12-hydroxy-5-8-10-14-eicosatetraenoic-acid and 15-hydroxy-5-8-11-13-eicosatetraenoic-acid

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Amino Acid Sequence; Animals; Hydroxyeicosatetraenoic Acids; Isoenzymes; Lipoxygenase; Mammals; Molecular Sequence Data; Substrate Specificity

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Fatty Acids, Nonesterified; Fish Oils; Humans; Hydroxyeicosatetraenoic Acids; Lipids; Prostaglandin-Endoperoxide Synthases; Psoriasis

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

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

There are considerable data implicating a pancreatic islet 12-lipoxy-genase in glucose-induced insulin secretion. This enzyme traditionally is conceived as converting unesterified arachidonic acid to "free" hydroperoxyeicosatetraenoic acid and metabolites thereof. However, studies employing the provision of exogenous metabolites of arachidonic acid to islet tissue fail to identify convincingly the mediator of insulin release. It is proposed that the islet lipoxygenase directly peroxidizes unsaturated fatty acids esterified within membrane phospholipids, leading to changes in ion flux and enzyme activity (particularly phospholipase A2) at the membrane level. The release of unesterified metabolites of arachidonate, although reflecting islet lipoxygenase activity, may be an epiphenomenon.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5,8,11,14-Eicosatetraynoic Acid; Arachidonate Lipoxygenases; Arachidonic Acids; Enzyme Activation; Fatty Acids, Unsaturated; Glucose; Hydroxyeicosatetraenoic Acids; Insulin; Islets of Langerhans; Leukotrienes; Lipid Peroxides; Lipoxygenase; Membrane Lipids; Models, Biological; Phospholipases A; Phospholipases A2; Phospholipids

Resident mouse peritoneal macrophages when exposed to zymosan during the first day of cell culture synthesize and secrete large amounts of prostaglandin E2 and leukotriene (LT) C4, the respective products of cyclooxygenase- and 5-lipoxygenase-catalyzed oxygenations of arachidonic acid. Under these conditions of cell stimulation only small amounts of hydroxyeicosatetraenoic acids (HETEs) are concomitantly produced. However, exogenously added arachidonic acid is metabolized to large amounts of 12- and 15-HETE. No LTC4 is formed under these conditions. Inasmuch as 12- and 15-HETE have been shown to modulate certain lymphocyte responses, further study of the regulation of their production by macrophages is warranted.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Leukotrienes; Lipid Peroxides; Lipoxygenase; Lymphocyte Activation; Macrophages; Mice; Prostaglandin-Endoperoxide Synthases; Rats; SRS-A; T-Lymphocytes; Zymosan

Disease-specific treatment options for autosomal dominant polycystic kidney disease are limited. Clinical intervention early in life is likely to have the greatest effect. In a 3-year randomized double-blind placebo-controlled phase 3 clinical trial, the authors recently showed that pravastatin decreased height-corrected total kidney volume (HtTKV) progression of structural kidney disease over a 3-year period. However, the underlying mechanisms have not been elucidated.. Participants were recruited nationally from July 2007 through October 2009. Plasma and urine samples collected at baseline, 18 months, and 36 months from 91 pediatric patients enrolled in the above-mentioned clinical trial were subjected to mass spectrometry-based biomarker analysis. Changes in biomarkers over 3 years were compared between placebo and pravastatin-treated groups. Linear regression was used to evaluate the changes in biomarkers with the percent change in HtTKV over 3 years.. Changes in plasma concentrations of proinflammatory and oxidative stress markers (9- hydroxyoctadecadienoic acid, 13-hydroxyoctadecadienoic acid, and 15-hydroxyeicosatetraenoic acid [HETE]) over 3 years were significantly different between the placebo and pravastatin-treated groups, with the pravastatin group showing a lower rate of biomarker increase. Urinary 8-HETE, 9-HETE, and 11-HETE were positively associated with the changes in HtTKV in the pravastatin group.. Pravastatin therapy diminished the increase of cyclooxygenase- and lipoxygenase-derived plasma lipid mediators. The identified biomarkers and related molecular pathways of inflammation and endothelial dysfunction may present potential targets for monitoring of disease severity and therapeutic intervention of autosomal dominant polycystic kidney disease.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adolescent; Biomarkers; Child; Female; Humans; Hydroxyeicosatetraenoic Acids; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Linoleic Acids; Male; Organ Size; Oxidative Stress; Polycystic Kidney, Autosomal Dominant; Pravastatin; 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

The polarization of macrophages is critical to inflammation and tissue repair, with unbalanced macrophage polarization associated with critical dysfunctions of the immune system. Cytochrome P450 1A1 (CYP1A1) is a hydroxylase mainly controlled by the inflammation-limiting aryl hydrocarbon receptor (AhR), which plays a critical role in mycoplasma infection, oxidative stress injury, and cancer. Arginase-1 (Arg-1) is a surrogate for polarized alternative macrophages and is important to the production of nitric oxide (NO) by the modulation of arginine. In the present study, we found CYP1A1 to be upregulated in IL-4-stimulated mouse peritoneal macrophages (PMs) and human peripheral blood monocytes. Using CYP1A1-overexpressing RAW264.7 cells (CYP1A1/RAW) we found that CYP1A1 augmented Arg-1 expression by strengthening the activation of the JAK1/STAT6 signaling pathway in macrophages treated with IL-4. 15(S)-HETE, a metabolite of CYP1A1 hydroxylase, was elevated in IL-4-induced CYP1A1/RAW cells. Further, in macrophages, the loss-of-CYP1A1-hydroxylase activity was associated with reduced IL-4-induced Arg-1 expression due to impaired 15(S)-HETE generation. Of importance, CYP1A1 overexpressing macrophages reduced the inflammation associated with LPS-induced peritonitis. Taken together, these findings identified a novel signaling axis, CYP1A1-15(S)-HETE-JAK1-STAT6, that may be a promising target for the proper maintenance of macrophage polarization and may also be a means by which to treat immune-related disease due to macrophage dysfunction.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adoptive Transfer; Animals; Arachidonate 15-Lipoxygenase; Arginase; Cytochrome P-450 CYP1A1; Endotoxins; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-4; Janus Kinase 1; Leukocytes, Mononuclear; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C57BL; Peritonitis; RAW 264.7 Cells; Receptors, Cytoplasmic and Nuclear; RNA Interference; RNA, Messenger; Signal Transduction; STAT6 Transcription Factor; THP-1 Cells; Up-Regulation

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

The net vascular effect of estrogens on the vasculature is still under debate. Here we tested the effects of estradiol- 17β (E2) as well as estrogen-receptor subtype specific and non-specific agonists and antagonists on the expression and eicosanoid production of lipoxygenase (LO) enzymes expressed in culture human umbilical vascular smooth muscle cells (VSMC), the platelet type 12LO and 15LO type 2. E2 increased 12 and 15LO mRNA expression by 2-3 folds and elicited an acute 50% increase 12 and 15 hydroxyeicosatetraenoic acid (HETE) production. Neither estrogen receptor ERα nor ERβ-specific agonists were able to reproduce the induction of LO expression, but E2-induced expression was effectively blocked by ER non-specific and receptor subtype specific antagonists. Because 12 and 15HETE can increase reactive oxygen species in other cell types, we tested the possibility that E2 could raise ROS through LO. Indeed, E2 as well as the LO products 12 and 15HETE increased reactive oxygen species (ROS) in VSMC. E2-dependent and HETE-induced ROS could be blocked by NAD (P) H-oxidase inhibitors and by the ER general antagonist ICI. E2-induced ROS was partially (∼50%) blocked by the LO inhibitor baicalein, but the LO blocker had no effect on 12 or 15HETE- induced ROS formation, thus suggesting that part of E2-dependent ROS generation resulted from E2-induced 12 and 15HETE. Collectively these findings unveil an unrecognized effect of E2 in human VSMC, to induce 12 and 15LO type 2 expression and activity and suggest that E2-dependent ROS formation in VSMC may be partially mediated by the induction of 12 and 15HETE.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Flavanones; Gene Expression Regulation; Humans; Hydroxyeicosatetraenoic Acids; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; NADPH Oxidases; Nitriles; Phenols; Piperidines; Primary Cell Culture; Propionates; Pyrazoles; Pyrimidines; Raloxifene Hydrochloride; Reactive Oxygen Species; RNA, Messenger; Umbilical Veins

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

We recently demonstrated that 12/15-lipoxygenase (LOX) derived metabolites, hydroxyeicosatetraenoic acids (HETEs), contribute to diabetic retinopathy (DR) via NADPH oxidase (NOX) and disruption of the balance in retinal levels of the vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). Here, we test whether PEDF ameliorates retinal vascular injury induced by HETEs and the underlying mechanisms. Furthermore, we pursue the causal relationship between LOX-NOX system and regulation of PEDF expression during DR. For these purposes, we used an experimental eye model in which normal mice were injected intravitreally with 12-HETE with/without PEDF. Thereafter, fluorescein angiography (FA) was used to evaluate the vascular leakage, followed by optical coherence tomography (OCT) to assess the presence of angiogenesis. FA and OCT reported an increased vascular leakage and pre-retinal neovascularization, respectively, in response to 12-HETE that were not observed in the PEDF-treated group. Moreover, PEDF significantly attenuated the increased levels of vascular cell and intercellular adhesion molecules, VCAM-1 and ICAM-1, elicited by 12-HETE injection. Accordingly, the direct relationship between HETEs and PEDF has been explored through in-vitro studies using Müller cells (rMCs) and human retinal endothelial cells (HRECs). The results showed that 12- and 15-HETEs triggered the secretion of TNF-α and IL-6, as well as activation of NFκB in rMCs and significantly increased permeability and reduced zonula occludens protein-1 (ZO-1) immunoreactivity in HRECs. All these effects were prevented in PEDF-treated cells. Furthermore, interest in PEDF regulation during DR has been expanded to include NOX system. Retinal PEDF was significantly restored in diabetic mice treated with NOX inhibitor, apocynin, or lacking NOX2 up to 80% of the control level. Collectively, our findings suggest that interfering with LOX-NOX signaling opens up a new direction for treating DR by restoring endogenous PEDF that carries out multilevel vascular protective functions.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Acetophenones; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Cells, Cultured; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Endothelial Cells; Ependymoglial Cells; Eye Proteins; Gene Expression Regulation; Humans; Hydroxyeicosatetraenoic Acids; Intercellular Adhesion Molecule-1; Interleukin-6; Intravitreal Injections; Membrane Glycoproteins; Mice; Mice, Knockout; NADPH Oxidase 2; NADPH Oxidases; Nerve Growth Factors; NF-kappa B; Retina; Retinal Neovascularization; Serpins; Signal Transduction; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1; Zonula Occludens-1 Protein

The enzyme 12/15-lipoxygenase (LOX) oxidizes various free fatty acids, including arachidonic acid (AA). In the brain, the principal 12/15-LOX metabolites of AA are 12(S)-HETE and 15(S)-HETE. PPARγ is a nuclear receptor whose activation is neuroprotective through its anti-inflammatory properties. In this study, we investigate the involvement of 12(S)- and 15(S)-HETE in the regulation of PPARγ following cerebral ischemia and their effects on ischemia-induced inflammatory response. We show here the increased expression of 12/15-LOX, predominantly in neurons, and elevated production of 12(S)-HETE and 15(S)-HETE in ischemic brain. The exogenous 12(S)- and 15(S)-HETE increase PPARγ protein level, nuclear translocation, and DNA-binding activity in ischemic rats, suggesting the activation of PPARγ. This effect was further confirmed by showing the increased PPARγ transcriptional activity in primary cortical neurons when incubated with 12(S)- or 15(S)-HETE. Moreover, both 12(S)- and 15(S)-HETE potently inhibited the induction of nuclear factor-κB, inducible NO synthase, and cyclooxygenase-2 in ischemic rats, and elicited neuroprotection. The reversal of the effects of 12(S)- and 15(S)-HETE on pro-inflammatory factors by PPARγ antagonist GW9662 indicated their actions were mediated via PPARγ. Thus, the induction of 12(S)- and 15(S)-HETE during brain ischemia suggests that endogenous signals of neuroprotection may be generated.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Active Transport, Cell Nucleus; Anilides; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Brain Ischemia; Cell Nucleus; Enzyme Activation; Hydroxyeicosatetraenoic Acids; Male; Neurons; PPAR gamma; Rats; Rats, Sprague-Dawley

The metabolic pathway disturbances associated with hepatocellular carcinoma (HCC) remain unsatisfactorily characterized. Determination of the metabolic alterations associated with the presence of HCC can improve our understanding of the pathophysiology of this cancer and may provide opportunities for improved disease monitoring of patients at risk for HCC development. To characterize the global metabolic alterations associated with HCC arising from hepatitis C (HCV)-associated cirrhosis using an integrated non-targeted metabolomics methodology employing both gas chromatography/mass spectrometry (GC/MS) and ultrahigh-performance liquid chromatography/electrospray ionization tandem mass spectrometry (UPLC/MS-MS).. The global serum metabolomes of 30 HCC patients, 27 hepatitis C cirrhosis disease controls and 30 healthy volunteers were characterized using a metabolomics approach that combined two metabolomics platforms, GC/MS and UPLC/MS-MS. Random forest, multivariate statistics and receiver operator characteristic analysis were performed to identify the most significantly altered metabolites in HCC patients vs. HCV-cirrhosis controls and which therefore exhibited a close association with the presence of HCC.. Elevated 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, sphingosine, γ-glutamyl oxidative stress-associated metabolites, xanthine, amino acids serine, glycine and aspartate, and acylcarnitines were strongly associated with the presence of HCC. Elevations in bile acids and dicarboxylic acids were highly correlated with cirrhosis.. Integrated metabolomic profiling through GC/MS and UPLC/MS-MS identified global metabolic disturbances in HCC and HCV-cirrhosis. Aberrant amino acid biosynthesis, cell turnover regulation, reactive oxygen species neutralization and eicosanoid pathways may be hallmarks of HCC. Aberrant dicarboxylic acid metabolism, enhanced bile acid metabolism and elevations in fibrinogen cleavage peptides may be signatures of cirrhosis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Amino Acids; Bile Acids and Salts; Carcinoma, Hepatocellular; Chromatography, High Pressure Liquid; Dicarboxylic Acids; Gas Chromatography-Mass Spectrometry; Hepatitis C; Humans; Hydroxyeicosatetraenoic Acids; Liver Cirrhosis; Liver Neoplasms; Metabolome; Metabolomics; Multivariate Analysis; ROC Curve; Sphingosine; Tandem Mass Spectrometry; Xanthine

The influence of the inclusion of a stable isotopic labeled internal standard (SIL-IS) on the quantitative analysis of hydroxyeicosatetranoic acids (HETEs) in human serum is evaluated in this research. A solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) platform, one of the preferred approaches for targeted analysis of biofluids through the selected reaction monitoring (SRM) operational mode, was used to determine HETEs. These compounds were chosen as targeted metabolites because of their involvement in cardiovascular disease, cancer and osteoporosis. 15HETE-d8 was chosen as internal standard to evaluate matrix effects. Thus, the physico-chemical properties of the SIL-IS were the basis to evaluate the analytical features of the method for each metabolite through four calibration models. Two of the models were built with standard solutions at different concentration levels, but one of the calibration sets was spiked with an internal standard (IS). The other two models were built with the serum pool from osteoporotic patients, which was spiked at different concentrations with the target analytes. In this case, one of the serum calibration sets was also spiked with the IS. The study shows that the IS allowed noticeable correction of matrix effects for some HETE isomers at certain concentration levels, while accuracy was decreased at low concentration (15ng/mL) of them. Therefore, characterization of the method has been properly completed at different concentration levels.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Calibration; Chromatography, Liquid; Female; Humans; Hydroxyeicosatetraenoic Acids; Molecular Structure; Postmenopause; Reference Standards; Reproducibility of Results; Solid Phase Extraction; Tandem Mass Spectrometry

The purpose of the current study was to evaluate the effect of 12/15-lipoxygenase (12/15-LOX) metabolites on retinal endothelial cell (REC) barrier function. FITC-dextran flux across the REC monolayers and electrical cell-substrate impedance sensing (ECIS) were used to evaluate the effect of 12- and 15-hydroxyeicosatetreanoic acids (HETE) on REC permeability and transcellular electrical resistance (TER). Effect of 12- or 15-HETE on the levels of zonula occludens protein 1 (ZO-1), reactive oxygen species (ROS), NOX2, pVEGF-R2 and pSHP1 was examined in the presence or absence of inhibitors of NADPH oxidase. In vivo studies were performed using Ins2(Akita) mice treated with or without the 12/15-LOX inhibitor baicalein. Levels of HETE and inflammatory mediators were examined by LC/MS and Multiplex Immunoassay respectively. ROS generation and NOX2 expression were also measured in mice retinas. 12- and 15- HETE significantly increased permeability and reduced TER and ZO-1 expression in REC. VEGF-R2 inhibitor reduced the permeability effect of 12-HETE. Treatment of REC with HETE also increased ROS generation and expression of NOX2 and pVEGF-R2 and decreased pSHP1 expression. Treatment of diabetic mice with baicalein significantly decreased retinal HETE, ICAM-1, VCAM-1, IL-6, ROS generation, and NOX2 expression. Baicalein also reduced pVEGF-R2 while restored pSHP1 levels in diabetic retina. Our findings suggest that 12/15-LOX contributes to vascular hyperpermeability during DR via NADPH oxidase dependent mechanism which involves suppression of protein tyrosine phosphatase and activation of VEGF-R2 signal pathway.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Biological Transport; Cell Membrane Permeability; Dextrans; Diabetic Retinopathy; Electric Impedance; Endothelial Cells; Enzyme Inhibitors; Flavanones; Fluorescein-5-isothiocyanate; Gene Expression; Hydroxyeicosatetraenoic Acids; Membrane Glycoproteins; Mice; Mice, Transgenic; NADPH Oxidase 2; NADPH Oxidases; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Reactive Oxygen Species; Retina; Signal Transduction; Vascular Endothelial Growth Factor Receptor-2; Zonula Occludens-1 Protein

Human bone cell line (SaOS2) express different mRNAs involved in bone biology and physiology such as estrogen receptor α (ERα), estrogen receptor β (ERβ), vitamin D receptor (VDR), 1α, 25 hydroxy vitamin D(3) hydroxylase (1OHase) as well as 12 and 15 lipoxygenases (12LO and 15LO). These mRNAs are modulated by estrogenic compounds. Since the skeletal protective effects of estrogens are not discernible in diabetic women, we tested whether the expression of the parameters measured here and their modulations by estrogens, in SaOS2 cells grown in growth medium containing high glucose (HG; 9.0 g/L; 44 mM) compared to normal glucose (NG; 4.5 g/L; 22 mM). High Glucose (HG) significantly increased DNA synthesis and creatine kinase (CK) specific activity in SaOS2 cells. Stimulations of DNA but not of CK by E(2), by 4, 4', 4''-[4-propyl-(1H)-pyrazol-1, 3, 5- triyl] tris-phenol (PPT, ERα specific agonist), or by 2, 3-bis (4-hydroxyphenyl)-propionitrile (DPN, ERβ specific agonist), were abolished by HG. HG itself upregulated the expression of mRNA of 12LO and 15LO and upregulated to much less extent of ERβ and VDR, but had no effect on the expression of mRNA of ERα and 1OHase. The different hormonal treatments modulated the expressions of 12LO and 15LO mRNAs which was reduced in HG, whereas the induction of their products 12HETE and 15HETE was only slightly affected by HG. The exact mechanism of HG effects on bone cell responses is yet to be investigated and its relationship to human bone physiology is not yet clear.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Bone and Bones; Cell Line; Creatine Kinase; DNA; Estrogens; Female; Gene Expression Regulation; Glucose; Humans; Hydroxyeicosatetraenoic Acids; Hypoglycemia; RNA, Messenger

12/15-Lipoxygenase (12/15-LOX) is a non-haeme iron-containing dioxygenase that forms 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) or 15(S)-HETE. Several biological mediators including cytokines, growth factors and lipid metabolites released during tumour cell-endothelial cell adhesion are associated with malignant tumour progression. Here we found that HETEs released from the host organ played a critical role in tumour metastasis. Intravenous injection of B16F10 melanoma cells caused lung nodule formation, which was markedly attenuated in 12/15-LOX null mice. Co-injection of melanoma cells with 12(S)-HETE increased the lung homing activity of B16F10 melanoma cells. In vitro studies showed that 12(S)-HETE and 15(S)-HETE treatment resulted in a concentration-dependent increase of adhesion of B16F10 cells on collagen or fibronectin. The melanoma cell adhesion was then evaluated in pulmonary primary cell culture isolated from wild-type (WT) and 12/15-LOX knockout (KO) mice. It was found that the adhesion of melanoma cells on the epithelial cells isolated from 12/15-LOX null mice was reduced in comparison with those isolated from WT mice. Treatment of 12(S)-HETE increased the pFAK in melanoma cells adhering on collagen-coated slide. The enhancement of adherence elicited by 12(S)-HETE in B16F10 cells could be antagonised by focal adhesion kinase (FAK) inhibitor 14 (FAK inhibitor) or PD98059 (extracellular signal-regulated kinase (ERK) inhibitor). 12(S)-HETE increased the phosphorylation of FAK and ERK in adhering melanoma cells. The FAK phosphorylation induced by 12(S)-HETE was further inhibited by PD98059, indicating that FAK is the downstream target of ERK. The adhesion and lung metastasis of human melanoma cells of C32 in NOD/SCID mice were also potentiated by co-treatment with 12(S)-HETE. These results demonstrate that 12(S)-HETE/15(S)-HETE activates ERK and FAK signalling pathways, thereby upregulates the adhesion and metastatic potential of melanoma cells. The endogenous release of 12(S)-HETE/15(S)-HETE in the host organ may affect the metastatic potential of melanoma.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Blotting, Western; Cell Adhesion; Cell Line, Tumor; Collagen; Disease Progression; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Fibronectins; Focal Adhesion Protein-Tyrosine Kinases; Humans; Hydroxyeicosatetraenoic Acids; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Mice, Inbred NOD; Mice, Knockout; Mice, SCID; Signal Transduction; Transplantation, Heterologous; Tumor Cells, Cultured

