Eosinophil peroxidase-mediated inactivation of leukotrienes B4, C4, and D4

The slow-reacting substance (SRS) bioactivity of leukotrienes C4 (LTC4) and D4 (LTD4) was rapidly decreased by incubation with eosinophil peroxidase (EPO), H2O2, and iodide, bromide, or to a lesser degree, chloride, LTB4 chemotactic activity was also decreased by the EPO-H2-H2-halide system, although at a slower rate. Myeloperoxidase could substitute for EPO in these reactions. Leukotriene inactivation was greatly decreased or abolished by deletion of any of the components of the system or by the addition of the hemeprotein inhibitors, azide, cyanide, or aminotriazole, indicating a requirement for peroxidase. The H2O2 concentration employed in the above studies was 10(-4) M. H2O2 at higher concentrations (5 x 10(-4) to 10(-2) M) inactivated LTC4 and LTD4 in the absence of EPO and a halide but had no effect on the chemotactic activity of LTB4. We have previously shown that horse eosinophils stimulated with the calcium ionophore A23187 generate SRS. In the present study, eosinophils stimulated in this way were found to release extracellularly both H2O2 and EPO. Incubation of eosinophils with azide that inhibits EPO, and catalase that degrades H2O2, significantly increased the amount of SRS activity detected in the extracellular medium after A23187 stimulation. These findings suggests eosinophils may play an important modulating role in hypersensitivity reactions both by the production of leukotrienes and by their inactivation through the release of H2O2 and EPO.     

Endothelial cell leukotriene C4 synthesis results from intercellular transfer of leukotriene A4 synthesized by polymorphonuclear leukocytes

Leukotriene (LT) synthesis and metabolism were studied in porcine aortic endothelial cells. Leukotrienes were identified by combinations of guinea pig lung parenchymal strip bioassay, radioimmunoassay, and UV spectrophotometry with high performance liquid chromatography. Endothelial cells stimulated with the calcium ionophore, A23187, were unable to convert arachidonic acid to detectable levels of LTA4-derived products including the biologically active metabolites, LTB4 or LTC4. However, these cells readily converted exogenous LTA4 to the potent slow-reacting substance, LTC4. Smaller quantities of 11-trans-LTC4 and LTD4 were also observed. LTB4 was not detectable in these incubations nor was LTB4 metabolism observed. The possible intercellular transfer of LTA4 between polymorphonuclear leukocytes (PMNL) and endothelial cells was tested since PMNL release LTA4 when stimulated and have significant contact with endothelium. When A23187-stimulated neutrophils were coincubated with endothelial cells, a significant increase in LTC4 levels was detected over PMNL alone. LTC4 is formed by the enzymatic conjugation of glutathione (GSH) with LTA4. Therefore in some experiments, endothelial cells were prelabeled with [35S]cysteine to allow intracellular synthesis of [35S]GSH. When unlabeled PMNL were added, as a source of LTA4 to the prelabeled endothelial cells, substantial levels of [35S] LTC4 were recovered. The data indicate that endothelial cells synthesize LTC4 from LTA4. They also demonstrate a specific PMNL-endothelial cell interaction in which endothelial cell LTC4 synthesis results from the intercellular transfer of LTA4 produced by PMNL.     

Generation of leukotrienes by human monocytes pretreated with cytochalasin B and stimulated with formyl-methionyl-leucyl-phenylalanine

