calcimycin and 15-hydroxy-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

15-hydroxyeicosatetraenoic acid (15-HETE) is an arachidonic acid derived lipid mediator which can originate both from 15-lipoxygenase (15-LOX) activity and cyclooxygenase (COX) activity. The enzymatic source determines the enantiomeric profile of the 15-HETE formed. 15-HETE is the most abundant arachidonic acid metabolite in the human lung and has been suggested to influence the pathophysiology of asthma. Mast cells are central effectors in asthma, but there are contradictory reports on whether 15-HETE originates from 15-LOX or COX in human mast cells. This prompted the current study where the pathway of 15-HETE biosynthesis was examined in three human mast cell models; the cell line LAD2, cord blood derived mast cells (CBMC) and tissue isolated human lung mast cells (HLMC). Levels and enantiomeric profiles of 15-HETE and levels of the downstream metabolite 15-KETE, were analyzed by UPLC-MS/MS after stimulation with anti-IgE or calcium ionophore A23187 in the presence and absence of inhibitors of COX isoenzymes. We found that 15-HETE was produced by COX-1 in human mast cells under these experimental conditions. Unexpectedly, chiral analysis showed that the 15(R) isomer was predominant and gradually accumulated, whereas the 15(S) isomer was metabolized by the 15-hydroxyprostaglandin dehydrogenase. We conclude that during physiological conditions, i.e., without addition of exogenous arachidonic acid, both enantiomers of 15-HETE are produced by COX-1 in human mast cells but that the 15(S) isomer is selectively depleted by undergoing further metabolism. The study highlights that 15-HETE cannot be used as an indicator of 15-LOX activity for cellular studies, unless chirality and sensitivity to pharmacologic inhibition is determined.

Topics: Calcimycin; Cell Line; Cyclooxygenase 1; Humans; Hydroxyeicosatetraenoic Acids; Immunoglobulin E; Lung; Mast Cells

The accumulation of neutrophils and proinflammatory mediators, such as leukotriene B4 (LTB4), is a classic marker of inflammatory disease. The clearance of apoptotic neutrophils, inhibition of proinflammatory signaling, and production of proresolving lipids (including lipoxins, such as lipoxin A4 [LXA4]) are imperative for resolving inflammation. Tulathromycin (TUL), a macrolide used to treat bovine respiratory disease, confers immunomodulatory benefits via mechanisms that remain unclear. We recently reported the anti-inflammatory properties of TUL in bovine phagocytes in vitro and in Mannheimia haemolytica-challenged calves. The findings demonstrated that this system offers a powerful model for investigating novel mechanisms of pharmacological immunomodulation. In the present study, we examined the effects of TUL in a nonbacterial model of pulmonary inflammation in vivo and characterized its effects on lipid signaling. In bronchoalveolar lavage (BAL) fluid samples from calves challenged with zymosan particles (50 mg), treatment with TUL (2.5 mg/kg of body weight) significantly reduced pulmonary levels of LTB4 and prostaglandin E2 (PGE2). In calcium ionophore (A23187)-stimulated bovine neutrophils, TUL inhibited phospholipase D (PLD), cytosolic phospholipase A2 (PLA2) activity, and the release of LTB4. In contrast, TUL promoted the secretion of LXA4 in resting and A23187-stimulated neutrophils, while levels of its precursor, 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], were significantly lower. These findings indicate that TUL directly modulates lipid signaling by inhibiting the production of proinflammatory eicosanoids and promoting the production of proresolving lipoxins.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Bronchoalveolar Lavage Fluid; Calcimycin; Cattle; Dinoprostone; Disaccharides; Heterocyclic Compounds; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxins; Lung; Male; Neutrophils; Particulate Matter; Phospholipases A2; Pneumonia; Primary Cell Culture; Zymosan

The arachidonic acid (AA) metabolic network produces key inflammatory mediators which have been considered as hallmark contributors in various inflammatory related diseases. Enzymes in this network, such as 5-lipoxygenase (5-LOX), cyclooxygenase (COX), leukotriene A(4) hydrolase (LTA4H) and prostaglandin E synthase (PGES), have been used as targets for anti-inflammatory drug discovery. Multi-target drugs and drug combinations have also been developed for this network. However, how the inhibitors alter the dynamics of metabolite production and which combinatorial target intervention solutions are better needs further exploration. We did a system based intervention analysis on the AA metabolic network. Using an LC-MS/MS method, we quantitatively studied the eicosanoid metabolites responses of AA metabolic network during stimulation of Sprague Dawley rat blood samples with the calcium ionophore. Our results indicate that inhibiting the upstream rather than the downstream target of 5-LOX pathway will simultaneously alter the AA metabolism to the COX pathway (and vice versa). Therefore, single-target inhibitors cannot control all the inflammatory mediators at the same time. We also suggest that in the case of multiple-target anti-inflammatory solutions, the combination of inhibitors of the downstream enzymes may have stronger inhibition efficiency and cause less side-effects compared to the other solutions. One therapeutic strategy, LTA4H/COX inhibition solution, was found promising for the intervention of inflammatory mediator biosynthesis and at the same time stimulating the production of anti-inflammatory agents.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Biosynthetic Pathways; Calcimycin; Chromatography, Liquid; Cyclooxygenase Inhibitors; Dinoprostone; Eicosanoids; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Intramolecular Oxidoreductases; Leukotriene B4; Lipoxygenase Inhibitors; Male; Mass Spectrometry; Metabolic Networks and Pathways; Models, Biological; Prostaglandin-E Synthases; Rats; Rats, Sprague-Dawley; Time Factors

Resolution of inflammation is an active temporally orchestrated process demonstrated by the biosynthesis of novel proresolving mediators. Dysregulation of resolution pathways may underlie prevalent human inflammatory diseases such as cardiovascular diseases and periodontitis. Localized Aggressive Periodontitis (LAP) is an early onset, rapidly progressing form of inflammatory periodontal disease. Here, we report increased surface P-selectin on circulating LAP platelets, and elevated integrin (CD18) surface expression on neutrophils and monocytes compared to healthy, asymptomatic controls. Significantly more platelet-neutrophil and platelet-monocyte aggregates were identified in circulating whole blood of LAP patients compared with asymptomatic controls. LAP whole blood generates increased pro-inflammatory LTB4 with addition of divalent cation ionophore A23187 (5 µM) and significantly less, 15-HETE, 12-HETE, 14-HDHA, and lipoxin A(4). Macrophages from LAP subjects exhibit reduced phagocytosis. The pro-resolving lipid mediator, Resolvin E1 (0.1-100 nM), rescues the impaired phagocytic activity in LAP macrophages. These abnormalities suggest compromised resolution pathways, which may contribute to persistent inflammation resulting in establishment of a chronic inflammatory lesion and periodontal disease progression.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Blood Platelets; Calcimycin; Calcium Ionophores; Cells, Cultured; Chromatography, Liquid; Eicosapentaenoic Acid; Female; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxins; Macrophages; Male; Middle Aged; Monocytes; Neutrophils; P-Selectin; Periodontitis; Phagocytosis; Tandem Mass Spectrometry

Rat intestinal epithelial cells that permanently express the cyclooxygenase-2 (COX-2) gene (RIES cells) were used to investigate COX-2-mediated arachidonic acid (AA) metabolism. A targeted chiral lipidomics approach was employed to quantify AA metabolites that were secreted by the cells into the culture media. When intact RIES cells were treated with calcium ionophore A-23187 (1 microM) for 1 h, 11-(R)-hydroxyeicosatetraenoic acid (HETE) was the most abundant metabolite, followed by prostaglandin (PG) E 2, 15-(S)-HETE, 15-oxo-eicosatetraenoic acid (ETE), and 15-(R)-HETE. Incubation for a further 23 h after the calcium ionophore was removed resulted in a substantial increase in PGE 2 concentrations while HETE and 15-oxo-ETE concentrations decreased to almost undetectable levels. A similar metabolic profile was observed when RIES cells were treated with increasing concentrations of AA for 24 h. Incubation of the RIES cells with 10 microM AA revealed that maximal concentrations of 11-(R)-HETE, 15-(S)-HETE, and 15-oxo-ETE occurred after 10 min of incubation when the 15-( S)-HETE concentrations were approximately twice that of PGE 2. There was a gradual decrease in the concentrations of HETE and 15-oxo-ETE over time, whereas PGE 2 concentrations increased steadily until they reached a maximum after 24 h of incubation. The ratio of PGE 2 to 15-(S)-HETE was then approximately 20:1. 15-(S)-HETE and 15-oxo-ETE concentrations declined in the cell media during prolonged incubations with pseudo-first-order rate constants of 0.0121 and 0.0073 min(-1), respectively. 15-(S)-HETE was shown to undergo metabolism primarily to 15-oxo-ETE, which was further metabolized to a glutathione (GSH) adduct. The GSH adduct of 15-oxo-ETE was further metabolized in the extracellular milieu to a cysteinylglycine adduct. Thus, we have established for the first time that 15-oxo-ETE can be formed biosynthetically from AA, that 15-(S)-HETE is its immediate precursor, and that 15-oxo-ETE forms a GSH adduct. For ionophore-A-23187-stimulated cells and at early time points for AA-stimulated cells, 11-(R)-HETE was the major eicosanoid to be secreted into the media. Adding increasing concentrations of AA to cells in culture made it possible to estimate with surprising accuracy endogenous eicosanoid production using regression analyses. Thus, after 24 h in the absence of added AA, 11-(R)-HETE and 15-(R)-HETE were estimated to be present at concentrations close to the detection limit of our very se

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cells, Cultured; Cyclooxygenase 2; Dipeptides; Glutathione; Hydroxyeicosatetraenoic Acids; Intestinal Mucosa; Mass Spectrometry; Rats

Topics: Arachidonic Acid; Asthma; Calcimycin; Cell Culture Techniques; Eicosanoids; Glucocorticoids; Humans; Hydroxyeicosatetraenoic Acids; Ionophores; Macrophages, Alveolar; Monocytes; Neutrophils

The supposed 5-LO inhibitory activity of two N-omega-ethoxycarbonyl-4-quinolones was tested determining leukotriene B4 (LTB4) in RBL-1 cell cultures, pretreated with the two compounds of interest. LTB4, obtained by solid-phase extraction (SPE) from cell cultures supernatants, was determined by micellar electrokinetic chromatography (MEKC). The analysis was performed using an uncoated capillary, filled with borate buffer at pH 8.3, containing 12.5 mM SDS as micelles generator. Therefore, following the decreasing of LTB4 it was possible to verify the 5-LO inhibitory activity of two quinolone derivatives. To asses the suitability of the use of LTB4 as marker of the activity of the new compounds, the analysis was repeated using quercetin, a well known 5-LO inhibitor.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 5-Lipoxygenase; Biomarkers; Calcimycin; Chromatography, High Pressure Liquid; Chromatography, Micellar Electrokinetic Capillary; Culture Media, Conditioned; Electrophoresis, Capillary; Enzyme Activation; Evaluation Studies as Topic; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Leukotriene B4; Lipoxygenase Inhibitors; Prostaglandins B; Quercetin; Quinolones; Rats; Sodium Dodecyl Sulfate; Tumor Cells, Cultured

