Biosynthesis of eicosanoids and transcellular metabolism of leukotrienes in murine bone marrow cells

Leukotriene B(4) (LTB(4)) biosynthesis by polymorphonuclear leukocytes (PMNs) is an important factor of inflammatory responses. PMNs also release LTA(4), an unstable intermediate that can be taken up by neighboring cells and metabolized into LTC(4). Most studies of LT synthesis have been carried out using human PMNs, but very little information is available about mouse PMNs. Mouse bone marrow PMNs were found to synthesize eicosanoids upon stimulation with A23187, fMLP, or zymosan. The major eicosanoids produced are LTB(4) and 5-hydroxyeicosatetraenoic acid, with some nonenzymatic products of LTA(4) hydrolysis. No cysteinyl leukotrienes were produced, in contrast to what was observed with human blood neutrophil preparations. Human megakaryoblast-like MEG-01 cells synthesized thromboxane B(2) and prostaglandin E(2) in response to A23187 but produced no 5-lipoxygenase (5-LO)-derived eicosanoids. When mouse bone marrow cells (mBMCs) and MEG-01 cells were stimulated during coincubation, LTC(4) and LTD(4) were produced. Mouse peritoneal macrophages from 5-LO-deficient mice were able to synthesize LTC(4) when incubated with mBMCs from wild-type mice, demonstrating transcellular exchange of LTA(4) from mBMCs into murine peritoneal macrophages. These data demonstrate that murine bone marrow PMNs are a valid model for the study of LT biosynthesis, which now offers the possibility to investigate specific biochemical pathways through the use of transgenic mice.     

Specificity of receptors for leukotrienes A4, B4, C4, D4, E4 and histamine on the guinea-pig lung parenchyma. Effect of FPL-55712 and desensitization of the myotropic activity

The novel metabolites of arachidonic acid, leukotriene (LT) A4, B4, C4, D4 and E4 have potent myotropic activity on guinea-pig lung parenchymal strip in vitro. The receptors responsible for their action were characterized using desensitization experiments and the selective SRS-A antagonist, FPL-55712. During the continuous infusion of LTB4, the tissues became desensitized to LTB4 but were still responsive to histamine, LTA4, LTC4, LTD4 and LTE4. When LTD4 was infused continuously, the lung strips contracted to LTB4 and histamine but were no longer responsive to LTA4, LTC4, LTD4 and LTE4. Furthermore, FPL-55712 (10 ng ml-1 - 10 ug ml-1) produced dose-dependent inhibitions of LTA4, LTC4, LTD4 and LTE4 without inhibiting the contraction to LTB4 and histamine. On the basis of these results, it appears that the guinea-pig lung parenchyma may have one type of receptor for LTB4 and another for LTD4; LTA4, LTC4 and LTE4 probably act on the LTD4 receptor.     

Attenuated zymosan-induced peritoneal vascular permeability and IgE-dependent passive cutaneous anaphylaxis in mice lacking leukotriene C4 synthase

Leukotriene C(4) synthase (LTC(4)S), the terminal 5-lipoxygenase pathway enzyme that is responsible for the biosynthesis of cysteinyl leukotrienes, has been deleted by targeted gene disruption to define its tissue distribution and integrated pathway function in vitro and in vivo. The LTC(4)S (-/-) mice developed normally and were fertile. LTC(4)S activity, assessed by conjugation of leukotriene (LT) A(4) methyl ester with glutathione, was absent from tongue, spleen, and brain and > or = 90% reduced in lung, stomach, and colon of the LTC(4)S (-/-) mice. Bone marrow-derived mast cells (BMMC) from the LTC(4)S (-/-) mice provided no LTC(4) in response to IgE-dependent activation. Exocytosis and the generation of prostaglandin D(2), LTB(4), and 5-hydroxyeicosatetraenoic acid by BMMC from LTC(4)S (-/-) mice and LTC(4)S (+/+) mice were similar, whereas the degraded product of LTA(4), 6-trans-LTB(4), was doubled in BMMC from LTC(4)S (-/-) mice because of lack of utilization. The zymosan-elicited intraperitoneal extravasation of plasma protein and the IgE-mediated passive cutaneous anaphylaxis in the ear were significantly diminished in the LTC(4)S (-/-) mice. These observations indicate that LTC(4)S, but not microsomal or cytosolic glutathione S-transferases, is the major LTC(4)-producing enzyme in tissues and that its integrated function includes mediation of increased vascular permeability in either innate or adaptive immune host inflammatory responses.     