Cyclooxygenase (COX)-, lipoxygenase (LOX)-, and cytochrome P450 monooxygenase (CYP)-derived eicosanoids have been implicated in ocular surface inflammation and neovascularization. These eicosanoids are subjected to regulation by enzymes, such as heme oxygenases (HOs) and ferritin.. Quantitative polymerase chain reaction and lipidomics based on liquid chromatography-tandem mass spectrometry were performed on pterygia from patients undergoing surgical pterygium excision. Control tissues consisted of donor corneas. In addition, lipidomics based on liquid chromatography-tandem mass spectrometry was performed on tears collected from patients before the surgery.. Messenger RNA (mRNA) expression of HO-2, the constitutive HO isoform, was upregulated by 40% in pterygia compared with control tissue, whereas the mRNA level of the inducible form, HO-1, was downregulated by more than 50%. Levels of CYP4B1 mRNA showed an approximate 2-fold increase in pterygia compared with control. Lipidomic analysis of tissues indicated a moderate elevation in Prostaglandin E2 and thromboxane B2 levels in pterygia compared with control. Among the LOX-derived metabolites, the antiinflammatory-hydroxyeicosatetraenoic acid (15-HETE) levels were significantly reduced in pterygia (79.3 ± 48.11 pg/mg protein) compared with control (586.2 ± 213.5 pg/mg protein), whereas the proinflammatory LOX- and CYP4B1-derived 12-HETE levels were 10-fold higher in pterygia (2768 ± 832.3 pg/mg protein) compared with control (231.4 ± 87.35 pg/mg protein). Prostaglandin E2 and HETEs were also present in tears from patients with pterygium but were not detected in tears from healthy volunteers. The mRNA expression levels of both light and heavy chain ferritin were 60% and 30% lower, respectively, in pterygia compared with control.. We believe that a dysfunctional HO-ferritin system leads to increased levels of proinflammatory mediators, thus contributing to the inflammation characteristic of pterygia.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Aryl Hydrocarbon Hydroxylases; Chromatography, High Pressure Liquid; Dinoprostone; Female; Ferritins; Gene Expression Regulation, Enzymologic; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Humans; Hydroxyeicosatetraenoic Acids; Isoenzymes; Male; Middle Aged; Pterygium; Real-Time Polymerase Chain Reaction; RNA, Messenger; Tears; Thromboxane B2

Mammalian lipoxygenases (LOXs) play an important role in physiological and pathological processes through the biosynthesis of lipid mediators-leukotrienes, lipoxins and other arachidonic acid derivatives.There are four major families of LOXs that can be analyzed through the production of hydroxyeicosatetraenoic acids (HETEs). No analytical method to detect 5-, 8-, 12- and 15-HETE in one run has been published to date. The HPLC method combines reversed-phase separative column Nucleosil 120-5 C18 and NP column Zorbax Rx.SIL for identification. This conjunction enables separation of 12-HETE and 15-HETE to the baseline which is essential in 12/15-LOX research and elution of all four HETEs in one run. The method was successfully tested on partially purified LOXs from rat lung cytosol.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Lung; Male; Rats; Rats, Wistar

Acute transplant rejection is the leading cause of graft loss in the first months after kidney transplantation. Lipoxygenase products mediate pro- and anti-inflammatory actions and thus we aimed to correlate the histological reports of renal transplant biopsies with urinary lipoxygenase products concentrations to evaluate their role as a diagnostic marker. This study included a total of 34 kidney transplant recipients: 17 with an acute transplant rejection and 17 controls. LTE4, LTB4, 12-HETE and 15-HETE concentrations were measured by enzyme immunoassay. Urinary lipoxygenase product concentrations were not significantly changed during an acute allograft rejection. Nevertheless, LTB4 concentrations correlated significantly with the body temperature (P ≤ 0.05) 3 months after transplantation, and 12- and 15-HETE concentrations correlated significantly with renal function (P ≤ 0.05) 2 weeks after transplantation. In conclusion, our data show a correlation for LTB4 with the body temperature 3 months after transplantation and urinary 12- and 15-HETE concentrations correlate positively with elevated serum creatinine concentrations but do not predict acute allograft rejection.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Acute Disease; Adult; Body Temperature; Female; Graft Rejection; Humans; Hydroxyeicosatetraenoic Acids; Kidney; Kidney Transplantation; Leukotriene B4; Leukotriene E4; Lipoxygenase; Male; Middle Aged

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

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

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

Vitamin D metabolites or its less-calcemic analogs (JKF or QW) are beneficial for bone biology. We analyzed whether or not 25(OH)D3 (25), 1,25(OH)2D3 (1,25), JKF or QW regulate lipooxygenase (LO) enzymes expression and their products hydroxyeicosatetraenoic acid (12 and 15 HETE) formation as well as reactive oxygen species (ROS) production in human bone cell lines (SaOS2 and hFOB) and primary cultured human bone cells (Obs) from males or females. All compounds except 25 increased LOs mRNA expression and HETE production in female or male Obs. ROS formation was induced by JKF and QW in both cell lines, and was inhibited by different inhibitors. Baicalein (baic) an inhibitor of 12 and 15 LO activity, inhibited partially ROS formation by JKF or QW in SaSO2 and hFOB. JKF-stimulated DNA synthesis in female Obs was inhibited by baic but unchanged by addition of HETE or HETE with baic. These results indicate that vitamin D increased oxidative stress in bone cells is in part via induction of LO expression and activity. This new feature of vitamin D is probably mediated by intracellular and/or membranal receptors and its potential hazard could lead to potential damage due to increased lipid oxidation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Bone and Bones; Cell Line, Tumor; Cell Proliferation; Female; Gene Expression Regulation, Enzymologic; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Male; Models, Biological; Oxidative Stress; Reactive Oxygen Species; RNA, Messenger; Vitamin D

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

Several lines of evidence suggest that aldosterone excess may have detrimental effects in the cardiovascular system, independent of its interaction with the renal epithelial cells. Here we examined the possibility that aldosterone modulates 12- and/or 15-lipoxygenase (LO) expression/activity in human vascular smooth muscle cells (VSMC), in vitro, thereby potentially contributing to both vascular reactivity and atherogenesis. Following 24 h treatment of VSMC with aldosterone (1 nmol/L), there was a approximately 2-fold increase in the generation rate of 12 hydroxyeicosatetraenoic acid (12-HETE), 70% increase in platelet type 12-LO mRNA expression (P < 0.001) along with a approximately 3-fold increase in 12-LO protein expression, which were blocked by the mineralocorticoid receptor (MR) antagonists spironolactone (100 nmol/L) and eplerelone (100 nmol/ml). Additionally, aldosterone (1 nmol/L; 24 h) increased the production of 15-HETE (50%; P < 0.001) and the expression of 15-LO type 2 mRNA (50%; P < 0.05) (in VSMC). Aldosterone also increased the 12- and 15-LO type 2 mRNA expression in a line of human aortic smooth muscle cells (T/G HA-VSMC) (60% and 50%, respectively). Aldosterone-induced 12- and 15-LO type 2 mRNA expressions were blocked by the EGF-receptor antagonist AG 1478 and by the MAPK-kinase inhibitor UO126. Aldosterone-treated VSMC also showed increased LDL oxidation, (approximately 2-fold; P < 0.001), which was blocked by spironolactone. In conclusion, aldosterone increased 12- and 15-LO expression in human VSMC, in association with increased 12- and 15-HETE generation and enhanced LDL oxidation and may directly augment VSMC contractility, hypertrophy, and migration through 12-HETE and promote LDL oxidation via the pro-oxidative properties of these enzymes.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Aldosterone; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Cell Line; ErbB Receptors; Humans; Hydroxyeicosatetraenoic Acids; Lipoproteins, LDL; Mineralocorticoid Receptor Antagonists; Mitogen-Activated Protein Kinase Kinases; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Oxidation-Reduction; Receptors, Mineralocorticoid; Up-Regulation

Several reports have demonstrated an important role of leukotriene B(4) (LTB(4)) in the immune system. We investigated whether leukemic blasts from acute myeloid leukemic (AML) and acute lymphoid leukemic (ALL) patients produced LTB(4), 12- and 15-hydroxyeicosatetraenoic acids (12-HETE and 15-HETE) and whether these compounds affected blast proliferation and apoptosis. Leukemic blasts from AML M(0-2) and ALL patients expressed 5-LOX, 12-LOX and 15-LOX transcripts. Quantitative polymerase chain reaction indicated that 5-LOX transcripts were far more abundant than 12-LOX and 15-LOX ones. Leukemic blasts expressed 5-LOX activating protein (FLAP) transcripts and produced LTB(4) in response to calcium ionophore. In contrast no 15-HETE production was found. Calcium ionophore-stimulated leukemic blasts produced 12-HETE but also released thromboxane A(2) suggesting that contaminating platelets accounted for the release of these compounds. No significant effect of LTB(4), 12-HETE or 15-HETE could be documented on leukemic blast growth and on their apoptose rate. Results of the present study indicate that immature form of leukemic blasts produce LTB(4). However, the three major lipoxygenase metabolites of arachidonic acid; i.e., LTB(4), 12-HETE or 15-HETE, had no evident effect on their growth and apoptosis. We may speculate that LTB(4)-derived blast cells might initiate, augment or prolong tissue inflammation and damages by affecting the marrow and blood cytokine network.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Apoptosis; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Blast Crisis; Calcium; Cell Proliferation; Humans; Hydroxyeicosatetraenoic Acids; Ionophores; Leukemia, Myeloid, Acute; Leukotriene B4; Polymerase Chain Reaction; Precursor Cell Lymphoblastic Leukemia-Lymphoma; RNA, Messenger; Thromboxane A2

Lipoxygenase (LOX) are enzymes implicated in a broad range of inflammatory diseases, cancer, asthma and atherosclerosis. These diverse biological properties lead to the interesting target for the inhibition of this metabolic pathway of LOX. The drugs available in the market against LOX reported to have various side effects. To develop potent and selective therapeutic agents against LOX, it is essential to have the knowledge of its active site. Due to the lack of structural data of human LOX, researchers are using soybean LOX (sLOX) because of their availability and similarities in the active site structure. Based on the crystal structure of sLOX-3 and its complex with known inhibitors, we have designed a tripeptide, FWY which strongly inhibits sLOX-3 activity. The inhibition by peptide has been tested with purified sLOX-3 and with LOX present in blood serum of breast cancer patients in the presence of substrate linoleic acid and arachidonic acid respectively. The dissociation constant (K(D)) of the peptide with sLOX-3 as determined by Surface Plasmon Resonance (SPR) was 3.59x10(-9) M. The kinetic constant (K(i)) and IC(50), as determined biochemical methods were 7.41x10(-8) M and 0.15x10(-6) M respectively.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Breast Neoplasms; Drug Design; Drug Screening Assays, Antitumor; Enzyme Activation; Female; Glycine max; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Lipoxygenase; Lipoxygenase Inhibitors; Protein Binding; Surface Plasmon Resonance

PTH-induced osteoblast proliferation may contribute to its anabolic effects in bone. Since PTH-dependent osteoblast-like cell (Ob) growth is mediated via protein kinase C (PKC) and MAP kinase-kinase (MEK) and since lipoxygenase (LO) products activate PKC in a number of cell types, we assessed the expression of LO pathways in primary human cultured Ob. Ob from pre- or post-menopausal women were cultured and were treated with PTH and assayed for the expression of 12-LO and both type I and type II 15-LO mRNA and for the release their enzymatic products, 12- and 15-hydroxyeicosatetraenoic acid (HETE). Cells were also treated with PTH for stimulation DNA synthesis. First, Ob express platelet type- 12-LO and both type I and type II 15-LO mRNA and release their enzymatic products, 12- and 15-hydroxyeicosatetraenoic acid (HETE). Second, in female Ob, PTH induced a rapid increase in 12-HETE (50 fold increase) and 15-HETE (80 fold increase) and increased the expression of 12-LO mRNA but not of the two isoforms of 15-LO. PTH as well as 12 and 15-HETE stimulated DNA synthesis in Ob. The LO inhibitor baicalein inhibited PTH-stimulated DNA synthesis, which was reversed in the presence of either 12- or 15-HETE. A PKC inhibitor (bisindolylmaleimide I) as well as a MEK inhibitor (PD 98059) completely inhibited the stimulation of DNA synthesis by PTH, 12-HETE and the combination of PTH and 12-HETE. In contrast, 15-HETE-induced DNA synthesis was not abolished by these inhibitors. Further, 15-HETE partially restored the stimulatory effect of PTH on DNA synthesis in cells treated with PKC or MEK inhibitors. Finally, PTH- induced ERK1/2 phosphorylation, was blocked by a MEK inhibitor. These results demonstrate a novel mechanism of PTH-induced human bone cell proliferation operating through LO enzymes.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Cells, Cultured; DNA; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Hydroxyeicosatetraenoic Acids; Isoenzymes; MAP Kinase Kinase 1; Osteoblasts; Parathyroid Hormone; Protein Kinase C

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a member of the nuclear receptor family that has been implicated in cell differentiation and proliferation, glucose metabolism, macrophage development, and inflammatory responses. PPAR-gamma can be activated by a range of synthetic substances and also by products of lipid oxidation such as oxidized low-density lipoprotein, 13-hydroxyoctadecadienoic acid (13-HODE) and 15-hydroxyeicosatetraenoic acid (15-HETE). Since 12- and 15-lipoxygenase (12- and 15-LO) are expressed in human vascular smooth muscle cells (VSMCs), we set out to investigate the possible relation between PPAR-gamma and LO system in these cells.. In vitro experiments in human VSMC using standard methods.. The LO products, 12-HETE (10(-7) mol/l), 15-HETE (10(-7) mol/l) and 13-HODE (10(-7) mol//l) increased the expression of PPAR-gamma-2 messenger RNA (mRNA) in VSMC (by 100, 50, and 100%, respectively. Rosiglitazone (1-10 micromol/l) was found to upregulate both the mRNA expression of two LO enzymes, platelet-type 12-lipoxygenase (12-LO; +70%) and 15-lipoxygenase type 2 (15-LO; +60%), and the secretion of their eicosanoid products 12- and 15-HETE. In addition, rosiglitazone-induced a threefold increase in PPAR-gamma-2 mRNA expressions and modest 50% rise in PPAR-gamma-1 mRNA expression. The effect of rosiglitazone on PPAR-gamma-2 could be entirely blocked by the LO inhibitor baicalein and restored by the addition of exogenous 12-HETE.. These results suggest a novel amplification cycle in which PPAR-gamma activation induces production of 12- and 15-LO-derived metabolites which in turn feed back to upregulate PPAR-gamma-2's own expression. The implications of this link in VSMC pathophysiology remain to be elucidated.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Cells, Cultured; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Hypoglycemic Agents; Muscle, Smooth, Vascular; PPAR gamma; RNA, Messenger; Rosiglitazone; Thiazolidinediones; Umbilical Cord; Up-Regulation

Monocyte chemoattractant protein (MCP)-1 plays a key role in atherosclerosis and inflammation associated with visceral adiposity by inducing mononuclear cell migration. Evidence shows that mouse peritoneal macrophages (MPM) express a 12-lipoxygenase (12/15-LO) that has been clearly linked to accelerated atherosclerosis in mouse models and increased monocyte endothelial interactions in both rodent and human cells. However, the role of 12/15-LO products in regulating MCP-1 expression in macrophages has not been clarified. In this study, we tested the role of 12/15-LO products using MPM and the mouse macrophage cell line, J774A.1 cells. We found that 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] increased MCP-1 mRNA and protein expression in J774A.1 cells and MPM. In contrast, 12(R)-HETE, a lipid not derived from 12/15-LO, did not affect MCP-1 expression. 15(S)-HETE also increased MCP-1 mRNA expression, but the effect was less compared with 12(S)-HETE. MCP-1 mRNA expression was upregulated in a macrophage cell line stably overexpressing 12/15-LO (Plox-86 cells) and in MPM isolated from a 12/15-LO transgenic mouse. In addition, the expression of MCP-1 was downregulated in MPM isolated from 12/15-LO knockout mice. 12(S)-HETE-induced MCP-1 mRNA expression was attenuated by specific inhibitors of protein kinase C (PKC) and p38 mitogen-activated protein kinase (p38). 12(S)-HETE also directly activated NADPH oxidase activity. Two NADPH oxidase inhibitors, apocynin and diphenyleneiodonium chloride, blocked 12(S)-HETE-induced MCP-1 mRNA. Apocynin attenuated 12(S)-HETE-induced MCP-1 protein secretion. These data show that 12(S)-HETE increases MCP-1 expression by inducing PKC, p38, and NADPH oxidase activity. These results suggest a potentially important mechanism linking 12/15-LO activation to MCP-1 expression that induces inflammatory cell infiltration.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Acetophenones; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Cell Line; Chemokine CCL2; Enzyme Activation; Hydroxyeicosatetraenoic Acids; Imidazoles; Indoles; Macrophages; Male; Maleimides; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; NADPH Oxidases; Naphthalenes; Onium Compounds; p38 Mitogen-Activated Protein Kinases; Protein Kinase C; Protein Kinase Inhibitors; Pyridines; RNA, Messenger; Signal Transduction; Transfection; Up-Regulation

12/15-Lipoxygenase (LOX) mediates immune-regulatory activities not accounted for by its known free acid eicosanoids, suggesting that additional lipids may be generated by activated cells. To characterize novel LOX-derived lipids, a lipidomic approach was utilized. Ionophore-activated interleukin-4-treated human peripheral monocytes generated up to 10-fold more esterified 15-hydroxyeicosatetraenoic acid (15-HETE) than free in a phosphatidylinositol 3-kinase- and protein kinase C-sensitive manner. Precursor scanning electrospray ionization/tandem spectroscopy for m/z 319 (HETE, [M-H](-)) showed 4 ions at m/z 738, 764, 766, and 782 that were identified using tandem spectroscopy and MS3 as specific diacyl and plasmalogen 15-HETE phosphatidylethanolamines. Using H (18)(2)O water, the compounds were shown to form by direct oxidation of endogenous phosphatidylethanolamine (PE) by 15-LOX, with PE being the preferred phospholipid pool containing 15-HETE. Similarly, human platelets generated 4 analogous PE lipids that contained 12-HETE and increased significantly in response to ionophore, collagen, or convulxin. These products were retained in the cells, in contrast to free acids, which are primarily secreted. Precursor scanning of platelet extracts for the major platelet-derived prostanoid, thromboxane B2 (m/z 369.2), did not reveal PE esters, indicating that this modification is restricted to the LOX pathway. In summary, we show formation of PE-esterified HETEs in immune cells that may contribute to LOX signaling in inflammation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Blood Platelets; Coculture Techniques; Collagen; Crotalid Venoms; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-4; Ionophores; Lectins, C-Type; Monocytes; Oxidation-Reduction; Phosphatidylethanolamines; Platelet Activation; Signal Transduction; Spectrometry, Mass, Electrospray Ionization

Arachidonic acid (AA) metabolism in the non-pregnant sheep uterus was studied in vitro using conventional chromatographic and HPLC techniques. High expression of both lipoxygenase (LOX) as well as cyclooxygenase (COX) enzymes and their activities was found in the uterine tissues. On incubation of uterine enymes with AA, the LOX products formed were identified as 5-hydroperoxyeicosatetraenoic acid (5-HPETE), 12- and 15-hydroxyeicosatetraenoic acids (12- and 15-HETEs), based on their separation on TLC and HPLC. By employing differential salt precipitation techniques, the LOXs generating products 5-HPETE (5-LOX), 12-HETE and 15-HETE (12- and 15-dual LOX) were isolated. Based on their analysis on TLC, the COX products formed were identified as prostaglandins - PGF2alpha and prostacyclin derivative 6-keto PGF1alpha. The study forms the first report on the comprehensive analysis on the metabolism of AA in sheep uterus in vitro via the LOX and COX pathways.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 6-Ketoprostaglandin F1 alpha; Animals; Arachidonic Acid; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Dinoprost; Female; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipoxygenase; Prostaglandin-Endoperoxide Synthases; Sheep; Uterus

The 12/15 lipoxygenase (12/15LOX) enzyme is increased in pathologically affected frontal and temporal regions of Alzheimer's disease (AD) brains compared with controls. Herein, we measured 12(S)-HETE and 15(S)-HETE levels, products of 12/15LOX, in cerebrospinal fluid (CSF) of normal individuals, subjects with mild cognitive impairment (MCI) and AD. Compared with controls, there was a significant increase of both metabolites in CSF from AD and MCI, which correlated with lipid peroxidation and tau protein levels. These results suggest that the activation of this enzyme occurs early in the course of AD, before the onset of overt dementia, thereby implicating 12/15LOX-mediated lipid peroxidation in the pathogenesis of AD.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Aged; Alzheimer Disease; Apolipoproteins E; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Case-Control Studies; Cognition Disorders; Enzyme-Linked Immunosorbent Assay; Female; Humans; Hydroxyeicosatetraenoic Acids; Male; Statistics as Topic; tau Proteins

Mammalian lipoxygenases (LOXs) are categorized with respect to their positional specificity of arachidonic acid oxygenation. Site-directed mutagenesis identified sequence determinants for the positional specificity of these enzymes, and a critical amino acid for the stereoselectivity was recently discovered. To search for sequence determinants of murine (12R)-LOX, we carried out multiple amino acid sequence alignments and found that Phe(390), Gly(441), Ala(455), and Val(631) align with previously identified positional determinants of S-LOX isoforms. Multiple site-directed mutagenesis studies on Phe(390) and Ala(455) did not induce specific alterations in the reaction specificity, but yielded enzyme species with reduced specific activities and stereo random product patterns. Mutation of Gly(441) to Ala, which caused drastic alterations in the reaction specificity of other LOX isoforms, failed to induce major alterations in the positional specificity of mouse (12R)-LOX, but markedly modified the enantioselectivity of the enzyme. When Val(631), which aligns with the positional determinant Ile(593) of rabbit 15-LOX, was mutated to a less space-filling residue (Ala or Gly), we obtained an enzyme species with augmented catalytic activity and specifically altered reaction characteristics (major formation of chiral (11R)-hydroxyeicosatetraenoic acid methyl ester). The importance of Val(631) for the stereo control of murine (12R)-LOX was confirmed with other substrates such as methyl linoleate and 20-hydroxyeicosatetraenoic acid methyl ester. These data identify Val(631) as the major sequence determinant for the specificity of murine (12R)-LOX. Furthermore, we conclude that substrate fatty acids may adopt different catalytically productive arrangements at the active site of murine (12R)-LOX and that each of these arrangements may lead to the formation of chiral oxygenation products.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Amino Acid Sequence; Amino Acid Substitution; Animals; Arachidonate 12-Lipoxygenase; Binding Sites; Humans; Hydroxyeicosatetraenoic Acids; Mice; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Rabbits; Recombinant Proteins; Sequence Homology, Amino Acid; Stereoisomerism; Substrate Specificity; Valine