To investigate the cooperation of chondrocytes and polymorphonuclear cells (PMN) in the biosynthesis of leukotrienes (LT).. PMN, resting and interleukin-1beta-stimulated cultured human chondrocytes, and mixtures of both cell types were incubated with A23187 and/or 14C-arachidonic acid (14C-AA). To explore the presence of LTC4 synthase and LTA4 hydrolase, the chondrocytes were incubated with authentic LTA4. Eicosanoids were analyzed using high performance liquid chromatography techniques.. Chondrocytes formed only prostaglandin E2 and minor amounts of 15-HETE and 11-HETE, the production of all of which was inhibited by 1 microM indomethacin. Incubation of PMN and chondrocytes produced more LTC4 from endogenous and exogenous AA, and more LTB4 from endogenous AA, than incubation of PMN alone, which was consistent with the presence of LTC4 synthase and LTA4 hydrolase activities in chondrocytes. Chondrocytes also slightly increased the level of PMN production of all 5-lipoxygenase (5-LO)-derived products from endogenous AA.. Human chondrocytes form eicosanoids from AA only by the cyclooxygenase pathway. Chondrocytes cooperate in the transcellular biosynthesis of LT since they possess LTA4 hydrolase and LTC4 synthase activities and increase metabolism by the 5-LO pathway in PMN.

Topics: Arachidonic Acid; Calcimycin; Cartilage, Articular; Cells, Cultured; Chondrocytes; Chromatography, High Pressure Liquid; Culture Media, Conditioned; Dinoprostone; Epoxide Hydrolases; Glutathione Transferase; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Knee Joint; Leukotriene B4; Leukotriene C4; Neutrophils

Serum-cultured rat W256 carcinosarcoma cells of the monocytoid origin undergo rapid apoptosis in response to the lipoxygenase inhibitor NDGA (nordihydroguaiaretic acid). Exogenous arachidonic acid (AA), in a time- and dose-dependent fashion, suppressed NDGA-induced W256 cell apoptosis as well as DNA fragmentation, with the maximal effect observed at approximately 25 microM. Mobilization of endogenous AA by calcium ionophore A23187 provided an even stronger and longer-lasting protection against NDGA-caused cell death. The A23187 effect on AA release as well as W256 cell death can be blocked by bromophenacyl bromide, thus suggesting involvement of phospholipase A2 activation. Serum withdrawal similarly caused W256 cells to undergo typical apoptosis, which was not rescued by several growth factors commonly found in serum. However, exogenous AA suppressed serum starvation-induced W256 cell apoptosis and significantly extended cell survival in a dose-dependent manner. Lipoxygenase products, 12(S)- and 15(S)-, but not 5(S)-hydroxyeicosatetraenoic acid (HETE), in a dose-dependent fashion, also prevented both NDGA- and serum-starvation-induced W256 cell apoptosis. AA appears to suppress W256 cell apoptosis via distinct signaling pathway(s) since it does not prevent cell death triggered by several other inducers. Examination of a panel of polyunsaturated fatty acids revealed that alpha-linolenic and linoleic acid can also suppress NDGA-induced W256 cell apoptosis. Our data suggest that AA and other polyunsaturated fatty acids and/or their metabolites may enhance tumor growth not only by promoting cell proliferation but also by suppressing apoptosis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Acetophenones; Animals; Apoptosis; Arachidonic Acid; Calcimycin; Carcinosarcoma; Cell Line; Cell Survival; Cytokines; DNA Fragmentation; Fatty Acids, Nonesterified; Growth Substances; Hydroxyeicosatetraenoic Acids; Kinetics; Masoprocol; Rats; Tumor Cells, Cultured

(15S)-Hydroxy-(5Z,8Z,11Z,13E)-eicosatetraenoic acid (15-HETE) suppresses in ionophore-A23187-stimulated human polymorphonuclear leucocytes (PMN) the conversion of exogenous arachidonic acid into leukotriene B(4) (LTB4) and (5S)-hydroxy-(6E,8Z,11Z,14Z)-eicosatetraenoic acid (5-HETE). However, contrary to earlier suggestions, 15-HETE is not a genuine 5-lipoxygenase inhibitor under these conditions, but rather suppresses the 5-lipoxygenation of arachidonic acid by switching-over of substrate utilization, as judged from a sizeable formation of labelled (5S,15S)-dihydroxy-(6E,8Z,11Z,13E)-eicosatetr aen oic acid (5,15-diHETE) from 15-[1(-14)C]HETE. Identical results were obtained with human recombinant 5-lipoxygenase. In PMN the formation of 5,15-diHETE is strongly stimulated by either hydroperoxypolyenoic fatty acids or arachidonic acid, suggesting a crucial role of the hydroperoxide tone of the cell. A comparison of a selection of hydroxypolyenoic fatty acids with respect to their capability of suppressing 5-lipoxygenation of arachidonic acid revealed that 15-mono-hydroxyeicosanoids throughout exhibit the highest inhibitory potencies, whereas the other HETEs, 5,15-diHETE as well as octadecanoids, are modest or poor inhibitors. The R and S enantiomers of 15-HETE do not differ from each other, excluding a receptor-like binding of the 15-hydroxy group.

Topics: Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Calcimycin; Fatty Acids, Unsaturated; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Lipoxygenase Inhibitors; Neutrophils; Recombinant Proteins; Structure-Activity Relationship; Substrate Specificity

5-Lipoxygenase activation of human blood polymorphonuclear cells (PMN) from asthmatic patients (asthmatics) was studied to investigate whether differences may exist with healthy subjects (controls). The respective cell capacities to produce lipoxins (LXs), leukotrienes, and 5(S), 15(S)-dihydroxyeicosatetraenoic acid [5(S),15(S)-diHETE] were compared under in vitro stimulation by ionophore A23187, with or without exogenous 15(S)-hydroxyeicosatetraenoic acid [15(S)-diHETE]. Eicosanoids were analyzed by elution with an isocratic reverse-phase high performance liquid chromatography system, and their profiles, detected by simultaneous monitoring at 302, 280, and 246 nm, were evaluated on the basis of chromatographic behavior: UV spectral characteristics and coelution with synthetic standards. In the presence of exogenous 15(S)-HETE, human PMN were able to produce LXs and 5(S),15(S)-diHETE, PMN from asthmatics were able to produce 5(S), 5(S),15(S)-diHETE, and LXs from endogenous sources, whereas in the same experimental conditions, no detectable amounts of these compounds were released by PMN from controls. The levels of 5(S),15(S)-diHETE, and LXs biosynthesized from endogenous arachidonic acid were highly correlated. Two different LX patterns were observed involving two possible metabolic pathways: (a) via the intermediate 5,6-epoxytetraene alone for LXs generation from exogenous 15(S)-HETE; and (b) via 5,6- and/or 14,15-epoxytetraenes leading to the formation of an enzyme-bound delocalized carbocation for LXs generation from endogenous arachidonate, respectively. The enhanced 5-lipoxygenase activation of blood PMN from asthmatics and the metabolism of exogenous 15(S)-HETE may reflect a priming induced by various mediators released from environmental cells, and could be considered as a model of transcellular signalization between PMN and endothelial cells.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acids; Asthma; Calcimycin; Chromatography, High Pressure Liquid; Female; Humans; Hydroxyeicosatetraenoic Acids; Male; Models, Biological; Neutrophil Activation; Substrate Specificity

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

The effects of tumor necrosis factor alpha (TNF-alpha) on arachidonic acid (AA) metabolism were investigated by prelabeling the human osteoblastic osteosarcoma cell line, G292, with [3H]AA. TNF-alpha differentially stimulates cyclooxygenase and lipoxygenase pathways of AA metabolism in a dose response manner in the cells. The highest concentration of TNF-alpha (10(-8)M) significantly increased the cyclooxygenase pathway, with prostaglandin E2 (PGE2) being a major product. However, at the lowest concentration (10(-10)M) of TNF-alpha, 15-hydroxyeicosatetraenoic acid (HETE) production was significantly increased, with no significant effects on the other identifiable products. When the concentration of TNF-alpha was increased to 10(-9) M leukotriene B4 (LTB4), 15-, 12-, and 5-HETE were significantly increased. The calcium ionophore A23187 (10(-6) M) significantly increased 15-HETE production, without significantly affecting cyclooxygenase metabolites. However, a combination of TNF-alpha (10(-8)M) and A23187 (10(-6)M) caused an inhibitory effect on each agent-induced PGE2 or 15-HETE production.

Topics: Arachidonic Acid; Calcimycin; Dinoprostone; Dose-Response Relationship, Drug; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Leukotriene B4; Lipoxygenase; Osteoblasts; Osteosarcoma; Prostaglandin-Endoperoxide Synthases; Tritium; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Topics: Animals; Arachidonic Acid; Aspirin; Calcimycin; Carbon Radioisotopes; Cell Line; Chromatography, High Pressure Liquid; Dinoprostone; Fibrosarcoma; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Interleukin-1; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Studies were made on the effects of stilbene derivatives isolated from medicinal plants on arachidonate metabolism and degranulation in human polymorphonuclear leukocytes (PMN-L). Resveratrol (3,4',5-trihydroxystilbene) isolated from the roots of Reynoutria japonica was found to inhibit the 5-lipoxygenase products 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid (5,12-diHETE) and leukotriene C4(LTC4); its concentrations for 50% inhibition (IC50) were 8.90 x 10(-6) M, 6.70 x 10(-6) M and 1.37 x 10(-6) M, respectively. The IC50 of 5-HETE, 5,12-diHETE and LTC4 formations of synthetic 3,3',4-trihydroxystilbene were 5.90 x 10(-6) M, 6.30 x 10(-7) M and 8.80 x 10(-7) M, respectively. Moreover, they inhibited the release of lysosomal enzyme such as lysozyme and beta-glucuronidase induced by calcium ionophore A 23187 from human PMN-L at 10(-3)-10(-4) M.