The N-formylated tripeptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) initiated the generation of immunoreactive C-6 sulfidopeptide leukotrienes and of leukotriene B4 (LTB4) in a dose-dependent manner from monolayers of human monocytes pretreated for 10 min with 5 micrograms/ml of cytochalasin B. The EC50 for the immunoreactive C-6 sulfidopeptide leukotrienes was 10(-8) M FMLP and for immunoreactive LTB4 was 5 X 10(-8) M FMLP. The maximal response to FMLP occurred within 10 min, and the sum of the two classes of leukotrienes generated was about 1/6 that obtained from monocytes stimulated with calcium ionophore A23187. The requirement for cytochalasin B in order for FMLP, but not the calcium ionophore, to stimulate leukotriene generation is compatible with the ability of cytochalasin B to augment in other cells certain stimulus-specific transmembrane responses that are not dependent on the integrity of the cytoskeleton. Resolution by reverse phase high performance liquid chromatography of the products released from monocytes pretreated with cytochalasin B and stimulated with FMLP or calcium ionophore yielded a single peak of immunoreactive LTB4 eluting at the same retention time as the synthetic standard; immunoreactive C-6 sulfidopeptide leukotrienes eluted at the retention times of leukotriene C4 (LTC4) and leukotriene D4 (LTD4). [3H]LTB4 was not metabolically altered by monocytes pretreated with cytochalasin B and activated with FMLP in comparison with cells treated with buffer alone, whereas [3H]LTC4 was partially converted to [3H]LTD4. The leukotriene-generating response of monolayers of human monocytes pretreated with cytochalasin B to FMLP is receptor-mediated, as indicated by the inactivity of the structural analog N-acetyl-methionyl-leucyl-phenylalanine and by the capacity of the FMLP receptor antagonist carbobenzoxyphenylalanyl-methionine to inhibit the agonist action of FMLP in a dose-response fashion.     

Synthesis and metabolism of leukotrienes by human endothelial cells: influence on prostacyclin release

The synthesis and metabolism of leukotrienes (LTs) by endothelial cells was investigated using reverse-phase high-performance liquid chromatography. Cells were incubated with [14C]arachidonic acid. LTA4 or [3H]LTA4 and stimulated with ionophore A23187. The cells did not synthesize leukotrienes from [14C]arachidonic acid. LTA4 and [3H]LTA4 were converted to LTC4, LTD4, LTE4 and 5,12-diHETE. Endothelial cells metabolized [3H]LTC4 to [3H]LTD4 and [3H]LTE4. The metabolism of [3H]LTC4 was inhibited by L-serine-borate complex, phenobarbital and acivicin in a concentration-related manner, with maximal inhibition occurring at a concentration of 0.1 M, 0.01 M and 0.01 M, respectively. LTC4, LTB4 and LTD4 stimulated the synthesis of prostacyclin, measured by radioimmunoassays as 6-keto-PGF1 alpha. The stimulation by LTC4 was greater than that by LTD4 or LTB4. LTE4, 14,15-LTC4 and 14,15-LTD4 failed to stimulate the synthesis of prostacyclin. LTD4 and LTB4 also stimulated the release of PGE2, whereas LTC4 did not. Serine-borate and phenobarbital inhibited LTC4-stimulated synthesis of prostacyclin in a concentration-related manner. They also inhibited the release of prostacyclin by histamine, A23187 and arachidonic acid. Acivicin had no effect on the release of prostacyclin by LTC4, histamine or A23187. Furthermore, FPL-55712, an LT receptor antagonist, inhibited LTC4-stimulated prostacyclin synthesis but had no effect on histamine-stimulated release of prostacyclin or PGE2. Indomethacin inhibited both LTC4- and histamine-stimulated release. The results show that (a) endothelial cells metabolize LTA4, LTC4 and LTD4 but do not synthesize LTs from arachidonic acid; (b) LTC4 act directly at the leukotriene receptor to stimulation prostacyclin synthesis; (c) the presence of the glutathione moiety at the C-6 position of the eicosatetraenoic acid skeleton is necessary for leukotriene stimulation of prostacyclin release; and (d) the metabolism of LTC4 to LTD4 and LTE4 does not appear to alter the ability of LTC4 to stimulate the synthesis of PGI2.     

Influence of hypoxia on 5-lipoxygenase pathway in rat alveolar macrophages

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.     

