Metabolism of leukotriene B4 in hepatic microsomes

Leukotriene B4 was metabolized in rat hepatic microsomes to two products. Mass spectral analysis of these two metabolites indicated that the major metabolite was the 20-hydroxy metabolite while the minor metabolite was the 19-hydroxy metabolite. The formation of these metabolites required NADPH and was linear with time (20 min) and protein (1.6 mg/ml). The Km apparent and Vmax for omega hydroxylation of LTB4 was 14 uM and 0.138 nmol/min/mg protein. In contrast, the km and Vmax for omega minus one hydroxylation was 54 uM and 0.093 nmol/min/mg protein. These results suggest that omega and omega minus one hydroxylations of LTB4 may be mediated by different isozymes of hepatic P-450.     

Molecular cloning and characterization of another leukotriene B4 receptor

Leukotriene B(4) is a potent lipid mediator known to be implicated mainly in inflammatory actions. Previous pharmacological studies indicated the existence of only one class of G protein-coupled receptor for leukotriene B(4), for which a candidate gene, namely BLT, had been identified. Here we report the isolation of another gene encoding a functional G protein-coupled receptor for leukotriene B(4), named JULF2. JULF2 is a novel G protein-coupled receptor of 358 amino acids that shares 36.6% amino acid identity with human BLT. According to genomic information, the JULF2 gene is located on the chromosome 14, about 4 kilobases upstream of the BLT gene. During screening of endogenous ligands for JULF2, we found that leukotriene B(4) induced inhibition of forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells, stably expressing JULF2. Additionally, Chinese hamster ovary cells expressing exogenous JULF2 showed chemotactic responses with leukotriene B(4) in a pertussis toxin-sensitive manner. A large amount of JULF2 mRNA was detected in the human spleen and the peripheral blood leukocytes. Furthermore, JULF2 mRNA was expressed in mononuclear lymphocytes, in which BLT mRNA was barely detected. The discovery of this second leukotriene B(4) receptor will eventually lead to a better understanding of the classification of leukotriene B(4) receptors and reconsideration of the pathophysiological role of leukotriene B(4).     

Inhibition of leukotriene B4-induced neutrophil degranulation by leukotriene B4-dimethylamide

Leukotriene B₄ (LTB₄) is a potent mediator of pro-inflammatory responses including neutrophil degranulation. Leukotriene B₄ dimethylamide has been synthesized and shown to inhibit neutrophil degranulation induced by LTB₄. The inhibition required time to develop (～60 secs), and had a K_D of circa 2 × 10^?7M, and occurred at concentrations where LTB₄ dimethylamide had negligible agonist activity.     

Human B and T lymphocytes convert leukotriene A4 into leukotriene B4

Incubation of human tonsillar B lymphocytes and peripheral blood T lymphocytes with leukotriene A4 led to the formation of leukotriene B4. The purity of these cell suspensions was more than 99%, containing less than 0.5% monocytes. Incubation of purified B or T lymphocytes with the calcium ionophore A23187 did not lead to the formation of any detectable amounts of leukotrienes. Several established cell lines of B and T lymphocytic origin were also found to convert leukotriene A4 into leukotriene B4, showing that monoclonal lymphocytic cells possess leukotriene A4 hydrolase activity.     

Purification and characterization of hepatic steroid hydroxylases from untreated rainbow trout

Purification of cytochrome P450 from liver microsomes of untreated juvenile male rainbow trout yielded five fractions designated LMC1 to LMC5. All fractions, except LMC4 and LMC5, appeared homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed minimum molecular weights of 50,000 (LMC1), 54,000 (LMC2), 56,000 (LMC3), 58,000 (LMC4), and 59,000 (LMC5). Specific contents ranged from 2.8 (LMC3) to 14.9 (LMC5) nmol heme/mg protein. The catalytic activity of LMC1, LMC2, and LMC5 toward various substrates was examined. LMC2 exhibited the highest estradiol 2-hydroxylase activity and progesterone 16 alpha-hydroxylase activity. LMC2 also was most active in the metabolic activation of aflatoxin B1 (AFB1). In contrast, LMC5 was most active in catalyzing the 6 beta- and 16 beta-hydroxylation of testosterone and the 6 beta-hydroxylation of progesterone. LMC1 showed the highest lauric acid hydroxylase activity. The three isozymes tested had low activity (for LMC2 and LMC5) or no activity (for LMC1) toward benzphetamine or benzo[a]pyrene. Polyclonal antibodies to all five isozymes were raised in rabbits and the antibodies were used to examine the contribution of the P450s to microsomal enzyme activities. The results of microsomal enzyme inhibition studies with polyclonal antibodies showed that anti-LMC2 IgG significantly inhibited the oxidative metabolism of testosterone, lauric acid, AFB1, and benzphetamine. Anti-LMC5 IgG inhibited the oxidation of progesterone, estradiol, benzo[a]pyrene, and benzphetamine. Anti-LMC1 IgG slightly inhibited the microsomal hydroxylation of lauric acid. Anti-LMC3 and anti-LMC4 IgG did not inhibit any of the measured microsomal enzyme activities. These findings suggest that individual constitutive isozymes of trout cytochrome P450 have well-defined contributions to the microsomal metabolism of steroids, fatty acids, and xenobiotics.     

