5-hydroxy-6-8-11-14-eicosatetraenoic-acid and 15-hydroxy-5-8-11-13-eicosatetraenoic-acid

High-cholesterol and high-fat diets alter biochemical composition and anti-oxidant properties of high-density lipoproteins (HDL) in animals. Whether this occurs in humans is unknown. Therefore, we examined the effect of a short-term elevation in dietary cholesterol and fat intake on HDL composition in healthy subjects.. In a randomized, crossover clinical trial, 14 healthy young volunteers followed a 14-day low-cholesterol/low-fat diet (LChF) and a 14-day isocaloric high-cholesterol/high-fat diet (HChF) in a random order. After each diet, we measured HDL concentrations of hydroxyeicosatetraenoic acids (HETE), hydroxyoctadecadienoic acids (HODE), and haptoglobin, as well as serum amyloid A (SAA) and paroxonase-1 activity (PON-1). HDL concentrations of 15-HETE (+254%, p = 0.002), 5-HETE (+116%, p = 0.004), 13-HODE (+102%, p = 0.049), and SAA levels (+75%, p = 0.007) were significantly higher after the HChF than after the LChF. Furthermore, haptoglobin was marginally increased (+32%, p = 0.091) while PON-1 activity was unaffected (-16%, p = 0.366) by the HChF.. In healthy subjects, a short-term elevation in dietary cholesterol and fat intake increases HDL lipid hydroperoxide content (15-HETE, 5-HETE, 13-HODE) and SAA levels, which are key features of dysfunctional HDL. This is the first study showing that a physiologic manipulation of dietary cholesterol and fat intake affects HDL lipidome and proteome in healthy subjects independently of weight changes.. NCT02549144.

Topics: Adult; Biomarkers; Cholesterol, Dietary; Cross-Over Studies; Diet, High-Fat; Female; Healthy Volunteers; Humans; Hydroxyeicosatetraenoic Acids; Italy; Linoleic Acids; Lipid Peroxides; Lipoproteins, HDL; Male; Postprandial Period; Prospective Studies; Serum Amyloid A Protein; Time Factors; Young Adult

Eicosanoids, active metabolites of arachidonic acid (AA), play an important role in the regulation of renal haemodynamics and glomerular filtration. Our study verified the hypothesis on the positive action of exogenously administered PGE(1) on renal function during an operation with temporary ischaemia of the lower half of the body. Also the effect of alprostadil (prostaglandin E(1) analogue) administered during the operation of an abdominal aorta aneurysm on the postoperative systemic metabolism of AA and the glomerular filtration rate (GFR) was investigated. The study included 42 patients with a diagnosed abdominal aorta aneurysm who have been qualified for the operation of implantation of the aortic prosthesis. The patients were randomly assigned to two groups: the study group (I) receiving alprostadil and the control group (II) without alprostadil. The levels of hydroxyeicosatetraenoic acids (15-HETE, 12-HETE, 5-HETE) were determined by RP-HPLC and the level of thromboxane B(2) (TxB(2)) was determined by ELISA in the plasma of the blood drawn from vena cava superior immediately before aortic clamping (A) and 5 min after aortic declamping (B). The administration of PGE(1) affects the metabolism of 15-HETE in a manner dependent on the baseline value of GFR but does not significantly change the postoperative renal function. The metabolism of 15-HETE is affected by the baseline value of GFR1 and a longer period of ischaemia is correlated with lower concentrations of 5-HETE during reperfusion. The results of our studies indicate that TxB(2) influences the postoperative function of kidneys.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Aged; Alprostadil; Aortic Aneurysm; Eicosanoids; Female; Glomerular Filtration Rate; Humans; Hydroxyeicosatetraenoic Acids; Kidney; Male; Middle Aged; Postoperative Period; Reperfusion Injury; Thromboxane B2

To determine the association of polyunsaturated fatty acid (PUFA)-derived lipid mediators with progression from rheumatoid arthritis (RA)-related autoimmunity to inflammatory arthritis (IA).. We conducted a prospective cohort study using data from the Studies of the Etiology of Rheumatoid Arthritis (SERA). SERA enrolled first-degree relatives (FDRs) of individuals with RA (FDR cohort) and individuals who screened positive for RA-related autoantibodies at health fairs (screened cohort). We followed up 133 anti-cyclic citrullinated peptide 3.1 (anti-CCP3.1)-positive participants, 29 of whom developed IA. Lipid mediators selected a priori were quantified from stored plasma samples using liquid chromatography tandem mass spectrometry. We fit multivariable Cox proportional hazards models for each lipid mediator as a time-varying variable. For lipid mediators found to be significantly associated with IA, we then examined interleukin-1β (IL-1β), IL-6, IL-8, and tumor necrosis factor (TNF) as potential statistical mediators.. For every 1 natural log pg/ml increase in the circulating plasma levels of proinflammatory 5-HETE, the risk of developing IA increased by 241% (hazard ratio 2.41 [95% confidence interval 1.43-4.07]) after adjusting for age at baseline, cohort (FDR or screened), and shared epitope status. The models examining 15-HETE and 17-HDHA had the same trend but did not reach significance. We did not find evidence that the association between 5-HETE and IA risk was influenced by the proinflammatory cytokines tested.. In a prospective cohort of anti-CCP-positive individuals, higher levels of 5-HETE, an important precursor to proinflammatory leukotrienes, is associated with subsequent IA. Our findings highlight the potential significance of these PUFA metabolites in pre-RA populations.

Topics: Adult; Aged; Anti-Citrullinated Protein Antibodies; Arthritis, Rheumatoid; Autoimmunity; Cohort Studies; Disease Progression; Docosahexaenoic Acids; Family; Fatty Acids, Unsaturated; Female; Humans; Hydroxyeicosatetraenoic Acids; Incidence; Interleukin-1beta; Interleukin-6; Interleukin-8; Male; Mediation Analysis; Middle Aged; Multivariate Analysis; Proportional Hazards Models; Prospective Studies; Tumor Necrosis Factor-alpha

Reproducibility issues regarding in vitro cell culture experiments are related to genetic fluctuations and batch-wise variations of biological materials such as fetal calf serum (FCS). Genome sequencing may control the former, while the latter may remain unrecognized. Using a U937 macrophage model for cell differentiation and inflammation, we investigated whether the formation of effector molecules was dependent on the FCS batch used for cultivation. High resolution mass spectrometry (HRMS) was used to identify FCS constituents and to explore their effects on cultured cells evaluating secreted cytokines, eicosanoids, and other inflammatory mediators. Remarkably, the FCS eicosanoid composition showed more batch-dependent variations than the protein composition. Efficient uptake of fatty acids from the medium by U937 macrophages and inflammation-induced release thereof was evidenced using C13-labelled arachidonic acid, highlighting rapid lipid metabolism. For functional testing, FCS batch-dependent nanomolar concentration differences of two selected eicosanoids, 5-HETE and 15-HETE, were balanced out by spiking. Culturing U937 cells at these defined conditions indeed resulted in significant proteome alterations indicating HETE-induced PPARγ activation, independently corroborated by HETE-induced formation of peroxisomes observed by high-resolution microscopy. In conclusion, the present data demonstrate that FCS-contained eicosanoids, subject to substantial batch-wise variation, may modulate cellular effector functions in cell culture experiments.

Topics: Cell Culture Techniques; Eicosanoids; Fatty Acids; Humans; Hydroxyeicosatetraenoic Acids; Macrophages; Peroxisomes; Reproducibility of Results; Serum Albumin, Bovine; U937 Cells

Post-exercise hypotension (PEH) is the phenomenon of lowered blood pressure after a single bout of exercise. Only a fraction of people develops PEH but its occurrence correlates well with long-term effects of sports on blood pressure. Therefore, PEH has been suggested as a suitable predictor for the effectivity of exercise as therapy in hypertension. Local vascular bioactive lipids might play a potential role in this context. We performed a cross-over clinical pilot study with 18 healthy volunteers to investigate the occurrence of PEH after a single short-term endurance exercise. Furthermore, we investigated the plasma lipid profile with focus on arachidonic acid (AA)-derived metabolites as potential biomarkers of PEH. A single bout of ergometer cycling induced a significant PEH in healthy volunteers with the expected high inter-individual variability. Targeted lipid spectrum analysis revealed significant upregulation of several lipids in the direct post-exercise phase. Among these changes, only 15- hydroxyeicosatetranoic acid (HETE) correlated significantly with the extent of PEH but in an AA-independent manner, suggesting that 15-HETE might act as specific PEH-marker. Our data indicate that specific lipid modulation might facilitate the identification of patients who will benefit from exercise activity in hypertension therapy. However, larger trials including hypertonic patients are necessary to verify the clinical value of this hypothesis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Arachidonic Acid; Biological Variation, Population; Blood Pressure; Cross-Over Studies; Dinoprostone; Exercise; Female; Healthy Volunteers; Heart Rate; Humans; Hydroxyeicosatetraenoic Acids; Hypertension; Lipid Metabolism; Male; Pilot Projects; Post-Exercise Hypotension; Thromboxanes

The accumulation of non-polar lipids arachidonic acid, 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE and 15-HETE during storage of transfusion products may play a role in the onset of transfusion-related acute lung injury (TRALI), a syndrome of respiratory distress after transfusion.. We investigated non-polar lipid accumulation in red blood cells (RBCs) stored for 42 days, plasma stored for 7 days at either 4 or 20°C and platelet (PLT) transfusion products stored for 7 days. Furthermore, we investigated whether transfusion of RBCs with increased levels of non-polar lipids induces TRALI in a 'two-hit' human volunteer model. All products were produced following Dutch Blood Bank protocols and are according to European standards. Non-polar lipids were measured with high-performance liquid chromotography followed by mass spectrometry.. All non-polar lipids increased in RBCs after 21 days of storage compared to baseline. The non-polar lipid concentration in plasma increased significantly, and the increase was even more pronounced in products stored at 20°C. In platelets, baseline levels of 5-HETE and 15-HETE were higher than in RBCs or plasma. However, the non-polar lipids did not change significantly during storage of PLT products. Infusion of RBCs with increased levels of non-polar lipids did not induce TRALI in LPS-primed human volunteers.. We conclude that non-polar lipids accumulate in RBC and plasma transfusion products and that accumulation is temperature dependent. Accumulation of non-polar lipids does not appear to explain the onset of TRALI (Dutch Trial Register - NTR4455).

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Acute Lung Injury; Adolescent; Adult; Arachidonic Acid; Blood Platelets; Blood Preservation; Blood Transfusion, Autologous; Chromatography, High Pressure Liquid; Erythrocytes; Humans; Hydroxyeicosatetraenoic Acids; Lipids; Lipopolysaccharides; Male; Models, Theoretical; Platelet Transfusion; Registries; Tandem Mass Spectrometry; Temperature; Time Factors; Transfusion Reaction; Young Adult

Recent studies have established the role of mid-chain hydroxyeicosatetraenoic acids (HETEs) in the development of cardiovascular disease. Mid-chain HETEs have been reported to have vasoconstrictive and pro-inflammatory effects. However, whether mid-chain HETEs can induce cardiac hypertrophy remains unclear. Therefore, the overall objective of the present study was to elucidate the potential hypertrophic effect of mid-chain HETEs in the human ventricular cardiomyocytes, RL-14 cells, and to explore the mechanisms involved. For this purpose, RL-14 cells were treated with increasing concentrations of mid-chain HETEs (2.5, 5, 10 and 20 µM). Thereafter, the cardiac hypertrophy markers and cell size were determined using real-time polymerase chain reaction and phase contrast imaging, respectively. Phosphorylated mitogen-activated protein kinase (MAPK) level and nuclear factor kappa B (NF-κB) binding activity were determined. Our results showed that mid-chain HETEs induced cellular hypertrophy in RL-14 cells as evidenced by the induction of cardiac hypertrophy markers, α- and β-myocin heavy chain and atrial and brain natriuretic peptide as well as the increase in cell size. Mechanistically, all mid-chain HETEs were able to induce the binding activity of NF-κB to its responsive element in a HETE-dependent manner, and they significantly induced the phosphorylation of ERK 1/2. The induction of cellular hypertrophy was associated with proportional increase in the formation of dihydroxyeicosatrienoic acids parallel to the increase of soluble epoxide hydrolase enzyme activity. In conclusion, our study provides the first evidence that mid-chain HETEs induce cellular hypertrophy in RL-14 cells through MAPK- and NF-κB-dependent mechanism.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Atrial Natriuretic Factor; Cardiomegaly; Cell Line; Cell Size; Cell Survival; Dose-Response Relationship, Drug; Heart Ventricles; Humans; Hydroxyeicosatetraenoic Acids; Mitogen-Activated Protein Kinases; Myocytes, Cardiac; Natriuretic Peptide, Brain; NF-kappa B

Pseudomonas aeruginosa is an opportunistic pathogen that can cause nosocomial and chronic infections in immunocompromised patients. P. aeruginosa secretes a lipoxygenase, LoxA, but the biological role of this enzyme is currently unknown. LoxA is poorly similar in sequence to both soybean LOX-1 (s15-LOX-1) and human 15-LOX-1 (37 and 39%, respectively) yet has kinetics comparably fast versus those of s15-LOX-1 (at pH 6.5, Kcat = 181 ± 6 s(-1) and Kcat/KM = 16 ± 2 μM(-1) s(-1)). LoxA is capable of efficiently catalyzing the peroxidation of a broad range of free fatty acid (FA) substrates (e.g., AA and LA) with high positional specificity, indicating a 15-LOX. Its mechanism includes hydrogen atom abstraction [a kinetic isotope effect (KIE) of >30], yet LoxA is a poor catalyst against phosphoester FAs, suggesting that LoxA is not involved in membrane decomposition. LoxA also does not react with 5- or 15-HETEs, indicating poor involvement in lipoxin production. A LOX high-throughput screen of the LOPAC library yielded a variety of low-micromolar inhibitors; however, none selectively targeted LoxA over the human LOX isozymes. With respect to cellular activity, the level of LoxA expression is increased when P. aeruginosa undergoes the transition to a biofilm mode of growth, but LoxA is not required for biofilm growth on abiotic surfaces. However, LoxA does appear to be required for biofilm growth in association with the host airway epithelium, suggesting a role for LoxA in mediating bacterium-host interactions during colonization.

Topics: Amino Acid Sequence; Animals; Antibody Formation; Arachidonate 15-Lipoxygenase; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Lipoxygenase Inhibitors; Pseudomonas aeruginosa; Rabbits; Substrate Specificity

Zishen pill (ZSP) is a traditional Chinese medicine (TCM) used to treat benign prostatic hyperplasia (BPH). The study used a metabolomic approach based on UHPLC-MS/MS to profile arachidonic acid (AA) metabolic changes and to investigate the interventional mechanisms of ZSP in testosterone- induced BPH rats. In order to explore the potential therapeutic effect of ZSP, rat models were constructed and orally administrated with ZSP. Plasma and urine samples were collected after four weeks and then eleven potential biomarkers (15-HETE, 12-HETE, TXA2, 5-HETE, AA, PGI2, PGF2α, 8-HETE, PGD2, PGE2 and LTB4) were identified and quantified by UHPLC-MS/MS. The chromatographic separation was carried out with gradient elution using a mobile phase comprised of 0.05% formic acid aqueous solution (pH=3.3) (A) and acetonitrile: methanol (80:20, V/V) (B), and each AA metabolites was measured using electrospray ionization source with negative mode and multiple reaction monitoring. The eleven biomarkers in BPH group rat plasma and urine were significant higher than those in sham group rats. Using the potential biomarkers as a screening index, the results suggest that ZSP can potentially reverse the process of BPH by partially regulating AA metabolism through refrain the expression of cyclooxygenase (COX) and lipoxygenase (LOX). This study demonstrates that a metabolomic strategy is useful for identifying potential BPH biomarkers and investigating the underlying mechanisms of a TCM in BPH treatment.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Biomarkers; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Male; Medicine, Chinese Traditional; Metabolomics; Plasma; Prostaglandin-Endoperoxide Synthases; Prostatic Hyperplasia; Rats; Rats, Sprague-Dawley; Tandem Mass Spectrometry

The balance of putative pro- and antiinflammatory lipoxygenase (LOX)-derived S-hydroxyeicosatetraenoic acids (S-HETEs) in colon mucosa is a potential target for modulating colon cancer risk and progression. The biological effects of S-HETEs and R-hydroxyeicosatetraenoic acids (produced by distinct pathways) may differ, but levels of these compounds in the colon are unknown. The objective of this study was to develop chiral methods to characterize hydroxyeicosatetraenoic (HETE) enantiomers in colonic mucosa and evaluate the effects of fish oil on HETE formation. C57BL/6 mice (COX-1 null, COX-2 null, wild-type) were fed a diet supplemented with either olive oil or menhaden oil for 11 wk, and R-/S-HETEs in colonic mucosa were quantified by chiral LC-MS/MS. The R-enantiomer comprised 60-72% of 5-HETE, 18-58% of 15-HETE, and 1-16% of 12-HETE in colonic mucosa, suggesting that non-LOX sources contribute to HETE profiles. Fish oil reduced levels of both R- and S-HETEs, and increased the preponderance of the R-enantiomers (particularly 12- and 15-HETEs). There was apparent shunting of arachidonic acid to 12-/15-LOX in the COX-1 null animals. This is the first report of the enantiomeric composition of HETEs in the colon in vivo and shows large effects of fish oil in the normal colon.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Chromatography, High Pressure Liquid; Colon; Cyclooxygenase 1; Cyclooxygenase 2; Fatty Acids; Female; Fish Oils; Hydroxyeicosatetraenoic Acids; Intestinal Mucosa; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Stereoisomerism

