formation of leukotriene E5 by murine peritoneal cells

Resident mouse peritoneal cells, stimulated with opsonized zymosan, produced leukotriene C₄ and E₄, with LTE₄ being the major (80–90%) product. When mice were placed on diets containing increasing amounts of fish oil, four additional sulfidopeptide leukotrienes (SP-LT), LTC₅, LTE₅, 11-trans LTC₅ and 11-trans LTE₅, were identified. The identity of LTE₅ was confirmed by spectrophotometric, chromatographic and enzymatic methods. When equivalent amounts of n-6 and n-3 polyunsaturated fatty acids (PUFA) were included in the diet, the stimulated peritoneal cells ( ) produced higher quantities of LTE₅ (30.2 ± 5.4 ng/10⁶ cells) than LTE₄ (22.8 ± 7.3 ng/10⁶ cells). In addition, studies demonstrated a 60% reduction in LTC₄ (42.0 ± 10.8 ng/10⁶ cells to 16.7 ± 6.2 ng/10⁶ cells) and the appearance of LTC₅ (2.1 ± 0.9 ng/10⁶ cells) in resident macrophages (stimulated with A23187) from mice maintained on a fish oil diet compared to mice fed the control diet. This study demonstrated that formation of the pentaenyl SP-LT , in particular LTE₅, by peritoneal cells can significantly contribute to the endogenous SP-LT pool in response to an inflammatory stimulus following a dietary regimen containing fish oil.     

In vivo formation of beta-oxidized metabolites of leukotriene E4 in the rat

Intraperitoneal administration of [3H]-leukotriene E4 in the rat resulted in the appearance of radiolabel in urine and feces. Separation of polar urinary metabolites and chromatographic comparison of synthetic metabolites indicated the in vivo formation of omega-oxidized metabolites of LTE4 with sequential beta-oxidation. Furthermore, the metabolite identified as 16-carboxy-17,18,19,20-tetranor-14,15-dihydro-N-acetyl-LTE4 substantiates the biochemical pathway of beta-oxidation in vivo involving the 2,4-dienoyl CoA reductase as an integral step. These results substantiate beta-oxidation of sulfidopeptide leukotrienes in vivo and these metabolites account for some of the major urinary metabolites of this class of lipid mediator.     

Metabolism of leukotriene E4 by rat tissues: formation of N-acetyl leukotriene E4

Leukotriene E4 was incubated with subcellular fractions from rat liver homogenates. A product identified as 5-hydroxy-6-S-(2-acetamido-3-thiopropionyl)-7,9-trans-11,14- cis-eicosatetraenoic acid (N-acetyl leukotriene E4) was formed. Enzymes catalyzing the reaction were associated with particulate fractions sedimenting between 600 and 8500 g and 20,000 and 105,000 g. Acetyl coenzyme A served as the donor of the acetyl group. N-Acetyl leukotriene E4 was also formed by the 105,000g sediment fractions from kidney, spleen, skin, and lung. The myotropic activity of N-acetyl leukotriene E4 on isolated guinea pig ileum was reduced over 100-fold compared to that of leukotriene D4.     

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.     

In vivo formation of omega-oxidized metabolites of leukotriene C4 in the rat

[3H8]Leukotriene C4 was administered to germfree rats and to conventional rats having a bile duct cannula. Several radioactive metabolites were isolated. Two polar biliary metabolites from conventional rats were identified as N-acetyl-omega-carboxy-leukotriene E4 and N-acetyl-omega-hydroxy-leukotriene E4. A polar fecal metabolite from germfree rats was found to be N-acetyl-omega-carboxy-leukotriene E4. Chemical identities were established using UV spectroscopy and cochromatographies with authentic compounds in several HPLC systems. The fecal metabolite was further characterized by reductive desulfurization followed by gas-liquid-radiochromatography. The yield of the two biliary metabolites was 5% of the administered tritium after three hours and the yield of fecal N-acetyl-omega-carboxy-leukotriene E4 was 3.5% after three days.     

Metabolism of leukotriene D4 by rat peritoneal mast cells

Metabolism of sulfidopeptide leukotrienes, leukotrienes (LT) C4 and D4 by rat peritoneal mast cells was studied. Rat peritoneal mast cells converted LTD4 to LTE4 but not LTC4 to LTD4. The LTD4-metabolizing activity was equally distributed on the cell surface and inside cells, but not released to the extracellular milieu even when a considerable portion of histamine and the secretory granule enzymes were released. Among various enzyme inhibitors examined, o-phenanthroline, a metal chelator, and dithiothreitol significantly suppressed the LTD4-metabolizing activity of mast cell. These results would suggest that some metalloenzyme located on the cell surface is involved in the conversion of LTD4 to LTE4 by rat peritoneal mast cells.     

