases

It has been previously shown that the cytochrome P450 (P450) modulator, fenofibrate, protects against cardiovascular diseases. P450 and their metabolites, epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) were found to play an important role in cardiovascular diseases. Therefore, it is important to examine whether fenofibrate would modulate the cardiac P450 and its associated arachidonic acid metabolites and whether this modulation protects against isoproterenol-induced cardiac hypertrophy. For this purpose, male Sprague-Dawley rats were treated with fenofibrate (30 mg·kg·d), isoproterenol (4.2 mg·kg·d), or the combination of both. The expression of hypertrophic markers and different P450s along with their metabolites was determined. Our results showed that fenofibrate significantly induced the cardiac P450 epoxygenases, such as CYP2B1, CYP2B2, CYP2C11, and CYP2C23, whereas it decreased the cardiac ω-hydroxylase, CYP4A3. Moreover, fenofibrate significantly increased the formation of 14,15-EET, 11,12-EET, and 8,9-EET, whereas it decreased the formation of 20-HETE in the heart. Furthermore, fenofibrate significantly decreased the hypertrophic markers and the increase in heart-to-body weight ratio induced by isoproterenol. This study demonstrates that fenofibrate alters the expression of cardiac P450s and their metabolites and partially protects against isoproterenol-induced cardiac hypertrophy, which further confirms the role of P450s, EETs, and 20-HETE in the development of cardiac hypertrophy.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Cardiomegaly; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Fenofibrate; Gene Expression Regulation, Enzymologic; Hydroxyeicosatetraenoic Acids; Hypolipidemic Agents; Isoproterenol; Male; Rats; Rats, Sprague-Dawley

The environmental toxin and carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) binds and activates the transcription factor aryl hydrocarbon receptor (AHR), inducing CYP1 family cytochrome P450 enzymes. CYP1A2 and its avian ortholog CYP1A5 are highly active arachidonic acid epoxygenases. Epoxygenases metabolize arachidonic acid to four regioisomeric epoxyeicosatrienoic acids (EETs) and selected monohydroxyeicosatetraenoic acids (HETEs). EETs can be further metabolized by epoxide hydrolases to dihydroxyeicosatrienoic acids (DHETs). As P450-arachidonic acid metabolites affect vasoregulation, responses to ischemia, inflammation, and metabolic disorders, identification of their production in vivo is needed to understand their contribution to biologic effects of TCDD and other AHR activators. Here we report use of an acetonitrile-based extraction procedure that markedly increased the yield of arachidonic acid products by lipidomic analysis over a standard solid-phase extraction protocol. We show that TCDD increased all four EETs (5,6-, 8,9-, 11,12-, and 14,15-), their corresponding DHETs, and 18- and 20-HETE in liver in vivo and increased 5,6-EET, the four DHETs, and 18-HETE in heart, in a chick embryo model. As the chick embryo heart lacks arachidonic acid-metabolizing activity, the latter findings suggest that arachidonic acid metabolites may travel from their site of production to a distal organ, i.e., heart. To determine if the TCDD-arachidonic acid-metabolite profile could be altered pharmacologically, chick embryos were treated with TCDD and the soluble epoxide hydrolase inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA). Cotreatment with AUDA increased hepatic EET-to-DHET ratios, indicating that the in vivo profile of P450-arachidonic acid metabolites can be modified for potential therapeutic intervention.

Topics: 8,11,14-Eicosatrienoic Acid; Adamantane; Animals; Chick Embryo; Enzyme Inhibitors; Epoxide Hydrolases; Gene Expression Regulation, Enzymologic; Heart; Hydroxyeicosatetraenoic Acids; Lauric Acids; Liver; Polychlorinated Dibenzodioxins; RNA, Messenger

Fullerenes are molecules being investigated for a wide range of therapeutic applications. We have shown previously that certain fullerene derivatives (FDs) inhibit mast cell (MC) function in vitro, and here we examine their in vivo therapeutic effect on asthma, a disease in which MCs play a predominant role.. We sought to determine whether an efficient MC-stabilizing FD (C(70)-tetraglycolate [TGA]) can inhibit asthma pathogenesis in vivo and to examine its in vivo mechanism of action.. Asthma was induced in mice, and animals were treated intranasally with TGA either simultaneously with treatment or after induction of pathogenesis. The efficacy of TGA was determined through the measurement of airway inflammation, bronchoconstriction, serum IgE levels, and bronchoalveolar lavage fluid cytokine and eicosanoid levels.. We found that TGA-treated mice have significantly reduced airway inflammation, eosinophilia, and bronchoconstriction. The TGA treatments are effective, even when given after disease is established. Moreover, we report a novel inhibitory mechanism because TGA stimulates the production of an anti-inflammatory P-450 eicosanoid metabolites (cis-epoxyeicosatrienoic acids [EETs]) in the lung. Inhibitors of these anti-inflammatory EETs reversed TGA inhibition. In human lung MCs incubated with TGA, there was a significant upregulation of CYP1B gene expression, and TGA also reduced IgE production from B cells. Lastly, MCs incubated with EET and challenged through FcεRI had a significant blunting of mediator release compared with nontreated cells.. The inhibitory capabilities of TGA reported here suggest that FDs might be used a platform for developing treatments for asthma.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Asthma; Bronchoconstriction; Eosinophilia; Female; Fullerenes; Immunoglobulin E; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL

Epoxyeicosatrienoic acids (EETs) are vasodilator, natriuretic, and antiinflammatory lipid mediators. Both cis- and trans-EETs are stored in phospholipids and in red blood cells (RBCs) in the circulation; the maximal velocity (V(max)) of trans-EET hydrolysis by soluble epoxide hydrolase (sEH) is threefold that of cis-EETs. Because RBCs of the spontaneously hypertensive rat (SHR) exhibit increased sEH activity, a deficiency of trans-EETs in the SHR was hypothesized to increase blood pressure (BP). This prediction was fulfilled, since sEH inhibition with cis-4-[4-(3-adamantan-1-ylureido)cyclohexyloxy]benzoic acid (AUCB; 2 mg·kg(-1)·day(-1) for 7 days) in the SHR reduced mean BP from 176 ± 8 to 153 ± 5 mmHg (P < 0.05), whereas BP in the control Wistar-Kyoto rat (WKY) was unaffected. Plasma levels of EETs in the SHR were lower than in the age-matched control WKY (16.4 ± 1.6 vs. 26.1 ± 1.8 ng/ml; P < 0.05). The decrease in BP in the SHR treated with AUCB was associated with an increase in plasma EETs, which was mostly accounted for by increasing trans-EET from 4.1 ± 0.2 to 7.9 ± 1.5 ng/ml (P < 0.05). Consistent with the effect of increased plasma trans-EETs and reduced BP in the SHR, the 14,15-trans-EET was more potent (ED(50) 10(-10) M; maximum dilation 59 ± 15 μm) than the cis-isomer (ED(50) 10(-9) M; maximum dilation 30 ± 11 μm) in relaxing rat preconstricted arcuate arteries. The 11,12-EET cis- and trans-isomers were equipotent dilators as were the 8,9-EET isomers. In summary, inhibition of sEH resulted in a twofold increase in plasma trans-EETs and reduced mean BP in the SHR. The greater vasodilator potency of trans- vs. cis-EETs may contribute to the antihypertensive effects of sEH inhibitors.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acids; Benzoic Acid; Blood Pressure; Disease Models, Animal; Epoxide Hydrolases; Erythrocytes; Hypertension; Male; Rats; Rats, Inbred SHR; Rats, Inbred WKY

Sodium reabsorption via the epithelial Na(+) channel (ENaC) in the aldosterone-sensitive distal nephron plays a central role in the regulation of body fluid volume. Previous studies have indicated that arachidonic acid (AA) and its metabolite 11,12-EET but not other regioisomers of EETs inhibit ENaC activity in the collecting duct. The goal of this study was to investigate the endogenous metabolism of AA in cultured mpkCCD(c14) principal cells and the effects of these metabolites on ENaC activity. Liquid chromatography/mass spectrometry analysis of the mpkCCD(c14) cells indicated that these cells produce prostaglandins, 8,9-EET, 11,12-EET, 14,15-EET, 5-HETE, 12/8-HETE, and 15-HETE, but not 20-HETE. Single-channel patch-clamp experiments revealed that 8,9-EET, 14,15-EET, and 11,12-EET all decrease ENaC activity. Neither 5-, 12-, nor 15-HETE had any effect on ENaC activity. Diclofenac and ibuprofen, inhibitors of cyclooxygenase, decreased transepithelial Na(+) transport in the mpkCCD(c14) cells. Inhibition of cytochrome P-450 (CYP450) with MS-PPOH activated ENaC-mediated sodium transport when cells were pretreated with AA and diclofenac. Coexpression of CYP2C8, but not CYP4A10, with ENaC in Chinese hamster ovary cells significantly decreased ENaC activity in whole-cell experiments, whereas 11,12-EET mimicked this effect. Thus both endogenously formed EETs and their exogenous application decrease ENaC activity. Downregulation of ENaC activity by overexpression of CYP2C8 was PKA dependent and was prevented by myristoylated PKI treatment. Biotinylation experiments and single-channel analysis revealed that long-term treatment with 11,12-EET and overexpression of CYP2C8 decreased the number of channels in the membrane. In contrast, the acute inhibitory effects are mediated by a decrease in the open probability of the ENaC. We conclude that 11,12-EET, 8,9-EET, and 14,15-EET are endogenously formed eicosanoids that modulate ENaC activity in the collecting duct.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Cell Line; CHO Cells; Cricetinae; Cricetulus; Cytochrome P-450 Enzyme System; Diclofenac; Epithelial Sodium Channels; Female; Ibuprofen; Kidney Tubules, Collecting; Mice; Models, Animal; Ovary; Patch-Clamp Techniques; Transfection

Epoxyeicosatrienoic acids (EETs) are generated from arachidomic acid by cytochrome P450(CYP). Previous studies revealed very strong and selective expression of CYP expoxygenase in human cancer tissues, but almost none in adjacent normal tissues. This study was to investigate the promotive effect of EETs on proliferation of tumor cells and the possible mechanisms.. Four tumor cell lines, Tca-8113, A549, Ncl-H446 and HepG2, were treated with different concentrations of EETs (8,9-EET, 11,12-EET and 14,15-EET) for 12, 24, 48 and 72 h, respectively. Cell proliferation was measured using the MTT assay. The effect of exogenous EETs on cell cycle of Tca-8113 cells was assessed by flow cytometry. Signal transduction inhibitors of PI3K (LY294002), MAPKK (PD98059), MAPK (apigenin) and PKC (H7) were used to block EETs-induced cell proliferation. Expressions of the total protein and phosphorylated ERK1/2 and Akt were determined by Western blot.. EETs promoted proliferation of tumor cells compared with the control and vehicle group in a dose-and time-dependent manner (P<0.01). Incubation of tumor cells with EETs markedly increased the cell number at S/G2-M phase. The percentages of Tca-8113 cells at S and G2-M phases were (49.7+/-7.5%) vs. (17.2+/-9.7%) (P<0.01) and (21.0+/-5.3%) vs. (4.9+/-7.3%), respectively(P<0.01) with and without the treatment of 11,12-EET. EETs incubation significantly enhanced phosphorylation of MARK as well as PI3K/Akt in tumor cells. LY294002, PD98059, apigenine and H7 reduced the stimulative effect of EETs on cell proliferation.. EETs possess the promotive effect on proliferation of tumor cells via activation of MAPK and PI3K/Akt signal pathways.