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

Inflammation is a multi-staged process whose expansive phase is thought to be driven by acutely released arachidonic acid (AA) and its metabolites. Inhibition of cyclooxygenase (COX), lipoxygenase (LOX), or soluble epoxide hydrolase (sEH) is known to be anti-inflammatory. Inhibition of sEH stabilizes the cytochrome P450 (CYP450) products epoxyeicosatrienoic acids (EETs). Here we used a non-selective COX inhibitor aspirin, a 5-lipoxygenase activation protein (FLAP) inhibitor MK886, and a sEH inhibitor t-AUCB to selectively modulate the branches of AA metabolism in a lipopolysaccharide (LPS)-challenged murine model. We used metabolomic profiling to simultaneously monitor representative AA metabolites of each branch. In addition to the significant crosstalk among branches of the AA cascade during selective modulation of COX, LOX, or sEH, we demonstrated that co-administration of t-AUCB enhanced the anti-inflammatory effects of aspirin or MK886, which was evidenced by the observations that co-administration resulted in favorable eicosanoid profiles and better control of LPS-mediated hypotension as well as hepatic protein expression of COX-2 and 5-LOX. Targeted disruption of the sEH gene displayed a parallel profile to that produced by t-AUCB. These observations demonstrate a significant level of crosstalk among the three major branches of the AA cascade and that they are not simply parallel pathways. These data illustrate that inhibition of sEH by both pharmacological intervention and gene knockout enhances the anti-inflammatory effects of aspirin and MK886, suggesting the possibility of modulating multiple branches to achieve better therapeutic effects.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Aspirin; Benzoates; Blood Pressure; Dose-Response Relationship, Drug; Enzyme Activation; Epoxide Hydrolases; Gene Expression Regulation, Enzymologic; Hydroxyeicosatetraenoic Acids; Indoles; Inflammation; Lipopolysaccharides; Lipoxygenase Inhibitors; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Prostaglandin-Endoperoxide Synthases; Urea

Active metabolites of arachidonic acid (AA), eicosanoids, strongly influence renal homeostasis. The aims of this study were to measure perioperative variations in lipoxygenase (LOX)-derived 5-, 12- and 15-hydroxyeicosatetraenoic (HETE) acids levels, and to examine whether (i) dynamics of these eicosanoid generation changes during the first 5 min of renal allograft reperfusion, (ii) examined HETE acids may influence perioperative 20-HETE generation, and (iii) LOX HETE may serve as perioperative markers of early post-transplant allograft function.. Sixty-nine kidney recipients were divided into early, slow and delayed graft function (EGF, SGF and DGF, respectively) groups. Blood was taken directly before, and in the consecutive minutes of graft reperfusion. HETE concentrations were measured using liquid chromatography. Creatinine levels were measured during the perioperative period, as well as during follow-up visits (first post-transplant year).. Our results demonstrated significant differences in the concentrations and dynamics of HETE changes between the examined groups. Moreover, observed changes in HETE concentrations were strongly associated with post-transplant graft function and perioperative 20-HETE synthesis. Application of cut-off limits for newly introduced markers, that is 71.72 ng/mL for 5-HETE(5), 12.3 ng/mL for 12-HETE△(5-0) and -6.1 ng/mL for 15-HETE△(5-0), resulted in 72.5-81.5% sensitivity and 50-54% specificity for SGF/DGF prediction. Moreover, mixed model analysis revealed that recipients classified according to results of 5-HETE(5) and 15-HETE△(5-0) significantly differ in 1-year post-transplant allograft function (P  =  0.03 and P  < 0.05, respectively), however, not in the frequency of acute rejections' episodes (P = 0.91 and P = 0.31, respectively).. We hereby report that human kidney transplantations are accompanied by significant changes in LOX AA metabolism, which strongly influences and predicts early (1 year) post-transplant graft function.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Arachidonic Acid; Biomarkers; Female; Graft Survival; Humans; Hydroxyeicosatetraenoic Acids; Kidney Transplantation; Lipoxygenase; Male; Middle Aged; Perioperative Period; Retrospective Studies; Time Factors; Transplantation, Homologous

To determine in vivo if L-4F differentially alters plasma levels of oxidized fatty acids resulting in more anti-inflammatory HDL. Injecting L-4F into apoE null mice resulted in a significant reduction in plasma levels of 15-HETE, 5-HETE, 13-HODE and 9-HODE. In contrast, plasma levels of 20-HETE were not reduced and plasma levels of 14,15-EET, which are derived from the cytochrome P450 pathway, were elevated after injection of L-4F. Injection of 13(S)-HPODE into wild-type C57BL/6J mice caused an increase in plasma levels of 13-HODE and 9-HODE and was accompanied by a significant loss in the anti-inflammatory properties of HDL. The response of atherosclerosis resistant C3H/HeJ mice to injection of 13(S)-HPODE was similar but much more blunted. Injection of L-4F at a site different from that at which the 13(S)-HPODE was injected resulted in significantly lower plasma levels of 13-HODE and 9-HODE and significantly less loss of HDL anti-inflammatory properties in both strains. i) L-4F differentially alters plasma levels of oxidized fatty acids in vivo. ii) The resistance of the C3H/HeJ strain to atherosclerosis may in part be mediated by a reduced reaction of this strain to these potent lipid oxidants.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Anti-Inflammatory Agents; Apolipoproteins E; Atherosclerosis; Chromatography, Liquid; Enzyme-Linked Immunosorbent Assay; Fatty Acids; Female; Hydroxyeicosatetraenoic Acids; Injections, Subcutaneous; Linoleic Acids; Linoleic Acids, Conjugated; Lipid Peroxides; Lipoproteins, HDL; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; Oxidation-Reduction; Peptides; Species Specificity; Tandem Mass Spectrometry; Time Factors; Up-Regulation

The ligand activated nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma) induces transcriptional repression of pro-inflammatory factors. Activation of PPARgamma is followed by amelioration of colitis in animal models of inflammatory bowel disease (IBD). A reduced expression of PPARgamma was found in epithelial cells of patients with ulcerative colitis. The eicosanoids 13-HODE and 15-HETE are products of 12/15-lipoxygenase (LOX) and endogenous ligands for PPARgamma. Dehydrogenation of 13-HODE by 13-HODE dehydrogenase results in formation of the 13-Oxo-ODE. Highest activity of 13-HODE dehydrogenase is found in colonic epithelial cells (CECs). We therefore investigated whether 13-Oxo-ODE is a new endogenous ligand of PPARgamma in CECs.. LOX activity and 13-HODE dehydrogenase in CECs were investigated after stimulation with arachidonic or linoleic acid. LOX metabolites were identified by RP-18 reversed-phase HPLC. Binding of (14)C-labelled 13-Oxo-ODE was demonstrated using a His-tagged PPARgamma.. Stimulation of HT-29 and primary CECs homogenates with and without Ca-ionophor was followed by the formation of high amounts of the linoleic acid metabolite 13-Oxo-ODE (155 and 85 ng/ml). The decrease of IL-8 secretion from IEC was more pronounced after pre-incubation with 13-Oxo-ODE compared to the PPARgamma agonist troglitazone and higher as with the known PPARgamma ligands 13-HODE and 15-HETE. Binding assays with (14)C-labelled 13-Oxo-ODE clearly demonstrated a direct interaction.. High amounts of 13-Oxo-ODE can be induced in CECs by stimulation of linoleic acid metabolism. 13-Oxo-ODE binds to PPARgamma and has anti-inflammatory effects. 13-HODE dehydrogenase might be a therapeutic target in IBD.

Topics: Animals; Arachidonic Acid; Blotting, Western; Carbon Radioisotopes; Cattle; Chromatography, High Pressure Liquid; Colon; Epithelial Cells; HT29 Cells; Humans; Hydroxyeicosatetraenoic Acids; Immunoprecipitation; Interleukin-8; Ligands; Linoleic Acid; Linoleic Acids; Linolenic Acids; Molecular Structure; PPAR gamma; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; Spectrometry, Mass, Electrospray Ionization; Transfection

Conjugated linoleic acid (CLA) consists of a group of linoleic acid geometric isomers that have been shown to reduce tumor growth and metastasis in animal models of breast, prostate and colon cancer. To delineate a possible mechanism of action for CLA, we have recently shown that the 5-lipoxygenase product, 5-hydroxyeicosatetraenoic acid (5-HETE), could play a role in CLA alteration of mammary tumorigenesis. In this study, we determined how CLA could modulate 5-lipoxygenase activity. The t10, c12-CLA isomer reduced production of 5-HETE but not 12- and 15-HETE in MDA-MB-231 human breast tumor cells. That isomer and the c9, t11-CLA isomer decreased 5-HETE production by competition with the lipoxygenase substrate, arachidonic acid (AA). Interestingly, t10, c12-CLA reduced the expression of five-lipoxygenase activating protein (FLAP) but not the 5-lipoxygenase enzyme. Over-expression of FLAP abrogated t10, c12-CLA-reduced viability of MDA-MB-231 cells. These data suggest that the reduction of 5-HETE by t10, c12-CLA was due to competition with AA and the reduction of FLAP expression.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5-Lipoxygenase-Activating Proteins; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Breast Neoplasms; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Female; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Isomerism; Linoleic Acids; Linoleic Acids, Conjugated; Membrane Proteins; Transfection

The purpose of this research was to evaluate the effects of targeted arterial delivery of the branched chain fatty acid 12-methyltetradecanoic acid (12-MTA) on the VX2 squamous cell carcinoma in rabbits. An intramuscular VX2 squamous cell carcinoma was induced at a single site in the right thigh of 39 New Zealand white rabbits. Approximately 10 days after inoculation, a 3-French catheter was introduced into the right common carotid artery and positioned using fluoroscopic guidance in the right deep femoral artery, which was the main, if not exclusive, artery supplying the tumor. Ethiodol alone (targeting agent), Ethiodol containing 12-MTA, or Ethiodol containing myristic acid was then injected through the catheter. Tumor growth and histopathology were evaluated 7-8 days after treatment. Caspase-3 activity was evaluated 2 days after therapy, and tumor tissues were assayed for eicosanoid metabolites 2 and 7 days after treatment to assess the effects of the branched chain fatty acid on the lipoxygenase (LOX) and cyclooxygenase-2 (COX-2) enzyme systems. Targeted arterial delivery of 12-MTA resulted in dose-dependent growth inhibition of intramuscular rabbit VX2 tumors while myristic acid, a saturated fatty acid of the same carbon length as 12-MTA, was found to stimulate tumor growth. Two and 7 days following treatment, tumors treated with 12-MTA showed a significant decrease in 5-hydroxyeicosatetraenoic acid (5-HETE) and a concomitant increase in 15-HETE levels while tumors treated with myristic acid exhibited a significant increase in prostaglandin E2 (PGE2) levels. Western blot as well as immunohistochemical analysis showed that 5-LOX and COX-2 proteins were present in the VX2 tumors. No alterations in tumor/tumor cell morphology or caspase-3 activity were evident on microscopic examination following treatment. These studies suggest that targeted arterial delivery of branched chain fatty acids such as 12-MTA may be considered as a potential new therapy for treatment of solid tumors. The exact mechanism(s) responsible for the observed inhibition of VX2 tumor growth by 12-MTA is unclear. Additional in vivo studies are warranted to elucidate 12-MTA's mechanism of action and further investigate the branched chain fatty acid's antitumor effects.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Antineoplastic Agents; Arachidonate 5-Lipoxygenase; Blotting, Western; Caspase 3; Caspases; Cell Line, Tumor; Cyclooxygenase 2; Dinoprostone; Enzyme Inhibitors; Fatty Acids; Humans; Hydroxyeicosatetraenoic Acids; Immunohistochemistry; Lipoxygenase Inhibitors; Neoplasm Transplantation; Neoplasms; Rabbits; Tissue Fixation

The influence of inhibitors of different lipoxygenases (LOX) on the growth of human tumor cells with different profiles of synthesized eicosanoids was studied. The studied LOX inhibitors had virtually no influence on the growth of A549 cells actively synthesizing cyclooxygenase and lipoxygenase metabolites of arachidonic acid (AA). The inhibitor of 12-LOX, baicalein, significantly inhibited proliferation in cultures of A431 epidermoid carcinoma cells with a characteristic domination of the major lipoxygenase metabolite of AA, 12-hydroxyeicosatetraenoic acid (12-HETE), in the profile of synthesized eicosanoids and reduced to 70% the incorporation of [3H]thymidine into DNA. Treatment of these cultures with 12-HETE virtually restored the growth potential of the tumor cells. The findings suggest that the lipoxygenase metabolite of AA, 12-HETE, is a growth-limiting factor for tumor cells of definite type.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adenocarcinoma; Arachidonate Lipoxygenases; Arachidonic Acid; Carcinoma, Squamous Cell; Cell Proliferation; Flavanones; Humans; Hydroxyeicosatetraenoic Acids; Lung Neoplasms; Nitrobenzenes; Salicylamides; Sulfonamides; Tumor Cells, Cultured; Umbelliferones

Human esophageal adenocarcinoma (EAC) develops in a sequence from gastroesophageal reflux disease (GERD), columnar-lined esophagus (CLE), dysplasia, and eventually to EAC. We established a rat surgical EAC model with esophagogastroduodenal anastomosis (EGDA) to mimic the staged process of esophageal adenocarcinogenesis. Profiling of the AA metabolites with mass spectrometry showed that prostaglandin E2 (PGE2), leukotriene B4 (LTB4), 15-hydroeicosatetraenoic acid (HETE), 12-HETE, 8-HETE and 5-HETE all increased at the esophagoduodenal junction after EGDA as compared with the proximal esophagus, with PGE2 as the major metabolite. Consistent with this profile, cyclooxygenase 2 (Cox2) was overexpressed in the basal cell layer of esophageal squamous epithelium, CLE cells and EAC tumor cells of the EGDA rats, as compared with the normal esophageal epithelium. Sulindac (a Cox inhibitor), nordihydroguaiaretic acid (NDGA, a lipoxygenase inhibitor) and alpha-difluoromethylornithine (DFMO, an ornithine decarboxylase inhibitor) were tested for their possible inhibitory actions against the formation of EAC in the rat EGDA model. In a short-term study (for 4 weeks after surgery), dietary administration of both sulindac (300 and 600 p.p.m.) and NDGA (100 p.p.m.) effectively reduced the EGDA-induced inflammation. In a long-term chemoprevention study (for 40 weeks after surgery), 300 p.p.m. sulindac, alone or in combination with 100 p.p.m. NDGA or 0.5% DFMO, decreased the tumor incidence from 57.7 to 26.9%, or 16.7 or 20%, respectively (P < 0.05). NDGA alone (100 and 200 p.p.m.) slightly decreased the tumor incidence to 52.4 and 37%, respectively, although the difference was not statistically significant. DFMO alone did not show significant effects on tumor incidence. Inhibition of tumor formation by sulindac was correlated with lowered levels of PGE2. In conclusion, sulindac exerted its chemopreventive effect against the formation of EAC in the rat EGDA model possibly through its inhibition of Cox.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Body Weight; Cyclooxygenase 2; Dinoprostone; Eflornithine; Esophageal Neoplasms; Esophagus; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Immunoenzyme Techniques; In Situ Hybridization; Inflammation; Isoenzymes; Leukotriene B4; Male; Masoprocol; Mass Spectrometry; Neoplasms; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Sprague-Dawley; Sulindac; Time Factors

We have examined the effects of endogenous lipoxygenase products on basal progesterone (P4) production by cultured bovine mid-luteal cells. The involvement of lipoxygenase products in the stimulatory effect of LH on luteal cAMP accumulation and P4 production was also examined. Bovine luteal cells from mid-cycle corpora lutea (CL) were exposed for 16 h to a lipoxygenase inhibitor (nordihydroguaiaretic acid: NDGA; 0.33-33 microM). For the last 4 h of incubation, the cells were exposed to LH and/or three different lipoxygenase products, 5-, 12- and 15-hydroxyeicosatetraenoic acid (HETE). NDGA inhibited P4 production by the cells in a dose-dependent manner (P < 0.05). NDGA-reduced P4 production was reversed by the addition of 12-HETE, but not 5- or 15-HETE, whereas 5-, 12- and 15-HETE alone showed no significant effect on P4 production in the intact cells. Furthermore, NDGA (33 microM) blocked the stimulatory action of LH on P4 production (P < 0.05), without changing cAMP accumulation (P > 0.1). When the cells were exposed to 5-, 12- or 15-HETE with LH and NDGA, only 15-HETE maintained the stimulatory effect of LH on P4 production in the cells (P < 0.05). These results suggest that endogenous lipoxygenase products play important roles in P4 production by bovine CL, i.e. basal P4 production is supported by 12-HETE, and LH-stimulated P4 production is partially mediated via the activation of lipoxygenase and subsequent 15-HETE formation downstream of the LH-activated cAMP-PKA-phosphorylation pathway.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Cattle; Cells, Cultured; Corpus Luteum; Dose-Response Relationship, Drug; Female; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Lipoxygenase Inhibitors; Luteinizing Hormone; Masoprocol; Progesterone; Time Factors

Dendritic cell (DC) differentiation from human CD34(+) hematopoietic progenitor cells (HPCs) can be triggered in vitro by a combination of cytokines consisting of stem cell factor, granulocyte-macrophage colony-stimulating factor, and tumor necrosis factor alpha. The immune response regulatory cytokines, IL-4 and IL-13, promote DC maturation from HPCs, induce monocyte-DC transdifferentiation, and selectively up-regulate 15-lipoxygenase 1 (15-LO-1) in blood monocytes. To gain more insight into cytokine-regulated eicosanoid production in DCs we studied the effects of IL-4/IL-13 on LO expression during DC differentiation. In the absence of IL-4, DCs that had been generated from CD34(+) HPCs in response to stem cell factor/granulocyte-macrophage colonystimulating factor/tumor necrosis factor alpha expressed high levels of 5-LO and 5-LO activating protein. However, a small subpopulation of eosinophil peroxidase(+) (EOS-PX) cells significantly expressed 15-LO-1. Addition of IL-4 to differentiating DCs led to a marked and selective down-regulation of 5-LO but not of 5-LO activating protein in DCs and in EOS-PX(+) cells and, when added at the onset of DC differentiation, also prevented 5-LO up-regulation. Similar effects were observed during IL-4- or IL-13-dependent monocyte-DC transdifferentiation. Down-regulation of 5-LO was accompanied by up-regulation of 15-LO-1, yielding 15-LO-1(+) 5-LO-deficient DCs. However, transforming growth factor beta1 counteracted the IL-4-dependent inhibition of 5-LO but only minimally affected 15-LO-1 up-regulation. Thus, transforming growth factor beta1 plus IL-4 yielded large mature DCs that coexpress both LOs. Localization of 5-LO in the nucleus and of 15-LO-1 in the cytosol was maintained at all cytokine combinations in all DC phenotypes and in EOS-PX(+) cells. In the absence of IL-4, major eicosanoids of CD34(+)-derived DCs were 5S-hydroxyeicosatetraenoic acid (5S-HETE) and leukotriene B(4), whereas the major eicosanoids of IL-4-treated DCs were 15S-HETE and 5S-15S-diHETE. These actions of IL-4/IL-13 reveal a paradigm of eicosanoid formation consisting of the inhibition of one and the stimulation of another LO in a single leukocyte lineage.

