Bioconversion of leukotriene D4 by lung dipeptidase

Sheep lung dipeptidase was released from a lung membrane preparation by digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. The total enzyme activity released into the supernatant was 4- to 5-fold greater than that measured in the intact membrane prior to solubilization. The release of the peptidase from the membrane by this treatment is typical of proteins anchored to the lipid bilayer by a covalent attachment of phosphatidylinositol via a C-terminal glycolipid extension. The solubilized lung peptidase was further purified by ammonium sulfate fractionation followed by affinity chromatography and high-pressure liquid chromatography. A linear relationship between log molecular weight and elution volume for proteins of known molecular weight was established using a Toya Soda TSK 3000 high-pressure liquid chromatography column, and the molecular weight of the lung dipeptidase was estimated at 105,000. The peptidase activity against glycyldehydrophenylalanine of the purified enzyme co-chromatographed in high-pressure liquid chromatography with the activity that converted leukotriene D4 to leukotriene E4. In kinetic studies using leukotriene D4 as substrate, the relationship between the rate of hydrolysis and enzyme concentration was shown to be linear over the range 20 ng to 98 ng enzyme. Values of Km and Vmax for the dipeptidase using leukotriene D4 as substrate were 43 +/- 6 microM and 11,200 +/- 400 nmol/min per mg, respectively. Inhibition of the conversion of leukotriene D4 to leukotriene E4 was observed with a series of inhibitory agents. Cilastatin, bestatin and chloracetyldehydrophenylalanine were all effective at the micromolar level with cilastatin proving to be the most effective inhibitor. Dithiothreitol was effective within the millimolar range.     

The excretion of leukotriene E4 into urine following inhalation of leukotriene D4 by human individuals

Healthy volunteers underwent bronchial challenge with increasing doses of nebulized leukotriene D4 (0.007 - 200 nmol) at 15 min intervals. Total amounts of 200 nmol (females) and 400 nmol (males) were inhaled, corresponding to approximately 100 nmol and 200 nmol deposited in the lung, respectively. Of the latter amounts 3 +/- 1% (mean +/- S.E.M., n = 5) was found to be excreted as leukotriene E4 into the urine within 12 h. No further excretion after this period was observed. Approximately 50% of the total urinary leukotriene E4 was excreted during the first 2 h. These results suggest that a possible formation of sulfidopeptide leukotrienes in the lung in vivo can be monitored by measuring leukotriene E4 excretion into the urine.     

Enzymatic hydrolysis of leukotriene A4 into 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid and LTB4 by mammalian kidney

Homogenates from rat and pig kidney converted leukotriene A4 to 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid as well as leukotriene B4. Both hydrolyses were enzymatic as judged by the effects of heat treatment and proteolytic digestion. Upon subcellular fractionation, conversion of leukotriene A4 to 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid occurred both in the 105,000xg supernatant and the 20,000xg pellet from rat kidney, whereas conversion to leukotriene B4 was confined to the 105,000xg supernatant. We also found production of 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid and leukotriene B4 in isolated rat renal epithelial cells, either from exogenous leukotriene A4 or from this substrate supplied by human leukocytes.     

In vivo metabolism of leukotriene C4 in man: urinary excretion of leukotriene E4

Five - 20 nmoles of [5,6,8,9,11,12,14,15-3H8]leukotriene C4 was injected into three male volunteers. Forty-eight percent of the administered 3H was recovered from urine and 8% from feces, within a 72 hr period. Of the total urinary radioactivity 44% was excreted during the first hour after injection. This activity was mainly found in one compound, designated "I". The radioactivity excreted into urine later than one hour after injection, consisted partly of Compound I and two additional components, and partly of polar, non-volatile material. Compound I was identified as leukotriene E4 by UV-spectroscopy and cochromatographies in three high performance liquid chromatography systems with synthetic reference compounds. A total of 13% of administered radioactivity was excreted in urine as leukotriene E4.     