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.     

Relationship between leukotriene A4 metabolism and biological activity

The data on the pharmacology of leukotrienes showed that LTA4, LTC4 and LTD4 were equipotent on the guinea-pig lung parenchyma whereas LTB4 was slightly less active. However, on the trachea, the myotropic activity of LTC4 and LTD4 was equivalent and higher than LTB4 and LTA4. The potency of these compounds was also different on the ileum where LTD4 was more active than LTC4; at the concentration used, LTA4 and LTB4 were inactive on this tissue. These results suggested that the transformation of leukotrienes by the smooth muscle preparations was a prerequisite for its biological activity. To verify this hypothesis, LTA4 (100 ng) was incubated for 10 min. with 20,000 g supernatants of homogenates of guinea-pig lung parenchyma, trachea and ileum; the metabolites were analysed by bioassay using strips of guinea-pig ileum and lung parenchyma in a cascade superfusion system and by RP-HPLC. Homogenates of lung parenchyma rapidly transformed LTA4 to LTB4, LTC4, LTD4 and LTE4, which is in agreement with the myotropic potency of these leukotrienes on the lung parenchymal strip. Conversely, incubation of LTA4 with homogenates of guinea-pig ileum showed the formation of LTB4 and its isomers which are inactive on this preparation. Similarly, incubation of homogenates of trachea with LTA4 led to the formation of LTB4; this finding is again in agreement with the potency of these two leukotrienes on the trachea. Our results suggest that the myotropic activity and potency of LTA4 is related to the tissue levels of enzymes which catalyse its transformation.     

Tetraene and pentaene leukotrienes: selective production from murine mastocytoma cells after dietary manipulation

A neoplastic mast cell tumor was grown in mice which had been raised since birth on a diet enriched with eicosapentaenoic acid. Intact harvested mastocytoma cells were stimulated with calcium ionophore A23187 to produce lipoxygenase products from the polyunsaturated fatty acids liberated from the cellular membranes. Leukotriene B4, B5, C4, and C5 were isolated and characterized by HPLC retention time, ultraviolet absorption spectrometry and mass spectrometry. The arachidonic acid content of the mast cell tumor lipids was altered from 9.2 to 3.9 mole % while eicosapentaenoic acid increased from 0.5 to 4.5 mole % in response to the fish oil-supplemented diet. The relative amounts of arachidonic and eicosapentaenoic acids (3.9 and 4.5 mole % respectively) were associated with similar amounts of LTB4 and LTB5 synthesized by the cells. These results suggest that the epoxide leukotriene (LIA) derivative can be made efficiently from either arachidonic or eicosapentaenoic acids when both are present in cellular lipids. In contrast, the ratio of LTC4 to LTC5 (10 to 1) indicates that the reaction of LTA with glutathione may be critically dependent upon the structure of the unsaturated fatty acid with the ratio of LTC4/LTB4 (2.0) more than 10 times greater than that (0.16) for LTC5/LTB5.     

Comparison of leukotriene A4 metabolism into leukotriene C4 by human platelets and endothelial cells.