Topics: Antigens, CD34; Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Cell Differentiation; Cells, Cultured; Dendritic Cells; Down-Regulation; Eicosanoids; Enzyme Induction; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-13; Interleukin-4; Microscopy, Confocal; Monocytes; Stem Cell Factor; Stem Cells; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tumor Necrosis Factor-alpha

Butyrate, a short-chain fatty acid, modulates proliferation and differentiation of normal and neoplastic colonocytes. We examined the expression of 5-lipoxygenase (5-LO) and its metabolites in human colorectal carcinoma (Caco-2) cells, exposed to differentiation-inducing doses of butyrate. Treatment with butyrate significantly increased 5-lipoxygenase mRNA and protein in comparison to nontreated cells. Cyclooxygenases (COX)-1 and COX-2 mRNA were not significantly influenced by the treatment. However, 5-LO activity, low in nontreated cells, increased only minimally after butyrate, and its metabolic product (5-HETE) was detectable neither in control nor in treated cells. In contrast, 15-HETE (a product of 15-LO, which is also upregulated by butyrate) rose significantly. We conclude that, although being overexpressed by butyrate on mRNA and protein level, 5-LO remains inactive in differentiating Caco-2 cells. This is likely to be due either to some associated actions of butyrate, or to 5-LO-inhibition by 15-HETE, concomitantly induced by butyrate treatment.

Topics: 5-Lipoxygenase-Activating Proteins; Arachidonate 5-Lipoxygenase; Butyric Acid; Caco-2 Cells; Carrier Proteins; Cell Differentiation; Cell Division; Colorectal Neoplasms; Cyclooxygenase Inhibitors; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase Inhibitors; Membrane Proteins; RNA, Messenger; RNA, Neoplasm; Transforming Growth Factor beta; Up-Regulation

Capsaicin, a pungent ingredient of hot peppers, causes excitation of small sensory neurons, and thereby produces severe pain. A nonselective cation channel activated by capsaicin has been identified in sensory neurons and a cDNA encoding the channel has been cloned recently. However, an endogenous activator of the receptor has not yet been found. In this study, we show that several products of lipoxygenases directly activate the capsaicin-activated channel in isolated membrane patches of sensory neurons. Among them, 12- and 15-(S)-hydroperoxyeicosatetraenoic acids, 5- and 15-(S)-hydroxyeicosatetraenoic acids, and leukotriene B(4) possessed the highest potency. The eicosanoids also activated the cloned capsaicin receptor (VR1) expressed in HEK cells. Prostaglandins and unsaturated fatty acids failed to activate the channel. These results suggest a novel signaling mechanism underlying the pain sensory transduction.

Topics: Animals; Capsaicin; Cell Line; Cells, Cultured; Dinoprostone; Eicosanoids; Ganglia, Spinal; Humans; Hydroxyeicosatetraenoic Acids; Inflammation; Ion Channel Gating; Leukotriene B4; Leukotrienes; Ligands; Lipid Peroxides; Lipoxygenase; Molecular Structure; Neurons, Afferent; Prostaglandin D2; Prostaglandin H2; Prostaglandins H; Rats; Receptors, Drug; Structure-Activity Relationship

12-Lipoxygenase and cyclooxygenase 1 are the dominating enzymes that metabolize arachidonic acid in human platelets. In addition to the conversion of arachidonic acid to 12(S)-hydroxyeicosatetraenoic acid, 12-lipoxygenase can also utilize 5(S)-hydroxyeicosatetraenoic acid and 15(S)-hydroxyeicosatetraenoic acid to form 5(S), 12(S)-dihydroxyeicosatetraenoic acid and 14(R), 15(S)-dihydroxyeicosatetraenoic acid, respectively. Furthermore, 15(S)-hydroxyeicosatetraenoic acid works as an inhibitor for 12-lipoxygenase. In the present paper we have studied the influence of albumin on the in vitro metabolism of 5 - and 15 -hydroxyeicosatetraenoic acids, and 5,15 -dihydroxyeicosatetraenoic acid by the platelet 12-lipoxygenase. The presence of albumin reduced the formation of 5(S),12(S)- dihydroxyeicosatetraenoic acid from 5(S)-hydroxyeicosatetraenoic acid, however, it had no effect on the 12(S)-hydroxyeicosatetraenoic acid production from endogenous arachidonic acid. In contrast, when 15(S)-hydroxyeicosatetraenoic acid was incubated with activated platelets, the formation of 14(R), 15(S)- dihydroxyeicosatetraenoic acid was stimulated by the presence of albumin. Furthermore, albumin reduced the inhibitory action 15(S)-hydroxyeicosatetraenoic acid had on 12(S)-hydroxyeicosatetraenoic acid formation from endogenous arachidonic acid. However, addition of exogenous arachidonic acid (20 microm) to the incubations inverted the effects of albumin on the conversion of 15(S)-hydroxyeicosatetraenoic acid to 14(R),15(S)- dihydroxyeicosatetraenoic acid and the production of 12(S)-hydroxyeicosatetraenoic acid in these incubations. Based on the Scatchard equation, the estimates of the binding constants to albumin were 1.8 x 10(5) for 15 -HETE, 1.4 x 10(5) for 12-HETE, and 0.9 x 10(5) for 5 -HETE respectively. These results suggest an important role of albumin for the regulation of the availability of substrates for platelet 12-lipoxygenase.

Topics: Arachidonate 12-Lipoxygenase; Blood Platelets; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Protein Binding; Serum Albumin; Substrate Specificity

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Binding, Competitive; Blood Platelets; Eicosanoids; Enzyme Inhibitors; Estrenes; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Lipoxygenase Inhibitors; Phospholipases A; Platelet Activation; Pyrrolidinones; Serum Albumin; Type C Phospholipases

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 examine the effects of ropivacaine, currently being investigated for treatment of ulcerative colitis, on the release of arachidonic acid metabolites.. Human granulocytes and endothelial cells.. Ropivacaine, lidocaine, hydrocortisone, 5-aminosalicylic acid or acetylsalicylic acid (10-1000 microM).. Leukotriene B4, 5-hydroxyeicosatetraenoic acid, 6-keto PGF1 alpha and 15-hydroxyeicosatetraenoic acid were measured using immuno assays. Wilcoxon signed rank test was used for statistical calculations.. Ropivacaine dose-dependently inhibited zymosan-induced release of leukotriene B4 and 5-hydroxyeicosatetraenoic acid whereas the release after ionophore stimulation was not affected. Ropivacaine was more potent than 5-aminosalicylic acid but less potent compared to hydrocortisone. Ropivacaine had only a weak inhibitory effect on the release of 15-hydroxyeicosatetraenoic acid from zymosan- or ionophore-stimulated cells. In contrast to hydrocortisone and 5-aminosalicylic acid, ropivacaine only weakly affected the release of 6-keto PGF1 alpha after stimulation with thrombin.. The inhibited release of 5-lipoxygenase products may account for some of the anti-inflammatory effects of ropivacaine seen in the treatment of ulcerative colitis.

Topics: 6-Ketoprostaglandin F1 alpha; Amides; Anesthetics, Local; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Cells, Cultured; Eicosanoids; Endothelium, Vascular; Granulocytes; Humans; Hydrocortisone; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lidocaine; Mesalamine; Ropivacaine; Thrombin; Zymosan

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

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Cell Division; Cell Line; Cricetinae; DNA; DNA Replication; Embryo, Mammalian; Epidermal Growth Factor; Hydroxyeicosatetraenoic Acids; Linoleic Acids; Linoleic Acids, Conjugated; Mesocricetus; Oxidation-Reduction; Thymidine

(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

Many arachidonic metabolites have been shown to have marked potent biological effects in animals. The cyclooxygenase and lipoxygenase products of arachidonate metabolism are known to play a key role in the development of inflammatory symptoms and signs. Reports published during past decades indicate that cyclooxygenase products of arachidonic acid metabolism are present in much higher concentrations in inflamed than in healthy periodontal tissues. Since information about the role of lipoxygenase products of arachidonic acid metabolism in human periodontal disease is lacking, the objective of this study was to determine the ability of diseased and non-diseased gingival tissue to synthesize lipoxygenase products from the precursor arachidonic acid. Twenty-six samples of diseased tissue and nine samples of non-diseased tissue were included in our data analysis. After incubation of the tissue with 100,000 cpm [3H]-arachidonic acid, lipoxygenase products were separated by high performance liquid chromatography (HPLC) and identified by comparison with cochromatographed standards. Our results showed that inflamed gingival tissue synthesized significantly larger amounts, of LTB4(p < 0.01), LTC4(p < 0.01), LTD4(p < 0.01), LTE4(p < 0.01), 5-HETE(p < 0.05), 12-HETE(p < 0.01), and 15-HETE(p < 0.01), compared to non-diseased tissue. The lipoxygenases are more active in inflamed gingival tissue than in non-diseased gingival tissue. 12-HETE and 15-HETE were the the major metabolites formed by lipoxygenases in diseased and non-diseased human gingiva. Since we did not functionally determine the fractions separated by HPLC, our present data may only provided indirect evidence for the existence of lipoxygenase products in periodontal tissue. However, our study did establish a research model for the investigation of arachidonic acid metabolism in the pathogenesis of periodontal disease.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Humans; Hydroxyeicosatetraenoic Acids; Leukotrienes; Periodontal Diseases; Periodontium

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

Arachidonic acid (AA) seems to play an important role in testicular steroidogenesis, although controversial data exist in the literature. In the present study AA induced a dose related increase of testosterone (T) formation and, at the highest dose, stimulated the production of prostaglandin E2 (PGE2), leukotrienes B4 (LTB4) and C4 (LTC4) by purified rat Leydig cells. The contemporary addition of the prostaglandin synthesis blocker, indomethacin (IND), and AA further increased T formation, decreased PGE2 levels and did not modify LTB4 and LTC4 concentrations. The addition of a lipoxygenase inhibitor, nordihydroguaiaretic acid (NDGA, 5 microM), did not influence the stimulatory effect of AA on T and PGE2 formation while it decreased the output of LTB4 and LTC4. When 20 microM NDGA was used in addition to AA the expected reduction of leukotrienes release was observed together with a surprising impairment of T and PGE2 secretion. PGE2 and PGF2 alpha did not modify basal T production but reduced HCG-stimulated T secretion at the 10 nM dose. When 5-12- and 15-HETE were tested an enhancement of basal T formation was observed at the 10nM dose. 5-HETE (10nM) stimulated HCG-induced T production. LTA4, LTB4 and LTE4 did not influence basal T output while LTC4 and LTD4 inhibited it. LTC4 (10nM) induced a decrease of HCG-stimulated T production. These findings suggest that: 1) exogenous AA stimulates T secretion; 2) conversion of AA to cycloxygenated and lipoxygenated metabolites is not required for its steroidogenic effect; 3) cycloxygenated and lipoxygenated compounds play a diverse modulatory role on testicular steroidogenesis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Chorionic Gonadotropin; Dinoprostone; Hydroxyeicosatetraenoic Acids; Indomethacin; Leukotriene B4; Leukotriene C4; Leydig Cells; Male; Masoprocol; Rats; Rats, Sprague-Dawley; Testosterone

An assay for the quantitative determination of the hydroxylation profile of long-chain fatty acids is described for gas chromatography negative-ion chemical ionization mass spectrometry and stable isotope dilution using [carboxyl-18O2]-labeled internal standards. The assay has been applied to the study of fatty acids isolated from body fluids, tissue, and cultured cells. Examples for the analyses of biological systems expressing 5-, 8-, 12-, or 15-lipoxygenase activity are given and the most important sources of analytical errors are addressed. Increased specificity compared to analysis by negative-ion chemical ionization, at the cost of sensitivity, can be achieved by the use of positive-ion electron impact ionization for the investigation of hydrogenated pentafluorobenzylester/trimethylsilylether derivatives. The method described provides complete, specific, and quantitative profiles of hydroxylated fatty acids originally present in biological samples or generated in vitro by incubation with polyunsaturated fatty acid substrates such as linoleic or arachidonic acid.

Topics: Animals; Cell Line; Female; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase; Mice

Lipoxygenase products were quantified in human mixed saliva and in saliva fractions obtained from a parotid or submandibular gland using gas chromatography-mass spectrometry and stable isotope dilution. In glandular saliva, only linoleic acid was detected at levels of 20-30 ng/ml. In contrast, mixed saliva showed a linoleic acid concentration of around 300 ng/ml, arachidonic acid levels of around 30 ng/ml, hydroxyoctadecadienoic acid (HODE) levels between 5 and 10 ng/ml, and hydroxyeicosatetraenoic acid (HETE) levels up to 25 ng/ml. By far the most abundant HETE was 12-HETE, and incubation experiments with arachidonic acid showed the presence of a substantial 12-lipoxygenase activity in human mixed saliva, but not in saliva fractions. This activity was identified as 12(S)-lipoxygenase activity by chiral analysis of the reaction product. Investigating mixed saliva and glandular saliva of patients with squamous cell carcinoma in the upper aerodigestive tract and of controls, most patients showed elevated levels of free arachidonic acid and elevated HETE levels. Besides a moderate increase in 12-HETE levels, markedly elevated concentrations of 5-HETE and 15-HETE were observed for the carcinoma patients. The level of free arachidonic acid and the quantitative HETE profile appear to be good markers for the inflammatory processes occurring in the oral mucosa and in saliva in response to the development of squamous cell carcinoma.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase; Mouth Neoplasms; Parotid Gland; Saliva; Submandibular Gland

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

The investigation was undertaken to characterize the profile of arachidonic acid metabolites in different spontaneous and transplantable tumors in mice. The five metabolites via the cyclooxygenase pathway (PGE2, PGF2 alpha, PGD2, TxB2, 6-keto-PGF1 alpha), as well as the three lipoxygenase products (5-HETE, 12-HETE, and 15-HETE) were monitored by thin layer chromatography and high performance liquid chromatography after "ex vivo" metabolism of exogenous [1-C14]-arachidonic acid by homogenates of tumor tissues. It was shown that all tumors had a unique profile of eicosanoids. The most cyclooxygenase activity along with the significant synthesis of PGE2, PGF2 alpha, and 6-keto-PGF1 alpha was noted in lung tumors. The antitumor effect of indomethacin was directly related to the ability of tumors to produce PGE2. On the other hand, there were varying lipoxygenase activities in tumors. In some cases, the extremely high levels of 15- and 12-HETE synthesis in neoplastic tissue could indicate that there was a basic possibility of using lipoxygenase inhibitors for suppressing malignant tumors.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adenocarcinoma; Animals; Arachidonic Acid; Carcinoma, Lewis Lung; Carcinoma, Squamous Cell; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Dinoprostone; Eicosanoids; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Indomethacin; Leukemia L1210; Lipoxygenase Inhibitors; Mammary Neoplasms, Experimental; Melanoma, Experimental; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Inbred DBA; Neoplasm Transplantation; Neoplasms, Experimental; Skin Neoplasms

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

Endothelial cells actively regulate their environment by secreting humoral substances, including endothelin-1 and a variety of eicosanoids, that have local actions. To elucidate interactions among these local mediators, we measured release of cyclooxygenase and lipoxygenase pathway products of arachidonate metabolism by human aortic endothelial cells after incubation with endothelin-1. Supernatants were collected, extracted, and fractionated using high-performance liquid chromatography. Radioimmunoassays for eicosanoids were performed on the appropriate fractions. After endothelin stimulation, production of the prostacyclin metabolite 6-ketoprostaglandin F1 alpha (6-keto-PGF1 alpha), the thromboxane (Tx) metabolite TxB2, and prostaglandin E2 (PGE2) were increased (307 +/- 48, 320 +/- 91, and 315 +/- 74% of control, P < 0.05). Leukotriene B4 (LTB4) release was modestly increased (195 +/- 19% of control, P < 0.05). The release of 5-hydroxyeicosatetraenoic acid (5-HETE) was increased (300 +/- 57% of control, P < 0.05); production of 12-HETE and 15-HETE was unchanged. Production of eicosanoids peaked between 30 and 120 min. Preincubation with pertussis toxin prevented increased production of PGE2, LTB4, and 5-HETE after endothelin-1 stimulation; pretreatment with sphingosine had no effect. Interactions between endothelin and eicosanoids may be important components of the complex network that regulates vascular tone, coagulation, and inflammation at the local level.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 6-Ketoprostaglandin F1 alpha; Aorta; Arachidonic Acid; Cells, Cultured; Dinoprostone; Eicosanoids; Endothelins; Endothelium, Vascular; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Pertussis Toxin; Thromboxane B2; Virulence Factors, Bordetella

Our previous investigations demonstrated that 5- and 15-lipoxygenase metabolites of arachidonic acid are synthesized in bovine adrenal fasciculata cells, although their exact role in the regulation of adrenal steroidogenesis is unknown. Thus we attempted to investigate their direct effects on cholesterol side-chain cleavage in bovine adrenal mitochondria. We also examined Ca2+ efflux in mitochondria, based on a reported correlation between pregnenolone formation and Ca2+ release in adrenal mitochondria. The present experiments showed that 5-HETE increased pregnenolone generation in the isolated intact mitochondria, but not in the inner mitochondrial membrane. Thus it is suggested that 5-HETE may activate cholesterol side-chain cleavage by inducing the translocation of cholesterol from the outer to the inner mitochondrial membrane. The present experiments also demonstrated that 5-HPETE, 5-HETE, 15-HPETE, and 15-HETE, but not leukotriene B4, activated cholesterol side-chain cleavage and Ca2+ efflux from mitochondria, suggesting that these substances may activate cholesterol side-chain cleavage by regulating Ca2+ movement in mitochondria. These effects were additively enhanced when mitochondria were stimulated simultaneously with these substances and GTP. Therefore, both GTP and lipoxygenase metabolites seem to play crucial roles in the regulation of pregnenolone generation. The direct effect of 5- and 15-lipoxygenase metabolites other than leukotriene B4 on the regulation of pregnenolone synthesis, which is known to be the rate-limiting step of steroidogenesis, were clearly observed in the present experiments.