U937 and THP-1 cells do not release LTB4, LTC4, or LTD4 in response to A23187

U937 and THP-1 cells possess some characteristics of human mononuclear phagocytes, cells which synthesize and release LTB4, LTC4, and LTD4. Incubation of these cells with recombinant human interferon-gamma (IFN-gamma) or Phorbol Myristate Acetate (PMA) induces a more differentiated cell state. We hypothesized that U937 and THP-1 cells would release LTB4, LTC4, and LTD4 in response to stimulation with the non-physiologic agonist, calcium ionophore A23187 and that preincubation with IFN-gamma or PMA might alter leukotriene release by these cells. We cultured both cell lines for 48 hours in the presence and absence of IFN-gamma (1000 units/ml) and for 120 hours in the presence and absence of PMA (160 nM) and then challenged them with A23187 (5uM) for 30 minutes at 37 degrees C. The supernatants were deproteinated and assayed by RIA for LTB4 and LTC4 and by RP-HPLC for LTB4, LTC4, and LTD4. Neither U937 nor THP-1 cells released quantities of leukotrienes detectable by RIA, less than 0.3ng/5 X 10(6) cells. Peripheral blood mononuclear phagocytes from normal volunteers, cultured and challenged in vitro at under identical conditions, released 11.3 +/- 2.9 ng LTB4 and 2.0 +/- 1.5 ng LTC4/10(6) viable monocytes. The lack of leukotriene production by U937 and THP-1 cells was not altered by preincubation for 48 hours with IFN-gamma (n = 3) nor by preincubation with PMA for 120 hours (n = 3). We conclude 1) U937 and THP-1 cells do not appear to be appropriate in vitro models for the examination of leukotriene release from normal mononuclear phagocytes. 2) Pre-incubation of U937 and THP-1 cells with IFN-gamma or PMA under the conditions tested, does not induce the ability of these cell lines to release leukotrienes.     

The effect of vasoactive intestinal peptide and calcitonin gene-related peptide on peptidoleukotriene release from platelet activating factor stimulated rat lungs and ionophore stimulated guinea pig lungs

The effect of four neuropeptides and acetylcholine on the release of leukotrienes LTC4, LTD4 and LTE4 from platelet activating factor-stimulated rat lung and ionophore A23187-stimulated guinea pig lung, as detected by the combined use of HPLC and radioimmunoassay, was studied. Both vasoactive intestinal peptide and calcitonin gene-related peptide were found to inhibit the release of leukotrienes in both preparations. This effect was most marked in platelet activating factor-stimulated rat lung, where inhibition of LTC4 release was more pronounced than either inhibition of LTD4 or LTE4 production. The effect of vasoactive intestinal peptide on LTC4 biosynthesis was dose-related in rat lung. Neither substance P nor beta-endorphin were found to inhibit leukotriene release in rat lung. Vasoactive intestinal peptide inhibition of leukotriene release is independent from its actions on the muscarinic receptor, since acetylcholine was found to have no effect in the same preparation.     

Effects of immune complexes, zymosan preparations and ionophore A 23187 on leukotriene formation in human leukocytes

Incubation of human leukocytes with opsonized zymosan or IgG immune complexes led to a time dependent release of leukotrienes (LT) B4 and C4. After 3-4 min, the levels of LTB4 were 93 and 35 pmol/3*10(7) cells, respectively [corrected]. These amounts were 2-4 times lower than those released by leukocytes stimulated with the calcium ionophore A 23187. The levels of LTC4 were 8 and 20 times lower than those of LTB4 after incubation with opsonized zymosan or immune complexes, respectively. Heat-inactivation of the serum prior to zymosan coating decreased the effect of opsonized zymosan. Uncoated zymosan was an even weaker stimulus of leukotriene formation. These results suggest that both complement factors and immunoglobulins play a pivotal role in activating leukotriene synthesis in a mixed suspension of human leukocytes.     