Topics: Arachidonic Acid; Autoradiography; Calcimycin; Cell Degranulation; Cyclic AMP; Fatty Acids, Unsaturated; Glucuronidase; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Leukotriene C4; Lysosomes; Muramidase; Neutrophils; Plant Roots; Plants, Medicinal; Resveratrol; Stilbenes; Structure-Activity Relationship

Products of arachidonic acid (AA) metabolism via the lipoxygenase pathways may have key roles in the maintenance of pregnancy and the onset of labor. We have determined whether calcium ionophores can modulate the rate of biosynthesis within the uterus of five important arachidonate lipoxygenase metabolites, i.e. leukotriene B4 (LTB4), LTC4, 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE. Amnion, chorion, and decidual cells were isolated, grown to confluence and incubated with ionomycin. The production of LTB4, LTC4, 5-HETE, 12-HETE, and 15-HETE was determined using specific radioimmunoassays. Cell-specific, concentration-related stimulatory actions of ionomycin on 5-HETE, 12-HETE, 15-HETE, and LTC4 but not LTB4 production were found. A23187 had effects similar to ionomycin. Hence elevation of intracellular calcium levels can result in enhanced intrauterine production of arachidonate lipoxygenase metabolites that may affect pregnancy outcome.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Amnion; Arachidonic Acid; Calcimycin; Chorion; Decidua; Female; Humans; Hydroxyeicosatetraenoic Acids; Ionomycin; Leukotriene B4; Leukotriene C4; Lipoxygenase; Pregnancy; Uterus

15-Hydroxyeicosatetraenoic acid [15-(S)-HETE], a major arachidonic acid metabolite produced from the 15-lipoxygenase pathway, has been characterized as an antiinflammatory cellular mediator since it can inhibit the in vivo and in vitro formation of the proinflammatory leukotrienes via the 5-lipoxygenase pathway in various cells. 15-HETE has been confirmed to inhibit the 5-lipoxygenase in rat basophilic leukemia cell (RBL-1) homogenates with an I50 = 7.7 microM. The I50 of the 12-HETE isomer was 6 microM whereas prostaglandin F2 alpha was ineffective. In order to examine the mechanistic basis underlying the inhibitory action of 15-HETE, association assays of [3H]-15-HETE with RBL-1 subcellular fractions were carried out. The presence of the zwitterionic detergent CHAPS enhanced specific [3H]-15-HETE binding in the membrane fractions three-fold and specific 15-HETE binding was distributed among the nuclear (32%)-, granule (19%)-, plasma membrane (35%)-, and cytosol (14%)-enriched fractions. Studies using combined granule and plasma membrane enriched-, CHAPS treated-fractions showed that [3H]-15-HETE binding was time-dependent, specific and reversible, sensitive to pertussis toxin treatment, and indicated a single class of binding sites with a Kd = 460 +/- 160 nM and Bmax = 5.0 +/- 1.1 nM. Competition experiments showed that the order of 15-HETE or analogs in inhibiting the binding of [3H]-15-HETE was: 15(S)-HETE > or = 12-(S)-HETE = 5-(S)-HETE > 15-(R)-HETE > arachidonic acid. Prostaglandin F2 alpha and lipoxin B4 were ineffective as competitors. The similar profiles of the binding assays and inhibition of the 5-lipoxygenase suggest that 15-HETE binding sites may mediate this inhibitory action of 15-HETE.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Binding Sites; Binding, Competitive; Calcimycin; Cell Line; Cholic Acids; Dinoprost; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Lipoxygenase Inhibitors; Rats; Subcellular Fractions; Tumor Cells, Cultured

5-Lipoxygenase products, such as leukotrienes, are important stimuli for leukocyte-mediated tissue injury in acute inflammation. 15-Hydroxyeicosatetraenoic acid (15-HETE) is an eicosanoid generated by a variety of cell types via the actions of 15-lipoxygenases and, in addition, cyclooxygenases and epoxygenases. 15-HETE levels are frequently elevated at sites of inflammation, and extracellular 15(S)-HETE is esterified rapidly into neutrophil (PMN) phospholipids in vitro to levels that are comparable with arachidonic acid. We present evidence that remodeling of PMN phospholipids with 15(S)-HETE stereoselectively inhibits PMN migration across endothelium in response to leukotriene B4 (LTB4) and other chemoattractants. Esterified 15(S)-HETE causes a striking reduction in the affinity of LTB4 cell-surface receptors for their ligand and inhibition of LTB4-triggered stimulus-response coupling. As a result of these actions, esterified 15(S)-HETE attenuates the cytoskeletal rearrangements and CD11/CD18-mediated adhesive events that subserve directed locomotion of PMN across endothelium. These observations indicate that products of the 5-lipoxygenase and 15-lipoxygenase pathways can exert counterbalancing influences on PMN trafficking across endothelium. They suggest that 15(S)-HETE may be a potent endogenous inhibitor of PMN-endothelial interactions in vivo and serve to limit or reverse acute inflammation.

Topics: Antigens, CD; Calcimycin; CD11 Antigens; CD18 Antigens; Cells, Cultured; Chemotaxis, Leukocyte; Dose-Response Relationship, Drug; Endothelium, Vascular; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phospholipids; Receptors, Leukocyte-Adhesion; Tetradecanoylphorbol Acetate; Umbilical Veins

Both IFN-alpha/beta and IFN-gamma have recently been demonstrated to induce a rapid but transient activation of phospholipase A2 (PLA2) in BALB/c 3T3 fibroblasts and a human neuroblastoma cell line. We report that IFN-gamma induces the synthesis and prolonged activation of cytosolic phospholipase A2 (cPLA2) in a human bronchial epithelial cell line (BEAS 2B). Treatment of the cells with IFN-gamma (300 U/ml) increased the release of [3H]arachidonic acid (AA) from prelabeled cells with a maximal effect at 12 h after stimulation. The increased [3H]AA release was inhibited by the PLA2 inhibitor p-bromophenacyl bromide (10(-5) M). Calcium ionophore A23187 (10(-5) M) further increased the [3H]AA release from the IFN-gamma-treated cells. Subcellular enzyme activity assay revealed that IFN-gamma increased PLA2 activity in both the cytosol and membrane fractions with a translocation of the cPLA2 to cell membranes in a Ca(2+)-free cell lysing buffer. Treatment with IFN-gamma also induced the release of 15-HETE, an arachidonic acid metabolite. Immunoblot showed that IFN-gamma induced the synthesis of cPLA2 protein. Nuclear run-on assay demonstrated that IFN-gamma initiated cPLA2 gene transcription within 15 min, and this effect was sustained at 4 h and returned to near control level at 12 h. The cPLA2 mRNA level was assayed by reverse transcription and PCR. IFN-gamma was found to increase the cPLA2 mRNA after 2-24 h treatment. Furthermore, the IFN-gamma induced cPLA2 mRNA increase was blocked by inhibitors of protein kinase C and calcium/calmodulin-dependent protein kinases, suggesting the involvement of these protein kinases in IFN-gamma-induced gene expression of cPLA2. This study shows that IFN-gamma induces the synthesis and prolonged activation of cPLA2.

Topics: Acetophenones; Arachidonic Acid; Base Sequence; Bronchi; Calcimycin; Cell Line; Cell Membrane; Cell Nucleus; Cytosol; DNA Primers; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Epithelium; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Interferon-gamma; Kinetics; Molecular Sequence Data; Phospholipases A; Phospholipases A2; Polymerase Chain Reaction; Protein Processing, Post-Translational; RNA, Messenger; Transcription, Genetic

Bronchial epithelial cells may be involved in the pathogenesis of asthma releasing several inflammatory mediators such as interleukins and lipoxygenase products. In this study we evaluated the spontaneous and A23128-induced release of 15-HETE, PGE2 and fibronectin as well as the spontaneous expression of HLA-DR and ICAM-1 molecules by bronchial epithelial cells obtained by bronchial brushing from 35 asthmatic and 27 normal subjects. The release of fibronectin and 15-HETE was studied using the EIA and RIA techniques. The expression of HLA-DR and ICAM-1 molecules was studied using the APAAP and the immunofluorescence methods. Bronchial epithelial cells from asthmatics released higher amounts of 15-HETE and fibronectin both spontaneously (p < 0.002, p < 0.05, respectively) or after stimulation with calcium ionophore compared with normals. On the other hand, PGE2 release was significantly higher only after stimulation with calcium ionophore (p < 0.002). The spontaneous expression of HLA-DR and ICAM-1 (p < 0.001) was significantly higher on epithelial cells from asthmatics than in normal subjects. Finally, the severity of asthma significantly correlated with the release of 15-HETE (p < 0.02) and the expression of HLA-DR and ICAM-1 respectively (p < 0.001 and p < 0.002, respectively). This study indicates that bronchial epithelial cells are in an activated state in asthma and that the degree of activation is correlated to the severity of the disease.

Topics: Adult; Alkaline Phosphatase; Asthma; Bronchi; Calcimycin; Cell Adhesion Molecules; Dinoprostone; Epithelium; Fibronectins; Fluorescent Antibody Technique; HLA-DR Antigens; Humans; Hydroxyeicosatetraenoic Acids; Immunoenzyme Techniques; Intercellular Adhesion Molecule-1

The purpose of the present study was to develop an ex vivo skin model to determine the capacity of lesional skin of psoriasis to form leukotriene B4 and other eicosanoids. Keratomed skin samples were incubated in the presence of the calcium ionophore A23187 and arachidonic acid for 45 min at 37 degrees C. After extraction of lipids, eicosanoids were determined by quantitative reversed-phase high-performance liquid chromatography in combination with specific radioimmunoassays. We found that stimulation of skin samples with A23187 and arachidonic acid increased the amount of leukotriene B4 4.0-fold. The 12-lipoxygenase product, 12-hydroxy-eicosatetraenoic acid, and the 15-lipoxygenase product, 15-hydroxy-eicosatetraenoic acid, were both increased 2.7-fold. The cyclooxygenase product, prostaglandin E2, was increased 8.0-fold. Similar incubations using psoriatic scales did not result in formation of eicosanoids. Incubations with the 5-lipoxygenase inhibitor RS43179 inhibited the formation of leukotriene B4 and prostaglandin E2 without significantly affecting the formation of 12-hydroxy-eicosatetraenoic acid and 15-hydroxy-eicosatetraenoic acid. These results reveal that lesional psoriatic skin ex vivo has the enzymatic capacity to increase the levels of eicosanoids. This provides an ex vivo skin model to determine whether putative lipoxygenase inhibitors are able to modulate the formation of eicosanoids in psoriatic skin.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Calcimycin; Chromatography, High Pressure Liquid; Dinoprostone; Eicosanoids; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Lipoxygenase Inhibitors; Naphthalenes; Psoriasis; Radioimmunoassay; Skin

Production of platelet-activating factor (PAF) and eicosanoids by human umbilical vein endothelial cells (HUVEC) after stimulation with different agonists has been studied. Significant amounts of PAF were measured in the cellular fraction after treatment with thrombin (2 NIHu/ml), calcium ionophore A23187 (2 microM) and histamine (100 microM) (110.3 +/- 14.3, 80.7 +/- 19.2 and 119.2 +/- 22.4 pg/10(5) cells, respectively). Only thrombin caused a partial release of PAF into the supernatant. IL-1 alpha (0.1 nM), TNF (1 nM), arachidonic acid (10 microM) and endothelin (0.1 microM) were not able to induce any PAF synthesis. High levels of 6-keto-PGF1 alpha were found after stimulation with thrombin and calcium ionophore A23187 (8641 +/- 2575 and 6715 +/- 3340 pg/10(5) cells, respectively). Cytokines IL-1 alpha and TNF were also able to stimulate PGI2 synthesis, although to a lesser extent. PGE2 production increased after treatment with thrombin and calcium ionophore A23187 three- and two-fold, respectively. Our results confirm that stimulated HUVEC are able to synthesize PAF and eicosanoids simultaneously, the relative amounts depending upon the agonist used. None of the agonists studied showed any significant effect on 15-HETE production.