Topics: Adrenal Cortex; Animals; Arachidonic Acid; Calcium; Cattle; Cholesterol; Drug Interactions; Guanosine Triphosphate; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase; Mitochondria; Pregnenolone

This study examines the effects of oxidized low density lipoprotein (ox-LDL) on mononuclear cell arachidonic acid (AA) metabolism. U937 and HL60 cells, employed as models for mononuclear cells and promyelocytic cells, respectively, were exposed to ox-LDL (20 micrograms protein/ml) for 24 hours. HPLC analysis of 1-14C-AA metabolites indicates that ox-LDL increases U937 and HL60 cell production of 15-hydroxyeicosatetraenoic acid (15-HETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). Northern analysis indicates that ox-LDL increases U937 cell FLAP transcript levels 10-times control levels but did not appear to alter 5-lipoxygenase (5-LO) mRNA levels. In contrast, ox-LDL increases HL60 cell transcript levels for FLAP and 5-LO 1.5 times and 10 times control levels, respectively. Thus, we propose that ox-LDL plays an important role in the up-regulation of the 5-LO pathway in mononuclear cells. Such activation may explain, in part, the mechanisms by which ox-LDL promotes atherogenesis.

Topics: 5-Lipoxygenase-Activating Proteins; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Blotting, Northern; Carrier Proteins; Cell Line; Enzyme Activation; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukemia; Lipoproteins, LDL; Lymphoma, Large B-Cell, Diffuse; Malondialdehyde; Membrane Proteins; Oxidation-Reduction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; RNA, Neoplasm; Transcription, Genetic; Tumor Cells, Cultured

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

The effects of interleukin-1 alpha (IL-1 alpha) on arachidonic acid (AA) metabolism were studied in the human osteosarcoma cell lines, G292 and SaOS-2. The cells were prelabeled with 3H-arachidonic acid. Radiolabeled metabolites were measured by reversed-phase high-pressure liquid chromatography with a radioactive detector. Indomethacin inhibited prostaglandin E2 (PGE2) production without affecting lipoxygenase (LO) products in G292 cells. In the G292 cells, IL-1 alpha (50 U/ml) induced a 10-fold increase in PGE2 production at all the incubation times tested, and a significant two-fold increase in 5 hydroxyeicosatetraenoic acid (HETE) formation after 48 h. These effects were not seen in SaOS-2 cells under identical conditions. These results suggest that, although some osteosarcomal cell lines may not respond directly to IL-1 with effects on AA metabolism, the mechanism of its action in others may involve modulation of both cyclooxygenase (CO) and LO pathways.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 6-Ketoprostaglandin F1 alpha; Arachidonic Acid; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Interleukin-1; Leukotriene B4; Lipoxygenase; Osteoblasts; Osteosarcoma; Tumor Cells, Cultured

1. Prostaglandin endoperoxide synthetase (PES) and lipoxygenase (Lox) activities were compared in the cerebella and cerebra of vitamin E-sufficient young chicks and in chicks in which nutritional encephalomalacia (NE) was induced by a diet deficient in vitamin E. 2. Eicosanoid production patterns were qualitatively similar in the brains of both groups of chicks, but prostaglandin production was 50-60% less in cerebella of ataxic chicks, compared to control cerebella, while the opposite trend was observed in the cerebellar Lox pathway, as measured by radioimmunoassay of 15-HETE. 3. Cerebellar phospholipase A2 activity was twice that of the cerebrum but was not affected by NE. 4. Purification of Lox activity from the cerebellar homogenates produced a lower yield and enrichment when the starting material was taken from ataxic chicks, compared to the controls. 5. In addition there were qualitative differences in the purified fractions from both groups, as seen by pH optima and kinetics. 6. The results are consistent with the view that the cerebellum has less antioxidant protection than the cerebrum and that its higher phospholipase A2 activity and greater propensity to oxygenate arachidonic acid via the Lox pathway at the expense of the PES pathway may render this region of the brain particularly vulnerable to oxidative damage in NE.

Topics: Animal Nutritional Physiological Phenomena; Animals; Arachidonic Acid; Brain; Cerebellum; Chickens; Encephalomalacia; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotrienes; Lipid Peroxides; Lipoxygenase; Male; Phospholipases A; Phospholipases A2; Poultry Diseases; Prostaglandin-Endoperoxide Synthases; Prostaglandins

The influence of the leukotoxin of Pasteurella haemolytica on the generation of arachidonic acid metabolites by bovine polymorphonuclear leukocytes (PMNs) was investigated. PMNs released 5-, 12-, and 15-hydroxyeicosatetraenoic acids (5-, 12-, and 15-HETE) and leukotriene B4 (LTB4) upon stimulation with arachidonic acid. The leukotoxin preparations dose dependently enhanced the release of the 5-lipoxygenase products 5-HETE and LTB4 in arachidonic acid-stimulated PMNs, whereas the release of 12- and 15-HETE was not affected. The enhanced release of LTB4 and 5-HETE was not due to a decreased cellular retention of the 5-lipoxygenase products. In addition, leukotoxin preparations by themselves were also able to induce LTB4 and 5-HETE production in the absence of exogenous arachidonic acid. Generation of 5-lipoxygenase products by PMNs stimulated by leukotoxin may represent an important cellular event that occurs during infections with P. haemolytica.

Topics: Animals; Bacterial Toxins; Cattle; Cells, Cultured; Exotoxins; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Mannheimia haemolytica; Neutrophils; Species Specificity

Rat basophilic leukemia cells exhibit 12-lipoxygenase activity only upon cell disruption. 12-Lipoxygenase may also possess 15-lipoxygenase activity, as is indicated by the formation of low amounts of 15(S)-HETE, in addition to the predominant product 12(S)-HETE, upon incubation of partially purified 12-lipoxygenase with arachidonic acid. With 5(S)-HPETE as substrate not only 5(S), 12(S)-diHETE and 5(S), 15(S)-diHETE are formed, but also LTA4, as was indicated by the presence of LTA4-derived LTB4-isomers. 12-Lipoxygenase from rat basophilic leukemia cells has many features in common with 12-lipoxygenase from bovine leukocytes. As was suggested for the latter enzyme, 12-lipoxygenase from rat basophilic leukemia cells may represent the remaining LTA4-synthase activity of 5-lipoxygenase, of which the 5-dioxygenase activity has disappeared upon cell disruption. Such a possible shift from 5-lipoxygenase activity to 12-lipoxygenase activity could not simply be induced by interaction of cytosolic 5-lipoxygenase with a membrane fraction after cell disruption, but may involve release of membrane-associated 5-lipoxygenase upon disruption of activated rat basophilic leukemia cells.

Topics: Animals; Arachidonate 12-Lipoxygenase; Arachidonate 5-Lipoxygenase; Cell Fractionation; Chromatography, High Pressure Liquid; Cytosol; Hydroxyeicosatetraenoic Acids; Kinetics; Leukemia, Basophilic, Acute; Rats; Tumor Cells, Cultured

The formation and role of arachidonic acid (AA) and its metabolites during gonadotropin releasing hormone- (GnRH-) induced gonadotropin secretion were investigated in primary cultures of rat pituitary cells. Prelabeled cells ([3H]AA) responded to GnRH challenge with increased formation (about 2-fold) of the leukotrienes LTC4, LTD4, and LTE4 as well as 5- and 15-eicosatetraenoic acids (5- and 15-HETE) as identified by HPLC. Formation of leukotrienes and 15-HETE was further verified by specific radioimmunoassays. No significant increase in the formation of 12-HETE or of the cyclooxygenase products prostaglandin E (PGE) and thromboxane A2 by GnRH was noticed. Addition of physiological concentrations of LTC4 enhanced basal LH release, while subphysiological concentrations of LTC4 (10(-15)-10(-12) M) inhibited GnRH-induced LH release by about 35% (p less than 0.02). Using specific lipoxygenase inhibitors L-656,224 and MK 886, we found inhibition of GnRH-induced LH release by about 40% at concentrations known to specifically inhibit the 5-lipoxygenase pathway. The peptidoleukotriene receptor antagonist ICI 198,615 inhibited LTC4- and LTE4-induced LH release and surprisingly also the effect of GnRH on LH release by 40%. The data strongly suggest a role for AA and its lipoxygenase metabolites in the on/off reactions of GnRH upon LH release. The data also present a novel amplification cycle in which newly formed leukotrienes become first messengers and establish an autocrine/paracrine loop.

Topics: Animals; Arachidonic Acid; Cells, Cultured; Eicosanoids; Exocytosis; Gonadotropin-Releasing Hormone; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Indazoles; Leukotrienes; Lipoxygenase; Lipoxygenase Inhibitors; Luteinizing Hormone; Pituitary Gland; Rats; Rats, Inbred Strains; Second Messenger Systems; Secretory Rate; Signal Transduction

Incubation of rocker-cultured neonatal rat heart cells with 3 mM L(+)-lactate led to a sharp increase in the sensitivity of cardiomyocytes to the beta-adrenergic agonist isoprenaline, as measured by their chronotropic response. This effect was accompanied by a reduction in the arachidonic acid content of the total phospholipids. The phospholipase A2-activator melittin as well as free arachidonic acid induced this supersensitivity to the same degree. On the other hand, the L(+)-lactate-evoked supersensitivity could be blocked by the phospholipase A2 inhibitors mepacrine and n-bromophenacyl-bromide, suggesting an involvement of phospholipase A2 in the process of beta-adrenergic sensitization. The sensitizing action of arachidonic acid was blocked by the lipoxygenase inhibitors esculetin and nordihydroguaiaretic acid, but not by the cyclo-oxygenase inhibitor indomethacin. Supersensitivity was likewise evoked by 15-S-hydroxyeicosatetraenoic acid (15-S-HETE), but not by 5-S-HPETE or 5-S-HETE. These findings suggest that the phospholipase A2-15-lipoxygenase pathway plays a role in the induction of beta-adrenergic supersensitivity in the cultured cardiomyocytes and point to a new physiological role of the lipoxygenase product 15-S-HETE.

Topics: Acetophenones; Animals; Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Chronobiology Phenomena; Dose-Response Relationship, Drug; Enzyme Activation; Heart; Hydroxyeicosatetraenoic Acids; Isoproterenol; Lactates; Leukotrienes; Melitten; Phospholipases A; Phospholipases A2; Prostaglandins D; Quinacrine; Rats; Rats, Inbred Strains; Receptors, Adrenergic, beta

The principal monohydroxyeicosatetraenoic acids (HETEs), 5-, 12-, and 15-HETE, which can be produced by rat basophilic leukemia (RBL-1) cells, are also esterified by these cells. Exogenously added 5-, 12-, and 15-HETE were rapidly incorporated as esters in RBL cells, reaching plateau levels within 25 min. In incubations in culture medium with protein added, all three HETEs were essentially completely metabolized within 24 h. 5-HETE was esterified more rapidly and to a greater extent than 12-HETE or 15-HETE when these were incubated together with RBL cells, indicating some degree of selectivity in the esterification pathways. When arachidonic acid (AA) was incubated in increasing concentrations with constant concentrations of 15-HETE and RBL cells, the free 15-HETE concentration increased and esterified 15-HETE concentration decreased markedly at AA: 15-HETE molar ratios above 9. 15-HETE esterification in RBL cells was also markedly inhibited by the polyunsaturated fatty acids, eicosatetraynoic and eicosapentanoic acids, but not by oleic or linoleic acids. In separate experiments with unlabeled and radiolabeled substrates, the extent of incorporation of esterified HETE in RBL cells decreased at higher concentrations of 15-HETE and AA, which showed that the pathway was saturable. The shapes of the curves for these fatty acid inhibitors suggest a concentration-dependent two-compartment pathway of esterification. These data indicate that the HETEs and other 20 carbon fatty acid substrates probably compete for activity of a specific arachidonyl-CoA synthetase, which is the first and rate-limiting step for esterification of arachidonic acid by many human cells. Esterified 15-HETE was found to be predominantly in the phosphatidylethanolamine fraction of RBL cell lipids.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Coenzyme A Ligases; Esterification; Fatty Acids; Hydroxyeicosatetraenoic Acids; Leukemia, Basophilic, Acute; Phospholipids; Rats; Tumor Cells, Cultured

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

Eugenol dose-dependently inhibited 5-HETE (5-hydroxy-5,8,10, 14-eicosatetraenoic acid) and 15-HETE formation by human polymorphonuclear leucocytes. p-Chlorophenol, guaiacol and phenol also inhibited the lipoxygenases. Formation of HETEs by rat dental pulp was inhibited by eugenol and p-chlorophenol. The concentrations of the phenolics required to inhibit lipoxygenases were in the similar range with those used for inhibiting cyclooxygenase. These results showed that phenolic compounds inhibited lipoxygenases and thus suggest that these compounds may be dual inhibitors of lipoxygenase and cyclooxygenase.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Dental Pulp; Eugenol; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Lipoxygenase Inhibitors; Male; Neutrophils; Phenols; Rats; Rats, Inbred Strains

Gallic esters with a varying chain length of its alcohol moiety produced strong inhibition of the conversion of [1-14C]-arachidonic acid to 5S-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-HETE) by isolated human polymorphonuclear leukocytes. Octyl gallate and decyl gallate were the most powerful inhibitors with a concentration of half-inhibition of about 1 mumol . 1-1. Additionally these compounds caused however at 10 mumol . 1-1 a complete inhibition of the incorporation of arachidonic acid in triacylglycerols and phospholipids which is assumed to be a consequence of the damage to the energy metabolism of the cells. In contrast, the other gallic esters enhance the incorporation of arachidonic acid in the ester lipids in addition to moderate inhibition of the 5-lipoxygenase pathway.

Topics: Arachidonic Acid; Arachidonic Acids; Gallic Acid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Neutrophils

Indomethacin, an inhibitor of cyclooxygenase that generates prostaglandins (PGs) from arachidonic acid, and 2 alpha,4 alpha,7-4,5-epoxy-17-hydroxy-4,17-dimethyl-3-oxoandrostane- 2-carbonitrile (epostane), an inhibitor of 3 beta-hydroxysteroid dehydrogenase that generates progesterone from pregnenolone, are both potent inhibitors of ovulation. This report compares the dose-dependent effects of these two inhibitors on ovarian levels of 5-, 12-, and 15-hydroxyeicosatetraenoic acid methyl ester (HETEs), prostaglandin E2 (PGE), prostaglandin F2 alpha (PGF), progesterone, 17 alpha-hydroxyprogesterone, 17 beta-estradiol, 4-androstene-3,17-dione, and testosterone during ovulation in 25-day-old immature Wistar rats. The ovulatory process was initiated by 10 IU of human chorionic gonadotropin (hCG). Indomethacin was given at 3 h after hCG in doses ranging from 0.0316 to 10.0 mg/rat. A dose of 0.1 mg/rat was the lowest dose to significantly reduce the ovulation rate from the control level of 70.5 +/- 5.8 ova/rat. This dose also reduced 15-HETE, but not 5-HETE, 12-HETE, or the steroids. PGE and PGF were strongly inhibited by an even lower dose of indomethacin (0.0316 mg/rat), but this dose did not affect the ovulation rate. Epostane was given at 3 h after hCG in doses ranging from 0.1 to 5.0 mg/rat. A dose of 1.0 mg/rat was the lowest dose to significantly inhibit ovulation. This dose also reduced the ovarian levels of 15-HETE and progesterone but not 5-HETE, 12-HETE, PGE, PGF, or the other steroids. The results indicate that the ovulation rate is most closely correlated to ovarian 15-HETE levels.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 17-alpha-Hydroxyprogesterone; 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Androstenedione; Androstenols; Animals; Chorionic Gonadotropin; Estradiol; Female; Hydroxyeicosatetraenoic Acids; Hydroxyprogesterones; Indomethacin; Masoprocol; Ovulation; Ovulation Induction; Progesterone; Prostaglandins E; Prostaglandins F; Rats; Rats, Inbred Strains; Steroids; Testosterone

The presence of polymorphonuclear leukocytes (PMNs) within the airways is a characteristic feature of a variety of lung diseases. Pulmonary alveolar macrophages (PAMs) and epithelial cells release many different factors which contribute to the recruitment of inflammatory cells into infected airways. PAMs and tracheal epithelial cells are able to produce linoleic acid metabolites (9-HODE and 13-HODE) besides arachidonic acid metabolites. The objective of the present study was to determine whether 9-HODE and 13-HODE possess chemotactic activity for isolated PMNs. It was found that 9-HODE and 13-HODE induced a chemotactic response of both human and bovine PMNs in vitro. The HODEs evoked chemotaxis with a linear dose response from 10(-10) to 10(-6) M to the same extent as the arachidonic acid metabolite 15-HETE. At 10(-8) M, 9-HODE and 13-HODE were approximately half as potent in inducing chemotaxis as compared to LTB4.

