8-11-14-eicosatrienoic-acid and 5-6-epoxy-8-11-14-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

Renal vascular effects of cyclooxygenase and cytochrome P450 metabolites of arachidonic acid have been extensively studied, with major advances having been made. More recently, studies indicate that arachidonic acid metabolites of the lipoxygenase and cytochrome P450 pathway, such as hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids, play novel roles in glomerular mesangial and epithelial cells that are relevant to the pathogenesis of kidney disease associated with diabetes and hypertension. These studies demonstrate that eicosanoids generated during the actions of growth factors and vasoconstrictors can modulate disease processes by affecting vascular homeostasis, inflammation, cellular growth, apoptosis and oxidant stress. In addition, they highlight the important roles played by these oxidized lipids in mediating multiple physiological and pathological functions in the kidney through activation of key signal transduction pathways and genes.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Epithelial Cells; Humans; Hydroxyeicosatetraenoic Acids; Kidney Glomerulus; Lipoxygenase

Arachidonic acid metabolites of the cyclooxygenase and lipoxygenase pathways have a variety of important lung functions. Recent observations indicate that cytochrome P-450 (P-450) monooxygenases are also expressed in the lung, localized to specific pulmonary cell types (e.g., epithelium, endothelium, and smooth muscle), and may modulate critical lung functions. This review summarizes recent data on the presence and biological activity of P-450-derived eicosanoids in the pulmonary vasculature and airways, including effects on pulmonary vascular and bronchial smooth muscle tone and airway epithelial ion transport. We hypothesize a number of potential functions of P-450-derived arachidonate metabolites in the lungs such as contribution to hypoxic pulmonary vasoconstriction, regulation of bronchomotor tone, control of the composition of airway lining fluid, and limitation of pulmonary inflammation. Finally, we describe a number of emerging technologies, including congenic and transgenic strains of experimental animals, P-450 isoform-specific inhibitors and inhibitory antibodies, eicosanoid analogs, and vectors for delivery of P-450 cDNAs and antisense oligonucleotides. These tools will facilitate further studies on the contribution of endogenously formed P-450 eicosanoid metabolites to lung function, under both normal and pathological conditions.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme System; Humans; Hydroxyeicosatetraenoic Acids; Lung; Microsomes; Mixed Function Oxygenases; Polymorphism, Genetic; Receptors, Cell Surface

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

Arachidonic acid is metabolized to biologically active substances by three major enzyme systems including cyclooxygenases, lipoxygenases and cytochrome P450s. The third pathway, P450-dependent pathway, includes allylic oxidation, omega-hydroxylation, and epoxidation of arachidonic acid. Of these metabolites, the physiological role of 20-hydroxyeicosatetraenoic acid (20-HETE) produced by CYP4A isoforms has been extensively studied. 20-HETE affects ion transport, constricts blood vessels and participates in tubuloglomerular feed back. Increased production of 20-HETE is a major factor in elevating blood pressure in spontaneously hypertensive rats (SHR). We have found that CYP4A2 level in SHR is much higher than that of normotensive rat. Recently, factors of endothelial origin other than nitric oxide and prostaglandins were reported. Inhibitors of P450-dependent arachidonic acid metabolism greatly reduce the vasodilator effect and this factor is speculated to be an epoxide of arachidonic acid. We have isolated CYP2C23 from rat kidney and have found that it produces arachidonic acid epoxides. We have investigated changes in the CYP2C23 levels in physiological and pathophysiological conditions. Multiple pathways of arachidonic acid metabolism by P450 have been reported and the diverse properties of these metabolites and the wide distribution of the P450 system make them prime candidates for participation in regulatory mechanisms of the circulation and transporting epithelia.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Blood Pressure; Cytochrome P-450 CYP2J2; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme System; Hydroxyeicosatetraenoic Acids; Ion Transport; Mixed Function Oxygenases; Rats; Rats, Inbred SHR; Vasoconstriction

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Blood Pressure; Cells, Cultured; Cytochrome P-450 Enzyme System; Hydroxyeicosatetraenoic Acids; Kidney; Sodium-Potassium-Exchanging ATPase; Vasodilator Agents

Cells of the thick ascending limb of the loop of Henle (TALH) metabolize arachidonic acid (AA) via the cytochrome P450 monooxygenase system to biologically active products that are resolved into two peaks, P1 and P2, on reverse-phase HPLC. Each peak contains materials that have characteristic biological activity. P1 contains a material that relaxes blood vessels and is structurally similar to a vasodilator, the 5,6 epoxyeicosatrienoic acid (EET). P2 contains a material that inhibits cardiac Na+-K+-ATPase, the major component of which has been identified as the 11,12 dihydroxyeicosatrienoic acid. In mTALH cells obtained from rabbits made hypertensive by aortic coarctation, there was a selective increase in P1 and P2 formation compared to other renomedullary cells. We have identified AA metabolites in bovine corneal epithelium with biological properties and chemical features similar to those of mTALH cells. 12(R)hydroxyeicosatetraenoic acid (12(R) HETE) a possible derivative of the 11,12-EET, is produced by the cornea and also has been shown to inhibit Na+-K+-ATPase activity. Renal microsomes obtained from spontaneously hypertensive rats (SHRs) also metabolize AA via a cytochrome P450 monooxygenase pathway to three principal biologically active metabolites that are formed in increased amounts during the developmental phase of hypertension.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acids; Blood Pressure; Chemical Phenomena; Chemistry; Cytochrome P-450 Enzyme System; Hypertension; Kidney; Kidney Tubules; Loop of Henle; Oxygenases; Sodium-Potassium-Exchanging ATPase

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

The usage of cisplatin, a highly potent chemotherapeutic, is limited by its severe nephrotoxicity. Arachidonic acid (ARA)-derived epoxyeicosatrienoic acids (EETs) and soluble epoxide hydrolase (sEH) inhibitors were shown to ameliorate this dose-limiting side effect, but both approaches have some pharmacological limitations. Analogues of EETs are an alternative avenue with unique benefits, but the current series of analogues face concerns regarding their structure and mimetic functionality. Hence, in this study, regioisomeric mixtures of four new ARA alkyl ethers were synthesized, characterized, and assessed as EET analogues against the concentration- and time-dependent toxicities of cisplatin in porcine proximal tubular epithelial cells. All four ether groups displayed bioisostere activity, ranging from marginal for methoxy- (

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Antineoplastic Agents; Caspase 3; Caspase 9; Cells, Cultured; Cisplatin; Dose-Response Relationship, Drug; Epithelial Cells; Humans; Kidney Tubules, Proximal; Mitochondria; Mitogen-Activated Protein Kinases; Molecular Structure; Oxidative Stress; Signal Transduction; Swine

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

The aim of the study was to investigate the role of cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in sustained hypoxic pulmonary vasoconstriction (HPV).. Vasomotor responses of isolated mouse intrapulmonary arteries (IPAs) were assessed using wire myography. Key findings were verified by haemodynamic measurements in isolated perfused and ventilated mouse lungs.. Pharmacological inhibition of EET synthesis with MS-PPOH, application of the EET antagonist 14,15-EEZE or deficiency of CYP2J isoforms suppressed sustained HPV. In contrast, knockdown of EET-degrading soluble epoxide hydrolase or its inhibition with TPPU augmented sustained HPV almost twofold. All EET regioisomers elicited relaxation in IPAs pre-contracted with thromboxane mimetic U46619. However, in the presence of KCl-induced depolarization, 5,6-EET caused biphasic contraction in IPAs and elevation of pulmonary vascular tone in isolated lungs, whereas other regioisomers had no effect. In patch-clamp experiments, hypoxia elicited depolarization in pulmonary artery smooth muscle cells (PASMCs), and 5,6-EET evoked inward whole cell currents in PASMCs depolarized to the hypoxic level, but not at their resting membrane potential.. The EET pathway substantially contributes to sustained HPV in mouse pulmonary arteries. 5,6-EET specifically appears to be involved in HPV, as it is the only EET regioisomer able to elicit not only relaxation, but also sustained contraction in these vessels. 5,6-EET-induced pulmonary vasoconstriction is enabled by PASMC depolarization, which occurs in hypoxia. The discovery of the dual role of 5,6-EET in the regulation of pulmonary vascular tone may provide a basis for the development of novel therapeutic strategies for treatment of HPV-related diseases.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Cytochrome P-450 Enzyme System; Hypoxia; Lung; Mice; Pulmonary Artery; Vasoconstriction; Vasodilation

Cutaneous wound pain causes physical and psychological stress for patients with wounds. Previous studies reported that stress induces hyperalgesia and deteriorates wound healing. However, the effect of the stress response such as in hypothalamic-pituitary-adrenal (HPA) axis on local wound area is unclear. We aimed to investigate the effects of a stress response on the mechanical withdrawal threshold in the local wound area and describe the identification of a wound pain exacerbation. We topically injected adrenocorticotropic hormone (ACTH) into the granulation tissue of full-thickness cutaneous wound model rats on the fifth day postwounding and measured the mechanical withdrawal thresholds, cytochrome P450 2Bs levels and concentration of 5,6-epoxyeicosatrienoic acid in wound exudate. We found that ACTH induced mechanical hypersensitivity at 4 and 6 hours after injection (P = .004 and .021, respectively), and increased gene expression of cytochrome P450 2B12 expression (P = .046). Concentration of 5,6-EET in the wound exudate was moderately correlated with the mechanical withdrawal threshold (r = -.630). Finally, the mechanical withdrawal threshold in the 5,6-EET group was significantly lower than that in the control group at 2 hours after the injection (P = .015). We propose that 5,6-EET is one of the most promising contributors to the wound pain exacerbation. These findings could guide clinical wound and pain management.

Topics: 8,11,14-Eicosatrienoic Acid; Adrenocorticotropic Hormone; Animals; Corticosterone; Cytochrome P-450 Enzyme System; Granulation Tissue; Hyperalgesia; Hypothalamo-Hypophyseal System; Ion Channels; Male; Models, Neurological; Pain; Pain Threshold; Pituitary-Adrenal System; Rats; Rats, Sprague-Dawley; Skin; Stress, Psychological; TRPV Cation Channels; Up-Regulation; Wound Healing

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

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

TRPV4 cation channel activation by cytochrome P450-mediated derivatives of arachidonic acid (AA), epoxyeicosatrienoic acids (EETs), constitute a major mechanisms of endothelium-derived vasodilatation. Besides, TRPV4 mechano/osmosensitivity depends on phospholipase A

Topics: 8,11,14-Eicosatrienoic Acid; Amino Acid Substitution; Binding Sites; HEK293 Cells; HeLa Cells; Humans; Ion Channel Gating; Molecular Docking Simulation; Protein Binding; TRPV Cation Channels

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

Adipocyte fatty acid-binding protein (FABP4) is a member of a highly conserved family of cytosolic proteins that bind with high affinity to hydrophobic ligands, such as saturated and unsaturated long-chain fatty acids and eicosanoids. Recent evidence has supported a novel role for FABP4 in linking obesity with metabolic and cardiovascular disorders. In this context, we identified FABP4 as a main bioactive factor released from human adipose tissue that directly suppresses heart contraction in vitro. As FABP4 is known to be a transport protein, it cannot be excluded that lipid ligands are involved in the cardiodepressant effect as well, acting in an additional and/or synergistic way.. We investigated a possible involvement of lipid ligands in the negative inotropic effect of adipocyte factors in vitro.. We verified that blocking the CYP epoxygenase pathway in adipocytes attenuates the inhibitory effect of adipocyte-conditioned medium (AM) on isolated adult rat cardiomyocytes, thus suggesting the participation of epoxyeicosatrienoic acids (EETs) in the cardiodepressant activity. Analysis of AM for EETs revealed the presence of 5,6-, 8,9-, 11,12- and 14,15-EET, whereas 5,6-EET represented about 45% of the total EET concentration in AM. Incubation of isolated cardiomyocytes with EETs in similar concentrations as found in AM showed that 5,6-EET directly suppresses cardiomyocyte contractility. Furthermore, after addition of 5,6-EET to FABP4, the negative inotropic effect of FABP4 was strongly potentiated in a concentration-dependent manner.. These data suggest that adipocytes release 5,6-EET and FABP4 into the extracellular medium and that the interaction of these factors modulates cardiac function. Therefore elevated levels of FABP4 and 5,6-EET in obese patients may contribute to the development of heart dysfunction in these subjects.

Topics: 8,11,14-Eicosatrienoic Acid; Adipose Tissue; Animals; Cardiovascular Diseases; Fatty Acid-Binding Proteins; Female; Humans; Male; Myocardial Contraction; Myocytes, Cardiac; Obesity; Rats

Paraoxonase 1 (PON1) is an antiatherogenic high density lipoprotein-associated lactonase. Recent findings revealed that PON1 knockout mice have low blood pressure, which is negatively correlated with the level of 5,6-epoxyeicosatrienoic acid (5,6-EET), a cytochrome P450 -derived arachidonic acid metabolite. 5,6-EET is an endothelium-derived hyperpolarizing factor that causes arterial dilation. Under physiological conditions, 5,6-EET is unstable, transforming to its δ-lactone (5,6-δ-DHTL) that evades the degradation by soluble epoxide hydrolase (sEH), arguing for the existence of yet another enzyme that is responsible specifically for its hydrolysis. We therefore hypothesized that PON1 degrades the 5,6-δ-DHTL, and this specific PON1 lactonase activity thus decreases endothelial vasodilatation. The aim of the present study was to investigate the PON1-5,6-δ-DHTL relationship. A liquid chromatography mass spectrometry based method for 5,6-EET derivatives identification was developed. Tracking the lactonization of 5,6-EET in a physiological solution revealed that 5,6-EET was fully converted into 5,6-δ-DHTL. Incubation of 5,6-δ-DHTL with rePON1 resulted in 85.1±3.4% degradation of the substrate to 5,6 dihydroxytrienoic acid (5,6-DHET), while only 12.0±8.7% hydrolysis was detected in the absence of PON1. Accordingly, the levels of 5,6-DHTL were found to be significantly higher in the PON1KO mice than in the wild type mice. Kinetic analysis revealed values of Vmax=0.021±0.01μM/s and Km=150.99±62.1μM. Calculation of the docking energy suggested possible interaction of the 5,6-δ-DHTL in the catalytic region of PON1 with free energy of-5.57 Kcal/mol, preferentially for the (S) enantiomer. These findings demonstrate that 5,6-δ-DHTL is a PON1 substrate and imply that the 5,6-EET vasodilation effect may be impaired by PON1.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Aryldialkylphosphatase; Catalytic Domain; Epoxide Hydrolases; Gene Expression; Hydroxyeicosatetraenoic Acids; Kidney; Kinetics; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Docking Simulation; Recombinant Proteins; Substrate Specificity; Thermodynamics; Vasodilation

Cytochrome P450 metabolites of arachidonic acid (AA) belong to eicosanoids and are potent lipid mediators of inflammation. It is well-known that eicosanoids play an important role in numerous pathophysiological processes. Therefore, quantitative analysis of cytochrome P450 metabolites of AA, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatreinoic acids (EETs), and dihydroxyeicosatrienoic acids (DHETs) can provide crucial information to uncover underlying mechanisms of cytochrome P450 metabolites of AA related diseases. Herein, we developed a highly sensitive method to identify and quantify HETEs, EETs, and DHETs in lipid extracts of biological samples based on stable isotope probe labeling coupled with ultra high-performance liquid chromatography/mass spectrometry. To this end, a pair of stable isotope probes, 2-dimethylaminoethylamine (DMED) and d4-2-dimethylaminoethylamine (d4-DMED), were utilized to facilely label eicosanoids. The heavy labeled eicosanoid standards were prepared and used as internal standards for quantification to minimize the matrix and ion suppression effects in mass spectrometry analysis. In addition, the detection sensitivities of DMED labeled eicosanoids improved by 3-104 folds in standard solution and 5-138 folds in serum matrix compared with unlabeled analytes. Moreover, a good separation of eicosanoids isomers was achieved upon DMED labeling. The established method provided substantial sensitivity (limit of quantification at sub-picogram), high specificity, and broad linear dynamics range (3 orders of magnitude). We further quantified cytochrome P450 metabolites of AA in rat liver, heart, brain tissues and human serum using the developed method. The results showed that 19 eicosanoids could be distinctly detected and the contents of 11-, 15-, 16-, 20-HETE, 5,6-EET, and 14,15-EET in type 2 diabetes mellitus patients and 5-, 11-, 12-, 15-, 16-, 20-HETE, 8,9-EET, and 5,6-DHET in myeloid leukemia patients had significant changes, demonstrating that these eicosanoids may have important roles on the pathogenesis of type 2 diabetes mellitus and myeloid leukemia.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Brain; Case-Control Studies; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Deuterium; Diabetes Mellitus, Type 2; Eicosanoids; Humans; Hydroxyeicosatetraenoic Acids; Isotope Labeling; Leukemia, Myeloid; Liver; Male; Myocardium; Organ Specificity; Rats, Sprague-Dawley; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

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

T-type calcium channels (T-channels) are important actors in neuronal pacemaking, in heart rhythm, and in the control of the vascular tone. T-channels are regulated by several endogenous lipids including the primary eicosanoid arachidonic acid (AA), which display an important role in vasodilation via its metabolism leading to prostanoids, leukotrienes, and epoxyeicosatrienoic acids (EETs). However, the effects of these latter molecules on T-currents have not been investigated. Here, we describe the effects of the major cyclooxygenase, lipoxygenase, and cytochrome P450 epoxygenase products on the three human recombinant T-channels (Cav3.1, Cav3.2, and Cav3.3), as compared to those of AA. We identified the P450 epoxygenase product, 5,6-EET, as a potent physiological inhibitor of Cav3 currents. The effects of 5,6-EET were observed at sub-micromolar concentrations (IC50 = 0.54 μM), occurred in the minute range, and were reversible. The 5,6-EET inhibited the Cav3 currents at physiological resting membrane potentials mostly by inducing a large negative shift in their steady-state inactivation properties. Using knockout mice for Cav3.1 and Cav3.2, we demonstrated that the vasodilation of preconstricted mesenteric arteries induced by 5,6-EET was specifically impaired in Cav3.2 knockout mice. Overall, our results indicate that inhibition of Cav3 currents by 5,6-EET is an important mechanism controlling the vascular tone.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Calcium Channels, T-Type; Humans; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle Tonus; Muscle, Smooth, Vascular; Patch-Clamp Techniques; Transfection

Cytochrome P450 (CYP) epoxygenases metabolize endogenous polyunsaturated fatty acids to their corresponding epoxides, generating bioactive lipid mediators. The latter play an important role in vascular homeostasis, angiogenesis, and inflammation. As little is known about the functional importance of extra-vascular sources of lipid epoxides, we focused on determining whether lipid epoxide-generating CYP isoforms are expressed in human monocytes/macrophages. Epoxides were generated by freshly isolated human monocytes and production increased markedly during differentiation to macrophages. Mass spectrometric analysis identified CYP2S1 as a novel macrophage CYP and CYP2S1-containing microsomes generated epoxides of arachidonic, linoleic and eicosapentaenoic acid. Macrophage CYP2S1 expression was increased by LPS and IFN-γ (classically activated), and oxidized LDL but not IL-4 and IL-13 (alternatively activated), and was colocalised with CD68 in inflamed human tonsils but not in breast cancer metastases. Prostaglandin (PG) E(2) is an immune modulator factor that promotes phagocytosis and CYP2S1 can metabolize its immediate precursors PGG(2) and PGH(2) to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT). We found that CYP inhibition and siRNA-mediated downregulation of CYP2S1 increased macrophage phagocytosis and that the latter effect correlated with decreased 12-HHT formation. Although no Cyp2s1 protein was detected in aortae from wild-type mice it was expressed in aortae and macrophage foam cells from ApoE(-/-) mice. Consistent with these observations CYP2S1 was colocalised with the monocyte marker CD68 in human atherosclerotic lesions. Thus, CYP2S1 generates 12-HHT and is a novel regulator of macrophage function that is expressed in classical inflammatory macrophages, and can be found in murine and human atherosclerotic plaques.