Studies on the leukotriene D4-metabolizing enzyme of rat leukocytes, which catalyzes the conversion of leukotriene D4 to leukotriene E4

Leukotriene D4-metabolizing enzyme was studied using rat neutrophils, lymphocytes and macrophages. These leukocyte sonicates converted leukotriene D4 to leukotriene E4. However, the leukotriene D4-metabolizing activity varied with cell type, and macrophages showed the highest activity among these leukocytes. The subcellular localization of the leukotriene D4-metabolizing enzyme of macrophages was examined, and the leukotriene D4-metabolizing activity was found to be present in the membrane fraction, but not in the nuclear, granular and cytosol fractions. When macrophages were modified chemically with diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell-surface enzymes selectively, the leukotriene D4-metabolizing activity of macrophages decreased significantly (about 95%) without any inhibition of marker enzymes of microsome, cytosol, lysosome and mitochondria. When neutrophils and lymphocytes were modified with diazotized sulfanilic acid, the leukotriene D4-metabolizing activity was also inhibited about 90% by the modification. Among various enzyme inhibitors used, o-phenanthroline, a metal chelator, remarkably inhibited the leukotriene D4-metabolizing activity of leukocytes, and the o-phenanthroline-inactivated enzyme activity was fully reactivated by Co2+ and Zn2+. These findings seem to indicate that rat neutrophils, lymphocytes and macrophages possess the leukotriene D4-metabolizing metalloenzyme which converts leukotriene D4 to leukotriene E4, on the cell surface, although macrophages have a higher enzyme activity than the other two.     

Metabolism of leukotriene A4 by human erythrocytes. A novel cellular source of leukotriene B4

Human erythrocytes transformed leukotriene A4 into leukotriene B4. Metabolism was proportional to the erythrocyte concentration, even at subphysiological levels (0.08-4 X 10(9) erythrocytes/ml). Comparative metabolic studies excluded the possibility that leukotriene B4 originated from trace amounts of polymorphonuclear leukocytes or platelets present in the purified erythrocyte suspensions. For example, suspensions of isolated platelets (100-500 X 10(6) cells/ml) failed to convert leukotriene A4 into leukotriene B4; and conversion by suspensions of isolated polymorphonuclear neutrophils was insufficient to account for the amounts of leukotriene B4 formed by erythrocytes. Leukotriene B4 formation was maximal within 2 min and substrate concentration dependent. Enzymatic activity originated from a 56 degrees C labile nondialyzable (Mr greater than 30,000) soluble component in the 100,000 X g supernatant obtained from lysed erythrocytes. In contrast to the contemporary view, our results indicate that human erythrocytes are not metabolically inert in terms of eicosanoid biosynthesis. The role of human erythrocytes during inflammatory or pulmonary disorders deserves re-examination in this context.     

Leukotriene A4. Enzymatic conversion into 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid by mouse liver cytosolic epoxide hydrolase

Mouse liver homogenates transformed leukotriene A4 into a 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acid. This novel enzymatic metabolite of leukotriene A4 was characterized by physical means including ultraviolet spectroscopy, high performance liquid chromatography, and gas chromatography-mass spectrometry. After subcellular fractionation, the enzymatic activity was mostly recovered in the 105,000 X g supernatant and 20,000 X g pellet. Heat treatment (80 degrees C, 10 min) or digestion with a proteolytic enzyme abolished the enzymatic activity in the high speed supernatant. A purified cytosolic epoxide hydrolase from mouse liver also transformed leukotriene A4 into a 5,6-dihydroxyeicosatetraenoic acid with the same physico-chemical characteristics as the compound formed in crude cytosol, but not into leukotriene B4, a compound previously reported to be formed in liver cytosol (Haeggstr?m, J., R?dmark, O., and Fitzpatrick, F.A. (1985) Biochim. Biophys. Acta 835, 378-384). These findings suggest a role for leukotriene A4 as an endogenous substrate for cytosolic epoxide hydrolase, an enzyme earlier characterized by xenobiotic substrates. Furthermore, they indicate that leukotriene A4 hydrolase in liver cytosol is a distinct enzyme, separate from previously described forms of epoxide hydrolases in liver.     

Metabolism of leukotriene E4 to 5-hydroxy-6-mercapto7,9-trans-11,14-cis-eicosatetraenoic acid by microfloral cysteine-conjugate beta-lyase and rat cecum contents

