leukotriene-a4 and 5-hydroxy-6-8-11-14-eicosatetraenoic-acid

The family of eicasanoids, biologically active metabolites of polyunsaturated C20 fatty acids such as arachidonic acid, has recently been enlarged by the recognition of a new biosynthetic pathway leading to the leukotrienes, including the compounds described two decades ago as 'slow reacting substances'. These biologically potent substances are involved in regulation of the immune response and also as mediators in various disease states. This account presents a brief history of this field, an overview of the biological relevance of leukotrienes, and a discussion of the investigations which led to the clarification of the molecular structures, pathway of biosynthesis and total chemical synthesis of the leukotrienes, including leukotrienes A, B, C, D and E (LTA-LTE). As a result of the synthetic work these rare substances are available for the first time in pure form and in quantities sufficient for biological and medical studies. Also reviewed are recent discoveries with regard to the development of inhibitors of leukotriene biosynthesis and anti-leukotrienes.

Topics: Animals; Arachidonic Acids; Asthma; Autacoids; Chemical Phenomena; Chemistry; Humans; Hydroxyeicosatetraenoic Acids; Hypersensitivity; Leukotriene A4; Leukotriene B4; Leukotriene E4; Leukotrienes; Lipoxygenase Inhibitors; Macrophages; Mast Cells; Molecular Conformation; Neutrophils; SRS-A; Stereoisomerism; Structure-Activity Relationship

Leukotriene A(4) (LTA(4)) is a chemically reactive conjugated triene epoxide product derived from 5-lipoxygenase oxygenation of arachidonic acid. At physiological pH, this reactive compound has a half-life of less than 3 s at 37 degrees C and approximately 40 s at 4 degrees C. Regardless of this aqueous instability, LTA(4) is an intermediate in the formation of biologically active leukotrienes, which can be formed through either intracellular or transcellular biosynthesis. Previously, epithelial fatty acid binding protein (E-FABP) present in RBL-1 cells was shown to increase the half-life of LTA(4) to approximately 20 min at 4 degrees C. Five FABPs (adipocyte FABP, intestinal FABP, E-FABP, heart/muscle FABP, and liver FABP) have now been examined and also found to increase the half-life of LTA(4) at 4 degrees C to approximately 20 min with protein present. Stabilization of LTA(4) was examined when arachidonic acid was present to compete with LTA(4) for the binding site on E-FABP. Arachidonate has an apparent higher affinity for E-FABP than LTA(4) and was able to completely block stabilization of the latter. When E-FABP is not saturated with arachidonate, FABP can still stabilize LTA(4). Several lipoxygenase products, including 5-hydroxyeicosatetraenoic acid, 5,6-dihydroxyeicosatetraenoic acid, and leukotriene B(4), were found to have no effect on the stability of LTA(4) induced by E-FABP even when present at concentrations 3-fold higher than LTA(4).

Topics: Animals; Arachidonic Acid; Binding Sites; Binding, Competitive; Biochemical Phenomena; Biochemistry; Carrier Proteins; Cell Line; Dose-Response Relationship, Drug; Fatty Acid-Binding Proteins; Hydrogen-Ion Concentration; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Lipoxygenase; Mass Spectrometry; Models, Biological; Protein Binding; Rats; Temperature; Time Factors