Topics: 8,11,14-Eicosatrienoic Acid; Amino Acid Sequence; Animals; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Apolipoproteins E; Cells, Cultured; Cytochrome P-450 Enzyme System; Fatty Acids, Unsaturated; Humans; Macrophages; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Monocytes; Phagocytosis; Plaque, Atherosclerotic

Serum paraoxonase 1 (PON1) is an HDL-associated lipolactonase and its association with hypertension is controversial. We studied the possible role of PON1 in blood pressure (BP) regulation, by using PON1 knockout (PON1KO) mice.. Both, systolic and diastolic BPs were lower in PON1KO compared to WT mice. Hypotension detected in PON1KO is probably neither related to nitric oxide/guanylate cyclase-mediated vasodilation nor to angiotensin II or aldosterone-mediated vasoconstriction. Surprisingly, when challenged by high-salt diet, BP was further reduced in PON1KO mice. The later, pointed to a possible involvement of transient receptor potential vanilloid 4 (TRPV4), and indeed, administration of ruthenium red, a TRPV4 blocker, resulted in a sharp rise in BP. The protein levels of TRPV4 in kidneys of PON1KO were not higher than in WT. However, the renal level of 5,6-epoxyeicosatrienoic acid (5,6-EET), a TRPV4 specific agonist, was significantly higher in PON1KO compared with WT mice. 5,6-EET levels were further elevated under high-salt diet or administration of arachidonic acid. Injection of inhibitor of CYP450 epoxygenase resulted in increased BP in PON1KO mice. Injection of recombinant human PON1 resulted in elevation of BP and a concomitant reduction in renal content of 5,6-EET. PON1, in vitro, metabolized 5,6-EET, but not other EETs, to its corresponding diol. Vasodilation, blocked by excess of dietary K(+) but not reversed by depletion of cellular Ca(2+) stores, point to endothelial-derived hyperpolarization-like response.. The present study shows causal, direct relationship between PON1 and blood pressure which is mediated, at least in part, by the regulation of 5,6-EET.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aryldialkylphosphatase; Blood Pressure; Humans; Hypotension; Male; Mice; Mice, Knockout; Sodium Chloride, Dietary; TRPV Cation Channels

Epoxyeicosatrienoic acids (EETs) are cytochrome P450-epoxygenase-derived metabolites of arachidonic acid that act as endogenous signaling molecules in multiple biological systems. Here we have investigated the specific contribution of 5,6-EET to transient receptor potential (TRP) channel activation in nociceptor neurons and its consequence for nociceptive processing. We found that, during capsaicin-induced nociception, 5,6-EET levels increased in dorsal root ganglia (DRGs) and the dorsal spinal cord, and 5,6-EET is released from activated sensory neurons in vitro. 5,6-EET potently induced a calcium flux (100 nm) in cultured DRG neurons that was completely abolished when TRPA1 was deleted or inhibited. In spinal cord slices, 5,6-EET dose dependently enhanced the frequency, but not the amplitude, of spontaneous EPSCs (sEPSCs) in lamina II neurons that also responded to mustard oil (allyl isothiocyanate), indicating a presynaptic action. Furthermore, 5,6-EET-induced enhancement of sEPSC frequency was abolished in TRPA1-null mice, suggesting that 5,6-EET presynaptically facilitated spinal cord synaptic transmission by TRPA1. Finally, in vivo intrathecal injection of 5,6-EET caused mechanical allodynia in wild-type but not TRPA1-null mice. We conclude that 5,6-EET is synthesized on the acute activation of nociceptors and can produce mechanical hypersensitivity via TRPA1 at central afferent terminals in the spinal cord.

Topics: 8,11,14-Eicosatrienoic Acid; Action Potentials; Afferent Pathways; Animals; Cells, Cultured; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Sensory Receptor Cells

Preeclampsia is a multisystem disorder of pregnancy, originating in the placenta. Cytochrome P450 (CYP)-dependent eicosanoids regulate vascular function, inflammation, and angiogenesis, which are mechanistically important in preeclampsia.. We performed microarray screening of placenta and decidua (maternal placenta) from 25 preeclamptic women and 23 control subjects. The CYP subfamily 2J polypeptide 2 (CYP2J2) was upregulated in preeclamptic placenta and decidua. Reverse-transcription polymerase chain reaction confirmed the upregulation, and immunohistochemistry localized CYP2J2 in trophoblastic villi and deciduas at 12 weeks and term. The CYP2J2 metabolites, 5,6-epoxyeicosatrienoic acid (EET), 14,15-EET, and the corresponding dihydroxyeicosatrienoic acids, were elevated in preeclamptic women compared with controls in the latter two thirds of pregnancy and after delivery. Stimulating a trophoblast-derived cell line with the preeclampsia-associated cytokine tumor necrosis factor-α enhanced CYP2J2 gene and protein expression. In 2 independent rat models of preeclampsia, reduced uterine-perfusion rat and the transgenic angiotensin II rat, we observed elevated EET, dihydroxyeicosatrienoic acid, and preeclamptic features that were ameliorated by the CYP epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide (MsPPOH). Uterine arterial rings of these rats also dilated in response to MsPPOH. Furthermore, 5,6-EET could be metabolized to a thromboxane analog. In a bioassay, 5,6-EET increased the beating rate of neonatal cardiomyocytes. Blocking thromboxane synthesis reversed that finding and also normalized large-conductance calcium-activated potassium channel activity.. Our data implicate CYP2J2 in the pathogenesis of preeclampsia and as a potential candidate for the disturbed uteroplacental remodeling, leading to hypertension and endothelial dysfunction.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cells, Cultured; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Fatty Acids, Unsaturated; Female; Humans; Hydrazines; Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; Oligonucleotide Array Sequence Analysis; Placenta; Polymorphism, Single Nucleotide; Pre-Eclampsia; Pregnancy; Rats; Rats, Sprague-Dawley

Oxidation of the endocannabinoid anandamide by cytochrome P450 (P450) enzymes has the potential to affect signaling pathways within the endocannabinoid system and pharmacological responses to novel drug candidates targeting this system. We previously reported that the human cytochromes P450 2D6, 3A4, and 4F2 are high-affinity, high-turnover anandamide oxygenases in vitro, forming the novel metabolites hydroxyeicosatetraenoic acid ethanolamides and epoxyeicosatrienoic acid ethanolamides. The objective of this study was to investigate the possible biological significance of these metabolic pathways. We report that the 5,6-epoxide of anandamide, 5,6-epoxyeicosatrienoic acid ethanolamide (5,6-EET-EA), is a potent and selective cannabinoid receptor 2 (CB2) agonist. The K(i) values for the binding of 5,6-EET-EA to membranes from Chinese hamster ovary (CHO) cells expressing either recombinant human CB1 or CB2 receptor were 11.4 microM and 8.9 nM, respectively. In addition, 5,6-EET-EA inhibited the forskolin-stimulated accumulation of cAMP in CHO cells stably expressing the CB2 receptor (IC(50) = 9.8 +/- 1.3 nM). Within the central nervous system, the CB2 receptor is expressed on activated microglia and is a potential therapeutic target for neuroinflammation. BV-2 microglial cells stimulated with low doses of interferon-gamma exhibited an increased capacity for converting anandamide to 5,6-EET-EA, which correlated with increased protein expression of microglial P450 4F and 3A isoforms. Finally, we demonstrate that 5,6-EET-EA is more stable than anandamide in mouse brain homogenates and is primarily metabolized by epoxide hydrolase. Combined, our results suggest that epoxidation of anandamide by P450s to form 5,6-EET-EA represents an endocannabinoid bioactivation pathway in the context of immune cell function.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acids; Binding, Competitive; CHO Cells; Cricetinae; Cricetulus; Cytochrome P-450 Enzyme System; Dose-Response Relationship, Drug; Endocannabinoids; Humans; Mice; Polyunsaturated Alkamides; Rats; Receptor, Cannabinoid, CB2

Fluid shear stress elicits endothelium-dependent vasodilatation via nitric oxide and prostacyclin-dependent and -independent mechanisms. The latter includes the opening of Ca(2+)-operated potassium channels by cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) leading to endothelial hyperpolarization. We previously reported that EETs activate the transient receptor potential (TRP) V4 channel in vascular endothelial cells and that Ca(2+) influx in these cells in response to mechanical stimuli is dependent on the activation of CYP epoxygenases. We therefore hypothesized that the TRPV4 channel is involved in the flow-induced vasodilatation attributed to the endothelium-derived hyperpolarizing factor (EDHF).. In the presence of N(omega)-nitro-l-arginine methyl ester and diclofenac, precontracted mouse carotid arteries displayed a considerable vasodilatation in response to step-wise increases in luminal flow. The EDHF-mediated, flow-induced vasodilatation could be inhibited by the epoxygenase inhibitor MS-PPOH, was abolished after down-regulation of CYP epoxygenases in tissue culture, and could be restored by viral expression of CYP2C9 in the endothelium. The TRPV4-channel inhibitor ruthenium red (RuR) inhibited the EDHF-mediated flow response, but the combination of MS-PPOH and RuR had no further effect. RuR also inhibited the response in CYP2C9-overexpressing vessels. Moreover, TRPV4-deficient mice demonstrated a blunted EDHF-mediated response to increases in luminal flow in comparison to their wild-type littermates, and the addition of MS-PPOH was without effect in these mice (up to 38 +/- 3% in TRPV4(-/-) vs. 57 +/- 6% in TRPV4(+/+), P < 0.01). In cultured human endothelial cells, exposure to fluid shear stress induced the translocation of the TRPV4 channel from a perinuclear localization to the cell membrane.. We conclude that the TRPV4 channel is involved in flow-induced, endothelium-dependent vasodilatation of murine carotid arteries. Moreover, the activation of the TRPV4 channel by flow requires an active CYP epoxygenase and the translocation of the channel to the cell membrane.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Biological Factors; Carotid Arteries; Cell Membrane; Cell Nucleus; Cells, Cultured; Cytochrome P-450 Enzyme System; Endothelium, Vascular; Epoprostenol; Humans; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide; Regional Blood Flow; TRPV Cation Channels; Vasodilation

Angiotensin II (Ang II) regulates renal proximal transport in a biphasic way via Ang II type 1 receptor (AT1). Whereas extracellular signal-regulated kinase (ERK) activation mediates the stimulatory effect, cytosolic phospholipase A2 (cPLA2) mediates the inhibitory effect independently of ERK. In this study, we tested the hypothesis that the cPLA2/P450 epoxygenase pathway might work to suppress the Ang II-mediated ERK activation. In the presence of arachidonic acid or 5,6-epoxyeicosatrienoic acid (EET), Ang II failed to stimulate the Na-HCO3 cotransporter activity in renal proximal tubules isolated from wild-type, AT1A-deficient, and cPLA2-alpha-deficient mice. In addition, Ang II failed to induce a significant ERK phosphorylation in the presence of arachidonic acid or 5,6-EET. Arachidonic acid or 5,6-EET also suppressed the stimulatory effect of Ang II on net proximal tubule bicarbonate absorption without changing cell Ca2+ concentrations. These results indicate that the cPLA2-alpha/P450/EET pathway blocks the stimulatory effect of Ang II by suppressing the ERK activation. Thus, the cPLA2-alpha/P450/EET pathway may operate as a unique negative feedback mechanism to attenuate excessive Ang II activity in the renal proximal tubules, where extremely high concentrations of Ang II are found.

Topics: 8,11,14-Eicosatrienoic Acid; Angiotensin II; Animals; Arachidonic Acid; Calcium; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Group IV Phospholipases A2; Kidney Tubules, Proximal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Phosphorylation; Receptor, Angiotensin, Type 1; Signal Transduction; Sodium-Bicarbonate Symporters

Protease-activated receptor (PAR(2)) is expressed by nociceptive neurons and activated during inflammation by proteases from mast cells, the intestinal lumen, and the circulation. Agonists of PAR(2) cause hyperexcitability of intestinal sensory neurons and hyperalgesia to distensive stimuli by unknown mechanisms. We evaluated the role of the transient receptor potential vanilloid 4 (TRPV4) in PAR(2)-induced mechanical hyperalgesia of the mouse colon. Colonic sensory neurons, identified by retrograde tracing, expressed immunoreactive TRPV4, PAR(2), and calcitonin gene-related peptide and are thus implicated in nociception. To assess nociception, visceromotor responses (VMR) to colorectal distension (CRD) were measured by electromyography of abdominal muscles. In TRPV4(+/+) mice, intraluminal PAR(2) activating peptide (PAR(2)-AP) exacerbated VMR to graded CRD from 6-24 h, indicative of mechanical hyperalgesia. PAR(2)-induced hyperalgesia was not observed in TRPV4(-/-) mice. PAR(2)-AP evoked discharge of action potentials from colonic afferent neurons in TRPV4(+/+) mice, but not from TRPV4(-/-) mice. The TRPV4 agonists 5',6'-epoxyeicosatrienoic acid and 4alpha-phorbol 12,13-didecanoate stimulated discharge of action potentials in colonic afferent fibers and enhanced current responses recorded from retrogradely labeled colonic dorsal root ganglia neurons, confirming expression of functional TRPV4. PAR(2)-AP enhanced these responses, indicating sensitization of TRPV4. Thus TRPV4 is expressed by primary spinal afferent neurons innervating the colon. Activation of PAR(2) increases currents in these neurons, evokes discharge of action potentials from colonic afferent fibers, and induces mechanical hyperalgesia. These responses require the presence of functional TRPV4. Therefore, TRPV4 is required for PAR(2)-induced mechanical hyperalgesia and excitation of colonic afferent neurons.

Topics: 8,11,14-Eicosatrienoic Acid; Action Potentials; Animals; Calcitonin Gene-Related Peptide; Colon; Electromyography; Female; Ganglia, Spinal; Hyperalgesia; Male; Mice; Mice, Inbred C57BL; Mice, Inbred Strains; Mice, Knockout; Neurons, Afferent; Nociceptors; Phorbol Esters; Rats; Rats, Sprague-Dawley; Receptor, PAR-2; Ruthenium Red; Serous Membrane; TRPC Cation Channels; Viscera

Mechanical and osmotic sensitivity of the transient receptor potential vanilloid 4 (TRPV4) channel depends on phospholipase A(2) (PLA(2)) activation and the subsequent production of the arachidonic acid metabolites, epoxyeicosatrienoic acid (EET). We show that both high viscous loading and hypotonicity stimuli in native ciliated epithelial cells use PLA(2)-EET as the primary pathway to activate TRPV4. Under conditions of low PLA(2) activation, both also use extracellular ATP-mediated activation of phospholipase C (PLC)-inositol trisphosphate (IP(3)) signaling to support TRPV4 gating. IP(3), without being an agonist itself, sensitizes TRPV4 to EET in epithelial ciliated cells and cells heterologously expressing TRPV4, an effect inhibited by the IP(3) receptor antagonist xestospongin C. Coimmunoprecipitation assays indicated a physical interaction between TRPV4 and IP(3) receptor 3. Collectively, our study suggests a functional coupling between plasma membrane TRPV4 channels and intracellular store Ca(2+) channels required to initiate and maintain the oscillatory Ca(2+) signal triggered by high viscosity and hypotonic stimuli that do not reach a threshold level of PLA(2) activation.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Calcium Signaling; Cricetinae; Female; HeLa Cells; Humans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Mechanotransduction, Cellular; Osmosis; Oviducts; Phospholipases A2; Temperature; TRPV Cation Channels; Type C Phospholipases

The acute physiologic release of tissue-type plasminogen activator (t-PA) from the endothelium is critical for vascular homeostasis. This process is prostacyclin- and nitric oxide (NO)-independent in humans. It has been suggested that calcium signaling and endothelial-derived hyperpolarizing factors (EDHF) may play a role in t-PA release. G-protein-coupled receptor-dependent calcium signaling is typically Galphaq-dependent. EDHFs have been functionally defined and in various tissues are believed to be various regioisomers of the epoxyeicosatrienoic acids (EETs). We tested the hypothesis in vitro that thrombin-stimulated t-PA release from human microvascular endothelial cells (HMECs) is both Galphaq- and EDHF-dependent. Conditioned media was harvested following thrombin stimulation, and t-PA antigen was measured by ELISA. Thrombin-induced t-PA release was limited by a membrane-permeable Galphaq inhibitory peptide, the PLC-beta antagonist U73122, and the IP3 receptor antagonist 2-aminoethoxyphenylborane, while the Galphaq agonist Pasteurella toxin modestly induced t-PA release. The cytochrome P450 (CYP450) inhibitor, miconazole, and the arachidonic acid epoxygenase inhibitor MS-PPOH inhibited thrombin-stimulated t-PA release, while 5,6-EET-methyl ester stimulated t-PA release. The 5,6- and 14,15-EET antagonist, 14,15-epoxyeicosa-5(Z)-enoic acid, inhibited t-PA release at the 100 microM concentration. However, thrombin-stimulated t-PA release was unaffected by the prostacyclin and NO inhibitors ASA and L-NAME, as well as the potassium channel inhibitors TEA, apamin and charybdotoxin. These studies suggest that thrombin-stimulated t-PA release is Galphaq-, PLC-beta-, IP3-, and 5,6-EET-dependent while being prostacyclin-, NO- and K+ channel-independent in HMECs.

