calcimycin and 15-hydroperoxy-5-8-11-13-eicosatetraenoic-acid

Recent data from our laboratory, as well as supporting evidence from other investigators, strongly suggest that the PMN 15-LO exists in a cryptic state. Several stimuli, including HETEs, can convert the inactive 15-LO to an active species that can metabolize AA to a variety of products. Many of these metabolites have been reported to modulate various components of the immune response.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 15-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Enzyme Activation; Humans; Hydroxyeicosatetraenoic Acids; Immune System; Leukotrienes; Lipid Peroxides; Models, Biological; Neutrophils

Selenium (Se) is an integral part of the Se-dependent glutathione peroxidase (Se-GSH-Px) catalytic domain. By modulating the cellular levels of fatty acid hydroperoxides, Se-GSH-Px can influence key enzymes of arachidonic acid cascade, in this case cyclooxygenase (COX) and lipoxygenase (LOX). To investigate this phenomenon, the effects of cellular Se status on the enzymatic oxidation of arachidonic acid were investigated in bovine mammary endothelial cells (BMEC), which were cultured in either Se-deficient (-Se) or Se-adequate (+Se) media. When stimulated with calcium ionophore A23187, BMEC produced eicosanoids of both COX and LOX pathways. Compared with the Se-adequate cells, the production of prostaglandin I(2) (PGI(2)), prostaglandin F(2) (PGF(2alpha)), and prostaglandin E(2) (PGE(2)) was significantly decreased in Se-deficient cells, whereas the production of thromboxane A(2) (TXA(2)) was markedly increased in the -Se BMEC cultures. Although the enzymatic oxidation of arachidonic acid by the LOX pathway was found to be relatively less than by the COX pathway, the BMEC cultured in -Se media produced significantly more 15-hydroperoxyeicosatetraenoic acid (15-HPETE) than the +Se cells produced. Based on these results, we postulate that cellular Se status plays an important regulatory role in the enzymatic oxidation of arachidonic acid by the COX and LOX pathways. The altered eicosanoid biosynthesis, especially the overproduction of 15-HPETE, in -Se BMEC may be one of the underlying biochemical phenomena responsible for vascular dysfunction during Se deficiency.

Topics: Animals; Arachidonic Acid; Calcimycin; Cattle; Cells, Cultured; Culture Media; Dinoprost; Dinoprostone; Eicosanoids; Endothelium, Vascular; Epoprostenol; Female; Leukotrienes; Lipid Peroxides; Lipoxygenase; Mammary Arteries; Oxidation-Reduction; Prostaglandin-Endoperoxide Synthases; Selenium; Thromboxane A2

The lipoxygenase product 15-hydroxyeicosatetraenoic acid (15-HETE) was shown to be the most important eicosanoid formed in the atherosclerotic rabbit aorta. The aim of the present study was to compare the effects of 15-HETE and its hydroperoxy precursor 15-HpETE with those of other vasoconstrictor and vasodilator agents in arteries from rabbits fed either a control or a cholesterol-rich diet for 16 and 30 weeks. 5-Hydroxytryptamine (5-HT) aggregated platelets and thrombin caused contractions of isolated rabbit aortas. The contractile responses elicited by platelets from control animals were similar to those evoked by platelets from atherosclerotic rabbits. After 16 weeks of hypercholesterolemia, the contractile responses were either augmented (5-HT), unchanged (platelets) or reduced (thrombin). After 30 weeks of hypercholesterolemia, the responses to all contractile agents used had decreased. In both aortas and pulmonary arteries the endothelium-dependent relaxations to the calcium ionophore, A23167, and to acetylcholine were progressively lost and the endothelium-independent relaxations to nitroglycerin were reduced by the progressing hypercholesterolemia. The 15-lipoxygenase metabolites contracted the isolated thoracic aorta and pulmonary artery from control rabbits and to a lesser extent those of the cholesterol-fed rabbits. After raising the tone in these vessels with prostaglandin F2 alpha PGF2 alpha) or noradrenaline, 15-HpETE induced relaxations which were not significantly influenced by the development of fatty streaks. Our data illustrate that the contractions of the blood vessel wall to 15-HETE, like those to other vasoconstrictors, are markedly reduced by developing atherosclerosis. In contrast, the relaxations to 15-HpETE in the rabbit arteries remain unaltered after 16 to 30 weeks of hypercholesterolemia. This is unlike the reactions to other vasodilators, which are markedly reduced.