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

Plasmodium falciparum digests up to 75% of erythrocyte (red blood cell [RBC]) hemoglobin and forms hemozoin. Phagocytosed hemozoin and trophozoites inhibit important monocyte functions. Delipidized trophozoites and hemozoin were remarkably less toxic to monocytes. Parasitized RBCs and hemozoin contained large amounts of mostly esterified monohydroxy derivatives (OH-PUFAs), the stable end products of peroxidation of polyenoic fatty acids. The concentrations of OH-PUFA were 1.8 micromoles per liter RBCs in nonparasitized RBCs, 11.1 micromoles per liter RBCs in rings, 35 micromoles per liter RBCs in trophozoites; and approximately 90 micromoles per liter RBC equivalents in hemozoin. In parasitized RBCs and hemozoin a complex mixture of monohydroxy derivatives of arachidonic (HETEs) and linoleic (HODEs) acid was determined. Respectively, 13- and 9-HODE and 9- and 12-HETE were predominant in hemozoin and parasitized RBCs. The estimated concentrations of all HETE isomers were 33 and 39 micromoles per liter RBCs or RBC equivalents in trophozoites and hemozoin, respectively. No evidence of lipoxygenase activity was found, whereas the large number of positional and optical isomers, the racemic structure, and their generation by incubation of arachidonic acid with hemozoin indicated nonenzymatic origin via heme-catalysis. Sub/low micromolar concentrations of 12- and 15-HETE were toxic to monocytes, whereas HODE isomers were ineffective. Low micromolar concentrations of HETE isomers were estimated to be similarly present in monocytes after phagocytosis of trophozoites or hemozoin. Thus, specific products of heme-catalyzed lipid peroxidation appear to contribute to hemozoin toxicity to phagocytes and may thus play a role in increased cytoadherence, vascular permeability, and chemotaxis, as well as in immunodepression in malaria.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Catalysis; Erythrocytes; Fatty Acids; Hemeproteins; Humans; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Lipid Peroxidation; Malaria; Malaria, Falciparum; Monocytes; Phagocytosis; Respiratory Burst

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

Norepinephrine (NE) stimulates phospholipase D (PLD) through a Ras/MAPK pathway in rabbit vascular smooth muscle cells (VSMC). NE also activates calcium influx and calmodulin (CaM)-dependent protein kinase II-dependent cytosolic phospholipase A(2) (cPLA(2)). Arachidonic acid (AA) released by cPLA(2)-catalyzed phospholipid hydrolysis is then metabolized into hydroxyeicosatetraenoic acids (HETEs) through lipoxygenase and cytochrome P450 4A (CYP4A) pathways. HETEs, in turn, have been shown to stimulate Ras translocation and to increase MAPK activity in VSMC. This study was conducted to determine the contribution of cPLA(2)-derived AA and its metabolites (HETEs) to the activation of PLD. NE-induced PLD activation was reduced by two structurally distinct CaM antagonists, W-7 and calmidazolium, and by CaM-dependent protein kinase II inhibition. Blockade of cPLA(2) activity or protein depletion with selective cPLA(2) antisense oligonucleotides abolished NE-induced PLD activation. The increase in PLD activity elicited by NE was also blocked by inhibitors of lipoxygenases (baicalein) and CYP4A (17-octadecynoic acid), but not of cyclooxygenase (indomethacin). AA and its metabolites (12(S)-, 15(S)-, and 20-HETEs) increased PLD activity. PLD activation by AA and HETEs was reduced by inhibitors of Ras farnesyltransferase (farnesyl protein transferase III and BMS-191563) and MEK (U0126 and PD98059). These data suggest that HETEs are the mediators of cPLA(2)-dependent PLD activation by NE in VSMC. In addition to cPLA(2), PLD was also found to contribute to AA release for prostacyclin production via the phosphatidate phosphohydrolase/diacylglycerol lipase pathway. Finally, a catalytically inactive PLD(2) (but not PLD(1)) mutant inhibited NE-induced PLD activity, and PLD(2) was tyrosine-phosphorylated in response to NE by a MAPK-dependent pathway. We conclude that NE stimulates cPLA(2)-dependent PLD(2) through lipoxygenase- and CYP4A-derived HETEs via the Ras/ERK pathway by a mechanism involving tyrosine phosphorylation of PLD(2) in rabbit VSMC.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Aorta; Benzylamines; Cells, Cultured; Cytosol; Enzyme Activation; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Imidazoles; Male; Muscle, Smooth, Vascular; Norepinephrine; Phospholipase D; Phospholipases A; Phospholipases A2; Rabbits; Recombinant Proteins; Sulfonamides; Transfection

Formation of the 12R-lipoxygenase product, 12R-hydroperoxyeicosatetraenoic acid (12R-HPETE), has been detected previously only in human skin (Boeglin et al. (1998) Proc. Natl. Acad. Sci. USA 95, 6744). The unexpected appearance of an EST sequence (AA649213) for human 12R-lipoxygenase from germinal center B lymphocytes purified from human tonsils prompted our search for the existence of the enzyme in this novel source. Incubation of [1-14C]arachidonic acid with homogenates of human tonsillar tissue yielded mixtures of radiolabeled 12-HETE and 15-HETE. Stereochemical analysis showed varying ratios of 12S- and 12R-HETE, while 15-HETE was exclusively of the S-configuration. Using stereospecifically labeled [10S-3H]- and [10R-3H]arachidonic acid substrates we detected pro-R hydrogen abstraction at carbon 10 associated with formation of 12R-HETE. This mechanistic evidence implicates a 12R-lipoxygenase in the biosynthesis of 12R-HETE. The mRNA for the enzyme was identified in tonsils by RT-PCR and Northern analysis. The cellular distribution was established by in situ hybridization. Unexpectedly, hybridization was not observed in the lymphocytes of the germinal centers. Specific reaction was restricted to squamous epithelial cells, including the epithelium lining the tonsillar crypts. In this location the 12R-lipoxygenase might help regulate differentiation of the epithelium or participate in lymphocyte- epithelial cell interactions.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Child; Child, Preschool; Chromatography, High Pressure Liquid; Epithelial Cells; Expressed Sequence Tags; Germinal Center; Humans; Hydroxyeicosatetraenoic Acids; In Situ Hybridization; Palatine Tonsil; Prostaglandins; RNA, Messenger

The monohydroxylated fatty acid content of peripheral blood mononuclear cells from 23 cleanup workers and 16 unexposed individuals was studied in relation to their immune status after the Chernobyl accident. Men with absorbed doses below 0.32 Gy showed higher levels of free and esterified 12-hydroxyeicosatetraenoic acid (12-HETE) than unexposed men, whereas 15-HETE and the 17-hydroxy derivative of C22 fatty acid (17-OH 22), either free or esterified in phospholipids, were increased in a dose-dependent manner. The percentage of CD4-positive cells was also increased significantly in heavily irradiated men, whereas the percentage of CD8-positive cells tended to decrease with dose. Furthermore, the absolute count of CD4-positive cells was correlated positively with the amount of esterified 15-HETE in the phospholipid fraction of the mononuclear cells and with the total 15-HETE. These results show for the first time that the accumulation of autoxidized/lipoxygenase products of polyunsaturated fatty acids in the mononuclear cells of irradiated individuals was associated with immune imbalance. This may be the basis for certain late effects of radiation such as autoimmune disorders, somatic and neoplastic diseases, and early aging.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Aged; CD4 Antigens; CD8 Antigens; HLA-DR Antigens; Humans; Hydroxyeicosatetraenoic Acids; Immunity; Leukocytes, Mononuclear; Middle Aged; Power Plants; Radioactive Hazard Release; Ukraine

Some cocoas and chocolates are rich in (-)-epicatechin and its related oligomers, the procyanidins. Fractions of these compounds, isolated from the seeds of Theobroma cacao, caused dose-dependent inhibition of isolated rabbit 15-lipoxygenase-1 with the larger oligomers being more active; the decamer fraction revealed an IC50 of 0.8 microM. Among the monomeric flavanols, epigallocatechin gallate (IC50 = 4 microM) and epicatechin gallate (5 microM) were more potent than (-)-epicatechin (IC50 = 60 microM). (-)-Epicatechin and procyanidin nonamer also inhibited the formation of 15-hydroxy-eicosatetraenoic acid from arachidonic acid in rabbit smooth muscle cells transfected with human 15-lipoxygenase-1. In contrast, inhibition of the lipoxygenase pathway in J774A.1 cells transfected with porcine leukocyte-type 12-lipoxygenase (another representative of the 12/15-lipoxygenase family) was only observed upon sonication of the cells, suggesting a membrane barrier for flavanols in these cells. Moreover, epicatechin (IC50 approx. 15 microM) and the procyanidin decamer inhibited recombinant human platelet 12-lipoxygenase. These observations suggest general lipoxygenase-inhibitory potency of flavanols and procyanidins that may contribute to their putative beneficial effects on the cardiovascular system in man. Thus, they may provide a plausible explanation for recent literature reports indicating that procyanidins decrease the leukotriene/prostacyclin ratio in humans and human aortic endothelial cells.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 15-Lipoxygenase; Cacao; Catechin; Cells, Cultured; Eicosanoic Acids; Enzyme Inhibitors; Flavonoids; Glycine max; Humans; Hydroxyeicosatetraenoic Acids; Lipoproteins, LDL; Lipoxygenase Inhibitors; Macrophages; Mice; Phenols; Plant Extracts; Polymers; Rabbits; Reticulocytes; Swine; Transfection

Electrospray ionization ion trap mass spectra of 5-, 12-, and 15-hydroperoxyeicosatetraenoic (HPETE), hydroxyeicosatetraenoic (HETE), and ketoeicosatetraenoic (KETE) acids were recorded. The HPETE were partly dehydrated to the corresponding KETE in the heated capillary of the mass spectrometer. 12-HPETE and 15-HPETE were also converted to KETE by collision-induced dissociation (CID) in the ion trap, whereas CID of 5-HPETE yielded little formation of 5-KETE. Subcellular fractions of bovine corneal epithelium were incubated with arachidonic acid (AA) and the metabolites were analyzed. 15-HETE and 12-HETE were consistently formed, whereas significant accumulation of HPETE and KETE was not detected. Biosynthesis of 12- and 15-HETE was quantified with octadeuterated 12-HETE and 15-HETE as internal standards. The average biosynthesis of 15-HETE and 12-HETE from 30 microM AA by the cytosol was 38 +/- 8 and below 3 ng/mg protein/30 min, respectively, which increased to 78 +/- 21 and 10 +/- 4 ng/mg protein/30 min in the presence of 1 mM free Ca2+. The microsomal biosynthesis was unaffected by Ca2+. The microsomes metabolized AA to 15-HETE as the main metabolite at a low protein concentration (0.3 mg/mL), whereas 12-HETE and 15-HETE were formed in a 2:1 ratio at a combined rate of 0.7 +/- 0.2 microg/mg protein/30 min at a high protein concentration (1.8 mg/mL). The level of 12-HETE in corneal epithelial cells was 50 +/- 13 pg/mg tissue, whereas the endogenous amount of 15-HETE was low or undetectable (<3 pg/mg tissue). Incubation of corneas for 20 min at 37 degrees C before processing selectively increased the amounts of 12-HETE in the epithelium fourfold to approximately 0.2 ng/mg tissue. We conclude that 12-HETE is the main endogenously formed lipoxygenase product of bovine corneal epithelium.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Cattle; Chromatography, Liquid; Epithelium, Corneal; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Lipoxygenase; Mass Spectrometry; Subcellular Fractions

The unstable epoxide leukotriene (LT) A(4) is a key intermediate in leukotriene biosynthesis, but may also be transformed to lipoxins via a second lipoxygenation at C-15. The capacity of various 12- and 15-lipoxygenases, including porcine leukocyte 12-lipoxygenase, a human recombinant platelet 12-lipoxygenase preparation, human platelet cytosolic fraction, rabbit reticulocyte 15-lipoxygenase, soybean 15-lipoxygenase and human eosinophil cytosolic fraction, to catalyze conversion of LTA(4) to lipoxins was investigated and standardized against the ability of the enzymes to transform arachidonic acid to 12- or 15-hydroxyeicosatetraenoic acids (HETE), respectively. The highest ratio between the capacity to produce lipoxins and HETE (LX/HETE ratio) was obtained for porcine leukocyte 12-lipoxygenase with an LX/HETE ratio of 0.3. In addition, the human platelet 100000xg supernatant 12-lipoxygenase preparation and the human platelet recombinant 12-lipoxygenase and human eosinophil 100000xg supernatant 15-lipoxygenase preparation possessed considerable capacity to produce lipoxins (ratio 0.07, 0.01 and 0.02 respectively). In contrast, lipoxin formation by the rabbit reticulocyte and soybean 15-lipoxygenases was much less pronounced (LX/HETE ratios <0.002). Kinetic studies of the human lipoxygenases revealed lower apparent K(m) for LTA(4) (9-27 microM), as compared to the other lipoxygenases tested (58-83 microM). The recombinant human 12-lipoxygenase demonstrated the lowest K(m) value for LTA(4) (9 microM) whereas the porcine leukocyte 12-lipoxygenase had the highest V(max). The profile of products was identical, irrespective of the lipoxygenase used. Thus, LXA(4) and 6S-LXA(4) together with the all-trans LXA(4) and LXB(4) isomers were isolated. Production of LXB(4) was not observed with any of the lipoxygenases. The lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate was considerably more efficient to inhibit conversion of LTA(4) to lipoxins, as compared to the inhibitory effect on 12-HETE formation from arachidonic acid (IC(50) 1 and 50 microM, respectively) in the human platelet cytosolic fraction.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Blood Platelets; Enzyme Inhibitors; Humans; Hydroxyeicosatetraenoic Acids; Leukocytes; Leukotriene A4; Lipoxins; Rabbits; Reticulocytes; Swine

The purpose of this study was to investigate the effects of aspirin-like drugs on nitric oxide (NO) synthesis in rat vascular smooth muscle cells (VSMCs). We measured the accumulation of nitrite, a stable oxidation product of NO, and the expression of inducible NO synthase (iNOS) mRNA and protein in rat cultured VSMCs. Sodium salicylate, aspirin, and indomethacin dose-dependently enhanced nitrite production by interleukin (IL)-1beta-stimulated VSMCs at therapeutic plasma concentration ranges. Increased nitrite production by aspirin-like drugs was accompanied by increased iNOS mRNA and protein accumulation in VSMCs. Addition of IL-1beta activated nuclear factor kappaB (NF-kappaB) in VSMCs, but sodium salicylate did not affect IL-1beta-induced NF-kappaB activation. The nonselective lipoxygenase (LO) inhibitor nordihydroguaiaretic acid inhibited sodium salicylate-induced nitrite production, whereas the selective 5-LO inhibitor caffeic acid did not influence production of nitrite. The 12-LO product 12-HETE dose-dependently enhanced nitrite production by IL-1beta-stimulated VSMCs, whereas the 15-LO product 15-HETE did not. Our study demonstrates that aspirin and the aspirin-like drugs, sodium salicylate and indomethacin, increase NO synthesis in IL-1beta-stimulated VSMCs by upregulation of iNOS transcription via a 12-LO pathway. These effects were independent of NF-kappaB activation. In addition to the direct inhibition of platelet function, aspirin-like drugs may contribute to the reduction of atherothrombotic risk in myocardial ischemia via enhancing NO production by VSMCs.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Anti-Inflammatory Agents, Non-Steroidal; Aspirin; Cells, Cultured; Dose-Response Relationship, Drug; Hydroxyeicosatetraenoic Acids; Indomethacin; Muscle, Smooth, Vascular; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Rats, Sprague-Dawley; Sodium Salicylate

This study investigates the production of interleukin (IL-)1beta by cultured human bone marrow stromal cells. RT-PCR experiments indicate that two-thirds of cultures constitutively express IL-1beta mRNA transcripts. Their cell-associated IL-1beta levels are elevated after stimulation with tumour necrosis factor (TNF-)alpha but not with cytokines such as IL-1alpha, IL-3, IL-4, IL-6, IL-7, IL-10, SCF, G-CSF, M-CSF and TGF-beta or lipid mediators such as PGE2, LTB4, LXA4, LXB4, 12-HETE, 15-HETE and PAF. Addition of IL-4, but not IL-10 or TGF-beta, reduces the TNF-alpha-induced cell-associated IL-1beta. IL-1beta is never detected in bone marrow stromal cell supernatants whatever the stimulant added. In conclusion the pro-inflammatory molecule TNF-alpha stimulates bone marrow stromal cell-associated IL-1beta levels while the anti-inflammatory cytokine IL-4 reduces the TNF-alpha-induced effect. These results strengthen the key regulatory role of IL-4 on the production of haematopoietic cytokines by human bone marrow stromal cells.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Bone Marrow Cells; Cells, Cultured; Cytokines; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-1; Leukotriene B4; Lipid Metabolism; Lipoxins; Platelet Activating Factor; Stromal Cells

The influence of lipoxygenase metabolites of arachidonic acid on proliferation and differentiation of CD34(+) cells was studied. Their effects on the CFU-GM and BFU-E progenitors were investigated by culture of CD34(+) cells in liquid or semisolid medium. Only 12-HETE (1 microM) stimulated the [(3)H]thymidine as well as BrdU incorporation and increased the number of cell divisions (PKH2 tracking). Addition of 12-HETE and 15-HETE but not of LXA(4), LXB(4), LTB(4), and LTC(4) to liquid cultures of CD34(+) cells for 3 and 8 days reduced in a time-dependent manner the number of CFU-GM and BFU-E. Both HETEs also increased the percentage of glycophorin A(+) cells while they reduced the percentage of CD34(-)/CD33(+) cells after 3 and 5 days of liquid cultures. These results show that HETE treatment stimulates proliferation and accelerates the differentiation of CD34(+) cells, mostly toward the erythroid lineage.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Antigens, CD34; Antigens, Differentiation; Arachidonic Acids; Cell Count; Cell Division; Colony-Forming Units Assay; Culture Media; Dose-Response Relationship, Drug; Erythroid Precursor Cells; Erythropoietin; Hematopoietic Stem Cells; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxins; Lipoxygenase; Methylcellulose; Solutions; Thymidine; Time Factors

Bone marrow stromal cells regulate marrow haematopoiesis by secreting interleukins (IL) such as IL-8. Lipid mediators modulate IL-8 synthesis in numerous cell types. We have investigated the effects of 5 lipid mediators (PAF, PGE(2), LTB(4), 12-HETE and 15-HETE) on the spontaneous and cytokine-induced IL-8 synthesis by human bone marrow stromal cells. By using reverse-transcriptase polymerase chain reaction (RT-PCR) we demonstrate that these cells constitutively express IL-8 transcripts. By using a specific ELISA, we found that the production of IL-8 by marrow stromal cells is enhanced after stimulation with 12-HETE (1 microM) both in serum-free and serum-containing culture medium. LTB(4)(1 microM) enhances IL-8 production only in serum-supplemented medium. PAF, PGE(2)and 15-HETE (1 microM to 0.1 nM) have no effect on the spontaneous and serum-induced production of IL-8 by human bone marrow stromal cells. PGE(2)(1 microM or 10 nM) reduces marrow stromal cell IL-8 synthesis in response to IL-1alpha or TNF-alpha. In contrast, PAF, 12-HETE, 15-HETE and LTB(4)have no effect. In conclusion, various lipid mediators modulate the spontaneous, serum- or cytokine-induced IL-8 synthesis by bone marrow stromal cells, highlighting, for the first time, their potential role in the regulation of IL-8 production within the human bone marrow.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acids; Bone Marrow Cells; Cells, Cultured; Dinoprostone; Gene Expression Regulation; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-8; Leukotriene B4; Reverse Transcriptase Polymerase Chain Reaction; Stromal Cells; Transcription, Genetic

The development of atherosclerosis is accelerated in individuals with type 2 diabetes. Adhesion of monocytes to the vascular endothelium is a key initial step in atherogenesis. We have previously shown that monocyte adhesion to human aortic endothelial cells (HAECs) cultured long-term in high-glucose medium (25 mmol/L, 2 passages) is increased compared with cells grown in normal glucose (5 mmol/L). One potential mechanism for increased monocyte adhesion to HAECs under hyperglycemic conditions is via the 12-lipoxygenase (12-LO) pathway. In this study, we demonstrated in HAECs that the major LO metabolite of arachidonic acid was the 12-LO product, 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], which was increased severalfold in HAECs cultured under high-glucose conditions. Furthermore, treatment of HAECs with 12(S)-HETE induced monocyte, but not neutrophil, adhesion an average of 3-fold (range of 1.5- to 5-fold) compared with untreated cells (75+/-5 versus 26+/-1 monocytes per field, respectively, P<0.001). Expression of the adhesion molecules vascular cell adhesion molecule-1, E-selectin, and intercellular adhesion molecule-1 was not significantly increased. However, both glucose and 12(S)-HETE induced a 60% increase in HAEC surface expression of connecting segment-1 (ie, CS-1) fibronectin, a ligand for very late-acting antigen-4 (VLA-4). The antibodies used to block monocyte integrin VLA-4 and leukocyte function-related antigen-1, a monocytic counterreceptor for intercellular adhesion molecule-1, inhibited the ability of both 12-LO products and high glucose to induce monocyte adhesion. These results definitively demonstrate for the first time in HAECs that the 12-LO pathway can induce monocyte-endothelial cell interaction and that the effects of glucose may be mediated, at least in part, through this pathway. Thus, these results suggest that the 12-LO pathway may play a role in the increased susceptibility of diabetics to atherosclerosis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Antibodies, Blocking; Antigens, CD; Aorta; Arteriosclerosis; CD18 Antigens; Cell Adhesion; Cells, Cultured; E-Selectin; Endothelium, Vascular; Glucose; Humans; Hydroxyeicosatetraenoic Acids; Hyperglycemia; Integrin alpha4; Intercellular Adhesion Molecule-1; Lipoxygenase; Monocytes; Neutrophils; Vascular Cell Adhesion Molecule-1