A novel hepatointestinal leukotriene B4 receptor. Cloning and functional characterization

Leukotriene B(4) (LTB(4)) is a product of eicosanoid metabolism and acts as an extremely potent chemotactic mediator for inflammation. LTB(4) exerts positive effects on the immigration and activation of leukocytes. These effects suggest an involvement of LTB(4) in several diseases: inflammatory bowel disease, psoriasis, arthritis, and asthma. LTB(4) elicits actions through interaction with one or more cell surface receptors that lead to chemotaxis and inflammation. One leukotriene B(4) receptor has been recently identified (LTB(4)-R1). In this report we describe cloning of a cDNA encoding a novel 358-amino acid receptor (LTB(4)-R2) that possesses seven membrane-spanning domains and is homologous (42%) and genetically linked to LTB(4)-R1. Expression of LTB(4)-R2 is broad but highest in liver, intestine, spleen, and kidney. In radioligand binding assays, membranes prepared from COS-7 cells transfected with LTB(4)-R2 cDNA displayed high affinity (K(d) = 0.17 nm) for [(3)H]LTB(4). Radioligand competition assays revealed high affinities of the receptor for LTB(4) and LTB(5), and 20-hydroxy-LTB(4), and intermediate affinities for 15(S)-HETE and 12-oxo-ETE. Three LTB(4) receptor antagonists, 14,15-dehydro-LTB(4), LTB(4)-3-aminopropylamide, and U-75302, had high affinity for LTB(4)-R1 but not for LTB(4)-R2. No apparent affinity binding for the receptors was detected for the CysLT1-selective antagonists montelukast and zafirlukast. LTB(4) functionally mobilized intracellular calcium and inhibited forskolin-stimulated cAMP production in 293 cells. The discovery of this new receptor should aid in further understanding the roles of LTB(4) in pathologies in these tissues and may provide a tool in identification of specific antagonists/agonists for potential therapeutic treatments.     

Leukotriene B3, leukotriene B4 and leukotriene B5; binding to leukotriene B4 receptors on rat and human leukocyte membranes

Specific high-affinity binding sites for [3H]-leukotriene B4 have been identified on membrane preparations from rat and human leukocytes. The rat and human leukocyte membrane preparations show linearity of binding with increasing protein concentration, saturable binding and rapid dissociation of binding by excess unlabelled leukotriene B4. Dissociation constants of 0.5 to 2.5 nM and maximum binding of 5000 fmoles/mg protein were obtained for [3H] leukotriene B4 binding to these preparations. Displacement of [3H]-leukotriene B4 by leukotriene B4 was compared with displacement by leukotriene B3 and leukotriene B5 which differ from leukotriene B4 only by the absence of a double bond at carbon 14 or the presence of an additional double bond at carbon 17, respectively. Leukotriene B3 was shown to be equipotent to leukotriene B4 in ability to displace [3H]-leukotriene B4 from both rat and human leukocyte membranes while leukotriene B5 was 20-50 fold less potent. The relative potencies for the displacement of [3H]-leukotriene B4 by leukotrienes B3, B4 and B5 on rat and human leukocyte membranes were shown to correlate well with their potencies for the induction of the aggregation of rat leukocytes and the chemokinesis of human leukocytes.     

Biosynthesis, characterization and inhibition of leukotriene B4 in human whole blood

We have evaluated the biosynthesis, characterization and inhibition of Leukotriene (LT) B4 in unstimulated and in A23187-stimulated human whole blood. LTB4 was assayed by radioimmunoassay (RIA) both in unextracted serum and after extraction and thin-layer chromatography (TLC). Unstimulated human whole blood allowed to clot at 37 degrees C for 60 min produced only trace amounts of LTB4 (0.16 +/- 0.05 ng/ml, mean +/- SD, n = 3). LTB4-like immunoreactivity (ir-LTB4) detectable in unstimulated serum samples was largely overestimated by direct RIA, most likely because of interfering substance(s) unrelated to cyclooxygenase or lipoxygenase activity. Incubation of human whole blood with A23187 (2-10 microM) resulted in a concentration-dependent stimulation of LTB4 production. At 10 microM A23187, ir-LTB4 was 18 +/- 2.4 ng/ml (mean +/- SEM, n = 28). In A23187-stimulated serum samples, LTB4 concentrations measured by direct RIA correlated in a statistically significant fashion with those measured after extraction and TLC. Nafazatrom added in vitro caused a dose-dependent inhibition of A23187-stimulated ir-LTB4 production with an IC50 of 17 microM.     