Polymorphonuclear leucocytes and blood platelets co-operate in several pathophysiological processes, and arachidonic acid (AA) metabolites produced in response to the activation of these cells are potent mediators of their functions. We studied the role of platelets in the formation of 5-lipoxygenase products from AA by autologous neutrophils, especially the chemotactic agent leucotriene (LT) B4. The formation of all products, namely 5-hydroxy-eicosatetraenoic acid (5-HETE), LTB4 and the other LTA4-derived metabolites, in response to the calcium ionophore A23187 was evaluated by high-performance liquid chromatography. All the 5-lipoxygenase products were significantly diminished by physiological concentrations of platelets. This inhibitory effect was lost when platelets were previously degranulated by thrombin in non-aggregating conditions. Peptides containing the Arg-Gly-Asp-Ser or His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val sequence, which prevent the adhesion of platelets to neutrophils via the fibrinogen released from platelet granules and the integrin glycoprotein IIb/IIIa, markedly decreased the inhibitory effect of non-degranulated platelets. The production of transcellular metabolites of AA such as LTC4, the dual 5- and 12-lipoxygenase product 5,12-diHETE and lipoxins could not account for the decreased formation of 5-HETE and LTA4-derived metabolites. It is concluded that platelets may inhibit the neutrophil 5-lipoxygenase activity at the integrin level and in turn may play a role in slowing down the production of LTB4 in the course of inflammation.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Blood Platelets; Calcimycin; Cells, Cultured; Chromatography, Gas; Humans; Hydroxyeicosatetraenoic Acids; Integrins; Ionophores; Leukotriene A4; Leukotriene B4; Leukotrienes; Neutrophils; Platelet Count

Activated neutrophils release a variety of eicosanoids into the extracellular medium including arachidonic acid, 5-hydroxyicosatetraenoic acid, and leukotriene A4 and B4. In this study, the mechanism of arachidonic acid export has been examined using inside-out plasma membrane vesicles from pig polymorphonuclear leukocytes. Tritiated arachidonic acid associated rapidly with the membrane vesicles and crossed the membrane into the intravesicular space in a time-dependent and saturable manner. Half the maximal influx rate was measured at an arachidonate concentration of 5.7 microM, and a maximal influx velocity of 3.0 nmol/mg x min was determined at pH 6.8. Influx into vesicles was sensitive to a number of common anion transport inhibitors including pentachlorophenol, phloretin, diiodosalicylic acid, and quercetin as well as to the proteases trypsin and Pronase, suggesting a protein-dependent process. Furthermore, influx was temperature-sensitive with an energy of activation of 11.6 kcal/mol. Varying extravesicular concentration of ATP, Na+, or K+ had no impact on arachidonate influx, whereas changes in pH had a profound effect; optimum transport activity was observed at an extravesicular pH of 6, whereas raising the pH to 9.5 essentially abolished uptake. These results indicate and initially characterize a novel protein-facilitated arachidonate export mechanism in pig neutrophils.

Topics: Adenosine Triphosphate; Animals; Arachidonic Acid; Calorimetry; Cell Fractionation; Ethylmaleimide; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Leukotriene A4; Leukotriene B4; Neutrophils; Phagocytosis; Phagosomes; Phloretin; Potassium; Quercetin; Sodium; Swine; Temperature

The effects of 1 microM concentrations of arachidonic acid hydroperoxide (HPETES) products of 5-, 12- and 15-lipoxygenase on Na+, K(+)-ATPase activity were investigated in synaptosomal membrane preparations from rat cerebral cortex. 5-HPETE inhibited Na+, K(+)-ATPase activity by up to 67 %. In contrast, 12-HPETE and 15-HPETE did not inhibit Na+, K(+)-ATPase activity. In addition, neither 5-HETE or LTA4 inhibited Na+, K(+)-ATPase activity. Dose-response studies indicated that 5-HPETE was a potent (IC25 = 10(-8) M) inhibitor of Na+, K(+)-ATPase activity. These findings indicate that 5-HPETE inhibits Na+, K(+)-ATPase activity by a mechanism that is dependent on the hydroperoxide position and independent of further metabolism by 5-lipoxygenase. It is proposed that 5-HPETE production by 5-lipoxygenase and subsequent inhibition of neuronal Na+, K(+)-ATPase activity may be a mechansim for modulating synaptic transmission.