Topics: 8,11,14-Eicosatrienoic Acid; Aorta; Biological Factors; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Endothelial Cells; Epoprostenol; GTP-Binding Protein alpha Subunits, Gq-G11; Humans; Inositol 1,4,5-Trisphosphate; Isoenzymes; Microcirculation; Nitric Oxide; Phospholipase C beta; Potassium; Signal Transduction; Thrombin; Time Factors; Tissue Plasminogen Activator; Type C Phospholipases; Umbilical Veins

Epoxyeicosatrienoic acids (EETs) have been reported to contract intralobar pulmonary arteries (PA) of the rabbit in a cyclooxygenase (COX)-dependent manner. In the present study, we observed that COX-1 and COX-2 isoforms were expressed in freshly isolated PA of healthy rabbits. We examined the hypothesis that both COX isoforms participate in 5,6-EET-induced contraction of rabbit intralobar PA. Selective inhibition of COX-1 with 300 nM 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazole (SC-560) prevented 5,6-EET (1x10(-8)-1x10(-5) M)-induced contractions of isolated intralobar rabbit PA rings in a manner similar to that observed with the nonselective cyclooxygenase inhibitor indomethacin at 10 microM. Selective inhibition of COX-2 with either 100 nM 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl) thiophene (DUP-697) or 3 microM N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (NS-398) shifted the EC50 value of 5,6-EET-induced PA contraction to the right but with considerably lower efficacy than SC-560. In rabbit PA, 5,6-EET-induced contraction was primarily dependent on COX-1 activity. Differential metabolism of 5,6-EET by COX-1 and COX-2 does not explain the primary dependence of PA contraction on COX-1 activity because 5,6-EET was metabolized similarly by both COX isoforms. COX-1 and -2 were expressed primarily in PA endothelium where COX-1 expression was dense and uniform, whereas COX-2 expression was sparse and nonuniform. 5,6-EET-induced PA contraction was endothelium-dependent. These results suggest that 5,6-EET-induced contraction is primarily dependent on COX-1 activity.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Cyclooxygenase 1; Cyclooxygenase 2; Immunohistochemistry; In Vitro Techniques; Pulmonary Artery; Rabbits; Vasoconstriction

The endocannabinoid anandamide is an arachidonic acid derivative that is found in most tissues where it acts as an important signaling mediator in neurological, immune, cardiovascular, and other functions. Cytochromes P450 (P450s) are known to oxidize arachidonic acid to the physiologically active molecules hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs), which play important roles in blood pressure regulation and inflammation. To determine whether anandamide can also be oxidized by P450s, its metabolism by human liver and kidney microsomes was investigated. The kidney microsomes metabolized anandamide to a single mono-oxygenated product, which was identified as 20-HETE-ethanolamide (EA). Human liver microsomal incubations with anandamide also produced 20-HETE-EA in addition to 5,6-, 8,9-, 11-12, and 14,15-EET-EA. The EET-EAs produced by the liver microsomal P450s were converted to their corresponding dihydroxy derivatives by microsomal epoxide hydrolase. P450 4F2 was identified as the isoform that is most probably responsible for the formation of 20-HETE-EA in both human kidney and human liver, with an apparent Km of 0.7 microM. The apparent Km values of the human liver microsomes for the formation of the EET-EAs were between 4 and 5 microM, and P450 3A4 was identified as the primary P450 in the liver responsible for epoxidation of anandamide. The in vivo formation and biological relevance of the P450-derived HETE and EET ethanolamides remains to be determined.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acids; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme System; Endocannabinoids; Epoxy Compounds; Humans; Hydrogen-Ion Concentration; Hydroxyeicosatetraenoic Acids; Kidney; Kinetics; Microsomes, Liver; Polyunsaturated Alkamides; Spectrometry, Mass, Electrospray Ionization

TRPV4, a Ca(2+)-permeable member of the vanilloid subgroup of the transient receptor potential (TRP) channels, is activated by cell swelling and moderate heat (>27 degrees C) as well as by diverse chemical compounds including synthetic 4 alpha-phorbol esters, the plant extract bisandrographolide A, and endogenous epoxyeicosatrienoic acids (EETs; 5,6-EET and 8,9-EET). Previous work identified a tyrosine residue located in the first half of putative transmembrane segment 3 (TM3) as a crucial determinant for the activation of TRPV4 by its most specific agonist 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), suggesting that 4 alpha-PDD interacts with the channel through its transmembrane segments. To obtain insight in the 4 alpha-PDD-binding site and in the mechanism of ligand-dependent TRPV4 activation, we investigated the consequences of specific point mutations in TM4 on the sensitivity of the channel to different chemical and physical stimuli. Mutations of two hydrophobic residues in the central part of TM4 (Leu(584) and Trp(586)) caused a severe reduction of the sensitivity of the channel to 4 alpha-PDD, bisandrographolide A, and heat, whereas responses to cell swelling, arachidonic acid, and 5,6-EET remained unaffected. In contrast, mutations of two residues in the C-terminal part of TM4 (Tyr(591) and Arg(594)) affected channel activation of TRPV4 by all stimuli, suggesting an involvement in channel gating rather than in interaction with agonists. Based on a comparison of the responses of WT and mutant TRPV4 to 4 alpha-PDD and different 4 alpha-phorbol esters, we conclude that the length of the fatty acid moiety determines the ligand binding affinity and propose a model for the interaction between 4 alpha-phorbol esters and the TM3/4 region of TRPV4.

Topics: 8,11,14-Eicosatrienoic Acid; Amino Acid Substitution; Animals; Carcinogens; Cell Line; Humans; Ion Channel Gating; Mice; Models, Molecular; Phorbol Esters; Point Mutation; Protein Binding; Protein Structure, Tertiary; TRPV Cation Channels; Vasodilator Agents

A major pathway for Ca(2+) entry in non-excitable cells is activated following depletion of intracellular Ca(2+) stores. A de novo conformational coupling between elements in the plasma membrane (PM) and Ca(2+) stores has been proposed as the most likely mechanism to activate this capacitative Ca(2+) entry (CCE) in several cell types, including platelets. Here we report that a cytochrome P450 metabolite, 5,6-EET, might be a component of the de novo conformational coupling in human platelets. In these cells, 5,6-EET induces divalent cation entry without having any detectable effect on Ca(2+) store depletion. 5,6-EET-induced Ca(2+) entry was sensitive to the CCE blockers 2-APB, lanthanum, SKF-96365 and nickel and impaired by incubation with anti-hTRPC1 antibody. Ca(2+) entry stimulated by low concentrations of thapsigargin, which selectively depletes the dense tubular system and induces EET production, was impaired by the cytochrome P450 inhibitor 17-ODYA, which has no effect on CCE mediated by depletion of the acidic stores using 2,5-di-(tert-butyl)-1,4-hydroquinone. We have found that 5,6-EET-induced Ca(2+) entry requires basal levels of H(2)O(2), which might maintain a redox state favourable for this event. Finally, our results indicate that 5,6-EET induces the activation of tyrosine kinase proteins and the reorganization of the actin cytoskeleton, which might provide a support for the transport of portions of the Ca(2+) store towards the PM to facilitate de novo coupling between IP(3)R type II and hTRPC1 detected by coimmunoprecipitation. We propose that the involvement of 5,6-EET in TG-induced coupling between IP(3)R type II and hTRPC1 and subsequently CCE is compatible with the de novo conformational coupling in human platelets.

Topics: 8,11,14-Eicosatrienoic Acid; Actins; Antibodies; Blood Platelets; Boron Compounds; Calcium; Calcium Channels; Cell Membrane; Cell Membrane Permeability; Cytochalasin D; Cytochrome P-450 Enzyme System; Cytoskeleton; Enzyme Activation; Humans; Hydrogen Peroxide; Imidazoles; Inositol 1,4,5-Trisphosphate Receptors; Lanthanum; Manganese; Nickel; Protein-Tyrosine Kinases; Receptors, Cytoplasmic and Nuclear; Thapsigargin; TRPC Cation Channels

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

Neuronal activity evokes localized changes in blood flow. Although this response, termed neurovascular coupling, is widely used to monitor human brain function and diagnose pathology, the cellular mechanisms that mediate the response remain unclear. We investigated the contribution of glial cells to neurovascular coupling in the acutely isolated mammalian retina. We found that light stimulation and glial cell stimulation can both evoke dilation or constriction of arterioles. Light-evoked and glial-evoked vasodilations were blocked by inhibitors of cytochrome P450 epoxygenase, the synthetic enzyme for epoxyeicosatrienoic acids. Vasoconstrictions, in contrast, were blocked by an inhibitor of omega-hydroxylase, which synthesizes 20-hydroxyeicosatetraenoic acid. Nitric oxide influenced whether vasodilations or vasoconstrictions were produced in response to light and glial stimulation. Light-evoked vasoactivity was blocked when neuron-to-glia signaling was interrupted by a purinergic antagonist. These results indicate that glial cells contribute to neurovascular coupling and suggest that regulation of blood flow may involve both vasodilating and vasoconstricting components.

Topics: 8,11,14-Eicosatrienoic Acid; Adenosine Triphosphate; Amidines; Animals; Arterioles; Calcium Signaling; Caproates; Cyclic N-Oxides; Cytochrome P-450 CYP2J2; Cytochrome P-450 CYP4A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Eye Proteins; Hydrazines; Hydroxyeicosatetraenoic Acids; Imidazoles; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Male; Miconazole; Neuroglia; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Photolysis; Proadifen; Purinergic Antagonists; Rats; Rats, Long-Evans; Retinal Vessels; Signal Transduction; Vasoconstriction; Vasodilation; Vasomotor System

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 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

5,6-epoxyeicosatrienoic acid (5,6-EET) is a cytochrome P450 epoxygenase metabolite of arachidonic acid that causes vasorelaxation. However, investigations of its role in biological systems have been limited by its chemical instability. We developed a stable agonist of 5,6-EET, 5-(pentadeca-3(Z),6(Z),9(Z)-trienyloxy)pentanoic acid (PTPA), in which the 5,6-epoxide was replaced with a 5-ether. PTPA obviates chemical and enzymatic hydrolysis. In bovine coronary artery rings precontracted with U46619, PTPA (1 nmol/L to 10 micromol/L) induced concentration-dependent relaxations, with maximal relaxation of 86+/-5% and EC50 of 1 micromol/L. The relaxations were inhibited by the cyclooxygenase inhibitor indomethacin (10 micromol/L; max relaxation 43+/-9%); the ATP-sensitive K+ channel inhibitor glybenclamide (10 micromol/L; max relaxation 49+/-6%); and the large conductance calcium-activated K+ channel inhibitor iberiotoxin (100 nmol/L; max relaxation 38+/-6%) and abolished by the combination of iberiotoxin with indomethacin or glybenclamide or increasing extracellular K+ to 20 mmol/L. Whole-cell outward K+ current was increased nearly 6-fold by PTPA (10 micromol/L), which was also blocked by iberiotoxin. Additionally, we synthesized 5-(pentadeca-6(Z),9(Z)-dienyloxy)pentanoic acid and 5-(pentadeca-3(Z),9(Z)-dienyloxy)pentanoic acid (PDPA), PTPA analogs that lack the 8,9 or 11,12 double bonds of arachidonic acid and therefore are not substrates for cyclooxygenase. The PDPAs caused concentration-dependent relaxations (max relaxations 46+/-13% and 52+/-7%, respectively; EC50 1micromol/L), which were not altered by glybenclamide but blocked by iberiotoxin. These studies suggested that PTPA induces relaxation through 2 mechanisms: (1) cyclooxygenase-dependent metabolism to 5-ether-containing prostaglandins that activate ATP-sensitive K+ channels and (2) activation of smooth muscle large conductance calcium-activated K+ channels. PDPAs only activate large conductance calcium-activated K+ channels.

Topics: 8,11,14-Eicosatrienoic Acid; Adenosine Triphosphate; Animals; Cattle; Coronary Vessels; Drug Stability; Electric Conductivity; Glyburide; In Vitro Techniques; Indomethacin; Patch-Clamp Techniques; Pentanoic Acids; Peptides; Potassium Channel Blockers; Potassium Channels; Potassium Channels, Calcium-Activated; Prostaglandin-Endoperoxide Synthases; Vasodilation

A novel, facile synthesis of 5,6-trans-epoxyeicosatrienoic acid (5,6-trans-EET) from 5,6-trans-arachidonic acid by iodolactonization and alkaline de-iodation is described along with characterization by mass spectrometry (LC-MS, negative ions) and NMR and comparison with 5,6-cis-EET.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acid; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism

5,6-trans-AA (5,6-TAA, where TAA stands for trans-arachidonic acid) is a recently identified trans fatty acid that originates from the cis-trans isomerization of AA initiated by the NO2 radical. This trans fatty acid has been detected in blood circulation and we suggested that it functions as a lipid mediator of the toxic effects of NO2. To understand its role as a lipid mediator, we studied the metabolism of 5,6-TAA by liver microsomes stimulated with NADPH. Profiling of metabolites by liquid chromatography/MS revealed a complex mixture of oxidized products among which were four epoxides, their respective hydrolysis products (dihydroxyeicosatrienoic acids), and several HETEs (hydroxyeicosatetraenoic acids) resulting from allylic, bis-allylic and (omega-1)/(omega-2) hydroxylations. We found that the C5-C6 trans bond competed with the three cis bonds for oxidative metabolism mediated by CYP (cytochrome P450) epoxygenase and hydroxylase. This was evidenced by the detection of 5,6-trans-EET (where EET stands for epoxyeicosatrienoic acid), 5,6-erythro-dihydroxyeicosatrienoic acid and an isomer of 5-HETE. A standard of 5,6-trans-EET obtained by iodolactonization of 5,6-TAA was used for the unequivocal identification of the unique microsomal epoxide in which the oxirane ring was of trans configuration. Additional lipid products originated from the metabolism involving the cis bonds and thus these metabolites had the trans C5-C6 bond. The 5,6-trans-isomers of 18- and 19-HETE were likely to be products of the CYP2E1, because a neutralizing antibody partially inhibited their formation without having an effect on the formation of the epoxides. Our study revealed a novel pathway of microsomal oxidative metabolism of a trans fatty acid in which both cis and trans bonds participated. Of particular significance is the detection of the trans-epoxide of AA, which may be involved in the metabolic activation of such trans fatty acids and probably contribute to their biological activity. Unlike its cis-isomer, 5,6-trans-EET was significantly more stable and resisted microsomal hydrolysis and conjugation with glutathione catalysed by hepatic glutathione S-transferase.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Cytochrome P-450 Enzyme System; Glutathione; Glutathione Transferase; Hydrolysis; Hydroxyeicosatetraenoic Acids; Hydroxylation; Microsomes, Liver; Molecular Structure; NADP; Oxidation-Reduction; Rats

The cytochrome P450 arachidonic acid epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs) are powerful, nonregioselective, stimulators of cell proliferation. In this study we compared the ability of the four EETs (5,6-, 8,9-, 11,12-, and 14,15-EETs) to regulate endothelial cell proliferation in vitro and angiogenesis in vivo and determined the molecular mechanism by which EETs control these events. Inhibition of the epoxygenase blocked serum-induced endothelial cell proliferation, and exogenously added EETs rescued cell proliferation from epoxygenase inhibition. Studies with selective ERK, p38 MAPK, or PI3K inhibitors revealed that whereas activation of p38 MAPK is required for the proliferative responses to 8,9- and 11,12-EET, activation of PI3K is necessary for the cell proliferation induced by 5,6- and 14,15-EET. Among the four EETs, only 5,6- and 8,9-EET are capable of promoting endothelial cell migration and the formation of capillary-like structures, events that are dependent on EET-mediated activation of ERK and PI3K. Using subcutaneous sponge models, we showed that 5,6- and 8,9-EET are pro-angiogenic in mice and that their neo-vascularization effects are enhanced by the co-administration of an inhibitor of EET enzymatic hydration, presumably because of reduced EET metabolism and inactivation. These studies identify 5,6- and 8,9-EET as powerful and selective angiogenic lipids, provide a functional link between the EET proliferative chemotactic properties and their angiogenic activity, and suggest a physiological role for them in angiogenesis and de novo vascularization.

Topics: 8,11,14-Eicosatrienoic Acid; Angiogenesis Inducing Agents; Animals; Cell Proliferation; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Eicosanoids; Endothelium, Vascular; Enzyme Inhibitors; Lipids; Mice; Neovascularization, Physiologic; Oxygenases; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases

The mechanism mediating epoxyeicosatrienoic acid (EET)-induced contraction of intralobar pulmonary arteries (PA) is currently unknown. EET-induced contraction of PA has been reported to require intact endothelium and activation of the thromboxane/endoperoxide (TP) receptor. Because TP receptor occupation with the thromboxane mimetic U-46619 contracts pulmonary artery via Rho-kinase activation, we examined the hypothesis that 5,6-EET-induced contraction of intralobar rabbit pulmonary arteries is mediated by a Rho-kinase-dependent signaling pathway. In isolated rings of second-order intralobar PA (1-2 mm OD) at basal tension, 5,6-EET (0.3-10 microM) induced increases in active tension that were inhibited by Y-27632 (1 microM) and HA-1077 (10 microM), selective inhibitors of Rho-kinase activity. In PA in which smooth muscle intracellular Ca(2+) concentration ([Ca(2+)](i)) was increased with KCl (25 mM) to produce a submaximal contraction, 5,6-EET (1 microM) induced a contraction that was 7.0 +/- 1.6 times greater than without KCl. 5,6-EET (10 microM) also contracted beta-escin permeabilized PA in which [Ca(2+)](i) was clamped at a concentration resulting in a submaximal contraction. Y-27632 inhibited the 5,6-EET-induced contraction in permeabilized PA. 5,6-EET (10 microM) increased phosphorylation of myosin light chain (MLC), increasing the ratio of phosphorylated MLC/total MLC from 0.10 +/- 0.03 to 0.30 +/- 0.02. Y-27632 prevented this increase in MLC phosphorylation. These data suggest that 5,6-EET induces contraction in intralobar PA by increasing Rho-kinase activity, phosphorylating MLC, and increasing the Ca(2+) sensitivity of the contractile apparatus.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 8,11,14-Eicosatrienoic Acid; Amides; Animals; Calcium Chloride; Enzyme Activation; Enzyme Inhibitors; In Vitro Techniques; Intracellular Signaling Peptides and Proteins; Myosin Light Chains; Phosphorylation; Potassium Chloride; Protein Serine-Threonine Kinases; Pulmonary Artery; Pyridines; Rabbits; rho-Associated Kinases; Vasoconstriction; Vasoconstrictor Agents