Generation of leukotriene B4 and leukotriene E4 from porcine pulmonary artery

When chopped porcine pulmonary arteries were incubated with calcium ionophore A23187 (1) in the presence of indomethacin there was a time dependent generation of a substance which produced contractions of superfused strips of guinea-pig ileum smooth muscle (GPISM) which were indistinguishable from those induced by LTD4. This material however had a different retention time from LTD4 when subjected to HPLC and co-chromatographed with synthetic LTE4. In addition to LTE4 a substance which had properties indistinguishable from those of LTB4 when assayed on a combination of guinea-pig lung parenchymal strips (GPP) and GPISM (2) was generated from the pulmonary artery. This substance co-chromatographed with synthetic LTB4. The adventitia and intima were the richest source of LTE4, the adventitia releasing slightly more than the intima. The output of LTB4 and LTE4 was inhibited by 6,9-deepoxy-6,9-(phenylimino)-delta 6,8 prostaglandin I (U-60,257). Nordihydroguaiaretic acid (NDGA) inhibited the generation of LTE4.     

A radioimmunoassay for leukotriene B4

A radioimmunoassay for leukotriene B4 has been developed. The assay is sensitive; 5 pg LTB4 caused significant inhibition of binding of [3H]-LTB4 and 50% displacement occurred with 30 pg. The specificity of the assay has been critically examined; prostaglandins, thromboxane B2 and arachidonic acid do not exhibit detectable cross-reactions (less than 0.03%). However, some non-cyclic dihydroxy- and monohydroxy-eicosatetraenoic acids do cross-react slightly (e.g. diastereomers of 5,12-dihydroxy-6,8,10-trans-14-cis-eicosatetraenoic and 12-hydroxy-5,8,10,14-eicosatetraenoic acids cross-react 3.3% and 2.0% respectively). The assay has been used to monitor the release of LTB4 from human neutrophils in response to the divalent cation ionophore, A23187. The immunoreactive material released during these incubations was confirmed as LTB4 by reverse phase high pressure liquid chromatography following solvent extraction and silicic acid chromatography.     

Solubilization and characterization of leukotriene B4 receptor-GTP binding protein complex from porcine spleen

A high amount of leukotriene B4 (LTB4) binding protein was observed in the porcine spleen. It was solubilized and partially purified from spleen membrane with 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS). Scatchard analysis indicated the presence of a single class of receptor with Kd and Bmax values of 0.26 nM and 120 fmol/mg protein, respectively. The receptor was specific for LTB4, and Ki values for 20-hydroxy- and 20-carboxy-LTB4, both inactive metabolites of LTB4, were 1.7 nM and over 1,000 nM, respectively. By the addition of 10 microM GTP gamma S, a low affinity binding site appeared with a Kd value of 390 nM. A pretreatment of the receptor-GTP binding protein complex with islet-activating protein (IAP) increased the inhibitory effect of GTP gamma S on LTB4 binding, indicating that the LTB4 receptor is coupled with an IAP-sensitive GTP-binding protein in the porcine spleen.     

Characterization of leukotriene A4 and B4 biosynthesis

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.     

Role of leukotriene B4 in the pathogenesis of hepatic ischemia-reperfusion injury in the rat.

A common feature to most models of ischemia-reperfusion injury is the accumulation of polymorphonuclear leukocytes (PMNs) into the post-ischemic tissue during the reperfusion period. Interventions that lead to decreased PMN infiltration protect against tissue injury and therefore a knowledge of the chemotactic mediators leading to PMN accumulation is essential to understanding the pathogenesis of the injury and to the development of successful therapeutic strategies. Leukotriene B4 (LTB4), a metabolite formed via the 5-lipoxygenase pathway from arachidonic acid, is one of the most potent chemotactic mediators known. We have investigated the formation of LTB4 in a well characterized model of hepatic ischemia-reperfusion injury in the rat and made use of a specific leukotriene biosynthesis inhibitor, L663,536, to determine the importance of LTB4 in the pathogenesis of the injury. LTB4 concentrations were measured with a specific and sensitive gas chromatographic-mass spectrometric method previously developed in our laboratory. In liver tissue LTB4 levels were below the detection limit of 20 pg/g before 45 min ischemia and did not increase during the first 6 h of reperfusion. However, at 15 h and 24 h reperfusion LTB4 concentrations had increased to levels 50-fold those in control liver (867 +/- 267 pg/g). The increase of plasma alanine aminotransferase (ALT) activities indicated two phases of injury, an initial phase during the first few hours of reperfusion, and a second more severe injury phase between 6 h and 24 h reperfusion. PMNs accumulated in tissue throughout the reflow period reaching 700 +/- 49 per 50 high power fields (HPF) at 24 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Studies on the conjugation of leukotriene B4 with proteins for development of a radioimmunoassay for leukotriene B4

Leukotriene B4 (LTB4) (I) has been converted to its N-(3-amino-propyl)amide derivative (III) and to its hydrazide derivative (VII) via LTB4 delta-lactone. The amide (III) was coupled with Bovine Serum Albumin using 1,5-difluoro-2,4-dinitrobenzene as coupling agent. The hydrazide (VII), was coupled with Hemocyanin (Keyhole Limpet) (KLH) using 6-N-maleimidohexanoic acid chloride as coupling agent.     