Topics: Animals; Arachidonate Lipoxygenases; Cerebral Cortex; Enzyme Inhibitors; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotrienes; Lipid Peroxides; Male; Neurons; Rats; Rats, Sprague-Dawley; Sodium-Potassium-Exchanging ATPase; Synaptic Transmission; Synaptosomes

We report that leukotriene A4, the electrophilic product of 5-lipoxygenase catalysis, irreversibly inactivates the enzyme. Leukotriene A4 inhibits 5-hydroxyeicosatetraenoic acid formation by human neutrophils and differentiated granulocytic HL-60 cells in a concentration-dependent manner with IC50 values = 22.4 +/- 2.5 and 29.0 +/- 8.0 microM, respectively. Recovery of cellular enzymatic activity is negligible (< 6%) following inactivation. Leukotriene A4 inactivates cellular 5-lipoxygenase without inhibiting its translocation from the cytosol to the membrane, suggesting that it impairs catalysis without impairing formation of the complex between 5-lipoxygenase and its membrane-associated activating protein. Consistent with this, leukotriene A4 inactivates purified 5-lipoxygenase from human neutrophils, via saturable, pseudo first-order kinetics with a rate constant, ki = 0.14 min-1 and a dissociation constant, Ki = 2.1 +/- 0.7 microM. Purified 5-lipoxygenase incubated with [3H]arachidonic acid incorporated a radiolabeled species that was not removed by electrophoresis under reduced denaturing conditions. Preincubation with leukotriene A4 diminished the incorporation of radiolabeled material, consistent with irreversible modification of 5-lipoxygenase by its metastable product, leukotriene A4. This unusual product inactivation mechanism may contribute to the decline in 5-lipoxygenase activity observed during catalysis.

Topics: Arachidonate 5-Lipoxygenase; Cell Differentiation; Cell Line; Humans; Hydroxyeicosatetraenoic Acids; Hydroxyurea; Kinetics; Leukemia, Promyelocytic, Acute; Leukocytes; Leukotriene A4; Lipoxygenase Inhibitors; Neutrophils; Tumor Cells, Cultured

The effect of arachidonic acid (AA) on Ca2+ transport in rat liver nuclei was investigated. Ca2+ uptake and release were determined with a Ca2+ electrode. Ca2+ uptake increased dependent on ATP (0.5-2.0 mM), while uptake was negligible in the presence of 2.0 mM ADP or AMP. AA (10-100 microM) caused a marked inhibition of Ca2+ uptake following the addition of 2.0 mM ATP. Also, Ca2+, which accumulated in the nuclei during 6 min after ATP addition, was clearly released by the addition of AA (10-100 microM). The alterations were concentration dependent. The nuclear Ca2+ uptake and release were not altered significantly by the presence of prostaglandin E2 (10 and 20 microM), prostaglandin H2 (1 and 4 microM), thromboxane B2 (1 and 4 microM), leukotriene A4 (1 and 4 microM), Ins(1,4,5)P3 (1 and 10 microM) or dibutyryl cAMP (10 and 50 microM). Only, 5-hydroxy-eicosatetraenoic acid (5-HETE) at 4 microM caused a significant inhibition of nuclear Ca2+ uptake and an appreciable increase in Ca2+ release; the 1 microM concentration had no effect. These results indicate that AA, one of the prostanoids, has a unique effect on Ca2+ uptake and release in rat liver nuclei. The finding suggests that AA has a regulatory effect on the Ca2+ transport system in liver nuclei.

Topics: Animals; Arachidonic Acid; Biological Transport; Calcium; Cell Nucleus; Dithiothreitol; Dose-Response Relationship, Drug; Drug Interactions; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotrienes; Liver; Male; Prostaglandins; Rats; Rats, Wistar; Thromboxane B2