TRPV4 is a broadly expressed Ca2+-permeable cation channel in the vanilloid subfamily of transient receptor potential channels. TRPV4 gates in response to a large variety of stimuli, including cell swelling, warm temperatures, the synthetic phorbol ester 4alpha-phorbol 12,13-didecanoate (4alpha-PDD), and the endogenous lipid arachidonic acid (AA). Activation by cell swelling and AA requires cytochrome P450 (CYP) epoxygenase activity to convert AA to epoxyeicosatrienoic acids (EETs) such as 5,6-EET, 8,9-EET, which both act as direct TRPV4 agonists. To evaluate the role of TRPV4 and its modulation by the CYP pathway in vascular endothelial cells, we performed Ca2+ imaging and patch-clamp measurements on mouse aortic endothelial cells (MAECs) isolated from wild-type and TRPV4(-/-) mice. All TRPV4-activating stimuli induced robust Ca2+ responses in wild-type MAECs but not in MAECs isolated from TRPV4(-/-) mice. Upregulation of CYP2C expression by preincubation with nifedipine enhanced the responses to AA and cell swelling in wild-type MAECs, whereas responses to other stimuli remained unaffected. Conversely, inhibition of CYP2C9 activity with sulfaphenazole abolished the responses to AA and hypotonic solution (HTS). Moreover, suppression of EET hydrolysis using 1-adamantyl-3-cyclo-hexylurea or indomethacin, inhibitors of soluble epoxide hydrolases (sEHs), and cyclooxygenases, respectively, enhanced the TRPV4-dependent responses to AA, HTS, and EETs but not those to 4alpha-PDD or heat. Together, our data establish that CYP-derived EETs modulate the activity of TRPV4 channels in endothelial cells and shows the unraveling of novel modulatory pathways via CYP2C modulation and sEH inhibition.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Calcium; Cells, Cultured; Cytochrome P-450 Enzyme System; Endothelial Cells; Epoxide Hydrolases; Mice; Nifedipine; TRPV Cation Channels

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

A novel eicosanoid, 5,6-trans-epoxy-8Z,11Z,14Z-eicosatrienoic acid (5,6-trans-EET), was identified in rat red blood cells. Characterization of 5,6-trans-EET in the sn-2 position of the phospholipids was accomplished by hydrolysis with phospholipase A(2) followed by gas chromatography/mass spectrometry as well as electrospray ionization-tandem mass spectrometry analyses. The electron ionization spectrum of 5,6-erythro-dihydroxyeicosatrienoic acid (5,6-erythro-DHET), converted from 5,6-trans-EET in the samples, matches that of the authentic standard. Hydrogenation of the extracted 5,6-erythro-DHET with platinum(IV) oxide/hydrogen resulted in an increase of the molecular mass by 6 daltons and the same retention time shift as an authentic standard in gas chromatography, suggesting the existence of three olefins as well as the 5,6-erythro-dihydroxyl structure in the metabolite. Match of retention times by chromatography indicated identity of the stereochemistry of the red blood cell 5,6-erythro-DHET vis à vis the synthetic standard. High pressure liquid chromatography-electrospray ionization-tandem mass spectrometry analysis of the phospholipase A(2)-hydrolyzed lipid extracts from red blood cells revealed match of the mass spectrum and retention time of the compound with the authentic 5,6-trans-EET standard, providing direct evidence of the existence of 5,6-trans-EET in red blood cells. The presence of other trans-EETs was also demonstrated. The ability of both 5,6-trans-EET and its product 5,6-erythro-DHET to relax preconstricted renal interlobar arteries was significantly greater than that of 5,6-cis-EET. In contrast, 5,6-cis-EET and 5,6-trans-EET were equipotent in their capacity to inhibit collagen-induced rat platelet aggregation, whereas 5,6-erythro-DHET was without effect. We propose that the red blood cells serve as a reservoir for epoxides which on release may act in a vasoregulatory capacity.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arteries; Blood Platelets; Chromatography, Gas; Chromatography, High Pressure Liquid; Collagen; Erythrocytes; Kidney; Lipids; Mass Spectrometry; Models, Chemical; Phospholipases A; Phospholipids; Platelet Aggregation; Rats; Rats, Sprague-Dawley; Spectrometry, Mass, Electrospray Ionization; Time Factors

In the rabbit, 5,6-epoxyeicosatrienoic acid (EET) was reported both to dilate and to constrict pulmonary blood vessels. We propose that these seemingly contradictory results could be explained by differences in responses to 5,6-EET in large-conductance pulmonary arteries (PA) compared with smaller PA and resistance vessels. Thus we found that in rings of extralobar PA [>2-mm outside diameter (OD)], in which active tension had been increased with PGF(2alpha), 5,6-EET produced relaxation in a concentration- and cyclooxygenase (COX)-dependent manner. In contrast, 5,6-EET increased tension in intralobar (1- to 2-mm OD) PA. Small extralobar PA (2- to 2.5-mm OD) exhibited intermediate responses. In the intact lung, the net effect of 5,6-EET (1 x 10(-8)-1 x 10(-5) M) was an increase in pulmonary vascular resistance (PVR) from 13.0 +/- 0.5 to 47.8 +/- 4.6 mmHg. 100 ml(-1) x min(-1) (EC(50) 5.9 +/- 1.7 x 10(-7) M). The increase in PVR was accompanied by a 10-fold increase in perfusate thromboxane (TX)B(2) concentration. The 5,6-EET-induced increase in PVR was prevented with indomethacin (100 microM), a cyclooxygenase inhibitor, or ONO-3708 (20 microM), a TX/PGH(2) (TP) receptor antagonist, but not with OKY-046 (700 microM), a TX synthase inhibitor. These results demonstrate that although 5,6-EET dilates large extralobar PA segments in a COX-dependent manner, in the intact rabbit lung 5,6-EET produces constriction that requires synthesis of a COX-dependent agonist of the TP receptor other than TX.

Topics: 6-Ketoprostaglandin F1 alpha; 8,11,14-Eicosatrienoic Acid; Animals; Cyclooxygenase Inhibitors; Epoprostenol; Immunoenzyme Techniques; In Vitro Techniques; Indomethacin; Methacrylates; Muscle Contraction; Muscle, Smooth, Vascular; Prostaglandin-Endoperoxide Synthases; Pulmonary Artery; Pulmonary Circulation; Rabbits; Receptors, Prostaglandin; Receptors, Thromboxane A2, Prostaglandin H2; Thromboxane A2; Thromboxane B2; Thromboxanes; Vascular Resistance

Human trophoblast cells are known to release a range of arachidonic acid metabolites into culture medium, including cyclo-oxygenase, lipoxygenase and epoxygenase products. In this study we investigated the effects of dibutyryl cyclic AMP (db cAMP) on arachidonic acid metabolism in human first trimester trophoblast cells, and also determined the distribution of metabolites between intracellular and extracellular compartments. db cAMP increased intracellular levels of radioactivity within 2 min, and extracellular levels of radioactivity were increased after 30 min. These changes were reflected in increased levels of arachidonic acid metabolites in both compartments, indicating that arachidonic acid was metabolised. db cAMP increased intracellular levels of 5,6-epoxyeicosatrienoic acid (5,6-EpETrE) within 2 min of addition to cultured cells. No changes were detected after 5-10 min, but substantial changes were found 30 min after the addition of db cAMP. The dihydroxyeicosatrienoic acid (DiHETrE) breakdown products also increased with similar kinetics. In contrast, levels of 14,15-EpETrE increased after 5-10 min.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acid; Bucladesine; Cells, Cultured; Chromatography, High Pressure Liquid; Female; Humans; Pregnancy; Pregnancy Trimester, First; Tritium; Trophoblasts

We have shown a cytochrome P450-dependent renal vasodilator effect of arachidonic acid in response to inhibition of cyclooxygenase and elevation of perfusion pressure, which was enhanced in the spontaneously hypertensive rat (SHR) and linked to increased production of and/or responsiveness to epoxyeicosatrienoic acids (EETs). In the SHR, vasodilation elicited by low doses of arachidonic acid was attenuated by the nitric oxide synthase inhibitor Nw-nitro-L-arginine (50 micromol/L), whereas the responses to high doses were unaffected. Inhibition of epoxygenases with miconazole (0.3 micromol/L) in the presence of Nw-nitro-L-arginine greatly reduced the renal vasodilator response to all doses of arachidonic acid. Tetraethylammonium (10 mmol/L), a nonselective K+ channel blocker, abolished the nitric oxide-independent renal vasodilator effect of arachidonic acid as well as the vasodilator effect of 5,6-EET, confirming that EET-dependent vasodilation involves activation of K+ channels. Under conditions of elevated perfusion pressure (200 mm Hg) and cyclooxygenase inhibition, 5,6-EET, 8, 9-EET, and 11,12-EET caused renal vasodilatation in both SHR and Wistar-Kyoto rats (WKY), whereas 14,15-EET produced vasoconstriction. 5,6-EET was the most potent renal vasodilator of the EET regioisomers in the SHR by a factor of 4 or more. In the SHR, 5,6-EET- and 11,12-EET-induced renal vasodilatation was >2-fold greater than that registered in WKY. Thus, the augmented vasodilator responses to arachidonic acid in the SHR is through activation of K+ channels, and 5,6-EET is the most likely mediator.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Hypertension; Kidney; Male; Potassium Channel Blockers; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Tetraethylammonium; Vasodilation; Vasodilator Agents

Endothelium-dependent hyperpolarizations and relaxation of vascular smooth muscle induced by acetylcholine and bradykinin are mediated by endothelium-derived hyperpolarizing factors (EDHFs). In bovine coronary arteries, arachidonic acid metabolites, epoxyeicosatrienoic acids (EETs), function as EDHFs. The 14,15-EET analog 14,15-epoxyeicosa-5(Z)-enoic-methylsulfonylimide (14,15-EEZE-mSI) was synthesized and tested for agonist and antagonist activity. In U46619-preconstricted bovine coronary arterial rings, 14,15-, 11,12-, 8,9-, and 5,6-EET induced maximal concentration-related relaxation averaging 75% to 87% at 10 micromol/L, whereas, 14,15-EEZE-mSI induced maximal relaxation averaging only 7%. 14,15-EEZE-mSI (10 micromol/L) preincubation inhibited relaxation to 14,15- and 5,6- EET but not 11,12- or 8,9- EET. 14,15-EEZE-mSI also inhibited indomethacin-resistant relaxation to arachidonic acid and indomethacin-resistant and l-nitroarginine-resistant relaxation to bradykinin and methacholine. It did not alter the relaxation to sodium nitroprusside, iloprost, or the K+ channel openers bimakalim or NS1619. In cell-attached patches of isolated bovine coronary arterial smooth muscle cells, 14,15-EEZE-mSI (100 nmol/L) blocked the 14,15-EET-induced (100 nmol/L) activation of large-conductance, calcium-activated K+ channels. Mass spectrometric analysis of rat renal cortical microsomes incubated with arachidonic acid showed that 14,15-EEZE-mSI (10 micromol/L) increased EET concentrations while decreasing the concentrations of the corresponding dihydroxyeicosatrienoic acids. Therefore, 14,15-EEZE-mSI inhibits relaxation to 5,6- and 14,15- EET and the K+ channel activation by 14,15-EET. It also inhibits the EDHF component of bradykinin-induced, methacholine-induced, and arachidonic acid-induced relaxation. These results suggest that 14,15- or 5,6 -EET act as an EDHF in bovine coronary arteries.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Cattle; Coronary Vessels; Culture Techniques; Patch-Clamp Techniques; Potassium Channels, Calcium-Activated; Rats; Sulfonamides; Vasodilator Agents

We examined the responses of newborn piglet pulmonary resistance arteries (PRAs) to 5,6-epoxyeicosatrienoic acid (5,6-EET), a cytochrome P-450 metabolite of arachidonic acid. In PRAs preconstricted with a thromboxane A(2) mimetic, 5,6-EET caused a concentration-dependent dilation. This dilation was partially inhibited by the combination of charybdotoxin (CTX) and apamin, inhibitors of large and small conductance calcium-dependent potassium (K(Ca)) channels, and was abolished by depolarization of vascular smooth muscle with KCl. Disruption of the endothelium significantly attenuated the dilation, suggesting involvement of one or more endothelium-derived vasodilator pathways in this response. The dilation was partially inhibited by nitro-L-arginine (L-NA), an inhibitor of nitric oxide synthase (NOS), but was unaffected by indomethacin, a cyclooxygenase (COX) inhibitor. The combined inhibition of NOS and K(Ca) channels with L-NA, CTX, and apamin abolished 5,6-EET-mediated dilation. Similarly, combined inhibition of NOS and COX abolished the response. We conclude that 5,6-EET is a potent vasodilator in newborn piglet PRAs. This dilation is mediated by redundant pathways that include release of nitric oxide (NO) and COX metabolites and activation of K(Ca) channels. The endothelium dependence of this response suggests that 5,6-EET is not itself an endothelium-derived hyperpolarizing factor (EDHF) but may induce the release of one or more endothelium-derived relaxing factors, such as NO and/or EDHF.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Animals, Newborn; Cyclooxygenase Inhibitors; Drug Combinations; Enzyme Inhibitors; In Vitro Techniques; Indomethacin; Nitric Oxide Synthase; Nitroarginine; Potassium Channel Blockers; Prostaglandin-Endoperoxide Synthases; Pulmonary Artery; Signal Transduction; Swine; Vascular Resistance; Vasodilation; Vasomotor System

Previous investigations have implicated epoxygenase metabolites of arachidonic acid in the control of steroidogenesis in luteinised granulosa cells. The aim of this study was to assess this hypothesis further. We first determined the responsiveness of the cells in vitro to three different stimuli, namely luteinising hormone (LH), interleukin-1beta (IL-1beta), and dibutyryl cyclic AMP (db. cyclic AMP). Their effects were time-dependent, in that progesterone production from cells incubated for 3 days prior to stimulation responded strongly to db. cyclic AMP, to a lesser extent to LH and not to IL-1beta. After 6 days of preincubation, all three stimuli increased progesterone production, and this preincubation period was used in the remainder of the study.LH and IL-1beta increased the intracellular levels of 5,6-epoxyeicosatrienoic acid (5,6-EpETrE) maximally after 10 min, whereas db. cyclic AMP had a more rapid effect within 2-5 min. There were no changes in levels of 14,15-epoxyeicosatrienoic acid (14,15-EpETrE), indicating that the effect was specific. Levels of dihydroxy derivatives of arachidonic acid were also increased, suggesting rapid metabolism of 5,6-EpETrE to inactive 5,6-DiHETrE. The effects of 5,6-EpETrE on progesterone production were transient, which may be due to the lability of this compound in solution, and limited passage into the granulosa-luteal cell cytoplasm. These results support a role for 5,6-EpETrE in the production of progesterone by human granulosa-luteal cells.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acids; Cells, Cultured; Cyclic AMP; Female; Granulosa Cells; Humans; In Vitro Techniques; Interleukin-1; Luteal Cells; Luteinizing Hormone; Ovarian Follicle; Progesterone

Application of glutamate to glial cell cultures stimulates the formation and release of epoxyeicosatrienoic acids (EETs) from arachidonic acid by cytochome P-450 epoxygenases. Epoxygenase inhibitors reduce the cerebral vasodilator response to glutamate and N-methyl-D-aspartate. We tested the hypothesis that epoxygenase inhibitors reduce the somatosensory cortical blood flow response to whisker activation. In chloralose-anesthetized rats, percent changes in cortical perfusion over whisker barrel cortex were measured by laser-Doppler flowmetry during whisker stimulation. Two pharmacologically distinct inhibitors were superfused subdurally: 1) N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH), an epoxygenase substrate inhibitor; and 2) miconazole, a reversible cytochrome P-450 inhibitor acting on the heme moiety. Superfusion with 5 micromol/l MS-PPOH decreased the hyperemic response to whisker stimulation by 28% (from 25 +/- 9 to 18 +/- 7%, means +/- SD, n = 8). With 20 micromol/l MS-PPOH superfusion, the response was decreased by 69% (from 28 +/- 9% to 9 +/- 4%, n = 8). Superfusion with 20 micromol/l miconazole decreased the flow response by 67% (from 31 +/- 6% to 10 +/- 3%, n = 8). Subsequent superfusion with vehicle restored the response to 26 +/- 11%. Indomethacin did not prevent MS-PPOH inhibition of the flow response, suggesting that EET-related vasodilation was not dependent solely on cyclooxygenase metabolism of 5,6-EET. Neither MS-PPOH nor miconazole changed baseline flow, reduced the blood flow response to an adenosine A(2) agonist, or decreased somatosensory evoked potentials. The marked reduction of the cortical flow response to whisker stimulation with two different types of epoxygenase inhibitors indicates that EETs play an important role in the physiological coupling of blood flow to neural activation.

Topics: 8,11,14-Eicosatrienoic Acid; Amides; Animals; Antifungal Agents; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Hyperemia; Laser-Doppler Flowmetry; Male; Miconazole; Rats; Rats, Wistar; Somatosensory Cortex; Vibrissae

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

We describe the biochemical properties of an eicosanoid-modulated Cl- channel and assess the mechanisms by which the epoxyeicosatrienoic acids (EETs) alter both its unitary conductance and its open probability (P(o)). After a purification protocol involving wheat-germ agglutinin affinity and anion-exchange chromatography, the proteins were sequentially inserted into liposomes, which were then fused into PLBs. Functional and biochemical characterization tests confirm that the Cl- channel is a 55-kDa glycosylated monomer with voltage- and Ca(2+) concentration-independent activity. 5,6- and 8,9-EET decreased the conductance of the native channel (control conductance: 70 +/- 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl) in a concentration-dependent manner, with respective 50% inhibitory concentration values of 0.31 and 0.42 microM. These regioisomers similarly decreased the conductance of the purified channel (control conductance value: 75 +/- 5 pS in asymmetrical 50 mM trans/250 mM cis CsCl), which had been stripped of its native proteic and lipidic environment. On the other hand, 5,6- and 8,9-EETs decreased the P(o) of the native channel with respective 50% inhibitory concentration values of 0.27 and 0.30 microM but failed to alter the P(o) of the purified protein. Thus we suggest that the effects of these EETs on channel conductance likely result from direct interactions of EET- anions with the channel pore, whereas the alteration of P(o) requires a lipid environment of specific composition that is lost on solubilization and purification of the protein.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Calcium; Cattle; Cell Fractionation; Chloride Channels; Electric Conductivity; Electrophysiology; Immunoblotting; Lipid Bilayers; Liposomes; Muscle, Smooth; Sarcolemma; Trachea; Vasodilator Agents

Notwithstanding extensive efforts, the mechanism of capacitative calcium entry (CCE) remains unclear. Two seemingly opposed theories have been proposed: secretion-like coupling (Patterson, R. L., van Rossum, D. B., and Gill, D. L. (1999) Cell 98, 487-499) and the calcium influx factor (CIF) (Randriamampita, C., and Tsien, R. Y. (1993) Nature 364, 809-814). In the current study, a combinatorial approach was taken to investigate the mechanism of CCE in corneal endothelial cells. Induction of cytochrome P-450s by beta-naphthoflavone (BN) enhanced CCE measured by Sr(2+) entry after store depletion. 5,6-Epoxyeicosatrienoic acid (5,6-EET), a proposed CIF generated by cytochrome P-450s (Rzigalinski, B. A., Willoughby, K. A., Hoffman, S. W., Falck, J. R., and Ellis, E. F. (1999) J. Biol. Chem. 274, 175-182), induced Ca(2+) entry. Both BN-enhanced CCE and the 5,6-EET-induced Ca(2+) entry were inhibited by the CCE blocker 2-aminoethoxydiphenyl borate, indicating a role for cytochrome P-450s in CCE. Treatment with calyculin A (CalyA), which causes condensation of cortical cytoskeleton, inhibited CCE. The actin polymerization inhibitor cytochalasin D partially reversed the inhibition of CCE by CalyA, suggesting a secretion-like coupling mechanism for CCE. However, CalyA could not inhibit CCE in BN-treated cells, and 5,6-EET caused a partial activation of CCE in CalyA-treated cells. These results further support the notion that cytochrome P-450 metabolites may be CIFs. The vesicular transport inhibitor brefeldin A inhibited CCE in both vehicle- and BN-treated cells. Surprisingly, Sr(2+) entry in the absence of store depletion was enhanced in BN-treated cells, which was also inhibited by 2-aminoethoxydiphenyl borate. An integrative model suggests that both CIF from cytochrome P-450 metabolism and secretion-like coupling mechanisms play roles in CCE in corneal endothelial cells.