Topics: Acetylcholine; Animals; Arachidonate 15-Lipoxygenase; Arteriosclerosis; Calcimycin; Dinoprost; Hydroxyeicosatetraenoic Acids; Hypercholesterolemia; In Vitro Techniques; Leukotrienes; Lipid Peroxides; Male; Muscle, Smooth, Vascular; Nitroglycerin; Platelet Aggregation; Rabbits; Thrombin; Vasoconstrictor Agents; Vasodilator Agents

Rat basophilic leukemia (RBL-1) cells (1.5 x 10(9) were incubated with (15S), 15-hydroperoxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HPETE) (100 microM) and calcium ionophore A23187 (5 microM) for 30 min at 37 degrees C. The reaction products were extracted and separated on reverse-phase high performance liquid chromatography (RP-HPLC). The fraction of interest which showed the characteristic UV spectrum of lipoxins (LXs) (lambda max at 301 nm, shoulders at 289 nm and 317 nm) was further purified using a second RP-HPLC. This step produced four distinct peaks, three of which co-eluted with synthetic lipoxin A4 (LXA4), lipoxin B4 (LXB4), and authentic 6S-LXA4 standards, respectively. These fractions were analyzed by capillary gas chromatography and mass spectrometry (GC/MS) for structural identity. Based on these data (chromatographic profile, UV spectrum, mass spectra) and the reported criteria (Serhan/Samuelsson, 1984-88), these three fractions were identified with LXA4[(5S,6R,15S)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetra enoic acid)], LXB4[(5S,14R,15S)-5,14,15-trihydroxy-6,10,12-trans-8-cis-eic osa-tetraenoic acid)] and 6S-LXA4[(5S,6S,15S)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosate traenoic acid)]. It is concluded that RBL-1 cells have the capacity to generate LXs by metabolizing arachidonic acid (AA) derivatives (i.e., 15-HPETE) and that LXs produced by RBL-1 cells are indistinguishable from those derived from other cells.

Topics: Animals; Calcimycin; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Leukotrienes; Lipid Peroxides; Lipoxins; Tumor Cells, Cultured

Incubation of RBL-1 cells in the presence of 15-HPETE and various agonists generated lipoxins and several isomers. Addition of either A23187, fMLP or PMA modulated the number of isomers and amount of lipoxins produced. Administration of A23187 yielded the largest amount of product (5.3 +/- 1.6 micrograms per 10(8) cells) and generated a total of six and three isomers of LXA4 and B4, respectively. This was 2-fold greater than fMLP, which produced a total of two isomers of LXA4 and LXB4. Addition of PMA generated only LXA4 (0.68 +/- 0.26 micrograms). This is similar to the control receiving only 15-HPETE. Biologically derived LXA4 (3 nM) isolated from RBL-1 incubations contracted a rat tail artery preparation to 12% of the maximum induced by phenylephrine (0.125 microM), whereas LXA4 standard (3 nM) elicited 17.6% of the maximum contraction. These results indicate that RBL-1 cells can utilize exogenous 15-HPETE to generate biologically active lipoxins. Further, the yield and isomers of lipoxins can be modified by different agonists.

Topics: Animals; Calcimycin; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Lipoxins; N-Formylmethionine Leucyl-Phenylalanine; Rats; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