Topics: 8,11,14-Eicosatrienoic Acid; Apigenin; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Chromones; Dose-Response Relationship, Drug; Flavonoids; Humans; Mitogen-Activated Protein Kinase Kinases; Morpholines; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction

Endothelial nitric oxide synthase (eNOS) is a key enzyme in NO-mediated cardiovascular homeostasis and its activity is modulated by a variety of hormonal and mechanical stimuli via phosphorylation modification. Our previous study has demonstrated that epoxyeicosatrienoic acids (EETs), the cytochrome P450 (CYP)-dependent metabolites of arachidonic acid, could robustly up-regulate eNOS expression. However, the molecular mechanism underlying the effects of EETs on eNOS remains elusive. Particularly, whether and how EETs affect eNOS phosphorylation is unknown. In the present study, we investigated the effects of EETs on eNOS phosphorylation with cultured bovine aortic endothelial cells (BAECs). BAECs were either treated with exogenous EETs or infected with recombinant adeno-associated virus (rAAV) carrying CYP2C11-CYPOR, CYP102 F87V mutant and CYP2J2, respectively, to increase endogenous EETs. Both addition of EETs and CYP epoxygenase transfection markedly increased eNOS phosphorylation at its Ser1179 and Thr497 residues. Inhibition of phosphatidylinositol 3-kinase (PI3K) with LY294002 prevented EETs-induced increases of eNOS-Ser(P)1179 but had no effect on the phosphorylation status of Thr497. However, inhibitors of protein kinase B (Akt), mitogen-activated protein kinase (MAPK) and MAPK kinase could block phosphorylation of eNOS at both sites. Inhibition of these kinases also attenuated the up-regulation of eNOS expression by EETs. Finally, administration of viral CYP epoxygenases expression vectors into rats enhanced eNOS phosphorylation and function in vivo. Thus, in addition to up-regulating eNOS expression, EETs also augment eNOS function by enhancing eNOS phosphorylation. EETs-induced up-regulation of eNOS phosphorylation and expression appears to involve in both PI3K/Akt and MAPK pathways.

Topics: 8,11,14-Eicosatrienoic Acid; Amino Acid Sequence; Animals; Aorta, Thoracic; Apigenin; Cattle; Cells, Cultured; Chromones; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Epoxy Compounds; Flavonoids; Inositol; Male; MAP Kinase Signaling System; Morpholines; Nitric Oxide Synthase Type III; Oxygenases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Phosphothreonine; Proto-Oncogene Proteins c-akt; Rats; Serine

Cytochrome P450 (CYP) epoxygenases and their arachidonic acid (AA) metabolites, the epoxyeicosatrienoic acids (EETs), have been shown to produce reductions in infarct size in canine myocardium following ischemia-reperfusion injury via opening of either the sarcolemmal K(ATP) (sarcK(ATP)) or mitochondrial K(ATP) (mitoK(ATP)) channel. In the present study, we subjected intact rat hearts to 30 min of left coronary artery occlusion and 2 h of reperfusion followed by tetrazolium staining to determine infarct size as a percent of the area at risk (IS/AAR, %). The results demonstrate that the two major regioisomers of the CYP epoxygenase pathway, 11,12-EET (2.5 mg/kg, iv) and 14,15-EET (2.5 mg/kg, iv) significantly reduced myocardial infarct size (IS/AAR, %) in rats as compared with control (41.9+/-2.3%, 40.9+/-1.2% versus 61.5+/-1.6%, respectively), whereas, a third regioisomer, 8,9-EET (2.5 mg/kg, iv) had no effect (55.2+/-1.4). The protective effect of pretreatment with 11,12- and 14,15-EETs was completely abolished (61.9+/-0.7%, 58.6+/-3.1%, HMR; 63.3+/-1.2%, 63.2+/-2.5%, 5-HD) in the presence of the selective sarcK(ATP) channel antagonist, HMR 1098 (6 mg/kg, iv) or the selective mitoK(ATP) channel antagonist, 5-HD (10 mg/kg, iv) given 10 min after 11,12- or 14,15-EET administration but 5 min prior to index ischemia. Furthermore, concomitant pretreatment with 11,12- or 14,15-EET in combination with the free radical scavenger, 2-mercaptopropionyl glycine (2-MPG), at a dose (20 mg/kg, iv) that had no effect on IS/AAR (57.7+/-1.3%), completely abolished the cardioprotective effect of 11,12- and 14,15-EETs (58.2+/-1.6%, 61.4+/-1.0%), respectively. These data suggest that part of the cardioprotective effects of EETs in rat hearts against infarction is the result of an initial burst of reactive oxygen species (ROS) and subsequent activation of both the sarcK(ATP) and mitoK(ATP) channel.

Topics: 8,11,14-Eicosatrienoic Acid; Adenosine Triphosphate; Animals; Male; Myocardial Infarction; Potassium Channels; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Tiopronin

An HPLC method for the chiral analysis of the four regioisomeric epoxyeicosatrienoic acids (EETs) is described. The cytochrome P450 arachidonic acid epoxygenase metabolites are resolved, without the need for derivatization, by chiral-phase HPLC on a Chiralcel OJ column. Application of this methodology to the analysis of the liver endogenous EETs demonstrates stereospecific biosynthesis and corroborates the role of cytochrome P450 as the endogenous arachidonic acid epoxygenase.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Liver; Male; Microsomes, Liver; Oxygenases; Rats; Rats, Sprague-Dawley; Stereoisomerism; Vasodilator Agents

11,12-Epoxyeicosatrienoic acid (11,12-EET), a potent vasodilator produced by the endothelium, acts on calcium-activated potassium channels and shares biological activities with the heme oxygenase/carbon monoxide (HO/CO) system. We examined whether activation of HO mediates the dilator action of 11,12-EET, and that of the other EETs, on rat mesenteric arteries. Dose-response curves (10(-9) to 10(-6) M) to 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, and ACh (10(-9) to 10(-4) M) were evaluated in preconstricted (10(-6) mol/l phenylephrine) mesenteric arteries (<350 microm diameter) in the presence or absence of 1) the cyclooxygenase inhibitor indomethacin (2.8 microM), 2) the HO inhibitor chromium mesoporphyrin (CrMP) (15 microM), 3) the soluble guanylyl cyclase (GC) inhibitor ODQ (10 microM), and 4) the calcium-activated potassium channel inhibitor iberiotoxin (25 nM). The vasodilator response to 11,12-EET was abolished by CrMP and iberiotoxin, whereas indomethacin and ODQ had no effect. In contrast, the effect of ACh was attenuated by ODQ but not by CrMP. The vasodilator effect of 8,9-EET, like that of 11,12-EET, was greatly attenuated by HO inhibition. In contrast, the mesenteric vasodilator response to 5,6-EET was independent of both HO and GC, whereas that to 14,15-EET demonstrated two components, an HO and a GC, of equal magnitude. Incubation of mesenteric microvessels with 11,12-EET caused a 30% increase in CO release, an effect abolished by inhibition of HO. We conclude that the rat mesenteric vasodilator action of 11,12-EET is mediated via an increase in HO activity and an activation of calcium-activated potassium channels.

Topics: 8,11,14-Eicosatrienoic Acid; Acetylcholine; Animals; Carbon Monoxide; Dose-Response Relationship, Drug; Heme Oxygenase (Decyclizing); Male; Mesenteric Arteries; Mesoporphyrins; Organometallic Compounds; Oxadiazoles; Peptides; Potassium Channels, Calcium-Activated; Quinoxalines; Rats; Rats, Wistar; Vasodilation; Vasodilator Agents

Epoxyeicosatrienoic acids (EETs), the cytochrome P450 metabolites of arachidonic acid (AA), are potent and stereospecific activators of cardiac ATP-sensitive K(+)(K(ATP)) channels. EETs activate K(ATP) channels by reducing channel sensitivity to ATP. In this study, we determined the direct effects of EETs on the binding of ATP to K(ATP) channel protein. A fluorescent ATP analog, 2,4,6-trinitrophenyl (TNP)-ATP, which increases its fluorescence emission significantly upon binding with proteins, was used for binding studies with glutathione-S-transferase (GST) Kir6.2 fusion proteins. TNP-ATP bound to GST fusion protein containing the C-terminus of Kir6.2 (GST-Kir6.2C), but not to the N-terminus of Kir6.2, or to GST alone. 11,12-EET (5 muM) did not change TNP-ATP binding K(D) to GST-Kir6.2C, but B(max) was reduced by half. The effect of 11,12-EET was dose-dependent, and 8,9- and 14,15-EETs were as effective as 11,12-EET in inhibiting TNP-ATP binding to GST-Kir6.2C. AA and 11,12-dihydroxyeicosatrienoic acid (11,12-DHET), the parent compound and metabolite of 11,12-EET, respectively, were not effective inhibitors of TNP-ATP binding to GST-Kir6.2C, whereas the methyl ester of 11,12-EET was. These findings suggest that the epoxide group in EETs is important for modulation of ATP binding to Kir6.2. We conclude that EETs bind to the C-terminus of K(ATP) channels, inhibiting binding of ATP to the channel.

Topics: 8,11,14-Eicosatrienoic Acid; Adenosine Triphosphate; Animals; ATP-Binding Cassette Transporters; Cell Line; Glutathione Transferase; Humans; Ion Channel Gating; Mice; Potassium Channels, Inwardly Rectifying; Protein Binding; Receptors, Drug; Recombinant Fusion Proteins; Sulfonylurea Receptors

Epoxyeicosatrienoic acids (EETs), the cytochrome P-450 epoxygenase metabolites of arachidonic acid, are candidates of endothelium-derived hyperpolarizing factors. We have previously reported that EETs are potent activators of cardiac ATP-sensitive K(+) (K(ATP)) channels, but their effects on the vascular K(ATP) channels are unknown. With the use of whole cell patch-clamp techniques with 0.1 mM ATP in the pipette and holding at -60 mV, freshly isolated smooth muscle cells from rat mesenteric arteries had small glibenclamide-sensitive currents at baseline (13.1 +/- 3.9 pA, n = 5) that showed a 7.2-fold activation by 10 microM pinacidil (94.1 +/- 21.9 pA, n = 7, P < 0.05). 11,12-EET dose dependently activated the K(ATP) current with an apparent EC(50) of 87 nM. Activation of the K(ATP) channels by 500 nM 11,12-EET was inhibited by inclusion of the PKA inhibitor peptide (5 microM) but not by the inclusion of the PKC inhibitor peptide (100 microM) in the pipette solution. These results were corroborated by vasoreactivity studies. 11,12-EET produced dose-dependent vasorelaxation in isolated small mesenteric arteries, and this effect was reduced by 50% with glibenclamide (1 microM) preincubation. The 11,12-EET effects on vasorelaxation were also significantly attenuated by preincubation with cell-permeant PKA inhibitor myristoylated PKI(14-22), and, in the presence of PKA inhibitor, glibenclamide had no additional effects. These results suggest that 11,12-EET is a potent activator of the vascular K(ATP) channels, and its effects are dependent on PKA activities.

Topics: 8,11,14-Eicosatrienoic Acid; Adenosine Triphosphate; Animals; Cyclic AMP-Dependent Protein Kinases; In Vitro Techniques; Mesenteric Arteries; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Potassium Channels; Rats; Vasodilation; Vasodilator Agents

We have previously reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolites of arachidonic acid, are potent stereospecific activators of the cardiac K(ATP) channel. The epoxide group in EET is critical for reducing channel sensitivity to ATP, thereby activating the channel. This study is to identify the molecular sites on the K(ATP) channels for EET-mediated activation. We investigated the effects of EETs on Kir6.2delta C26 with or without the coexpression of SUR2A and on Kir6.2 mutants of positively charged residues known to affect channel activity coexpressed with SUR2A in HEK293 cells. The ATP IC50 values were significantly increased in Kir6.2 R27A, R50A, K185A, and R201A but not in R16A, K47A, R54A, K67A, R192A, R195A, K207A, K222A, and R314A mutants. Similar to native cardiac K(ATP) channel, 5 microM 11,12-EET increased the ATP IC50 by 9.6-fold in Kir6.2/SUR2A wild type and 8.4-fold in Kir6.2delta C26. 8,9- and 14,15-EET regioisomers activated the Kir6.2 channel as potently as 11,12-EET. 8,9- and 11,12-EET failed to change the ATP sensitivity of Kir6.2 K185A, R195A, and R201A, whereas their effects were intact in the other mutants. 14,15-EET had a similar effect with K185A and R201A mutants, but instead of R195A, it failed to activate Kir6.2R192A. These results indicate that activation of Kir6.2 by EETs does not require the SUR2A subunit, and the region in the Kir6.2 C terminus from Lys-185 to Arg-201 plays a critical role in EET-mediated Kir6.2 channel activation. Based on computer modeling of the Kir6.2 structure, we infer that the EET-Kir6.2 interaction may allosterically change the ATP binding site on Kir6.2, reducing the channel sensitivity to ATP.