The studies presented here explore intracellular signals resulting from the action of repellents on growth cones. Growth cone challenge with thrombin or thrombin receptor-activating peptide (TRAP) triggers collapse via a receptor-mediated process. The results indicate that this involves activation of cytosolic phospholipase A(2) (PLA(2)) and eicosanoid synthesis. The collapse response to repellents targets at least two functional units of the growth cone, the actin cytoskeleton and substratum adhesion sites. We show in a cell-free assay that thrombin and TRAP cause the detachment of isolated growth cones from laminin. Biochemical analyses of isolated growth cones reveal that thrombin and TRAP stimulate cytosolic PLA(2) but not phospholipase C. In addition, thrombin stimulates synthesis of 12- and 15-hydroxyeicosatetraenoic acid (HETE) from the released arachidonic acid via a lipoxygenase (LO) pathway. A selective LO inhibitor blocks 12/15-HETE synthesis in growth cones and inhibits thrombin-induced growth cone collapse. Exogenously applied 12(S)-HETE mimics the thrombin effect and induces growth cone collapse in culture. These observations indicate that thrombin-induced growth cone collapse occurs by a mechanism that involves the activation of cytosolic PLA(2) and the generation of 12/15-HETE.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Cells, Cultured; Cerebral Cortex; Eicosanoids; Embryo, Mammalian; Enzyme Activation; Growth Cones; Hydroxyeicosatetraenoic Acids; Peptide Fragments; Phospholipases A; Rats; Rats, Sprague-Dawley; Thrombin

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

In summary, we suggest that hyperglycemia causes upregulation of 12-lipoxygenase activity. The increased production of 12-LO products, 12(S) and 15(S)-HETE, activates monocyte integrins which result in enhanced adhesion of monocytes to endothelium. The binding of monocytes to endothelium is a key early event in development of atherosclerosis. Upregulation of this process by vascular cells exposed to chronic elevations in glucose may be one explanation for the accelerated atherosclerosis observed in patients with Type 2 diabetes.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Arteriosclerosis; Cell Adhesion; Cell Communication; Cells, Cultured; Diabetic Angiopathies; Endothelium, Vascular; Glucose; Humans; Hydroxyeicosatetraenoic Acids; Hyperglycemia; Lipoxygenase Inhibitors; Monocytes; Signal Transduction

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

The objectives of this work are to investigate the incorporation of arachidonic acid (AA) in the human myeloma cell lines OPM2, U266 and IM9, and to assess the effect of AA and lipoxygenase products of AA on their growth. The kinetics of acylation of [3H]AA indicates that myeloma cells incorporate AA into their membrane phospholipids and triglycerides. PLA2-treatment and base hydrolysis experiments confirm that [3H]AA is incorporated unmodified in U266, IM9 and OPM2 phospholipids, and is linked by an ester bond. Prelabeling-chase experiments indicate no trafficking of labeled AA among the various phospholipid species. Addition of AA and lipoxygenase products of AA (leukotriene B4 and C4, lipoxin A4 and B4, 12- and 15-hydroxyeicosatetraenoic acid) have no effect on U266, IM9 and OPM2 proliferation assessed by [3H]thymidine incorporation into DNA. In conclusion, while human myeloma cells readily incorporate AA in their membrane phospholipids and triglycerides, AA and lipoxygenase products are not important modulators of their proliferation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Acylation; Arachidonic Acid; Cell Division; Cell Membrane; Humans; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Leukotriene C4; Lipoxins; Lipoxygenase; Membrane Lipids; Multiple Myeloma; Phospholipids; Triglycerides; Tumor Cells, Cultured

The purpose of our studies was to examine differentiation-dependent expression of 15-lipoxygenase (15-LO) and prostaglandin H synthase (PGHS) isoforms in cultured normal human tracheobronchial epithelial cells. In the presence of retinoic acid (RA) the cultures differentiated into a mucociliary epithelium. When cultured in RA-depleted media, the cultures differentiated into a squamous epithelium. In the absence of RA the cultures did not express 15-LO or either of the PGHS isoforms. The PGHS-1 isoform was not expressed in RA-sufficient cultures, but both PGHS-2 messenger RNA (mRNA) and protein were strongly expressed, and prostaglandin E2 (PGE2) was produced during the predifferentiation phase. No PGHS-2 expression or PGE2 could be detected in fully differentiated mucociliary cultures. 15-LO showed the opposite expression pattern: neither mRNA nor protein were detected during the predifferentiation stage, but both were strongly expressed once mucous differentiation had occurred. Cytosolic phospholipase A2 protein was expressed throughout all stages of growth and differentiation. The cultures generated no 15-LO metabolites when incubated with 10 microM to 50 microM arachidonic acid (AA) and stimulated with ionophore. However, lysates prepared from such cultures generated 15-hydroxyeicosatetraenoic acid (15-HETE) and 12-HETE from AA, indicating that the cells contained active enzyme. When cultures expressing 15-LO protein were incubated with 10 microM linoleic acid (LA) instead of AA, and were stimulated with ionophore, they generated 13-hydroxy-9,11-octadecadienoic acid. LA rather than AA appeared to be the preferred substrate for the 15-LO enzyme. Our studies indicated that the expression of 15-LO and PGHS-2 is differentiation dependent in airway epithelial cells.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Antithrombins; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Bronchi; Cell Differentiation; Cells, Cultured; Chromatography, High Pressure Liquid; Cilia; Cytosol; Epithelial Cells; Gene Expression Regulation, Enzymologic; Humans; Hydroxyeicosatetraenoic Acids; Isoenzymes; Linoleic Acid; Linoleic Acids; Mucous Membrane; Phospholipases A; Phospholipases A2; Prostaglandin-Endoperoxide Synthases; Retinoids; Trachea

The ubiquitous hydroxylated fatty acids derived from arachidonic acid (HETEs) or linoleic acid (HODEs) exhibit diverse biological effects including chemotaxis, cell proliferation, and modulation of several enzymatic pathways, including the 5-lipoxygenase leading to the inflammatory leukotrienes. It was observed that 12(S)- and 15(S)-HETE and 13(S)-HODE (12- and 15-lipoxygenase-derived metabolites, respectively) inhibited the 5-lipoxygenase present in rat basophilic leukemia (RBL-1) cell homogenates whereas the 15(R) chiral enantiomer and the nonhydroxylated linoleic, oleic, and stearic acids were either less potent or ineffective. In examining the mechanism of this inhibition, the relative effectiveness of several fatty acids in displacing [3H]15-HETE bound to cytosol preparations were compared and the results indicated that these (S) hydroxy fatty acids and 5(S)-HETE were significantly more potent than either the 15(R) enantiomer, 15(S)-HETE methyl ester, arachidonic acid, or prostaglandin F2alpha. In order to identify the protein(s) that specifically binds HETEs, 15(S)-HETE biotin hydrazide was used as a probe to detect any HETE-protein complexes as this compound both inhibited the 5-lipoxygenase and interfered with the binding of [3H]15-HETE to cytosol preparations. SDS-PAGE analysis and chemiluminescent detection revealed that the major cytosolic proteins that bound this biotinylated probe had molecular masses of 43 and 51 kD. Fatty acid competition experiments indicated that the order of effectiveness in displacing this probe from these proteins was 13(S)-HODE > 5(S)-HETE approximately equal to 15(S)-HETE > > stearic acid approximately equal to arachidonic acid approximately equal to 15(R)-HETE. Amino acid sequence analysis showed that the 43 kD protein was actin. These findings suggest the possibility that actin may play a major role in the biological effects of monohydroxylated metabolites derived from cellular 5-, 12-, and 15-lipoxygenases.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Actins; Amino Acid Sequence; Animals; Arachidonate 15-Lipoxygenase; Biotinylation; Carrier Proteins; Cytosol; Dinoprost; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Hydroxyeicosatetraenoic Acids; Kinetics; Leukemia, Basophilic, Acute; Ligands; Linoleic Acid; Linoleic Acids; Molecular Sequence Data; Myelin P2 Protein; Neoplasm Proteins; Nerve Tissue Proteins; Oleic Acid; Rats; Stearic Acids; Stereoisomerism; Structure-Activity Relationship; Tumor Cells, Cultured

In previous studies, we reported that vascular wall cells such as endothelial cells metabolize linoleic acid to 13-hydroxyoctadecadienoic acid (13-HODE) via the 15-lipoxygenase pathway. Endothelial cell 13-HODE levels vary inversely with endothelial cell reactivity to platelets, which, in turn, varies directly with the expression of the vitronectin receptor (VnR) on the apical surface of endothelial cells. We and others have also found that tumour cell adhesivity is dependent, in part, upon the relative amounts of intracellular 13-HODE and the arachidonic acid monohydroxide(s), 12- and/or 15-hydroxyeicosatetraenoic acids (12-, 15-HETE). In addition, we and others have found that platelet adhesivity is dependent upon the intraplatelet level of its major lipoxygenase metabolite, 12-HETE. Finally, we have demonstrated that 13-HODE and VnR co-localize in nonadhesive endothelial cells but dissociate following endothelial cell injury, at which time, the VnR relocates on the endothelial cell apical surface. These data suggest to us that lipoxygenase-derived monohydroxides regulate the ability of various receptors to recognize their specific ligands. The latter data also suggest that these monohydroxides act directly by a physiochemical mechanism. The present study supports this possibility. Thus, we demonstrate that 13-HODE downregulates VnR binding with vitronectin (Vn) > fibronectin (Fn) > fibrinogen (Fgn), whereas 12- and 15-HETE upregulate specific VnR/ligand binding, using purified VnR/liposomes and purified ligands in an adhesion assay; and that 12- and 15-HETE upregulate GPIIb/IIIa:liposome binding of Fgn > Fn > Vn. We conclude that cell-specific monohydroxides influence cell-specific receptor-ligand binding directly through a physiochemical mechanism.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Chromatography, Liquid; Endothelium, Vascular; Fibrinogen; Fibronectins; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acids; Liposomes; Platelet Glycoprotein GPIIb-IIIa Complex; Receptors, Vitronectin; Vitronectin

When human monocytes or alveolar macrophages are cultured in the presence of interleukin (IL)-4 or IL-13, the expression of the reticulocyte-type 15-lipoxygenase is induced. In mice a 15-lipoxygenase is not expressed, but a leukocyte-type 12-lipoxygenase is present in peritoneal macrophages. To investigate whether both lipoxygenase isoforms exhibit a similar regulatory response toward cytokine stimulation, we studied the regulation of the leukocyte-type 12-lipoxygenase of murine peritoneal macrophages by interleukins and found that the activity of this enzyme is upregulated in a dose-dependent manner when the cells were cultured in the presence of the IL-4 or IL-13 but not by IL-10. When peripheral murine monocytes that do not express the lipoxygenase were treated with IL-4 expression of 12/15-lipoxygenase mRNA was induced, suggesting pretranslational control mechanisms. In contrast, no upregulation of the lipoxygenase activity was observed when the macrophages were prepared from homozygous STAT6-deficient mice. Peritoneal macrophages of transgenic mice that systemically overexpress IL-4 exhibited a threefold to fourfold higher 12-lipoxygenase activity than cells prepared from control animals. A similar upregulation of 12-lipoxygenase activity was detected in heart, spleen, and lung of the transgenic animals. Moreover, a strong induction of the enzyme was observed in red cells during experimental anemia in mice. The data presented here indicate that (1) the 12-lipoxygenase activity of murine macrophages is upregulated in vitro and in vivo by IL-4 and/or IL-13, (2) this upregulation requires expression of the transcription factor STAT6, and (3) the constitutive expression of the enzyme appears to be STAT6 independent. The cytokine-dependent upregulation of the murine macrophage 12-lipoxygenase and its induction during experimental anemia suggests its close relatedness with the human reticulocyte-type 15-lipoxygenase despite their differences in the positional specificity of arachidonic acid oxygenation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Anemia; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Cells, Cultured; Enzyme Induction; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-10; Interleukin-13; Interleukin-4; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Monocytes; Organ Specificity; Polymerase Chain Reaction; Signal Transduction; Species Specificity; STAT6 Transcription Factor; Stimulation, Chemical; Trans-Activators

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Humans; Hydroxyeicosatetraenoic Acids; Lung Neoplasms; Neoplasm Metastasis

Human bone marrow stromal cells regulate haematopoiesis by releasing cytokines such as leukaemia inhibitory factor (LIF) and interleukin 6 (IL-6). We have investigated the effects of 5 lipid mediators (i.e. 12-HETE, 15-HETE, platelet-activating factor (PAF), LTB4 and prostaglandin E2 (PGE2)) on their LIF and IL-6 synthesis. 12-HETE, 15-HETE and LTB4 (both at 1 microM) stimulate the LIF production by human bone marrow stromal cells grown in 10% fetal calf serum (FCS). In contrast, PAF and PGE2 have no effect. 12-HETE (1 microM) enhances LIF synthesis in serum free medium 7.7-fold and stimulates IL-1 induced LIF production. 12-HETE, 15-HETE, PAF and LTB4 have no effect on the spontaneous, serum- and cytokine-induced IL-6 synthesis by bone marrow stromal cells. In contrast PGE2 significantly stimulates serum-induced IL-6 synthesis. This study reports for the first time that lipid mediators may act on human haematopoiesis by modulating LIF and IL-6 synthesis by bone marrow stromal cells. Particularly, 12-HETE enhances LIF but not IL-6 synthesis. The different regulation of IL-6 and LIF synthesis in response to lipid mediators highlights the complexity of the cytokine regulation into the human bone marrow.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Bone Marrow Cells; Cells, Cultured; Culture Media, Serum-Free; Dinoprostone; Enzyme-Linked Immunosorbent Assay; Growth Inhibitors; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-1; Interleukin-6; Leukemia Inhibitory Factor; Leukotriene B4; Lipids; Lymphokines; Platelet Activating Factor; Stromal Cells; Tumor Necrosis Factor-alpha

It has been proposed that 5-lipoxygenase (5-LO)-activating protein (FLAP) is an arachidonate transfer protein for leukotriene biosynthesis. Using the Spodoptera frugiperda (Sf9) insect cells, we demonstrate that FLAP causes a large stimulation (190-fold) of the conversion of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) to 5, 12-diHETE when co-expressed with 5-lipoxygenase. We also demonstrate that FLAP can stimulate (2-2.5-fold) the oxygenation of 15(S)-HETE by 5-LO to 5,15-diHETE. The stimulation of both 12(S)-HETE and 15(S)-HETE oxygenation by 5-LO is completely inhibitable by the FLAP inhibitor, MK-886. In order to determine which residues of FLAP are important for 12(S)-HETE and arachidonic acid utilization by 5-LO, various mutants of FLAP were co-expressed with 5-LO in Sf9 cells. The FLAP deletion mutants del 37-53, del 52-58, del 106-108, and del 148-161 and the point mutant D62N were analyzed. The D62N mutation, which reduces the binding of indole inhibitors to FLAP, had no effect on the stimulation of substrate utilization by 5-LO. In contrast to wild type FLAP, the mutant proteins del 37-53, del 106-108, and del 148-161 failed to stimulate 12(S)-HETE and arachidonic acid utilization by 5-LO. Only one of the latter three mutations (del 37-53) has been shown to abolish the binding of indole inhibitors to FLAP. These results suggest that the lipid binding site of FLAP overlaps the inhibitor binding site and occupies several regions of the protein not essential for inhibitor binding. Because FLAP can stimulate the utilization of 12(S)-HETE, 15(S)-HETE, and arachidonic acid by 5-LO, FLAP may also function as a more general lipid carrier protein for the biosynthesis of multiple oxygenation products of arachidonic acid in addition to its role in leukotriene biosynthesis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5-Lipoxygenase-Activating Proteins; Animals; Arachidonate 5-Lipoxygenase; Carrier Proteins; Cell Line; Humans; Hydroxyeicosatetraenoic Acids; Membrane Proteins; Oxygen; Spodoptera

Cytokine induced pancreatic beta-cell destruction seen in Type 1 diabetes and islet graft rejection involves multiple intracellular signaling pathways that directly or indirectly lead to inflammatory damage or programmed cell death. IL-1beta has been shown to stimulate the 12-lipoxygenase pathway product 12-HETE, in RIN m5F cells; however, the precise role of 12-LO activation in mediating cytokine effects is not clear. Since the stress-activated protein kinase, JNK, has been linked to cytokine mediated inflammatory actions, we studied the effect of two LO products, 12-HETE and 15-HETE, on JNK activity. We demonstrate that 1 nM 12-HETE stimulates JNK activity, while 1 nM 15-HETE, the 15-lipoxygenase pathway product, does not. These results suggest 12-HETE is a novel upstream signal for IL-1beta induced JNK activation in RIN m5F cells.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line; Enzyme Activation; Hydroxyeicosatetraenoic Acids; Interleukin-1; Islets of Langerhans; JNK Mitogen-Activated Protein Kinases; Kinetics; Mitogen-Activated Protein Kinases; Phosphorylation; Rats

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

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Cell Communication; Drug Carriers; Endothelium, Vascular; Fibrinogen; Fibronectins; Hydroxyeicosatetraenoic Acids; Kinetics; Linoleic Acids; Liposomes; Platelet Glycoprotein GPIIb-IIIa Complex; Receptors, Vitronectin; Vitronectin

Diets rich in linoleic acid (LA) stimulate the metastasis of MDA-MB-435 human breast cancer cells from the mammary fat pads of nude mice. This omega-6 fatty acid is metabolized to various cyclo-oxygenase and lipoxygenase products, several of which have been previously associated with tumor cell invasion and metastasis. We now report that MDA-MB-435 cells secreted increased levels of prostaglandin E2 (PGE2), and 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE when cultured in the presence of 2.7 microM (0.75 micrograms/ml) LA; 5-HETE secretion was unchanged. The 12-lipoxygenase inhibitor esculetin (20 microM) completely blocked the LA-stimulated 12-HETE secretion. Linoleic acid also increased MDA-MB-435 cell invasion in an in vitro assay; this stimulation was abolished by 20 microM esculetin, but was unaffected by piroxicam, a selective cyclooxygenase inhibitor. The effect of LA on invasion was replicated by 0.1 microM 12-HETE, but not by 5-HETE or PGE2; 15-HETE was stimulatory only at a concentration of 1.0 microM. Zymographic and Northern blot analyses showed that these events are accompanied by the induction of 92 kDa isoform type IV collagenase (metalloproteinase-9) enzymic activity and mRNA expression by exogenous LA and 12-HETE, and their suppression by the 12-lipoxygenase inhibitor. These results suggest that the effects of dietary LA on breast cancer cell metastasis in the nude mouse model are due, at least in part, to enhanced 12-HETE biosynthesis, with an associated increase in proteolytic enzyme activity and tumor cell invasiveness.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Breast Neoplasms; Collagenases; Eicosanoids; Gene Expression; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Lipoxygenase Inhibitors; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; Piroxicam; RNA, Messenger; Tumor Cells, Cultured; Umbelliferones

Recent evidence suggests that lipoxygenase (LO) metabolites inhibit renin production in vitro. However, the physiological significance of this effect has not been determined. This study examined the role of the LO pathway in the regulation of plasma renin concentration (PRC) in vivo. The acute administration of two structurally unrelated LO inhibitors, phenidone (30 and 60 mg/kg) and esculetin (60 mg/kg), resulted in suppression of platelet 12 hydroxyeicosatetraenoic acid (12HETE) production, reduction in systemic arterial pressure and a 2- to 3-fold increase in PRC. To determine whether the esculetin-induced increase in PRC was secondary to hypotension, esculetin was also administered to rats preinfused with a pressor dose of norepinephrine. In these acutely hypertensive rats, esculetin still induced a 2.5-fold increase in PRC, whereas blood pressure remained over 40 mm Hg above basal levels. Further, esculetin (10(-6)M) increased renin release in renal slices from 150 +/- 10 to 310 +/- 20 ng/ml.h (P < 0.05) and this rise was entirely blocked in the presence of 12HETE (10(-7)M; 130 +/- 40 ng/ml.h). In rats placed on high salt intake, 12HETE concentration in renal slices from the outer cortex was considerably higher than in renal slices from salt-restricted rats (116.5 +/- 15.7 vs. 65 +/- 12 pg/mg protein; P < 0.05). Chronic administration of the LO inhibitor phenidone also resulted in an increase of PRC, which was independent of changes in blood pressure. On either high salt (3.15%0 or low salt (0.05%) diet phenidone-treated rats had higher PRC levels than the respective control groups [high salt 9.7 +/- 3.5 vs. 1.9 +/- 1.4 ng/ml.h; P < 0.05; low salt 33.2 +/- 5.3 vs. 19.4 +/- 3.10 ng/ml.h; P < 0.05]. The finding that LO blockers are potent stimulators of PRC in vivo suggests the existence of a physiological tonic inhibition of renin secretion by LO products that is operative under a wide range of salt intake. High salt intake enhances this inhibitory tone by increasing renal cortical 12 LO activity and, in fact, normal suppression of PRC during high salt diet does not occur in LO-blocked animals. Thus, the LO pathway exerts a tonic inhibitory effect on renin release, which appears particularly important for renin suppression during high salt intake.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Blood Platelets; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Kidney; Lipoxygenase Inhibitors; Male; Norepinephrine; Pyrazoles; Rats; Rats, Sprague-Dawley; Renin; Sodium, Dietary; Umbelliferones

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Carbon Radioisotopes; Humans; Hydroxyeicosatetraenoic Acids; Lung Neoplasms; Neoplasm Metastasis

Arachidonic acid (AA) metabolites derived from both cyclooxygenase (COX) and lipoxygenase (LOX) pathways transduce a variety of signals related to cell growth. Here, we report that the AA LOX pathway also functions as a critical regulator of cell survival and apoptosis. Rat Walker 256 (W256) carcinosarcoma cells express 12-LOX and synthesize 12(S)- and 15(S)-hydroxyeicosatetraenoic acids as their major LOX metabolites. W256 cells transfected with 12-LOX-specific antisense oligonucleotide or antisense oligonucleotides directed to conserved regions of LOXs underwent time- and dose-dependent apoptosis. Likewise, treatment of W256 cells with various LOX but not COX inhibitors induced apoptotic cell death, which could be partially inhibited by exogenous 12(S)- or 15(S)-hydroxyeicosatetraenoic acids. The W256 cell apoptosis induced by antisense oligos and LOX inhibitors was followed by a rapid downregulation of bcl-2 protein, a dramatic decrease in the bcl-2/bax ratio, and could be suppressed by bcl-2 overexpression. In contrast, p53, which is wild type in W256 cells, did not undergo alterations during apoptosis induction. The results suggest that the LOX pathway plays an important physiological role in regulating apoptosis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Apoptosis; Arachidonate 12-Lipoxygenase; Base Sequence; Cell Division; Cell Line; Cell Survival; Female; Homeostasis; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Leukotriene C4; Leukotrienes; Lipid Peroxides; Lipoxins; Masoprocol; Molecular Sequence Data; Oligonucleotides, Antisense; Plasmids; Rats; Transfection