Effect of leukotrienes, bradykinin and calcium ionophore (A 23187) on bovine endothelial cells: release of prostacyclin

Incubation of the bovine endothelial cell line, CPAE, with the calcium ionophore (A23187), bradykinin (BK), leukotriene D4 (LTD4) or leukotriene C4 (LTC4) resulted in concentration dependent increases in prostacyclin release measured as 6-keto-prostaglandin F1 alpha. The kinetics of induction of prostacyclin synthesis differed among the agents studied. Statistically significant increases in prostacyclin were observed one minute after treatment, with A23187, at slightly longer times with bradykinin and after approximately three minutes with the leukotrienes. Two other leukotrienes were tested. Both leukotriene B4 and leukotriene E4 (LTE4) were inactive at concentrations up to 10 microM. The induction of prostacyclin synthesis by LTC4 and LTD4 was inhibited by cycloheximide and actinomycin-D. The effect of BK was inhibited by cycloheximide but not by actinomycin-D. Induction by A23187 was not inhibited by either actinomycin-D or cycloheximide. The results suggest that these agents induced the increases in prostacyclin synthesis by different mechanisms.     

Inhibition of leukotriene actions by the calcium channel blocker, nisoldipine

Nisoldipine, a calcium channel blocker having a highly potent effect on vascular smooth muscle relative to cardiac muscle, was tested to determine its anti-leukotriene properties. Nisoldipine, at concentrations from 1 to 300 ng/ml, significantly attenuated the vasoconstrictor effects of both LTC4 and LTD4 in isolated perfused cat coronary arteries and in isolated Langendorff perfused cat hearts. In isolated perfused coronary arteries, nisoldipine exerted a greater percentage inhibition of LTC4- and LTD4-induced constriction than of the constriction induced by the thromboxane analog, carbocyclic thromboxane A2 (CTA2). In isolated cat lung fragments, higher concentrations of nisoldipine were required to inhibit leukotriene formation (i.e., 10-200 microM). These concentrations of nisoldipine markedly inhibited the formation of the chemotactic leukotriene (LTB4) as well as the peptide leukotrienes (LTC4 and LTD4) stimulated by A-23187. Both types of leukotrienes were inhibited to a comparable degree. Thus, nisoldipine has significant anti-leukotriene actions. At normally employed concentrations, nisoldipine inhibits leukotriene actions on vascular smooth muscle, and at higher concentrations, it inhibits leukotriene formation.     

A simple and sensitive radioreceptor assay for leukotrienes

A simple and sensitive radioreceptor assay (RRA) for leukotrienes (LTs) was developed using a highly specific [3H]leukotriene D4 (LTD4) binding to guinea pig lung membrane homogenates. The assay can detect down to 0.15 pmol of LTD4. The values for fifty percent inhibition of bound [3H]LTD4 was 1.5 nM for LTD4, 45 nM for LTC4 and 24 nM for LTE4. LTB4 at 3.0 X 10(-5)M had no effect on [3H]LTD4 binding. The RRA for LTs in the absence of serine-borate complex was bi-specific for both LTC4 and LTD4. However, in the presence of 20 mM serine-borate this method was highly specific for LTD4. Recovery rate averaged 87.2% after ethanol extraction and evaporation of known amounts of LTD4. When the radioreceptor assay and radioimmunoassay data for leukotriene levels in the samples were compared to each other, an excellent correlation was observed with a correlation coefficient 'r' of 0.992. The assay was also validated by quantitation of Lts released from human granulocytes stimulated with calcium ionophore, A23187. The method is simpler, less expensive, and more specific for LTD4 than the other methods such as high pressure liquid chromatography and radioimmunoassay and is suitable for routine measurement of either LTD4 specifically or LTC4 plus LTD4 simultaneously in one cell system.     