Topics: 8,11,14-Eicosatrienoic Acid; Adenosine Triphosphate; Allosteric Site; Animals; Arachidonic Acid; ATP-Binding Cassette Transporters; Binding Sites; Cell Line; Electrophysiology; Gene Deletion; Humans; Inhibitory Concentration 50; Mice; Models, Chemical; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Potassium Channels; Potassium Channels, Inwardly Rectifying; Protein Structure, Secondary; Protein Structure, Tertiary; Receptors, Drug; Software; Sulfonylurea Receptors; Vasodilator Agents

Shell-less culture of chick chorioallantoic membrane (CAM) of developing chicken embryos is a useful model to evaluate the effects of vascular agents. We assessed the response of CAM vessels to epoxyeicosatrienoic acids (EETs), derivatives of the essential fatty acid arachidonic acid, that have a number of important biological functions, including dilation of microvessels in the coronary, cerebral, renal, and mesenteric circulations. Three of four regioisomers of EETs, 14,15-, 11,12-, and 8,9-EET, induced a characteristic dose-dependent acute hyperemia within 4 min after application on 10-day-old CAMs. This response was marked in early stages of development (between days 8 and 10), but the frequency and intensity of the response were reduced after 11 days of development. Histological examination demonstrated that the hyperemia was not due to extravasation of erythrocytes. However, many capillaries were distended and contained densely packed erythrocytes as compared to uniformly arranged vessels and erythrocytes in untreated CAMs. Transmission electron microscopy showed the basal laminae surrounding capillaries remained intact, similar to those in vehicle-treated or untreated CAM tissue. The hyperemia was specific to EETs since we did not observe it to be induced by other vasodilators such as nitric oxide or prostacyclin. In conclusion, we report a novel vascular response to EETs using the CAM as an in vivo model. These lipids specifically distend a subset of capillaries in a dose- and development-dependent manner.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Capillaries; Chick Embryo; Chorioallantoic Membrane; Culture Techniques; Disease Models, Animal; Dose-Response Relationship, Drug; Hyperemia; Neovascularization, Physiologic; Nitric Oxide Donors; Time Factors; Vasodilator Agents; Vitelline Membrane

This study tested the hypothesis that epoxyeicosatrienoic acids (EETs) derived from arachidonic acid via P-450 epoxygenases are soluble factors linking depletion of endoplasmic reticulum Ca(2+) stores and store-dependent regulation of endothelial cell (EC) permeability in rat lung. EC permeability was measured via the capillary filtration coefficient (K(f,c)) in isolated, perfused rat lungs. 14,15-EET and 5,6-EET increased EC permeability, a response that was significantly different from that of 8,9-EET, 11,12-EET, and vehicle control. The permeability response to 14,15-EET was not significantly attenuated by the nonspecific Ca(2+) channel blocker Gd(3+) (P = 0.068). In lungs perfused with low [Ca(2+)], 14,15-EET tended to increase EC permeability, although a significant increase in K(f,c) was observed only following Ca(2+) add-back. As positive control, we showed that the 3.7-fold increase in K(f,c) evoked by thapsigargin (TG), a known activator of store depletion-induced Ca(2+) entry, was blocked by both Gd(3+) and low [Ca(2+)] buffer. Nonetheless, the permeability response to TG could not be blocked by the phospholipase A(2) inhibitors mepacrine or methyl arachidonyl fluorophosphonate or the P-450 epoxygenase inhibitors 17-octadecynoic acid or propargyloxyphenyl hexanoic acid. Similarly, combined pretreatment with ibuprofen and dicyclohexylurea to block EET metabolism had no effect on the permeability response to TG. We conclude that EETs have a heterogeneous impact on EC permeability. Despite a requirement for Ca(2+) entry with both TG and 14,15-EET, our data suggest that distinct signaling pathways or heterogeneity in EC responsiveness is responsible for the observed EC injury evoked by EETs and store depletion in the isolated rat lung.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Calcium; Capillary Permeability; Endothelium, Vascular; Lung; Male; Pulmonary Circulation; Rats; Rats, Inbred Strains; Vasodilator Agents

Epoxyeicosatrienoic acids (EETs) are potent vasodilators produced by endothelial cells. In many vessels, they are an endothelium-derived hyperpolarizing factor (EDHF). However, it is unknown whether they act as an EDHF on platelets and whether this has functional consequences.. Flow cytometric measurement of platelet membrane potential using the fluorescent dye DiBac4 showed a resting potential of -58+/-9 mV. Different EET regioisomers hyperpolarized platelets down to -69+/-2 mV, which was prevented by the non-specific potassium channel inhibitor charybdotoxin and by use of a blocker of calcium-activated potassium channels of large conductance (BK(Ca) channels), iberiotoxin. EETs inhibited platelet adhesion to endothelial cells under static and flow conditions. Exposure to EETs inhibited platelet P-selectin expression in response to ADP. Stable overexpression of cytochrome P450 2C9 in EA.hy926 cells (EA.hy2C9 cells) resulted in release of EETs and a factor that hyperpolarized platelets and inhibited their adhesion to endothelial cells. These effects were again inhibited by charybdotoxin and iberiotoxin.. EETs hyperpolarize platelets and inactivate them by inhibiting adhesion molecule expression and platelet adhesion to cultured endothelial cells in a membrane potential-dependent manner. They act as an EDHF on platelets and might be important mediators of the anti-adhesive properties of vascular endothelium.

Topics: 8,11,14-Eicosatrienoic Acid; Apamin; Aryl Hydrocarbon Hydroxylases; Biological Factors; Blood Platelets; Cells, Cultured; Charybdotoxin; Cytochrome P-450 CYP2C9; Endothelial Cells; Endothelium, Vascular; Humans; Hydroxyeicosatetraenoic Acids; Ion Channels; Membrane Potentials; Peptides; Platelet Adhesiveness; Platelet Aggregation; Potassium Channels; Recombinant Fusion Proteins; Transfection; Umbilical Veins

Cytochrome P450 (CYP) epoxygenase products, such as 11,12-epoxyeicosatrienoic acid (EET), stimulate endothelial cell proliferation. We set out to identify the signal transduction cascade linking EET generation to enhanced proliferation and angiogenesis. In human endothelial cells overexpressing CYP 2C9, cell number was increased compared with control cells and was inhibited by the CYP 2C9 inhibitor, sulfaphenazole. CYP 2C9 overexpression was associated with the activation of Akt and an increase in cyclin D1 expression, effects that were abolished by the epidermal growth factor (EGF) receptor inhibitor, AG1478, which also prevented the CYP 2C9-induced increase in cell proliferation. Stimulation of EGF receptor overexpressing cells with 11,12-EET or transfection of these cells with CYP 2C9 enhanced the tyrosine phosphorylation of the EGF receptor. Endothelial tube formation in a fibrin gel was significantly enhanced (6-fold) in CYP 2C9 overexpressing cells and was comparable with the tube formation induced by EGF. In the chick chorioallantoic membrane, 11,12-EET stimulated vessel formation (3.5-fold) and induced vessel convergence, an effect that was abolished by cotreatment with either an EGF receptor-neutralizing antibody or AG1478. These results indicate that CYP 2C9-derived EETs stimulate angiogenesis by a mechanism involving the activation of the EGF receptor.

Topics: 8,11,14-Eicosatrienoic Acid; Allantois; Animals; Aryl Hydrocarbon Hydroxylases; Cell Division; Cell Line; Chick Embryo; Chorion; Cyclin D1; Cytochrome P-450 CYP2C9; Endothelium, Vascular; Enzyme Inhibitors; ErbB Receptors; Humans; Neovascularization, Physiologic; Phosphorylation; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Quinazolines; Receptor Cross-Talk; Tyrphostins

Background and Purpose- Epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 epoxygenation of arachidonic acid. We have previously demonstrated that astrocyte-conditioned medium induced mitogenesis in brain capillary endothelial cells. The goals of the present studies are to further define the mechanism through which this can occur and to confirm that EETs are derived from astrocytes, through which astrocytic activity can regulate cerebral angiogenesis in response to neuronal activation.. Astrocytes and cerebral capillary endothelial cells in primary cultures were cocultured to examine the interaction of the 2 cell types. We used multiple immunohistochemical techniques to characterize the multicellular nature of the capillaries, which is not simply an artifact related to the culture conditions. The mitogenic effect of EETs was determined by (3)H-thymidine incorporation and cell proliferation assay. Endothelial tube formation was examined in vitro and in vivo with the use of a reconstituted basement membrane (Matrigel) assay.. In cocultures of astrocytes and capillary endothelium, we observed morphological changes in both cell types such that each assumed certain physiological characteristics, ie, endothelial networks and astrocytes with "footlike" projections as well as intermittent gap junctions forming within the endothelial cells. EETs from astrocytes as well as synthetic EETs promoted mitogenesis of endothelial cells, a process sensitive to inhibition of tyrosine kinase with genistein. Treatments with exogenous EETs were sufficient for endothelial cells to differentiate into capillary-like structures in culture as well as in vivo in a Matrigel matrix.. The 2 major conclusions from these data are that astrocytes may play an important role in regulating angiogenesis in the brain and that cytochrome P450-derived EETs from astrocytes are mitogenic and angiogenic.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Astrocytes; Brain; Capillaries; Cell Differentiation; Cells, Cultured; Coculture Techniques; Culture Media, Conditioned; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Endothelial Growth Factors; Endothelium, Vascular; Enzyme Inhibitors; Intercellular Signaling Peptides and Proteins; Lymphokines; Mitosis; Neovascularization, Physiologic; Rats; Thymidine; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Topics: 8,11,14-Eicosatrienoic Acid; Antibodies; Arachidonic Acids; Cross Reactions; Enzyme-Linked Immunosorbent Assay; Hydroxylation; Isomerism; Sensitivity and Specificity

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

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Benzimidazoles; Benzopyrans; Bradykinin; Cattle; Coronary Vessels; Dihydropyridines; Dose-Response Relationship, Drug; Endothelium, Vascular; Iloprost; In Vitro Techniques; Kidney Cortex; Male; Microsomes; Muscle, Smooth, Vascular; Nitroprusside; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship; Vasoconstriction; Vasoconstrictor Agents; Vasodilation

We tested the hypothesis that cyclooxygenase-independent vasodilation produced by arachidonic acid (AA) is mediated by epoxyeicosatrienoic acids (EETs) and is blunted in the spontaneously hypertensive rat (SHR). At normal perfusion pressure (PP; 70 to 90 mm Hg), AA constricted the renal vasculature in both SHR and normotensive Wistar-Kyoto rats, an effect abolished by cyclooxygenase inhibition, and converted to vasodilation when PP was raised to approximately 200 mm Hg. Unexpectedly, renal vasodilation elicited by AA was greater in the SHR at high PP; for example, 2.5, 5, and 10 microg of AA produced PP declines of 54+/-9, 92+/-10, and 112+/-5 mm Hg, respectively, in SHR compared with 26+/-3, 45+/-5, and 77+/-6 mm Hg in Wistar-Kyoto rats (P:<0.01). However, the renal vasodilator responses to acetylcholine (0.1 microg) and sodium nitroprusside (1 microg) did not differ between strains, indicating that vascular responsiveness to AA was independent of intrinsic changes in vascular smooth muscle. Hyperresponsiveness of the renal vasculature to AA may be unique for the SHR, because it did not occur in Sprague-Dawley rats with angiotensin II-induced hypertension. 5,8,11,14-Eicosatetraynoic acid (ETYA; 4 micromol/L), an inhibitor of all AA pathways, attenuated the vasodilator responses to AA, as did treatment with stannous chloride, which depletes cytochrome P450 enzymes, suggesting that a cytochrome P450 AA metabolite mediated the renal vasodilation. N:-Methylsulfonyl-12,12-dibromododec-11-en-amide (DDMS; 2 micromol/L), a selective omega-hydroxylase inhibitor, did not affect AA-induced vasodilation, whereas selective inhibition of epoxygenases with either miconazole (0.3 micromol/L) or N:-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH; 12 micromol/L) did, indicating that one or more EETs were involved in the renal vasodilator action of AA at high PP. This conclusion was supported by the demonstration that AA greatly enhanced the renal efflux of EETs at high PP but not at basal PP.