Eicosanoids have been implicated in colon carcinogenesis, but very little is known on the potential role of leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) in this process; such compounds are produced by colonocytes and tumor infiltrating leukocytes. We studied the effect of LTB4, LTB4 methyl ester, LTB5, 12(R)-HETE, 12(S)-HETE and 15(S)-HETE (10(-10), 10(-8), 10(-6) M) on the proliferation rate, the cell cycle distribution, and the rate of apoptosis in HT-29 and HCT-15 human colon carcinoma cells. Our data show that LTB4, a lipoxygenase product, increased the proliferation rate of both cell lines in a time- and concentration-dependent manner. In HT-29 cells the concentration-response curve was bell-shaped (maximal effect at 10(-8) M). The proliferative effects of LTB4 in HT-29 cells were inhibited by SC-41930, a competitive antagonist of LTB4, suggesting the existence of an LTB4 receptor in epithelial cells. The methyl ester of LTB4 stimulated the proliferation of these cells, but LTB5, an isomer of LTB4 derived from eicosapentaenoic acid, did not. Of the HETEs, only 12(R)-HETE, a P-450 product, stimulated the proliferation of both cell lines; the other HETEs, all lipoxygenase products, failed to affect the proliferation of these cells. None of these eicosanoids had any effect on cell cycle distribution or apoptosis in either cell line. Taken together with our previous data showing that PGs stimulate colon cancer cell proliferation (Qiao et al. (1995) Biochim. Biophys. Acta 1258, 215-223), these findings indicate that arachidonic acid products synthesized via at least three different pathways (cyclooxygenase, lipoxygenase, P-450) may not be able to modulate the growth of colon cancer, and suggest a potential role in human colon carcinogenesis for LTB4 and 12(R)-HETE.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Anti-Inflammatory Agents, Non-Steroidal; Apoptosis; Benzopyrans; Cell Division; Colonic Neoplasms; Eicosanoids; Flow Cytometry; Humans; Hydroxyeicosatetraenoic Acids; Interphase; Leukotriene B4; Mitosis; Receptors, Leukotriene; S Phase; Tumor Cells, Cultured

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

Arachidonic acid (AA) metabolism via the lipoxygenase (LOX) pathway in rat hearts and in cultured rat cardiomyocytes was investigated using 1-[14C]AA. LOX activity was detected in the microsomal fraction, in the high speed supernatant prepared from rat hearts and in rat cardiomyocyte supernatant. LOX products from all fractions comigrated in thin layer chromatography as 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE. Enzyme linked immunosorbent assay for 12-HETE showed its formation by the microsomal fraction, the ammonium sulfate (AS) pellet, and by rat cardiomyocyte supernatant, while radioimmunoassay for 15-HETE showed its formation only by the AS pellet. The properties of LOX in each fraction are reported here.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Cells, Cultured; Cytosol; Female; Hydroxyeicosatetraenoic Acids; Kinetics; Lipoxygenase; Microsomes; Myocardium; Rats

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

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

These studies characterized a method to measure 15-lipoxygenase (15-LO) activity in human monocytes (HMC) exposed to interleukin-4 (IL-4) and compare the activity with that of soybean 15-LO. 15-LO activity was quantitated by measuring 15-[14C]hydroxyeicosa-5Z,8Z,11Z,13E-tetraenoic acid (15-HETE) production from the substrate [14C]arachidonic acid (AA) after high-performance liquid chromatographic or thin-layer chromatographic separation. 15-HETE production by HMC was significantly elevated after continuous exposure to a single dose (10 ng/ml) of IL-4 for 4 days, was maximal at 5 days, and remained elevated at 6 days. At 6 days 15-LO activity in IL-4-treated HMC was increased significantly (2.81 +/- 0.70 fmol 15-HETE/cell) compared with background levels of 15-HETE in untreated HMC (0.098 +/- 0.31 fmol 15-HETE/cell). 15-HETE production was linear in the range of 5 x 10(4) to 7 x 10(5) HMC/assay, from 2 to 160 microM AA, and during 5-10 min of incubation with AA. Ethyl 2-cyano-3-(3',4'-dihydroxyphenyl)propenoate (a caffeic acid analogue), N-methyl-4-benzyloxy-phenyl acetohydroxamate (RG-6866), esculetin, and four novel lipoxygenase inhibitors, a phenylcyanomethylene analogue (RP-27493) and three benzoxadiazine analogues (RP-64835, RP-65047, and RP-65208), inhibited HMC 15-LO, with concentrations eliciting 50% of maximal inhibition (microM) of 0.21, 0.8, 6.3, 10, 22, 20, 6.3, 3, and 20 and 5.8, 5, 12, 1, 30, 0.8, 0.15, 0.05, and 2.8 for inhibition of soybean 15-LO, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 15-Lipoxygenase; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Glycine max; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-4; Lipoxygenase Inhibitors; Monocytes

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

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

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

The extent of epidermal fatty acid oxygenase activation in non-psoriatic dermatoses and the nature of these oxygenases are not known. The monohydroxylated fatty acid derivatives produced in vivo and trapped in skin scales or produced in vitro by oxygenases preserved in scales were analyzed by high performance liquid chromatography in 10 patients with non-psoriatic dermatoses. Evidence for 15-lipoxygenase activation included the finding of 15(S)-hydroxyeicosatetraenoic acid (HETE) in scales from seven patients and the production of 15(S)-[14C]HETE and 13(S)-[14C]hydroxyoctadecadienoic acid (HODE) during scale incubations, respectively, with [14C]arachidonic and [14C]linoleic acid. Evidence for the activation of an arachidonic acid 12(R)-oxygenase included the finding of 12(R)-HETE in scales from eight patients and the production of 12(R)-[14C]HETE during scale incubations with [14C]arachidonic acid. 13-HODE was the predominant fatty acid derivative present in scale extracts; its lack of enantiopurity (mean S/R = 3.1) and the substantial formation of 9-HODE (mean S/R = 0.6; 9/13-HODE = 0.43) suggest its derivation from 15-lipoxygenase and a second oxygenase. The levels of 15(S)-HETE and 12(R)-HETE had a 125- to 144-fold range and were highest in scales from a patient with erythroderma and in three psoriatic scale samples similarly analyzed. These findings indicate that 15-lipoxygenase, most likely of keratinocyte origin, and an arachidonic acid 12(R)oxygenase of unknown type and cell origin are activated in diverse dermatoses.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Carbon Radioisotopes; Dermatitis, Exfoliative; Enzyme Activation; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Skin; Skin Diseases

Arachidonate 12-lipoxygenase is an enzyme that oxygenates the 12 position of arachidonic acid to produce its 12-hydroperoxy derivative. We were interested in the tissue distribution and subcellular localization of the platelet-type 12-lipoxygenase, which is distinguished from the leukocyte type by several criteria. Antiserum was raised in rabbits against purified recombinant arachidonate 12-lipoxygenase of human platelets. When mouse bone marrow cells and lung were immunostained and observed by light and electron microscopy, the positively stained cells were platelets, megakaryocytes, and eosinophils. 12-Lipoxygenase was localized in the cytoplasm of platelets but was hardly detectable in the plasma membrane and intracellular organelles. The enzyme was found in the cytoplasm of immature megakaryocytes with kidney-shaped nuclei and a few profiles of demarcation membranes, as well as in the mature form with well-developed demarcation membranes. These results indicated the expression of 12-lipoxygenase at an early stage in the course of megakaryocytopoiesis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Antibodies; Arachidonate 12-Lipoxygenase; Blood Platelets; Bone Marrow Cells; Chromatography, High Pressure Liquid; Eosinophils; Hematopoietic Stem Cells; Humans; Hydroxyeicosatetraenoic Acids; Immunohistochemistry; Linoleic Acids; Lung; Megakaryocytes; Mice; Microscopy, Immunoelectron; Recombinant Proteins

15-Lipoxygenase is expressed in foamy macrophages of atherosclerotic lesions and has been implicated in the oxidative modification of low density lipoprotein during early stages of atherogenesis. To establish an animal model of 15-lipoxygenase overexpression, we created transgenic rabbits that express at high level the human 15-lipoxygenase in monocyte-derived macrophages but not in liver, heart, kidney, lung, or other tissues. The expression level of the enzyme in monocyte-derived macrophages is comparable to that of interleukin 4 (IL4)-treated human monocytes, but more than 20-fold higher than in macrophages of normal rabbits. The transgenic enzyme oxygenates linoleic acid to 13S-hydroperoxy-9, 11 (Z,E)-octadecadienoic acid (13-HODE), and arachidonic acid to a mixture of 12S-hydroperoxy-5, 8, 10, 14 (Z,Z,E,Z)-eicosatetraenoic acid (12S-HETE), and 15S-hydroperoxy-5, 8, 11, 14 (Z,Z,Z,E)-eicosatetraenoic acid (15S-HETE). The 12-HETE/15-HETE ratio varied between 0.3 and 5.4, indicating a remarkable variability in the positional specificity of the transgenic enzyme. Macrophages from normal rabbits consistently produced 12S-HETE as the major oxygenation product. 15-Lipoxygenase-overexpressing rabbits may be used for further mechanistic studies on the implication of lipoxygenase in atherogenesis; they are also an ideal model for testing the in vivo action of 15-lipoxygenase inhibitors.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Animals, Genetically Modified; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Base Sequence; Enzyme Induction; Female; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Macrophages; Male; Molecular Sequence Data; Monocytes; Muramidase; Organ Specificity; Polymerase Chain Reaction; Promoter Regions, Genetic; Rabbits; Recombinant Fusion Proteins; Substrate Specificity

We examined the ability of rIL-13 to regulate rIL-l alpha induced IL-1 beta, IL-1 receptor antagonist (IL-1ra) and IL-8 production in cultured peripheral blood mononuclear cells (PBMC), endothelial cells and fibroblasts. Furthermore we examined whether rIL-13 could influence the production of the arachidonic acid products LTB4, 12-HETE and 15-HETE by PBMC. rIL-1 alpha-stimulated PBMC cultures secreted high levels of IL-1 beta and IL-8; this could be inhibited to the level of unstimulated control cells by co-incubation with rIL-13 (10 ng/ml). IL-13 induced a 3-fold increase of the IL-1ra secretion which was inhibited by rIFN-gamma. In the presence of both rIL-1 alpha and rIL-13, endothelial cells increased IL-8 secretion, whereas dermal fibroblasts remained unchanged. Of the arachidonic acid metabolites examined, the greatest change was observed in the formation of 15-HETE. In unstimulated PBMC cultures the amount of 15-HETE was less than 4 ng/10(6) cells, whereas after addition of rIL-13 we measured a formation of 139 +/- 6.2 ng/10(6) cells. The effect of rIL-13 on the 15-HETE formation in PBMC was abolished by addition of 100 U/ml rIFN-gamma. rIL-13 only induced minor changes in the LTB4 and 12-HETE formation. Compared to IL-4, IL-13 induced a similar alteration of the cytokine cascade and arachidonic acid metabolism, supporting the hypothesis that the two cytokines use a common receptor complex or signal pathway.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Anti-Inflammatory Agents, Non-Steroidal; Antigens, CD; Arachidonic Acid; Cells, Cultured; Drug Synergism; Endothelium, Vascular; Fibroblasts; Humans; Hydroxyeicosatetraenoic Acids; Interferon-gamma; Interleukin 1 Receptor Antagonist Protein; Interleukin-1; Interleukin-13; Interleukin-13 Receptor alpha1 Subunit; Interleukin-4; Interleukin-8; Leukocytes, Mononuclear; Leukotriene B4; Mice; Receptors, Interleukin; Receptors, Interleukin-13; Receptors, Interleukin-4; Recombinant Proteins; Sialoglycoproteins; Signal Transduction

Arachidonic acid metabolites have been implicated in the regulation of ACTH secretion. To define further which eicosanoid(s) is primarily involved, we examined the effects of both inhibitors of the three arachidonate metabolic pathways (cyclooxygenase, lipoxygenase, and epoxygenase) and specific eicosanoid products on ACTH secretion by rat pituitary corticotrophs in a microperifusion system. CRF stimulates sustained ACTH release that is mediated by protein kinase-A-induced extracellular Ca2+ (Cae2+) influx via L-type voltage-sensitive calcium channels (VSCC). Arginine vasopressin (AVP) stimulates an initial spike phase of ACTH release that presumably is mediated by inositol 1,4,5-trisphosphate-induced intracellular Ca2+ (Cai2+) release, followed by a sustained plateau phase of ACTH release that is mediated by protein kinase-C-induced Cae2+ influx via L-type VSCC. Pretreatment for 15 min with the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 50 microM), but not the cyclooxygenase inhibitor indomethacin (10 microM) or the epoxygenase inhibitor SKF525A (100 microM) inhibited the sustained response to CRF by 48% and the initial spike response to AVP by 38%. NDGA-induced inhibition was not reversed by indomethacin or SKF525A, alone or in combination, precluding arachidonate shunting into other pathways. However, the results suggested that epoxygenase metabolites may have a minor stimulatory and cyclooxygenase metabolites may have a minor inhibitory effect on ACTH secretion. Preexposure to NDGA suppressed by 43% the sustained response to 8-bromo-cAMP, which directly activates protein kinase-A; by 57% the sustained response to dioctanolglycerol, which directly activates protein kinase-C; and by 59% the spike-type response to ionomycin, which releases Cai2+ by an inositol 1,4,5-trisphosphate-independent mechanism. These results suggest that NDGA either inhibits the production of a lipoxygenase metabolite involved in Cae2+ influx and/or Cai2+ release or acts other than by inhibiting lipoxygenase, such as by directly blocking membrane transport of Cae2+. The three major lipoxygenase metabolites tested, 5(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acid (HETE), all stimulated sustained ACTH release in a dose-dependent manner. At a concentration of 2 microM, 12(S)-HETE was 4.7 and 2.5 times more potent than 5(S)- and 15(S)-HETE, respectively, and completely reversed NDGA inhibition of both CRF- and AVP-stimulated ACTH secretion. The ACTH-releasing activi

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; 8-Bromo Cyclic Adenosine Monophosphate; Adrenocorticotropic Hormone; Animals; Arachidonic Acid; Arginine Vasopressin; Calcium; Cells, Cultured; Corticotropin-Releasing Hormone; Diglycerides; Eicosanoids; Hydroxyeicosatetraenoic Acids; Indomethacin; Ionomycin; Lipoxygenase; Male; Masoprocol; Pituitary Gland, Anterior; Potassium Chloride; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Tritium

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

We have previously demonstrated that peroxisomal-deficient human skin fibroblasts and mutant Chinese hamster ovary cells do not convert 12- and 15-hydroxyeicosatetraenoic acids (HETEs) to chain-shortened, polar metabolites, suggesting that peroxisomes are the intracellular location for beta-oxidation of these compounds. This implies that mitochondria do not beta-oxidize HETEs. To test this hypothesis we incubated highly purified rat liver mitochondria with [3H]12-(S)- and [3H]15-(S)-HETE in the presence of carnitine and an acylcoenzyme A-generating system. Extracts obtained from these incubations were analyzed for radiolabeled polar metabolites. Both HETEs were converted to apparent products of beta-oxidation, although the 12-HETE compound was a markedly better substrate. The presence of 50 microM 2-tetradecyloxirane carboxylate, a potent inhibitor of carnitine palmitoyl transferase, completely blocked 12- and 15-HETE conversion to these metabolites as did omission of carnitine from the medium. These data demonstrate carnitine-dependent beta-oxidation of HETEs in isolated mitochondria and suggest that mitochondria are competent to carry out this metabolic process in eukaryotic cells. Prevailing metabolic conditions in subcellular compartments may have precluded observation of mitochondrial activity in our earlier work with cultured cells. Alternatively, transport mechanisms may exist in the cell types studied that distribute 12-(S)- and 15-(S)-HETEs specifically to peroxisomes.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Carnitine; Chromatography, High Pressure Liquid; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Liver; Male; Mitochondria, Liver; Rats; Rats, Sprague-Dawley

High extracellular Ca2+ (Ca2+ ec) stimulates the formation of inositol phosphates and diacylglycerol and activates phospholipase A2 in porcine parathyroid cells. Ca2+ ec action is also coupled to the formation of arachidonic acid, the precursor of both the cyclooxygenase and lipoxygenase (LO) pathways. We previously reported that LO pathway products might act as second messengers and play a part in regulating PTH secretion by Ca2+ ec. We have now investigated the effects of hydroxyeicosatetranoic acids (HETEs) on PTH secretion. Collagenase-dispersed porcine parathyroid cells were incubated in low [Ca2+] (0.5 mM, maximal stimulation) with or without HETEs for three 15-min periods. 12- and 15-HETEs inhibited PTH secretion in a dose-dependent manner from 10(-12) to 10(-9) M. Maximal inhibition was with 10(-9) M. Since 12- and 15-HETEs are the metabolic reduction products of 12- and 15-HPETEs, we also examined the effect of those precursors on PTH release. 12- and 15-hydroxyperoxyeicosatetranoic acids (HPETEs) were more potent inhibitors of PTH secretion. The threshold concentrations of both HPETEs that inhibited PTH release were lower than those for HETEs: 10(-9) M suppressed PTH secretion. This effect is comparable to that of high [Ca2+] (2 mM). This provides new evidence that products of 12-LO and 15-LO pathways are potent inhibitors of PTH secretion.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Hydrogen Peroxide; Hydroxyeicosatetraenoic Acids; Leukotrienes; Lipid Peroxides; Lipoxygenase; Parathyroid Hormone; Structure-Activity Relationship; Swine; Time Factors

5-Lipoxygenase-activating protein is required for cellular leukotriene synthesis and is the target of the leukotriene biosynthesis inhibitors MK-886 (3-[1-(p-chlorophenyl)-5-isopropyl-3-tert-butylthio-1H- indol-2-yl]-2,2-dimethylpropanoic acid) and MK-591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-ylmethoxy)-indol-2-yl] - 2,2-dimethylpropanoic acid). Recent studies demonstrate that 5-lipoxygenase-activating protein binds arachidonic acid and stimulates the utilization of this substrate by 5-lipoxygenase. The present study utilizes a radioligand binding assay to assess the affinity of 5-lipoxygenase-activating protein for arachidonic acid and the specificity of the fatty acid binding site on 5-lipoxygenase-activating protein. Our findings demonstrate that the presence of a free carboxyl group on fatty acids or leukotriene biosynthesis inhibitors which interact with 5-lipoxygenase-activating protein is not required for specific binding to the protein. However, the degree of saturation significantly affects the affinity of fatty acids for 5-lipoxygenase-activating protein.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5-Lipoxygenase-Activating Proteins; Arachidonic Acid; Binding Sites; Carrier Proteins; Humans; Hydroxyeicosatetraenoic Acids; Indoles; Leukocytes; Leukotrienes; Membrane Proteins; Quinolines; Radioligand Assay

We have examined the ability of interleukin-4 (IL-4), interleukin-10 (IL-10) and interleukin-1 receptor antagonist protein (IL-1ra) to regulate spontaneous interleukin-8 (IL-8) production in cultured SF mononuclear cells (SFMC) from RA. Furthermore, we examined whether IL-4, IL-10, or IL-1ra could influence the production of the arachidonic acid products leukotriene B4 (LTB4), 12-hydroxy-eicosatetraenoic acid (12-HETE) and 15-hydroxy-eicosatetraenoic acid (15-HETE). IL-4 induced a maximal suppression of 75% in the IL-8 secretion in SFMC from 10.0 ng/ml down to 2.5 ng/ml after 24 h and from 17.2 ng/ml to 4.2 ng/ml after 72 h of culture. IL-10 induced a 55% inhibition of the IL-8 secretion at 24 h and a 40% inhibition at 72 h. IL-1ra did not change the spontaneous IL-8 secretion from rheumatoid SFMC. We also examined, whether addition of IL-4, IL-10 or IL-1ra was able to modulate formation of the arachidonic acid products LTB4, 12-HETE and 15-HETE in cultured SF cells, stimulated with the calcium ionophore A23187. 15-HETE was not detected in untreated cultures, nor in IL-10 or IL-1ra treated cultures. IL-4, however, stimulated the formation of the anti-inflammatory mediator; 15-HETE (23 ng/10(6) cells). These results suggest that IL-4 or IL-10, could have beneficial anti-inflammatory effects in RA.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arthritis, Rheumatoid; Humans; Hydroxyeicosatetraenoic Acids; Interleukin 1 Receptor Antagonist Protein; Interleukin-10; Interleukin-4; Interleukin-8; Leukotriene B4; Sialoglycoproteins; Synovial Fluid

Inhibition of the formation of pro-inflammatory eicosanoids such as leukotrienes and 12-hydroxyeicosatetraenoic acid by 15-hydroxyeicosatetraenoic acid (15-HETE) has been reported. Psoriatic dermis synthesizes reduced levels of 15-HETE and it has been postulated to play a role in the pathophysiology of this disease. Interleukin-1 stimulates the production of prostaglandin E2 in fibroblasts, but its effect on the synthesis of 15-HETE is at present unknown. The aim of this study was to investigate the modulation of 15-HETE formation by interleukin-1 in dermal fibroblasts. Cells were treated with recombinant interleukin-1 alpha or beta prior to incubation with exogenous 14C-arachidonic acid, and eicosanoids were analyzed by HPLC. Interleukin-1 significantly increased the production of 15-HETE, but also 12-hydroxy-heptadecatrienoic acid, 11-hydroxyeicosatetraenoic acid, and prostaglandins, in a concentration- and time-dependent fashion. No significant differences between the two types of interleukin-1 were found. Dexamethasone (10 nM), and the protein synthesis inhibitors actinomycin D (1 microM) and cycloheximide (3 micrograms/ml) completely abolished the effect of interleukin-1 on 15-HETE formation. Whereas indomethacin (0.5-25 microM) strongly inhibited the synthesis of 15-HETE, aspirin (100-1000 microM) was unable to significantly inhibit its formation in both untreated and interleukin-treated fibroblasts. Aspirin inhibited the 15-HETE produced by cyclooxygenase from ram seminal vesicles, although to a lesser extent than indomethacin. In cell-free extracts, the activity concerning the synthesis of 15-HETE was associated with the microsomal fraction (100,000 x g pellet). Overall, these results strongly suggest that interleukin-1 increases 15-HETE formation mainly through the expression of new cyclooxygenase.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Cells, Cultured; Chromatography, High Pressure Liquid; Cycloheximide; Dactinomycin; Dexamethasone; Dose-Response Relationship, Drug; Eicosanoids; Epidermal Cells; Epidermis; Female; Fibroblasts; Humans; Hydroxyeicosatetraenoic Acids; Interleukin-1; Time Factors