1. The mechanism by which incubation of human peripheral blood neutrophils with exogenous arachidonic acid leads to 5-lipoxygenase product synthesis was investigated. 2. Incubation of neutrophils with arachidonic acid caused a concentration- and time-dependent synthesis of leukotriene B4, its omega-oxidation products, and 5-hydroxyeicosatetraenoic acid. 3. The threshold concentration of arachidonic acid required for this effect was equal to, or greater than 3.3 microM and the synthesis increased with up to 33 microM arachidonic acid, the highest concentration used. Synthesis induced by arachidonic acid increased with time for up to 15 min and the major products detected were the omega-oxidation products of leukotriene B4. 4. Pre-incubation of neutrophils with pertussis toxin inhibited the synthesis of 5-lipoxygenase products induced by arachidonic acid by 75% or more, but had no effect on either arachidonic acid-induced synthesis of the 15-lipoxygenase product, 15-hydroxyeicosatetraenoic acid, or activation of the 5-lipoxygenase induced by the calcium ionophore A23187. 5. Pre-incubation of neutrophils with granulocyte-macrophage colony-stimulating factor lead to enhanced leukotriene synthesis in response to arachidonic acid. 6. These results imply that exogenous arachidonic acid is not only used as a substrate, but also activates the 5-lipoxygenase. Possible mechanisms of action are discussed.

Topics: Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Blood Cells; Calcimycin; Chromatography, High Pressure Liquid; Colony-Stimulating Factors; Enzyme Activation; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; GTP-Binding Proteins; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotrienes; Lipid Peroxides; Neutrophils; Pertussis Toxin; Virulence Factors, Bordetella

Topics: Animals; Calcimycin; Cell Line; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Leukotrienes; Lipid Peroxides; Lipoxins; N-Formylmethionine Leucyl-Phenylalanine; Rats; Tetradecanoylphorbol Acetate

Incubation of rat mesangial cells with leukotriene A4 in the presence of calcium ionophore A23187 led to a substrate dependent formation of lipoxin and its isomers. The major metabolite coeluted with authentic lipoxin A4 (LXA4) and lipoxin B4 (LXB4) in RP-HPLC system, and possessed a characteristic U.V. spectrum and C-value which were identical to authentic standards. GC/MS analysis on LXA4 further demonstrates that the mesangial cell derived LXA4 was identical to that reported by Serhan et al. (1) as LXA4 [5(S), 6,(R), 15(S)-trihydroxy7,9,13-trans-11-cis-eicosatetraenoic acid]. The formation of LXA4 was linear with substrate (LTA4) concentration. No similar products occurred in boiled controls. Incubation of mesangial cell with 15-HPETE failed to produce any lipoxin-like material. The absence of LX-like substance following incubation of 15-HPETE with mesangial cells suggested that 5-lipoxygenase activity is not expressed in mesangial cells under these conditions. The generation of LXA4 from LTA4 in mesangial cells suggested that there is an active 15- or 12- lipoxygenase activity in the kidney. The production of LX may play an important role in the regulation of renal function and the response to inflammatory stimuli.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cells, Cultured; Chromatography, High Pressure Liquid; Glomerular Mesangium; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotrienes; Lipid Peroxides; Lipoxins; Male; Mass Spectrometry; Rats; Rats, Inbred Strains; Spectrophotometry, Ultraviolet

Rat alveolar macrophages incubated with (15S), 15-hydroperoxy-5,8, 11-cis-13-trans-eicosatetraenoic acid (15-HPETE) in the presence of the calcium ionophore A23187 produced a series of polar compounds. Reverse-phase high performance liquid chromatography (RP-HPLC) was used to purify these compounds and their identities were characterized by physical criteria (UV spectroscopy, GC/MS) and by co-elution with synthetic compounds. Alveolar macrophage-derived materials proved to be lipoxin A4 [LXA4; (5S, 6R, 15S)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid], lipoxin B4[LXB4; (5S, 14R, 15S)-5,14,15-trihydroxy-6,10,12-trans-8-cis- eicosatetraenoic acid], and a structural isomer. These results indicate that rat alveolar macrophages have the capacity to generate lipoxins and related compounds.