Topics: 5,8,11,14-Eicosatetraynoic Acid; 8,11,14-Eicosatrienoic Acid; Acetylcholine; Amides; Animals; Arachidonic Acid; Enzyme Inhibitors; Hypertension; Indomethacin; Male; Nitroprusside; Perfusion; Pressure; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley; Renal Circulation; Sulfones; Tin Compounds; Vasodilation

The epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 (CYP) epoxygenases that have vasodilatory and anti-inflammatory properties. Here we report that EETs have additional fibrinolytic properties. In vascular endothelial cells, physiological concentrations of EETs, particularly 11,12-EET, or overexpression of the endothelial epoxygenase, CYP2J2, increased tissue plasminogen activator (t-PA) expression by 2.5-fold without affecting plasminogen activator inhibitor-1 expression. This increase in t-PA expression correlated with a 4-fold induction in t-PA gene transcription and a 3-fold increase in t-PA fibrinolytic activity and was blocked by the CYP inhibitor, SKF525A, but not by the calcium-activated potassium channel blocker, charybdotoxin, indicating a mechanism that does not involve endothelial cell hyperpolarization. The t-PA promoter is cAMP-responsive, and induction of t-PA gene transcription by EETs correlated with increases in intracellular cAMP levels and, functionally, with cAMP-driven promoter activity. To determine whether increases in intracellular cAMP levels were due to modulation of guanine nucleotide-binding proteins, we assessed the effects of EETs on Galpha(s) and Galpha(i2). Treatment with EETs increased Galpha(s), but not Galpha(i2), GTP-binding activity by 3.5-fold. These findings indicate that EETs possess fibrinolytic properties through the induction of t-PA and suggest that endothelial CYP2J2 may play an important role in regulating vascular hemostasis.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aorta; Atropine Derivatives; Cattle; Cells, Cultured; Cyclic AMP; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Endothelium, Vascular; Gene Expression Regulation, Enzymologic; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gs; Humans; Oxygenases; Polymerase Chain Reaction; Proadifen; Promoter Regions, Genetic; Saphenous Vein; Tissue Plasminogen Activator; Transcription, Genetic; Transfection

Epoxyeicosatrienoic acids (EETs) are produced from arachidonic acid via the cytochrome P-450 epoxygenase pathway. EETs are able to modulate smooth muscle tone by increasing K(+) conductance, hence generating hyperpolarization of the tissues. However, the molecular mechanisms by which EETs induce smooth muscle relaxation are not fully understood. In the present study, the effects of EETs on airway smooth muscle (ASM) were investigated using three electrophysiological techniques. 8,9-EET and 14,15-EET induced concentration-dependent relaxations of the ASM precontracted with a muscarinc agonist (carbamylcholine chloride), and these relaxations were partly inhibited by 10 nM iberiotoxin (IbTX), a specific large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel blocker. Moreover, 3 microM 8,9- or 14,15-EET induced hyperpolarizations of -12 +/- 3.5 and -16 +/- 3 mV, with EC(50) values of 0.13 and 0.14 microM, respectively, which were either reversed or blocked on addition of 10 nM IbTX. These results indicate that BK(Ca) channels are involved in hyperpolarization and participate in the relaxation of ASM. In addition, complementary experiments demonstrated that 8,9- and 14,15-EET activate reconstituted BK(Ca) channels at low free Ca(2+) concentrations without affecting their unitary conductance. These increases in channel activity were IbTX sensitive and correlated well with the IbTX-sensitive hyperpolarization and relaxation of ASM. Together these results support the view that, in ASM, the EETs act through an epithelium-derived hyperpolarizing factorlike effect.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Biological Factors; Bronchoconstriction; Cattle; Cyclooxygenase Inhibitors; Dose-Response Relationship, Drug; Enzyme Inhibitors; Guinea Pigs; In Vitro Techniques; Large-Conductance Calcium-Activated Potassium Channels; Male; Membrane Potentials; Muscarinic Agonists; Muscle, Smooth; Nitric Oxide Synthase; Peptides; Potassium Channels; Potassium Channels, Calcium-Activated; Rabbits; Trachea

Epoxyeicosatrienoic acids (EETs) are released from endothelial cells and potently dilate small arteries by hyperpolarizing vascular myocytes. In the present study, we investigated the structural specificity of EETs in dilating canine and porcine coronary microvessels (50-140 microm ID) and activating large-conductance Ca2+-activated K+ (BK(Ca)) channels. The potencies and efficacies of EET regioisomers and enantiomers were compared with those of two EET homologs: epoxyeicosaquatraenoic acids (EEQs), which are made from eicosapentaenoic acid by the same cytochrome P-450 epoxygenase that generates EETs from arachidonic acid, and epoxydocosatetraenoic acids (EDTs), which are EETs that are two carbons longer. With EC50 values of 3-120 pM but without regio- or stereoselectivity, EETs potently dilated canine and porcine microvessels. Surprisingly, the EEQs and EDTs had comparable potencies and efficacies in dilating microvessels. Moreover, 50 nM 13,14-EDT activated the BK(Ca) channels with the same efficacy as either 11,12-EET enantiomer at 50 nM. We conclude that coronary microvessels and BK(Ca) channels possess low structural specificity for EETs and suggest that EEQs and EDTs may thereby also be endothelium-derived hyperpolarizing factors.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arterioles; Calcium Channel Agonists; Coronary Vessels; Dogs; Dose-Response Relationship, Drug; Endothelium, Vascular; Fatty Acids, Unsaturated; Female; In Vitro Techniques; Large-Conductance Calcium-Activated Potassium Channels; Male; Microcirculation; Muscle, Smooth, Vascular; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Calcium-Activated; Stereoisomerism; Swine; Vasodilation; Vasodilator Agents

Epoxygenase metabolites produced by the kidney affect renal blood flow and tubular transport function and 11,12-epoxyeicosatrienoic acid (11,12-EET) has been putatively identified as an endothelium-derived hyperpolarizing factor. The current studies were performed to determine the influence of 11,12-EET on the regulation of afferent arteriolar diameter in angiotensin II-infused hypertensive rats.. Male Sprague-Dawley rats received angiotensin II (60 ng/min) or vehicle via an osmotic minipump. Angiotensin II-infused hypertensive and vehicle-infused normotensive rats were studied for 2 weeks following implantation of the minipump. Renal microvascular responses to the sulfonimide analog of 11,12-EET (11,12-EET-SI) and angiotensin II were observed utilizing the in-vitro juxtamedullary nephron preparation. Renal cortical epoxygenase enzyme protein levels were quantified by Western blot analysis. Renal microvessels were also isolated and epoxygenase metabolite levels measured by negative ion chemical ionization (NICI)/gas chromatography-mass spectroscopy.. Systolic blood pressure averaged 118 +/- 2 mmHg prior to pump implantation and increased to 185 +/- 7 mmHg in rats infused with angiotensin II for 2 weeks. Afferent arteriolar diameters of 2-week normotensive animals averaged 22 +/- 1 microm. Diameters of the afferent arterioles were 17% smaller in hypertensive rats (P< 0.05); however, arterioles from both groups responded to 11,12-EET-SI (100 nmol) with similar 15-17% increases in diameter. As we previously demonstrated, the afferent arteriolar reactivity to angiotensin II was enhanced in angiotensin II-infused animals. Interestingly, elevation of 11,12-EET-SI levels to 100 nmol reversed the enhanced vascular reactivity to angiotensin II associated with angiotensin II hypertension. Renal microvascular EET levels were not different between groups and averaged 81 +/- 9 and 87 +/- 13 pg/mg per 30 min in normotensive and hypertensive animals, respectively. Renal cortical microsomal levels of the epoxygenase CYP2C23 and CYP2C11 proteins were also similar in normotensive and angiotensin II hypertensive rats.. Taken together, these data support the concept that renal microvascular 11,12-EET activity and levels may not properly offset the enhanced angiotensin II renal vasoconstriction during angiotensin II hypertension.

Topics: 8,11,14-Eicosatrienoic Acid; Angiotensin II; Animals; Arterioles; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Drug Synergism; Hypertension; Kidney Cortex; Male; Microcirculation; Microsomes; Rats; Rats, Sprague-Dawley; Renal Circulation; Sulfonamides; Vasoconstriction

An effective synthesis is described for the preparation of all four mono trans isomers of arachidonic acid via deoxidation of epoxide precursors with lithium diphenylphosphide and quaternization with methyl iodide.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acid; Biochemistry; Chromatography, High Pressure Liquid; Magnetic Resonance Spectroscopy; Stereoisomerism

1. We examined the effects of epoxyeicosatrienoic acids (EETs), which are cytochrome P450 metabolites of arachidonic acid (AA), on the activities of the ATP-sensitive K(+) (K(ATP)) channels of rat cardiac myocytes, using the inside-out patch-clamp technique. 2. In the presence of 100 microM cytoplasmic ATP, the K(ATP) channel open probability (P(o)) was increased by 240 +/- 60 % with 0.1 microM 11,12-EET and by 400 +/- 54 % with 5 microM 11,12-EET (n = 5-10, P < 0.05 vs. control), whereas neither 5 microM AA nor 5 microM 11,12-dihydroxyeicosatrienoic acid (DHET), which is the epoxide hydrolysis product of 11,12-EET, had any effect on P(o). 3. The half-maximal activating concentration (EC(50)) was 18.9 +/- 2.6 nM for 11,12-EET (n = 5) and 19.1 +/- 4.8 nM for 8,9-EET (n = 5, P = n.s. vs. 11,12-EET). Furthermore, 11,12-EET failed to alter the inhibition of K(ATP) channels by glyburide. 4. Application of 11,12-EET markedly decreased the channel sensitivity to cytoplasmic ATP. The half-maximal inhibitory concentration of ATP (IC(50)) was increased from 21.2 +/- 2.0 microM at baseline to 240 +/- 60 microM with 0.1 microM 11,12-EET (n = 5, P < 0.05 vs. control) and to 780 +/- 30 microM with 5 microM 11,12-EET (n = 11, P < 0.05 vs. control). 5. Increasing the ATP concentration increased the number of kinetically distinguishable closed states, promoting prolonged closure durations. 11,12-EET antagonized the effects of ATP on the kinetics of the K(ATP) channels in a dose- and voltage-dependent manner. 11,12-EET (1 microM) reduced the apparent association rate constant of ATP to the channel by 135-fold. 6. Application of 5 microM 11,12-EET resulted in hyperpolarization of the resting membrane potential in isolated cardiac myocytes, which could be blocked by glyburide. 7. These results suggest that EETs are potent activators of the cardiac K(ATP) channels, modulating channel behaviour by reducing the channel sensitivity to ATP. Thus, EETs could be important endogenous regulators of cardiac electrical excitability.