Topics: Animals; Cattle; Chemotaxis, Leukocyte; Fatty Acids, Unsaturated; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Linoleic Acids; Linoleic Acids, Conjugated; Neutrophils; Structure-Activity Relationship

Topics: Adapalene; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Arachidonic Acids; Carrageenan; Cell Differentiation; Chemotaxis, Leukocyte; Croton Oil; Erythema; Guinea Pigs; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Inflammation; Lipoxygenase; Luminescent Measurements; Mice; Naphthalenes; Neutrophils; Passive Cutaneous Anaphylaxis; Rabbits; Rats; Ultraviolet Rays

Mammalian 5-lipoxygenase systems exist in inactive or cryptic states and have to be stimulated in order to metabolize exogenous [14C]arachidonic acid to 5-HETE and leukotrienes. In most cells, both the activation process and the 5-lipoxygenase activity are calcium-dependent. However, the cryptic 5-lipoxygenase system in the murine PT-18 mast/basophil cell line, which can be stimulated by 15-hydroxyeicosatetraenoic acid (15-HETE), is unusual. Studies with fura-2 loaded PT-18 cells indicate that increases in cytosolic calcium do not appear to correlate with enhanced 5-lipoxygenase product formation. Thus, both the calcium ionophore ionomycin and arachidonic acid increase cytosolic calcium levels but have very little effect on [14C]5-HETE formation, whereas 15-HETE induces large increases in [14C]5-HETE production but no concomitant enhancement in cytosolic calcium is observed. Chelation of extracellular calcium by 3 mM EGTA resulted in a 30-40% inhibition of [14C]5-HETE formation induced by 15 HETE, whereas 3 mM EGTA has no appreciable effect on a crude PT-18 5-lipoxygenase homogenate. These results indicate that in PT-18 cells, calcium does not appear to play an important role in either the 15-HETE-induced activation process, or the enzymatic activity of the cryptic 5-lipoxygenase system.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Basophils; Calcium; Calcium Channel Blockers; Cell Line; Cytosol; Egtazic Acid; Gallic Acid; Hydroxyeicosatetraenoic Acids; Ionomycin; Mast Cells; Mice

Pulmonary epithelial cells may be responsible for regulating airway smooth muscle function, in part by release of fatty acid-derived mediators. Incubation of isolated guinea pig tracheal epithelial cells with radiolabeled arachidonic acid (AA) leads to the production of 5- and 15-hydroxyeicosatetraenoic acid (5- and 15-HETE) and smaller amounts of leukotriene (LT) B4 and C4 and 12-hydroxyheptadecatrienoic acid (HHT). Epithelial cells also are able to release linoleic acid (LA) metabolites. Incubation with radiolabeled linoleic acid leads to the formation of 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE). The biological significance of these mediators produced by epithelial cells is discussed.

Topics: Animals; Antithrombins; Arachidonic Acid; Arachidonic Acids; Carbon Radioisotopes; Epithelial Cells; Epithelium; Fatty Acids; Guinea Pigs; Hydroxyeicosatetraenoic Acids; Linoleic Acids; Linoleic Acids, Conjugated; Male; Perfusion; Trachea

Products of the lipoxygenase pathway have been implicated in the development of the cerebrovascular spasm that arises after subarachnoid hemorrhage. In particular the hydroperoxyeicosatetranenoic acids (HPETEs), which are unstable and break down rapidly to the corresponding 5-hydroxy acids (HETEs), are vasoconstrictor agents that mimic some aspects of cerebrovascular spasm. It is not, however, well established whether segments of cerebral artery can manufacture these products. We have studied the lipoxygenase product profile of cerebral arteries stimulated with arachidonic acid. Rings of bovine cerebral arteries were incubated in Krebs solution containing arachidonic acid. The lipoxygenase products were studied using high performance liquid chromatography. The largest peaks had the retention times of 5- and 15-HETEs, and the identity of these peaks was confirmed using specific radioimmunoassays. Stimulation with arachidonic acid resulted in a time- and dose-dependent increase in the formation of both HETEs, with 15-HETE being most abundant. The release of both HETEs was markedly reduced in the presence of AA-861, an inhibitor of lipoxygenase, but not with the cyclooxygenase inhibitor indomethacin. These data are thus consistent with our previous suggestion that the contractile activity of arachidonic acid in cerebral arteries arises, at least in part, from HPETE formation and with a possible role for these compounds in cerebral vasospasm.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Benzoquinones; Cattle; Cerebral Arteries; Chromatography, High Pressure Liquid; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Indomethacin; Lipoxygenase Inhibitors; Muscle, Smooth, Vascular; Quinones; Radioimmunoassay

The role of specific products of the lipoxygenase pathway of arachidonic acid metabolism has been investigated in the Friend erythroleukemia cell line, a model system for erythroid cell differentiation. When triggered with agents such as hexamethylene-bis-acetamide, these cells mature as normal erythroid cells. 15-Hydroxyeicosatetraenoic acid (15-HETE) was identified by reverse-phase high-performance liquid chromatography and by radioimmunoassay as the principal lipoxygenase metabolite produced by Friend cells. Its production was significantly lower (903 +/- 73 pg/ml) in stationary-phase cells compared with logarithmic-phase cells (1,496 +/- 24 pg/ml). In addition, inhibitors of both the cyclooxygenase and lipoxygenase pathways (phenidone, BW 755C, caffeic acid, nordihydroguaiaretic acid and BW 4AC) significantly blocked DNA synthesis (P less than 0.05), whereas neither specific inhibitor of the cyclooxygenase pathway (aspirin or sodium meclofenate) blocked DNA synthesis. The addition of 15-hydroperoxyeicosatetraenoic acid as well as 15-HETE to Friend cells produced an increase in DNA synthesis as assessed by [3H]thymidine incorporation in differentiating cells but not in proliferating cells. These data support a role for 15-lipoxygenase products of arachidonic acid metabolism in maintaining DNA synthesis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 15-Lipoxygenase; Arachidonic Acids; Cell Differentiation; Cell Division; Cell Line; Cell Survival; Clone Cells; DNA Replication; Hydroxyeicosatetraenoic Acids; Kinetics; Leukemia, Experimental; Lipoxygenase Inhibitors; Mice; Pyrazoles; Thymidine

A variety of lipoxygenase products such as 12- and 15-hydroxyeicosatetraenoic acid (12- and 15-HETE) inhibit thromboxane A2 (TXA2) mimetic induced human platelet aggregation in a stereoselective manner. The mechanism of this inhibition remains unclear. To determine if this inhibition is due to a receptor level interaction of the lipoxygenase products at the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor, radioligand binding studies were performed using a new [125I]-labelled thromboxane mimetic [125I]BOP. The mono-HETES 5(S), 12(R), 12(S) and 15(S) inhibited binding of the radioligand to the TXA2/PGH2 receptor in washed human platelets with IC50 values of greater than 25, 0.73, 2.06 and 2.0 microM respectively. LTB4 and its positional isomer 5(S), 12(S)-diHETE were less potent with IC50 values greater than 10 microM for LTB4 and 9.38 microM for 5(S), 12(S)-diHETE. Thus, stereoselective inhibition of the binding of the radioligand was demonstrated between 12(R)- and 12(S)-HETE. These lipoxygenase products also inhibited IBOP (10nM) induced platelet aggregation in a concentration dependent fashion with a similar rank order of potency as that obtained in the competition binding assay. These results suggest that, at least in part, the platelet inhibitory properties of these HETEs may be mediated through their interaction at the TXA2/PGH2 receptor.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Blood Platelets; Bridged Bicyclo Compounds; Bridged Bicyclo Compounds, Heterocyclic; Fatty Acids, Unsaturated; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Platelet Aggregation; Platelet Aggregation Inhibitors; Receptors, Prostaglandin; Receptors, Thromboxane

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

The effects of complete ischemia on cerebral arachidonic acid (AA) metabolism were investigated in the isolated perfused rat brain. During 12.5 min of ischemia, AA, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and 15-hydroxy-5,8,11,13-eicosatetraenoic acid increased 129-, 4-, and 10-fold, respectively, while subsequent reperfusion for 30 min resulted in normalized levels independently of the duration of preceding ischemia. Prostaglandin (PG) F2 alpha, PGE2, PGD2, 6-keto-PGF1 alpha, and thromboxane (Tx) B2 remained at preischemic levels during 12.5 min of complete ischemia. However, at the end of subsequent reperfusion for 30 min, the levels of the prostanoids PGF2 alpha, PGE2, PGD2, 6-keto-PGF1 alpha, and TxB2 increased according to the preceding ischemic time. The levels reached a maximum after 7.5 min of ischemia and were elevated by 7-, 14-, 48-, 3-, and 30-fold, respectively. A prolongation of ischemia of up to 12.5 min was not associated with further increases of prostanoids at the end of reperfusion. The mechanisms underlying the metabolism of eicosanoids are discussed in relation to the changes of cortical direct current potential.

Topics: Animals; Arachidonic Acids; Brain Ischemia; Cell Membrane; Electrophysiology; Fatty Acids, Nonesterified; Hydroxyeicosatetraenoic Acids; Male; Prostaglandins; Rats; Rats, Inbred Strains

The role of several lipoxygenase metabolites of arachidonic acid in the action of luteinizing hormone-releasing hormone (LHRH) on ovarian hormone production was investigated. Like LHRH, treatment of rat granulosa cells with 5-HETE, 5-HPETE, 12-HETE, 15-HETE or 15-HPETE stimulated progesterone (P) and prostaglandin E2 (PGE2) production. 12-HEPE was most potent and stimulated P and PGE2 equally well. By contrast, 5-HETE stimulated P better than PGE2, while 15-HETE was a potent stimulator of PGE2 but not of P. Stimulation of P and PGE2 by LHRH or 12-O-tetradecanoylphorbol 13-acetate (TPA) was further augmented by several HETEs and HPETEs. Like protein kinase C, arachidonic acid metabolites appear to mediate the multiple actions of LHRH in the ovary.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Dinoprostone; Drug Interactions; Female; Gonadotropin-Releasing Hormone; Granulosa Cells; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Lipoxygenase; Progesterone; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate

Liver fatty acid binding protein (L-FABP) binds avidly the arachidonic acid metabolites, hydroperoxyeicosatetraenoic acids (HPETEs) and hydroxyeicosatetraenoic acids (HETEs). Binding of 15-[3H]HPETE was specific, saturable, reversible, and rapid. Protein specificity was indicated by the following order: L-FABP greater than bovine serum albumin greater than ovalbumin = beta-lactoglobulin greater than ribonuclease. Ligand specificity was evidenced by the following order of apparent competition: 15-HPETE greater than or equal to 5-HETE greater than or equal to 5-HPETE = oleic acid greater than 12-HETE greater than 12-HPETE greater than or equal to 15-HETE greater than prostaglandin E1 much greater than leukotriene C4 greater than prostaglandin E2 much greater than thromboxane B2 = leukotriene B4. Once bound, 15-HPETE was reversibly displaced. Ligand was recovered from the protein complex and confirmed to be 15-[3H]HPETE by TLC. L-FABP bound HPETE with a dissociation constant of 76 nM,5-HETE at 175 nM, and 15-HETE at 1.8 microM, and the reference fatty acids oleic acid at 1.2 microM and arachidonic acid at 1.7 microM. Thus, the affinity was approximately 16-fold greater for 15-HPETE, and 7-fold higher for 5-HETE, than for oleic acid. The need exists for studies of complexes of L-FABP with the HPETEs and HETEs in hepatocytes, especially since L-FABP has previously been associated with mitosis in normal hepatocytes, and shown to be the target protein of two liver carcinogens, and these arachidonic acid metabolites have been found to be able to modulate activities related to cell growth.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Binding, Competitive; Carrier Proteins; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Leukotrienes; Ligands; Lipid Peroxides; Lipoxygenase; Liver; Neoplasm Proteins; Nerve Tissue Proteins; Oleic Acid; Oleic Acids; Protein Binding; Rats; Structure-Activity Relationship

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

Upon activation, human neutrophils generate 5-lipoxygenase products which are involved in inflammation as well as other physiological and pathophysiological processes. We have examined the influence of red cells on the generation of lipoxygenase-derived products by neutrophils utilizing high pressure liquid chromatography system which permitted quantitation of 5-HETE, leukotriene B4 (and its isomers) and the omega oxidation products of leukotriene B4 (20-hydroxyleukotriene B4, 20-carboxyleukotriene B4) within the same sample. Co-incubation of red cells with neutrophils (50:1, red cells:neutrophils) resulted in a 722 percent increase in 5-hydroxyeicosatetraenoic acid production and a slight increase in leukotriene B4 and its omega oxidation products which were not accompanied by increases in 15-hydroxyeicosatetraenoic acid production. The role of the sulfhydryl status of the red cell and its ability to scavenge hydrogen peroxide were assessed in relationship to the interaction of red cells on the neutrophil-derived lipoxygenase products. Together, these findings indicate that red cells can regulate the levels of lipid-derived mediators produced by neutrophils. Moreover, they suggest that red cell-neutrophil interactions may be of importance in inflammatory reactions.

Topics: Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Erythrocytes; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Neutrophils

The effect of intravenous injections of 5-, 12- and 15-hydroxyeicosatetraenoic acids (HETE), leukotrienes D4 and E4 (LTD4, LTE4) on tracheal mucous gel layer (TMGL) thickness was assessed in rats. When administered in doses ranging from 0.03 pg to 33 ng per rat, the lipoxygenase metabolites produced significant increases in TMGL thickness. The order of potency of the metabolites was 15-HETE greater than 12-HETE greater than or equal to 5-HETE greater than LTD4 greater than or equal to LTE4. Imidazole (31.6 mg/kg), intravenously, significantly decreased this response. These findings suggest that the mono-HETEs, especially 15-HETE, may be important modulators of airway mucus in the rat.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Hydroxyeicosatetraenoic Acids; Imidazoles; In Vitro Techniques; Injections, Intravenous; Leukotriene E4; Leukotrienes; Mucous Membrane; Mucus; Rats; SRS-A; Trachea

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

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

In view of the likely production of monohydroxyeicosatetraenoic acids (HETEs) in bronchial asthma, the role of these lipoxygenase products in the development of a classical clinical element of airway disease, namely airway hyperreactivity, has been investigated. Tracheas removed from guinea-pigs actively sensitized to ovalbumin produced, upon antigenic challenge (0.01 microgram/ml), a 17-fold increase (0.97 +/- 0.34 ng/ml to 16.73 +/- 1.58 ng/ml) in the amount of 5-hydroxyeicosatetraenoic acid (5-HETE) as measured by radioimmunoassay of the tissue-bath fluid, indicating that this tissue is capable of producing 5-HETE. While 5-HETE alone, at concentrations equal to or greater than those found during the above antigenic response (0.001 to 1.0 microM), failed to produce intrinsic contractions of normal, nonsensitized guinea-pig trachea, a 30 min pretreatment with 5-HETE (1.0 microM) enhanced subsequent LTD4-induced contractions. Pretreatment with either 12- or 15-HETE, at similar concentrations and conditions, failed to potentiate LTD4 concentration-response curves. The effect of 5-HETE was time-dependent, since pretreatment for either 15 or 60 min had little or no effect on subsequent LTD4 responses. Also, the 5-HETE-induced enhancement seemed specific for LTD4, since contractions to LTC4 (in the presence of I-serine borate), acetylcholine, histamine, PGD2 or U-46619 were unaffected by 5-HETE. Therefore, 5-HETE may have a role in the development of airway hyperreactivity by interacting with released LTD4 to exacerbate airway smooth muscle contraction in asthma.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Antigens; Drug Synergism; Guinea Pigs; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Male; Muscle Contraction; Ovalbumin; SRS-A; Trachea

The effects of lipoxygenase products (5-, 12-, 15-HETE, LTB4) and superoxide radicals on human colonic (Na+ + K+)-ATPase and specific ouabain binding were measured. No significant inhibition in concentrations up to 3 x 10(-5) M was observed. The results are discussed with regard to a possible role of lipoxygenase products and radicals in the pathogenesis of water and electrolyte disturbances in various diarrheal states including inflammatory bowel disease.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Colon; Free Radicals; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase; Receptors, Drug; Sodium-Potassium-Exchanging ATPase; Superoxides

A high-performance liquid chromatographic method was developed to determine arachidonic 5-lipoxygenase products in calcium ionophore-stimulated neutrophils. This procedure allows the simultaneous measurement of leukotriene B4 (LTB4) and its omega-oxidation products without using a gradient elution system. 20-Carboxy-LTB4, 20-hydroxy-LTB4, 6-trans-LTB4, 12-epi-6-trans-LTB4, LTB4 and 5s,12s-dihydroxyeicosatetraenoic acid can be separated and quantitated by reversed-phase chromatography using isocratic elution. The generation and degradation of 5-lipoxygenase products by human neutrophils following stimulation with calcium ionophore have been examined by this method.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Indicators and Reagents; Isomerism; Leukotriene B4; Neutrophils; Oxidation-Reduction; Spectrophotometry, Ultraviolet

Mouse peritoneal macrophages metabolize dihomogammalinolenic acid (20:3n-6) primarily to 15-hydroxy-8,11,13-eicosatrienoic acid (15-OH-20:3). Since the biological properties of this novel trienoic eicosanoid remain poorly defined, the effects of increasing concentrations of 15-OH-20:3 and its arachidonic acid (20:4n-6) derived analogue. 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE), on mouse macrophage 20:4n-6 metabolism were investigated. Resident peritoneal macrophages were prelabeled with [3H]-20:4n-6 and subsequently stimulated with zymosan in the presence of either 15-OH-20:3 or 15-HETE (1-30 microM). After 1 hr, the radiolabeled soluble metabolites were analyzed by reverse phase high performance liquid chromatography. 15-OH-20:3 inhibited zymosan-induced leukotriene C4 (IC50 = 2.4 microM) and 5-HETE (IC50 = 3.1 microM) synthesis. In contrast to the inhibition of macrophage 5-lipoxygenase, 15-OH-20:3 enhanced 12-HETE synthesis (5-30 microM) and had no measurable effect on cyclooxygenase metabolism (1-10 microM) i.e., 6-keto-prostaglandin F1 alpha and prostaglandin E2 synthesis. Addition of exogenous 15-HETE produced similar effects. These results suggest that the manipulation of macrophage 15-OH-20:3n-6 levels may provide a measure of cellular control over 20:4n-6 metabolism, specifically, leukotriene production.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 8,11,14-Eicosatrienoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Fatty Acids, Unsaturated; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Macrophages; Mice; SRS-A