Leukotriene E4 was incubated with cysteine-conjugate beta-lyase isolated from the intestinal bacterium Eubacterium limosum. The reaction was terminated by addition of iodoacetic acid or dimethyl sulfate, and the products formed were isolated by reverse-phase high-performance liquid chromatography. The structures of two adducts of a metabolite were determined by uv spectroscopy, by gas-liquid radiochromatography, and by comparisons with chemically synthesized reference compounds. They were 5-hydroxy-6-S-carboxymethylthio-7,9-trans-11,14-cis-eicosatetraeno ic acid (iodoacetic acid adduct) and 5-hydroxy-6-S-methylthio-7,9-trans-11,14-cis-eicosatetraenoic acid (dimethyl sulfate adduct) indicating that the structure of the underivatized metabolite was 5-hydroxy-6-mercapto-7,9,11,14-eicosatetraenoic acid (5,6-HMETE). The latter product is formed by beta-lyase-catalyzed cleavage of the cysteine C-S bond in leukotriene E4. Leukotriene E4 was also metabolized to 5,6-HMETE by rat cecal contents. A product formed was trapped as the iodoacetic acid derivative and identified as 5-hydroxy-6-S-carboxy-methylthio-7,9,11,14-eicosatetraenoic acid. It is concluded that intestinal leukotriene E4, originating from biliary excretion of systemic cysteinyl leukotrienes or produced in the intestine, is converted by microfloral cysteine-conjugate beta-lyase to 5,6-HMETE.     

Human endothelial cells stimulate leukotriene synthesis and convert granulocyte released leukotriene A4 into leukotrienes B4, C4, D4 and E4

Incubation of human endothelial cells with leukotriene A4 resulted in the formation of leukotrienes B4, C4, D4 and E4. Endothelial cells did not produce leukotrienes after stimulation with the ionophore A23187 and/or exogenously added arachidonic acid. However, incubation of polymorphonuclear leukocytes with ionophore A23187 together with endothelial cells led to an increased synthesis of cysteinyl-containing leukotrienes (364%, mean, n = 11) and leukotriene B4 (52%) as compared to leukocytes alone. Thus, the major part of leukotriene C4 recovered in mixed cultures was attributable to the presence of endothelial cells. Similar incubations of leukocytes with fibroblasts or smooth muscle cells did not cause an increased formation of leukotriene C4 or leukotriene B4. The increased biosynthesis of cysteinyl-containing leukotrienes and leukotriene B4 in coincubation of leukocytes and endothelial cells appeared to be caused by two independent mechanisms. First, cell interactions resulted in an increased production of the total amount of leukotrienes, suggesting a stimulation of the leukocyte 5-lipoxygenase pathway, induced by a factor contributed by endothelial cells. Secondly, when endothelial cells prelabeled with [35S]cysteine were incubated with either polymorphonuclear leukocytes and A23187, or synthetic leukotriene A4, the specific activity of the isolated cysteinyl-containing leukotrienes were similar. Thus, transfer of leukotriene A4 from stimulated leukocytes to endothelial cells appeared to be an important mechanism causing an increased formation of cysteinyl-containing leukotrienes in mixed cultures of leukocytes and endothelial cells. In conclusion, the present study indicates that the vascular endothelium, when interacting with activated leukocytes, modulates both the quantity and profile of liberated leukotrienes.     

Prostaglandin E2 and leukotriene C4-induced luteinizing hormone-releasing hormone release from immature and adult male rat median eminences in vitro: eicosanoid formation and binding parameters

The amounts of prostaglandin E2 formed in vitro by the median eminences of adult male rats were greater than those produced by the median eminences of immature, 22 day-old rats. However, the amount of leukotriene C4 produced by the adult rat median eminences was lower than that produced by the immature rat median eminences. Analysis of the prostaglandin E2 binding parameters of hypothalamic P2 membrane fractions indicates that there are two binding components, one high affinity (RH) and one low affinity (RL) in both adult and immature rats. The maximal binding capacity of RH from adult rat membranes was significantly lower than that of immature rat membranes, correlating with greater prostaglandin E2 production by the adult rat median eminence. Only one leukotriene C4 binding site was detected in both adult and immature rat membranes. Exogenous prostaglandin E2 and leukotriene C4 both stimulated, the release of luteinizing hormone-releasing hormone to the same extent from both the adult and immature median eminences.     

Formation and metabolism of leukotriene C4 in macrophages exposed to bacterial lipopolysaccharide

Lipopolysaccharide (10 micrograms/ml) was found to stimulate resident mouse peritoneal macrophages to produce leukotriene C4 (36 +/- 1.3 ng/10(6) cells, SEM, n = 20) within 16 h. Spontaneous synthesis in control cultures without lipopolysaccharide was less than 1.6 ng/10(6) cells. Leukotriene C4 was characterized by reversed-phase high-performance liquid chromatography, ultraviolet spectrometry and radioimmunoassay. When the macrophages, prelabeled with [3H]arachidonic acid, were treated with lipopolysaccharide radioactivity was incorporated into leukotriene C4. The amount produced varied with the method of macrophage preparation and incubation conditions and was dependent on the amount of lipopolysaccharide added (0.5-60 micrograms/ml), on cell counts and on the incubation time (4-16 h). The released leukotriene C4 was converted to a compound identified as a C6-cysteinylleukotriene, indicating metabolism of the leukotriene by the macrophages. Parallel determinations of prostaglandins E2 and F2 alpha by radioimmunoassay demonstrated that leukotriene C4 and prostaglandin E2 are formed by mouse peritoneal macrophages to a similar degree.     