Topics: 8,11,14-Eicosatrienoic Acid; Adenosine Triphosphate; Animals; beta-Naphthoflavone; Boron Compounds; Brefeldin A; Calcium; Cattle; Cornea; Cytochalasin D; Cytochrome P-450 Enzyme System; Cytoskeleton; Endothelium, Corneal; Enzyme Inhibitors; Marine Toxins; Models, Biological; Oxazoles; Protein Conformation; Strontium; Time Factors

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

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

We have investigated the role of cAMP in nitric oxide (NO)- and prostanoid-independent vascular relaxations evoked by acetylcholine (ACh) in isolated arteries and perfused ear preparations from the rabbit. These EDHF-type responses are shown to be associated with elevated cAMP levels specifically in smooth muscle and are attenuated by blocking adenylyl cyclase or protein kinase A (PKA). Relaxations are amplified by 3-isobutyl-1-methylxanthine, which prevents cAMP hydrolysis, while remaining susceptible to inhibition by the combination of two K(Ca) channel blockers, apamin and charybdotoxin. Analogous endothelium- and cAMP-dependent relaxations were evoked by cyclopiazonic acid (CPA) which stimulates Ca(2+) influx via channels linked to the depletion of Ca(2+) stores. Responses to ACh and CPA were both inhibited by interrupting cell-to-cell coupling via gap junctions with 18alpha-glycyrrhetinic acid and a connexin-specific Gap 27 peptide. The findings suggest that EDHF-type responses are initiated by capacitative Ca(2+) influx into the endothelium and propagated by direct intercellular communication to effect relaxation via cAMP/PKA-dependent phosphorylation events in smooth muscle.

Topics: 1-Methyl-3-isobutylxanthine; 8,11,14-Eicosatrienoic Acid; Acetylcholine; Animals; Arteries; Biological Factors; Cyclic AMP; Gap Junctions; In Vitro Techniques; Indoles; Male; Muscle Relaxation; Muscle, Smooth, Vascular; Phenylephrine; Rabbits; Vasodilation

Localized aggressive periodontitis (LAgP) is associated with neutrophil dysfunction including defective chemotaxis and reduced calcium influx factor activity. Nitric oxide (NO) and its enzyme, nitric oxide synthase (NOS), have been suggested to be involved in chemotaxis. Some reports, however, were unable to detect either NO or NOS in human neutrophils. In this study, we focused on NOS activity in LAgP neutrophils and examined the involvement of NOS in chemotaxis of normal neutrophils and NOS activity in neutrophils from normal subjects and patients with LAgP.. Neutrophils from 10 normal subjects and 10 LAgP patients were isolated from peripheral venous blood. Membrane associated-NOS (MA-NOS) and soluble NOS (S-NOS) were extracted from cells with or without FMLP stimulation. NOS activity was measured using the radiolabeled L-arginine to L-citrulline conversion assay.. N(omega)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS, significantly inhibited FMLP-induced chemotaxis (P<0.01) and dibutyryl cGMP, an activator of cGMP-dependent protein kinase, significantly attenuated the inhibition by L-NAME (P<0.01). Unstimulated and FMLP-stimulated MA-NOS activity in LAgP neutrophils was statistically significantly higher than that in normal neutrophils (P<0.05). S-NOS activity in LAgP neutrophils was higher than that in normal neutrophils.. This study suggests that NOS is present in human neutrophils and may be involved in FMLP-induced chemotaxis in normal neutrophils. NOS activity is increased in LAgP and is negatively correlated to chemotaxis response.

Topics: 8,11,14-Eicosatrienoic Acid; Adolescent; Adult; Aggressive Periodontitis; Analysis of Variance; Calcium; Calcium Channel Agonists; Case-Control Studies; Chemotaxis, Leukocyte; Female; Humans; Male; Neutrophils; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase

To characterize the role of K(+) channels, the cytochrome P-450 (CYP) metabolite 5,6-EET, and gap junctions in modulation of arteriolar myogenic tone by a non-nitric oxide nonprostaglandin mediator, termed "endothelium-dependent hyperpolarizing factor" (EDHF), released to acetylcholine (ACh) in skeletal muscle arterioles.. In isolated rat gracilis arterioles, simultaneous changes in smooth muscle (aSM) [Ca(2+)](i) (assessed by changes in fura-2 ratiometric signal, R(Ca)) and diameter were measured in response to ACh in the presence of indomethacin and L-NAME.. ACh, the K(ATP) channel opener pinacidil, and the Ca(2+) channel inhibitor verapamil elicited comparable decreases in aSM [Ca(2+)](i) (max.: -32 +/- 3%, 29 +/- 3%, and -30 +/- 3%, respectively) and arteriolar dilations (max.: 90 +/- 4%, 96 +/- 2%, and 95 +/- 2%, respectively). ACh-induced responses were inhibited by KCl-depolarization, K(Ca) channel blockers (TEA, charybdotoxin), or gap junction inhibitors (18alpha-glycyrrhetinic acid, hyperosmolar sucrose). The K(ATP) channel inhibitor glibenclamide, the K(IR) channel inhibitor barium chloride, or the CYP inhibitor 17-octadecynoic acid (ODYA) were without effect. The putative EDHF analogue 5,6-EET elicited constrictions in the presence of the endothelium that could be prevented by indomethacin or a TxA(2) receptor antagonist, whereas in the absence of the endothelium, EDHF elicited only small, charybdotoxin-insensitive decreases in aSM R(Ca) and dilations (max.: -8 +/- 2% and 27 +/- 4%, respectively).. In skeletal muscle arterioles, EDHF 1) substantially and rapidly reduces myogenic tone by decreasing aSM [Ca(2+)](i) via opening K(Ca) channels, 2) it is unlikely to be 5,6-EET or other CYP metabolites, but 3) requires functional gap junctions.

Topics: 8,11,14-Eicosatrienoic Acid; Acetylcholine; Animals; Arterioles; Biological Factors; Calcium; Endothelium, Vascular; Gap Junctions; In Vitro Techniques; Kinetics; Microscopy, Fluorescence; Muscle, Skeletal; Muscle, Smooth, Vascular; Potassium Channels; Rats; Rats, Wistar; Vasodilation

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

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

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

We present evidence in astrocytes that 5,6-epoxyeicosatrienoic acid, a cytochrome P450 epoxygenase metabolite of arachidonic acid, may be a component of calcium influx factor, the elusive link between release of Ca2+ from intracellular stores and capacitative Ca2+ influx. Capacitative influx of extracellular Ca2+ was inhibited by blockade of the two critical steps in epoxyeicosatrienoic acid synthesis: release of arachidonic acid from phospholipid stores by cytosolic phospholipase A2 and cytochrome P450 metabolism of arachidonic acid. AAOCF3, which inhibits cytosolic phospholipase A2, blocked thapsigargin-stimulated release of arachidonic acid as well as thapsigargin-stimulated elevation of intracellular free calcium. Inhibition of P450 arachidonic acid metabolism with SKF525A, econazole, or N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide, a substrate inhibitor of P450 arachidonic acid metabolism, also blocked thapsigargin-stimulated Ca2+ influx. Nano- to picomolar 5, 6-epoxyeicosatrienoic acid induced [Ca2+]i elevation consistent with capacitative Ca2+ influx. We have previously shown that 5, 6-epoxyeicosatrienoic acid is synthesized and released by astrocytes. When 5,6-epoxyeicosatrienoic acid was applied to the rat brain surface, it induced vasodilation, suggesting that calcium influx factor may also serve a paracrine function. In summary, our results suggest that 5,6-epoxyeicosatrienoic acid may be a component of calcium influx factor and may participate in regulation of cerebral vascular tone.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Astrocytes; Calcium; Cytochrome P-450 Enzyme Inhibitors; Enzyme Inhibitors; Ion Transport; Phospholipases A; Phospholipases A2; Rats; Signal Transduction; Thapsigargin

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

We recently reported that canine pulmonary microsomes metabolize arachidonic acid to all four regioisomeric epoxyeicosatrienoic acids (EET). 5,6-EET dilates blood vessels in several nonpulmonary vascular beds, often in a cyclooxygenase-dependent manner. The present study was designed to determine whether 5,6-EET can decrease pulmonary vascular resistance (PVR) in the intact pulmonary circulation. In isolated canine lungs perfused with physiological salt solution, a constant infusion of U-46619 (3.28 +/- 0.99 nmol/min) increased PVR 62.1 +/- 4.5%. Administration of 5,6-EET (10(-5) M) into the perfusate reduced the U-46619-mediated increase in PVR by 23.6 +/- 6.1%. These effects of U-46619 and 5,6-EET were limited to changes in resistance solely in the pulmonary venous segment. In contrast, venous as well as arterial segmental resistances were increased in 5-hydroxytryptamine (5-HT)-treated lungs. However, in the latter instance, 5,6-EET reduced arterial but not venous segmental resistance. 5,6-EET increased pulmonary PGI2 synthesis from 70.5 +/- 18.4 to 675.9 +/- 125.4 ng/min. In the presence of indomethacin (10(-4) M), 5,6-EET did not increase PGI2 synthesis nor did it decrease U-46619- or 5-HT-mediated increases in PVR. In canine intrapulmonary vessels, 5,6-EET decreased active tension in veins contracted with U-46619. 5,6-EET decreased active tension in arteries but not veins contracted with 5-HT, consistent with results in the perfused lungs. These results demonstrate that 5, 6-EET is a vasodilator in the intact pulmonary circulation. Its dilator activity depends on the constrictor agent present, the segmental resistance, and cyclooxygenase activity.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 6-Ketoprostaglandin F1 alpha; 8,11,14-Eicosatrienoic Acid; Animals; Blood Pressure; Dogs; Indomethacin; Male; Muscle, Smooth, Vascular; Pulmonary Artery; Pulmonary Circulation; Pulmonary Veins; Regional Blood Flow; Serotonin; Thromboxane B2; Vascular Resistance

The present study of newborn pig cerebral circulation investigated the role of pertussis toxin (PTX)-sensitive GTP binding proteins in the permissive action of prostacyclin in specific dilator responses. Pial arterioles of anesthetized piglets were observed through closed cranial windows. The piglets were treated topically with PTX and intravenously with indomethacin. The effects of hypercapnia (10% CO2 ventilation) and topical 5,6-epoxyeicosatrienoic acid (5,6-EET) on pial arteriolar diameter were noted before and after the intervention. Samples of the artificial cerebrospinal fluid (aCSF) were collected from beneath the cranial windows for determination of the cAMP concentration. After administration of PTX, indomethacin still abolished pial arteriolar dilation to both hypercapnia and 5, 6-EET and also inhibited the cAMP elevation caused by hypercapnia. The addition of phorbol 12-myristate 13-acetate (PMA), but not iloprost, restored the increase in cAMP and vascular responses to hypercapnia and 5,6-EET. Therefore, in the newborn pig cerebral microvasculature, PTX appears to inhibit a G protein involved in the permissive action of prostacyclin. However, the protein kinase C (PKC) activator PMA appears to act downstream from the block, and, therefore, the permissive action of PMA is not affected by PTX. We suggest that the prostacyclin IP receptor may be coupled to phospholipase C via a PTX-sensitive G protein that normally permits vasodilation to specific stimuli via activation of a PKC, resulting in phosphorylation of a component of the adenylyl cyclase pathway.

Topics: 8,11,14-Eicosatrienoic Acid; Adenylate Cyclase Toxin; Animals; Animals, Newborn; Arterioles; Carbon Dioxide; Cerebrovascular Circulation; Cyclic AMP; Endothelium, Vascular; GTP-Binding Proteins; Hydrogen-Ion Concentration; Iloprost; Indomethacin; Models, Cardiovascular; Muscle, Smooth, Vascular; Partial Pressure; Pertussis Toxin; Pia Mater; Swine; Tetradecanoylphorbol Acetate; Vasodilation; Virulence Factors, Bordetella

The 5,6 epoxyeicosatrienoic acid (5,6-EET) exhibits a range of biological activities but the functional significance of this labile eicosanoid is unknown due, in part, to difficulties of quantitation in biological samples. We have developed a sensitive and specific method to measure 5,6-EET utilizing its selective capacity to form a lactone. The initial conversion of 5,6-EET and 5,6-dihydroxyeicosatrienoic acid (5,6-DHT) to 5,6-delta-lactone is followed by selective purification using reverse phase high performance liquid chromatography (HPLC), reconversion to 5,6-DHT and quantitation by gas chromatography-mass spectrometry (GCMS). In oxygenated Krebs' buffer, 5,6-EET degrades to 5,6-delta-lactone and 5,6-DHT with a t1/2 approximately 8 min. In the presence of camphorsulfonic acid, 5,6-EET and 5,6-DHT convert to a single HPLC peak (lambda = 205) comigrating with 5,6-delta-lactone. Incubation of 5,6-delta-lactone with triethylamine resulted in a single HPLC peak with the retention time of 5,6-DHT. In the perfusate from the isolated kidney, release of 5,6-EET (20 +/- 5 pg/ml), measured indirectly via conversion to 5,6-DHT, was approx. 6-fold less than that reported for prostaglandin E2 (PGE2) and 20-HETE. The coronary perfusate concentration of 5,6 EET was 9 +/- 2 pg/ml. 5,6-EET recovered from renal and coronary perfusates was increased 2-fold to 45.5 +/- 5.5 pg/ml and 21.6 +/- 6.3 pg/ml, respectively, by arachidonic acid.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Chromatography, High Pressure Liquid; Dinoprostone; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney; Male; Myocardium; Perfusion; Rats; Rats, Wistar; Reference Standards; Sensitivity and Specificity

Using microelectrode potential measurements, we tested the involvement of Cl- conductances in the hyperpolarization induced by 5,6- and 11,12-epoxyeicosatrienoic acid (EET) in airway smooth muscle (ASM) cells. 5,6-EET and 11,12-EET (0.75 microM) caused -5.4 +/- 1.1- and -3.34 +/- 0.95-mV hyperpolarizations, respectively, of rabbit tracheal cells (from a resting membrane potential of -53.25 +/- 0.44 mV), with significant residual repolarizations remaining after the Ca2+-activated K+ channels had been blocked by 10 nM iberiotoxin. In bilayer reconstitution experiments, we demonstrated that the EETs directly inhibit a Ca2+-insensitive Cl- channel from bovine ASM; 1 microM 5,6-EET and 1.5 microM 11,12-EET lowered the unitary current amplitude by 40 (n = 6 experiments) and 44.7% (n = 4 experiments), respectively. Concentration-dependent decreases in channel open probability were observed, with estimated IC50 values of 0.26 microM for 5,6- and 1.15 microM for 11,12-EET. Furthermore, pharmacomechanical tension measurements showed that both regioisomers induced significant bronchorelaxations in epithelium-denuded ASM strips. These results suggest that 5,6- and 11,12-EET can act in ASM as epithelium-derived hyperpolarizing factors.

Topics: 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Carbachol; Cattle; Cesium; Chloride Channels; Chlorides; Epithelial Cells; In Vitro Techniques; Membrane Potentials; Microelectrodes; Microsomes; Muscle, Smooth; Peptides; Rabbits; Trachea

Epoxyeicosatrienoic acids (EETs) relax various smooth muscles by increasing outward K+ movement, but the molecular mode of action of EET regioisomers remains to be clarified. The effects of EETs were investigated on bovine airway smooth muscle tone and on reconstituted Ca2+-activated K+ (KCa) channels. 5,6-EET and 11, 12-EET induced dose-dependent relaxations of precontracted bronchial spirals. These effects were partly abolished by 10 nM iberiotoxin. Bilayer experiments have shown that 0.1-10 microM 11,12-EET produced up to fourfold increases in the open probability of KCa channels from the cis (extracellular) side by enhancing the mean open time constant and reducing the long closed time constant, without affecting the unitary conductance. EET-induced activations were blocked by 10 nM iberiotoxin. Addition of vehicles or other lipids as well as of GTP and guanosine 5'-O-(3-thiotriphosphate) in the absence of EET had no effect on channel activity. Thus EETs directly activate KCa channels from airway smooth muscle through an interaction with the extracellular face of the channel. We propose that EETs could represent candidate molecules as epithelium-derived hyperpolarizing factors.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Bronchi; Carbachol; Cattle; Guinea Pigs; Histamine; In Vitro Techniques; Ion Channel Gating; Male; Membrane Potentials; Microsomes; Muscle Relaxation; Muscle, Smooth; Peptides; Potassium Channels; Potassium Chloride; Sarcolemma; Tetraethylammonium; Trachea

Products of cytochrome P-450 enzymes may play a role in capacitative Ca2+ entry in endothelial cells, which can promote a rise in vascular permeability. Thapsigargin (150 nM) stimulated capacitative Ca2+ entry and increased the capillary filtration coefficient (Kf,c) in isolated normal canine lung lobes. Pretreatment of the lobes with cytochrome P-450 inhibitors clotrimazole (10 microM) or 17-octadecynoic acid (5 microM) abolished the thapsigargin-induced increases in Kf,c. Because clotrimazole also blocks Ca2+-activated K+ channels, the K+-channel blocker tetraethylammonium (10 mM) was used to ensure that permeability was not influenced by this mechanism. Tetraethylammonium did not affect thapsigargin-induced permeability. The effects of the cytochrome P-450 arachidonic acid metabolite 5,6-epoxyeicosatrienoic acid (EET) were also investigated in lobes taken from control dogs and dogs with pacing-induced heart failure (paced at 245 beats/min for 4 wk). 5,6-EET (10 microM) significantly increased Kf,c in lobes from the control but not from the paced animals. We conclude that cytochrome P-450 metabolites are involved in mediating microvascular permeability in normal canine lungs, but an absence of 5,6-EET after heart failure does not explain the resistance of lungs from these animals to permeability changes.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Capillary Permeability; Clotrimazole; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dogs; Endothelium, Vascular; Fatty Acids, Unsaturated; In Vitro Techniques; Lung; Microcirculation; Potassium Channels; Pulmonary Circulation; Tetraethylammonium; Thapsigargin