Leukotriene (LT) A4 metabolism was studied in human platelets and endothelial cells, since both cells could be involved in transcellular formation of LTC4. Upon addition of exogenous LTA4, both cells produced LTC4 as a major metabolite at various incubation times, and no LTB4, LTD4, or LTE4 was detected. Kinetic studies revealed a higher apparent Km for LTA4 in endothelial cells as compared to platelets (5.8 microM for human umbilical vein endothelial cells (HUVEC) versus 1.3 microM for platelets); platelets were more efficient in this reaction with a higher Vmax (174 pmol/mg protein/min) versus 15 pmol/mg protein/min in HUVEC. The formation of LTC4 and corresponding kinetic parameters were not modified when platelets or endothelial cells were stimulated by thrombin prior to or simultaneously with the addition of LTA4. In both cells LTC4 synthase activity was not modified by repeated addition of LTA4 showing that it is not a suicide-inactivated enzyme. Furthermore, in platelets and endothelial cells, the enzyme activity was localized in the membrane fraction and was distinct from cytosolic glutathione-S-transferases. Platelet membrane fractions showed apparent Km values of 31 microM and 1.2 mM for LTA4 and GSH, respectively. Inhibition of LTC4 formation from platelets and endothelial cells preparations by S-substituted glutathione derivatives was correlated to the length of the S-alkyl chain. The same substances inhibited cytosolic glutathione-S-transferases with significantly lower IC50, confirming the distinct nature of the two enzymes. These results show that platelets and HUVEC possess similar enzymes for the production of LTC4 from LTA4; however, platelets seem to have a higher efficiency than HUVEC in performing this reaction.     

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.     

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.     

Determination of the contribution of cysteinyl leukotrienes and leukotriene B4 in acute inflammatory responses using 5-lipoxygenase- and leukotriene A4 hydrolase-deficient mice

Arachidonic acid metabolism by 5-lipoxygenase leads to production of the potent inflammatory mediators, leukotriene (LT) B4 and the cysteinyl LT. Relative synthesis of these subclasses of LT, each with different proinflammatory properties, depends on the expression and subsequent activity of LTA4 hydrolase and LTC4 synthase, respectively. LTA4 hydrolase differs from other proteins required for LT synthesis because it is expressed ubiquitously. Also, in vitro studies indicate that it possesses an aminopeptidase activity. Introduction of cysteinyl LT and LTB4 into animals has shown LTB4 is a potent chemoattractant, while the cysteinyl LT alter vascular permeability and smooth muscle tone. It has been impossible to determine the relative contributions of these two classes of LT to inflammatory responses in vivo or to define possible synergy resulting from the synthesis of both classes of mediators. To address this question, we have generated LTA4 hydrolase-deficient mice. These mice develop normally and are healthy. Using these animals, we show that LTA4 hydrolase is required for the production of LTB4 in an in vivo inflammatory response. We show that LTB4 is responsible for the characteristic influx of neutrophils accompanying topical arachidonic acid and that it contributes to the vascular changes seen in this model. In contrast, LTB4 influences only the cellular component of zymosan A-induced peritonitis. Furthermore, LTA4 hydrolase-deficient mice are resistant to platelet-activating factor, identifying LTB4 as one mediator of the physiological changes seen in systemic shock. We do not identify an in vivo role for the aminopeptidase activity of LTA4 hydrolase.     

Metabolism of arachidonic acid through the 5-lipoxygenase pathway in normal human peritoneal macrophages

Peritoneal macrophages (PM), obtained from 39 healthy women with normal laparoscopy findings, were stimulated with the ionophore A23187 or/and arachidonic acid (AA) both in adherence and in suspension. AA lipoxygenase metabolites were determined by reversed-phase HPLC. The major metabolites identified were 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene (LT)B4 and LTC4. The 20-hydroxy-LTB4, 20-carboxy-LTB4, and 15-HETE were not detected. Incubations of adherent PM with 2 microM A23187 induced the formation of LTB4, 110 +/- 19 pmol/10(6) cells, 5-HETE, 264 +/- 53 pmol/10(6) cells and LTC4, 192 +/- 37 pmol/10(6) cells. When incubated with 30 microM exogenous AA, adherent PM released similar amounts of 5-HETE (217 +/- 67 pmol/10(6) cells), but sevenfold less LTC4 (27 +/- 12 pmol/10(6) cells) (p less than 0.01). In these conditions LTB4 was not detectable. These results indicate that efficient LT synthesis in PM requires activation of the 5-lipoxygenase/LTA4 synthase, as demonstrated previously for blood phagocytes. When stimulated with ionophore, suspensions of Ficoll-Paque-purified PM produced the same lipoxygenase metabolites. The kinetics of accumulation of the 5-lipoxygenase/LTA4 synthase products in A23187-stimulated adherent cells varied for the various metabolites. LTB4 reached a plateau by 5 min, whereas LTC4 levels increased up to 60 min, the longest incubation time studied. Levels of 5-HETE were maximal at 5 min, and then slowly decreased with time. Thus, normal PM, in suspension or adherence, have the capacity to produce significant amounts of 5-HETE, LTB4, and LTC4. The profile of lipoxygenase products formed by the PM and the reactivity of this cell to AA and ionophore A23187 are similar to those of the human blood monocyte, but different from those of the human alveolar macrophage.     