Lipoxygenases of bovine and human corneal epithelia were investigated. The bovine epithelium contained an arachidonate 12-lipoxygenase and a 15-lipoxygenase. The 12-lipoxygenase was found in the microsomal fraction, while the 15-lipoxygenase was mainly present in the cytosol (100,000 x g supernatant). 12S-Hydroxyeicosatetraenoic acid (12S-HETE) and 15S-hydroxyeicosatetraenoic acid (15S-HETE) were identified by GC-MS and chiral HPLC. BW A4C, an acetohydroxamic acid lipoxygenase inhibitor, reduced the biosynthesis of 12S-HETE and 15S-HETE by over 90% at 10 microM. IC50 for the 12-lipoxygenase was 0.3 microM. The bovine corneal 12-lipoxygenase was compared with the 12-lipoxygenases of bovine platelets and leukocytes. All three enzymes metabolized 14C-labelled linoleic acid and alpha-linolenic acid poorly (5-16%) in comparison with [14C]arachidonic acid. [14C]Docosahexaenoic acid and [14C]4,7,10,13,16-docosapentaenoic acid appeared to be less efficiently converted by the corneal enzyme than by the platelet and leukocyte enzymes. Immunohistochemical analysis of the bovine corneal epithelium using a polyclonal antibody against porcine leukocyte 12-lipoxygenase gave positive staining. The cytosol of human corneal epithelium converted [14C]arachidonic acid to one prominent metabolite. The product co-chromatographed with 15S-HETE on reverse phase HPLC, straight phase HPLC and chiral HPLC. Our results suggest that human corneal epithelium contains a 15-lipoxygenase and that bovine corneal epithelium contains both a 15-lipoxygenase and a 12-lipoxygenase. The corneal 12-lipoxygenase appears to differ catalytically from earlier described bovine 12-lipoxygenases.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonic Acid; Cattle; Chromatography, High Pressure Liquid; Cornea; Epithelium; Humans; Hydroxyeicosatetraenoic Acids; Immunohistochemistry; Subcellular Fractions

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

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

Monohydroxy derivatives of polyunsaturated fatty acids such as arachidonic acid (AA) and linoleic acid (LA) can modulate inflammation and epidermal proliferation. The purpose of this study was to determine the in vivo distribution of the AA derivatives; 12- and 15-hydroxyeicosatetraenoic acid (12-HETE and 15-HETE) and the LA derivatives; 9- and 13-hydroxyotadecadienoic acid (9-HODE and 13-HODE) in specific phospholipids of normal human skin. Lipids were extracted from 6 normal keratome skin biopsies and phospholipids were separated into the major classes by two-dimensional thin layer chromatography. Monohydroxy fatty acids (MHFAs) released from specific phospholipids after treatment with phospholipase A2 were identified by reversed phase and straight phase high-performance liquid chromatography and UV-absorption spectra. Unesterified MHFAs were determined in a similar way. 9-HODE, 13-HODE and 15-HETE were detectable in phosphatidylcholine (PC), phosphatidylinositol (PI) and phosphatidylethanolamine (PE). Interestingly, 12-HETE was not detectable in these phospholipids, although the unesterified 12-HETE was detectable in amounts similar to unesterified 15-HETE. Esterified 15-HETE was equally distributed between PI and PC, in which 15-HETE was predominant, accounting for 60% and 69% of the total MHFAs, respectively (p < 0.05). These results demonstrate that the LA derivatives 9-HODE and 13-HODE, as well as the AA derivative 15-HETE, are esterified to PC, PI and PE of normal human epidermis in vivo. The possibility remains that 9-HODE, 13-HODE and 15-HETE, may mediate their biological effects by being incorporated into specific phospholipids.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Epidermis; Fatty Acids, Unsaturated; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acids; Linoleic Acids, Conjugated; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylinositols; Phospholipids

A homogenate of epidermal cells isolated from human skin converted arachidonic acid to 12S-hydroxy-5, 8,10,14-eicosatetraenoic acid and 15-hydroxy-5, 8,11,13-eicosatetraenoic acid as the main lipoxygenase products. The production of these hydroxy acids was not stimulated by the addition of 1 mM NADPH required for cytochrome P-450 reaction, but inhibited by 65-75% with 40 microM nordihydroguaiaretic acid, a nonspecific lipoxygenase inhibitor. In addition to these lipoxygenase products, the epidermal cell homogenate converted arachidonic acid to prostaglandin E2 together with minor amounts of prostaglandins D2 and F2a and 12-hydroxy-5,8,10-heptadecatrienoic acid. Thromboxane B2 was not detected. This finding rules out the possible contamination of platelet 12-lipoxygenase in the epidermal cells. After subcellular fractionation of the epidermal cell homogenate, the 12-lipoxygenase activity was found in the 164,000 x g supernatant, the 164,000 x g pellet, and the 10,000 x g pellet. The cytosolic enzyme and the enzymes solubilized from the two pellets produced 12S-hydroperoxy-5,8,10,14-eicosatetraenoic acid as the primary product in contrast to cytochrome P-450 which produces primarily hydroxy acids. The 12-lipoxygenase in the 164,000 x g supernatant and the solubilized enzymes from the 164,000 x g pellet and 10,000 x g pellet were precipitable by antibodies raised against human platelet 12-lipoxygenase, but not by antibodies against porcine leukocyte 12-lipoxygenase. The immunoprecipitated 12-lipoxygenase from each fraction was almost inactive with linoleic acid as substrate, characteristic of 12-lipoxygenase of platelet-type. Furthermore, 12-lipoxygenase mRNA in the epidermal cells could be reverse-transcribed and amplified by polymerase chain reaction with the primers specific for human platelet 12-lipoxygenase cDNA, but not with those for porcine leukocyte 12-lipoxygenase cDNA. Thus, the 12-lipoxygenase of human epidermal cells is similar to human platelet 12-lipoxygenase in terms of immunogenicity, catalytic property, and primary structure, and distinct from leukocyte 12-lipoxygenase.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 12-Lipoxygenase; Base Sequence; Blood Platelets; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; DNA; Epidermal Cells; Epidermis; Humans; Hydroxyeicosatetraenoic Acids; Molecular Sequence Data; Polymerase Chain Reaction; Precipitin Tests; Solubility

N-phenyllinoleamide (NPLA) has been detected as extraneous compound in adulterated cooking oils associated with a unique epidemic disease known as the Toxic Oil Syndrome (TOS). In this communication we report on the action of NPLA on the endogenous cyclooxygenase and lipoxygenase arachidonate metabolism. Results show that mouse peritoneal macrophages (MPM) exposed to 1 mM NPLA for 2 h undergo significant increases of 6-keto prostaglandin F1a, prostaglandin E2, leukotriene B4, 12- and 15-hydroxyeicosatetraenoic acids. MPM prelabelled with 3H-AA showed an enhanced release when exposed to NPLA. Thus, it is concluded that NPLA potentiates AA release from cell membrane phospholipids and the subsequent cyclooxygenase and lipoxygenase oxidative metabolism of this precursor to various eicosanoids. This is in agreement with the implication of peroxidative process mediated by fatty acids anilides in TOS.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Analysis of Variance; Anilides; Animals; Arachidonic Acid; Brassica; Cells, Cultured; Fatty Acids, Monounsaturated; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Linoleic Acids; Lipoxygenase; Macrophages; Male; Mice; Peritoneal Cavity; Plant Oils; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Radioimmunoassay; Rapeseed Oil; Thromboxanes

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

The lipoxygenase (LO) pathway of arachidonate metabolism has been suggested to play a key role in atherosclerosis and in mediating several actions of angiotensin II (AII). However, the relationship between LO activation and factors linked to accelerated diabetic vascular disease such as hyperglycemia and AII is not known. We have investigated the effect of high glucose (HG; 25 mM) and AII on LO activity as well as LO protein and mRNA expression in porcine aortic vascular smooth muscle cells (PVSMCs). We observed that cells cultured in HG had significantly higher levels of the cell-associated LO products 12- and 15-hydroxyeicosatetraenoic acids (HETEs). AII added to cells grown in HG specifically further increased only cell-associated 12-HETE levels. Using immunoblot analysis and reverse transcriptase PCRs, we demonstrated the presence in PVSMCs of porcine leukocyte-type 12-LO protein and mRNA, respectively. Furthermore, the levels of both were markedly upregulated by AII as well as by HG. These studies suggest that enhanced 12-LO activity and expression are mechanisms for accelerated vascular disease produced by HG and AII in diabetes mellitus.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Angiotensin II; Animals; Aorta; Arachidonate 12-Lipoxygenase; Base Sequence; Blotting, Western; Cells, Cultured; Cyclooxygenase Inhibitors; Flavanones; Flavonoids; Gene Expression Regulation, Enzymologic; Glucose; Hydroxyeicosatetraenoic Acids; Kinetics; Leukocytes; Lipoxygenase Inhibitors; Meclofenamic Acid; Molecular Sequence Data; Muscle, Smooth, Vascular; Oligodeoxyribonucleotides; Oligonucleotide Probes; Polymerase Chain Reaction; RNA, Messenger; Swine

The arachidonic acid metabolite 12-hydroxyeicosatetraenoic acid (12-HETE) inhibits renin secretion both in vivo and in vitro, but the enzymatic origin of the 12-HETE responsible for renin inhibition is unknown. These studies examined the effect of the 12-HETE stereoisomers (R)-12-HETE (a product of the cytochrome P-450 monooxygenase enzyme system) and (S)-12-HETE (a product of the lipoxygenase enzyme system) on basal and stimulated renin secretion from superficial cortical slices in the rat. First, rat cortex was shown to produce 12-HETE; chiral-phase high-performance liquid chromatography revealed that cortex produced 81% (S)-12-HETE and 19% (R)-12-HETE. (R)-12-HETE reduced basal renin release by 28 +/- 4% to 49 +/- 5% at concentrations of 10(-9) to 10(-7) M (P < 0.05 to 0.01). (S)-12-HETE did not significantly affect renin release. (R)-12-HETE also blunted isoproterenol-stimulated renin secretion (P < 0.05) at a concentration of 10(-6) M. 20-HETE, another cytochrome P-450 product, did not exert a significant effect on renin release. In summary, both (R)-12-HETE and (S)-12-HETE are synthesized by renal cortical tissue. Only (R)-12-HETE directly inhibits in vitro renin release. These findings suggest that the renal cytochrome P-450 enzyme system is capable of directly influencing local renin secretion.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Aorta; Arachidonic Acid; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Isoproterenol; Kidney Cortex; Leukotrienes; Lipid Peroxides; Rats; Rats, Sprague-Dawley; Renin; Stereoisomerism

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

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

N-phenyllinoleamide (NPLA) is a useful marker for adulterated oil samples associated with cases of toxic oil syndrome (TOS). To date, NPLA has not reproduced the human poisoning episode in experimental animal models and, thus, its pathological role in the syndrome remains controversial. The present report describes the effect of NPLA on the lipoxygenase metabolism of exogenous arachidonic acid (AA) in mouse peritoneal macrophages (MPM). Results show that MPM cells exposed to 1mM NPLA for 2 h, when subsequently incubated with exogenous 3H-AA, undergo a significant increase in the biosynthesis of 3H-12-hydroxyeicosatetraenoic acid (3H-12-HETE) whereas levels of 3H-15-HETE are relatively stable. These data indicate that NPLA selectively potentiates the lipoxygenase metabolism of exogenous AA, supporting the possible implication of lipid peroxidative processes in the ethiopathology of TOS, although the relatively high NPLA concentration required 'in vitro' makes it unlikely that this xenobiotic could be directly related to human toxicity.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Anilides; Animals; Arachidonic Acid; Enzyme Activation; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Linoleic Acids; Lipoxygenase; Macrophages; Mice; Oils; Peritoneal Cavity

Uncovered fetal rabbit excisional wounds do not exhibit any classic signs of healing; wounds covered with an impermeable cover do contract, reepithelialize, and exhibit inflammation. Prostaglandin E2 (PGE2) is elevated in amniotic fluid, acting as an immunosuppressant at the maternal-fetal interface. Full-thickness excisional wounds were made on 25-day gestational age rabbit fetuses. Half the wounds were covered with an impermeable cover. Tissue from covered, uncovered, and nonwounded fetuses was examined 72 h after wounding for arachidonic acid metabolites. Uncovered wounds had significantly (P less than or equal to 0.05) elevated levels of PGE2, PGF2 alpha, and 12-HETE versus covered wounds and control tissue. Covered wounds had significantly elevated levels of 15-HETE compared to uncovered and control tissue. The elevated PGE2 in uncovered wounds may act as a fetal immunosuppressant; covered wounds (lower PGE2) developed cellular inflammation. Further investigations of these interactions may permit modulation of adult inflammation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Amniotic Fluid; Animals; Arachidonic Acids; Dinoprost; Dinoprostone; Female; Fetus; Gestational Age; Hydroxyeicosatetraenoic Acids; Inflammation; Leukotrienes; Occlusive Dressings; Pregnancy; Prenatal Injuries; Rabbits; Silicones; Sutures; Wound Healing; Wounds, Penetrating

The peptide hormone angiotensin-II (AII) is a potent vasoconstrictor and major regulator of aldosterone synthesis. In addition, AII also has growth-promoting effects. We have recently shown that the lipoxygenase (LO) pathway of arachidonic acid plays a major role in AII-induced aldosterone synthesis in adrenal glomerulosa cells. The LO pathway is also involved in the vasopressor and renin-inhibitory effects of AII. However, the role of LO products in AII-induced mitogenic effects have not yet been investigated. In the present studies we have evaluated the role of the LO pathway in AII-induced proliferative responses in a bovine adrenocortical cell clone termed AC1 cells. In addition, the potential receptor type and mechanism of AII-induced proliferation was studied by evaluating the effect of specific nonpeptide type 1 and type 2 AII receptor antagonists and the role of protein kinase-C (PKC). AII-induced DNA synthesis was significantly attenuated by two structurally dissimilar LO inhibitors, baicalein and phenidone. In addition, the LO product 12-hydroxyeicosatetraenoic acid (12-HETE) itself caused a significant increase in DNA synthesis, suggesting that the 12-LO pathway in part plays a role in AII-mediated mitogenesis. AII-induced proliferative responses were blocked by the type 1 AII receptor antagonist. Both AII- and 12-HETE-induced increases in DNA synthesis were markedly inhibited by two PKC blockers, staurosporine and sangivamycin. Further, both AII and 12-HETE could activate PKC by translocating it from the cytosol to the membrane fraction, as determined by Western immunoblotting. These results suggest that both 12-LO activation and protein kinase-C have an important role in AII-induced adrenal cell proliferation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adrenal Cortex; Angiotensin II; Angiotensin Receptor Antagonists; Biphenyl Compounds; Cell Division; Cells, Cultured; DNA Replication; Dose-Response Relationship, Drug; Flavanones; Flavonoids; Humans; Hydroxyeicosatetraenoic Acids; Imidazoles; Losartan; Protein Kinase C; Pyrazoles; Pyridines; Pyrimidine Nucleosides; Tetrazoles; Thymidine

Oxidatively-modified low density lipoprotein (LDL) is thought to play a significant role in the formation of lipid-laden macrophages, the primary cellular component of atherosclerotic fatty lesions. Recently, lipoxygenases have been implicated as a major enzymatic pathway involved in rabbit endothelial cell-mediated LDL modification. We investigated the effect of LDL on porcine aortic endothelial cell (PAEC) and human umbilical vein (HUVEC) and aortic endothelial cell (HAEC) lipoxygenase activity. By thin layer chromatography, we observed that human LDL stimulated the metabolism of radiolabeled arachidonic acid to 12 + 15-hydroxyeicosatetraenoic acid (HETE) in indomethacin-treated PAEC. Furthermore, radiolabeled linoleic acid, a specific substrate for the 15-lipoxygenase, was metabolized to its respective product 13-hydroxyoctadecadienoic acid (13-HODE) in the presence of LDL. Increased product formation in both studies was inhibited by the lipoxygenase blockers nordihydroguaiaretic acid (NDGA) and RG 6866. 15-HETE was confirmed as the predominant HETE product in LDL-treated cells by high performance liquid chromatography. Both porcine- and human-derived LDL stimulated the CL release of 15-HETE from cells as determined by radioimmunoassay. Release of immunoreactive 15-HETE was inhibited by NDGA, RG 6866, and 5,8,11,14-eicosatetraynoic acid (ETYA) but not by the selective 5-lipoxygenase inhibitor RG 5901. These lipoxygenase inhibitors had similar effects on the modification of LDL. Our results suggest that the oxidative modification of LDL by endothelial cells may be mediated in part through activation of 15-lipoxygenase.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5,8,11,14-Eicosatetraynoic Acid; Animals; Aorta; Arachidonate 15-Lipoxygenase; Benzyl Compounds; Cells, Cultured; Copper; Copper Sulfate; Endothelium, Vascular; Enzyme Activation; Hydroxamic Acids; Hydroxyeicosatetraenoic Acids; Indomethacin; Linoleic Acid; Linoleic Acids; Lipoproteins, LDL; Lipoxygenase Inhibitors; Masoprocol; Oxidation-Reduction; Quinolines; Swine; Umbilical Veins

MOLT-4 lymphocytes metabolize 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE via beta-oxidation with retention of the hydroxyl group at the omega 9 carbon atom. The isolation of 6-hydroxy-4,8-tetradecadienoic acid documents that these cells have the capacity to catabolize the conjugated diene system. 12(S)-HETE was also metabolized to 3,12-dihydroxy-8,10,14-eicosatrienoic acid and 1,9-dihydroxy-5,7,11-heptadecatriene as well as to 17- and 19-carbon aldehydes. When MOLT-4 cells were incubated with the beta-oxidation product, 10-hydroxy-6,8,12-octadecatrienoic acid, it was in part further catabolized but in addition it served as an anabolic precursor as defined by the accumulation 3,12-dihydroxy-8,10,14-eicosatrienoic acid as well as 1,11-dihydroxy-7,9,13-nonadecatriene. Neither 10-hydroxy-6,8,12-octadecatrienoic acid nor 13-hydroxy-5,8,11-octadecatrienic acid was as potent in inhibiting phytohemagglutin-induced lymphocyte mitogenesis as were their parent compounds--i.e., 12(S)- and 15(S)-HETE. These findings argue against the hypothesis that beta-oxidation products of 12(S)- and 15(S)-HETE are the potential modulators of lymphocyte function. However, neither the pathway for synthesis, nor the role of odd chain aldehydes and diols as potential lipid mediators was determined in this study.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Cell Line; Humans; Hydroxyeicosatetraenoic Acids; Lymphocyte Activation; Oxidation-Reduction; T-Lymphocytes

We investigated the interactions of exogenous leukotriene A4 (LTA4) with isolated cells in the presence or absence of cellular stimuli. The majority of isolated cells are able to transform exogenous LTA4 into LTB4 as well as LTC4. In eosinophils, LTA4 induced 15-hydroxy-eicosatetranoic acid formation and was converted into LTB4. The Ca-ionophore-induced generation of LTB4 from polymorphonuclear leukocytes or from the cell fraction containing lymphocytes, monocytes and basophils was significantly suppressed with LTA4 while the formation of LTC4 was increased. Conversely, the Na-fluoride- and fMLP-induced generation of LTB4 was significantly increased. Our results suggest that the stimulus and the cellular composition determine the pattern of the generated inflammatory mediators.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene A4; Leukotrienes

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

Rat epidermal microsomes were incubated with [1-14C]-arachidonic acid for 30 min at 37 degrees C in the absence and presence of NADPH. The arachidonate metabolites that eluted in the "monohydroxy acid fraction" on reverse-phase high performance liquid chromatography (HPLC) were methylated, purified by straight-phase HPLC and analyzed by chromatography with standard compounds, UV spectroscopy and/or gas chromatography-mass spectrometry (GC-MS). In the absence of NADPH, epidermal microsomes converted arachidonic acid to two major products identified as 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15(S)-HETE) and 12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid (12(S)-HETE). In the presence of NADPH, the microsomal reaction produced, besides 15(S)- and 12(S)-HETE, two less polar metabolites which were characterized as 15-hydroxy-5,8,11,-eicosatrienoic acid (15-HETrE) and 12-hydroxy-5,8,14-eicosatrienoic acid (12-HETrE). Stereochemical analysis by chiral-phase HPLC showed that the biosynthesized 12-HETrE consisted of a mixture of optical isomers in a S/R ratio of 65:35. Formation of 15- and 12-HETrE was blocked by the mixed cyclooxygenase-lipoxygenase inhibitors quercetin and phenidone but was not affected by the cyclooxygenase inhibitor indomethacin or the cytochrome P-450 monooxygenase inhibitor metyrapone. These data indicate that rat epidermal microsomes, supplemented with NADPH, are capable of metabolizing arachidonic acid to 15- and 12-HETrE. The production of these compounds may be initiated by lipoxygenase-mediated hydroperoxidation of arachidonic acid.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Animals, Newborn; Arachidonic Acids; Chromatography, High Pressure Liquid; Epidermis; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Mass Spectrometry; Microsomes; NADP; Rats; Rats, Inbred Strains; Stereoisomerism

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate 15-Lipoxygenase; Bronchi; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotrienes; Lipoxins; Nasal Polyps; Neutrophils

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

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

The principal in vivo oxygenase products of arachidonic acid and linoleic acid in psoriatic skin scales are 12-hydroxyeicosatetraenoic acid (R/S ratio = 5.7), 13-hydroxyoctadecadienoic acid (S/R = 1.9), and 9-hydroxyoctadecadienoic acid (R/S = 2.4). Definition of the enzymatic origin of these fatty acid derivatives is an important step in assessing their possible role in the pathogenesis of psoriasis. Psoriatic skin scales were incubated with radiolabeled arachidonic acid and linoleic acid and the monohydroxylated derivatives produced in vitro were characterized. The products of incubation with [3H]arachidonic acid were an enantiopure 15(S)-[3H]hydroxyeicosatetraenoic acid and a nonracemic mixture of the 12-[3H]hydroxyeicosatetraenoic acid steroisomers (R/S ratio = 4.5). An enantiopure 13(S)-[14C]hydroxyoctadecadienoic acid was produced from [14C]linoleic acid. No radiolabeled products were derived from incubations with heat-denatured scales. These results provide evidence for two distinct oxygenase activities that are preserved in psoriatic skin scales. One is that of an omega-6 oxygenase with strict (S) stereospecificity, consistent with the activity of a lipoxygenase. This enzyme activity appears to be similar to that of the 15-lipoxygenase which has been described in cultured human keratinocytes. The second activity is that of an arachidonic acid 12(R)-oxygenase that has not been observed in normal human epidermis but which appears to be expressed in psoriatic epidermis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Oxidation-Reduction; Oxygenases; Psoriasis; Skin; Stereoisomerism