Topics: 8,11,14-Eicosatrienoic Acid; Adenosine Triphosphate; Animals; Arachidonic Acid; Dose-Response Relationship, Drug; Electrophysiology; Glyburide; Heart; Kinetics; Male; Membrane Potentials; Myocardium; Potassium Channel Blockers; Potassium Channels; Rats; Rats, Sprague-Dawley

Little information is available regarding the vasoactive effects of epoxyeicosatrienoic acids (EETs) in the lung. We demonstrate that 5, 6-, 8,9-, 11,12-, and 14,15-EETs contract pressurized rabbit pulmonary arteries in a concentration-dependent manner. Constriction to 5,6-EET methyl ester or 14,15-EET is blocked by indomethacin or ibuprofen (10(-5) M), SQ-29548, endothelial denuding, or submaximal preconstriction with the thromboxane mimetic U-46619. Constriction of pulmonary artery rings to phenylephrine is blunted by treatment with the epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide. Pulmonary arteries and peripheral lung microsomes metabolize arachidonate to products that comigrate on reverse-phrase HPLC with authentic regioisomers of 5,6-, 8,9-, 11,12-, and 14,15-EETs, but no cyclooxygenase products of EETs could be demonstrated. Proteins of the CYP2B, CYP2E, CYP2J, CYP1A, and CYP2C subfamilies are present in pulmonary artery and peripheral lung microsomes. Constriction of isolated rabbit pulmonary arteries to EETs is nonregioselective and depends on intact endothelium and cyclooxygenase, consistent with the formation of a pressor prostanoid compound. These data raise the possibility that EETs may contribute to regulation of pulmonary vascular tone.

Topics: 8,11,14-Eicosatrienoic Acid; Amides; Animals; Arachidonic Acid; Cytochrome P-450 Enzyme System; Dogs; In Vitro Techniques; Male; Pressure; Pulmonary Artery; Rabbits; Vasoconstriction; Vasoconstrictor Agents; Vasomotor System

Lipoxygenase (LO) and cytochrome P450 monooxygenase products of arachidonic acid (AA) have been implicated in a large number of vasoregulatory processes. In intact, blood-free, perfused and ventilated human lungs (n = 8), isolated during surgery for bronchial carcinoma, we analyzed leukotrienes (LTs), hydroxyeicosatetraenoic acids (HETEs), and epoxyeicosatrienoic acids (EETs) by sequential sampling of the recirculating buffer fluid. For the analysis we used multistep, solid-phase extraction, isocratic reversed-phase high-performance liquid chromatography, with elution of all metabolites within one run and photodiode array detection to obtain full UV spectra of eluting compounds. We detected no LT release in a 15-min baseline period, but the admixture of the calcium ionophore A23187 with the buffer fluid provoked the rapid appearance of all LTs. Some baseline release of 15-HETE was observed, and in response to A23187, maximum buffer concentrations were noted for 5-HETE, with 8-HETE, 9-HETE, 11-HETE, and 12-HETE being detected at lower levels. Marked baseline liberation of 11,12-EET and 8,9-EET was observed. In response to A23187, high oxirane buffer concentrations were registered, which far surpassed those of LTs and HETEs. The eicosanoid release was paralleled by a limited pulmonary artery pressor response and progressive vascular leakage. We conclude that ex-vivo-perfused human lungs release EETs > LTs > HETEs into the vascular compartment in response to inflammatory challenge. The marked oxirane synthesis in the lung vasculature may have major impact on lung vasoregulation when considering the possible function of these AA epoxides as endothelium-derived hyperpolarizing factors.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acid; Cytochrome P-450 Enzyme System; Endothelium, Vascular; Ethylene Oxide; Homeostasis; Humans; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipoxygenase; Lung; Perfusion; Vascular Resistance

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(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(-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.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Bradykinin; Cattle; Cells, Cultured; Chromatography, High Pressure Liquid; Coronary Vessels; Endothelium, Vascular; Methacholine Chloride; Microchemistry; Spectrometry, Fluorescence

The cytochrome P450-derived epoxyeicosatrienoic acids (EETs) have potent effects on renal vascular reactivity and tubular sodium and water transport; however, the role of these eicosanoids in the pathogenesis of hypertension is controversial. The current study examined the hydrolysis of the EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs) as a mechanism for regulation of EET activity and blood pressure. EET hydrolysis was increased 5- to 54-fold in renal cortical S9 fractions from the spontaneously hypertensive rat (SHR) relative to the normotensive Wistar-Kyoto (WKY) rat. This increase was most significant for the 14,15-EET regioisomer, and there was a clear preference for hydrolysis of 14, 15-EET over the 8,9- and 11,12-EETs. Increased EET hydrolysis was consistent with increased expression of soluble epoxide hydrolase (sEH) in the SHR renal microsomes and cytosol relative to the WKY samples. The urinary excretion of 14,15-DHET was 2.6-fold higher in the SHR than in the WKY rat, confirming increased EET hydrolysis in the SHR in vivo. Blood pressure was decreased 22+/-4 mm Hg (P:<0.01) 6 hours after treatment of SHRs with the selective sEH inhibitor N:, N:'-dicyclohexylurea; this treatment had no effect on blood pressure in the WKY rat. These studies identify sEH as a novel therapeutic target for control of blood pressure. The identification of a potent and selective inhibitor of EET hydrolysis will be invaluable in separating the vascular effects of the EET and DHET eicosanoids.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acids; Blood Pressure; Cytosol; Eicosanoids; Enzyme Inhibitors; Epoxide Hydrolases; Epoxy Compounds; Hydrolysis; Hypertension; Kidney Cortex; Male; Microsomes; Microsomes, Liver; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Sprague-Dawley; Species Specificity; Urea

1. Whole-cell Na+ currents (holding potential, -80 mV; test potential, -30 mV) in rat myocytes were inhibited by 8, 9-epoxyeicosatrienoic acid (8,9-EET) in a dose-dependent manner with 22+/-4% inhibition at 0.5 microM, 48+/-5% at 1 microM, and 73+/-5% at 5 microM (mean +/- S.E.M., n = 10, P<0.05 for each dose vs. control). Similar results were obtained with 5,6-, 11,12-, and 14,15-EETs, while 8,9-dihydroxyeicosatrienoic acid (DHET) was 3-fold less potent and arachidonic acid was 10- to 20-fold less potent. 2. 8,9-EET produced a dose-dependent, hyperpolarized shift in the steady-state membrane potential at half-maximum inactivation (V ), without changing the slope factor. 8,9-EET had no effect on the steady-state activation of Na+ currents. 3. Inhibition of Na+ currents by 8,9-EET was use dependent, and channel recovery was slowed. The effects of 8,9-EET were greater at depolarized potentials. 4. Single channel recordings showed 8,9-EET did not change the conductance or the number of active Na+ channels, but markedly decreased the probability of Na+ channel opening. These results were associated with a decrease in the channel open time and an increase in the channel closed times. 5. Incubation of cultured cardiac myocytes with 1 microM [3H]8,9-EET showed that 25% of the radioactivity was taken up by the cells over a 2 h period, and most of the uptake was incorporated into phospholipids, principally phosphatidylcholine. Analysis of the medium after a 2 h incubation indicated that 86% of the radioactivity remained as [3H]8,9-EET while 13% was converted into [3H]8,9-DHET. After a 30 min incubation, 1-2% of the [3H]8,9-EET uptake by cells remained as unesterified EET. 6. These results demonstrate that cardiac cells have a high capacity to take up and metabolize 8,9-EET. 8,9-EET is a potent use- and voltage-dependent inhibitor of the cardiac Na+ channels through modulation of the channel gating behaviour.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Animals, Newborn; Arachidonic Acid; Cells, Cultured; Heart; Heart Ventricles; Membrane Potentials; Myocardium; Rats; Rats, Sprague-Dawley; Sodium Channels; Structure-Activity Relationship

The epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 epoxygenases that have vasodilatory properties similar to that of endothelium-derived hyperpolarizing factor. The cytochrome P450 isoform CYP2J2 was cloned and identified as a potential source of EETs in human endothelial cells. Physiological concentrations of EETs or overexpression of CYP2J2 decreased cytokine-induced endothelial cell adhesion molecule expression, and EETs prevented leukocyte adhesion to the vascular wall by a mechanism involving inhibition of transcription factor NF-kappaB and IkappaB kinase. The inhibitory effects of EETs were independent of their membrane-hyperpolarizing effects, suggesting that these molecules play an important nonvasodilatory role in vascular inflammation.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Anti-Inflammatory Agents, Non-Steroidal; Carotid Arteries; Cattle; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Coronary Vessels; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; DNA-Binding Proteins; Endothelium, Vascular; Humans; Hydroxyeicosatetraenoic Acids; I-kappa B Kinase; I-kappa B Proteins; Mice; Mice, Inbred C57BL; NF-kappa B; NF-KappaB Inhibitor alpha; Oxygenases; Protein Serine-Threonine Kinases; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Epoxyeicosatrienoic acids (EETs) are cytochrome P450-derived metabolites of arachidonic acid. They are potent endogenous vasodilator compounds produced by vascular cells, and EET-induced vasodilation has been attributed to activation of vascular smooth muscle cell (SMC) K(+) channels. However, in some cells, EETs activate Ca(2+) channels, resulting in Ca(2+) influx and increased intracellular Ca(2+) concentration ([Ca(2+)](i)). We investigated whether EETs also can activate Ca(2+) channels in vascular SMC and whether the resultant Ca(2+) influx can influence vascular tone. The 4 EET regioisomers (1 micromol/L) increased porcine aortic SMC [Ca(2+)](i) by 52% to 81%, whereas arachidonic acid, dihydroxyeicosatrienoic acids, and 15-hydroxyeicosatetraenoic acid (1 micromol/L) produced little effect. The increases in [Ca(2+)](i) produced by 14,15-EET were abolished by removal of extracellular Ca(2+) and by pretreatment with verapamil (10 micromol/L), an inhibitor of voltage-dependent (L-type) Ca(2+) channels. 14,15-EET did not alter Ca(2+) signaling induced by norepinephrine and thapsigargin. When administered to porcine coronary artery rings precontracted with a thromboxane mimetic, 14,15-EET produced relaxation. However, when administered to rings precontracted with acetylcholine or KCl, 14,15-EET produced additional contractions. In rings exposed to 10 mmol/L KCl, a concentration that did not affect resting ring tension, 14,15-EET produced small contractions that were abolished by EGTA (3 mmol/L) or verapamil (10 micromol/L). These observations indicate that 14,15-EET enhances [Ca(2+)](i) influx in vascular SMC through voltage-dependent Ca(2+) channels. This 14,15-EET-induced increase in [Ca(i)(2+)] can produce vasoconstriction and therefore may act to modulate EET-induced vasorelaxation.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aorta, Thoracic; Calcium; Calcium Channel Blockers; Cell Membrane Permeability; Cells, Cultured; Chelating Agents; Coronary Vessels; Dose-Response Relationship, Drug; In Vitro Techniques; Intracellular Fluid; Muscle Contraction; Muscle, Smooth, Vascular; Structure-Activity Relationship; Swine; Vasoconstrictor Agents; Vasodilator Agents

The CYP monooxygenase, CYP2B12, is the first identified skin-specific cytochrome P450 enzyme. It is characterized by high, constitutive expression in an extrahepatic tissue, the sebaceous glands of cutaneous tissues. It is expressed exclusively in a subset of differentiated keratinocytes called sebocytes, as demonstrated by Northern blot analysis, in situ hybridization, and polymerase chain reaction. The onset of its expression coincides with the morphological appearance of sebaceous glands in the neonatal rat. Recombinant CYP2B12 produced in Escherichia coli epoxidizes arachidonic acid to 11,12- and 8,9-epoxyeicosatrienoic acids (80 and 20% of total metabolites, respectively). The identification of arachidonic acid as a substrate for this skin-specific CYP monooxygenase suggests an endogenous function in keratinocytes in the generation of bioactive lipids and intracellular signaling.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Animals, Newborn; Arachidonic Acid; Cloning, Molecular; Cytochrome P-450 Enzyme System; Escherichia coli; Keratinocytes; Organ Specificity; Rats; Rats, Sprague-Dawley; Recombinant Proteins; Sebaceous Glands; Skin; Substrate Specificity; Transcription, Genetic