Fluorescent leukotriene B4: potential applications

Leukotriene B4 (LTB4) is a potent lipid mediator of inflammation that acts primarily via a seven-transmembrane-spanning, G-protein-coupled receptor denoted BLT1. Here, we describe the synthesis and characterization of fluorescent analogs of LTB4 that are easy to produce, inexpensive, and without the disadvantages of a radioligand. Fluorescent LTB4 is useful for labeling LTB4 receptors for which no antibodies are available and for performing one-step fluorescence polarization assays conducive to high-throughput screening. We found that orange and green fluorescent LTB4 were full agonists that activated the LTB4 receptor BLT1 with EC50 values of 68 and 40 nM, respectively (4.5 nM for unmodified LTB4). Flow cytometric measurements and confocal imaging showed that fluorescent LTB4 colocalized with BLT1. Fluorescence polarization measurements showed that orange fluorescent LTB4 bound to BLT1 with a Kd of 66 nM and that this binding could be displaced by unlabeled LTB4 and other BLT1-specific ligands. Fluorescent LTB4 analogs were also able to displace tritiated LTB4. Orange fluorescent LTB4 binding to enhanced green fluorescent protein-tagged BLT1 could be observed using fluorescence resonance energy transfer. In addition to being a useful alternative to radiolabeled LTB4, the unique properties of fluorescently labeled LTB4 allow a variety of detection technologies to be used.     

The conversion of leukotriene C4 to isomers of leukotriene B4 by human eosinophil peroxidase

The smooth muscle contractile and vasoactive mediator leukotriene C₄ (5(S)-hydroxy-6(R)-sulfido-glutathionyl-eicosatetraenoic acid; LTC₄) is converted by phorbol ester-stimulated human eosinophils to two isomers of leukotriene B₄, 5(S),12(R)-6,8,10 trans-14 cis-eicosatetraenoic acid (5(S),12(R)-“all-trans”-LTB₄) and 5(S),12(S)-“all-trans”-LTB₄, which are leukocyte chemotactic factors lacking the humoral functions of LTC₄. Optimal conversion of LTC₄ to the “all-trans” isomers of LTB₄ by intact eosinophils and soluble eosinophil peroxidase requires both H₂O₂ and halide ions. Oxidative metabolism of leukotrienes may represent an important regulatory function of eosinophils in hypersensitivity reactions.     

Human monocytes convert leukotriene B4 to two dihydro-leukotriene B4-metabolites

Human monocytes metabolize LTB4 by an additional pathway different from omega-oxidation. Reverse-phase high performance liquid chromatography showed four metabolites: 20-COOH-LTB4, 20-OH-LTB4 and two metabolites less polar than LTB4 with an UV maximum at 232 nm. Gas-chromatography mass-spectrometry showed nearly identical mass spectra for both metabolites. The main mass fragments of the two metabolites were increased by two mass units compared to LTB4. Our findings suggest that LTB4 had been reduced to a known and a new dihydro-metabolite of LTB4. Both metabolites together amounted to 85% of total metabolites. The remaining 15% were omega-oxidation products. Thus, the major pathway of LTB4 metabolism by human monocytes is reduction to dihydro-LTB4.     

Comparative effect of leukotriene B4 and leukotriene B5 on calcium mobilization in human neutrophils

Leukotriene B4 (LTB4) is reported to exert its biological activity in neutrophils through the increase in cytosolic free calcium that follows binding to its specific receptor. Leukotriene B5 has been shown to be far less active than LTB4. Therefore we compared the capacity of LTB4 and LTB5 to stimulate the rise in cytosolic free calcium using fura-2-loaded human neutrophils, to assess the relationship between the calcium mobilizing activity and biological potency of LTB4 and LTB5. At any concentration tested, LTB5 was less active than LTB4 in increasing cytosolic free calcium. ED50 for LTB4 and LTB5 were 5 X 10(-10) M and 5 X 10(-9) M, respectively. The difference in the binding affinities of LTB4 and LTB5 to the LTB4 receptor has been reported to explain the difference in their biological activities. In the present study we further demonstrated that the calcium mobilizing activity of LTB4 and LTB5 also correlates the different biological activity of the two compounds.