Topics: 6-Ketoprostaglandin F1 alpha; Arachidonic Acid; Blood Platelets; Calcimycin; Cells, Cultured; Dinoprostone; Endothelins; Endothelium, Vascular; Epoprostenol; Histamine; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-1; Platelet Activating Factor; Platelet Aggregation; Prostaglandins; Serotonin; Thrombin; Tumor Necrosis Factor-alpha; Umbilical Veins

The present studies were conducted to understand better the regulation of phospholipase A2 (PLA2)-dependent mobilization of lipid mediators by arachidonic acid (C20:4). After stimulation of human neutrophils, g.l.c./m.s. analysis of non-esterified fatty acids indicated that the quantity of C20:4 increased as a function of time after stimulation, from undetectable quantities to > 800 pmol/10(7) cells. In contrast with C20:4, the quantities of other free fatty acids such as oleic and linoleic were high in resting cells and did not change after stimulation. Some 15% of the C20:4 released from cellular lipids remained cell-associated. To examine the effect of C20:4 on its own release, neutrophils were exposed to [2H8]C20:4, to differentiate it by g.l.c./m.s. from naturally occurring C20:4. In A23187-stimulated neutrophils, low concentrations (5-10 microM) of [2H8]C20:4 added just before A23187 increased the quantity of C20:4 produced by the cell, whereas higher concentrations (30-50 microM) decreased the quantity of C20:4 released from phospholipids. As other measures of PLA2 activity, the effects of C20:4 on production of platelet-activity factor (PAF) and leukotriene B4 (LTB4) were assessed. C20:4 treatment just before stimulation of neutrophils blocked PAF and LTB4 production in a concentration-dependent manner (IC50 10-20 microM). The effect of C20:4 was not blocked by the cyclo-oxygenase inhibitor naproxine (10 microM), nor could it be mimicked by 1 microM LTB4, 5-hydroxyeicosa-6,8,11,14-tetraenoic acid (5HETE), 5-hydroperoxyeicosa-6,8,11,14-tetraenoic acid (5HPETE) or 15-hydroxyeicosa-5,8,11,13-tetraenoic acid (15HETE). The 5-lipoxygenase (5LO) inhibitor zileuton induced a concentration-dependent decrease in PAF, with a maximal effect of a 50% decrease at 10-50 microM. The decrease in PAF by the 5LO inhibitor could not be circumvented by addition of 1 microM 5HETE, 5HPETE and LTB4, and may be attributed to the capacity of zileuton to increase the quantity of C20:4 in A23187-treated neutrophils. The inhibitory effect of C20:4 (20-40 microM) on PAF production could be antagonized by the protein kinase C inhibitor staurosporine (30 nM), but not by inhibitors of protein kinase A, tyrosine kinase or calmodulin kinase II. Taken together, these data demonstrate that C20:4 is selectively released from membrane phospholipids of A23187-stimulated neutrophils, and this C20:4 may play an important role in regulating the mobilization of C20:4 by altering PLA2 activi

Topics: Arachidonic Acid; Calcimycin; Fatty Acids; Fatty Acids, Nonesterified; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Leukotrienes; Neutrophils; Phospholipases A; Phospholipases A2; Platelet Activating Factor

Airways epithelial cells may be involved in the pathogenesis of asthma, but their role remains to be determined. Epithelial cells can release large amounts of 15-hydroxy-eicosatetranoic acid (15-HETE) and smaller amounts of prostaglandin E2 (PGE2) as well as fibronectin, a mediator involved in epithelial repair after injury. Epithelial cells obtained after bronchial brushing of 16 asthmatic (age 38 +/- 5 yr) and 11 normal subjects (age 36 +/- 5 yr) were studied. The percentage of epithelial cells was assessed by immunocytochemistry using an anti-cytokeratin antibody. The viability of the cells was assessed by trypan blue exclusion. The release of 15-HETE PGE2 and fibronectin was studied in resting cells and after A23187 calcium ionophore stimulation. Epithelial cells always comprised more than 86% of cells recovered, and the viability of epithelial cells was significantly (p < 0.001, Mann-Whitney U test) greater in normal subjects (54 +/- 5%) compared with asthmatic subjects (13 +/- 1%). The release of 15-HETE and fibronectin by resting epithelial cells was significantly greater in asthmatics (p < 0.05, Mann-Whitney U test) than in normal subjects. A23187 significantly (p < 0.05, Wilcoxon W test) increased the release of 15-HETE and fibronectin. There was no significant difference in the release of PGE2 by resting cells from either asthmatics or normal subjects, but challenge with A23187 induced a significant (p < 0.03, Wilcoxon W test) increase in PGE2 from cells of asthmatics but not from cells of normal subjects. This study shows that epithelial cells are activated and less viable in asthma and suggests a role for these cells in asthma.

Topics: Adult; Albuterol; Asthma; Bronchi; Bronchoalveolar Lavage Fluid; Bronchoscopy; Calcimycin; Cell Survival; Dinoprostone; Epithelial Cells; Epithelium; Fiber Optic Technology; Fibronectins; Humans; Hydroxyeicosatetraenoic Acids

Cells were incubated in the presence of the Ca2+ ionophore A23187 (10 microM) and arachidonic acid (AA, 80 microM). The release of eicosanoids from subcultivated cardiac endothelial and fibroblast-like cells amounted to 23.3 +/- 4.5 and 2.0 +/- 0.4 nmol/mg cellular protein per 30 min, respectively. The release from isolated cardiomyocytes remained below the detection limit of the high-performance liquid chromatography assay (< 0.00015 nmol/assay). When a very sensitive radioimmunoassay was applied, cardiomyocytes released 0.002 +/- 0.0001 nmol prostacyclin per milligram cellular protein per 30 min. Prostaglandin (PG) E2 and PGF2 alpha, 12-hydroxyheptadecatrienoic acid, 11- and 15-hydroxyeicosatetraenoic acid, and thromboxane B2 were the main eicosanoids released by endothelial cells. The stable product of prostacyclin, 6-keto-PGF1 alpha, contributed relatively little to the total amount of eicosanoids formed by endothelial cells. Fibroblast-like cells released predominantly PGE2 and 6-keto-PGF1 alpha and, to a lesser extent, 12-hydroxyheptadecatrienoic and 15-hydroxyeicosatetraenoic acids. Neither endothelial cells nor fibroblast-like cells released leukotrienes. A23187 stimulated eicosanoid release from endothelial cells when exogenous AA was below 40 microM. Addition of albumin reduced the amount of eicosanoids produced. Histamine and bradykinin did not influence 6-keto-PGF1 alpha and PGE2 production in cardiomyocytes. Histamine only gave rise to a slight but significantly higher release of 6-keto-PGF1 alpha in endothelial cells.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Arachidonic Acid; Calcimycin; Cells, Cultured; Dinoprost; Dinoprostone; Endothelium; Fatty Acids, Unsaturated; Fibroblasts; Heart; Hydroxyeicosatetraenoic Acids; Indomethacin; Male; Microscopy, Electron; Myocardium; Rats; Rats, Inbred Lew; Rats, Wistar

Human alveolar macrophages (AM) from bronchoalveolar lavage of asthmatic patients (AP) and healthy volunteers (HS) were compared for their respective capacities to produce lipoxins and leukotrienes when stimulated by calcium ionophore A23187 with or without 15(S)-HETE. The metabolites were analyzed using an isocratic RP-HPLC system and their formation profiles evaluated on the basis of chromatographic behaviour, UV spectral characteristics and co-elution with synthetic standards. Without 15-HETE, AM from AP produced more LTB4 and 5-HETE than those from HS. In the presence of 15-HETE, human AM were able to produce 5,15-diHETE and lipoxins. Moreover, the total amount of lipoxins synthesized by AM from AP was 2 fold higher than that synthesized by AM from HS, thus showing an enhanced cell activation via the 5-lipoxygenase (5-LO) pathway. These results presented AM as in vitro 15-HETE metabolizing cells and suggested some hypothesis about human AM 5-LO regulation mechanism. The enhanced 5-LO activity in AM from AP suggested that in vivo they could participate in cell to cell interaction mechanisms involved in inflammatory lung diseases and might also take up and transform 15-HETE predominantly released by airway epithelial cells.

Topics: Adult; Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Cells, Cultured; Chromatography, High Pressure Liquid; Epithelial Cells; Epithelium; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxins; Lung; Macrophage Activation; Macrophages, Alveolar; Middle Aged

Eicosanoids were measured in colonic biopsies from eleven patients with active ulcerative colitis and from thirteen controls. Eicosanoid formation was measured after the addition of arachidonic acid and stimulation with calcium ionophore A23187. The 15-lipoxygenase derivative 15-hydroxy-eicosatetraenoic acid (15-HETE) was the predominant product formed in all biopsies. The amount of 15-HETE formed was dependent on the site of biopsy and decreased in the controls in biopsies taken in an aboral direction in the colon. The formation of 15-HETE and that of prostaglandin E2 (PGE2) and PGF2 alpha was proportional to the histologically obtained inflammation score. The role of 15-H(P)ETE as a mediator in ulcerative colitis should, therefore, be considered in addition to the effects of known modulators such as leukotriene B4 (LTB4) and PGE2.