Topics: Animals; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotrienes; Lipid Peroxides; Macrophages; Rats; Rats, Inbred Strains; Spectrophotometry, Ultraviolet

Lipoxin B (LXB) was prepared by incubation of (15S)-15-hydroperoxy-5,8,11-cis-13-trans-icosatetraenoic acid (15-HPETE) with human leukocytes. Comparison with a number of trihydroxyicosatetraenes prepared by total synthesis showed that biologically derived LXB is (5S,14R,15S)-5,14,15-trihydroxy-6,10,12-trans-8-cis-icosatetraenoi c acid. Two isomers of LXB were identified by using an improved isolation procedure. These compounds were shown to be (5S,14R,15S)-5,14,15-trihydroxy-6,8,10,12-trans-icosatetraenoic acid (8-trans-LXB) and (5S,14S,15S)-5,14,15-trihydroxy-6,8,10,12-trans-icosatetraenoic acid [(14S)-8-trans-LXB]. Experiments with 18O2 showed that formation of LXB and its two isomers occurred with incorporation of molecular oxygen at C-5 but not at C-14. These results together with the finding that (15S)-hydroxy-5,8,11-cis-13-trans-icosatetraenoic acid (15-HETE) is a precursor of LXB compounds in activated leukocytes suggest that 15-hydroxy-5,6-epoxy-7,9,13-trans-11-cis-icosatetraenoic acid or its equivalent is a common intermediate in the biosynthesis of LXB and its two isomers.

Topics: Arachidonic Acids; Blood Platelets; Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Isomerism; Leukocytes; Leukotrienes; Lipid Peroxides; Lipoxins; Spectrophotometry, Ultraviolet; Stereoisomerism

The effect of 5-lipoxygenase products of arachidonic acid on 14-C-alkyl-acetyl-glycero-phosphocholine (14C-alkyl-acetyl GPC) production in rat peritoneal macrophages was investigated, using macrophages prelabeled with N-methyl-14C-alkyl-lyso-glycero-phosphocholine (14C-alkyl-lyso GPC) (prelabeled macrophages). Bromophenacyl bromide (BPB: phospholipase A2 inhibitor), and AA861 (5-lipoxygenase inhibitor) suppressed the production of 14C-alkyl-acetyl GPC in the A23187-stimulated prelabeled macrophages in a dose-dependent manner. A23187-induced hydrolysis of 14C-alkyl-acyl-glycero-phosphocholine (14C-alkyl-acyl GPC) and formation of 14C-alkyl-lyso GPC were also reduced by BPB and AA861. However, indomethacin (IND: cyclo-oxygenase inhibitor) had no significant effect on 14C-alkyl-acetyl GPC production in the A23187-stimulated prelabeled macrophages. Exogenously supplied 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) reversed the inhibitory effect of AA861 on 14C-alkyl-acetyl GPC production in A23187-stimulated prelabeled macrophages. Reduced hydrolysis of 14C-alkyl-acyl GPC and formation of 14C-alkyl-lyso GPC in A23187-stimulated prelabeled macrophages, which were pretreated with AA861, were also reversed by the addition of 5-HPETE and 5-HETE. However, LTB4 had no such effects. 5-HPETE and 5-HETE augmented the stimulatory effect of A23187 on 14C-alkyl-acetyl GPC production in prelabeled macrophages, while they could not stimulate alkyl-acetyl GPC production in the absence of A23187. These results suggest that 5-lipoxygenase products, especially 5-HPETE and 5-HETE, may play an important role in alkyl-acetyl GPC production in rat peritoneal macrophages.

Topics: Acetophenones; Animals; Arachidonic Acids; Benzoquinones; Calcimycin; Carbon Radioisotopes; Dose-Response Relationship, Drug; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotrienes; Lipid Peroxides; Macrophages; Male; Platelet Activating Factor; Quinones; Rats; Rats, Inbred Strains

Potentiation of chemical toxicity by hypoxia was studied in confluent hepatocyte monolayers. Addition of either hydroperoxyarachidonic acid (50 micrograms), leukotriene C4 (10 micrograms), or calcium ionophore A23187 (1.8 micrograms) to hepatocyte monolayers followed by incubation in 2% oxygen for 24 h killed 95% of the hypoxic cells, but was without effect on the normoxic cells. The greater than 10-fold increase in toxicity of A23187 suggests that hypoxic cells are less able to regulate intracellular calcium. The increased toxicity of hydroperoxyarachidonic acid and leukotriene C4 may be due to a related reduction in activity of protective enzymes.