A cytochrome P450-derived metabolite of arachidonic acid, namely an epoxyeicosatrienoic acid (EET), has many of the properties of a hyperpolarizing factor that mediates endothelium-dependent, nitric oxide-independent vasodilation. As there are four EET regioisomers, we used pharmacological criteria, based on previous observations with bradykinin (BK), to evaluate which, if any, of the EETs could be considered a potential mediator of vasodilator responses to BK in the rat isolated heart treated with indomethacin and nitroarginine to eliminate prostaglandin and nitric oxide components of the response. Nifedipine, used as a probe for dilator mechanisms dependent on closure of voltage-dependent Ca++ channels, almost abolished the vasodilator effect of cromakalim and attenuated those of BK and 5,6 EET. The vasodilator effects of the other EETs were not reduced and were excluded from consideration as mediators of BK-induced vasodilation. The vasodilator effect of 5,6 EET, as with that of BK, was markedly reduced by charybdotoxin but not iberiotoxin, suggesting the contribution of a similar type K+ channel to the vascular response to both agents. As expected for a putative endothelium- and cytochrome P450-derived mediator, the coronary vasodilator effect of 5,6 EET was not affected by either removal of the endothelium or inhibition of cytochrome P450 with clotrimazole, interventions that virtually abolished the vasodilator activity of BK. Thus, of the four EET regioisomers, 5,6 EET is the most likely mediator of the vasodilator effect of BK in the isolated heart under these experimental conditions.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Bradykinin; Calcium Channels; Cytochromes; Endothelium, Vascular; Enzyme Inhibitors; Heart; In Vitro Techniques; Ion Channel Gating; Male; Nitric Oxide; Potassium Channel Blockers; Potassium Channels; Rats; Rats, Wistar; 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

Recent studies from this laboratory demonstrated that bradykinin transiently elevates intracellular Ca2+ and inhibits Cl-reabsorption in the in vitro microperfused medullary thick ascending limb (mTAL) of the rat. The present study was designed to identify the intracellular signaling mechanism(s) that mediate this response. Preincubation with the intracellular calcium chelator BAPTA (10(-5) M) completely eliminated the bradykinin-dependent increase in intracellular Ca2+ and the suppression of Cl- transport. Preincubation with the cGMP-dependent protein kinase inhibitor H-89 (10(-5) M) had no effect on the transport response to bradykinin. In contrast, 17-octadecynoic acid (17-ODYA; 10(-5) M), a suicide-substrate inhibitor of renal cytochrome P450 omega-hydroxylase, completely blocked the transport response to bradykinin, while the cyclooxygenase inhibitor sodium meclofenamate (10(-5) M) had no effect. Finally, addition of the cytochrome P450 omega-hydroxylase metabolite 20-hydroxyeicosatetraenoic acid (20-HETE; 10(-8) M) to the bathing medium significantly inhibited Cl- transport in the mTAL (delta -39 +/- 6.0%; p < 0.05), while the epoxygenase metabolite 5,6-epoxyeicosatrienoic acid (5,6-EET; 10(-8) M) had no effect. These data suggest that the bradykinin-dependent inhibition of Cl- transport in the mTAL of the rat is mediated by cytochrome P450 dependent metabolite(s) of arachidonic acid.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Bradykinin; Calcium; Cyclic GMP; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney Tubules, Distal; Male; Rats; Rats, Sprague-Dawley; Sodium Chloride

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

We have previously found that 5,6-EET (epoxyeicosatrienoic acid)(50 nM) significantly dilates the vascular bed(42%) of the isolated, constantly perfused rabbit lung, which has been constricted with U46619(5-8 pM). We studied the role of EDRF-NO and prostaglandins in the 5,6-EET-induced vascular relaxation. Dilation to 5,6-EET was evident only when the pulmonary vascular tone was increased. L-NNA (N omega-nitro-L-arginine, 10(-4) M), an inhibitor of NO synthase(NOS); U46619(5-10 pM), a thromboxane mimetic; and L-NNA + INDO(indomethacin, 10(-5) M), a cyclooxygenase inhibitor, all increased the pressure of pulmonary artery(PPa) from baseline, to a peak range of 28-38mmHg(32.75 [symbol: see text] 2.2), whereas INDO alone increased Ppa only by 10mmHg. L-NNA + INDO,L-NNA alone, and INDO + U46619 attenuated the 5,6-EET relaxing effect by 100%, 88% and 64.5%, respectively. In the presence of L-NNA and 5,6-EET, SNAP(S-nitroso-N-acetyl-D,L-penicillamine, 10(-6) M), a NO donor, reduced Ppa by 75%. We conclude that the mechanism of vasodilation to 5,6-EET in the rabbit pulmonary circulation is via both EDRF-NO and PG pathways and that the vasodilation is largely EDRF-NO dependent.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Lung; Male; Nitric Oxide; Prostaglandins; Pulmonary Artery; Pulmonary Circulation; Rabbits; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents

5,6-Epoxyeicosatrienoic acid (5,6-EET), a cytochrome P450-dependent arachidonate product, is a substrate for cyclooxygenase (COX) and, in some vascular preparations, elicits COX-dependent vasodilation. In the blood perfused rat kidney, 5,6-EET causes COX-dependent renal vasoconstriction, whereas in the rat isolated kidney perfused with a physiological buffer, 5,6-EET produces dose-dependent vasodilation that is unaffected by indomethacin. We examined the possible contribution of platelet COX to the vasoconstrictor action of 5,6-EET. Incubation of labeled 5,6-EET with rat washed platelets yields additional products that elute between 14 to 17 min on high-performance liquid chromatography (HPLC) and cause constriction of the perfused kidney. Indomethacin decreased the formation of these products and reduced the vasoconstrictor capacity of the corresponding HPLC fractions. Thus, platelet COX can metabolize 5,6-EET to vasoconstrictor products that may contribute to the in vivo vasoconstrictor effect of this eicosanoid.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Blood Platelets; Chromatography, High Pressure Liquid; Kidney; Male; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Wistar; Vasoconstriction

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

Epoxygenase metabolites of arachidonic acid are produced by the kidney and have been implicated in the control of renal blood flow. This study examined the preglomerular actions of various epoxyeicosatrienoic acids (EET). By use of the in vitro blood-perfused juxtamedullary nephron preparation, interlobular and afferent arteriolar diameter responses to 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET were determined. Diameters of interlobular and afferent arterioles preconstricted with 0.5 microM norepinephrine averaged 24 +/- 1 microns (N = 27) and 17 +/- 1 microns (N = 32), respectively, at a renal perfusion pressure of 100 mm Hg. Superfusion with 0.01 to 100 nM 11,12-EET caused graded increases in diameters of the interlobular and afferent arterioles. At a dose of 100 nM, 11,12-EET increased the diameters of the interlobular and afferent arterioles by 18 +/- 2% (N = 10) and 20 +/- 3% (N = 9), respectively. The vasodilatory response to 11,12-EET was stereoselective because 11,12(R,S)-EET but not 11,12(S,R)-EET increased the diameters of the interlobular and afferent arterioles. 14,15-EET had a much smaller effect and increased the diameters of the these vessels by 10%; 8,9-EET did not significantly affect vascular diameters. In contrast, 5,6-EET constricted the interlobular and afferent arterioles by 16 +/- 3% (N = 6) and 21 +/- 3% (N = 7), respectively. The corresponding diols, 5,6-DIHETE and 11,12-DIHETE, had no effect on diameters of the interlobular and afferent arterioles at concentrations up to 1 microM. The vasodilatory response to 11,12-EET was not affected by removal of the endothelium or by inhibition of cyclooxygenase with indomethacin. In contrast, the vasoconstrictor response to 5,6-EET was abolished by both removal of the endothelium or cyclooxygenase inhibition. The thromboxane/ enderoperoxide receptor inhibitor, SQ 29,548, resulted in a 60% attenuation of the afferent arteriolar vasconstriction to 5,6-EET. These results indicate that the preglomerular vasoconstriction to 5,6-EET is cyclooxygenase dependent and requires an intact endothelium, whereas the vasodilation to 11,12-EET is stereoselective and is the result of direct action of the epoxide on the preglomerular vascular smooth muscle.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arterioles; Endothelium, Vascular; Juxtaglomerular Apparatus; Male; Microscopy, Video; Muscle, Smooth, Vascular; Oxygenases; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Sprague-Dawley; Vasoconstriction

The intravenous administration of ethchlorvynol (ECV), in dogs, resulted in an acute lung injury (ALI) characterized by a 200 +/- 80% increase in venous admixture and a 142 +/- 30% increase in extravascular lung water (EVLW). Pretreatment with the cytochrome P-450 inhibitor 8-methoxypsoralen prevented the ECV-induced increase in venous admixture but not the increased EVLW. These findings parallel those reported for cyclooxygenase inhibition in ECV-induced ALI and suggest that an arachidonic acid (AA) metabolite of pulmonary cytochrome P-450 activity may mediate the increase in venous admixture of ALI. We demonstrate that canine pulmonary microsomes metabolize [1-(14)C]AA to a variety of products, including the cytochrome P-450 metabolites 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid (EET). In prostaglandin F2 alpha-contracted, isolated pulmonary venous rings, 5,6-EET induced relaxation in a concentration-dependent manner. This action of 5,6-EET was prevented by indomethacin (10(-5) M). These results suggest that may serve as the cyclooxygenase-dependent endogenous pulmonary vasodilator responsible for the increase in venous admixture of ECV-induced ALI.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Cytochrome P-450 Enzyme Inhibitors; Dinoprost; Dogs; Ethchlorvynol; Hypoxia; Lung; Lung Injury; Male; Methoxsalen; Microsomes; Pharmaceutical Vehicles; Vasoconstriction

1. The vascular endothelium releases a hyperpolarizing factor (endothelium-derived hyperpolarizing factor, EDHF) tentatively identified as a cytochrome P450-derived arachidonic acid metabolite. However, there is still controversy concerning its transferability and identity. We designed a bioassay system for assessing EDHF release in which the membrane potential was recorded in cultured vascular smooth muscle cells located downstream from donor endothelial cells. 2. Under combined nitric oxide (NO) synthase and cyclo-oxygenase blockade with NG-nitro-L-arginine (100 mumol l-1) and diclofenac (10 mumol l-1), the superfusate from bradykinin (30 mumol l-1)-stimulated, cultured porcine coronary endothelial cells induced a distinct hyperpolarization followed by a depolarization. Direct application of bradykinin to the smooth muscle cells resulted solely in membrane depolarization. Similar results were obtained using bradykinin-stimulated porcine coronary arteries as donor. 3. Single-channel current measurements suggest that this EDHF-induced hyperpolarization was elicited by the activation of Ca(2+)-dependent K+ channels. 4. Increasing the transmural pressure within the donor segment significantly enhanced the duration, but not the amplitude of the hyperpolarization induced by the effluate from bradykinin-stimulated donor segments. 5. Inhibition of P450 oxygenase activity with clotrimazole (3 mumol l-1) or 17-octadecynoic acid (3 mumol l-1) abolished EDHF release from the coronary endothelium, while the P450-derived arachidonic acid metabolite, 5,6-epoxyeicosatrienoic acid, induced a hyperpolarization of detector smooth muscle cells almost identical to that induced by EDHF. Moreover, induction of P450 activity by beta-naphthoflavone (3 mumol l-1, 48 h), significantly increased the bradykinin-induced release of EDHF. 6. These findings suggest that the vascular endothelium releases a transferable hyperpolarizing factor, chemically distinct from NO and prostacyclin, in response to agonists and mechanical stimulation. This beta-naphthoflavone-inducible EDHF appears to be a cytochrome P450-derived metabolite of arachidonic acid, which elicits hyperpolarization by activation of Ca(2+)-dependent K+ channels.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; beta-Naphthoflavone; Biological Factors; Cytochrome P-450 Enzyme System; Endothelium, Vascular; Epoprostenol; Large-Conductance Calcium-Activated Potassium Channels; Muscle, Smooth, Vascular; Nitric Oxide; Potassium Channels; Potassium Channels, Calcium-Activated; Swine

1. We tested the hypothesis that agonist-stimulated Ca2+ entry, and thus formation of endothelium-derived nitric oxide (EDNO) in vascular endothelial cells, is related to activation of microsomal P450 mono-oxygenase (P450 MO) and the biosynthesis of 5,6-epoxyeicosatrienoic acid (5,6-EET). 2. Several P450 inhibitors diminished the sustained [Ca2+]i plateau response to agonist or intracellular Ca2+ store depletion with ATPase inhibitors by 31-69% (fura-2 technique). Mn2+ influx stimulated by agonists or ATPase inhibitors was prevented by P450 inhibitors. 3. Histamine- or ATPase inhibitor-stimulated formation of EDNO was strongly attenuated (50-83%) by P450 inhibitors, without any effect on EDNO formation by the Ca2+ ionophore A23187, indicating that decreased EDNO synthesis is due specifically to the inhibition of Ca2+ entry by these compounds. 4. Induction of P450 MO by beta-naphthoflavone potentiated agonist-induced Ca2+ and Mn2+ influx by 60 and 53%, respectively. Intracellular Ca2+ release remained unchanged. 5. The P450 MO product, 5,6-EET (< 156 nmol l-1), activated Ca2+/Mn2+ entry without any depletion of intracellular Ca2+ stores. The 5,6-EET-stimulated Ca2+/Mn2+ entry was not affected by P450 inhibitors. 6. As with the bradykinin-stimulated Ca2+ entry pathway, the 5,6-EET-activated Ca2+ entry pathway was permeable to Mn2+ and Ba2+, sensitive to Ni2+, La3+ and membrane depolarization, and insensitive to the removal of extracellular Na+ or the organic Ca2+ antagonist, nitrendipine. 7. In the presence of 5,6-EET, stimulation with bradykinin only transiently increased [Ca2+]i. Vice versa, 5,6-EET failed to increase [Ca2+]i further in bradykinin-stimulated cells. The sustained [Ca2+]i plateau phase induced by a co-stimulation with bradykinin and 5,6-EET was identical to that observed with bradykinin or 5,6-EET alone. 8. These results demonstrate that Ca2+ entry induced by the P450 MO product, 5,6-EET, is indistinguishable to that observed by stimulation with bradykinin. 9. All data support our hypothesis that depletion of endothelial Ca2+ stores activates microsomal P450 MO which in turn synthesizes 5,6-EET. We propose that the arachidonic acid metabolite 5,6-EET or one of its metabolites is a second messenger for activation of endothelial Ca2+ entry.

Topics: 8,11,14-Eicosatrienoic Acid; Adenosine Triphosphatases; Amino Acid Oxidoreductases; Animals; Barium; Benzoflavones; beta-Naphthoflavone; Bradykinin; Calcium; Cattle; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Econazole; Endothelium, Vascular; Female; Histamine; Humans; Magnesium; Nitric Oxide Synthase; Oxygenases; Potassium Channels; Second Messenger Systems

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

We examined the mechanism by which the cytochrome P-450 metabolite of arachidonate, 5,6-epoxyeicosatrienoic acid (5,6-EET), modulates electrogenic transport in the rabbit cortical collecting duct (CCD). 5,6-EET depolarized transepithelial voltage (VT) in a concentration-dependent manner with a maximal effect at 1 microM. None of the other EET regioisomers (8,9-, 11,12-, or 14,15-EET; all at 1 microM) affected VT, This action was also stereoselective, with 5(S),6(R)-EET producing a 2.5-fold greater effect on VT than 5(R),6(S)-EET (1 microM each). Like basolateral prostaglandin E2 (PGE2), both luminal and basolateral 5,6-EET increased cytosolic Ca2+ concentration ([Ca2+]i) in the rabbit CCD. Pretreatment with cyclooxygenase inhibitors (10 microM ibuprofen or 5 microM indomethacin) completely blocked both the [Ca2+]i increase and the change in VT. Neither 5,6-epoxy-PGE1 nor 5-hydroxy-PGI1, cyclooxygenase metabolites of 5,6-EET, affected VT. However, when added to primary cultures of rabbit CCDs, 5,6-EET stimulated endogenous PGE2 synthesis. We propose that 5,6-EET stimulates endogenous prostaglandin synthesis, which inhibits electrogenic ion transport in the CCD.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Biological Transport; Dinoprostone; Electrophysiology; Epithelium; Female; Ions; Kidney Tubules, Collecting; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Rabbits; Signal Transduction; Stereoisomerism

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

These studies were designed to determine the role of arachidonic acid metabolites in catecholamine secretion from adrenal chromaffin cells. Inhibitors of the cytochrome P450-dependent metabolism of arachidonic acid were shown to interfere with stimulus-secretion coupling in cultured chromaffin cells. Ketoconazole (10 microM), clotrimazole (20 microM), and piperonyl butoxide (50 microM) inhibited carbachol-dependent catecholamine secretion by 44%, 83%, and 100%, respectively; histamine-dependent secretion by 25%, 60%, and 81%, and secretion induced by 59 mM KCl depolarization by 25%, 55%, and 89%. Uptake of 45Ca2+ into the cells in response to carbachol was inhibited 63% by ketoconazole, 86% by clotrimazole, and 95% by piperonyl butoxide; KCl-dependent uptake was inhibited 7%, 56%, and 85%, respectively. However, cytochrome P450 inhibitors did not inhibit catecholamine secretion when cells were stimulated with the calcium ionophores ionomycin or lasalocid. These results indicated the involvement of a cytochrome P450 product in controlling Ca2+ influx in response to membrane depolarization. Cells prelabeled with [3H]arachidonic acid formed a 3H-labeled metabolite which comigrated with authentic 5,6-epoxyeicosatrienoic (5,6-EET) acid on reverse phase and normal phase HPLC. Pretreatment with clotrimazole inhibited the production of this 3H-labeled metabolite. Addition of synthetic 5,6-EET (1 nM) to cells pretreated with piperonyl butoxide resulted in catecholamine secretion. These data suggest a role for a cytochrome P450 metabolite of arachidonic acid in agonist-stimulated catecholamine secretion.