Cytochrome P450 epoxygenases convert arachidonic acid into 4 epoxyeicosatrienoic acid (EET) regioisomers, which were recently identified as endothelium-derived hyperpolarizing factors in coronary blood vessels. Both EETs and their dihydroxyeicosatrienoic acid (DHET) metabolites have been shown to relax conduit coronary arteries at micromolar concentrations, whereas the plasma concentrations of EETs are in the nanomolar range. However, the effects of EETs and DHETs on coronary resistance arterioles have not been examined. We administered EETs and DHETs to isolated canine coronary arterioles (diameter, 90.0+/-3.4 microm; distending pressure, 20 mm Hg) preconstricted by 30% to 60% of the resting diameter with endothelin. All 4 EET regioisomers produced potent, concentration-dependent vasodilation (EC50 values ranging from -12.7 to -10.1 log [M]) and were approximately 1000 times more potent than reported in conduit coronary arteries. The vasodilation produced by 14,15-EET was not attenuated by removal of the endothelium and indicated a direct action of 14,15-EET on microvascular smooth muscle. Likewise, 14,15-DHET, 11,12-DHET, 8,9-DHET, and the delta-lactone of 5,6-EET produced extremely potent vasodilation (EC50 values ranging from -15.8 to -13.1 log [M]). The vasodilation produced by these eicosanoids was highly potent in comparison to that produced by other vasodilators, including arachidonic acid (EC50=-7.5 log [M]). The epoxide hydrolase inhibitor, 4-phenylchalone oxide, which blocked the conversion of [3H]14,15-EET to [3H]14,15-DHET by canine coronary arteries, did not alter arteriolar dilation to 11,12-EET; thus, the potent vasodilation induced by EETs does not require formation of DHETs. In contrast, charybdotoxin (a KCa channel inhibitor) and KCl (a depolarizing agent) blocked vasodilation by 11,12-EET and 11,12-DHET. We conclude that EETs and DHETs potently dilate canine coronary arterioles via activation of KCa channels. The preferential ability of these compounds to dilate resistance blood vessels suggests that they may be important regulators of coronary circulation.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Calcimycin; Coronary Vessels; Dogs; Dose-Response Relationship, Drug; Female; Hydroxyeicosatetraenoic Acids; Male; Microcirculation; Potassium Channels; Vasodilator Agents

In spite of the large quantities of epoxyeicosatrienoic acids (EEts) released by reproductive tissues, their function has not yet been determined. In order to analyze the influence of epoxygenase products on isolated uterine function, Clotrimazole, a cytochrome P450 inhibitor was used. The drug decreased isolated rat uterine isometric developed tension (IDT) and frequency (FC). 14,15 EEt induced a contractile response when added at 10(11) M, 8,9 EEt and 11,12 EEt produced an increment of IDT when added to 10(-7) M and 5,6 EEt did not modify IDT values. A contractile stimulatory effect was observed when 14,15 EEt (10(-7) M) was added to a tissue bath preparation containing Clotrimazole (20 microM). On the other hand, uterine contractile response to 14,15 EEt addition was partially abolished by indomethacin (10(-6) M), a well known cyclooxygenase inhibitor. Uterine response to 5,6; 8,9 and 11,12 EEts was not modified by indomethacin. This is the first evidence of 14-15 EEt uterotonic properties, possibly exerted in part through the cyclooxygenase pathway.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Clotrimazole; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme Inhibitors; Dose-Response Relationship, Drug; Estradiol; Female; In Vitro Techniques; Indomethacin; Isometric Contraction; Ovariectomy; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; Uterine Contraction

Epoxyeicosatrienoic acids (EETs) are potent endothelium-derived vasodilators formed from cytochrome P-450 metabolism of arachidonic acid. EETs and their diol products (DHETs) are also avidly taken up by endothelial cells and incorporated into phospholipids that participate in signal transduction. To investigate the possible functional significance of EET and DHET incorporation into cell lipids, we examined the capacity of EETs and DHETs to relax porcine coronary arterial rings and determined responses to bradykinin (which potently activates endothelial phospholipases) before and after incubating the rings with these eicosanoids. 14,15-EET and 11,12-EET (5 mumol/L) produced 75 +/- 9% and 52 +/- 4% relaxation, respectively, of U46619-contracted rings, whereas 8,9-EET and 5,6-EET did not produce significant relaxation. The corresponding DHET regioisomers produced comparable relaxation responses. Preincubation with 14,15-EET, 11,12-EET, 14,15-DHET, and 11,12-DHET augmented the magnitude and duration of bradykinin-induced relaxation, whereas endothelium-independent relaxations to aprikalim and sodium nitroprusside were not potentiated. Pretreatment with 2 mumol/L triacsin C (an inhibitor of acyl coenzyme A synthases) inhibited [3H]14,15-EET incorporation into endothelial phospholipids and blocked 11,12-EET- and 14,15-DHET-induced potentiation of relaxation to bradykinin. Exposure of [3H]14,15-EET-labeled endothelial cells to the Ca2+ ionophore A23187 (2 mumol/L) resulted in a 4-fold increased release of EET and DHET into the medium. We conclude that incorporation of EETs and DHETs into cell lipids results in potentiation of bradykinin-induced relaxation in porcine coronary arteries, providing the first evidence that incorporated EETs and DHETs are capable of modulating vascular function.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Bradykinin; Cells, Cultured; Coronary Vessels; Endothelium, Vascular; Prostaglandin Endoperoxides, Synthetic; Swine; Thromboxane A2; Vasoconstrictor Agents; Vasodilation

The present study on the newborn pig cerebral microcirculation determined the vasoactive properties of epoxyeicosatrienoic acids (EETs) and the contributions of prostaglandin cyclooxygenase to these properties. Pial arterioles of anesthetized piglets were observed through closed cranial windows, EETs were applied topically, and artificial cerebrospinal fluid from beneath the cranial windows was collected for the determination of adenosine 3',5'-cyclic monophosphate and 6-ketoprostaglandin F1 alpha. EETs caused dilation of pial arterioles and increased adenosine 3',5'-cyclic monophosphate. 5,6-EET produced a dose-dependent dilation at 10(-8) M and above, whereas 10(-6) M was required for 8,9-EET, 11,12-EET, and 14,15-EET. Indomethacin abolished pial arteriolar dilation to the EETs. However, EETs did not increase cortical 6-ketoprostaglandin F1 alpha concentration. Treatment of indomethacin-treated piglets with iloprost (10(-12) M topically) restored dilation to 5,6-EET. Neither isoproterenol nor sodium nitroprusside allowed vasodilation to 5,6-EET in indomethacin-treated piglets. Therefore, in the newborn pig cerebral microvasculature. EETs are potent vasodilators and prostacyclin-receptor agonists are necessary to allow this dilation to occur.

Topics: 6-Ketoprostaglandin F1 alpha; 8,11,14-Eicosatrienoic Acid; Animals; Animals, Newborn; Arterioles; Carbon Dioxide; Cyclic AMP; Dose-Response Relationship, Drug; Iloprost; Indomethacin; Muscle, Smooth, Vascular; Nitroprusside; Pia Mater; Structure-Activity Relationship; Swine; Vasodilation; Vasodilator Agents

1. Epoxyeicosatrienoic acids (EETs) have been described as endothelium-derived hyperpolarizing factors (EDHFs), based on their stimulatory effects on smooth muscle K+ channels. In order to reveal a putative autocrine effect of EETs on endothelial channels, we have studied the effects of the four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET) on the high-conductance, Ca(2+)-dependent K+ (BKCa) channel recorded in inside-out patches of primary cultured pig coronary artery endothelial cells. Currents were recorded in the presence of either 500 nm or 1 microM free Ca2+ on the cytosolic side of the membrane. 2. In 81% of experiments, EETs at < 156 nM, applied on the cytosolic side of the membrane, transiently increased BKCa channel open state probability (PO) without affecting its unitary conductance, thus providing evidence for direct action of EETs, without involvement of a cytosolic transduction pathway. 3. The four EET regioisomers appeared to be equally active, multiplying the BKCa channel PO by a mean factor of 4.3 +/- 0.6 (n = 15), and involving an increase in the number and duration of openings. 4. The EET-induced increase in BKCa channel activity was more pronounced with low initial PO. When the BKCa channel was activated by 500 nM Ca2+, application of EETs increased the initial PO value of below 0.1 by a factor of 5. When the channel was activated by 1 microM Ca2+, application of EETs increased the initial PO value by a factor of 3. 5. Our results show that EETs potentiate endothelial BKCa channel activation by Ca2+. The autocrine action of EETs on endothelial cells, which occurs in the same concentration range as their action on muscle cells, should therefore fully participate in the vasoactive effects of EETs, and thus be taken into account when considering their putative EDHF function.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Calcium; Cells, Cultured; Coronary Vessels; Endothelium, Vascular; Membrane Potentials; Potassium Channels; Swine

Cytochrome P450 (P450) monooxygenases catalyze the epoxidation of arachidonic acid to form epoxyeicosatrienoic acids, which modulate bronchial smooth muscle tone and airway transepithelial ion transport. We recently described a new human P450 arachidonic acid epoxygenase (CYP2J2) and the corresponding rat homologue (CYP2J3). Northern analysis of lung RNA using CYP2J cDNA probes demonstrated that CYP2J2 and CYP2J3 mRNAs were expressed in the lung. Immunoblotting of microsomal fractions prepared from human and rat lungs using a polyclonal antibody raised against recombinant human CYP2J2 revealed a single 56-kDa band confirming abundant pulmonary CYP2J2 and CYP2J3 protein expression. Immunohistochemical analysis of formalin-fixed paraffin-embedded human and rat lung sections using the anti-human CYP2J2 IgG and avidin/biotin/peroxidase detection showed that CYP2J proteins were primarily expressed in ciliated epithelial cells lining the airway. Prominent staining was also noted in nonciliated airway epithelial cells, bronchial and pulmonary vascular smooth muscle cells, pulmonary vascular endothelium, and alveolar macrophages, whereas less intense staining was noted in alveolar epithelial cells. Endogenous epoxyeicosatrienoic acids were detected in both human and rat lung using gas chromatography/mass spectrometry, thus providing direct evidence for the in vivo human and rat pulmonary P450 metabolism of arachidonic acid. Based on these data, we conclude that CYP2J2 and CYP2J3 are abundant pulmonary arachidonic acid epoxygenases and that CYP2J products, the epoxyeicosatrienoic acids, are endogenous constituents of human and rat lung. In addition to known effects on airway smooth muscle tone and transepithelial electrolyte transport, the localization of CYP2J proteins to vascular smooth muscle and endothelium suggests that epoxyeicosatrienoic acids may also be involved in the modulation of pulmonary vascular tone.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Blotting, Northern; Cytochrome P-450 Enzyme System; Endothelium, Vascular; Gas Chromatography-Mass Spectrometry; Humans; Immunoblotting; Immunohistochemistry; Isoenzymes; Lung; Macrophages, Alveolar; Muscle, Smooth, Vascular; Rats

Epoxyeicosatrienoic acids (EETs) are eicosanoids synthesized from arachidonic acid by the cytochrome P450 eposygenase pathway. The present studies demonstrate that 8,9-, 11,12-, and 14,15-EET are rapidly taken up by porcine aortic smooth muscle cells. About half of the uptake is incorporated into phospholipids, and saponification indicates that most of this remains in the form of EET. The EETs also are converted to the corresponding dihydroxyeicosatrienoic acids (DHETs) and during prolonged incubations, additional metabolites that do not retain the EET carboxyl group are formed. Most of these products are released into the medium. However, some DHET and metabolites less polar than EET are incorporated into the phospholipids, and a small amount of unesterified EET is also present in the cells. The incorporation of 14,15-EET and its conversion to DHET did not approach saturation until the concentration exceeded 10-20 microM, indicating that vascular smooth muscle has a large capacity to utilize this EET. These findings suggest that certain vasoactive effects of EETs may be due to their incorporation by smooth muscle cells. Furthermore, through conversion to DHET and other oxidized metabolites, smooth muscle apparently has the capacity to inactivate EETs that are either formed in or penetrate into the vascular wall.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aorta; Cell Division; Cells, Cultured; Chromatography, High Pressure Liquid; Endothelium, Vascular; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Kinetics; Muscle, Smooth, Vascular; Phospholipids; Swine; Tritium