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.     

In vivo expression of a nonselected gene transferred into murine hematopoietic stem cells by electroporation

Mouse bone marrow cells were subjected to electroporation in the presence of RSVCAT and SV2NEO plasmids. CAT activity was detected in the G-418 resistant granulocyte-macrophage colonies. RSVCAT electroporated into primary bone marrow cells, repopulated lethally irradiated mice as demonstrated by the persistence of CAT activity in the hematopoietic tissues showing that electroporation can offer a powerful mode of gene transfer into bone marrow cells.     

In vitro interactions of murine peritoneal macrophages and sarcoma cells. I. Promotion of tumor cells proliferation by macrophages

An ascites subline (AA) of the murine sarcoma MC1M grows in vivo in the peritoneal cavity but dies in vitro when cultured on glass or collagen. The viability of AA cells in vitro is not influenced in cocultures with fibroblast cell line L929, and is diminished in cocultures supplemented with macrophage culture supernatant or in cocultures with non-adherent peritoneal cells. However, AA cells proliferate in vitro on glass or collagen when cocultured with syngeneic, semisyngeneic, and allogeneic peritoneal macrophages. This was demonstrated by tritiated thymidine incorporation assay, by AA cell number counting, and by measuring AA cell protein content. Proliferation also occurs when AA cells are separated from the macrophage monolayer by millipore filters.     

In vivo radioprotection by 5-aminosalicylic acid

The radioprotective effect of 5-aminosalicylic acid (5ASA) was investigated in mouse bone marrow. The present study was aimed at investigating the radioprotective effect of pre-irradiation treatment with 5ASA against a range of whole-body lethal (8-11 Gy) and sublethal (1-4 Gy) doses of gamma-radiation (RT) in adult Swiss albino mice. Protection against lethal irradiation was evaluated from 30-day mouse survival and against sublethal doses was assessed from chromosomal aberrations in the bone marrow 24 h after irradiation. An intraperitoneal injection of 5ASA at a dose of 25mg/kg body weight (b. wt.) 30 min before lethal RT increased survival, giving a dose modification factor (DMF) of 1.08. Injection of 5ASA (25 mg/kg b. wt.) 60 or 30 min before or within 15 min after 3 Gy whole body RT resulted in a significant decrease in the radiation-induced aberrant metaphases, at 24 h post-irradiation. Maximum effect was seen when the drug was administered 30 min before irradiation. 5ASA (25 mg/kg b. wt.) significantly reduced the number of aberrant metaphases and the different types of aberrations at all the radiation doses (1-4 Gy) tested, giving a DMFs of 1.43 for number of aberrant metaphases. 5ASA pretreatment also significantly enhanced the endogenous spleen colonies in mouse exposed to 11 Gy RT. Pretreatment with 5ASA, protected plasmid DNA (pGEM-7Zf) against breakage induced by RT and Fenton reactants. Using nanosecond pulse radiolysis technique, the bimolecular rate constant of the reaction of 5ASA with hydroxyl radical was found to be 6.7x10(9)M(-1)s(-1). The p53 and p21 protein levels of bone marrow and spleen were evaluated to identify the specific molecular mechanisms. Both p53 and p21 increased 24h after 6 Gy irradiation, while treatment with 5ASA inhibited this RT-induced increase. Therefore, the present data suggest that 5ASA pretreatment decreases death caused by RT-induced gastrointestinal and hemopoeitic syndromes. The proposed mechanism of radioprotection by 5ASA is through the inhibition of damage to DNA, lipids, and proteins; and prevention of RT-induced increased expression of p53 and p21.     

In vivo and in vitro phosphorylation of murine lymphocyte differentiation antigen CD5

Ly-1, the murine lymphocyte differentiation antigen CD5, is phosphorylated constitutively in vivo. This phosphorylation is enhanced by phorbol 12-myristate 13-acetate (PMA) treatment, but not by concanavalin A, Ca2+ ionophore or dibutyryl cAMP. Prolonged PMA treatment abolished PMA-induced Ly-1 phosphorylation but not constitutive phosphorylation, suggesting that protein kinase C (PKC) is responsible for this enhanced phosphorylation, but not the basal phosphorylation of Ly-1. Ly-1 is phosphorylated by PKC added to membranes, further supporting a role for protein kinase C in the in vivo phosphorylation of Ly-1.     

In vivo polyclonal B-lymphocyte activation elicited by murine viruses.