It has previously been reported that rat embryonic tissue produces various prostanoids. This report demonstrates that rat embryo homogenates synthesized various lipoxygenase metabolites, including 12-hydroxyeicosatetraenoic acid (12-HETE) as the major metabolite, 5-HETE, and 15-HETE. The cyclooxygenase product 11-HETE was also formed. Product identification was based on radioimmunoassay and comparison of reverse-phase- and straight-phase-high-pressure liquid chromatography retention times with authentic standards. Additional evidence was the observation that the lipoxygenase inhibitor nordihydroguaiaretic acid inhibited HETE formation. It appears that, under the same (though not necessarily optimal) experimental conditions, lipoxygenase metabolites predominate quantitatively over cyclooxygenase pathway products and that 11-day embryonic tissue produces more HETEs than either 12-day or 13-day embryo homogenates.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Embryo, Mammalian; Gestational Age; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Lipoxygenase Inhibitors; Masoprocol; Radioimmunoassay; Rats; Rats, Inbred Strains

The rate-limiting step of the lipoxygenase reaction involves the abstraction of a hydrogen from the methylene carbon of a 1,4-diene. One possibility for the mechanism of the enzyme is the abstraction of this hydrogen as a proton to generate a carbanionic intermediate or transition state. In order to investigate this possibility, 5-, 8-, 12-, and 15-hydroxy-eicosatetraenoic acid were oxidized to the corresponding ketones and these ketones were assayed as substrates of the 5-, 12-, and 15-lipoxygenases from rat neutrophils, rat platelets, and soybeans, respectively. The ketones were in no case better substrates than arachidonic acid and in some cases the hydroxyeicosatetraenoic acids were equally active as the corresponding ketones. Since no increased rate of oxidation for these electrophilic substrates was observed, it is concluded that no transition state with carbanionic character is generated in the rate-determining step of the lipoxygenase reaction.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Hydroxyeicosatetraenoic Acids; Ketones; Kinetics; Lipoxygenase; Rats

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

Cultured murine cerebromicrovascular endothelial cells were employed to study the metabolism of 12-hydroxyeicosatetraenoic acid (12-HETE) in an in vitro model of the blood-brain barrier. These endothelial cells convert 12-HETE to at least four, more polar compounds. Analysis of the least polar and predominant metabolite by gas chromatography combined with chemical ionization and electron impact mass spectrometry of reduced and nonreduced derivatives indicate that the compound is 8-hydroxyhexadecatrienoic acid (8-HHDTrE). The uptake of 12-HETE into cell phospholipids peaks at 2 hr, and is not saturable up to the highest concentration tested, 5 microM. Seventy-five to 92% of this 12-HETE is incorporated into phosphatidylcholine, while the remainder is divided between the inositol and ethanolamine phospholipids. Incorporation into neutral lipids is slower, with radioactivity gradually accumulating in triglycerides over 24 hr. Saponification of cell lipids demonstrated that not only 12-HETE, but also its major metabolite, 8-HHDTrE, is incorporated into the cell lipids. Prostacyclin and prostaglandin E2 production by the cerebral endothelial cells is inhibited by up to 56% with 1 microM and 90% with 5 microM 12-HETE. These data demonstrate that 12-HETE is actively metabolized by cerebral endothelium and suggest at least two mechanisms through which 12-HETE may alter cerebromicrovascular function: 1) incorporation into cerebral endothelial membranes and 2) inhibition of cerebral endothelial prostaglandin production. Conversion of 12-HETE to more polar compounds, particularly 8-HHDTrE, may be interpreted as either the inactivation of 12-HETE or the production of additional, biological mediators.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Blood-Brain Barrier; Brain; Cells, Cultured; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Endothelium, Vascular; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Lipid Metabolism; Lipids; Mice; Microcirculation; Radioimmunoassay

Hydroxyeicosatetraenoic acids (HETE) are major arachidonic acid metabolites of a number of cells found in blood and blood vessels. These products have been implicated in physiologic responses as diverse as platelet aggregation, cell migration, and cell proliferation. Using a sensitive and specific assay, GC/selected ion monitoring after high-performance liquid chromatography separation, we have measured the levels of three HETE isomers of biologic significance 12-HETE, 15-HETE, and 5-HETE in plasma, serum and stimulated serum (formed in the presence of arachidonic acid and calcium ionophore), obtained from normal adults and cord blood from normal neonates. Whereas there were no significant differences between the two groups for 5- or 15-HETE in any of the samples, stimulated serum from adults produced 12 times as much 12-HETE when compared to cord blood. When platelets were isolated from adult and cord blood, 12-HETE production by neonatal platelets, stimulated with 10 microM arachidonic acid, was less than one-fourth that of adults. Although no role for 12-HETE in normal platelet responses has yet been established, it has been reported that those individuals with myeloproliferative syndromes who demonstrate a concomitant decrease in platelet 12-HETE synthetic ability have an increased bleeding tendency. It needs to be further evaluated if this already depressed level of 12-lipoxygenase in neonatal platelets may contribute to pathologic bleeding in those infants subjected to additional stress (such as prematurity or birth asphyxia).

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Female; Fetal Blood; Humans; Hydroxyeicosatetraenoic Acids; Infant, Newborn; Isomerism; Male

Linoleate metabolism via the cyclooxygenase pathway enhances the proliferation of mammary epithelial cells in serum-free culture in the presence of epidermal growth factor and insulin (Bandyopadhyay, G.K., Imagawa, W., Wallace, D., and Nandi, S. (1987) J. Biol. Chem. 262, 2750-2756). Prostaglandin E2 (PGE2) can fully substitute for linoleic acid provided endogenous hydroxyeicosatetraenoic acids (HETEs, lipoxygenase metabolites) are available. The PGE2 effect is partial if lipoxygenase activity is inhibited by nordihydroguaiaretic acid. Any combination of two HETEs out of three tested (5-, 12-, and 15-HETEs) stimulates growth synergistically with PGE2; and together (i.e. PGE2 + HETEs), they completely substitute for linoleate. In the absence of PGE2, maximal stimulation cannot be attained with HETEs. Exogenous 5-HETE, compared with 12- or 15-HETE, is preferentially incorporated by the mammary epithelial cells, and about 25-30% of it is retained esterified in phospholipids. The cellular level of nonesterified, free HETE is low. Radioimmunoassay revealed that the concentrations of 12- and 15-HETEs in the culture media (with or without added linoleate) were always higher than that of 5-HETE. Both intra- and extracellular free HETEs are rapidly metabolized by the cells. Since these cells are capable of producing eicosanoids from linoleate, periodic supplementation of the cultures with linoleate allows maintenance of higher HETE and PGE2 levels. Thus, it appears that not only are HETEs short-lived in the cell cultures, but cells handle 5-HETE differently than 12- and 15-HETEs. Whatever may be the pathways of interaction, synergism between HETEs and PGE2 seems to explain how linoleate stimulates the growth of mammary epithelial cells in the presence of epidermal growth factor and insulin.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Cell Division; Cells, Cultured; Dinoprostone; Drug Synergism; Epidermal Growth Factor; Epithelium; Hydroxyeicosatetraenoic Acids; Insulin; Linoleic Acid; Linoleic Acids; Mammary Glands, Animal; Mice; Mice, Inbred BALB C; Prostaglandins E

5-, 15-, and 12-HETE (monohydroxyeicosatetraenoic acids) are products of the lipoxygenation of arachidonic acid. We investigated their role as possible mediators of pulmonary vasoactivity and pulmonary edema. Pulmonary artery pressure (Ppa), capillary pressure (Pcap), the change in lung wet weight (delta wt) from baseline, and capillary filtration coefficient (Kf) (as a measure of vascular permeability) were determined following an intravenous injection of each mono-HETE in lungs perfused at constant flow with either a phosphate-buffered Ringer's-albumin solution (PBR) or diluted blood. Injection of 2 micrograms of each compound into the pulmonary artery of lungs perfused with either PBR or diluted blood did not produce any effect. However, in PBR-perfused lungs, 4 micrograms 15-HETE induced increases in Ppa, Pcap, and lung wet weight (p less than 0.05), which were greater than the increases observed after 4 micrograms 5-HETE. Kf increased following both 5- and 15-HETE. The pulmonary vasoconstrictor and edemagenic responses were attenuated by increasing perfusate albumin concentration from 0.5 to 1.5 g%. In contrast, 12-HETE (4 micrograms) had no effect on these parameters. In blood-perfused lungs, the pulmonary vascular responses to all HETE compounds (4 micrograms) were attenuated. In both Ringer's-albumin-perfused and blood-perfused lungs, the relative magnitude of the hemodynamic and fluid filtration responses to each mono-HETE were as follows: 15-HETE greater than 5-HETE greater than 12-HETE. In conclusion, the pulmonary vasoconstrictor and edemagenic effects of 5- and 15-HETE occur independently of blood-formed elements. 15-HETE causes greater pulmonary vasoconstriction and edema than 5-HETE. Both 5- and 15-HETE induce pulmonary edema, probably as a result of increased lung vascular permeability. The results indicate that 5- and 15-HETE are potent pulmonary inflammatory mediators.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Pulmonary Circulation; Pulmonary Edema; Vasoconstriction

Some studies report that endothelial cells preferentially take up the lipoxygenase-derived arachidonic acid metabolite, 5-hydroxyeicosatetraenoic acid (5-HETE), released from stimulated leukocytes (polymorphonuclear leukocytes, PMNs), whereas others report that endothelial cells preferentially take up 12-HETE released from platelets. The biological relevance of these observations, however, is unknown. Recently, we and others have found that, under basal conditions, endothelial cells, PMNs and tumor cells metabolize linoleic acid via the lipoxygenase enzyme to 13-hydroxyoctadecadienoic acid (13-HODE). We propose that endogenous levels of these metabolites regulate blood-vessel wall cell adhesion. In this study, we have measured (1) the relative binding of 5-, 12- and 15-HETE, and 13-HODE to endothelial cell monolayers, and (2) their effects on endothelial cell adhesivity with platelets, PMNs and tumor cells. There was a dose-related and specific binding of 5-[3H]HETE to endothelial cells but no binding of 12- or 15-HETE or 13-HODE. Platelet or PMN adhesion to endothelial cells was unaffected by the 5-HETE binding, but tumor cell adhesion was blocked by 40% (P less than 0.01). Interestingly, preincubation of endothelial cells with 13-HODE, 12-HETE or 15-HETE decreased platelet adhesion to endothelial cells (P less than 0.05), even though these metabolites did not bind to the endothelial cells. We conclude that 5-HETE preferentially binds to endothelial cells and interferes with a specific receptor for tumor cells, whereas the other metabolites neither bind to cells nor affect cell adhesion.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Cell Adhesion; Endothelium, Vascular; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acids; Neoplasms; Neutrophils; Platelet Adhesiveness; Tumor Cells, Cultured

Ten frogs (Xenopus laevis) were injected with mixed bacteria to produce a septic peritonitis. Peritoneal inflammatory cells of eight animals were studied for monohydroxyeicosanoid and leukotriene production from exogenous arachidonic acid. Large amounts of 12-hydroxyeicosatetraenoic acid were produced; smaller amounts of 5- and 15-hydroxyeicosatetraenoic and leukotriene B4 were produced. Identifications were confirmed by retention times on HPLC, ultraviolet spectroscopy on all products, and gas chromatograph/mass spectrometry in the case of 12-hydroxyeicosatetraenoic acid.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Chromatography, High Pressure Liquid; Female; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Male; Peritoneal Cavity; Peritonitis; Xenopus laevis

Luteinizing hormone and follicle stimulating hormone secretion was stimulated by 4 min pulses of arachidonic acid (3 X 10(-5) to 10(-4)M) in superfused rat pituitary cells. The effect of its lipoxygenase metabolites, 5-hydroxy-6,8,11,14-eicosatetranoic acid (5-HETE) and 15-hydroxy-5,8,10,14-eicosatetranoic acid (15-HETE) was more potent on hormone release when added in the same dose. Using 3 X 10(-5)M 5-HETE, its releasing activity on gonadotropins was comparable to that of GnRH (10(-9)M). 15-HETE (3 X 10(-5)M) was even more potent on LH and FSH secretion than 5-HETE. The secretory profile induced by 5-HETE and 15-HETE was also similar to that shown for GnRH, resulting in a rapid increase and a more prolonged decline of the hormone release. The addition of these fatty acids to superfused pituitary cells did not alter the response of the cells to their physiological ligand. These findings give further support to the proposal that metabolites of arachidonic acid may be involved in receptor-mediated mechanisms of gonadotropin release in pituitary cells.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Female; Follicle Stimulating Hormone; Hydroxyeicosatetraenoic Acids; Kinetics; Lipoxygenase; Luteinizing Hormone; Perfusion; Pituitary Gland, Anterior; Rats; Rats, Inbred Strains

The direct effect of leukotrienes and other lipoxygenase products on prolactin release has been assessed. Arachidonic acid and its lipoxygenase metabolites 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 15-hydroxy-5,8,10,14-eicosatetranoic acid (15-HETE) stimulated the release of prolactin in superfused rat pituitary cells in a dose-dependent manner. Leukotrienes (LT) A4, B4, C4 and E4 provoked a very marked biphasic and dose-dependent secretion of prolactin from superfused cells. Maximal effects were achieved with leukotrienes at a concentration of 3 X 10(-11) to 3 X 10(-10) M but LTD4 did not affect peptide release under these conditions. The metabolites were more potent than arachidonic acid in affecting hormone secretion. Pulses of 4 minutes duration of these fatty acids may even elicit a more pronounced response than thyrotrophin-releasing hormone (TRH). Nordihydroguaiaretic acid (NDGA 10(-6) M), a lipoxygenase inhibitor, prevented the effect of arachidonic acid on peptide secretion. Repeated TRH (10(-7) M) administration to pituitary cells led to a reduction in cell response, which may also be observed in cells pre-treated with pulsatile 5-HETE or 15-HETE. These data support previous findings that arachidonic acid and its lipoxygenase metabolites may play a role in the secretory mechanism of prolactin release in pituitary cells.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Dose-Response Relationship, Drug; Female; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Masoprocol; Pituitary Gland; Prolactin; Rats; Rats, Inbred Strains; Thyrotropin-Releasing Hormone

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

A stable isotope dilution gas chromatography-negative ion chemical ionization-mass spectrometry assay for simultaneous quantitative measurement of 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, 15-HETE, and 12-hydroxyheptadecatrienoic acid in one single GC/MS run was established. 18O2-Labeled analogs as internal standards, prepared according to conventional procedures, were found to be useful for this application. A sample processing and derivatization sequence providing highly purified compounds with a recovery of 42.7% was elaborated. The detection limit was in the femtomole range.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Indicator Dilution Techniques; Isotope Labeling; Oxygen Isotopes

Selected ion monitoring of mass fragments of peaks from capillary gas chromatograms permits a sensitive and selective analysis of positional isomers of hydroxy-eicosanoids (as their methyl ester, trimethylsilyl ether derivatives). Because deuterated analogs of these HETEs are not readily available, stable isotope dilution cannot be easily performed. We have developed a method for the quantitation of HETEs using ricinoleic acid (12-hydroxy-oleic acid) as an internal standard. Ricinoleic acid is monitored at m/z 187, while 5-, 12-, and 15-HETEs (those commonly found in biological systems) are monitored at m/z 255, 295 and 225 respectively. The procedure has proven linear for levels from 0.03 to greater than 3.0 nmol HETE per nmol standard. There is however significant "crossover" among the HETEs, requiring preliminary resolution of the various isomers by reverse phase HPLC in samples containing more than one HETE. Recovery of authentic standards added to various blood samples through the extraction, resolution and analyses averaged over 90%. Using this procedure, mean plasma concentrations were found to be less than 0.1 microM for all HETEs tested, while serum levels were approximately 0.3, 0.8 and 0.1 microM for 5-, 12-, and 15-HETE respectively. Serum formed in the presence of 2mM arachidonic acid plus 25 microM A23187, to maximize HETE production, contained an average of 4, 22 and 1 microM 5-, 12- and 15-HETE respectively. Thus while normal human plasma contains sufficient quantities of the various HETEs to produce some of the biological effects attributed to these metabolites, there is potential for an even greater production when sufficient substrate is available.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Chromatography, Gas; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Mass Spectrometry; Ricinoleic Acids; Spectrophotometry, Ultraviolet

The gas chromatographic and mass spectrometric properties of the monohydroxy acids 5-hydroxyeicosatetraenoic acid (5-HETE), 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) as their methyl ester trimethylsilyl, methyl ester allyldimethylsilyl and methyl ester tert.-butyldimethylsilyl ethers were investigated. The gas chromatographic properties of the trimethylsilyl and tert.-butyldimethylsilyl derivatives were found to be excellent while the allyldimethylsilyl derivative required a well deactivated column. The mass spectra of these silyl derivatives with the exception for 12-HETE did not exhibit particularly intense ions in the upper mass region. A quantitative analysis by selected-ion monitoring of the most intense ion in the upper mass region of respective mass spectrum demonstrated that a detection limit in the low picogram range could only be obtained for 12-HETE. Since the mass spectra indicated that the double bonds exerted a strong influence on the fragmentation pattern, the trimethylsilyl, allyldimethylsilyl and tert.-butyldimethylsilyl ethers of the methyl esters of the reduced analogues of the monohydroxy acids were prepared. The saturation of the double bonds completely altered the fragmentation patterns and very intense ions carrying a high percentage of the total ion abundance were found in all of the mass spectra. The developed technique was utilized for measurements of 5-HETE in lung tissue samples from patients with lung cancer.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Allyl Compounds; Catalysis; Chromatography, Gas; Esters; Gas Chromatography-Mass Spectrometry; Humans; Hydrogenation; Hydroxyeicosatetraenoic Acids; Lung; Organosilicon Compounds; Silicon; Trimethylsilyl Compounds