Generation of leukotrienes by antigen and calcium ionophore A23187 stimuli from a mouse mastocytoma cell line, MMC-16

The generation of leukotrienes and histamine release by the mouse mastocytoma cell line MMC-16 was investigated. These cells produced leukotriene C4 (LTC4) and released histamine upon calcium ionophore A23187 and antigen stimulation. The ionophore also stimulated the biosynthesis of leukotriene B4 (LTB4) by MMC-16. Generation of LTC4 was confirmed by its characteristic UV absorption spectrum, fast atom bombardment-MS, equivalent HPLC retention time with an authentic standard and radioimmunoassay. Leukotriene B4 was characterized by its distinctive UV spectrum and HPLC retention time compared with synthetic material. IgE-mediated LTC4 generation was also observed in a dose dependent fashion with MMC-16 cells passively sensitized with monoclonal IgE specific for ovalbumin. LTC4 biosynthesis was effectively inhibited by the lipoxygenase inhibitor NDGA.     

Time-dependent alterations of leukotriene production and catabolism in rat peritoneal macrophages following intraperitoneal injection of thioglycollate broth.

Alterations of leukotriene (LT) productivity in peritoneal macrophages (PM) from untreated rats (control) as well as from rats treated i.p. with thioglycollate broth (TG) were investigated on days 3, 7 and 14 after TG administration. The resident PM from the untreated rats produced mainly LTB4 and 5-HETE with small amounts of 12-HETE and LTD4 with only a trace of LTC4 when stimulated with the calcium ionophore A23187. The PM elicited from rats on days 3 and 7 produced more LTC4 than did the resident PM but fewer other lipoxygenase metabolites. On day 14, however, the elicited PM resembled the resident PM in terms of lipoxygenase metabolite production. Similar results were achieved in the presence of arachidonic acid and A23187. A decrease in lipoxygenase metabolism in the elicited PM was also suggested by using opsonized zymosan. Catabolism studies indicated a reduction in r-glutamyl transpeptidase activity in the elicited PM and suggested a reduction in catabolism for LTB4 in the former cells. The authors conclude that the TG-elicited PM generate fewer lipoxygenase metabolites than the resident PM following stimulation, but show a preferential conversion of LTA4 to sulfidopeptide LTs rather than to LTB4. The elicited PM also show a reduced catabolism for LTC4 and LTB4.     

Characteristics of formation and further metabolism of leukotrienes in the chopped human lung

The bronchoconstrictive leukotrienes (LTs) LTC4, LTD4 and LTE4 (cysteinyl-LTs) and the chemoattractant LTB4 were formed in chopped human lung stimulated by the calcium ionophore A23187, or supplied with the precursor LTA4. In contrast, challenge with anti-IgE exclusively induced release of cysteinyl-LTs, indicating that LTB4 is not released as a primary consequence of IgE-mediated reactions in the human lung. Furthermore, several differences were observed with respect to formation and further conversion of LTB4 and LTC4 in the chopped lung preparation. Thus, exogenous [1-14C]arachidonic acid was dose-dependently converted to radioactive LTB4, whereas the cysteinyl-LTs released were not radiolabeled and the amounts of LTC4, D4 and E4 were not influenced by addition of increasing concentrations of arachidonic acid. LTC4 was rapidly and completely converted into LTD4 and LTE4, with no further catabolism of LTE4 within 90 min. The metabolism of LTB4 was much slower than that of LTC4. Thus, following a 60 min incubation approx. 25% of the material remained as LTB4, whereas 35% was omega-oxidized and 40% eluted on RP-HPLC as two unidentified peaks.     

Arachidonic acid metabolism among human mononuclear leukocytes. Lipoxygenase-related pathways