Topics: 8,11,14-Eicosatrienoic Acid; Blood Platelets; Gas Chromatography-Mass Spectrometry; Humans; Mass Spectrometry; Phospholipids

Topics: 8,11,14-Eicosatrienoic Acid; Amiloride; Animals; Biological Transport; Cell Line; Epithelium; Kidney; Rubidium; Rubidium Radioisotopes; Structure-Activity Relationship

Epoxyeicosatrienoic acids (EETs), normally present in brain and blood, appear to be released from atherosclerotic vessels in large amounts. Once intravascular, EETs can constrict renal arteries in vivo and dilate cerebral and coronary arteries in vitro. Whether EETs in blood will alter cerebral blood flow (CBF) in vivo is unknown. In the present study, the chemical synthesis of four EET regioisomers was optimized, and their identity and structural integrity established by chromatographic and mass spectral methods. The chemically labile EETs were converted to a sodium salt, complexed with albumin, and infused into anesthetized rats via the common carotid. The objective was to test whether sustained, high levels of intravascular EETs alter CBF. The CBF (cortical H2 clearance) was measured before and 30 min after the continuous infusion of 14,15- (n = 5), 11,12- (n = 5), 8,9- (n = 7) and 5,6-EET (unesterified or as the methyl ester, n = 5 for each). Neither the CBF nor the systemic blood pressure was affected by EETs. Because the infusions elevated the plasma concentrations of EETs about 700-fold above normal levels (1.0 nM), it is unlikely that EETs released from atherosclerotic vessels will alter CBF.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Cerebrovascular Circulation; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Infusions, Intravenous; Male; Rats; Rats, Wistar

In the present study, the regio- and stereoselective epoxidation of arachidonic acid by cytochromes P450 2C8 and 2C9, two members of the CYP2C gene subfamily expressed in human liver, was determined. Purified P450 isozymes, reconstituted with NADPH:P450 oxidoreductase, cytochrome b5 and lipid, or microsomes isolated from human liver, were incubated with [1-14C]-arachidonic acid. For regioselective analysis, the epoxide metabolites formed, 14,15-, 11,12- and 8,9-epoxyeicosatrienoic acids (EETs), were resolved by reverse-phase high-performance liquid chromatography. P450 2C8 produces only the 14,15- and 11,12-EETs in a 1.25:1.00 ratio. The two epoxides represent 68% of the total metabolites. P450 2C9 produces 14,15-, 11,12- and 8,9-EETs in a 2.3:1.0:0.5 ratio. The three epoxides represent 69% of the total metabolites. Neither P450 isoform catalyzes the formation of 5,6-EET. For chiral analysis, the two major epoxide metabolites, 14,15- and 11,12-EETs, were derivatized to methyl and pentafluorbenzyl esters, respectively. Enantiomers of 14,15- and 11,12-EET esters were subsequently resolved on Chiralcel OB and OD columns (J.T. Baker, Phillipsburg, PA), respectively. Both P450 2C8 and 2C9 are stereoselective at the 14,15- position, preferentially producing 14(R), 15(S)-EET with 86.2% and 62.5% selectivity, respectively. Both enzymes are also stereoselective at the 11,12-position but have the opposite selectivity. P450 2C8 is 81.1% selective for 11(R), 12(S)-EET; P450 2C9 is 69.4% selective for the 11(S), 12(R)-EET. Immunoinhibition studies performed with anti-2C9 immunoglobulin G (which also reacts with P450 2C8) and hepatic microsomes indicate that these two P450s are important arachidonic acid epoxygenases in human liver.

Topics: 8,11,14-Eicosatrienoic Acid; Amino Acid Sequence; Animals; Arachidonic Acid; Aryl Hydrocarbon Hydroxylases; Cytochrome P-450 Enzyme System; Humans; In Vitro Techniques; Male; Microsomes, Liver; Molecular Sequence Data; Rabbits; Stereoisomerism; Steroid 16-alpha-Hydroxylase; Steroid Hydroxylases

Effects of cytochrome P-450 metabolites of arachidonic acid, epoxyeicosatrienoic acids (EETS; 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET), were examined in isolated guinea pig hearts and ventricular myocytes. Addition of 1-16 ng/ml EETs to normal isolated hearts produced no effects on contractility or coronary pressure. In hearts subjected to 60 min of low-flow ischemia, impairment of contractility and declines in heart rate and coronary perfusion pressure were similar in the presence or absence of 1 ng/ml EETs. However, in the presence of either 5,6- or 11,12-EET, recovery was delayed for the first 10 min only. No significant differences were found in any group regarding heart rate, coronary perfusion pressure, or energy metabolite content after 30 min of reperfusion. In myocytes, both 5,6- and 11,12-EET (100 pg/ml, 1.0 ng/ml, and 20 ng/ml) significantly increased cell shortening as well as intracellular calcium concentrations, whereas 8,9- or 14,15-EET was without effect on these parameters. These results describe for the first time the direct effects of various EETs on cardiac cell function as well as their ability to modulate some of the myocardial responses to postischemic reperfusion. The results suggest a potential role for these substances in the response of the heart to pathological insult.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Calcium; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Guinea Pigs; Heart; Heart Rate; Heart Ventricles; Male; Myocardium; Reperfusion Injury; Time Factors

Microsomal preparations of cat brain incubated with [14C]arachidonic acid produced epoxyeicosatrienoic acids (EETs) that eluted with the same retention times as synthetically prepared 5,6-, 8,9-, and 11,12-EETs. These compounds dilated serotonin-preconstricted, pressurized cat cerebral arteries in a dose-dependent fashion. Epoxide formation was not found in mitochondrial fractions and was dependent on the presence of NADPH. The maximum effects of 8,9-EET and 11,12-EET were greater than those of 5,6-EET. The cellular basis of this vasodilation was further investigated by examining the effects of 8,9-EET and 11,12-EET on K+ channel activity in vascular muscle cells freshly isolated from cat cerebral arteries. Both 8,9-EET and 11,12-EET increased the frequency of opening, mean open time, and open-state probability of a 98-pS K+ channel recorded in the cell-attached mode with 145 mM KCl in the pipette and 4.7 mM KCl in the bath. Blockade of K+ channel activity with tetraethylammonium attenuated the vasodilatory effects of 11,12-EET on serotonin-preconstricted cat cerebral arteries. These results suggest that endogenously formed EETs may participate in local regulation of cerebral blood flow by dilating cerebral arteries through a mechanism that involves activation of K+ channels.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Brain; Cats; Cerebral Arteries; Electrophysiology; Female; In Vitro Techniques; Male; Microsomes; Muscle, Smooth, Vascular; Potassium; Potassium Channels; Vasodilator Agents

Gas chromatographic/mass spectroscopic and chiral analysis showed the presence of enzymatically derived 8,9-, 11,12- and 14,15-EET in rat plasma (2.8:1:3.4 molar ratio, respectively; 10.2 +/- 0.4 ng total EET/ml plasma). Greater than 90% of the plasma EETs was esterified to the phospholipids of circulating lipoproteins. The lipoprotein fraction with the highest EET concentration was LDL (8.1 +/- 0.9 ng/mg of protein) followed by HDL and VLDL (3.5 +/- 0.1 and 1.9 +/- 0.3 ng/mg of protein, respectively). In light of the biological activities of the EETs, these results suggest a potential systemic function for the cytochrome P-450 epoxygenase.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Gas Chromatography-Mass Spectrometry; Isomerism; Lipoproteins; Lipoproteins, HDL; Lipoproteins, LDL; Lipoproteins, VLDL; Male; Oxygenases; Rats; Rats, Inbred Strains

The renovascular effects of cytochrome P450-dependent arachidonic acid (P450-AA) metabolites synthesized by rat and rabbit kidneys were studied in the rabbit isolated kidney under conditions of constant flow and examined for their dependency on cyclooxygenase relative to their expression of vasoactivity. Kidneys were perfused with Krebs-Henseleit solution, and perfusion pressure was raised to levels of 90 to 110 mm Hg with the addition of 2 to 3 microM phenylephrine to the perfusate. Close arterial injection of 1 to 20 micrograms of 5,6-, 8,9- and 11,12-epoxyeicosatrienoic acid (EET) dose-dependently decreased perfusion pressure. The 5,6-EET was the most potent and the only epoxide dependent on cyclooxygenase for expression of vasoactivity, being inhibited by indomethacin (2.8 microM). In contrast, 14,15-EET resulted in dose-dependent increases in perfusion pressure. The vasodilator effects of the omega- and omega-1 oxidation products, 20-hydroxyeicosatetraenoic acid (HETE) and the stereoisomers of 19-HETE, were also inhibited by indomethacin. Furthermore, the renal vasodilator responses to 5,6-EET were not inhibited by either superoxide dismutase (10 U) or catalase (40 U) and, therefore, were unrelated to the formation of oxygen radicals generated during transformation of the epoxide by cyclooxygenase. As 5,6-EET and 19- and 20-HETE are synthesized by the renal tubules and can affect movement of salt and water, expression of vasoactivity by P450-dependent arachidonic acid metabolites, and after release from a nephron segment, may represent a mechanism that couples altered renal tubular function to appropriate changes in local blood flow.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Blood Pressure; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme System; Eicosanoids; Free Radicals; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney; Male; Prostaglandin-Endoperoxide Synthases; Rabbits; Renal Circulation; Vascular Resistance

Topics: 1-Methyl-3-isobutylxanthine; 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 8,11,14-Eicosatrienoic Acid; Angina, Unstable; Angioplasty, Balloon, Coronary; Arachidonic Acid; Arachidonic Acids; Colforsin; Cyclic AMP; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Female; Humans; Oxygenases; Platelet Activation; Pre-Eclampsia; Pregnancy; Prostaglandin Endoperoxides, Synthetic; Thrombin

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Brain Chemistry; Cerebrovascular Circulation; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Free Radicals; Indomethacin; Mice; Oxygen; Oxygenases; Prostaglandins; Vasodilation

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Cattle; Cells, Cultured; Coronary Vessels; Dogs; Endothelium, Vascular; Epoprostenol; Hydroxyeicosatetraenoic Acids; Muscle, Smooth, Vascular; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Vasodilation

Arachidonic acid metabolism via cyclooxygenase, lipoxygenase, and cytochrome P-450 epoxygenase was investigated in thoracic aortic tissue obtained from rabbits fed either standard rabbit chow or chow containing 2% cholesterol. Aortic strips were incubated with [14C]arachidonic acid and A23187. Metabolites from extracted media were resolved by high-pressure liquid chromatography (HPLC). Normal and cholesterol-fed rabbit aortas synthesized prostaglandins (PGs) and hydroxyeicosatetraenoic acids (HETEs). The major cyclooxygenase products were 6-keto-PGF1 alpha and PGE2. Basal aortic 6-keto-PGF1 alpha production was slightly reduced in cholesterol-fed compared with normal rabbits. 12(S)- and 15(S)-HETE were the major aortic lipoxygenase products from both normal and cholesterol-fed rabbits. The structures were confirmed by gas chromatography-mass spectrometry (GC-MS). Only cholesterol-fed rabbit aortas metabolized arachidonic acid via cytochrome P-450 epoxygenase to the epoxyeicosatrienoic acids (EETs). 14,15-, 11,12-, 8,9-, and 5,6-EET were identified based on comigration on HPLC with known 14C-labeled standards and typical mass spectra. Incubation of normal aorta with 14,15-EET decreased the basal synthesis of 6-keto-PGF1 alpha. The other EETs were without effect. The four EET regioisomers relaxed the norepinephrine-precontracted normal and cholesterol-fed rabbit aorta. The relaxation response to 14,15-EET was greater in aortas from cholesterol-fed rabbits. These studies demonstrate that hypercholesterolemia, before the development of atherosclerosis, alters arachidonic acid metabolism via both the cyclooxygenase and epoxygenase pathways.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; 6-Ketoprostaglandin F1 alpha; 8,11,14-Eicosatrienoic Acid; Animals; Aorta, Thoracic; Arachidonic Acids; Carbon Radioisotopes; Cholesterol, Dietary; Clotrimazole; Diet, Atherogenic; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Indomethacin; Kinetics; Masoprocol; Metyrapone; Muscle, Smooth, Vascular; Rabbits; Reference Values; Stereoisomerism