Topics: Adult; Arachidonic Acid; Biopsy; Calcimycin; Colitis, Ulcerative; Colon; Eicosanoids; Female; Humans; Hydroxyeicosatetraenoic Acids; Intestinal Mucosa; Male; Middle Aged

The synthesis of 14C labelled arachidonic acid metabolites was measured in colonic tissues obtained from mice, rats, guinea pigs, rabbits, piglets and in colonic biopsies from humans during colonoscopy. The main eicosanoids formed after stimulation with calcium ionophore A23187 were: in humans, 15-hydroxy-eicosatetraenoic acid (15-HETE); in mice, 12-HETE; in rats, 12-HETE, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 6-keto-prostaglandin F1 alpha (6kPGF1 alpha); in guinea pigs, PGD2; in rabbits, 6kPGF1 alpha, PGE2 and 15-HETE; and in pigs PGE2 and 12-HETE. In inflamed 15-HETE production was increased in man, HHT and 12-HETE production in rats and overall eicosanoid production in mice.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 6-Ketoprostaglandin F1 alpha; Animals; Calcimycin; Colitis, Ulcerative; Colon; Dinoprostone; Eicosanoids; Fatty Acids, Unsaturated; Guinea Pigs; Humans; Hydroxyeicosatetraenoic Acids; Intestinal Mucosa; Mice; Mice, Inbred BALB C; Prostaglandin D2; Rabbits; Rats; Rats, Wistar; Species Specificity

Heaves is a respiratory disorder of horses and ponies characterized by bouts of acute airway obstruction and airway hyperresponsiveness. We measured prostaglandin E2 (PGE2) and 15-hydroxyeicosatetraenoic acid (15-HETE) production in vitro in tracheal epithelium obtained from six affected horses at the time of acute airway obstruction as compared with six matched control horses. Strips of epithelium and subepithelial tissue were prepared and stimulated with A23187, histamine, and bradykinin. The PGE2 and 15-HETE in media from strips was quantitated by radioimmunoassay. 15-HETE above the limits of accurate detection was found in epithelial strips of only two principal animals and in none of the control horses, and the amount of 15-HETE was not increased when strips were stimulated. Epithelial strips from affected horses tended to produce less PGE2 than did strips from control horses, and there was a significant correlation between epithelial PGE2 production and the time taken for affected animals to develop airway obstruction. Subepithelial tissue strips from control horses produced significantly more PGE2 in response to A23187 and bradykinin than did strips from affected horses. We conclude that equine tracheal epithelium is not a significant source of 15-HETE. Airway mucosal PGE2 production is reduced in horses with heaves, which suggests that a relative decrease in this bronchorelaxant substance may be a factor in the pathogenesis of this model of asthma.

Topics: Airway Obstruction; Animals; Bradykinin; Calcimycin; Dinoprostone; Epithelium; Histamine; Horse Diseases; Horses; Hydroxyeicosatetraenoic Acids; Mucous Membrane; Radioimmunoassay; Respiratory Function Tests; Trachea

Topics: Asthma; Bronchi; Calcimycin; Cell Survival; Dinoprostone; Epithelium; Fibronectins; HLA-DR Antigens; Humans; Hydroxyeicosatetraenoic Acids; Middle Aged

Topics: Acylation; Calcimycin; Cell Aggregation; Esterification; Humans; Hydroxyeicosatetraenoic Acids; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phosphatidylinositols; Tetradecanoylphorbol Acetate

Topics: Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Asthma; Bronchi; Calcimycin; Epithelium; Humans; Hydroxyeicosatetraenoic Acids; Lung

The acrosome reaction (AR) in bull spermatozoa was induced by the Ca2(+)-ionophore A23187, by dilauroylphosphatidylcholine or by arachidonic acid in the presence of Ca2+ in the incubation medium. The occurrence of AR was determined by following the release of acrosin from the cells. Nordihydroguaiaretic acid (NDGA), an inhibitor of both lipoxygenase and prostaglandin-synthetase, caused 35%, 43% and 69% inhibition of AR at concentrations of 1, 10 or 100 microM, respectively. Eicosatetraynoic acid (ETYA), an analogue of arachidonic acid, caused 17%, 61% and 77% inhibition of AR at concentrations of 20, 40 or 80 micrograms/ml, respectively. When AR was induced by arachidonic acid, ETYA, causes 36% and 58% inhibition at concentrations of 2 or 20 micrograms/ml, respectively. Under identical conditions, 100 microM indomethacin, a specific inhibitor of prostaglandin-synthetase, showed no inhibition but rather 35% stimulation at acrosin release rate. The fact that AR is inhibited by NDGA and not by indomethacin indicates that the lipoxygenase, rather than prostaglandin-synthetase, is involved in the mechanism of AR. Since the inhibition by NDGA is seen in the presence of the Ca-ionophore, we suggest that lipoxygenase activity is not involved in enhancing calcium transport into the cell, but rather at other steps in AR mechanism. A thin-layer chromatography revealed the presence of 15-HETE, the classical product of 15-lipoxygenase activity, which was identified by HPLC. Under AR conditions, there is an elevation of lipoxygenase products and the addition of NDGA caused a reduction in their levels. The inhibition of acrosin release by NDGA can be eliminated by adding 15-HETE or 15-HPETE to the incubation medium. In conclusion, we suggest here for the first time, a physiological role for 15-lipoxygenase in the mechanism of AR in mammalian spermatozoa.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Acrosome; Animals; Arachidonate 15-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Cattle; Cyclooxygenase Inhibitors; Hydroxyeicosatetraenoic Acids; Indomethacin; Lipoxygenase Inhibitors; Male; Masoprocol; Phosphatidylcholines; Spermatozoa

Alveolar macrophages (AM) obtained both from specific viral antibody-positive (VA+) rats and from specific viral antibody-negative (VA-) rats were compared for their respective capacity to produce lipoxins (LX) in a given experimental condition. Both cell preparations were incubated with (15S), 15-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HETE) in the presence of calcium ionophore A23187. The reaction mixtures were extracted and chromatographed on reverse-phase high performance liquid chromatography (RP-HPLC). Profiles of LX formation in each group were evaluated on the basis of chromatographic behavior, UV spectral characteristics (lambda max at 301 nm with shoulders at 289 nm and 317 nm), and co-elution with synthetic lipoxin A4 (LXA4) and lipoxin B4 (LXB4) standards as well as with authentic materials. The AM harvested from VA+ rats (AM-VA+) produced consistently detectable levels of LX (i.e., LXA4 and LXB4), whereas the AM from VA- rats (AM-VA-) produced no detectable LX under the same experimental condition. These results offer a possibility that elevated capacity of the AM-VA+ to produce LX may be associated with previous exposures to selected specific viral pathogens and/or with viral infections. Considering the distinct biologic properties of LX, the apparent production of the enhanced levels of LX as shown in this model system may play an important role in both pathophysiology and immunoregulatory network in virus-induced pulmonary disorder.

Topics: Animals; Antibodies, Viral; Calcimycin; Chromatography, High Pressure Liquid; Hydroxyeicosatetraenoic Acids; Lipoxins; Macrophages; Male; Pulmonary Alveoli; Rats; Rats, Inbred Strains; Spectrophotometry, Ultraviolet

The lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA) and 15S-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-HETE) have been found to suppress the rise in free cytoplasmic Ca2+ concentration [( Ca2+]i) induced by the Ca2+ ionophores ionomycin and A23187 in rat thymocytes. Bromophenacyl bromide (BPB), a phospholipase A2 (PLA2) inhibitor, produced a much weaker inhibitory effect, and indomethacin, a cyclo-oxygenase inhibitor, practically did not influence the [Ca2+]i response to ionomycin. These findings implicate the involvement of LO product(s) in the [Ca2+]i rise triggered by the Ca2+ ionophores. The contribution of the NDGA-sensitive component to the ionomycin-induced [Ca2+]i rise was significant in the ionomycin concentration range of 0.1 nM to 0.1 microM whereas at higher doses of the ionophore it gradually diminished. By contrast, the [Ca2+]i rise induced by exogenous arachidonic acid (AA) or melittin, a PLA2 activator, was not suppressed but potentiated by NDGA. Ionomycin and exogenous AA also elicited opposite changes in thymocyte cytoplasmic pH (pHi): the former elevated the pHi while the latter induced a pronounced acidification of the cytoplasm. This difference in the pHi responses may account for the different sensitivity of ionomycin- and AA-elicited [Ca2+]i signal to LO inhibitors.

Topics: Acetophenones; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Cyclooxygenase Inhibitors; Hydroxyeicosatetraenoic Acids; Indomethacin; Ion Channel Gating; Ionomycin; Masoprocol; Melitten; Phospholipases A; Phospholipases A2; Rats; Rats, Inbred Strains; T-Lymphocytes

In the presence of high concentrations of exogenous arachidonic acid (greater than or equal to 10 microM), eosinophils produced 15-hydroxyeicosatetraenoic acid (15-HETE) in the absence of stimuli. The calcium ionophore A23187, as well as the chemotaxins used in this study--complement split product C5a, platelet-activating factor (PAF), and N-formyl-methionyl-leucyl-phenylalanine (FMLP)--failed to increase 15-HETE production, indicating that eosinophil 15-lipoxygenase is already active. Production of 15-HETE from eosinophils increased with increasing concentrations of arachidonic acid, exogenously added. Maximal 15-HETE production was observed to be 1111 +/- 380 ng per 10(6) eosinophils at the concentration of 100 microM of arachidonic acid. With low concentrations of exogenous arachidonic acid (below 2 microM), eosinophils were considered to incorporate exogenous arachidonic acid into their cell membrane, and did not produce 15-HETE. In contrast, 15-HETE formation in highly purified neutrophils (eosinophils less than 1%) was negligible compared with that in eosinophils (300-fold less), suggesting that 15-HETE-forming activity in granulocytes is derived from the eosinophil 15-lipoxygenase pathway and that neutrophils may lack 15-lipoxygenase activity.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Cell Separation; Complement C5a; Eosinophils; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Platelet Activating Factor

Chopped human nasal polyps and bronchial tissue produced lipoxin A4 and isomers of lipoxins A4 and B4, but not lipoxin B4, after incubation with exogenous leukotriene A4. In addition, these tissues transformed arachidonic acid to 15-hydroxyeicosatetraenoic acid. The capacity per gram of tissue to produce lipoxins and 15-hydroxyeicosatetraenoic acid was 3-5-times higher in the nasal polyps. Neither tissue produced detectable levels of lipoxins or leukotrienes after incubation with ionophore A23187 and arachidonic acid. Co-incubation of nasal polyps and polymorphonuclear granulocytes with ionophore A23187 led to the formation of lipoxins, including lipoxins A4 and B4. The results indicate the involvement of an epithelial 15-lipoxygenase in lipoxin formation in human airways.