Human peripheral blood mononuclear cells were isolated and assessed for the presence of contaminating polymorphonuclear leukocytes and platelets. Incubations of these cell isolates were performed in the presence or absence of the calcium ionophore A23187 and/or 1-14C-labeled or unlabeled arachidonic acid. Using reverse phase high pressure liquid chromatography with simultaneous monitoring of ultraviolet light absorption at 229 and 280 nm and, where appropriate, of radioactivity, our studies reveal that human peripheral blood mononuclear cells generate leukotrienes C4 and B4 (LTC4 and LTB4) and 5-hydroxyeicosatetraenoic acid (5-HETE) following stimulation with A23187. The ratio of LTC4 to LTB4 was approximately 10-fold greater among the mononuclear cells than among similar incubations of polymorphonuclear leukocytes. Furthermore, the mononuclear cells failed to metabolize LTB4 into the omega-hydroxy or omega-carboxy derivatives that were always present in, and very characteristic of incubations of polymorphonuclear leukocytes. Depletion of monocytes from the mononuclear cells by double adherence resulted in virtual loss of the generation of 5-lipoxygenase-derived products by the remaining nonadherent cells, supporting the conclusion that the monocytes and not the lymphocytes were the source of LTC4, LTB4, and 5-HETE. The presence of both 12-HETE and the cyclooxygenase-derived 12-hydroxyheptadecatrienoic acid correlated with the degree of platelet contamination, suggesting that the platelets account for the presence of these compounds.     

Leukotriene C release by macrophages

Leukotriene C (LTC) and its metabolites leukotriene D and leukotriene E collectively make up the biological activity known as slow-reacting substance of anaphylaxis. Murine macrophages are potent sources of LTC (5(S)-hydroxy-6(R)-gamma-glutamylcysteinylglycyl-7,9-trans-11,14-cis-eicosatetr aenoic acid). Peritoneal and pulmonary tissue macrophages synthesize LTC and other arachidonic acid (20:4) metabolites in response to inflammatory stimuli such as unopsonized zymosan and IgG immune complexes. Peritoneal macrophages, in addition, release 20:4 when challenged with IgE immune complexes. These results suggest that macrophages may be a major source of leukotrienes in acute inflammation and also in immediate-type hypersensitivity reactions.     

Release of peptide leukotrienes from rat Kupffer cells

Kupffer cells isolated from the normal rat liver were incubated with calcium ionophore A23187, and the levels of peptide leukotrienes (LTC4, LTD4, and LTE4) contained in the culture supernatant were determined by the combined technique of reverse-phase high-performance liquid chromatography and radioimmunoassay. In response to A23187, Kupffer cells released LTC4, LTD4, and LTE4. After 10 min-preincubation of Kupffer cells with AA861, a 5-lipoxygenase inhibitor, the generation of LTC4, LTD4, and LTE4 from A23187-stimulated Kupffer cells was significantly suppressed. Platelet activating factor (PAF), a phospholipid mediator, significantly enhanced the release of LTC4, LTD4, and LTE4 from Kupffer cells stimulated with A23187. These results suggested that Kupffer cells may participate in inflammatory and immunologic events in the liver tissue by the release of peptide leukotrienes.     

Arachidonic acid metabolism by canine tracheal epithelial cells. Product formation and relationship to chloride secretion

Canine tracheal epithelial cells freshly isolated from mongrel dog trachea were used to study relationships between arachidonic acid metabolism and chloride ion movement. High performance liquid chromatography (HPLC) analysis of the cell incubation media after the addition of A23187 showed the presence of prostaglandin H synthase and lipoxygenase-derived metabolites. The major prostaglandin H synthase metabolite identified by HPLC, gas chromatography, and mass spectrometry was prostaglandin (PG) D2. The major lipoxygenase metabolites were leukotriene (LT) C4 and LTB4. LTB4 was identified by HPLC, UV spectroscopy, and gas chromatography. Straight phase HPLC of the methyl esters indicated only a minor formation of LTB4 isomers. LTC4 was identified by HPLC, UV spectroscopy, and conversion to LTD4 by gamma-glutamyl transpeptidase. Analysis by radioimmunoassays indicated approximately 1-2 ng of LTB4 and peptide LT formed by 10(6) cells after A23187 stimulation. The addition of ionophore A23187 caused a rapid release of arachidonic acid metabolites which was completed within 5 min of stimulation. Cl- secretion was measured in parallel studies of excised tracheas in Ussing chambers. Cl- secretion occurred at 2-3 min after the addition of ionophore, and the most rapid change occurred with the highest PGD2 concentrations. Indomethacin produced a concentration-dependent inhibition of PGD2 formation and Cl- movement. The addition of PGE2, PGD2, and PGH2 effectively stimulated Cl- secretion. LTC4 also stimulated Cl- secretion, but the stimulation was inhibited by indomethacin. These results indicate that canine tracheal epithelial cells metabolize arachidonic acid via both prostaglandin H synthase and lipoxygenase enzymes. It appears that endogenous PGD2 formation is the important variable controlling the Cl- ion movement in canine trachea.     