Arachidonic acid is metabolized by means of P450 isoenzyme(s) to form epoxyeicosatrienoic acids (EETs) and their corresponding dihydroxy derivatives (DHETs). In the present study, we established the presence in human urine of 8,9-, 11,12-, and 14,15-EETs and their corresponding DHETs by developing quantitative assays and using negative ion, chemical ionization GC/MS and octadeuterated internal standards. Urinary excretion of 8,9- and 11,12-DHET increased in healthy pregnant women compared with nonpregnant female volunteers. By contrast, excretion of 11,12-DHET and 14,15-DHET, but not the 8,9-DHET regioisomer, increased even further in patients with pregnancy-induced hypertension. Intravenous administration of [3H]14,15-EET to three dogs markedly increased its DHET in plasma. The terminal half-life ranged from 7.9-12.3 min and the volume of distribution (3.5-5.3 liters) suggested limited distribution outside the plasma compartment. Negligible radioactivity was detected in urine; this fact infers that under physiological circumstances, urinary DHETs largely derive from the kidney. That P450 metabolites of arachidonic acid are formed in humans supports the hypothesis that these metabolites contribute to the physiological response to normal pregnancy and the pathophysiology of pregnancy-induced hypertension.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Dogs; Fatty Acids, Unsaturated; Female; Gas Chromatography-Mass Spectrometry; Humans; Hypertension; Pre-Eclampsia; Pregnancy; Pregnancy Complications, Cardiovascular; Radioisotope Dilution Technique; Reference Values; Tritium

Four triene monoepoxides of arachidonic acid have been identified as endogenous components of human plasma, the epoxy groups being in the 5,6-, 8,9-, 11,12- and 14,15-positions. Prior to trimethylsilylation and gas chromatographic-mass spectrometric analysis, both the expoxy and ester functions were reduced to hydroxy groups and the double bonds were hydrogenated catalytically. Saturation of the double bonds gave diagnostic spectra that were suitable for elucidating the position of the epoxy group. The shift in the fragmentation of a deuteriated sample verified the presence of the intact epoxides prior to chemical reduction. The presence of the double bonds in the epoxy molecules was demonstrated by reduction using homogeneous catalysis with tris(triphenylphosphine)rhodium(I) chloride and deuterium.

Topics: 8,11,14-Eicosatrienoic Acid; Aluminum; Aluminum Compounds; Deuterium; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Humans; Lithium; Lithium Compounds; Molecular Structure; Oxidation-Reduction

Chiral analysis of the rat liver microsomal arachidonic acid epoxygenase metabolites shows enantioselective formation of 8,9-, 11,12-, and 14,15-cis-epoxyeicosatrienoic acids in an approximately 2:1, 4:1, and 2:1 ratio of antipodes, respectively. Animal treatment with the cytochrome P-450 inducer phenobarbital increased the overall enantiofacial selectivity of the microsomal epoxygenase and caused a concomitant inversion in the absolute configurations of its metabolites. These effects of phenobarbital were time-dependent and temporally linked to increases in the concentration of microsomal cytochrome P-450 enzymes. Reconstitution of the epoxygenase reaction utilizing several purified cytochrome P-450 demonstrated that the asymmetry of epoxidation is under cytochrome P-450 enzyme control. These results established that the chirality of the hepatic arachidonic acid epoxygenase is under regulatory control and confirm cytochromes P-450 IIB1 and IIB2 as two of the endogenous epoxygenases induced in vivo by phenobarbital.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Benzoflavones; beta-Naphthoflavone; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Male; Microsomes, Liver; Oxygenases; Phenobarbital; Rats; Rats, Inbred Strains; Stereoisomerism; Substrate Specificity

Mass spectral and chromatographic analysis demonstrates the presence of 14,15-, 11,12- and 8,9-epoxyeicosatrienoic acids (44%, 33% and 23% of the total, respectively) in human kidney cortex. Chiral analysis of the human renal epoxyeicosatrienoic acids shows the formation of 8,9-, 11,12- and 14,15-epoxyeicosatrienoic acids in a 1:1, 4:1 and 2:1 ratio of antipodes, respectively. These results demonstrate the biosynthetic origin of the human kidney 11,12- and 14,15-epoxyeicosatrienoic acids and suggest a role for renal cytochrome P-450 in the bioactivation of endogenous pools of arachidonic acid.

Topics: 8,11,14-Eicosatrienoic Acid; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Fatty Acids, Unsaturated; Humans; Kidney Cortex; Male; Mass Spectrometry; Oxygenases; Stereoisomerism

Growth hormone secretion was stimulated in vitro by products of arachidonic acid epoxygenase, the epoxyeicosatrienoic acids. 5,6-Epoxyeicosatrienoic and 14,15-epoxyeicosatrienoic acid stimulated growth hormone release from an enriched population of somatotrophs (approximately 85%) by twofold. Inhibition of arachidonic acid metabolism by indomethacin did not affect growth hormone-releasing hormone stimulation of growth hormone release. In contrast, pretreatment of somatotrophs with an 11,12-isonitrile analogue of arachidonic acid that inhibits arachidonic acid epoxygenase, resulted in a 20-25% inhibition of growth hormone-releasing hormone-stimulated growth hormone release. 14,15-Epoxyeicosatrienoic acid stimulated a concentration-dependent increase (twofold) in the cytoplasmic concentration of adenosine 3',5'-cyclic monophosphate (cAMP) in the somatotrophs. 14,15-Epoxyeicosatrienoic acid also rapidly increased the intracellular free calcium concentration in somatotrophs from resting levels (approximately 80 nM) to greater than 250 nM. Growth hormone-releasing hormone increased the free intracellular calcium to 160-180 nM. Preincubation of somatotrophs with somatostatin inhibited growth hormone-releasing hormone-stimulated growth hormone secretion, cAMP accumulation, and 14,15-epoxyeicosatrienoic acid stimulated cAMP accumulation. These data are suggestive that the epoxyeicosatrienoic acids may have a role in the secretion of growth hormone.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Cells, Cultured; Fatty Acids, Unsaturated; Growth Hormone; Growth Hormone-Releasing Hormone; Indomethacin; Kinetics; Male; Pituitary Gland, Anterior; Rats; Rats, Inbred Strains; Somatostatin

A chromatographic method is described for the direct enantiomeric characterization of all four regioisomeric epoxyeicosatrienoic acid (EET) metabolites generated by the cytochrome P450 arachidonate epoxygenase pathway. Following esterification, the individual methyl or pentafluorobenzyl esters are resolved by chiral phase HPLC utilizing a Chiralcel OB or OD column. This methodology will find analytical and preparative applications for chiral epoxides since it is convenient and efficient and does not destroy the epoxide functionality.

Topics: 8,11,14-Eicosatrienoic Acid; Chromatography, High Pressure Liquid; Fatty Acids, Unsaturated; Stereoisomerism

Purified synthetic products from the cytochrome P450 pathway of arachidonate metabolism were applied to the intestinal serosa. Arteriolar blood flow was calculated using video microscopy. After a steady-state baseline, a bolus containing 10-60 micrograms 14,15-epoxyeicosatrienoic acid/ml (14,15-EET) had no detectable effect on blood flow. However, 25 +/- 3 micrograms 11,12-EET/ml and 36 +/- 2 micrograms 8,9-EET/ml caused increases (134 +/- 8% and 127 +/- 6%) that were similar to those elicited by 8 +/- 2 micrograms adenosine/ml (138 +/- 12%). Furthermore, the increases (275 +/- 38%) produced by 32 +/- 6 micrograms 5,6-EET/ml exceeded those elicited (160 +/- 10%) by a similar concentration (27 +/- 3 micrograms/ml) of adenosine. Thus, a structure-activity relationship is suggested. Nevertheless, these values probably underestimate the potency of the EETs because the vasoactivity was reduced by contact with water. The activity of the cyclooxygenase pathway seemed to limit the formation of vasoactive quantities of EETs, or other nonprostanoids, from exogenous arachidonate in the serosa but not the mucosa. A bolus (1.3 +/- 0.2 mg/ml) or continuous application (122 +/- 45 micrograms/ml) of arachidonate caused blood flow increases (236 +/- 14% or 229 +/- 27%) that were almost eliminated (129 +/- 5% or 121 +/- 9%) by a cyclooxygenase inhibitor; the residual response was abolished by a cytochrome P450 inhibitor. However, cytochrome P450 inhibitors alone did not attenuate the arachidonate response. In contrast, a continuous application of 194 micrograms arachidonate/ml to the mucosa caused a markedly smaller blood flow increase (119 +/- 8%) and cyclooxygenase inhibitors potentiated (132 +/- 8%), rather than reduced, this response. We conclude that EETs are a labile class of vasodilators with a potency comparable to adenosine in the intestinal microcirculation. Indirect evidence suggests regional differences in the formation of vasoactive quantities of arachidonate metabolites within the intestinal wall.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Fatty Acids, Unsaturated; Intestinal Mucosa; Intestines; Male; Rats; Regional Blood Flow; Structure-Activity Relationship; Vasodilation

Arachidonic acid (AA) can be metabolized to epoxides and their corresponding diols via the cytochrome P450 epoxygenase pathway. We have compared the vascular activity of four synthetically prepared epoxyeicosatrienoic acids, i.e. 5,6-, 8,9-, 11,12- and 14,15-EET (2-20 microM) on the isolated perfused rat tail artery. The 5,6-EET was equipotent with acetylcholine in dose dependently reducing vascular resistance (ED50 = 3.4 +/- 0.5 microM). The 8,9-, 11,12- and 14,15-EETs of AA did not affect vascular resistance; neither did the 5,6-DHET and delta-lactone, hydrolysis products of 5,6-epoxide. We suggest that the 5,6-epoxide, in contrast to other cytochrome P450-derived products, contributes to the regulation of regional vascular tone.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acids; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Hemodynamics; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Rats; Vasodilator Agents

Certain epoxyeicosatrienoic acids (EETs) that were not cyclooxygenase substrates were effective cyclooxygenase inhibitors. Both (+/-)-14,15-cis-EET and (+/-)-8,9-cis-EET inhibited purified enzyme at concentrations from 1 to 50 microM; (+/-)-11,12-cis-EET was ineffective at concentrations below 100 microM. For the case of 14,15-cis-EET, only the (14R,15S)-stereoisomer was active. Other isomers including (14S,15R)-cis-EET, (14R,15R)-trans-EET, (14S,15S)-trans-EET, and the erythro and threo vicinal 14,15-diols were inactive. In addition to their effects on isolated enzyme preparations, cyclooxygenase activity in platelet suspensions, reflected by thromboxane B2 formation, was also inhibited by (14R,15S)-cis-EET and (+/-)-8,9-cis-EET but not by the other isomers. Thus potency and stereospecificity requirements were maintained for cyclooxygenase within intact platelets. Unlike the stereospecific inhibition of the cyclooxygenase enzyme, platelet aggregation induced by arachidonic acid was inhibited by all EET isomers at concentrations from 1 to 10 microM with no evident stereospecificity. Inhibition of aggregation was not uniformly associated with inhibition of thromboxane B2 formation; ordinarily, these two parameters correlate closely. This dissociation was not maintained for another biochemical process involved in platelet activation. For instance, there was a uniform correlation between inhibition of phosphorylation of a 40-kDa platelet protein and inhibition of aggregation. Our results suggest that effects of EET may originate from either stereospecific or nonspecific mechanisms. Definition of such mechanisms may be important to appreciate any physiological relevance of these substances.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Cyclooxygenase Inhibitors; Fatty Acids, Unsaturated; Humans; Kinetics; Male; Platelet Aggregation; Seminal Vesicles; Sheep; Structure-Activity Relationship