Viruses such as lactate dehydrogenase-elevating virus and adenovirus induce in vivo a polyclonal activation of murine B lymphocytes, followed by a marked increase in the production of immunoglobulin G2a (IgG2a). The role of T lymphocytes in this phenomenon was studied by injection of an anti-CD4 monoclonal antibody able to inhibit the T-helper function. This treatment profoundly depressed the production of IgG2a, whereas it had no effect on the proliferation of B cells. Activated B cells obtained from such infected and treated mice remained able to produce various immunoglobulin isotypes after exposure to an appropriate stimulus. In particular, gamma interferon, which is known to be secreted after viral infection, induced the production of IgG2a. These observations support the hypothesis that the influence of viruses on the switch of immunoglobulins is mediated by T-helper lymphocytes.     

Characterization of leukotriene C4 synthetase in mouse peritoneal exudate cells

Cell lysates of mouse peritoneal macrophages, in the presence of reduced glutathione, converted leukotriene LTA4 to LTC4, and neither LTD4 nor LTE4 was detected. Therefore, like cultured rat basophilic leukemia cells (RBL cells), the peritoneal macrophage contains LTC4 synthetase and appears to contain little, if any, gamma-glutamyl transpeptidase. When LTA4 was added to subcellular fractions of mouse macrophage lysate, the highest specific activity of LTC4 synthetase (nmol LTC4/mg protein per 10 min) was associated with the particulate or membrane fractions (i.e., 10(4) and 10(5) X g pellets). The 10(5) X g supernatant contains approx. 1% of the specific activity and 6% of the total LTC4 synthetase activity compared with that of the 10(5) X g pellet. Conversely, the 10(5) X g supernatant had four-times more specific activity and 19-times more total GSH S-transferase activity than did the 10(5) X g pellet when evaluated using 1-chloro-2,4-dinitrobenzene (DNCB) as the substrate. LTA4 was converted to LTC4 by the membrane enzyme LTC4 synthetase in a dose-dependent manner at low LTA4 concentrations (3-50 microM) and reached a plateau of approx. 30 microM LTA4 using the macrophage 10(5) X g pellet as an enzyme source. The apparent Km value of LTC4 synthetase for LTA4 was estimated to be 5 microM based on Lineweaver-Burk plots. Enzyme in the 10(5) X g supernatant produced negligible quantities of LTC4 (1% or less of the particulate fractions) over a wide range of LTA4 concentrations. However, an enzyme in the 10(5) X g supernatant fraction presumed to be GSH S-transferase effectively catalyzes the conjugation of glutathione (GSH) with the aromatic compound DNCB. The apparent Km value of GSH S-transferase for DNCB was estimated to be 1.0-1.5 mM. On the other hand, enzyme from the membrane fraction (i.e., 10(5) X g pellet) catalyzed this reaction at a negligible rate over a wide range of DNCB concentrations. The apparent Km value of LTC4 synthetase for GSH was estimated to be 0.36 mM and the corresponding Km value estimated for the glutathione S-transferase was 0.25-0.76 mM. These values indicate similar kinetics for GSH utilization by both enzymes. These Km values are also significantly lower than the intracellular GSH levels of 2 to 5 mM. Therefore, it is suggested that the substrate limiting LTC4 synthetase activity is LTA4 and not GSH. Our results indicate that LTC4 synthetase from mouse peritoneal macrophages is a particulate or membrane-bound enzyme, as was reported by Bach et al.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vivo metabolism of leukotriene C4 in germ-free and conventional rats. Fecal excretion of N-acetylleukotriene E4

[5,6,8,9,11,12,14,15-3H8]Leukotriene C4 was subcutaneously injected into rats. Substantial amounts of the administered radioactivity were excreted in feces of germ-free and conventional animals during a 72-h period (78 and 64%, respectively). Analyses of fecal extracts by high performance liquid chromatography showed eight radioactive components for each type of animal. One metabolite amounted to 4.6% of the injected radioactivity in germ-free and 0.6% in conventional rats. Its chemical structure, 5-hydoxy-6-S-(2-acetamido-3-thiopropionyl)-7,9-trans-11,14-c is-eicosatetraenoi c acid (N-acetylleukotriene E4) was determined by ultraviolet spectroscopy, fast atom bombardment mass spectrometry, chemical and enzymatic transformations, and confirmed by chemical synthesis. Another metabolite (2.7% of the administered radioactivity in germ-free and 0.5% in conventional rats) was characterized as the 11-trans isomer of the former metabolite. The pathway of formation of these compounds appears to be analogous to the pathway of mercapturic acid biosynthesis.