Biosynthesis and metabolism of leukotrienes

Leukotrienes are metabolites of arachidonic acid derived from the action of 5-LO (5-lipoxygenase). The immediate product of 5-LO is LTA4 (leukotriene A4), which is enzymatically converted into either LTB4 (leukotriene B4) by LTA4 hydrolase or LTC4 (leukotriene C4) by LTC4 synthase. The regulation of leukotriene production occurs at various levels, including expression of 5-LO, translocation of 5-LO to the perinuclear region and phosphorylation to either enhance or inhibit the activity of 5-LO. Several other proteins, including cPLA2a (cytosolic phospholipase A2a) and FLAP (5-LO-activating protein) also assemble at the perinuclear region before production of LTA4. LTC4 synthase is an integral membrane protein that is present at the nuclear envelope; however, LTA4 hydrolase remains cytosolic. Biologically active LTB4 is metabolized by w-oxidation carried out by specific cytochrome P450s (CYP4F) followed by b-oxidation from the w-carboxy position and after CoA ester formation. Other specific pathways of leukotriene metabolism include the 12-hydroxydehydrogenase/15-oxo-prostaglandin-13-reductase that forms a series of conjugated diene metabolites that have been observed to be excreted into human urine. Metabolism of LTC4 occurs by sequential peptide cleavage reactions involving a g-glutamyl transpeptidase that forms LTD4 (leukotriene D4) and a membrane-bound dipeptidase that converts LTD4 into LTE4 (leukotriene E4) before w-oxidation. These metabolic transformations of the primary leukotrienes are critical for termination of their biological activity, and defects in expression of participating enzymes may be involved in specific genetic disease.     

The mechanism of action of leukotrienes C4 and D4 in guinea-pig isolated perfused lung and parenchymal strips of guinea pig, rabbit and rat

The biological actions of pure slow-reacting substance of anaphylaxis (SRS-A) from guinea-pig lung, pure slow-reacting substance (SRS) from rat basophilic leukaemia cells (RBL-1) and synthetic leukotrienes C4 (LTC4) and D4 (LTD4) have been investigated on lung tissue from guinea pig, rabbit and rat. In the guinea pig, the leukotrienes released cyclo-oxygenase products from the perfused lung and contracted strips of parenchyma. The effects of SRS-A, SRS and LTD4 were indistinguishable. LTC4 and LTD4 had similar actions although LTD4 was more potent than LTC4. Indomethacin (1 microgram/ml) inhibited the release of cyclo-oxygenase products from perfused guinea-pig lung and caused a marked reduction in contractions of guinea-pig parenchymal strips (GPP) due to LTC4 and LTD4. The residual contraction of the GPP was abolished by FPL 55712 (0.5 - 1.0 microgram/ml). It appears, therefore, that a major part of the constrictor actions of LTC4 and LTD4 in guinea-pig lung are mediated by myotropic cyclo-oxygenase products, i.e. thromboxane A2 (TxA2) and prostaglandins (PGs). In rabbit and rat lung, however, SRS-A, SRS and the leukotrienes were much less potent in contracting parenchymal strips and there was little evidence of the release of cyclo-oxygenase products. FPL 55712 at a concentration of 1 microgram/ml failed to antagonise leukotriene-induced contractions.