Topics: Arachidonate 15-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Bronchi; Calcimycin; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotrienes; Lipoxins; Nasal Polyps; Neutrophils

Hydroxyeicosatetraenoic acids (HETEs) have previously been reported to stimulate the relatively inactive 15-lipoxygenase in A23187-activated human neutrophils to metabolize exogenously added arachidonic acid to 15-HETE. Several aspects of this HETE-induced activation process were examined. Pretreatment of intact PMNs with 3-20 microM 15-HETE, A23187 and the 5-lipoxygenase inhibitor NDGA (or the dual cyclooxygenase/lipoxygenase inhibitor BW755C) followed by [14C]arachidonic acid addition resulted in an unexpected synergistic activation of the cryptic 15-lipoxygenase activity. The ability of several HETE derivatives or analogues to stimulate the inactive 15-lipoxygenase was also investigated. The presence of the hydroxyl group but not its position was essential since 5-, 12- and 15-HETE were approximately equipotent and about ten times more effective than arachidonic acid. 5-HETE was more potent than 5-HETE methyl ester which suggested that a free carboxyl group was important. Both 5-HPETE and 5.15-diHETE were found to be less potent than 5-HETE. The C18 hydroxy fatty acid analogues 9- and 13-HODE were the least effective activators of the PMN 15-lipoxygenase activity that were tested.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Calcimycin; Humans; Hydroxyeicosatetraenoic Acids; Neutrophils

Mammalian tissues contain 5-, 12- and 15-lipoxygenases. Only the 15-lipoxygenase can act on linoleic acid, the predominant essential fatty acid of tissues and plasma, producing 13-hydroxyoctadecadienoic acid (13-HODE). Intracellular production of 13-HODE renders endothelial cells resistant to platelet adhesion, while its hydroperoxy precursor, 13-HPODE, synergises with the platelet anti-aggregatory factor prostacyclin. We have found that a 15-lipoxygenase activity is induced in aortas of cholesterol-fed and Watanabe Heritable Hyperlipidemic (WHHL) rabbits. Aortic tissue from WHHL rabbits incubated with 3H-linoleic acid produced a major metabolite identified as 13-HODE, which was formed with an efficiency comparable to the synthesis 15-HETE from arachidonic acid. These findings indicate that the increased aortic 15-lipoxygenase in vascular tissue is capable of producing 13-HODE in vivo. Since platelet adhesion is increased in atherogenesis, and thrombogenesis is a major complication of advanced atherosclerosis, it is suggested that induction of this enzyme may be a protective response to hypercholesterolemia.

Topics: Animals; Antithrombins; Aorta; Arachidonate 15-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acids; Arteriosclerosis; Calcimycin; Chromatography, High Pressure Liquid; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Linoleic Acids; Rabbits

Granulocytes from human eosinophilic donors were incubated with arachidonic acid or 15-hydroxyeicosatetraenoic acid (15-HETE) and stimulated with the ionophore A23187. The eicosanoids were extracted with reversed-phase cartridges and subjected to RP-HPLC analysis. When extracts from eosinophil-enriched populations were analysed and compared with extracts from human neutrophils, three additional peaks were detected which coeluted with 15-hydroxy-delta 13-trans-15H derivatives of leukotriene C4, D4 and E4 in different HPLC systems. The recorded absorbance spectra of the eluted compounds and the standards were identical and showed a maximum at 307 nm which is characteristic for a conjugated tetraene system with a bathochromic shift by the sulfur moiety in alpha-position to the tetraene system. The compound which coeluted with the 15-hydroxy-LTC4 standard was treated with gamma-glutamyltransferase and converted to the corresponding leukotriene D4 derivative. The results indicate that interaction between the 5- and 15-lipoxygenase pathways leads to the formation of a new series of arachidonic acid metabolites in human eosinophils. Since the biosynthetic route is similar to that of lipoxin A4 and lipoxin B4, we suggest the trivial names lipoxin C4, D4 and E4.

Topics: Arachidonic Acid; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Eosinophils; Granulocytes; Humans; Hydroxyeicosatetraenoic Acids; Lipoxins; Spectrophotometry, Ultraviolet

The capacity of bovine tracheal epithelial cells to convert arachidonic acid to oxygenation products with potential biologic activity was studied in homogeneous preparations of isolated cells. Purified epithelial cell suspensions were incubated with radiolabeled arachidonic acid, and oxygenated metabolites were identified using high-pressure liquid chromatography and gas chromatography-mass spectrometry. The cells released predominantly two products during incubation with 0.3 to 150 microM arachidonic acid for 1 to 60 min at 37 degrees C: prostaglandin E2 (PGE2) and 12-hydroxyeicosatetraenoic acid (12-HETE). Concentration-response curves for the two products yielded half-maximal effects at 2 and 45 microM arachidonic acid, respectively. Stereochemical analysis by chiral-phase high-pressure liquid chromatography demonstrated that the epithelial 12-HETE consisted exclusively of the 12(S) isomer, providing supporting evidence that it was derived from an arachidonate 12-lipoxygenase. Epithelial cells prelabeled with arachidonic acid and incubated with 5 microM A23187 to stimulate endogenous arachidonic acid metabolism also released two predominant products with the chromatographic properties of PGE2 and 12-HETE. The findings demonstrate that bovine tracheal epithelial cells express both a cyclooxygenase:PGE isomerase and a 12-lipoxygenase pathway and therefore implicate this pathway as a new source of epithelial cell mediators.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cattle; Chromatography, High Pressure Liquid; Dinoprostone; Epithelium; Hydroxyeicosatetraenoic Acids; Mass Spectrometry; Prostaglandin-Endoperoxide Synthases; Trachea

Lipoxygenase (LO) products generated by human PMN were examined utilizing a gradient-HPLC and rapid spectral detector which permitted continuous UV-spectral monitoring of leukotrienes, lipoxins and related oxygenated products of arachidonic acid. When exposed to the ionophore A23187, PMN generated LTB4 and its omega-oxidation products as well as LXA4, LXB4, and 7-cis-11-trans-LXA4 from endogenous sources. Addition of 15-HETE changed the profile of products generated by activated PMN and led to a time- and dose-dependent increase in lipoxins and related compounds while the production of LTB4 and its omega-oxidation products was inhibited. Results of time-course and radiolabel studies revealed that 15-HETE is rapidly transformed within 15 s to 5,15-DHETE and conjugated tetraene-containing products, and that the inhibition of leukotriene formation followed a similar time-course. In contrast, PMN did not generate either lipoxins or related products from 5-[3H]HETE, nor did 5-HETE block leukotriene formation. Stimulated PMN generated 5,15-DHETE from exogenous 5-HETE, while in the absence of ionophore, 5-HETE was transformed to 5,20-HETE. These results indicate that PMN can generate lipoxins and related products from endogenous sources and that 15-HETE and 5-HETE are transformed by different routes.

Topics: Calcimycin; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Leukotrienes; Lipoxins; Neutrophils; Spectrophotometry, Ultraviolet

Auranofin (AF), a lipophilic chrysotherapeutic agent, was investigated for its effect on the formation of lipoxygenase products and the activity of protein kinase C in human neutrophils. We have previously shown that inhibition of LTB4 formation by 5-lipoxygenase (5-LO) inhibitors is intimately associated with a marked increased in 15-HETE in excess of arachidonic acid. The calcium- and phospholipid-dependent protein kinase, protein kinase C, is activated in FMLP- and A23187-stimulated neutrophils, is hypothesized to stimulate superoxide generation, and plays an essential role in eicosanoid production. AF dose-dependently inhibited the generation of leukotriene B4(LTB4) in FMLP-stimulated neutrophils, the ID50 was approximately 4.5 micrograms/ml. Unlike known 5-LO inhibitors, AF did not enhance the production of 15-HETE. In neutrophils stimulated with the calcium ionophore, A23187, AF did not inhibit the generation of LTB4 nor did AF change the 15-HETE levels. AF inhibited superoxide generation in FMLP-stimulated neutrophils dose-dependently, but did not change the activation of protein kinase C in the cells. We therefore conclude, that AF inhibition of LTB4 production in neutrophils is different from 5-lipoxygenase inhibitors and is elicited at a step distal to protein kinase C activation.

Topics: Auranofin; Calcimycin; Chromatography, High Pressure Liquid; Enzyme Activation; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Masoprocol; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Protein Kinase C; Radioimmunoassay

Human neutrophils can aggregate, degranulate, and release mediators of inflammation including oxygen radicals and lipoxygenase (LO)-derived products of arachidonic acid. The regulation of 5- and 15-lipoxygenases appears to be important since their products (e.g. leukotrienes and lipoxins) display unique spectra of bioactions. Addition of 15-HETE, a product of the 15-LO, to neutrophils in suspension dramatically shifted the LO products generated and led to a dose-dependent increase in lipoxins, while the production of leukotriene B4 and its omega-oxidation products (i.e. 20-COOH-LTB4 and 20-OH-LTB4) was inhibited. Exogenous 15-HETE also dose-dependently inhibited the generation of superoxide anions induced by either the chemotactic peptide f-met-leu-phe or the divalent cation ionophore A23187. Neither lipoxin A4 nor lipoxin B4 (10(-8)-10(-6) M) inhibited O2-. generation induced by either f-met-leu-phe or A23187. These results indicate that in addition to serving as a substrate for lipoxin generation, 15-HETE also inhibits superoxide anion generation by human neutrophils. Together they provide further evidence to suggest that products of the 15-lipoxygenase may serve a regulatory role at inflammatory loci.

Topics: Calcimycin; Free Radicals; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Lipoxins; Neutrophils; Superoxides

To determine identities of mediators and mechanisms for their release from pulmonary airway epithelial cells, we examined the capacities of epithelial cells from human, dog and sheep airways to incorporate, release and oxygenate arachidonic acid. Purified cell suspensions were incubated with radiolabeled arachidonic acid and/or ionophore A23187; fatty acid esterification and hydrolysis were traced chromatographically, and oxygenated metabolites were identified using high-pressure liquid chromatography and mass-spectrometry. In each species, cellular uptake of 10 nM arachidonic acid was concentrated in the phosphatidylcholine, phosphatidylinositol and phosphatidylethanolamine fractions, and subsequent incubation with 5 microM A23187 caused release of 10-12% of the radiolabeled pool selectively from phosphatidylcholine and phosphatidylinositol. By contrast, the products of arachidonic acid oxygenation were species-dependent and in the case of human cells were also novel: A23187-stimulated human epithelial cells converted arachidonic acid predominantly to 15-hydroxyeicosatetraenoic acid (15-HETE) and two distinct 8,15-diols in addition to prostaglandin (PG) E2 and PGF2 alpha. Cell incubation with exogenous arachidonic acid (2.0-300 microM) led to progressively larger amounts of 15-HETE and the dihydroxy, epoxyhydroxy and keto acids characteristic of arachidonate 15-lipoxygenase. Both dog and sheep cells converted exogenous or endogenous arachidonic acid to low levels of 5-lipoxygenase products, including leukotriene B4 without significant 15-lipoxygenase activity. In the cyclooxygenase series, sheep cells selectively released PGE2, while dog cells generated predominantly PGD2. The findings demonstrate that stereotyped esterification and phospholipase activities are expressed at uniform levels among airway epithelial cells from these species, but pathways for oxygenating arachidonic acid allow mediator diversity depending greatly on species and little on arachidonic acid presentation.