Topics: 8,11,14-Eicosatrienoic Acid; Adrenal Glands; Animals; Arachidonic Acid; Calcium; Catecholamines; Cattle; Cells, Cultured; Chromaffin Granules; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Histamine

Epoxyeicosatrienoic acid (EET) metabolism was studied in endothelial cells to determine whether this tissue may influence their vasoactive properties. Porcine aortic endothelial cells rapidly took up all four EET regioisomers. The uptake of [1-14C]14,15-EET reached a maximum in 15-30 min, and saturation was not observed with concentrations up to 5 microM. More than 70% of the incorporated 14,15-EET was contained in choline and inositol glycerophospholipids, most of it in the form of an EET ester. A metabolite, 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), accumulated in the medium during incubation, and products with similar chromatographic properties also were formed from 5,6-, 8,9-, and 11,12-EET. Much of the 14,15-EET taken up was only temporarily retained by the cells, and in 2 h half was released into the medium as 14,15-DHET. Bovine aortic and human umbilical vein endothelial cells also took up 14,15-EET, incorporated it into choline glycerophospholipids, and converted it to 14,15-DHET. These findings suggest that the endothelium may limit the vascular actions of EETs through rapid uptake, hydration, and release of DHETs into the circulation. Some vasoactive effects of EETs may result from their temporary accumulation in endothelial phospholipids involved in stimulus-response coupling.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aorta; Cattle; Cells, Cultured; Chromatography, High Pressure Liquid; Endothelium, Vascular; Humans; Kinetics; Lipid Metabolism; Phospholipids; Swine; Umbilical Veins

Angiotensin II (Ang II) receptors are coupled to a variety of signal transduction mechanisms. In the kidney, Ang II at nanomolar concentration binds to proximal tubular cells and stimulates phospholipase A2 (PLA2), which in turn catalyzes the hydrolysis of phosphatidylcholine into lysophosphatidylcholine (LPC) and fatty acid. This signal transduction pathway has been shown to be an important modulator of sodium transport. The kidney cortex possesses the enzyme necessary to convert angiotensin I (Ang I) directly to Ang-(1-7) bypassing Ang II as an intermediate. The present investigation was undertaken to determine whether Ang-(1-7) influences epithelial cell function by comparing this heptapeptide with Ang II as a modulator of PLA2 activity and sodium transport. Proximal tubular cells were labeled in tissue culture with 3H-choline and PLA2 activity was measured by quantitation of LPC. We found that Ang II (10(-9) M to 10(-6) M) significantly increased PLA2 activity (154 +/- 36% to 209 +/- 94%). Similar results were obtained with Ang-(1-7) (240 +/- 130% to 353 +/- 40%). The bioactivity of the peptides was assayed by its ability to regulate transcellular 22Na flux. Ang II (10(-9) M) inhibited 22Na flux by 12 +/- 2% while Ang-(1-7) (10(-9) M) inhibited 22Na flux by 20 +/- 5%. These results suggest that one potential role of Ang-(1-7) in the regulation of kidney epithelial electrolyte transport may involve activation of PLA2.

Topics: 8,11,14-Eicosatrienoic Acid; Angiotensin I; Angiotensin II; Animals; Biological Transport; Cells, Cultured; Kidney Tubules, Proximal; Lysophosphatidylcholines; Male; Peptide Fragments; Phospholipases A; Phospholipases A2; Rabbits; Sodium

Metabolites of arachidonic acid regulate several physiological processes, including vascular tone. The purpose of this study was to determine which metabolites of arachidonic acid are produced by bovine coronary arteries and which may regulate coronary vascular tone. Arachidonic acid induced a concentration-related, endothelium-dependent relaxation [one-half maximum effective concentration (EC50) of 2 x 10(-7) M and a maximal relaxation of 91 +/- 2% at 10(-5) M] of bovine coronary arteries that were contracted with U-46619, a thromboxane mimetic. The concentration of 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), a metabolite of prostaglandin I2 (PGI2), increased from 82 +/- 6 to 328 +/- 24 pg/ml with arachidonic acid (10(-5) M). Treatment with the cyclooxygenase inhibitor indomethacin attenuated arachidonic acid-induced relaxations by approximately 50% and blocked the synthesis of 6-keto-PGF1 alpha. PGI2 caused a concentration-related relaxation (EC50 of 10(-8) M and a maximal relaxation of 125 +/- 11% at 10(-7) M). BW755C, a cyclooxygenase and lipoxygenase inhibitor, inhibited arachidonic acid-induced relaxation to the same extent as indomethacin. When vessels were treated with both indomethacin and BW755C, the inhibition of relaxation was the same as either inhibitor alone. SKF 525a, a cytochrome P-450 inhibitor, reduced arachidonic acid-induced relaxation by approximately 50%. When SKF 525a was given in combination with indomethacin, the relaxation by arachidonic acid was almost completely inhibited. SKF 525a inhibited the synthesis of epoxyeicosatrienoic acids (EETs).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arteries; Cattle; Coronary Vessels; Epoprostenol; Vasodilation

The 5,6-epoxyeicosatrienoic acid (5,6-EET), a renal vasodilator metabolite of arachidonic acid via cytochrome P450 (P450) requires cyclooxygenase for expression of its vasoactivity as the responses are inhibited by indomethacin and other aspirin-like drugs. We now report on the metabolism of 5,6-EET by rabbit kidneys in order to characterize those metabolites that may account for its vasoactivity. The 5,6-EET was injected close-arterially into the rabbit isolated Krebs-Henseleit perfused kidney, preconstricted with phenylephrine, and the effluent collected throughout the response period. Basal collections, following injection of 100 microliters of vehicle, were made at 20-min intervals before each 5,6-EET injection. Prior to acidic extraction, deuterated 6-keto-prostaglandin (PG) F1 alpha and PGE2 were added as internal standards. The extracts were separated by TLC and prostaglandins were derivatized for gas chromatography-mass spectrometry analysis using a negative ion chemical ionization mode. Injection of 0.5, 1, 5, 10, and 20 micrograms of 5,6-EET (n = 4) resulted in dose-related decreases in perfusion pressure of 6 +/- 2, 12 +/- 4, 21 +/- 4, 26 +/- 4, and 27 +/- 7 mm Hg, respectively. Basal perfusates contained 6-keto-PGF1 alpha and PGE2, levels of which were increased by 2-fold or more by 5,6-EET. Perfusates, collected during 5,6-EET administration, also contained 5-hydroxy-PGI1 and 5,6-epoxy-PGE1, cyclooxygenase metabolites of 5,6-EET. This is the first report of the recovery and identification of these 5,6-EET metabolites from an intact organ. Since the responses to 5,6-EET are endothelial-dependent, we also studied the profile of eicosanoids formed following incubation of 5,6-EET with cultured bovine pulmonary endothelial cells. Endothelial cells metabolized 5,6-EET to products with a similar radioactive profile on reverse-phase high pressure liquid chromatography compared to kidney perfusates. We compared the vasodilator activity of 5,6-epoxy-PGE1 and 5-hydroxy-PGI1, chemically synthesized by us from PGE2 and PGF2 alpha, respectively, with PGE2 and PGI2 in the rabbit kidney. The 5,6-epoxy-PGE1 was equipotent to PGE2 as a vasodilator. The ED50 values for 5,6-EET, 5,6-epoxy-PGE1, and PGE2 were 4.69, 0.43, and 0.42 nmol, respectively. Although PGI2 was a potent vasodilator (ED50, 0.24 nmol), 5-hydroxy-PGI1 was devoid of activity. Thus, the cyclooxygenase-dependent vasoactivity of 5,6-EET in the rabbit kidney has two components: release of vasodilat

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Cattle; Cells, Cultured; Cytochrome P-450 Enzyme System; Endothelium, Vascular; Gas Chromatography-Mass Spectrometry; In Vitro Techniques; Kidney; Male; Perfusion; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Rabbits; Renal Circulation; Vasodilation; Vasodilator Agents

We have recently shown that brain slices are capable of metabolizing arachidonic acid by the epoxygenase pathway. The purpose of this study was to begin to determine the ability of individual brain cell types to form epoxygenase metabolites. We have examined the astrocyte epoxygenase pathway and have also confirmed metabolism by the cyclooxygenase and lipoxygenase enzyme systems. Cultured rat hippocampal astrocyte homogenate, when incubated with radiolabeled [3H]arachidonic acid, formed products that eluted in four major groups designated as R17-30, R42-50, R51-82, and R83-90 based on their retention times in reverse-phase HPLC. These fractions were further segregated into as many as 13 peaks by normal-phase HPLC and a second reverse-phase HPLC system. The principal components in each peak were structurally characterized by gas chromatography/electron impact-mass spectrometry. Based on HPLC retention times and gas chromatography/electron impact-mass spectrometry analysis, the more polar fractions (R17-30) contained prostaglandin D2 as the major cyclooxygenase product. Minor products included 6-keto prostaglandin F1 alpha, prostaglandin E2, prostaglandin F2 alpha, and thromboxane B2. Fractions R42-50, R51-82, and R83-90 contained epoxygenase and lipoxygenase-like products. The major metabolite in fractions R83-90 was 5,6-epoxyeicosatrienoic acid (EET). Fractions R51-82 contained 14,15- and 8,9-EETs, 12- and 5-hydroxyeicosatetraenoic acids, and 8,9- and 5,6-dihydroxyeicosatrienoic acids (DHETs). In fractions R42-50, 14,15-DHET was the major product. When radiolabeled [3H]14,15-EET was incubated with astrocyte homogenate, it was rapidly metabolized to [3H]14,15-DHET. The metabolism was inhibited by submicromolar concentration of 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase activity. Formation of other polar metabolites such as triols or epoxy alcohols from 14,15-DHET was not observed. In conclusion, astrocytes readily metabolize arachidonic acid to 14,15-EET, 5,6-EET, and their vicinal-diols. Previous studies suggest these products may affect neuronal function and cerebral blood flow.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Astrocytes; Cells, Cultured; Chromatography, Gas; Chromatography, High Pressure Liquid; Epoxide Hydrolases; Hippocampus; Hydroxyeicosatetraenoic Acids; Mass Spectrometry; Prostaglandins; Rats

Ovariectomized Fischer 344 rats were implanted with silastic capsules containing estradiol for approximately 4 weeks to increase the number of lactotrophs in the anterior pituitary gland. Anterior pituitary cells were enzymatically dispersed and cultured for 1 day and challenged with eicosanoids or other prolactin (PRL) secretagogues. The isolated pituitary cells from estradiol-pretreated animals exhibited an increase in the ability to secrete PRL in the presence of maximally effective concentrations of thyrotropin releasing hormone (TRH), arachidonic acid or 5,6-epoxyeicosatrienoic acid (5,6-EET). Anterior pituitary cells from estradiol-pretreated animals also showed an increased activity of cytochrome P-450. The possible involvement of cytochrome P-450 in PRL secretion from anterior pituitary animal cells isolated from animals pretreated with estradiol was shown by the fact that these anterior pituitary cells increased the synthesis of 5,6-EET, a potent PRL releasing agent. TRH and 5,6-EET increased the mobilization of both cyclic AMP and cytoplasmic calcium to about the same extent. The data suggest that cytochrome P-450 is important in the release of PRL from anterior pituitary cells isolated from estradiol-pretreated Fischer 344 rats and that a product of cytochrome P-450-catalyzed epoxidation of arachidonic acid, 5,6-EET, plays a significant role in the release of PRL.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Calcium; Cells, Cultured; Cyclic AMP; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Estradiol; Female; Kinetics; Pituitary Gland, Anterior; Prolactin; Rats; Rats, Inbred F344; Testosterone

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

The purpose of this study was to determine if whole brain makes epoxygenase metabolites of arachidonic acid and, if so, whether they are vasoactive on the cerebral microcirculation. Blood-free mouse brain slices were incubated with exogenous radiolabeled arachidonic acid, and the extracted metabolites were resolved by HPLC. Metabolite structures were confirmed by gas chromatography/mass spectrometry. In addition to prostaglandins, leukotriene B4, and hydroxyeicosatetraenoic acids, mouse brain metabolized arachidonic acid into several other compounds. Among them, we identified 5,6- and 14,15-epoxyeicosatrienoic acid. Next, we tested the effect of topical application of brain-synthesized 5,6-epoxyeicosatrienoic acid and synthetic epoxyeicosatrienoic acids on in vivo rabbit cerebral arteriolar diameter using the cranial window technique and in vivo microscopy. Brain-synthesized 5,6-epoxyeicosatrienoic acid caused a transient 28% arteriolar dilation, similar to that produced by 5 micrograms/ml of synthetic 5,6-epoxyeicosatrienoic acid. A concentration of synthetic 14,15- and 11,12-epoxyeicosatrienoic acid of 5 micrograms/ml CSF had little or no effect on diameter, whereas 8,9-epoxyeicosatrienoic acid caused a maximum dilation of 8%. These studies suggest that brain-synthesized 5,6-epoxyeicosatrienoic acid may play a role in the normal or pathophysiological regulation of the cerebral microcirculation.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Brain; Cerebrovascular Circulation; Chromatography, High Pressure Liquid; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Gas Chromatography-Mass Spectrometry; Male; Mice; Mice, Inbred ICR; Oxygenases

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

The vasodilatory effect of epoxyeicosatrienoic acids (EpETrE), especially 5(6)-EpETrE, has been reported recently and a role of P-450-dependent arachidonic acid monooxygenase metabolites was suggested in vasoregulation. Accordingly, the presence of P-450-dependent arachidonic acid monooxygenase was investigated in rat aortic smooth muscle cells. Incubation of the microsomes of rat cultured aortic smooth muscle cells with 14C-arachidonic acid in the presence of 1 mM NADPH resulted in the formation of oxygenated metabolites. The metabolites were separated and purified by reverse phase and straight phase high performance liquid chromatography and identified by gas chromatography-mass spectrometry. Identified metabolites were 5(6)-EpETrE, 5,6-dihydroxyeicosatrienoic acid (DiHETrE), and 14,15-DiHETrE. The formation of these metabolites was totally dependent on the presence of NADPH, and inhibitors of cytochrome P-450-dependent enzymes, SKF-525A and metyrapone, reduced the formation of these metabolites. This is the first report that cytochrome P-450-dependent arachidonic acid metabolites, especially 5(6)-EpETrE and 14(15)-EpETrE, can be produced in the microsomes of vascular smooth muscle cells of rats.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Aorta, Thoracic; Arachidonic Acid; Arachidonic Acids; Bacterial Proteins; Cells, Cultured; Chromatography, High Pressure Liquid; Cytochrome P-450 Enzyme System; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Male; Metyrapone; Microsomes; Mixed Function Oxygenases; Muscle, Smooth, Vascular; NADP; NADPH-Ferrihemoprotein Reductase; Pyridines; Rats; Rats, Inbred WKY

Previous studies from this and other laboratories have shown that angiotensin II (AII) induces [Ca2+]i transients in proximal tubular epithelium independent of phospholipase C. AII also stimulates formation of 5,6-epoxyeicosatrienoic acid (5,6-EET) from arachidonic acid by a cytochrome P450 epoxygenase and decreases Na+ transport in the same concentration range. Because 5,6-EET mimics AII with regard to Na+ transport, it effects on calcium mobilization were evaluated. [Ca2+]i was measured by video microscopy with the fluorescent indicator fura-2 employing cultured rabbit proximal tubule. AII-induced [Ca2+]i transients were enhanced by arachidonic acid and attenuated by ketoconazole, an inhibitor of cytochrome P450 epoxygenases. Arachidonic acid also elicited a [Ca2+]i transient that was attenuated by ketoconazole. 5,6-EET augmented [Ca2+]i similar to that seen with AII, but was unaffected by ketoconazole. By contrast, the other regioisomers (8,9-, 11,12-, and 14,15-EET) were much less potent. [Ca2+]i transients resulted from influx through verapamil- and nifedipine-sensitive channels. These results suggest a novel mechanism for AII-induced Ca mobilization in proximal tubule involving cytochrome P450-dependent arachidonic acid metabolism and Ca influx through voltage-sensitive channels.

Topics: 8,11,14-Eicosatrienoic Acid; Angiotensin II; Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Calcium Channels; Cells, Cultured; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Cytosol; Ketoconazole; Kidney Tubules, Proximal; Male; Oxygenases; Rabbits; Signal Transduction

Radiolabeled cis-(+-)-5,6-epoxyeicosatrienoic acid (5(6)-EpETrE) was incubated with a suspension of isolated human platelets in order to study its metabolic fate. The epoxide slowly disappeared from the suspension and was completely metabolized within 30 min. After extraction and analysis by reverse-phase high performance liquid chromatography, seven metabolites were found. Addition of either indomethacin (0.01 mM, cyclooxygenase inhibitor) or BW755C (0.1 mM, cyclooxygenase/lipoxygenase inhibitor) to the incubations blocked the formation of four and six metabolites, respectively, 1,2-Epoxy-3,3,3-trichloropropane (inhibitor of microsomal epoxide hydrolase) failed to inhibit the formation of 5,6-dihydroxyeicosatrienoic acid (5,6-DiHETrE), a hydrolysis product of the precursor 5(6)-EpETrE. The metabolites were characterized by UV spectroscopy, negative ion chemical ionization liquid chromatography/mass spectrometry, gas chromatography/mass spectrometry and, in one instance, coelution with synthetic standard. Three primary platelet metabolites were structurally determined to be 5,6-epoxy-12-hydroxyeicosatrienoic acid, 5,6-epoxy-12-hydroxyheptadecadienoic acid, and a unique bicyclic metabolite, 5-hydroxy-6,9-epoxy-thromboxane B1, which originated from intramolecular hydrolysis of 5,6-epoxythromboxane-B1. This thromboxane analog was partially separated into stereoisomers and coeluted with the racemic synthetic standard in gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Three other metabolites were characterized as 5,6,12-trihydroxyeicosatrienoic acid, 5,6,12-trihydroxyheptadecadienoic acid, and 5,6-dihydroxythromboxane-B1, and resulted from the hydrolysis of the corresponding epoxides rather than from the metabolism of 5,6-DiHETrE. The latter was not metabolized by platelet cyclooxygenase or lipoxygenase. The biosynthesis of two cyclooxygenase metabolites indicated the formation of unstable 5,6-epoxythromboxane-A1 as an intermediate precursor. Platelet aggregation was not induced by 5(6)-EpETrE, although responsiveness to arachidonic acid was reduced following preincubation with the epoxide. The platelet metabolites of 5(6)-EpETrE might be useful in assessing its in vivo production in humans.

Topics: 8,11,14-Eicosatrienoic Acid; Arachidonic Acid; Blood Platelets; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Humans; In Vitro Techniques; Kinetics; Mass Spectrometry; Platelet Aggregation; Platelet Aggregation Inhibitors; Thromboxanes; Tritium

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

Several laboratories have documented that angiotensin II (AII) induces natriuresis in proximal tubular epithelium by a mechanism that has as yet not been disclosed. The present studies were designed to test the hypothesis that cytochrome P450-dependent metabolites of arachidonic acid mediate this effect. In cultured rabbit proximal tubule a ketoconazole-sensitive product comigrating with 5,6-epoxy-eicosatrienoic acid (5,6-EET) on reverse phase and normal phase HPLC was stimulated two-fold by AII. We employed cultures of early S1 segments on a modified Ussing chamber as a bioassay to evaluate the effects of AII and 5,6-EET on unidirectional 22Na (apical to basolateral) flux (JNa). AII inhibited JNa, an effect that was abolished by ketoconazole. Furthermore, 5,6-EET decreased JNa in a manner analogous to AII, and the effect was potentiated by inhibition of endogenous 5,6-EET production. Employing the Ca2(+)-sensitive fluorescent probe, Fura 2, we observed a dose-dependent increase in [Ca2+]i with nM to microM 5,6-EET, effects that were abolished by depletion of extracellular Ca2+ and voltage-sensitive Ca-channel blockers. These observations support the hypothesis that 5,6-EET represents the second messenger that mediates AII-induced natriuresis via stimulation of Ca2+ influx through voltage-sensitive channels.