Profile and quantity of leukotriene (LT) and hydroxyeicosatetraenoic acid (HETE) generation upon selective stimulation of isolated polymorphonuclear neutrophils (PMN) compared with neutrophils in a model of pulmonary leukostasis were investigated. Freshly prepared human PMN (2 x 10(8) were injected into the pulmonary artery of isolated, ventilated, and bloodfree perfused rabbit lungs, resulting in nearly quantitative sticking in the microvasculature. The sequestered neutrophils and, in parallel, aliquots of isolated PMN were stimulated with mAb in the presence of C, known to activate PMN arachidonate metabolism via formation of membrane attack complexes. In the isolated cells, a typical LT profile including LTB4 and its omega-oxidation products, 5-HETE and nonenzymatic hydrolysis products of LTA4 was evoked. The latter indicate secretion of LTA4 in considerable amounts. In the model of pulmonary leukostasis, no nonenzymatic LTA4-derivatives were detected, coincident with a predominance of cysteinyl-LT. This finding gives indirect evidence for an efficient LTA4-transfer between PMN feeder cells and vascular acceptor cells with glutathione-S-transferase activity. Moreover, a threefold increase in the total amount of LTA4-derived products was noted in the model of leukostasis, paralleled by a marked decrease in 5-HETE liberation. This effect was further enhanced by inhibition of lung cyclooxygenase. These findings were corroborated in a homologous system, in which rabbit PMN, sticking in the rabbit lung microvasculature, were stimulated with calcium-ionophore A23187. Collectively, these data suggest a complex interaction between microvascular tissue and adhering neutrophils in LT synthesis, involving transcellular LTA4-shift, modulation of the PMN 5-lipoxygenase pathway, and amplification of LT generation. These findings may be relevant for inflammatory events with neutrophils involved.

Topics: Animals; Cell Adhesion; Endothelium, Vascular; Female; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukotriene A4; Leukotriene B4; Leukotrienes; Lung; Male; Microcirculation; Neutrophils; Rabbits

The effect of hypoxia was studied on the ionophore A23187-induced leukotriene production by rat alveolar macrophages. The production of LTB4 and LTC4 decreased with reducing oxygenation without change of cell viability. The synthesis of 5-HETE increased during hypoxia and the total production of LTB4, LTC4 and 5-HETE, the major metabolites of the 5-lipoxygenase pathway in rat alveolar macrophages, was equal during normoxia and hypoxia. Arachidonate release and LTA4-converting into LTB4 and LTC4 was unaffected by hypoxia. LTB4- and LTC4-degradating activities were not affected by hypoxia. These results suggest that LTA4 synthase reaction of leukotrienes biosynthesis might be suppressed by hypoxia.

Topics: Animals; Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Cattle; Hydroxyeicosatetraenoic Acids; Hypoxia; Leukotriene A4; Leukotriene B4; Leukotrienes; Macrophages; Male; Pulmonary Alveoli; Rats; Rats, Inbred Strains; SRS-A; Tritium

A method for the simultaneous single-step organic extraction from biological matrices of peptido- and dihydroxyleukotrienes as well as 5-hydroperoxy- and 5-hydroxyeicosatetraenoic acid followed by separation and quantitation in a single run on reversed-phase high-performance liquid chromatography was evaluated. Using an extraction system comprising 400/1200/4800 (v/v/v) aqueous phase/isopropanol/dichloromethane, pH 3.0, absolute recoveries of 82.3 +/- 2.0, 89.7 +/- 1.0, 93.7 +/- 1.4, 92.8 +/- 1.4, 90 +/- 4, and 90 +/- 4% for prostaglandin B1 (PGB1), leukotriene C4 (LTC4), leukotriene B4 (LTB4), leukotriene D4 (LTD4), 5-hydroperoxyeicosatetraenoic acid (5-HETE), respectively, were achieved. Separation and quantitation of products were performed on a Nucleosil 100 C18 column (5 microns, 4.6 X 250 mm) using, at pH 6.0, a gradient system comprising 72/28/0.02 (v/v/v) methanol/water/glacial acetic acid from 0 to 15 min, followed by a convex gradient to 76/24/0.02 (v/v/v) methanol/water/glacial acetic acid, followed by a 10-min hold at this methanol concentration. The method was used to investigate the profile of leukotrienes synthesized by rat hepatocyte homogenates from 5-HPETE or leukotriene A4 in absence or presence of glutathione (GSH). During a 5-min incubation with 100 microM 5-HPETE, 9.6 ng LTB4/mg protein and 2.2 micrograms 5-HETE/mg protein were formed in the absence of GSH. In the presence of 0.4 mM GSH, 3.7 ng LTB4/mg protein and 11.0 micrograms 5-HETE/mg protein were formed. Using 20 microM LTA4 as a substrate, 17.3 and 324.0 ng LTC4/mg protein X min and 14.3 and 19.3 ng LTB4/mg protein X min were formed in the presence of 0.4 and 10 mM GSH, respectively.