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.     

TNF-alpha compensates for the impaired host defense of IL-1 type I receptor-deficient mice during pneumococcal pneumonia.

To determine the role of IL-1 in the host defense against pneumonia, IL-1R type I-deficient (IL-1R(-/-)) and wild-type (Wt) mice were intranasally inoculated with Streptococcus pneumoniae. Pneumonia resulted in elevated IL-1alpha and IL-1beta mRNA and protein levels in the lungs. Survival rates did not differ between IL-1R(-/-) and Wt mice after inoculation with 5 x 10(4) or 2 x 10(5) CFU. At early time points (24 and 48 h) IL-1R(-/-) mice had 2-log more S. pneumoniae CFU in lungs than Wt mice; at 72 h bacterial outgrowth in lungs was similar in both groups. Upon histopathologic examination IL-1R(-/-) mice displayed a reduced capacity to form inflammatory infiltrates at 24 h after the induction of pneumonia. IL-1R(-/-) mice also had significantly less granulocyte influx in bronchoalveolar lavage fluid at 24 h after inoculation. Since TNF is known to enhance host defense during pneumonia, we determined the role of endogenous TNF in the early impairment and subsequent recovery of defense mechanisms in IL-1R(-/-) mice. All IL-1R(-/-) mice treated with anti-TNF rapidly died (no survivors (of 14 mice) after 4 days), while 10-day survival in IL-1R(-/-) mice (control Ab), Wt mice (anti-TNF), and Wt mice (control Ab) was 7 of 13, 3 of 14, and 12 of 13, respectively. These data suggest that TNF is more important for host defense against pneumococcal pneumonia than IL-1, and that the impaired early host defense in IL-1R(-/-) mice is compensated for by TNF at a later phase.     

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.     

Peritoneal macrophages of guinea pig possibly lack LTC4 synthetase

Peritoneal cells and adherent cells of mice and rats synthesized LTC4 and LTB4 when stimulated with A23187 in vitro. On the other hand, neither peritoneal cells nor adherent cells of guinea pigs generated LTC4, D4, and E4, but did the lower amounts of LTB4. Only generation of LTB4 was potentiated by simultaneous addition of 10 microM A.A. in this species. Enzyme solutions which were extracted from peritoneal cells of these three species were capable of converting DNCB to a colored product in the presence of glutathione and then these potencies were in the following order; guinea pig greater than mouse greater than rat. On the other hand, the potencies of converting LTA4 to LTC4 in the presence of glutathione were in the following order; mouse greater than rat much greater than guinea pig approximately equal to 0. These results suggest that macrophages of guinea pigs lack "LTC4 synthetase" and also this enzyme is different from usual GSH S-transferases.     

Role of multidrug resistance-associated protein 1 in the pathogenesis of allergic airway inflammation