Topics: Animals; Arachidonate 15-Lipoxygenase; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Dogs; Epithelium; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Oxidation-Reduction; Prostaglandins; Sheep; Species Specificity; Trachea

Leukotrienes, products of the 5-lipoxygenase pathway of arachidonic acid metabolism, have been suggested to play a pathogenic role in psoriasis, because of their ability to induce skin inflammation and to stimulate epidermal proliferation. The 15-lipoxygenase product 15-hydroxy-eicosatetraenoic acid (15-HETE) has no proinflammatory capacity. In contrast, it can inhibit the activity of the 5-lipoxygenase. The purpose of the present study was to study the effect of 5-lipoxygenase inhibitors on the formation of 15-HETE by human neutrophils in vitro. Purified neutrophils were incubated with A 23187 (5 microM) and arachidonic acid (25 microM) with and without different inhibitors of 5-lipoxygenase activity (RS 43179, benoxaprofen, NDGA, and CP 66248). Methods for identifying eicosanoids included RP-HPLC and radioimmunoassay. Formation of leukotriene B4 was inhibited in a dose-dependent way, which was strongly correlated with a concomitant increase in the formation of 15-HETE (r = 0.97, p less than 0.01). The cyclooxygenase inhibitor indomethacin did not change 15-HETE formation. The stimulation of 15-HETE formation was not associated with cell damage as assessed by LDH release. Furthermore, identical incubations of T lymphocytes, characterized by a low 5-lipoxygenase activity, did not result in increased 15-HETE formation. These results show that inhibition of 5-lipoxygenase activity can lead to increased formation of 15-HETE. Because 15-HETE inhibits formation of 5-LO products, it may amplify the effect of 5-lipoxygenase inhibitors.

Topics: Arachidonate Lipoxygenases; Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Lipoxygenase Inhibitors; Naphthalenes; Neutrophils

Cultured porcine aortic endothelial cells were conditioned through two passages to mimic euglycemic and hyperglycemic conditions (5.2 mM, normal glucose; 15.6 mM, elevated glucose). After incubation with 1 microM [14C]arachidonic acid for 24 h, the cells were stimulated with 1 microM A23187 for times up to 30 min. Uptake of [14C]arachidonic acid and its distribution among cell lipids were unaffected by the increased glucose concentration. The release of eicosanoids from labeled cells and unlabeled cells was measured by reverse-phase HPLC and by RIA, respectively. Compared with cells stimulated in the presence of normal glucose concentrations, cells stimulated in the presence of elevated glucose released 62.6% less free [14C]arachidonic acid, but released 129% more 14C-labeled 15-hydroxyeicosatetraenoic acid (HETE). Increased release of 15-HETE in the presence of elevated glucose in response to A23187, bradykinin, and thrombin was confirmed by RIA. A similar increase in 5-HETE release was observed by RIA after A23187 treatment. The release of both radiolabeled and unlabeled prostanoids was equal at both glucose concentrations. The data indicate that glucose may play an important role in the regulation of release and metabolism of arachidonic acid after agonist stimulation. In the presence of elevated glucose concentrations, such as those associated with diabetes mellitus, the extent and pattern of eicosanoid release from endothelial cells is markedly altered.

Topics: 6-Ketoprostaglandin F1 alpha; Animals; Aorta; Arachidonic Acid; Arachidonic Acids; Bradykinin; Calcimycin; Eicosanoic Acids; Endothelium, Vascular; Glucose; Hydroxyeicosatetraenoic Acids; Swine; Thrombin

Confluent cultures of porcine aortic endothelial cells were prelabeled with 1 microM [14C]arachidonic acid complexed to 1 microM bovine serum albumin. After washing, the cells were stimulated with 1 microM A23187 for time intervals between 30 s and 30 min. Cellular lipids were extracted and separated into major lipid classes and phospholipid subclasses. The external medium was analyzed for released radioactive eicosanoids. The time-course of total release of 14C radioactivity demonstrated a biphasic nature of A23187-induced changes in endothelial cell lipids. Early, from 30 s to 5 min, substantial losses of [14C]arachidonic acid from diacylphosphatidylethanolamine and phosphatidylinositol, as well as an abrupt increase in diacylphosphatidylcholine-associated radioactivity were observed. These initial changes coincided with the release of 14C-labeled cyclooxygenase products. Later changes (5-30 min) included a sustained progressive loss of 14C radioactivity from alkenyl (alk-1-enyl) acylphosphatidylethanolamine and diacylphosphatidylcholine. These later changes coincided with the elaboration of 14C-labeled lipoxygenase products. Although unequivocal assignments cannot be made, the data suggest that specific pools of arachidonic acid provide precursors for individual classes of eicosanoids.

Topics: 6-Ketoprostaglandin F1 alpha; Acylation; Animals; Aorta, Thoracic; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cells, Cultured; Endothelium, Vascular; Ethers; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Kinetics; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositols; Phospholipids; Swine

Topics: Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Lipoxygenase; Neutrophils; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Time Factors

Human umbilical arteries converted arachidonic acid to three hydroxyeicosatetraenoic acids (HETEs) as well as prostaglandins. The mono-HETEs have been identified by reverse-phase high pressure liquid chromatography and gas chromatography-mass spectroscopy as 15-HETE and 11-HETE. 15-HETE in arterial segments appears to be derived mainly via the 15-lipoxygenase pathway, whereas 11-HETE, and the presumed di-HETE(s) were products of cyclooxygenase. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, stimulated prostanoid production with a concomitant inhibition of 15-HETE formation. These results suggested that 15-HETE may function as an endogenous regulator of prostacyclin. In human umbilical arterial microsomes, 15-HETE was found to inhibit 6-keto-prostaglandin F1 alpha and total prostanoid production in a concentration-dependent manner (median inhibition constant [IC50] of 52 +/- 3 and 63 +/- 4 microM respectively). The relative distribution of prostaglandins, however, remained unaffected, indicating that the site of action was cyclooxygenase. Kinetic analysis revealed that 15-HETE was a competitive inhibitor of the enzyme. Although no changes in maximum velocity occurred, the apparent Km was significantly different (9.3 +/- 6.9 microM [1 SD] for control vs. 37.6 +/- 17.7 microM for the 15-HETE-treated enzyme). Furthermore, the inhibitory effect of 15-HETE on prostacyclin production was confirmed using cultured bovine endothelial cells. In this cell system, not only did 15-HETE inhibit endogenous prostacyclin production, but also the conversion of exogenous [1-14C]arachidonic acid to prostacyclin (IC50 of 40 +/- 17 microM). No effect on arachidonic acid release was noted. To investigate whether our in vitro finding that 15-HETE inhibited prostacyclin production could be relevant to the in vivo situation, our final studies were performed on vasculature obtained from the diabetic milieu. We found that the production of 15-HETE was significantly increased in vasculature obtained from the infant of the diabetic mother (1.14 +/- 0.26 pmol/mg) when compared to control neonates (0.77 +/- 0.22; P less than 0.01). A concomitant decrease in prostacyclin production was seen (51.6 +/- 12.6 pmol/mg in infants of diabetic mothers vs. 71 +/- 22.3 in controls). Moreover, an inverse correlation between these two eicosanoids was also noted. Our results suggest a potential in vivo regulatory role for 15-HETE on prostacyclin production.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Cattle; Cyclooxygenase Inhibitors; Diabetes Mellitus; Endothelium; Female; Humans; Hydroxyeicosatetraenoic Acids; Infant, Newborn; Muscle, Smooth, Vascular; Prostaglandins F; Umbilical Arteries

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

Rabbit hydronephrotic cortical interstitial cells in primary culture were labeled with [1-14C]arachidonic acid and the eicosanoids released after stimulation with bradykinin or A23187 were studied by reverse-phase high performance liquid chromatography. The major arachidonic acid metabolite formed was prostaglandin (PG)E2, comprising more than 30% of the total radioactivity released. 12-Hydroxyheptadecatrienoic acid, probably representing spontaneous breakdown of the cyclic endoperoxides PGG2 and/or PGH2, made up 10 to 15% of the radioactivity released. Other cyclooxygenase products that were released included PGF2 alpha, PGD2, 6-keto PGF1 alpha and only minute amounts of thromboxane B2. Small quantities of the lipoxygenase products 15-, 12- and 5-hydroxyeicosatetraenoic acids (HETEs) as well as leukotrienes (LT)B4, LTC4 and LTD4 were also identified. Significantly larger quantities of 15- and 5-HETEs were recovered at 2 to 5 min than after longer incubations with A23187, suggesting esterification of these HETEs into cellular phospholipids. The data indicate that interstitial cells of the hydronephrotic kidney synthesize a variety of cyclooxygenase and lipoxygenase products of arachidonic acid, which may contribute to the pathophysiology of hydronephrosis. Moreover, it is suggested that PGG2 and/or PGH2 that are released from these cells may be metabolized further by adjacent kidney cells or circulating blood elements to other eicosanoid products, thus increasing the diversity of eicosanoids synthesized in the hydronephrotic kidney.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Bradykinin; Calcimycin; Cells, Cultured; Chromatography, High Pressure Liquid; Dinoprostone; Eicosanoic Acids; Hydronephrosis; Hydroxyeicosatetraenoic Acids; Kidney Cortex; Leukotrienes; Male; Prostaglandins E; Prostaglandins G; Rabbits; SRS-A; Strontium

Reticulocytes from various species (rat, mouse, rhesus monkey) obtained by phenylhydrazine treatment of the animals metabolized polyenoic fatty acids via a lipoxygenase pathway. Linoleic acid was converted to 13-hydro(pero)xy-9,11(Z,E)octadecadienoic acid [13-H(P)ODE] and 9-hydro(pero)xy-10,12(E,Z)octadecadienoic acid [9-H(P)ODE], whereas arachidonic acid was oxygenated to 15-hydroxy-5,8,11,13(Z,Z,Z,E)eicosatetraenoic acid (15-HETE) as shown by straight-phase high-pressure liquid chromatography (SP-HPLC). Addition of calcium and ionophore A 23,187 strongly enhanced the formation of lipoxygenase products, whereas 5,8,11,14eicosatetraenoic acid (ETYA) completely inhibited their formation. Estimates of the specific radioactivities of the lipoxygenase products indicate differences in the metabolization of externally added and endogenously released polyenoic fatty acids. These results strongly suggest that lipoxygenases generally occur in immature red blood cells.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Lipoxygenase; Lipoxygenase Inhibitors; Macaca; Mice; Phenylhydrazines; Rats; Reticulocytes; Species Specificity