Topics: 1-Propanol; Animals; Chromatography, High Pressure Liquid; Glutathione; Hydrogen-Ion Concentration; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotriene B4; Leukotrienes; Liver; Male; Methylene Chloride; Prostaglandins B; Rats; Rats, Inbred Strains; SRS-A

We have examined the effects of co-culture and in vitro co-stimulation on lipoxygenase metabolism in monocytes and platelets. Monocytes were obtained from the peripheral blood of normal volunteers by discontinuous gradient centrifugation and adherence to tissue culture plastic. Platelets were obtained from the platelet-rich plasma of the same donor. When 10(9) platelets and 2.5 x 10(6) monocytes were co-stimulated with 1 microM A23187, these preparations released greater quantities of 12(S)-hydroxy-10-trans-5,8,14-cis-eicosatetraenoic acid, 5(S),12-(S)dihydroxy-6,10-trans-8,14-cis-eicosatetraenoic acid, and leukotriene C4, 5(S)-hydroxy-6(R)-S-glutathionyl-7,9-trans-11,14-cis-eicosatetraenoic (LTC4) when compared with monocytes alone. Release of arachidonic acid, 5-HETE, delta 6-trans-LTB4, and delta 6-trans-12-epi-LTB4 from monocytes was decreased in the presence of platelets. A dose-response curve was constructed and revealed that the above changes became evident when the platelet number exceeded 10(7). Dual radiolabeling experiments with 3H- and 14C-arachidonic acid revealed that monocytes provided arachidonic acid, 5-HETE, and LTA4 for further metabolism by the platelet. Monocytes did not metabolize platelet intermediates detectably. In addition, as much as 1.2 microM 12(S)-hydroxy-10-trans-5,8,14-cis-eicosatetraenoic acid and 12(S)-hydroperoxy-10-trans-5,8,14-cis-eicosatetraenoic acid had no effect on monocyte lipoxygenase metabolism. Platelets were capable of converting LTA4 to LTC4, but conversion of LTA4 to LTB4 was not detected. We conclude that the monocyte and platelet lipoxygenase pathways undergo a transcellular lipoxygenase interaction that differs from the interaction of the neutrophil and platelet lipoxygenase pathways. In this interaction monocytes provide intermediate substrates for further metabolic conversion by platelets in an unidirectional manner.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Cell Communication; Cell-Free System; Chromatography, High Pressure Liquid; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotrienes; Lipoxygenase; Monocytes; Tritium

Bovine polymorphonuclear leukocytes exhibit a 12-lipoxygenase activity upon sonication. In contrast to bovine platelet 12-lipoxygenase and other 12-lipoxygenases, this enzyme is unable to convert 5(S)-HETE (5(S)-hydroxy,6-trans-8,11,14-cis-eicosatetraenoic acid) or 5(S)-HPETE (5(S)-hydroperoxy,6-trans-8,11,14-cis-eicosatetraenoic acid) into 5(S),12(S)-dihydroxy-6,10-trans,8,14-cis-eicosatetraenoic acid. Surprisingly, the formation of leukotriene A4-derived products namely leukotriene B4 and the leukotriene B4-isomers 12-epi,6-trans- leukotriene B4 and 6-trans-leukotriene B4, was observed upon incubation of this enzyme with 5(S)-HPETE. Hence, the 12-lipoxygenase from bovine polymorphonuclear leukocytes possesses leukotriene A4-synthase activity.

Topics: Animals; Arachidonate 12-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acids; Cattle; Cell-Free System; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotriene B4; Leukotrienes; Neutrophils