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Diglycerides; Female; Guanylate Cyclase; Hydroxyeicosatetraenoic Acids; Interferon-gamma; Lymphocytes; Mice; Prostaglandins E; SRS-A; Structure-Activity Relationship

Ketoconazole inhibits in vitro (IC50:2.6 X 10(-5) M) the formation of 5-HETE and LTB4 by isolated, carrageenin-elicited rat peritoneal PMN leukocytes, challenged with the Ca2+-ionophore A23187 in the presence of [14C]-arachidonic acid ([14C]-AA). The relative potency of various compounds tested in this respect is NDGA greater than nafazatrom greater than phenidone greater than ketoconazole greater than BW 755C. In contrast to the other compounds studies, ketoconazole in vitro, up to 1 X 10(-4) M, has no effect on the fatty acid cyclo-oxygenase or the 12-lipoxygenase-mediated metabolism of [14C]-AA by isolated human platelets; however, it stimulates the 15-lipoxygenase activity in phenylhydrazine-induced rabbit reticulocytes. After oral administration (10-40 mg/kg, -2 hr), ketoconazole inhibits in a dose-dependent way, the leukotriene-mediated anaphylactic bronchoconstriction in guinea pigs. This study demonstrates that ketoconazole is a comparatively specific and orally active inhibitor of the 5-lipoxygenase activity bearing on the production of leukotrienes derived from arachidonic acid.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Animals; Antigens; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Bronchi; Catechols; Cyclooxygenase Inhibitors; Cytochrome P-450 Enzyme Inhibitors; Dose-Response Relationship, Drug; Guinea Pigs; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Ketoconazole; Leukotriene B4; Lipoxygenase Inhibitors; Male; Masoprocol; Neutrophils; Pyrazoles; Rabbits; Rats; Rats, Inbred Strains; Reticulocytes; SRS-A

The data on whether T cells produce leukotrienes or other 5-lipoxygenase metabolites of arachidonic acid is conflicting. We report that exogenous arachidonic acid added to phytohemagglutin-stimulated human T cells profoundly inhibits leukotriene B4 production, with 90% inhibition caused by 10(-6) M arachidonic acid. The 12- and 15-lipoxygenase pathways were also inhibited by arachidonic acid. Recent reports that human T cells produce no 5-lipoxygenase metabolites of arachidonic acid might be explained by the fact that the studies used greater than or equal to 10(-5)M arachidonic acid in the incubation media.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Lipoxygenase; Lipoxygenase Inhibitors; Lymphocyte Activation; Phytohemagglutinins; T-Lymphocytes

Liquid chromatography methods for quantitation of leukotrienes and HETEs (hydroxyeicosatetraenoic acids) in biological samples are described. Extraction is accomplished by acetonitrile precipitation of proteins followed by selective acetonitrile elution from a solid-phase C18 extraction cartridge. Isocratic elution of extracts from short reverse-phase columns with 3 microns C18-bonded silica results in elution of all components of interest in less than 10 min. The addition of the mobile-phase additives, trifluoroacetic acid and triethylamine, enable the peptido-leukotrienes to be eluted with excellent peak shape and unique elution times. As little as 1 pmol of each metabolite can be detected by uv spectrophotometry with a wavelength change from 280 to 235 nm midway through the chromatographic run. This method is demonstrated by the extraction and analysis of leukotriene and HETE standards from human serum.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Chromatography, High Pressure Liquid; Ethylamines; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase; SRS-A; Trifluoroacetic Acid

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

The profile of cyclooxygenase and lipoxygenase products in normal rat colonic epithelium and subepithelium was examined. Colons were thoroughly perfused to eliminate contamination with blood. Two preparations of colonic epithelium were employed. The first consisted of intact colonic crypts and epithelial sheets. The second yielded single cell suspensions of superficial versus proliferative epithelial cells. Lipoxygenase product formation by colonic epithelium as measured by hydroxyeicosatetraenoic acid (HETE) and leukotriene B4 (LTB4) production (5-HETE greater than 12-HETE greater than 15-HETE greater than LTB4) accounted for 58% of the total colonic production of these moieties, whereas epithelium accounted for only 20% of total colonic protein. By contrast, prostaglandin (PG) E2 and PGF2 alpha production occurred predominantly (greater than 97%) in the subepithelial layers. The present studies also demonstrate markedly higher levels of accumulation of lipoxygenase products in proliferative versus superficial epithelial cells, whereas prostaglandin accumulation was greater in superficial cells. Previous studies have supported a role for lipoxygenase and cyclooxygenase products in the control of colonic secretion, inflammatory cell infiltration and proliferative activity. The present results raise the possibility that the striking differences in the sites of production of these products within the colon has functional implications.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Colon; Dinoprost; Dinoprostone; Eicosanoic Acids; Epithelium; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase; Prostaglandin-Endoperoxide Synthases; Prostaglandins E; Prostaglandins F; Radioimmunoassay; Rats

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase Inhibitors; Naphthalenes; Psoriasis; Skin

The purpose of this study was to investigate the cellular composition of peritoneal fluid during post-surgical re-epithelialization and to determine the metabolism of arachidonic acid by these cells. Rabbits underwent a midline laparotomy followed by abrasion of the broad ligament. The presence of adhesions was graded and the peritoneal exudative cells collected up to 14 days thereafter. Ascitic fluid and cells were separated by centrifugation and the cellular percipitate incubated with [14C]arachidonic acid. The aliquot was separated by silica Gel G thin-layer chromatography and the specific radioactivity of each strip determined. To evaluate the reformation of adhesions 14 days after the first abrasion, rabbits underwent reabrasion of the same area and the pattern of arachidonic acid metabolism by the ascites cells was similarly evaluated. Six hours after the abrasion, PMNs comprised 87.5% of the peritoneal exudative cells (total 0.03 X 10(7) cells/rabbit). On Day 3, the total cell number increased to 2.92 X 10(7), 97.6% of which were large mononuclear cells. No significant change in the type of distribution of adhesions was evident from 6 hr through Day 2. After Day 7, the total number of adhesions was minimal; however, those that were present were primarily severe. The second-look evaluation of adhesion formation was not found to be consistent prior to the 7th postoperative day, since many filmy bands present prior to that time were not present later. The in vitro formation of 5-HETE by these cells increased from 6 hr through Day 11. Production of di-HETE increased beginning Day 3 and maintained high steady state levels thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Arachidonic Acids; Ascitic Fluid; Dinoprostone; Female; Hydroxyeicosatetraenoic Acids; Kinetics; Leukocytes; Peritoneum; Prostaglandins E; Rabbits; Thromboxane B2; Wound Healing

12-Hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase product released by activated platelets and macrophages, reduced prostacyclin (PGI2) formation in bovine aortic endothelial cultures by as much as 70%. Maximal inhibition required 1 to 2 h to occur and after 2 hr, a concentration of 1 microM 12-HETE produced 80% of the maximum inhibitory effect. 5-HETE and 15-HETE also inhibited PGI2 formation. The inhibition was not specific for PGI2; 12-HETE reduced the formation of all of the radioactive eicosanoids synthesized from [1-14C]arachidonic acid by human umbilical vein endothelial cultures. Inhibition occurred in the human cultures when PGI2 formation was elicited with arachidonic acid, ionophore A23187 or thrombin. These findings suggest that prolonged exposure to HETEs may compromise the antithrombotic and vasodilator properties of the endothelium by reducing its capacity to produce eicosanoids, including PGI2.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Aorta; Cattle; Cells, Cultured; Dose-Response Relationship, Drug; Endothelium; Epoprostenol; Humans; Hydroxyeicosatetraenoic Acids; Time Factors; Umbilical Veins

Lipoxygenase metabolites of arachidonic acid were shown to stimulate gonadotrophin release dose-dependently in rat pituitary cells. The secretory activity of the arachidonate metabolite leukotriene C4 (LTC4) was biphasic and 10-fold more potent than that of the physiological stimulus gonadotrophin-releasing hormone (GnRH). In pre-labelled, superfused pituitary cells, GnRH dose-dependently enhanced the release of [3H]arachidonic acid, which occurred simultaneously with the secretion of luteinizing hormone (LH). When cells were pre-treated with GnRH for 24 h no response to a further stimulus by GnRH (10(-7) M) could be observed for either [3H]arachidonate nor LH, demonstrating that also in desensitized cells these two mechanisms react similarly. In addition, a GnRH antagonist did not affect the release of arachidonate or LH. These results suggest that arachidonic acid may be involved in the mechanism of GnRH action on gonadotrophins via its lipoxygenase metabolites and LTC4 could act as a very potent intracellular stimulus of LH secretion.

Topics: Animals; Female; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Luteinizing Hormone; Pituitary Gland, Anterior; Pituitary Hormone-Releasing Hormones; Rats; SRS-A

Topics: Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Endothelium; Epoprostenol; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; SRS-A

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

Cercariae of Schistosoma mansoni are stimulated to penetrate skin by certain free fatty acids. The cercariae have an active arachidonate cascade, presumably using host skin essential fatty acids as cascade precursors. Exposing cercariae to 3.3 mM linoleate for 1, 10, and 60 min resulted in production of a wide variety of eicosanoids. Using high-performance liquid chromatography, eicosanoids coeluting with prostaglandin E2, D2, and A2, leukotriene B4, and 5-hydroxyeicosatetraenoic acid standards were identified, as well as unidentified peak positions. Radioimmunoassay confirmed the presence of immunoreactive prostaglandin E1, and E2, and 5- and 15-hydroxyeicosatetraenoic acids in cercarial extracts. No eicosanoid production occurred when cercariae were exposed to 3.3 mM oleate and 1 or 330 microM linoleate. Both high-performance liquid chromatography and radioimmunoassay data indicated that cercariae regulate the production of eicosanoids through time. It is postulated that arachidonate metabolism and subsequent eicosanoid production are required for successful cercarial penetration.

Topics: Alprostadil; Animals; Arachidonic Acid; Arachidonic Acids; Dinoprostone; Hydroxyeicosatetraenoic Acids; Kinetics; Linoleic Acid; Linoleic Acids; Oleic Acid; Oleic Acids; Prostaglandins E; Schistosoma mansoni

Isolated pancreatic islets from the rat have been demonstrated by stable isotope dilution-mass spectrometric methods to synthesize the 12-lipoxygenase product 12-hydroxyeicosatetraenoic acid (12-HETE) in amounts of 1.7 to 2.8 ng per 10(3) islets. No detectable amounts of 5-HETE and only trace amounts of 15-HETE could be demonstrated by these methods. Nordihydroguaiaretic acid (NDGA) and BW755C have been demonstrated to inhibit islet 12-HETE synthesis and also to inhibit glucose-induced insulin secretion. Inhibition of insulin secretion and of 12-HETE synthesis exhibited similar dependence on the concentration of these compounds. Eicosa-5,8,11,14-tetrynoic acid (ETYA) also inhibited glucose-induced insulin secretion, as previously reported, at concentrations which inhibit islet 12-HETE synthesis. Exogenous 12-HETE partially reversed the suppression of glucose-induced insulin secretion by lipoxygenase inhibitors, but exogenous 12-hydroperoxyeicosatetraenoic acid (12-HPETE), 15-HPETE, 5-HPETE, 15-HETE, or 5-HETE did not reverse this suppression. These observations argue against the recently suggested hypothesis that islet synthesis of 5-HETE modulates insulin secretion. Suppression of glucose-induced insulin secretion by ETYA, BW755C and NDGA may be due to inhibition of the islet 12-lipoxygenase by these compounds. The possibility that other processes involved in glucose-induced insulin secretion are inhibited by ETYA, BW755C and NDGA cannot yet be excluded.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Insulin; Insulin Secretion; Islets of Langerhans; Leukotrienes; Lipoxygenase; Male; Pyrazoles; Rats; Rats, Inbred Strains

The effects of 5-, 5-lactone, 12- and 15-hydroxyeicosatetraenoic acids (HETEs) on the synthesis of leukotriene C4 (LTC4), thromboxane B2 (TXB2) and prostaglandin E2 (PGE2) by mouse resident peritoneal macrophages incubated with zymosan particles (100 micrograms/ml) were investigated. Zymosan phagocytosis stimulated a 110-, 16-, and 16-fold increase in LTC4, TXB2 and PGE2 synthesis, respectively. 15-HETE inhibited zymosan-induced LTC4 (IC50 = 1.1 microM) and TXB2 (IC50 = 38.9 microM) synthesis; in contrast, 15-HETE induced a consistent but variable enhancement of PGE2 synthesis. 5-HETE (IC50 = 15 microM), 5-lactone HETE (IC50 = 10.4 microM) and 12-HETE (IC50 = 13 microM) also inhibited LTC4 synthesis but they were approximately an order of magnitude less potent than 15-HETE. Furthermore, 5-HETE, 5-lactone HETE and 12-HETE inhibited TXB2 (IC50 = 20.4, 16.9 and 11.8 microM, respectively) and PGE2 (IC50 = 38.6, 2.3 and 11.6 microM, respectively) synthesis. Thus, monoHETEs exert modulatory actions on arachidonic acid metabolism and the different isomers of HETE differ quantitatively and qualitatively in their actions.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Dinoprostone; Female; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Macrophages; Mice; Prostaglandins E; SRS-A; Thromboxane B2; Zymosan

The cytosolic fraction of human polymorphonuclear leukocytes precipitated with 60% ammonium sulfate produced 5-lipoxygenase products from [14C]arachidonic acid and omega-6 lipoxygenase products from both [14C]linoleic acid and, to a lesser extent, [14C]- and [3H]arachidonic acid. The arachidonyl 5-lipoxygenase products 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) derived from [14C]arachidonic acid, and the omega-6 lipoxygenase products 13-hydroperoxy-9,11-octadecadienoic acid (13-OOH linoleic acid) and 13-hydroxy-9,11-octadecadienoic acid (13-OH linoleic acid) derived from [14C]linoleic acid and 15-hydroxyperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE), and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) derived from [14C]- and [3H]arachidonic acid were identified by TLC-autoradiography and by reverse-phase high-performance liquid chromatography (RP-HPLC). Products were quantitated by counting samples that had been scraped from replicate TLC plates and by determination of the integrated optical density during RP-HPLC. The arachidonyl 5-lipoxygenase had a pH optimum of 7.5 and was 50% maximally active at a Ca2+ concentration of 0.05 mM; the Km for production of 5-HPETE/5-HETE from arachidonic acid was 12.2 +/- 4.5 microM (mean +/- S.D., n = 3), and the Vmax was 2.8 +/- 0.9 nmol/min X mg protein (mean +/- S.D., n = 3). The omega-6 linoleic lipoxygenase had a pH optimum of 6.5 and was 50% maximally active at a Ca2+ concentration of 0.1 mM in the presence of 5 mM EGTA. When the arachidonyl 5-lipoxygenase and the omega-6 lipoxygenase were separated by DEAE-Sephadex ion exchange chromatography, the omega-6 lipoxygenase exhibited a Km of 77.2 microM and a Vmax of 9.5 nmol/min X mg protein (mean, n = 2) for conversion of linoleic acid to 13-OOH/13-OH linoleic acid and a Km of 63.1 microM and a Vmax of 5.3 nmol/min X mg protein (mean, n = 2) for formation of 15-HPETE/15-HETE from arachidonic acid.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Calcium; Chromatography, Ion Exchange; Humans; Hydrogen-Ion Concentration; Hydroxyeicosatetraenoic Acids; Isoenzymes; Kinetics; Lipoxygenase; Neutrophils; Structure-Activity Relationship; Substrate Specificity

It was shown that 15-hydroxyeicosatetraenoic acid (15-HETE) but not 15-H(P)ETE or 5-HETE is a potent agonist for secretion of glycoprotein-containing mucus from the in vivo canine trachea. Given by aerosol into the lungs or by intra-arterial injection into the trachea, 15 HETE also caused the chemotaxis of inflammatory cells into the lumen of the airways. Accompanying this inflammatory cell infiltrate was an increase (183%, p less than 0.05) of expiration of fluid in the partially saturated air coming from the lung. The levels of 15-HETE extracted from tracheal mucus correlated well with hillocks and weight of secreted mucus found in the mucus after hypoxia or after arachidonic acid loading. Indomethacin and atropine blocked the mucus secretagogue effect of 15-HETE in the trachea. Indomethacin and U-52, 412 (a 15-lipoxygenase inhibitor) pretreatment abolished a portion of the 15-HETE-induced enhancement of mucus weight and 15-HETE level in the secretion.

Topics: Aerosols; Animals; Arachidonic Acid; Arachidonic Acids; Body Fluids; Cell Movement; Chemotaxis; Dogs; Hydroxyeicosatetraenoic Acids; Hypoxia; Inflammation; Leukotrienes; Lipid Peroxides; Lipoxygenase Inhibitors; Mucus; Respiration; Respiratory System; Respiratory Tract Diseases; Trachea

Several investigations have suggested that products of arachidonic acid metabolism have modulatory effects on the development of cellular immunity. In this report we have studied the role of arachidonic acid metabolism in the specific effects of interleukin 1 (IL 1) induction of interleukin 2 (IL 2), and also IL 2 stimulation of proliferation and interferon-gamma (IFN-gamma) production. Utilizing cell lines that are specifically responsive to IL 1 or IL 2, it was found that both interleukins stimulate lipoxygenation of arachidonic acid in their respective target cell. The ability of each interleukin to induce monohydroxyeicosatetraenoic acid (HETE) correlated with the induction of secondary lymphokine secretion. Utilizing selective and partially selective pharmacologic inhibitors of arachidonic acid metabolism, the data suggest that the participation of lipoxygenase activity is required for both IL 1 induction of IL 2 production and IL 2 regulation of proliferation and IFN-gamma secretion. The same requirement for lipoxygenase activity was seen when phorbol myristate acetate (PMA) was used as a secretory stimulant, suggesting a similar mode of action for stimulation-secretory activity between PMA and interleukins. Studies performed with an endogenous inhibitor of 5-lipoxygenase (15-HETE) demonstrated the requirement of this enzyme system for IL 2-dependent proliferation and IFN-gamma production. Although leukotrienes could replace IL 2 for IFN-gamma secretion, they had no effect on IL 2 growth promotion. The results suggest that both IL 1 and IL 2, and PMA, may share the lipoxygenase pathway of arachidonic acid metabolism which is a component of the intracellular signal transduction process that regulates secretory activity and/or cellular proliferation.