Topics: Animals; Arachidonic Acids; Calcimycin; Cell Survival; Cells, Cultured; Leukotriene B4; Leukotrienes; Lipid Peroxides; Liver; Male; Oxygen; Rats; Rats, Inbred Strains; SRS-A

Addition of 15L-hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) to human leukocytes led to the formation of a novel series of compounds containing four conjugated double bonds. The yield of tetraenes was increased approx. 100-fold when ionophore A23187 (5 microM) was added simultaneously with 15-HPETE. The structure of the major tetraene was established by physical methods as well as by chemical degradation and found to be 5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic acid.

Topics: Alkenes; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Calcimycin; Chemical Phenomena; Chemistry; Humans; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotrienes; Lipid Peroxides; Lipoxins

To address the hypothesis that metabolites of arachidonic acid are important regulators of prostaglandin (PG) synthesis in intact vascular tissue, we studied arachidonate metabolism in rabbit aortas in response to a continuous infusion of arachidonic acid, 10 micrograms/ml. Prostacyclin (PGI2; measured as 6-keto-PGF1 alpha) production rate accelerated during the first 2 min, reached peak velocity at 2 min, and then progressively decelerated. The velocity profile of PGI2 production was similar to that previously reported for cyclooxygenase holoenzyme assayed in vitro, and was consistent with progressive inactivation of the enzymes leading to PGI2 synthesis. We determined the specific inhibition of cyclooxygenase and prostacyclin synthetase by measuring PGI2 and PGE2 production rates and by infusing cyclic endoperoxides. Our results indicate preferential inactivation of cyclooxygenase during arachidonate metabolism, most likely due to cyclooxygenase-derived oxidative intermediates. This was a dose-dependent response and resulted in a progressive decrease in the 6-keto-PGF1 alpha/PGE2 ratio. Exogenously added 15-hydroperoxy eicosatetraenoic acid, on the other hand, actually stimulated cyclooxygenase activity at low doses, while markedly inhibiting prostacyclin synthetase. This finding, along with the accelerating nature of arachidonate metabolism, is consistent with the concept of "peroxide tone" as a mediator of cyclooxygenase activity in this system. These results demonstrate that arachidonate metabolites regulate PG synthesis in intact blood vessels. The progressive enzymatic inhibition intrinsic to arachidonate metabolism may be a model for similar changes occurring in states of enhanced lipid peroxidation. These metabolic alterations might greatly influence the numerous vascular functions known to involve arachidonic acid metabolism.

Topics: Animals; Aorta, Thoracic; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cytochrome P-450 Enzyme System; Dinoprostone; Dose-Response Relationship, Drug; Epoprostenol; Female; Intramolecular Oxidoreductases; Leukotrienes; Lipid Peroxides; Male; Perfusion; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Prostaglandins E; Prostaglandins F; Rabbits

In this paper, we examined the effect of two known inhibitors of prostacyclin synthetase, tranylcypromine and 15-hydroperoxyarachidonate, on bradykinin-stimulated prostacyclin synthesis and arachidonic acid release from cellular phospholipids in endothelial cells derived from calf aorta. These two inhibitors inhibit prostacyclin synthesis stimulated by bradykinin, arachidonic acid, and ionophore A23187. However, these two inhibitors also affect the release of arachidonic acid from cells. Using cells prelabeled with [3H]arachidonic acid, we found that bradykinin-stimulated arachidonic acid release is severely inhibited by tranylcypromine (500 microgram/ml) but is stimulated by 15-hydroxyperoxyarachidonate at a concentration as low as 1 microgram/ml. We also found that 15-hydroperoxyarachidonate inhibits not only prostacyclin formation but also prostaglandin formation from the released arachidonic acid. Under the conditions used, these two compounds have no effects on the cell viability as judged by trypan blue exclusion test. We conclude that tranylcypromine and 15-hydroperoxyarachidonate not only inhibit prostacyclin synthesis but also affect other steps in the metabolism of arachidonic acid in whole cells.

Topics: Animals; Aorta; Arachidonic Acids; Bradykinin; Calcimycin; Cattle; Endothelium; Epoprostenol; Leukotrienes; Lipid Peroxides; Peroxides; Prostaglandins; Tranylcypromine