Synthesis of 12- and/or 15-HETE by human epidermal cells was investigated after separating basal cells from suprabasal epidermal cell layers. We found that the main metabolite of 3H-arachidonic acid (3H-AA), formed by freshly prepared upper epidermal layers (stratum granulosum and spinosum), upon RP-HPLC co-eluted with authentic 3H-12-HETE. A 3H-15-HETE co-eluting peak selectively occurred in chromatograms obtained from supernatants of fractions containing basal cells. Supernatants of freshly prepared suspensions rich in basal keratinocytes appeared to contain 3H-15-HETE as their main 3H-AA metabolite, by far exceeding the recovered amounts of 3H-12-HETE. Moreover, keratinocytes cultured for 1 week or longer were found to produce predominantly a 3H-AA metabolite co-eluting with 3H-15-HETE. In supernatants of cultured cells, little if any 3H-12-HETE was detectable. Cultured human skin fibroblasts were not found to produce relevant amounts of HETE. Genuine tissue rich in basal cells, i.e., cells of hair follicles, were found to form twice as much 3H-15-HETE as 3H-12-HETE (3H-15-HETE/3H-12-HETE-ratio = 1.9 +/- 0.8; n = 7). Apparently, different epidermal layers are able to produce a characteristic pattern of 3H-AA metabolites. 3H-15-HETE generation seems to be a marker for proliferating keratinocytes, whereas 3H-12-HETE formation appears to be typical for differentiating suprabasal epidermal cells. Our results may explain the heretofore varying patterns of AA-metabolites by keratinocytes reported in the literature.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adolescent; Adult; Arachidonic Acid; Arachidonic Acids; Cell Differentiation; Cells, Cultured; Epidermal Cells; Epidermis; Female; Fibroblasts; Hair; Humans; Hydroxyeicosatetraenoic Acids; Keratinocytes; Skin; Subcellular Fractions; Trypsin

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 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

To determine whether the peroxisome is responsible for hydroxyeicosatetraenoic acid (HETE) oxidation, 12- and 15-HETE oxidation was measured in normal and peroxisomal deficient skin fibroblasts from patients with Zellweger's (cerebrohepatorenal) syndrome. When incubated for 1 h with normal fibroblasts, reverse phase HPLC indicated that 24% of the 12-HETE radioactivity was converted to one major polar metabolite. Chemical derivatization followed by reverse phase HPLC and TLC indicated that this metabolite is 8-hydroxyhexadecatrienoic acid [16:3(8-OH)]. Similarly, 33% of the added 15-HETE was also converted to a more polar metabolite. Neither 12- nor 15-HETE were converted to any metabolites by the peroxisomal deficient (Zellweger) cells. No defect in HETE oxidation was found in other human fibroblast cell lines with diverse metabolic abnormalities. Zellweger fibroblasts accumulated increased amounts of 12-HETE, compared with normal fibroblasts. As in the normal cells, most of the 12-HETE incorporated into Zellweger fibroblasts was present in the choline and ethanolamine phosphoglycerides. Protein synthesis, lysosomal acid lipase activity, and mitochondrial butyrate oxidation were not impaired in the Zellweger fibroblasts. Since the Zellweger cells do not convert 12- and 15-HETE to oxidative metabolites, peroxisomes appear to be the cellular organelle responsible for HETE oxidation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Cells, Cultured; Fatty Acids; Fibroblasts; Humans; Hydroxyeicosatetraenoic Acids; Lipid Metabolism; Microbodies; Oxidation-Reduction; Zellweger Syndrome

Recently, oocytes or eggs of two marine invertebrates have been found to metabolize arachidonic acid to specific monohydroxy products. These studies have prompted our examination of the oocytes of higher organisms. In the present study, oocytes of an amphibian, Xenopus laevis, were examined for their capacity to biosynthesize hydroxyeicosatetraenoic acids (HETEs) and related hydroxy fatty acids. Two hydroxyeicosanoids were formed during incubations of oocyte homogenates with [14C]arachidonic acid; their structures and stereochemistry were determined by high-pressure liquid chromatography, uv spectroscopy, and gas chromatography-mass spectrometry. The compounds were identified as 15(S)- and 12(S)-hydroxyeicosatetraenoic acids. The synthesis of the two HETEs was not blocked by a cyclooxygenase inhibitor, indomethacin (10 microM), or by prior exposure of the oocyte homogenates to carbon monoxide, an inhibitor of cytochrome P450. Furthermore, 12(S)- and 15(S)-hydroperoxyeicosatetraenoic acids were isolated from brief incubations of gel-filtered ammonium sulfate fraction of frog oocyte homogenates; isolation of the hydroperoxide is further support for the existence of 12(S)- and 15(S)-lipoxygenase activities in the oocytes of X. laevis. Other polyunsaturated acids, including C18.2, C18.3, C20.3, C20.5, and C22.6 were also substrates for the lipoxygenase, and in each case the major product was formed by omega 6 oxygenation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Carbon Monoxide; Chromatography, High Pressure Liquid; Fatty Acids, Unsaturated; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Indomethacin; Lipoxygenase; Oocytes; Oogenesis; Xenopus laevis

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

The lipoxygenase activities of cultured human buccal epithelial cells and cells taken ex vivo from the human buccal cavity were compared. Lipoxygenation by cultured cells exhibited exclusively omega-6 positional specificity. A membrane-damaging event such as freezing was required for activation. In contrast, after simple addition of arachidonic acid, the ex vivo buccal cells produced predominantly 12-hydroxyeicosatetraenoic acid. Chromatography on chiral columns yielded products which had an (S) configuration at the site of oxygen insertion. 5-hydroxyeicosatetraenoic acid was transformed by ex vivo buccal cells to 5(S),12(S)-dihydroxyeicosatetraenoic acid. These products could have a role in modulation of inflammatory states of the oral cavity.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Cell Differentiation; Cells, Cultured; Cheek; Chromatography, Gas; Epithelium; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase

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

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

Homogenates of normal human epidermis synthesized 12-hydroxyeicosatetraenoic acid (12-HETE) when incubated in vitro with arachidonic acid. The stereoconfigurations of the C-12 hydroxyl isomers were determined by incubation with potato 5-lipoxygenase. The synthesized substrate-specific diHETEs; 5S, 12R and 5S, 12S, were readily separated by high performance liquid chromatography. Using this novel methodology, the normal epidermis was found to synthesize predominantly 12-S-HETE while, in contrast, psoriatic scale was found to contain 12-R-HETE. The 5-lipoxygenase inhibitors, Merck L-651, 896, Takeda AA86I, and the active metabolite of Syntex lonapalene were found to inhibit 12-HETE formation in normal epidermal homogenates.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Epidermis; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Lipoxygenase Inhibitors; Methods; NADP; Phenothiazines; Psoriasis; Pyridines; Stereoisomerism

The biochemical changes underlying the clinical manifestations of psoriasis are unknown. Certain chemotactic eicosanoids derived from arachidonic acid metabolism have been suggested to play important roles in psoriasis, because of their presence in lesional psoriatic skin and their ability to elicit skin inflammation and to stimulate epidermal proliferation. The purpose of the present study was to elucidate which eicosanoids might be involved in the early phases of the inflammatory processes of psoriasis. Eicosanoids were analyzed in scale and in lesional skin without scale both in acute guttate and chronic plaque psoriatic lesions. Methods for identification of eicosanoids included reversed-phase high-performance liquid chromatography combined with radioimmunoassay. Leukotriene B4 was present in both acute guttate and chronic plaque skin lesions in biologically active amounts (acute guttate lesions: 18.7 +/- 7.1 ng/g wet tissue in scale and 3.2 +/- 1.5 ng/g wet tissue in lesional skin without scale; chronic plaque lesions: 33.1 +/- 9.7 ng/g wet tissue in scale and 5.3 +/- 2.0 ng/g wet tissue in lesional skin without scale). 12- and 15-hydroxy-eicosatetraenoic acid (HETE) reached biologically active concentrations only in scale of chronic plaque lesions (1,512 +/- 282 and 1,441 +/- 411 ng/g wet tissue, respectively). The level of prostaglandin E2 in chronic plaque lesions was similar to the level in normal skin, while the level in acute guttate lesions was increased twofold (71.0 +/- 14.8 ng/g wet tissue). These results demonstrate that leukotriene B4, but not 12-HETE, is present in acute guttate psoriatic skin lesions in concentrations able to exert biologic effects. Leukotriene B4 may therefore participate in inflammatory changes of acute psoriasis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Acute Disease; Chromatography, High Pressure Liquid; Chronic Disease; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Psoriasis; Radioimmunoassay; Skin

Modulation of renin synthesis by lipoxygenase products has been studied in cultured human mesangial cells under basal conditions and in the presence of prostaglandin (PG) E2. Total renin and cyclic AMP productions were stimulated in a dose-dependent manner (0.1-10 microM) by PGE2. The stimulatory effect of PGE2 on renin production was inhibited by 12-hydroxyeicosatetraenoic acid (12-HETE) between 0.1 and 100 nM. Extracellular and intracellular renin were affected similarly. Neither basal and PGE2-dependent cyclic AMP nor basal cyclic GMP productions were modified. 15-Hydroxyeicosatetraenoic acid (15-HPETE), 12-hydroperoxyeicosatetraenoic acid (12-HPETE) and 15-hydroperoxyeicosatetraenoic acid (15-HPETE) had the same effects as 12-HETE. Intracellular calcium concentration was not modified in the presence of 12-HETE. Since oleyl-2-acetylglycerol (OAG), an analog of diacylglycerol, also inhibited PGE2-stimulated renin production, it is hypothesized that the effect of the lipoxygenase products is mediated via protein kinase C stimulation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Angiotensin II; Calcium; Cyclic AMP; Cyclic GMP; Diglycerides; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kidney Cortex; Leukotrienes; Lipid Peroxides; Lipoxygenase; Renin

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

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

Specific receptors for [3H]-15 HETE have been identified on GH3 cells, a cloned strain of rat pituitary cells. With incremental inputs of radioligand and a constant cell number, specific [3H]-15 HETE binding reached a plateau indicative of saturable binding sites. Ligand analysis of the Scatchard plot demonstrated a single class of high affinity binding sites with a dissociation constant (Kd) of 0.75 nM. 12 HETE competed with radiolabeled 15 HETE (IC50 = 1 x 10(-6) +/- 0.8 M). In contrast, arachidonic acid, leukotriene B4, prostaglandins E2 and F2 alpha did not compete with [3H]-15 HETE.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Binding Sites; Dinoprost; Dinoprostone; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Pituitary Gland; Prostaglandins E; Prostaglandins F; Rats

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

The rabbit reticulocyte lipoxygenase is known to display an unusual facility for oxygenation of esterified polyunsaturated fatty acids, yet the precise structures of the products are not known. With free arachidonate as substrate the enzyme is known to catalyze 15S and 12S oxygenations, and demonstration of a facility for catalysis of these reactions on phospholipids would extend the potential scope of lipoxygenase reactions in cells. We elected to study in detail the reaction of the enzyme with a natural phospholipid, palmitoyl/arachidonoyl-phosphatidylcholine. We determined the nature of the products by initial isolation by RP-HPLC, followed by transesterification and identification of the oxygenated products by HPLC, uv, GC-MS, and steric analysis of hydroxyl configuration by HPLC. The major product was identified as a phosphatidylcholine in which the arachidonate component was converted to the 15(S)-hydroperoxy-eicosatetraenoate. A second oxygenated phospholipid was produced in smaller quantities (2-5% of the latter product) and identified as the 12(S)-oxygenated analog. These products were also identified after reaction of the reticulocyte lipoxygenase with human red cell membranes which were radiolabeled by preincubation with [3H]arachidonic acid. The finding of 12S oxygenation represents the first evidence that a lipoxygenase can control a reaction centered on the 10-carbon of an arachidonoyl phospholipid. This is an important precedent, because hydrogen abstraction from carbon-10 is a critical step in the lipoxygenase-catalyzed synthesis of 8- and 12-hydroperoxy-eicosatetraenoates (HPETEs) and for the conversion of 5- and 15-HPETEs to leukotrienes.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Chromatography, High Pressure Liquid; Erythrocyte Membrane; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Oxidation-Reduction; Phosphatidylcholines; Rabbits; Reticulocytes; Spectrophotometry, Ultraviolet

Biochemical evidence in support of a role for arachidonic acid 5-lipoxygenase activity in pancreatic islet insulin secretion has been obtained. Peptidyl leukotriene metabolism was studied in rat islets using a dual-labeling technique in extended culture, with analysis of arachidonic acid metabolites by reverse-phase high-performance liquid chromatography. The production of [3H]arachidonoyl/[35S]cysteinyl leukotrienes C4 and E4 by islets was compared with that by mouse resident peritoneal macrophages and with the lipoxygenase metabolism of rabbit polymorphonuclear leukocytes. The stimulus-specific nature of leukotriene biosynthesis was characterized by low basal biosynthesis in unstimulated islet cells with a calcium-mediated activation of 5-lipoxygenase product formation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Cysteine; Female; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Islets of Langerhans; Macrophages; Mice; Mice, Inbred ICR; Neutrophils; Rabbits; Rats; Spectrophotometry, Ultraviolet; SRS-A

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

15-lipoxygenase activity was investigated in sonicated polymorphonuclear leukocytes (PMNLs) from patients with juvenile and rapidly progressive periodontitis and adult periodontitis. The group with juvenile and rapidly progressive periodontitis had 17 patients (6 male, 11 female, mean age 27.4 years), and the age matched control group had 18 normal individuals (11 male, 7 female, mean age 26.3 years). The group with adult periodontitis had 14 patients with 9 male, 5 female, mean age 45.2 years and the age-matched control group had 6 normal subjects with 5 male, 1 female, mean age 43.7 years. 15-hydroxyeicosa-tetraenoic acid (15-HETE) synthesized in the group with juvenile and rapidly progressive periodontitis was 0.219 +/- 0.102 ng/mg protein (mean +/- S.D.), while it was 0.410 +/- 0.138 ng/mg protein in the age-matched control group. There was a significant difference between the two groups. The group with adult periodontitis produced 0.358 +/- 0.124 ng/mg protein and the age matched control group produced 0.448 +/- 0.176 ng/mg protein (no significant difference). These results are relevant to reports that PMNLs of patients with juvenile and rapidly progressive periodontitis have abnormal functions, while those of patients with adult periodontitis are normal.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Aggressive Periodontitis; Arachidonate 15-Lipoxygenase; Arachidonate Lipoxygenases; Female; Humans; Hydroxyeicosatetraenoic Acids; Male; Middle Aged; Neutrophils; Periodontal Diseases; Periodontitis

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

Intrauterine tissues from women at term of pregnancy metabolized arachidonic acid by way of lipoxygenase pathways that included 5-, 12-, and 15- lipoxygenases. The major lipoxygenase product formed by amnion obtained before labor was leukotriene B4 and after labor was 12- hydroxyeicosatetraenoic acid (12-HETE). Chorion laeve and decidua vera synthesized predominantly 15-HETE at all times and placenta produced mainly 12-HETE. Trends existed for increased prostaglandin formation with labor by amnion, chorion laeve and decidua vera and for increased lipoxygenase product formation by chorion laeve, decidua vera and placenta. It is suggested that products of arachidonic acid metabolism by way of lipoxygenase and cyclooxygenase pathways play significant roles in the control of fetal and uteroplacental hemodynamics and the mechanism(s) of parturition.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Amnion; Arachidonic Acid; Arachidonic Acids; Chorion; Decidua; Female; Humans; Hydroxyeicosatetraenoic Acids; Labor, Obstetric; Leukotriene B4; Lipoxygenase; Placenta; Pregnancy; Prostaglandin-Endoperoxide Synthases; Uterus

15-Hydroxyeicosatetraenoic acid (15-HETE), a major lipoxygenase metabolite of arachidonic acid in fetal bovine aortic endothelial cells, was a mitogen for these cells, stimulating both cell proliferation and DNA synthesis in the presence of serum and serum-deprived cells. In [14C]arachidonic acid-labeled confluent endothelial cell monolayers, 15-HETE (30 microM) caused an elevation of [14C]diacylglycerol (DAG) with a concomitant decrease in cellular [14C]phosphatidylinositol (PI) in both unstimulated and stimulated cells. 1-Oleoyl-2-acetylglycerol, a synthetic DAG analog, stimulated endothelial cell DNA synthesis in a concentration-dependent manner. In [3H]inositol-labeled cells, 15-HETE also caused a decrease in cellular PI content under both basal and stimulated conditions. 15-HETE, however, had no effect on either isolated phospholipase C activity or phosphoinositide turnover in lithium chloride-treated cells. In intact cells, 15-HETE (30 microM) inhibited the synthesis of [3H]PI from [3H]inositol (80% inhibition, p less than 0.001). In human red cell membranes, the production of phosphatidic acid from endogenous DAG was inhibited by 15-HETE in a concentration-dependent manner with an IC50 of 41 microM. Although 12-HETE had effects similar to those of 15-HETE, the parent compound arachidonic acid did not affect DNA synthesis or DAG kinase activity. Our study thus demonstrates that the mitogenic activity of 15- and 12-HETE on endothelial cells may be mediated via DAG kinase inhibition with the concomitant accumulation of cellular DAG.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Cattle; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Diacylglycerol Kinase; Diglycerides; Dose-Response Relationship, Drug; Endothelium, Vascular; Hydroxyeicosatetraenoic Acids; Indomethacin; Mitosis; Phosphotransferases; Thymidine

This study was undertaken to examine the effects of intrauterine infection and preterm labor on the amniotic fluid concentrations of arachidonate lipoxygenase metabolites in women with premature rupture of membranes. Amniotic fluid was obtained from four groups of women with premature rupture of membranes: group 1, women without labor or infection; group 2, women with labor but without infection; group 3, women with intra-amniotic infection but without labor; and group 4, women with both infection and labor. 12-Hydroxyeicosatetraenoic acid, 15-hydroxyeicosatetraenoic acid, and leukotriene B4 were measured by radioimmunoassays. Amniotic fluid concentrations of 12-hydroxyeicosatetraenoic acid were found not to differ significantly among the four groups. Amniotic fluid concentrations of 15-hydroxyeicosatetraenoic acid in group 4 were significantly higher than in women in groups 1 and 3 (p less than 0.05). In addition, amniotic fluid concentrations in leukotriene B4 were significantly higher in group 4 than in any of the other three groups (p less than 0.05). Leukotriene B4 concentrations were higher in groups 2 and 3 than in group 1, suggesting that the presence of both labor and infection increases the concentration of this metabolite in amniotic fluid. Infection and labor had an additive effect in the elevation of amniotic fluid concentrations of leukotriene B4. These results suggest that the amniotic fluid concentrations of arachidonate lipoxygenase metabolites are affected differently by the presence of infection and labor in women with premature rupture of membranes.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Amniotic Fluid; Arachidonate Lipoxygenases; Bacterial Infections; Female; Fetal Membranes, Premature Rupture; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Obstetric Labor, Premature; Pregnancy; Pregnancy Complications, Infectious

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Arachidonate 12-Lipoxygenase; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Psoriasis; Skin

The formation of arachidonate lipoxygenase products by uterine and intrauterine tissues of sheep in the last third of gestation has been evaluated. Maternal and fetal cotyledon, myometrium and fetal membrane exhibited evidence of arachidonate 5-, 12-, and 15- lipoxygenase activities. The major lipoxygenase product formed by fetal membrane and fetal cotyledon was leukotriene B4 (LTB4) whereas maternal cotyledon and myometrium produced mainly 12-hydroxyeicosatetraenoic acid (12-HETE). Arachidonate lipoxygenase products may play significant roles in the regulation of fetal and uteroplacental hemodynamics and it is speculated that the formation of leukotriene B4 predominantly by tissues of fetal origin may be of significance in the immunologic adaptations of pregnancy.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Extraembryonic Membranes; Fatty Acids, Unsaturated; Female; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Myometrium; Pregnancy; Pregnancy, Animal; Sheep

Cytosolic (100,000 g) fractions of fetal rabbit brain and placenta tissue convert [1-14C]arachidonic acid into several oxidation products identified with the lipoxygenase [12-hydroxyeicosatetraenoic acid (12-HETE) and 15-HETE] and cyclooxygenase [prostaglandin E2 (PGE2)] pathways. Formation of 12-HETE and 15-HETE by fetal brain is time-dependent, reaching a plateau after 40 min and is linear with protein concentration. An apparent affinity constant of 0.06 mM and a Vmax of 0.1 mumol/h/g wet weight are presumably responsible for the excessive accumulation of 12-HETE and 15-HETE in comparison to PGE2 (Km = 0.5 mM). The latter is synthesized by the placenta particulate fraction but almost exclusively by the brain cytosol. Compared to brain, the activity of the placenta tissue is exceedingly higher and in addition to 12-HETE and 15-HETE there is a substantial formation of 12-L-hydroxyheptadecatrienic acid. Formation of 12-HETE and 15-HETE at 21 days is as effective as at 31 days gestation and is strongly inhibited by nordihydroguaiaretic acid (93%), BW755c (99%), and AA861 (84%) but not by indomethacin. Placenta and brain tissues of intrauterine growth retarded fetuses after ligation of placental blood vessels fail to convert arachidonic acid into other eicosanoids. Loss of enzymatic activity also observed in normal tissue after prolonged storage cannot be restored by the addition of several SH agents, ascorbate, or ferric iron.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Brain; Dinoprostone; Female; Fetal Growth Retardation; Hydroxyeicosatetraenoic Acids; Kinetics; Lipoxygenase; Oxidation-Reduction; Placenta; Placenta Diseases; Placental Insufficiency; Pregnancy; Prostaglandin-Endoperoxide Synthases; Prostaglandins E; Rabbits

Two different lipoxygenases have been identified in human and rat epidermis. One lipoxygenase has a (n-9)-specificity, converts arachidonic acid into 12-hydroxyeicosatetraenoic acid (12-HETE), and has been described by several investigators. Linoleic acid is not a substrate for this enzyme. The other lipoxygenase, with (n-6)-specificity, converts arachidonic acid into 15-HETE and linoleic acid into 13-hydroxyoctadecadienoic acid (13-HOD). Especially the latter lipoxygenase is thought to be involved in the regulation of the differentiation of the skin cells into a proper water-barrier layer. Linoleate is supposed to be the physiological substrate; this fatty acid is especially present in characteristic sphingolipids with unique structures.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Epidermis; Humans; Hydroxyeicosatetraenoic Acids; Linoleic Acid; Linoleic Acids; Linoleic Acids, Conjugated; Lipoxygenase; Psoriasis; Skin