Leukotriene B4 (LTB4) release by calcium ionophore-stimulated human leucocytes was measured by use of selective solvent partition of reaction mixtures and an agarose microdroplet chemokinesis assay, and the inhibitory effects of four monohydroxy fatty acids were determined. 15-Hydroxy-eicosatetraenoic acid (15-HETE) was the most effective inhibitor of LTB4 production with an approximate IC50 value of 6 microM and 99% inhibition at 50 microM, whereas 13-hydroxy-octadecadienoic acid (13-HODD) and 12-HETE were weaker inhibitors with approximate IC50 values of 32 microM and 23 microM, and 59% and 68% inhibition at 50 microM, respectively. We suggest that 13-HODD and 12-HETE, which are present in large amounts in the lesions of the skin disease psoriasis, may act as endogenous modulators of 5-lipoxygenase activity in skin.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotriene B4; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase; Skin; Time Factors

We investigated the effects of monohydroxyeicosatetraenoic acids (monoHETEs) on lipoxygenase- and cyclooxygenase-catalyzed reactions in glycogen-elicited rat PMNs challenged with A23187 and exogenous [14C]arachidonic acid. A23187 (10 microM) stimulated a 10-, 4-, 1.7- and 1.8-fold increase in the synthesis of radiolabeled 5-HETE, LTB4, TxB2, and PGE2 by rat PMNs. Addition of 5-HETE, 5 lactone-HETE, 12-HETE, and 15-HETE led to a dose-related reduction in [14C]5-HETE and [14C]LTB4 synthesis by these cells. These monoHETEs also inhibited [14C]TxB2 synthesis, but only 5-HETE and 5 lactone-HETE inhibited the synthesis of [14C]PGE2. Both 12-HETE and 15-HETE failed to reduce the formation of [14C]PGE2. These results suggest that monoHETEs differ significantly in their effects on arachidonic acid metabolism in rat PMNs and may play a role in modulating the synthesis of both lipoxygenase and cyclooxygenase products.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Calcimycin; Carbon Radioisotopes; Dinoprostone; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Neutrophils; Prostaglandins E; Rats; Thromboxane A2

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

Arachidonic acid metabolism in ionophore A23187-activated human polymorphonuclear leukocytes (PMNs) proceeds predominantly via the 5-lipoxygenase pathway in comparison to metabolism by the 15-lipoxygenase route. Products of both lipoxygenase pathways appear to be involved in the mediation of inflammatory reactions. Pretreatment of polymorphonuclear leukocytes with micromolar amounts of the platelet-derived 12-lipoxygenase product 12-hydroxy-5,8,10,14- eicosatetraenoic acid (12-HETE) prior to the addition of A23187 and [14C]arachidonic acid resulted in the unexpected dose-dependent stimulation of the 15-lipoxygenase pathway, as evidenced by the formation of [14C]15-HETE. A concomitant inhibition of the 5-lipoxygenase pathway was also observed. The structural identity of 15-HETE was confirmed by retention times on straight-phase and reverse-phase high pressure liquid chromatography in comparison with an authentic standard, radioimmunoassay, and chemical derivatization. When other isomeric HETEs were tested, the order of stimulatory potencies was 15-HETE greater than 12-HETE greater than 5-HETE. When arachidonic acid metabolism via the 5-lipoxygenase route was inhibited by nordihydroguaiaretic acid, previously ineffective concentrations of exogenous 12-HETE were now able to stimulate the polymorphonuclear leukocyte 15-lipoxygenase. Thus, blockade of the 5-lipoxygenase pathway appeared to be a prerequisite for the activation of the 15-lipoxygenase. The HETE-induced activation of the 15-lipoxygenase occurred within 1-2 min, was a reversible process, and was enhanced in the presence of A23187. In nine donors tested, up to 14-fold stimulation of [14C]15-HETE production was observed. Our results indicate that endogenous HETEs can have a dual role in the post-phospholipase regulation of arachidonic acid metabolism since they can act as physiological stimulators of the 15-lipoxygenase as well as inhibitors of the 5-lipoxygenase.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Models, Chemical; Neutrophils; Time Factors

The effects of melittin on the synthesis of lipoxygenase metabolites of arachidonic acid in human leukocytes and platelets were studied using high performance liquid chromatography. Melittin was found to stimulate strongly the formation of leukotrienes and hydroxy-eicosatetraenoic acids (HETEs) in a concentration-dependent fashion. The metabolites detected were LTB4, omega-OH-LTB4, omega-COOH-LTB4, LTC4, 5-HETE, 12-HETE, 15-HETE, 5S,12S-DiHETE, and 5S,15S-DiHETE. These results suggest that the action of melittin on the formation of arachidonic acid metabolites might be involved in its ability to release endogenous substrates required for the synthesis of 5-, 15-, and 12-lipoxygenase products in leukocytes and platelets, respectively.

Topics: Arachidonic Acid; Arachidonic Acids; Bee Venoms; Blood Platelets; Calcimycin; Dose-Response Relationship, Drug; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukocytes; Lipoxygenase; Melitten; Phospholipases A

15-Hydroxyeicosatetraenoic acid (15-HETE) was prepared by soybean lipoxygenase-mediated oxygenation of arachidonic acid to 15-hydroperoxyeicosatetraenoic acid (15-HETE) and subsequent reduction by NaBH4. 15-HETE was identified, purified and proved as biologically active by thin layer chromatography, high pressure liquid chromatography, gas chromatography/mass spectrometry and biological experiments on horse thrombocytes and rabbit peritoneal leukocytes. 15-HETE (0.1-40 microM) was added to peripheral leukocytes of 48 human donors (33 atopics, 15 nonatopics) which were challenged with rabbit anti-human-IgE or calcium ionophore A 23187. Its effect was compared with the effects of nordihydroguaiaretic acid (NDGA) and eicosatetraynoic acid (ETYA). NDGA and ETYA markedly inhibited histamine release (17 microM: 74 +/- 11 and 39 +/- 14,3%, respectively), whereas 15-HETE neither stimulated nor inhibited spontaneous anti-IgE- or calcium ionophore A 23187-induced histamine release.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Adult; Animals; Antibodies, Anti-Idiotypic; Arachidonic Acids; Calcimycin; Catechols; Dose-Response Relationship, Immunologic; Histamine Release; Humans; Hydroxyeicosatetraenoic Acids; Hypersensitivity, Immediate; Immunoglobulin E; Immunosuppressive Agents; Leukocytes; Masoprocol; Rabbits

Suspensions of human blood leukocytes and platelets were stimulated with the ionophore A23187 in the presence of increasing concentrations of arachidonic acid. Nine metabolites of the 5-, 12- and 15-lipoxygenase and the cyclooxygenase pathways were analyzed by high performance liquid chromatography in order to study the rate-limiting steps and other characteristics of their biosynthesis. The data indicate that the LTA4 hydrolase and the LTA4 glutathione transferase activities are respectively the limiting factors in the synthesis of LTB4 and LTC4. At high substrate concentrations (5 X 10(-5)M and above), the 5-lipoxygenase activity was inhibited whereas the synthesis of the 15-HETE (15-lipoxygenase product) increased linearly up to the highest concentration tested (3 X 10(-4)M). The data also indicate that contrary to 5-HETE and LTs, 15-HETE is not formed upon stimulation with the ionophore, but only following incubation with exogenous arachidonic acid (20 microM and above), pointing out major differences in the synthesis of 5- and 15-lipoxygenase products.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Arachidonic Acids; Blood Platelets; Calcimycin; Humans; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotriene B4; Lipoxygenase; SRS-A

Isolated dog mastocytoma cells sensitized with dog anti-ragweed IgE and challenged with ragweed antigen or incubated with ionophore A23187 or the carboxy-terminal dodecapeptide of platelet factor 4, PF4(59-70), release histamine and concurrently generate leukotrienes B4, C4, and D4. In contrast, the exposure of mastocytoma cells to 0.1-3 micrograms/ml of 15-hydroxyeicosatetraenoic acid (15-HETE) stimulates selectively the generation of leukotrienes, in the absence of histamine release, while 0.1-1 micrograms/ml of compound 48/80 releases histamine without enhancing the generation of leukotrienes. That natural stimuli are capable of selectively activating one synthetic or secretory compartment of mast cells suggests that separate subsets of receptors as well as different biochemical events may serve to mobilize each class of mediators.

Topics: Allergens; Animals; Arachidonic Acids; Calcimycin; Cell Transformation, Neoplastic; Dogs; Histamine Release; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Mast-Cell Sarcoma; p-Methoxy-N-methylphenethylamine; Platelet Factor 4; SRS-A

Mono-hydroxy-eicosatetraenoic acids (HETE's) are frequently the principal lipoxygenase-derived products in a number of cell types. This paper describes the development of a selective and sensitive radioimmunoassay procedure for 15-HETE, a metabolite which has previously been shown to be both an activator and inhibitor of leukotriene formation in various cells. Initially, rabbits were immunized with 15-HETE conjugated to bovine serum albumin. After seven months, the anti-plasma showed significant binding of tritiated 15-HETE (40-45% binding with a 1:600 dilution of the anti-plasma) which was displaceable by cold 15-HETE. The sensitivity of the assay was approximately 20 pg. of 15-HETE. The anti-plasma exhibited very little (less than 1%) cross-reactivity with arachidonic acid, 5-, 8-, 9-, 11- and 12-HETE's, HHT, TXB2, PGE2 and 6-Keto-PGF1 alpha. Significant cross-reactivity was observed with 5,15-diHETE (53%), 8, 15-diHETE (6.6%), and several other 15-hydroxy-eicosanoids. Rabbit reticulocytes have a very active 15S-lipoxygenase and converted arachidonic acid (final concentration 7 microM) principally to 15-HETE. Unstimulated reticulocytes were found to release negligible amounts of 15-HETE as determined by radioimmunoassay. Treatment of these cells with the calcium ionophore A23187 (0.16 to 4.0 micrograms/ml) elicited a level of 15-HETE release (8 - 14 ng/ml) that was twenty to forty times less than that obtained with exogenous arachidonic acid (2.5 micrograms/ml). The radioimmunoassay reported here may be useful for identifying factors which stimulate cellular release of 15-HETE and other 15-hydroxy-eicosanoids from endogenous arachidonic acid.

Topics: Animals; Arachidonic Acids; Calcimycin; Cross Reactions; Hydroxyeicosatetraenoic Acids; Rabbits; Radioimmunoassay; Reticulocytes; Thromboxane B2; Time Factors

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotriene B4; Lipoxygenase; Substrate Specificity; Swine