Topics: 8,11,14-Eicosatrienoic Acid; Angiotensin II; Animals; Arachidonic Acid; Arachidonic Acids; Calcium; Cells, Cultured; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Epithelium; Kidney Tubules, Proximal; Natriuresis; Oxygenases; Phospholipases A; Rabbits; Second Messenger Systems; Sodium; Sodium-Potassium-Exchanging ATPase

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

Coronary vascular injury promotes blood cell-vessel wall interactions that influence arachidonic acid metabolism and coronary blood flow patterns. Since lipoxygenase and cytochrome P-450 epoxygenase metabolites of arachidonic acid are synthesized by vascular and inflammatory cells and have a variety of important biological actions, we investigated the metabolism of arachidonic acid by these pathways in normal and stenosed, endothelially injured canine coronary arteries. We found and confirmed by gas chromatography/mass spectrometry that primarily 12- and 15-hydroxyeicosatetraenoic acids (HETEs) are synthesized by both coronary artery segments. Lesser amounts of 11-, 9-, 8-, and 5-HETEs are also produced. 15-Ketoeicosatetraenoic acid is also synthesized. The synthesis of 14C-HETEs is fivefold to 10-fold greater by the stenosed than the normal coronary artery. Specific radioimmunoassays indicated that the stenosed coronary artery synthesized 93 +/- 14 and 1,102 +/- 154 ng/g of tissue of 15- and 12-HETE, respectively, while the normal coronary artery produced 17 +/- 3 and 162 +/- 68 ng/g of tissue of 15- and 12-HETE, respectively. Products comigrating with 14,15-; 11,12-; 8,9-; and 5,6-epoxyeicosatrienoic acids (EETs) and the corresponding dihydroxyeicosatrienoic acids (DHETs) were detected predominantly in stenosed coronary arteries by high-pressure liquid chromatography. The structures of the EETs were confirmed by GC/MS. The EETs and prostaglandin I2 produced endothelium-independent, concentration-related relaxations of dog coronary artery rings. These data indicate that normal and stenotic coronary arteries metabolize arachidonic acid to HETEs, DHETs, and EETs along with prostaglandins; however, the synthesis of these metabolites is greater in the stenosed, endothelially injured vessel. The EETs may be synthesized during the development of cyclic flow variations and counteract the vasoconstrictor effects of thromboxane A2.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Constriction, Pathologic; Coronary Disease; Coronary Vessels; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Dogs; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Oxygenases; Prostaglandins; Reference Values

Rat kidney cortical and medullary microsomal fractions catalyzed cytochrome P-450-linked metabolism of arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) (56 +/- 6% of total products in cortex and 10% in medulla) and 19- and 20-hydroxyeicosatetraenoic acids (19- and 20-OHAA) (36 +/- 4% in cortex and 90% in medulla). In addition, endogenous renal generation of EETs was established by negative ion-chemical ionization mass spectrometry. The total amount of EETs present in the rat kidney was approximately 1 microgram/g wet tissue. The responses to renal arterial administration of 20-OHAA and 5,6-EET were evaluated in anesthetized euvolemic rats. 20-OHAA resulted in ipsilateral dose-dependent natriuresis without affecting systemic or renal hemodynamics or glomerular filtration rate (GFR). Equimolar doses of 5,6-EET resulted in dose-dependent renal vasoconstriction and reduced GFR but were without effect on arterial pressure or sodium excretion. During cyclooxygenase inhibition, 5,6-EET caused renal vasodilatation and augmentation of GFR. These data establish the capacity of rat kidney to metabolize AA through cytochrome P-450-dependent oxygenases and demonstrate the endogenous formation of the resulting eicosanoids. Monooxygenase and epoxygenase products exert effects on renal blood flow, GFR, and urinary sodium excretion rate, suggesting their potential relevance in the regulation of renal function.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Cytochrome P-450 Enzyme System; Hemodynamics; Ibuprofen; Kidney; Male; Rats; Rats, Inbred Strains

We have recently shown that brain tissue can synthesize cytochrome P-450 monooxygenase metabolites of arachidonic acid (AA), including 5,6-epoxyeicosatrienoic acid (5,6-EET), and 14,15-EET. The purpose of this investigation was to determine the vasoactivity of EETs and AA on the cerebral microcirculation. Pial arteriolar diameter was measured in rabbits and cats using in vivo microscopy and the closed cranial window technique. Prostaglandin (PG) E2 and 6-keto-PGF1 alpha formed by the brain cortex during application of these fatty acids was measured in cerebrospinal fluid by use of radioimmunoassay. A transient dose-dependent dilation was produced by 5,6-EET (1-15 micrograms/ml), with the maximum being 23% of control in both species. Other EETs had little or no activity, and AA-induced dilation was greater in rabbits than in cats. Indomethacin or superoxide dismutase plus catalase prevented dilation by 5,6-EET and AA, indicating that both produce dilation via cyclooxygenase-dependent oxygen radicals. PGE2 and 6-keto-PGF1 alpha levels were increased by AA but not by EETs, implying that EETs do not directly activate AA metabolism. Since 5,6-EET, but not other EETs, is known to be a substrate for cyclooxygenase, our data are consistent with brain cyclooxygenase metabolism of 5,6-EET with concomitant generation of dilator oxygen radicals. An implication of these results is that many previous studies of the cerebral circulation which based conclusions on results with cyclooxygenase inhibitors may need to be additionally interpreted.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Arterioles; Brain; Cats; Cerebrovascular Circulation; Cytochrome P-450 Enzyme System; Rabbits; Vasodilation

We have reported that 5,6-epoxyeicosatrienoic acid (5,6-EET) was the only cytochrome P-450-dependent arachidonic acid (AA) epoxide to dilate the isolated, perfused caudal artery of the rat. We have investigated the mechanisms by which 5,6-EET dilates the rat-tail artery by studying the effect of deendothelialization and inhibition of AA metabolic pathways (cyclooxygenase, lipoxygenase, and cytochrome P-450 monooxygenase) on the vascular action of the epoxide. Rat isolated caudal arteries were perfused with Krebs-Henseleit solution at 37 degrees C, pH 7.4, and gassed with 95% O2-5% CO2. Arterial tone was elevated with phenylephrine; acetylcholine (0.5 nmol) was used to detect the presence of intact, functional endothelium. Doses of 5,6-EET, from 6.25 to 25.0 nmol, were injected close-arterially. After obtaining control responses, the same doses were randomly retested after deendothelialization or in the presence of inhibitors of AA metabolism. Removal of the endothelium decreased by 70% the vasodilator responses to 5,6-EET. The endothelial dependency was a function of the epoxide interacting with cyclooxygenase of the endothelium, because indomethacin (3 microM) and aspirin (50 microM) prevented the vasodilator response to 5,6-EET while not affecting the response to acetylcholine. SKF-525A (1.1 microM) and metyrapone (150 microM) did not affect the responses to the 5,6-EET, whereas clotrimazole (0.7 microM) and nordihydroguaiaretic acid (2.5 microM) had nonspecific effects, decreasing responses to 5,6-EET and acetylcholine. Because 5,6-EET failed to stimulate detectable release of prostanoids into the effluent from the caudal artery, we conclude that 5,6-EET requires conversion by cyclooxygenase for expression of its vasoactivity.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Arteries; Cytochrome P-450 Enzyme System; Immunoenzyme Techniques; In Vitro Techniques; Oxygenases; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Rats; Vasodilator Agents

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

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

The effect of arachidonic acid and some of its metabolites have been examined in rat anterior pituitary cells for their ability to release growth hormone. The cytochrome P-450 metabolite, 5,6-epoxyeicosatrienoic acid is a much more effective growth-hormone releasing agent than 15-hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid methyl ester, 5-hydroxyeicosatetraenoic acid or arachidonic acid. The release of growth hormone is rapid, dose-dependent and reaches an apparent saturation after eight minutes. These studies described herein provide evidence that lipoxygenase and cyclooxygenase products of arachidonic acid are less potent while cytochrome P-450 products are more potent in the release of growth hormone from anterior pituitary cells.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acids; Cells, Cultured; Fatty Acids, Unsaturated; Female; Growth Hormone; Hydroxyeicosatetraenoic Acids; Kinetics; Pituitary Gland, Anterior; Rats; Rats, Inbred F344

In addition to cyclooxygenase and lipoxygenase pathways, the kidney can also metabolize arachidonic acid by a NADPH-dependent cytochrome P-450 enzyme to epoxyeicosatrienoic acids (EETs); furthermore, 5,6-EET has been shown to alter electrolyte transport across isolated renal tubules. We examined the effects of three EETs (5,6-, 11, 12-, and 14,15-EET) on osmotic water flow across toad urinary bladder. All three EETs reversibly inhibited vasopressin-stimulated osmotic water flow with 5,6- and 11,12-EET being the most potent. The effects appeared to be independent of prostaglandins. EETs inhibited the water flow response to forskolin but not (with the exception of 11,12-EET) the response to adenosine 3',5'-cyclic monophosphate (cAMP) or 8-BrcAMP, consistent with an effect on cAMP generation. For 11,12-EET the question of an additional inhibition at a site beyond or independent of cAMP has to be considered. To determine whether these effects were due to the EETs or to products of their metabolism, we examined the effects of their vicinal diol hydrolysis products, the dihydroxyeicosatrienoic acids. Nonenzymatic conversion of labeled 5,6-EET to its vicinal diol occurred rapidly in the buffer, whereas 11,12-EET was hydrolyzed in a saturable manner only when incubated in the presence of bladder tissue. The dihydroxyeicosatrienoic acids formed inhibited water flow in a manner paralleling that of the EETs. Both 5,6-EET and 11,12-EET (10(-5) M) prevented the increase in intracellular cAMP content observed in control tissues after vasopressin stimulation. Finally, 11,12- and 14,15-dihydroxyeicosatrienoic acid inhibited vasopressin- and forskolin-stimulated adenylate cyclase in the same rank order as their inhibition of water flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 8,11,14-Eicosatrienoic Acid; Adenylyl Cyclases; Animals; Arachidonic Acid; Arachidonic Acids; Bufo marinus; Colforsin; Cyclic AMP; Epithelium; Fatty Acids, Unsaturated; Female; In Vitro Techniques; Urinary Bladder; Vasopressins; Water-Electrolyte Balance

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

Epoxyeicosatrienoic acids, metabolites of the cytochrome P-450-mediated epoxygenase reaction, were detected in human urine by gas chromatographic-mass spectroscopic techniques after conversion to their hydrogenated and non-hydrogenated methyl and pentafluorobenzyl esters. Initial analysis of the regioisomeric composition utilizing the corresponding hydrogenated pentafluorobenzyl esters revealed the presence of the 8,9- and 14,15-isomers.

Topics: 8,11,14-Eicosatrienoic Acid; Cytochrome P-450 CYP2J2; Cytochrome P-450 Enzyme System; Fatty Acids, Unsaturated; Female; Gas Chromatography-Mass Spectrometry; Humans; Isomerism; Oxygenases

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

Arachidonic acid is metabolized to three distinct classes of metabolites: cyclooxygenase produces prostaglandins, prostacyclins, and thromboxanes; lipoxygenase produces hydroperoxyeicosatetraenoic acids and, epoxygenase, a NADPH-dependent cytochrome P-450 enzyme, produces epoxyeicosatrienoic acids. Addition of 5,6-epoxyeicosatrienoic acid (5,6-EET) to GH3 cells, a rat anterior pituitary cell line, produces a rapid, dose-dependent stimulation of prolactin (PRL) release. Incubation with arachidonic acid (AA) was ineffective at increasing PRL release. The lipoxygenase metabolite 5-hydroxyeicosatetraenoic acid (5-HETE), however, increased PRL release from GH3 cells but with a much lower maximal response than 5,6-EET. We examined the role of metabolism inhibitors in 5,6-EET-mediated PRL release. Microsomal and cytosolic epoxide hydrolase (EH) inhibitors do not alter 5,6-EET-induced PRL release, suggesting that EH does not play a significant role in 5,6-EET mediated PRL release from GH3 cells. A chemical analog of 5,6-EET wherein the epoxide oxygen is replaced with a sulfur to afford 5,6-thioepoxyeicosatrienoic acid was also tested and found to stimulate the release of PRL, although not to the same extent as 5,6-EET. Although 5-HETE tends to increase PRL release from GH3 cells, 5,6-EET is significantly more potent at the stimulation of PRL release from GH3 cells.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acids; Cell Line; Epoxide Hydrolases; Epoxy Compounds; Hydroxyeicosatetraenoic Acids; Pituitary Gland, Anterior; Prolactin; Rats

Luteinizing hormone releasing hormone stimulates the concomitant release of luteinizing hormone and 45Ca2+ from prelabeled anterior pituitary cells. Indomethacin (10 microM) and nordihydroguaiaretic acid (10 microM) had no effect on the luteinizing hormone releasing hormone-stimulated release of either luteinizing hormone or 45Ca2+. Eicosatetraynoic acid (10 microM) blocked both luteinizing hormone releasing hormone-stimulated luteinizing hormone secretion and luteinizing hormone releasing hormone-stimulated 45Ca2+ efflux. 5,6-Epoxyeicosatrienoic acid stimulated both luteinizing hormone secretion and 45Ca2+ efflux from anterior pituitary cells. Additionally, 5,6-epoxyeicosatrienoic acid closely mimics the ability of luteinizing hormone releasing hormone to increase intracellular free calcium. These results are consistent with the hypothesis that 5,6-EET alters calcium homeostasis in a manner similar to that observed during luteinizing hormone releasing hormone stimulation of luteinizing hormone release.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Calcium; Cytosol; Ethers; Fatty Acids, Unsaturated; Gonadotropin-Releasing Hormone; Indomethacin; Ionomycin; Luteinizing Hormone; Male; Masoprocol; Pituitary Gland, Anterior; Rats; Rats, Inbred Strains

cis-5(6)Epoxy- and cis-14(15)epoxyeicosatrienoic acid are formed from arachidonic acid by monooxygenases. 5(6)Epoxyeicosatrienoic acid is metabolized by fatty acid cyclooxygenase of ram seminal vesicles and the major products were recently identified as 5(6)epoxy-PGE1 and two stereoisomers of 5-hydroxy-PGI1. The two isomers were likely formed from an unstable intermediate, 5(6)epoxy-PGF1 alpha. The isolation of 5(6)epoxy-PGF1 alpha is described here and 14(15)epoxyeicosatrienoic acid is shown to inhibit fatty acid cyclooxygenase of ram seminal vesicles, albeit less potently than eicosatetraynoic acid (IC50 0.18 and 0.05 mM, respectively).

Topics: 8,11,14-Eicosatrienoic Acid; Alprostadil; Animals; Chromatography, High Pressure Liquid; Fatty Acids, Unsaturated; In Vitro Techniques; Isomerism; Male; Mass Spectrometry; Microsomes; Prostaglandins E; Prostaglandins F; Seminal Vesicles; Sheep; Stereoisomerism

The catalysis of glutathione (GSH) conjugation to epoxyeicosatrienoic acids (EETs) by various purified isozymes of glutathione S-transferase was studied. A GSH conjugate of 14,15-EET was isolated by HPLC and TLC; this metabolite contained one molecule of EET and one molecule of GSH. Fast atom bombardment mass spectrometry of the isolated metabolite confirmed the structure as a GSH conjugate of 14,15-EET. Studies designed to determine the isozyme specificity of this reaction demonstrated that two isozymes, 3-3, and 5-5, efficiently catalyzed this conjugation reaction. The Km values for 14,15-EET were approximately 10 microM and the Vmax values ranged from 25 to 60 nmol conjugate formed min-1 mg-1 purified transferase 3-3 and 5-5. The 5,6-, 8,9-, and 11,12-EETs were also substrates for the reaction, albeit at lower rates. These results demonstrate that the EETs can serve as substrates for the cytosolic glutathione S-transferases.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Fatty Acids, Unsaturated; Glutathione; Glutathione Transferase; Isoenzymes; Kinetics; Male; Mass Spectrometry; Rats; Rats, Inbred Strains; Substrate Specificity

Cytochrome P-450 can metabolize arachidonic (5,8,11,14-eicosatetraenoic) acid to four epoxides. One of them, cis-5(6)oxido-8,11,14-eicosatrienoic acid, has been reported to possess biological activity. To ascertain whether this epoxide could be a substrate for the enzyme fatty acid cyclooxygenase, synthetic 3H-labeled cis-5(6)-oxido-8,11,14-eicosatrienoic acid was incubated with microsomes of ram seminal vesicles and incubated with microsomes of ram seminal vesicles and the products were separated by reversed phase high performance liquid chromatography. The substrate was enzymatically transformed into products, which were more polar than 5,6-dihydroxy-8,11,14-eicosatrienoic acid. The biosynthesis was strongly inhibited by indomethacin or diclofenac sodium, two inhibitors of fatty acid cyclooxygenase. Two of the major metabolites could be identified by capillary gas chromatography-mass spectrometry as two stereoisomers of 5-hydroxyprostaglandin I1, viz. (5R,6R)-5-hydroxyprostaglandin I1 and (5S,6S)-5-hydroxyprostaglandin I1. The structures were established by comparison with the mass spectra of authentic material and by the retention time on capillary gas chromatography using deuterated internal standards. The two stereoisomers were presumably formed nonenzymatically from the intermediate 5(6)oxidoprostaglandin endoperoxides or from 5(6)oxidoprostaglandin F1 alpha during the isolation procedure.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Chromatography, High Pressure Liquid; Epoprostenol; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Male; Microsomes; Seminal Vesicles; Sheep; Stereoisomerism; Structure-Activity Relationship

5(6)-Epoxy-8,11,14-eicosatrienoic acid was incubated with microsomes or ram seminal vesicles in the presence of glutathione (1 mM) for 2 min at 37 degrees C. Following extractive isolation on octadecasilane silica, the products were purified on straight-phase HPLC and separated into three major polar metabolites, which all showed maximal ultraviolet absorbance at 278 nm after treatment with alkali. The least-polar of the three metabolites was identified by capillary column gas chromatography-mass spectrometry as 5(6)- epoxyprostaglandin E1 and the structure was confirmed by comparison with authentic material. The most-polar metabolite was identified as 5,6- dihydroxyprostaglandin E1, while the metabolite of medium polarity was identified as its delta 5-lactone. When glutathione was omitted, 5-hydroxyprostaglandin I 1 alpha and 5-hydroxyprostaglandin I 1 beta were previously identified as the two major metabolites. These results indicate that the postulated epoxyprostaglandin endoperoxide intermediates, 5(6)- epoxyprostaglandin G1 and 5(6)- epoxyprostaglandin H1, might be substrates for the endoperoxide E isomerase enzyme, since this enzyme requires glutathione as a cofactor.

Topics: 8,11,14-Eicosatrienoic Acid; Alprostadil; Animals; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Glutathione; Male; Microsomes; Prostaglandins B; Prostaglandins E; Seminal Vesicles; Sheep