A reliable system for evaluating 5-lipoxygenase (5-LO) pathway inhibitors employing human whole blood stimulated by the calcium ionophore, A-23187, and yeast cell walls (YCW) is described. In developing this system, we have shown that leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) can be recovered quantitatively from whole blood, and can be measured with accuracy and a precision (standard deviation) of +/- 12%. Apparent differences in LTB4/5-HETE levels between donors can be minimized by normalizing the LTB4/5-HETE production to neutrophil number. Variability in LTB4/5-HETE production among different donors was reduced by increasing the ionophore concentration. The kinetics of ionophore stimulated product production display a 1-4 min lag which is dependent on ionophore concentration. The lag is removed by pretreatment of blood with 5 micrograms/ml cytochalasin B. Likewise, the kinetics of product formation after stimulation with yeast cell walls demonstrated a lag period, which could be shortened by prior opsonization of the YCW. The amount of LTB4 metabolism to 20-OH-LTB4 and 20-COOH-LTB4 in this system is approximately 20%. Phenidone, nordihydroguaiaretic acid, and nafazatrom, known inhibitors of the 5-LO pathway, display half-maximal inhibition points of 0.4, 1.5, and 9 micrograms/ml, respectively. In summary, we believe that this assay offers a guide for predicting systemic levels of drug needed to be achieved for effective inhibition of cellular LTB4/5-HETE synthesis/release in humans.

Topics: Arachidonate 5-Lipoxygenase; Arachidonate Lipoxygenases; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Cytochalasin B; Egtazic Acid; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Kinetics; Leukotriene A4; Leukotriene B4; Lipoxygenase Inhibitors; Radioimmunoassay; Spectrophotometry, Ultraviolet; Yeast, Dried

Substitution of dietary fatty acids has potential for altering the inflammatory response. The purpose of the present study was to define the metabolites of arachidonic acid (AA) and eicosapentaenoic acid (EPA) secreted by bovine peripheral blood neutrophils and platelets. High performance liquid chromatography was used to characterize cyclooxygenase and lipoxygenase metabolites secreted in response to the calcium ionophore A23187. Cells were prelabelled with 3H-AA or 3H-EPA prior to challenge with the calcium ionophore. Bovine neutrophils secreted leukotriene B4 (LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) as the major metabolites of AA, as well as the corresponding leukotriene B5 (LTB5) and 5-hydroxyeicosapentaenoic acid (5-HEPE) metabolites of EPA. Peptidoleukotrienes derived from 3H-AA or 3H-EPA were not detected under these conditions. The major tritiated metabolites secreted from bovine platelets were: thromboxane A2, measured as the stable metabolite thromboxane B2 (TXB2); hydroxyheptadecatrienoic acid (HHT) and 12-HETE derived from 3H-AA; and the omega-3 analogs TXB3 and 12-HEPE, derived from 3H-EPA. Preferred substrate specificities existed amongst the AA- and EPA-derived metabolites for the intermediary enzymes involved in the arachidonic acid cascade. These findings support the hypothesis that substitution of membrane-bound AA by EPA has potential for modulation of the host inflammatory response following cellular phospholipid mobilization.

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Animals; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Calcimycin; Cattle; Eicosapentaenoic Acid; Gas Chromatography-Mass Spectrometry; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotriene B4; Male; Neutrophils; Tritium

Polymorphonuclear neutrophils, when stimulated, rapidly form platelet-activating factor (PAF) and metabolize their arachidonate into 5-hydroperoxyeicosatetraenoate (5-HPETE), 5-hydroxyeicosatetraenoate (5-HETE), leukotriene (LT)A4, and LTB4. PAF and LTB4 degranulate neutrophils; 5-HETE, while lacking intrinsic degranulating actions, potentiates these responses. We now find that: a) 5-HPETE similarly potentiates the two lipids and has weak degranulating actions, b) LTA4 and LTB4 degranulate neutrophils by a common pathway, c) PAF degranulates neutrophils by a distinctly different pathway, d) the actions of either LT are additive to those of PAF, e) 5-HETE is particularly effective in potentiating response to combinations of PAF and LTB4, and f) combinations of the lipids partially circumvent requirements for cytochalasin B in these degranulation responses. Thus, the five lipids can be classified into potentiators (i.e., 5-HPETE and 5-HETE) and two types of independently acting agonists (i.e., LT's are one type, PAF a second type). At low concentrations, potentiator, LT, and PAF can all interact to produce prominent responses. They may similarly interact to promote function within their cells of origin.