Formation of leukotrienes E3, E4 and E5 in rat basophilic leukemia cells

Rat basophilic leukemia (RBL-1) cells incubated with ionophore A23187 and 5,8,11-eicosatrienoic acid produced three slow-reacting substances identified as leukotrienes C3, D3 and E3 by spectroscopic, chromatographic and enzymatic methods. 5,8,11,14,17-Eicosapentaenoic acid was similarly converted by RBL-1 cells to leukotrienes C5, D5. and E5. Leukotrienes C4, D4 and E4 were also formed in these experiments from endogenous arachidonic acid. Time-course studies, incubations with 3H-labeled leukotriene C3 and effects of acivicin [L-(alpha S, 5S)-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid; a gamma-glutamyl transpeptidase inhibitor] indicated that leukotrienes C and D are intermediates in the formation of leukotrienes E. L-Cysteine enhanced the conversion of leukotriene C3 to leukotriene D3 and inhibited further degradation of leukotriene D3 to leukotriene E3.     

w-oxidation products of leukotriene E4 in bile and urine of the monkey

The intravenous administration of [3H]leukotriene C4 in the monkey Macaca fascicularis results in the biliary and urinary elimination of [3H]leukotriene D4 and [3H]leukotriene E4 in addition to more-polar metabolites. Separation of these polar metabolites and chromatographic comparison with synthetic w-oxidized leukotrienes indicated the in vivo formation of w-hydroxy-[3H]leukotriene E4 and w-carboxy-[3H]leukotriene E4. Time course studies of the [3H]leukotriene metabolite pattern in bile and urine showed that w-hydroxy-leukotriene E4 was decreasing as w-carboxy-leukotriene E4 and additional polar derivatives were increasing.     

The effect of revascularisation of an ischaemic lower limb on leukotriene E4 production

It has previously been shown that leukotriene E4 production is increased both in acute and chronic lower limb ischaemia. The aim of this study was to measure the effect of revascularisation on leuktriene E4 excretion in chronic lower limb ischaemia. Revascularisation did not affect significantly on leukotriene E4 excretion (preop. 34.9+/-7.1 pg/mg creatinine, postop. 24.5+/-4.7 pg/mg creatinine, n=10, P<0.238). We suggest that the enhanced leukotriene E4 production continues after revascularisation which may have a therapeutical implication.     

N-acetyl[3H]leukotriene D4: a stable internal standard for automated BIO-FAST HPLC extraction and separation of LTE4 from human urine.

The BIO-FAST (Fully Automated Sample Treatment) HPLC can be used for the isolation and separation of leukotriene E4 (LTE4) from the urine of asthmatic patients. A chemically related leukotriene, N-acetyl[14,15-3H]leukotriene D4 (NAc[3H]LTD4), has been evaluated as an internal standard to allow full automation of the BIO-FAST method. NAcLTD4 is not a human metabolite, does not co-elute with endogenously produced LTs and is stable in native urine at 37 degrees C for at least 18 h. Recovery and stability studies were conducted by adding NAc[3H]LTD4 and [3H]LTE4 to the baseline urine of four asthmatic patients. Automated extraction of these four samples over 22 hours, using the BIO-FAST system, yielded recoveries of 80.5% (6.6 %CV, n = 12) and 72.4% (10.0 %CV, n = 12) for the NAc[3H]LTD4 and [3H]LTE4, respectively. The ratio of NAc[3H]LTD4 to [3H]LTE4 was 1.12 (6.3 %CV, n = 12) demonstrating the consistent relative extraction of these two leukotrienes.     

Kinetic resolution of racemic leukotriene A4 by mammalian cytosolic epoxide hydrolases

Cytosols of rat and guinea pig liver and of human placenta were screened for their capacity to catalyze the conversion of racemic leukotriene A4 into 5S, 12R-dihydroxy-(Z,E,E,Z)-6,8,10,14-eicosatetraenoic acid (leukotriene B4). The epoxide hydrolase activities showed some specificity for the 5S,6S-oxido-(E,E,Z,Z)-7,9,11,14-eicosatetraenoic acid (LTA4) and produced mixtures of leukotriene B4 and its enantiomer containing up to 78-87% of leukotriene B4.     