Erythroid progenitor cells synthesize 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) when stimulated by erythropoietin (Ep). Maximal stimulation of 12-HETE production occurred at one hour, whereas 15-HETE activity remained constant in response to Ep for 24 hours. Lipoxygenase-selective inhibitors of arachidonic acid metabolism blocked HETE production and Ep-stimulated growth and differentiation of erythroid progenitor cell-derived colonies (CFU-E). On the other hand, specific inhibitors of cyclooxygenase (aspirin and meclofenamate) did not significantly inhibit Ep-induced erythroid colony formation. It is hypothesized that the stimulation of HETE production from arachidonic acid (AA) is an essential step in the mechanism of action of Ep.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Animals; Arachidonic Acid; Arachidonic Acids; Aspirin; Butylated Hydroxyanisole; Erythropoiesis; Erythropoietin; Hydroxyeicosatetraenoic Acids; Liver; Masoprocol; Meclofenamic Acid; Mice; Phosphatidylinositols; Pyrazoles

Release of arachidonic acid from membrane phospholipids is a limiting step in the synthesis of both cyclooxygenase products and lipoxygenase products. The direct effects of prostacyclin and some lipoxygenase products on renin release were studied using rat renal cortical slices. Prostacyclin, at concentrations of 10(-5) M, stimulated renin secretion, but this effect was short-lived. Leukotrienes or their precursor, 5-hydroperoxyeicosatetraenoic acid, did not affect basal renin release. In contrast, 10(-9) M 12-hydroperoxyeicosatetraenoic acid and 10(-8) M 12-hydroxyeicosatetraenoic acid were potent inhibitors of renin secretion. Similarly, 15-hydroperoxyeicosatetraenoic acid and its hydroxy derivative, 15-hydroxyeicosatetraenoic acid, at somewhat higher molar concentrations (10(-6) M) also reduced basal renin. These studies confirm prostacyclin as a potential renin secretagogue; however, its action in vitro is transient, probably because of its rapid degradation. Our studies provide new evidence that products of the 12-lipoxygenase and 15-lipoxygenase pathways, reported to be present in renal vascular tissue, are potent inhibitors of renin secretion and much more active on a molar basis on renin secretion than is prostacyclin. These studies suggest the potential presence of a dual system of stimulation and suppression that may regulate renin secretion in normal and clinical states.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acids; Epoprostenol; Hydroxyeicosatetraenoic Acids; Kidney Cortex; Leukotrienes; Lipid Peroxides; Male; Rats; Rats, Inbred Strains; Renin; SRS-A

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

This study was undertaken to examine whether spontaneous labor at term is associated with changes in the amniotic fluid concentrations of arachidonate lipoxygenase metabolites. Amniotic fluid was obtained from 15 women at term in active labor (with cervical dilatation of at least 6 cm) and from 15 nonlaboring control women matched for maternal age, parity, and gestational age. Cultures of amniotic fluid for bacteria and mycoplasma were negative. Products of arachidonate lipoxygenase metabolism--12-hydroxyeicosatetraenoic acid (12-HETE), 15-hydroxyeicosatetraenoic acid (15-HETE), and leukotriene B4--were measured by radioimmunoassay. The median concentrations of 12-HETE, 15-HETE, and leukotriene B4 in the amniotic fluid of nonlaboring women were 11.50 ng/mL, 0.45 ng/mL, and 21 pg/mL, respectively, and in the amniotic fluid of laboring women, 24.63 ng/mL, 4.34 ng/mL, and 96 pg/mL, respectively. The differences between labor and nonlabor amniotic fluid concentrations of all three lipoxygenase products were significant (P less than .05, Wilcoxon test). These observations are consistent with involvement of products of the lipoxygenase pathway of arachidonic acid metabolism in the mechanism of human parturition.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Amniotic Fluid; Arachidonate Lipoxygenases; Female; Gestational Age; Humans; Hydroxyeicosatetraenoic Acids; Labor, Obstetric; Maternal Age; Parity; Pregnancy; Specimen Handling

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

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

Interactions between vascular endothelial cells and blood platelets have been investigated using a model microcirculation consisting of microcarrier beads colonized with human umbilical vein endothelial cells (HUVECs) and perfused with washed platelet suspensions. To simulate the effects of endothelial desquamation and exposure of subendothelium, fibrillar collagen in suspension was coinjected with the platelets. In this model, neither the passage of platelets alone nor collagen alone stimulated prostacyclin (PGI2) production by the HUVECs. Platelets activated by coinjection with collagen released thromboxane A2 (TXA2), and this was associated with the simultaneous production of PGI2 by the HUVECs. By means of double-isotope experiments with [3H]arachidonic acid (AA) incorporated into platelets and [14C]-AA into HUVECs, it was shown that all the PGI2 generated was derived from platelet AA and/or endoperoxides. This interpretation was strengthened by the finding that PGI2 production was not prevented by treatment of HUVECs with indomethacin followed by perfusion with collagen-stimulated platelets. AA metabolites in double-isotope label experiments were further characterized by reverse-phase chromatography, and it was shown that both cyclooxygenase and lipoxygenase products of the HUVECs were derived from platelet membrane lipid. Thrombin regularly produced transient PGI2 release, but showed rapid tachyphylaxis. Platelet-derived compounds including ADP, ATP, and platelet-activating factor (PAF) did not produce PGI2 release by HUVECs in this system. Thus, the transfer of AA and metabolites from collagen-stimulated platelets is likely to be the mechanism for PGI2 production in the context of minor degrees of endothelial desquamation.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Collagen; Endothelium; Epoprostenol; Female; Humans; Hydroxyeicosatetraenoic Acids; Pregnancy; Thromboxane B2; Time Factors; Umbilical Cord

Certain arachidonic acid (AA) metabolites have been detected in psoriatic skin lesions. In this study the capacity of normal epidermis and clinically uninvolved psoriatic epidermis to transform AA into lipoxygenase products was determined in vitro. After incubating homogenized epidermis with exogenous AA, the extracted lipids were isolated by reverse-phase high-performance liquid chromatography. Each chromatographic peak was characterized by its UV absorption spectrum and identified by its coelution with the appropriate authentic standard and by radioimmunoassay of its eluate fraction. Identified compounds were quantitated by integrated UV absorbance. Leukotriene B4 (LTB4) was also identified by neutrophil chemokinesis. Normal epidermis generated 15-hydroxy-eicosatetraenoic acid (15-HETE) and 12-HETE, the latter being more abundant. 5-Lipoxygenase products (LTB4, LTC4, and 5-HETE) were not detected. However, an unknown compound exhibiting a triplet UV absorbtion spectrum with maximum at 274 mm was formed. Its formation was inhibited by 5,8,11,14-eicosatetraynoic acid, but not by indomethacin or a specific 5-lipoxygenase inhibitor (REV 5901). These data suggest that a di-HETE with a triene structure is one possible candidate for the unknown compound. Compared with normal epidermis, the formation of 12-HETE and the unknown di-HETE by uninvolved psoriatic epidermis was increased by 54% and 63%, respectively. The formation of 12-HETE and the unknown di-HETE in uninvolved psoriatic epidermis was stimulated to the same degree in the presence of the phospholipase inhibitor quinacrine. These results indicate that uninvolved psoriatic epidermis has an increased capacity to metabolize free AA into 12-lipoxygenase products.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adult; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Chemotaxis, Leukocyte; Chromatography, High Pressure Liquid; Epidermis; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Lipoxygenase; Neutrophils; Psoriasis; Radioimmunoassay

Epidermis of psoriatic skin lesions is characterized by elevated 5-lipoxygenase and 12-lipoxygenase products. 15-hydroxyeicosatetraenoic acid (15-HETE), the predominant lipoxygenase product in normal dermis, has the potential to inhibit 5-lipoxygenase and 12-lipoxygenase. The purpose of the present study was to determine the capacity of homogenized dermis from uninvolved psoriatic skin to form 15-HETE in vitro. Extracted lipids were separated by reversed-phase high-performance liquid chromatography. Each chromatographic peak was identified by its coelution with authentic standards, by ultraviolet spectrometry, and by radioimmunoassay. Dermis from uninvolved psoriatic skin generated on average 48% less 15-HETE than normal dermis (P less than .01). In contrast, the formation of 12-hydroxyeicosatetraenoic acid was increased by 56% in psoriatic dermis (P less than .01). Prostaglandin E2 formation was similar in normal and psoriatic dermis. Since 15-HETE can inhibit the synthesis of 5-lipoxygenase and 12-lipoxygenase products that possess inflammatory and proliferative capacities, a defective 15-HETE generation in dermis may be of importance for the development of psoriasis.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Dinoprostone; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Prostaglandins E; Psoriasis; Radioimmunoassay; Skin

The purpose of the present study was to analyze the metabolism of arachidonic acid (AA) in normal human dermis. After incubating homogenized dermis with exogenous AA, the extracted lipids were isolated by reversed-phase high-performance liquid chromatography. Each chromatographic peak was characterized by its UV absorption spectrum and identified by its coelution with the appropriate authentic standard and by radioimmunoassay of its eluate fraction. Identified compounds were quantitated by integrated optical density. Homogenized human dermis transformed AA into both cyclooxygenase and lipoxygenase products, but predominantly 15-hydroxy-eicosatetraenoic acid (15-HETE). Cultured fibroblasts from normal human dermis also mainly metabolized AA into 15-HETE. To determine whether dermis-derived 15-HETE might modify the AA metabolism of epidermis, normal human epidermis was incubated with dermis. Increasing amounts of dermis resulted in an increasing inhibition of epidermal 12-HETE formation. Similarly, 15-HETE alone induced a dose-dependent decrease of epidermal 12-HETE formation, while the formation of prostaglandin E2 was unchanged. Since 12-HETE possess the ability to elicit skin inflammation and to stimulate epidermal DNA synthesis, 15-HETE formation may be a way whereby dermis regulates important epidermal activities.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Chromatography, High Pressure Liquid; Epidermis; Humans; Hydroxyeicosatetraenoic Acids; Lipoxygenase Inhibitors; Skin; Skin Physiological Phenomena

There is evidence suggesting a role of eicosanoids in the growth of certain tumors. In this study, tissue samples were collected from basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) of the skin. Both BCCs and SCCs contained more prostaglandin E2 and F2 alpha (PGE2 and PGF2 alpha) than normal epidermis. In vitro incubation of tumor samples with arachidonic acid also resulted in PGE2 and PGF2 alpha formation. Basal cell carcinomas exhibiting a histologically aggressive growth pattern contained higher levels of prostaglandins than those with a nonaggressive growth pattern, both in vivo and after in vitro incubation. Lipoxygenase products (12- and 15-hydroxyeicosatetraenoic acid) were present in smaller amounts than cyclo-oxygenase products (PGE2 and PGF2 alpha) in vivo. Compared with normal epidermis, SCCs and, particularly, BCCs produced smaller amounts of 12-hydroxyeicosatetraenoic acid during in vitro incubation with arachidonic acid. The levels of lipoxygenase products were not related to the tumor growth pattern. These results indicate that excessive prostaglandin levels in BCCs may be associated with an aggressive growth pattern.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Breast Neoplasms; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Chromatography, High Pressure Liquid; Dinoprost; Dinoprostone; Head and Neck Neoplasms; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Mice; Prostaglandins; Prostaglandins E; Prostaglandins F; Rabbits; Radioimmunoassay; Skin Neoplasms

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

Current strict O2 management may be precipitating more severe retinopathy of prematurity than would occur with a more lenient approach. Hypoxemia in an animal model has also been found to worsen retinal neovascularization. It has recently been shown that the hydroxyeicosatetraenoic acids can modulate angiogenesis. 15-Hydroxyeicosatetraenoic acid is proangiogenic, whereas 12-hydroxyeicosatetraenoic acid is an antiangiogenic metabolite. In vitro exposure of paired human neonatal vessels (n = 7) to hypoxia enhanced the production of total vascular hydroxyacids (232 +/- 36 pmol/mg of protein [experimental group] nu 168 +/- 31 pmol [control group]; P less than .01). The increase in vascular 15-hydroxyeicosatetraenoic acid under hypoxic conditions was even more significant (P less than .001). However, platelet production of 12-hydroxyeicosatetraenoic acid was not significantly affected by hypoxia. These observations suggest a possible biochemical basis for the abnormal angiogenic process that occurs during the proliferation phase of the retinopathy of prematurity. The production of local hydroxyeicosatetraenoic acids in tissues manifesting abnormal neovascularization needs to be further evaluated.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acids; Blood Platelets; Carbon Radioisotopes; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Neovascularization, Pathologic; Oxygen; Retinopathy of Prematurity; Umbilical Arteries; Vasoconstrictor Agents

Cytotoxic T lymphocyte (CTL)-mediated lysis of target cells was inhibited by 5,8,11,14-eicosatetraynoic acid (ETYA) and other inhibitors of the lipoxygenase pathway at concentrations that inhibited arachidonic acid metabolism in mixed lymphocyte cultures. Inhibition was reversible and selective for the "lethal hit" stage in the CTL-target interaction. Studies to define CTL-specific arachidonic acid metabolites demonstrated that cloned CTL populations have little or no capacity to metabolize arachidonic acid. Therefore, inhibitor actions appear to be independent of the effects on CTL arachidonic acid metabolism. Alternative explanations for inhibitory effects are discussed.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Binding, Competitive; Clone Cells; Cytotoxicity, Immunologic; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lipoxygenase Inhibitors; Lymphocyte Culture Test, Mixed; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Inbred DBA; T-Lymphocytes, Cytotoxic

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

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

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

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

AA and its derivatives are a family of potent mediators and regulators of inflammation. There are several pieces of evidence to suggest a pathophysiologic role of certain AA derivatives in psoriasis. The complexity of the pathways in the generation of LTs and other LO products suggests that carefully designed systems will be needed to establish the exact role of these compounds in the pathogenesis of diseases such as psoriasis, and to test the effects of pharmacological inhibitors.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Fish Oils; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Leukotrienes; Lipoxygenase; Lipoxygenase Inhibitors; Psoriasis; Skin; SRS-A

In vivo studies have shown that inhibitors of cyclooxygenase metabolism of arachidonic acid may diminish growth and metastasis of certain tumors. Because cyclooxygenase inhibition may increase the production of lipoxygenase products of arachidonic acid metabolism, we have investigated the effect of two such products, 12-hydroxyeicosatetraenoic acid (12-HETE) and 15-hydroxyeicosatetraenoic acid (15-HETE) on tumor cell proliferation in vitro. When neuroblastoma cells (SK-N-SH) in culture were treated with 12-HETE for 18 hr, incorporation of [3H]thymidine was inhibited up to 64% at concentrations from 20 to 50 microM. Under the same conditions, 15-HETE resulted in inhibition of up to 46%, while arachidonic acid had no apparent effect. When evaluated in the presence of serum, 12-HETE at a concentration of 120 microM produced a 20.6 +/- 2.8% (S.E.) inhibition of the increase in total DNA content over 48 hr, while 15-HETE at this concentration produced a 16.5 +/- 5.3% inhibition. We conclude that 12-HETE, the product of platelet lipoxygenase, and 15-HETE, a product of neutrophil and lymphocyte lipoxygenases, can inhibit human neuroblastoma cell growth in vitro and may play a role in the effect of cyclooxygenase inhibitors on tumor growth in vivo.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Cell Line; DNA Replication; Hydroxyeicosatetraenoic Acids; Lipoxygenase; Neuroblastoma

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

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 metabolism of [14C]arachidonic acid into cyclooxygenase and lipoxygenase products by homogenates of primary squamous carcinomas of head and neck in 12 patients was studied in vitro. The lipoxygenase pathway was predominant in all samples. The major metabolites were 12-hydroxy-5,8,11-14-eicosatetraenoic acid, (12-HETE) and 15-HETE. 5-HETE, 5,12-diHETE, 8-HETE and 9-HETE were also detected. The cyclooxygenase products detected were in the following order: PGE2 greater than PGF2 alpha greater than TxB2 greater than 15-keto-PGE2 greater than 6-keto-PGF1 alpha greater than PGD2. Literature review of the biological activities of these oxygenated metabolites of arachidonic acid suggest important modulatory roles in the pathophysiology of head and neck cancer.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Carcinoma, Squamous Cell; Head and Neck Neoplasms; Humans; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Prostaglandins

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

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

Minced rabbit pericardium actively converts [1-14C]arachidonic acid into the known prostaglandins (6-[1-14C]ketoprostaglandin F1 alpha, [1-14C]prostaglandin E2 and [1-14C]prostaglandin F2 alpha) and into several unidentified metabolites. The major metabolite was separated by C18 reverse-phase high-pressure liquid chromatography (HPLC) and identified by gas chromatography-mass spectrometry (GC-MS) to be 6,15-[1-14C]diketo-13,14-dihydroprostaglandin F1 alpha. The other nonpolar metabolites were 15-[1-14C]hydroxy-5,8,11,13-eicosa-tetraenoic acid (15-HETE), 11-[1-14C]hydroxy-5,8,12,14-eicosatetraenoic acid (11-HETE) and 12-[1-14C]hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE). Arachidonic acid metabolites actively produced by the pericardium could influence the tone of surface blood vessels on the myocardium.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 6-Ketoprostaglandin F1 alpha; Animals; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Pericardium; Rabbits

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

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

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acids; Hydroxyeicosatetraenoic Acids; Lipopolysaccharides; Lipoxygenase Inhibitors; Lymphocyte Activation; Male; Mice; Mice, Inbred C57BL; Phytohemagglutinins; T-Lymphocytes; Tetradecanoylphorbol Acetate

Particulate fractions and slices from fetal calf aorta convert arachidonic acid to 6-oxoprostaglandin F1 alpha (6-oxoPGF1 alpha), 6,15-dioxoPGF1 alpha, 12-hydroxy-5,8,10-heptadecatrienoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid (11h-20:4), and 15-hydroxy-5,8,11,13-icosatetraenoic acid (15h-20:4). In some cases, small amounts of 12-hydroxy-5,8,10,14-icosatetraenoic acid (12h-20:4) were also detected. The products were all identified by gas chromatography-mass spectrometry after purification by normal phase and argentation high pressure liquid chromatography. Both 11h-20:4 and 15h-20:4 appeared to be formed by prostaglandin endoperoxide synthetase rather than by lipoxygenases, since their formation was inhibited by indomethacin but not by nordihydroguaiaretic acid. The formation of 12h-20:4, on the other hand, was stimulated by indomethacin, probably due to increased substrate availability. The formation of hydroxyicosatetraenoic acids was markedly stimulated by adrenaline. Substantial amounts of 6,15-dioxoPGF1 alpha were formed from arachidonic acid by particulate fractions from fetal calf blood vessels, especially in the presence of relatively high substrate concentrations. The formation of this product was stimulated by methemoglobin and inhibited by adrenaline, glutathione, and tryptophan. It would appear that particulate fractions from fetal calf aorta convert arachidonic acid to 15-hydroperoxyPGI2, which can either be reduced in the presence of various cofactors to form PGI2 or dehydrated to give 15-oxoPGI2. The formation of hydroperoxides from arachidonic acid could be an important factor in regulating PGI2 synthesis in aorta, since PGI2 synthetase is strongly inhibited by such intermediates.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; 6-Ketoprostaglandin F1 alpha; Animals; Aorta; Arachidonic Acids; Cattle; Ductus Arteriosus; Fatty Acids, Unsaturated; Hydroxy Acids; Hydroxyeicosatetraenoic Acids; Kinetics; Lipoxygenase; Prostaglandin-Endoperoxide Synthases; Prostaglandins F, Synthetic

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

The present study of compares the effects of various inhibitors of arachidonate metabolism on mouse thyroid cyclo-oxygenase and lipoxygenase activities and thyrotropin-augmented cyclic-AMP accumulation. Mouse thyroid homogenate converts [1-14C]- arachidonate to several products of the cyclo-oxygenase pathway as well as one major product of the lipoxygenase pathway, 12-L-hydroxyeicosatetraenoic acid (12-Hete). Prostaglandin (PG) formation in thyroid homogenates is inhibited by 1-10 microM indomethacin and etya. 12-HETE accumulation is reduced by 91%, 83% and 20% by 5 microM ETYA, 15-HETE, and indomethacin, respectively. Thyrotropin-stimulated cyclic-AMP accumulation, measured in whole thyroid lobes by radioimmunoassay, is reduced by 45% and 73% by 50 microM and 100 microM ETYA, respectively; indomethacin is without effect at these concentrations. 15-HETE reduces thyrotropin-augmented cyclic-AMP accumulation by 57% and 100 microM. In product inhibition studies, 10 microM 12-HETE reduced the formation of radiolabeled 12-HETE by 20%. 10 microM PGE2, PGF2 alpha or PGD2 had no effect on [1-14C]-PG formation. 12-HETE, however, reduced PG synthesis by 76% at 10 microM. This is the first report implicating the arachidonate lipoxygenase pathway in thyrotropin action at the level of cyclic-AMP regulation. Additionally, our finding that 12-HETE inhibits prostaglandin synthesis suggests that the cyclo-oxygenase and lipoxygenase pathways in the mouse thyroid may be highly integrated.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Cyclic AMP; Hydroxyeicosatetraenoic Acids; Indomethacin; Lipoxygenase; Mice; Prostaglandin-Endoperoxide Synthases; Thyroid Gland; Thyrotropin

Arachidonic acid was incubated with 10,000 X g and 100,000 X g supernatants prepared from the medullae of hydronephrotic, normal, and contralateral rabbit kidneys. Metabolites whose production was calcium dependent but not inhibited by indomethacin were identified by thin layer chromatography. Preparative separation was carried out by silicic acid high pressure liquid chromatography. The methyl ester trimethylsilyl derivatives were prepared and examined by gas chromatography-mass spectrometry. The mass spectra matched previously published mass spectra for 12-hydroxy-5,8,10,14-eicosatetraenoic acid and 15-hydroxy-5,8,11,13-eicosatetraenoic acid. The renal cortex does not metabolize arachidonic acid through the lipoxygenase pathway. Thus, there is regional metabolism of arachidonate to monohydroxy eicosanoids in the kidney.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Hydronephrosis; Hydroxyeicosatetraenoic Acids; Kidney Medulla; Lipoxygenase; Mass Spectrometry; Prostaglandin-Endoperoxide Synthases; Rabbits; Ureter