Topics: Arachidonic Acids; Cytoplasmic Granules; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene A4; Leukotriene B4; Leukotrienes; Neutrophils; Platelet Activating Factor; Stimulation, Chemical

Ten eicosanoid compounds (3, 6, 9, 11, 12, 15, 18, 21, 23, and 25), methyl (6E,8Z,11Z,14Z)-5-hydroxy-6,8,11,14-eicosatetraenoate (5-HETE, 10), leukotriene A4 (26), and (5S,6E,8E,10E,12RS,14E)-5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid (5,12-diHETE, 27) were prepared and their inhibitory activities against the 5-lipoxygenase from guinea pig polymorphonuclear leukocytes (PMNL) were tested. 5,6-Methanoleukotriene A4 (18) was especially a potent and specific inhibitor of the 5-lipoxygenase without inhibiting the cyclooxygenase and the 12-lipoxygenase. Leukotriene A4, 5-HETE, and 5,12-diHETE also have inhibitory activities against the 5-lipoxygenase at micromolar concentrations, which can regulate the formation of slow-reacting substance of anaphylaxis intracellulary.

Topics: Animals; Arachidonate Lipoxygenases; Arachidonic Acids; Blood Platelets; Chemical Phenomena; Chemistry; Fatty Acids, Unsaturated; Guinea Pigs; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Leukocytes; Leukotriene A4; Leukotriene B4; Lipoxygenase Inhibitors

We have studied LTA4 and LTB4 synthesis in a cell-free system from RBL-1 cells. All the enzymes leading to the formation of LTB4 from arachidonic acid are localized in the soluble fraction (100,000 x g supernatant) of these cells. The formation of LTA4 and LTB4 is complete by 10 min. When we varied the arachidonic acid concentration from 1 to 300 microM, the synthesis of LTB4 leveled off at 30 microM and of LTA4 at 100 microM while 5-HETE had not reached a plateau at 300 microM. This enzyme system has the capacity to generate relatively large amounts of 5-HETE and LTA4 and only a relatively small amount of LTB4. Therefore, the rate limiting step is not the 5-lipoxygenase, the first step in the pathway, but the conversion of LTA4 to LTB4. This is in contrast to cyclooxygenase pathway where the first step is rate limiting. A second addition of arachidonic acid at submaximal concentration for LTA4 synthesis did not produce any additional LTA4 or LTB4. Further study of this phenomenon showed that the 5-lipoxygenase and LTA-synthase were inactivated with time by preincubation with arachidonic acid and that peroxy fatty acids seem to be the inactivating species.

Topics: Animals; Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Cells, Cultured; Hydroxyeicosatetraenoic Acids; Kinetics; Leukemia, Experimental; Leukotriene A4; Leukotriene B4; Lipoxygenase; Rats; Subcellular Fractions; Time Factors

Utilizing a variety of fatty acids, differing in chain length, degree and position of unsaturation, we investigated the substrate specificity for the enzymatic production of biologically active slow reacting substances (SRS) and of the other leukotrienes. A cell-free enzyme system obtained from RBL-1 cells was used in this study. The primary structural requirement observed for the conversion by this lipoxygenase enzyme system was a delta 5,8,11 unsaturation in a polyenoic fatty acid. Such fatty acids as 20:4 (5,8,11,14) 20:5 (5,8,11,14,17), 20:3 (5,8,11), 19:4 (5,8,11,14) and 18:4 (5,8,11,14) were readily converted to compounds that comigrated with 5-HETE and 5,12-DiHETE and to biologically active SRS. Chain length did not have an influence on the formatin of these hydroxyacids. Fatty acids with the initial unsaturation at delta 4, delta 6, delta 7, or delta 8 were a poor substrate for the leukotriene enzyme system. Therefore, this lipoxygenase pathway in leukocytes is quite different from the lipoxygenase in platelets which does not exhibit this specificity.

Topics: Animals; Arachidonic Acids; Autacoids; Basophils; Cells, Cultured; Fatty Acids, Unsaturated; Hydroxyeicosatetraenoic Acids; Ileum; Leukemia; Leukotriene A4; Leukotriene B4; Lipoxygenase; Rats; Substrate Specificity