Topics: Animals; Arachidonic Acid; Arachidonic Acids; Cell Line; Humans; Hydroxyeicosatetraenoic Acids; Indomethacin; Interleukin-1; Interleukin-2; Lipoxygenase; Lymphocyte Activation; Lymphoma; Mice; T-Lymphocytes

Human platelets metabolize arachidonic acid via cyclooxygenase (E.C. 1.14.99.1) to thromboxane A2 and the 12-lipoxygenase to 12-hydroxyeicosatetraenoic acid (12-HETE). Aspirin inhibits cyclooxygenase while the neutrophil product 15-Hydroxyeicosatetraenoic acid (15-HETE) is a selective inhibitor of platelet 12-lipoxygenase. The unexpected observation was made that the platelet 12-lipoxygenase of individuals who had ingested aspirin showed up to a twenty-fold increase in sensitivity to inhibition by 15-HETE. This observation was confirmed in platelets treated with aspirin in vitro. Aspirin pretreatment consistently resulted in a decrease in the I50 for 15-HETE from an average of 21.5 microM to only 5.2 +/- 1.5 microM, indicating a probable interaction between the cyclooxygenase and lipoxygenase pathways.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Aspirin; Blood Platelets; Drug Synergism; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase Inhibitors

The ability of lipoxygenase products to become incorporated into islet cell phospholipids and to affect fatty acid mobilization was investigated. Isolated intact islets or homogenized islets were incubated with tritiated 5-hydroxyeicosatetraenoic acid (HETE), 12-HETE, 15-HETE, the leukotrienes C4 and D4, or prostaglandin E2. Tritiated 5-HETE and 12-HETE were largely esterified into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) of intact islet membranes. Glucose stimulation increased [3H]5-HETE esterification to islet PC and PE. In islet homogenates, tritiated 5-HETE, 12-HETE, 15-HETE and arachidonic acid (AA) were also esterified into endogenous PC and PE, with less incorporation in phosphatidylinositol (PI) or phosphatidylserine. Addition of exogenous lysophospholipid acceptors potentiated the esterification of [3H]5-HETE to PC especially; [3H]AA was uniformly esterified to exogenous lysophospholipids. In addition, unlabeled 5-HETE (40 nM to 8 microM) affected the incorporation of [3H]AA into PC and PE of homogenates in a biphasic manner, whereas unlabeled AA inhibited [3H]AA incorporation into phospholipids in a concentration-dependent manner. Glucose (8.5 mM) stimulated the loss of labeled AA from prelabeled islet PC and PI. On the other hand, 5-HETE (40 nM) increased AA recovery in PC, PI, and PE of prelabeled islets, and HETE antagonized the glucose-stimulated release of AA from PC and PE. A 100-fold higher concentration of 5-HETE increased the glucose-stimulated loss of AA from phospholipids in prelabeled islets. Nanomolar concentrations of 5-HETE elicited a rapid and transient increase in insulin release, which was additive to the release response to a submaximal stimulatory concentration of glucose, whereas micromolar 5-HETE did not affect insulin release. Thus, pancreatic islets not only esterify HETE into phospholipids, but HETE also alters the turnover of AA in membrane phospholipids. HETE-induced changes in islet membrane fatty acid composition and/or AA mobilization may modulate stimulus-secretion coupling.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcium; Egtazic Acid; Fatty Acids; Glucose; Hydroxyeicosatetraenoic Acids; Insulin; Insulin Secretion; Islets of Langerhans; Lipoxygenase; Male; Phospholipids; Rats; Rats, Inbred Strains; SRS-A

When blood-free mouse brain slices were incubated with exogenous radiolabeled arachidonic acid, gas chromatography/mass spectrometry confirmed that the major radioactive lipoxygenase enzyme product of arachidonic acid was 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), with lesser amounts of 5-hydroxy-5,6,8,11,14-eicosatetraenoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid. When 12-[2H]HETE was used to measure endogenous 12-HETE in brain tissue frozen with liquid nitrogen, the level of 12-HETE was 41 +/- 6 ng/g of wet weight tissue. This frozen tissue level was not due to the presence of blood. When brain slices were incubated in vitro for 20 min, the 12-HETE level increased to 964 +/- 35 ng/g of wet weight tissue. Elimination of residual intravascular blood before tissue incubation reduced the brain slice 12-HETE concentration by one-half.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Brain; Brain Chemistry; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Guinea Pigs; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Male; Mice; SRS-A

The metabolism of arachidonic acid by cholesterol-enriched resident mouse peritoneal macrophages was investigated. The amounts of monohydroxyeicosatetraenoic acid (mono-HETE) produced by the cholesterol-rich macrophages were 2.5-fold greater when compared to control macrophages. The major lipoxygenase product, identified by high-performance liquid chromatography in both macrophages was 12-HETE. Since macrophages are important participants in the formation of atheromatous lesions, the increased metabolism of arachidonic acid to HETE products by cholesterol-rich macrophages could contribute to the initiation and progression of the atherosclerotic process.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Arteriosclerosis; Cells, Cultured; Cholesterol; Chromatography, Thin Layer; Cyclooxygenase Inhibitors; Hydroxyeicosatetraenoic Acids; Ibuprofen; Indomethacin; Lipoxygenase; Macrophages; Male; Mice

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 present study demonstrated that acid-extracted platelet phospholipase A2 (PLA2) exhibited marked hydrolytic activity against both [1-14C]oleic acid- and [1-14C]arachidonic acid-labeled Escherichia coli. The rate of hydrolysis was linear up to 30 min and was directly proportional to the amount of enzyme added to the reaction mixture. The data further indicated that 5-hydroxy-6,8,11,15-eicosatetraenoic acid (5-HETE) inhibited platelet PLA2 in a dose-dependent manner (IC50 = 42 microM), whereas 5-lactone HETE had no inhibitory effect up to 100 microM. The degree of inhibition of PLA2 activity was unaffected by Ca2+ concentrations but was reduced in the presence of increasing amounts of E. coli substrate. Both 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) and 15-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE) also inhibited platelet PLA2 activity (IC50 = 26 and 72 microM respectively). Furthermore, the inhibitory effects of these monoHETEs were confirmed with a PLA2 preparation derived from rat neutrophils. Thus, these data suggest a novel pharmacological action of HETEs on PLA2 which may have potential ramifications in the regulation of arachidonic acid metabolism.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Blood Platelets; Calcium; Fatty Acids, Nonesterified; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Neutrophils; Phospholipases; Phospholipases A; Phospholipases A2

The effects of prostaglandin-generating factor of anaphylaxis (PGF-A) upon the lipoxygenation of arachidonic acid and the promotion of mucous glycoprotein secretion by human airways were analyzed concurrently in order to determine the role that lipoxygenase products play in the secretion of mucus which accompanies immediate hypersensitivity reactions of airways. PGF-A enhanced both mucous glycoprotein release and the 5- and 15-lipoxygenation of arachidonic acid as well as the formation of leukotriene B4 (LTB4) with similar dose-response relationships. The capacity of PGF-A to stimulate mucous glycoprotein release was inhibited by ETYA but not by indomethacin, suggesting that PGF-A stimulated lipoxygenase products may be involved. Lipoxygenase products of arachidonic acid thus may serve as mediators of the enhancement of mucus secretion from human airways in response to PGF-A.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Anti-Inflammatory Agents; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Glycoproteins; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene B4; Lipoxygenase; Lung; Mast Cells; Mucus; Oligopeptides

Arachidonic acid is metabolized in neutrophils by lipoxygenase to leukotrienes, which are suggested to play a central role in inflammation. The antirheumatic drug auranofin (4 micrograms/ml) was found not to inhibit neutrophil production of the lipoxygenase products 5-HETE-, 15-HETE and LTB4, in vitro when stimulated with the calcium ionophore A23187. Auranofin, however, modulated neutrophil aggregation, enzyme release and chemotaxis induced by LTB4. The results suggest that auranofin may exert some of its antirheumatic effects through affecting neutrophil responses to leukotrienes.

Topics: Anti-Inflammatory Agents; Auranofin; Aurothioglucose; Cell Aggregation; Chemotaxis, Leukocyte; Glucuronidase; Gold; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils

Our previous data suggested that lipoxygenation of endogenously released arachidonic acid (AA) is a critical step in stimulus-secretion coupling in the pancreatic beta cell. In the current study using monolayer cultures of neonatal rat islet cells, exogenous arachidonic acid (AA) (5 micrograms/ml) potently stimulated insulin release in the presence of a substimulatory glucose concentration, and potentiated release induced by glucose. Since the latter stimulatory effect of AA is prevented by inhibitors of the lipoxygenase pathway, we examined the effects of various lipoxygenase pathway products on glucose-induced insulin secretion. The mediator was not one of the stable end-products of either limb of the lipoxygenase pathway: 12- or 5-hydroxyeicosatetraenoic acid (HETE) (0.5-2000 ng/ml) did not alter insulin release, whereas 11-HETE, 15-HETE, leukotriene (LT)B4 and the delta 6 trans isomers of LTB4, LTC4 and 11-trans LTC4 all inhibited insulin release. Furthermore, diethylcarbamazine, a selective leukotriene synthesis inhibitor, did not prevent AA- or glucose-induced insulin release, arguing against a role for LTs as the mediator of AA's stimulatory effect. However, the unstable intermediate 12-hydroperoxyeicosatetraenoic acid (12-HPETE), and positional isomers of 12-HPETE, potentiated glucose-induced insulin secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Diethylcarbamazine; Glucose; Hydroxyeicosatetraenoic Acids; Insulin; Insulin Secretion; Islets of Langerhans; Leukotriene B4; Lipoxygenase; Rats

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

Human peripheral blood polymorphonuclear leukocytes (PMNs) metabolized [14C]arachidonic acid predominantly by lipoxygenase pathways. The major products were 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and 15-HETE. These and other lipoxygenase products, including their derived leukotrienes, have been implicated as mediators of inflammatory and allergic reactions. In human platelets, the nonsteroidal anti-inflammatory drug ibuprofen inhibited production of the cyclooxygenase product thromboxane B2 (I50 = 65 microM), whereas the lipoxygenase product 12-HETE was not appreciably affected even at 5 mM ibuprofen. The 5-lipoxygenase of human PMNs (measured by 5-HETE formation) was inhibited by ibuprofen but was about six times less sensitive (I50 = 420 microM) than the platelet cyclooxygenase. The unexpected observation was made that the human PMN 15-lipoxygenase/leukotriene pathway was selectively activated by 1-5 mM ibuprofen. Metabolites were identified by ultraviolet spectroscopy, by radioimmunoassay, or by retention times on high pressure liquid chromatography in comparison with authentic standards. The major product was 15-HETE; and in all of 19 donors tested, 15-HETE formation was stimulated up to 20-fold by 5 mM ibuprofen. Other identified products included 12-HETE and 15- and 12-hydroperoxyeicosatetraenoic acid. Activation of the 15-lipoxygenase by ibuprofen occurred within 1 min and was readily reversible. The effects of aspirin, indomethacin, and ibuprofen on the PMN 15-lipoxygenase were compared in six donors. Ibuprofen produced an average 9-fold stimulation of the enzyme, whereas aspirin and indomethacin resulted in an average 1.5- and 2-fold enhancement, respectively.

Topics: Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Enzyme Activation; Humans; Hydroxyeicosatetraenoic Acids; Ibuprofen; Lipoxygenase; Neutrophils; Prostaglandin-Endoperoxide Synthases; SRS-A

The following high performance liquid chromatography system was found suitable for separating most lipoxygenase metabolites of arachidonic acid: Techsphere 5-C18 column, eluting solvent methanol:water:acetic acid (65:35:0.06 v/v), pH 5.3. Comparisons with other packing materials and solvent systems are described. The method could be used to identify lipoxygenase products released from mouse macrophage cells stimulated with gamma-hexachlorocyclohexane. Detection limits between 1 and 10 ng were obtained.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Macrophages; Mice; SRS-A

Cells of a mouse macrophage-like tumor cell line, J774.2, were incubated with 0.6 microM radiolabeled mono- and di-hydroxyfatty acids. Monohydroxyfatty acid products of the neutrophil and platelet lipoxygenase pathways (5-HETE, 15-HETE, and 12-HETE) were rapidly taken up (42-64% of the counts cell associated at 1 min) and esterified into triglycerides and phospholipids. 5-HETE and 12-HETE were found in triglycerides and distributed among phospholipid classes while 50% of added 15-HETE was esterified into phosphatidyl inositol. Treatment of phospholipids from cells incubated with 5-HETE, 12-HETE, and 15-HETE with phospholipase A2 resulted in release of the respective monohydroxyfatty acid. HHT, a monohydroxyfatty acid product of the cyclooxygenase pathway, was taken up and esterified more slowly than the lipoxygenase products. In addition, HHT was not released when the phospholipids from cells incubated with HHT were treated with phospholipase A2. LTB4, a dihydroxyfatty acid product of neutrophil lipoxygenase, was not taken up by J774.2 cells. The unique patterns of uptake and intracellular distribution of the different monohydroxyfatty acids suggests that the enzymes involved in the esterification of these compounds have substrate specificity and may also relate to the specific biologic effects of these compounds.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Cell Line; Fatty Acids; Hydroxyeicosatetraenoic Acids; Lipid Metabolism; Macrophages; Mice; Phospholipids

During allergic and inflammatory reactions, arachidonic acid is oxidized by lipoxygenases to a variety of biologically active products, including leukotrienes. The mechanisms for regulation of the different lipoxygenase activities are not well defined. We report here that [14C]arachidonic acid metabolism by the 5- and 15-lipoxygenase activities in rabbit leukocytes and the 12-lipoxygenase in rabbit platelets is inhibited by various hydroxyeicosatetraenoic acids (HETEs). 15-HETE was the most effective inhibitor of the 5- and 12-lipoxygenases, whereas similar inhibitory potencies were observed for 5-HETE and 12-HETE acting on the 15-lipoxygenase. These three enzyme pathways were all least sensitive to their own products HETEs. To determine which structural characteristics of 15-HETE are essential for inhibition of the 5-lipoxygenase, various derivatives were prepared and purified by high pressure liquid chromatography, and their structures were confirmed by gas chromatography-mass spectrometry. The inhibitory potencies of 15-HETE analogs with different degrees of unsaturation were in the order of three double bonds greater than 4 greater than 2 greater than 0. 15-Hydroperoxy-5,8,11,13-eicosatetraenoic acid (15-HPETE) was four times more potent than 15-HETE. The 15-acetoxy, 15-keto and methyl ester derivatives were of comparable activity to 15-HETE, and the 15-acetoxy methyl ester derivative was less potent. Based upon the observed patterns of inhibition, we postulate that complex interregulatory relationships exist between the various lipoxygenases, and that cells containing these lipoxygenases may interact with each other via their lipoxygenase metabolites.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Humans; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Lipoxygenase; Lipoxygenase Inhibitors; Neutrophils; Rabbits; Species Specificity; Structure-Activity Relationship

Within 5 min, resting macrophages metabolize microM quantities of exogenous arachidonic acid (20:4) to cyclooxygenase and lipoxygenase products. Mono-HETEs represent a major class of metabolites recovered from the medium. However, the quantity of mono-Hetes progressively decreases over a 60-min incubation period, with a concomitant increase in more polar lipoxygenase products, suggesting additional metabolic fates for these hydroxy acids. This was directly confirmed by exposing resident macrophage cultures to radiolabeled 15-, 12-, and 5-HETEs (1 microM). 12-30% of the recovered HETEs were cell-associated and predominantly esterified into phospholipid. High pressure liquid chromatography analyses of medium extracts indicated that 50% of each HETE was also converted to 10 or more metabolites over a 60-min time-course, a rate slower than for 20:4. The major metabolite generated from each mono-HETE had the elution characteristics of a di-HETE. The 5-HETE product has a triene spectrum similar to that of 5(S), 12(S)-di-HETE, whereas the 15- and 12-HETE products exhibited single ultraviolet absorption maxima, indicating a metabolic pathway for 5-HETE distinct from the other mono-HETEs. None of the stable cyclooxygenase products of 20:4 (6-keto PGF1 alpha, PGF2 alpha, PGE2, TXB2) nor polar metabolites of mono-HETEs are either incorporated or metabolized. The results indicate that macrophages have the capacity to specifically metabolize 20:4 and mono-HETEs to polar oxygenated products in the absence of a discernible trigger.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Dinoprost; Dinoprostone; Female; Humans; Hydroxyeicosatetraenoic Acids; Macrophage Activation; Macrophages; Male; Mice; Mice, Inbred ICR; Phosphatidylcholines; Phospholipids; Prostaglandins E; Prostaglandins F; Thromboxane B2; Time Factors

In view of the likelihood that monohydroxyeicosatetraenoic acids (HETEs) are produced in asthma we investigated the actions of these lipoxygenase products on a range of tracheobronchial smooth muscle preparations. 5- and 15-HETE contracted human isolated bronchial muscle and guinea-pig lung strip but were less potent and less effective than histamine. In contrast 5-, 12- and 15-HETE were inactive on guinea-pig and cat tracheae. In addition to its direct action, subthreshold doses of 5-HETE (0.001-0.01 micrograms ml-1) potentiated histamine-induced contractions of human isolated bronchial muscle. The latter effect may suggest that 5-HETE plays a significant role in the airway hyperreactivity commonly associated with asthma.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Bronchi; Cats; Dose-Response Relationship, Drug; Drug Synergism; Female; Guinea Pigs; Histamine; Humans; Hydroxyeicosatetraenoic Acids; Muscle Contraction; Muscle, Smooth; Trachea