Multidrug resistance-associated protein 1 (MRP1) is a cysteinyl leukotriene (CysLT) export pump expressed on mast cells. CysLTs are crucial mediators in allergic airway disease. However, biological significance of MRP1 in allergic airway inflammation has not yet been elucidated. In this study, we sensitized wild-type control mice (mrp1(+/+)) and MRP1-deficient mice (mrp1(-/-)) to ovalbumin (OVA) and challenged them with OVA by aerosol. Airway inflammation and goblet cell hyperplasia after OVA exposure were reduced in mrp1(-/-) mice compared with mrp1(+/+) mice. Furthermore, CysLT levels in bronchoalveolar lavage fluid (BALF) from OVA-exposed mrp1(-/-) mice were significantly lower than those from OVA-exposed mrp1(+/+) mice. Levels of OVA-specific IgE, IL-4, and IL-13 in BALF were also decreased in OVA-exposed mrp1(-/-) mice. IgE-mediated release of CysLTs from murine bone marrow-derived mast cells was markedly impaired by MRP1 deficiency. Our results indicate that MRP1 plays an important role in the development of allergic airway inflammation through regulation of IgE-mediated CysLT export from mast cells.     

Single-step organic extraction of leukotrienes and related compounds and their simultaneous analysis by high-performance liquid chromatography

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.     

Identification of an amino acid residue in multidrug resistance protein 1 critical for conferring resistance to anthracyclines

Murine multidrug resistance protein 1 (mrp1), unlike human MRP1, does not confer resistance to anthracyclines. Previously, we have shown that a human/murine hybrid protein containing amino acids 959-1187 of MRP1 can confer resistance to these drugs. We have now examined the functional characteristics of mutant proteins in which we have converted individual amino acids in the comparable region of mrp1 to those present at the respective locations in MRP1. These mutations had no effect on the drug resistance profile conferred by mrp1 with the exception of converting glutamine 1086 to glutamate, as it is in the corresponding position (1089) in MRP1. This mutation created a protein that conferred resistance to doxorubicin without affecting vincristine resistance, or the ability of mrp1 to transport leukotriene C(4) (LTC(4)) and 17beta-estradiol 17-(beta-d-glucuronide) (E(2)17betaG). Furthermore, mutation Q1086D conferred the same phenotype as mutation Q1086E while the mutation Q1086N did not detectably alter the drug resistance profile of mrp1, suggesting that an anionic side chain was required for anthracycline resistance. To confirm the importance of MRP1 E1089 for conferring resistance to anthracyclines, we mutated this residue to Gln, Asp, Ala, Leu, and Lys in the human protein. The mutation E1089D showed the same phenotype as MRP1, while the E1089Q substitution markedly decreased resistance to anthracyclines without affecting LTC(4) and E(2)17betaG transport. Conversion of Glu-1089 to Asn, Ala, or Leu had a similar effect on resistance to anthracyclines, while conversion to a positive amino acid, Lys, completely eliminated resistance to anthracyclines and vincristine without affecting transport of LTC(4), E(2)17betaG, and the GSH-dependent substrate, estrone-3-sulfate. These results demonstrate that an acidic amino acid residue at position 1089 in predicted TM14 of MRP1 is critical for the ability of the protein to confer drug resistance particularly to the anthracyclines, but is not essential for its ability to transport conjugated organic anions such as LTC(4) and E(2)17betaG.     

Interferons inhibit LTC4 production in murine macrophages

Mouse resident peritoneal macrophages (M phi) produce the highly bioactive eicosanoid LTC4 when stimulated in vitro with zymosan or with the calcium ionophore A23187. This production was dramatically inhibited in M phi pre-exposed to IFN-alpha, IFN-beta, or IFN-gamma. Although all IFN were able to decrease the availability in M phi of the LTC4 precursor AA, this decrease was not the only cause of the IFN-induced inhibition of LTC4. In fact, further analysis of the different steps of the LTC4 biosynthetic pathway revealed that IFN-gamma could inhibit the formation of LTA4, thus of its derivatives LTC4 and LTB4, possibly acting at the level of the enzyme LTA4-synthetase. In contrast, IFN-alpha and IFN-beta only depressed the ability of M phi to metabolize AA into LTC4, leaving unaltered the synthesis of LTB4. However, IFN-alpha and IFN-beta did not influence directly the activity of any of the enzymes involved in LTC4 biosynthesis, indicating that they may act through some indirect, as yet unidentified regulatory mechanism. These data suggest that IFN-alpha and IFN-beta and, in different situations, IFN-gamma can be potentially useful in vivo in antagonizing localized anaphylactic or inflammatory reactions.