Novel transcellular interaction: conversion of granulocyte-derived leukotriene A4 to cysteinyl-containing leukotrienes by human platelets

Human platelets dose-dependently converted exogenous leukotriene A4 to leukotriene C4 and efficiently metabolized this compound to leukotrienes D4 and E4. Neither of these compounds were produced after stimulation of human platelet suspensions with ionophore A23187. After LTA4 incubation of subcellular fractions, formation of leukotriene C4 was exclusively observed in the particulate fraction and was separable from the classical glutathione S-transferase activity. This suggested the presence of a specific leukotriene C4 synthase in human platelets. Addition of physiological amounts of autologous platelets to human granulocyte suspensions significantly increased ionophore A23187-induced formation of leukotriene C4. In contrast, the production of leukotriene B4 was decreased. After preincubation of platelets with [35S]cysteine, 35S-labeled leukotriene C4 was produced by A23187-stimulated platelet-granulocyte suspensions, strongly indicating a transcellular biosynthesis of this compound.     

A novel leukotriene formed by transpeptidation of leukotriene E

A new leukotriene 5(S)-hydroxy-6(R)-$\text{S}$-γ-glutamylcysteine-7,9-$\text{trans}$-11,14-$\text{cis}$-eicosatetraenoic acid (leukotriene F₄) was isolated after incubating leukotriene E₄ with γ-glutamyltranspeptidase and glutathione. Leukotriene F₄ induced contractions of the isolated quinea pig ileum and was less potent in this respect than leukotriene E₄.     

Saccharomyces cerevisiae leukotriene A4 hydrolase: formation of leukotriene B4 and identification of catalytic residues

Leukotriene A(4) hydrolase in mammals is a bifunctional zinc metalloenzyme that catalyzes the hydrolysis of leukotriene A(4) into the proinflammatory mediator leukotriene B(4), and also possesses an aminopeptidase activity. Recently we cloned and characterized an leukotriene A(4) hydrolase from Saccharomyces cerevisiae as a leucyl aminopeptidase with an epoxide hydrolase activity. Here we show that S. cerevisiae leukotriene A(4) hydrolase is a metalloenzyme containing one zinc atom complexed to His-340, His-344, and Glu-363. Mutagenetic analysis indicates that the aminopeptidase activity follows a general base mechanism with Glu-341 and Tyr-429 as the base and proton donor, respectively. Furthermore, the yeast enzyme hydrolyzes leukotriene A(4) into three compounds, viz., 5S,6S-dihydroxy-7,9-trans-11,14-cis-eicosatetraenoic acid, leukotriene B(4), and Delta(6)-trans-Delta(8)-cis-leukotriene B(4), with a relative formation of 1:0.2:0.1. In addition, exposure of S. cerevisiae leukotriene A(4) hydrolase to leukotriene A(4) selectively inactivates the epoxide hydrolase activity with a simultaneous stimulation of the aminopeptidase activity. Moreover, kinetic analyses of wild-type and mutated S. cerevisiae leukotriene A(4) hydrolase suggest that leukotriene A(4) binds in one catalytic mode and one tight-binding, regulatory mode. Exchange of a Phe-424 in S. cerevisiae leukotriene A(4) hydrolase for a Tyr, the corresponding residue in human leukotriene A(4) hydrolase, results in a protein that converts leukotriene A(4) into leukotriene B(4) with an improved efficiency and specificity. Hence, by a single point mutation, we could make the active site better suited to bind and turn over the substrate leukotriene A(4), thus mimicking a distinct step in the molecular evolution of S. cerevisiae leukotriene A(4) hydrolase toward its mammalian counterparts.     

Effect of leukotriene E4 on the central hemodynamics and vascular contractile activity

The effect of leukotriene E4 (LTE4) on the cardiac output and peripheral vascular resistance was studied. It was shown to damage the heart pump function and to cause spasms of peripheral resistive vessels. LTE4 was also found to induce constriction of heart and cerebral human arteries due to calcium entering myoplasm from the environment and intracellular depot. Low LTE4 concentrations enhanced vascular wall sensitivity to different vasoactive agents, including thromboxane A2. Prostacyclin appeared to be LTE4 antagonist, preventing its vasoconstrictive action. The role of LTE4 as a systemic vasoconstrictive agent is discussed.