Transformation of leukotriene A4 methyl ester to leukotriene C4 monomethyl ester by cytosolic rat glutathione transferases

Six major basic cytosolic glutathione transferases from rat liver catalyzed the conversion of leukotriene A4 methyl ester to the corresponding leukotriene C4 monomethyl ester. Glutathione transferase 4-4, the most active among these enzymes, had a Vmax of 615 nmol X min-1 X mg protein-1 at 30 degrees C in the presence of 5 mM glutathione. It was followed in efficiency by transferase 3-4 which had a Vmax of 160 nmol X min-1 X mg-1 under the same conditions. Transferases 1-1, 1-2, 2-2 and 3-3 had at least 30 times lower Vmax values than transferase 4-4.     

Enzymatic conversion of leukotriene B4 to 6-trans-leukotriene B4 by rat kidney homogenates

A novel isomerase reaction leading to conversion of leukotriene B4 to its 6-trans isomer was detected in rat kidney homogenates. The structure of the metabolite was determined by high performance liquid chromatography, ultraviolet spectrometry and gas-liquid chromatography-mass spectrometry. A recent report has shown that 6-trans-leukotriene B4 is transformed to a dihydro metabolite (6,7- or 10,11-dihydro 6-trans-leukotriene B4) and further omega-hydroxylated [Powell, W.S. (1986) Biochem, Biophys. Res. Commun. 136, 707-712]. The leukotriene B4 6-isomerase reaction reported here may therefore provide the first step in a novel pathway of biological degradation of leukotriene B4.     

Transformation of leukotriene D4 catalyzed by lysosomal cathepsin H of rat liver

Among several intracellular protease tested, cathepsin H transformed leukotriene D4 to E4 with a release of glycine in a stoichiometric quantity. Under the optimal conditions the rate of leukotriene D4 transformation by cathepsin H was about 3% of the hydrolysis rate of alpha-N-benzoyl-DL-arginine-2-naphthylamide which is commonly utilized as a very efficient substrate to test the peptidase activity of the enzyme. Leukotriene C4 was not transformed to leukotriene D4 by cathepsin H. Neither cathepsin B nor C was active with leukotrienes C4 and D4.     

Conversion of leukotriene C4 to leukotriene D4 by a cell-surface enzyme of rat macrophages

Leukotriene (LT) C4-metabolizing enzyme was studied using rat leukocytes. Neutrophils and lymphocytes hardly metabolized LTC4, whereas macrophages rapidly converted LTC4 to LTD4. The LTC4-metabolizing enzyme of macrophages was present in the membrane fraction but not in the nuclear, granular and cytosol fractions. When macrophages were modified chemically with diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell-surface enzymes selectively, the LTC4-metabolizing activity of macrophages decreased significantly (greater than 90%). These findings suggest that rat macrophages possess the LTC4-metabolizing enzyme which converts LTC4 to LTD4, on the cell surface membrane.     

A factor from damaged rat kidney stimulates collagen biosynthesis by mesangial cells

Rats were administered CCl4, a well-defined nephrotoxin, for 20 weeks to produce glomerular sclerosis. Tubular degeneration and necrosis with interstitial fibrosis was clearly evident by histological examination. Kidneys were homogenized in phosphate-buffered saline and a collagen synthesis-stimulating factor was isolated by Sephadex G-50 gel filtration. The 5 kDa component stimulated both type I and type IV procollagen synthesis by mesangial cells and type I procollagen synthesis by rat skin fibroblasts. In each cell type, 2-6-fold increases in procollagen protein production or cell proliferation was noted. The steady-state levels of mRNA encoding for procollagen alpha 1(I) and procollagen alpha 1(IV) chains in mesangial cells were determined by by hybridization to their corresponding cDNA clones. The type I procollagen mRNA was elevated 1.4-fold compared to a 1.6-fold increase in mRNA encoding for type IV procollagen. The similar properties and chemical characteristics of this fibrogenic factor with a factor from fibrotic liver suggests they are the same and that a common endogenous collagen synthesis stimulator may be present in fibrosing organs, thus providing a driving force for collagen over-production.     

Stabilization of leukotriene A4 by epithelial fatty acid-binding protein in the rat basophilic leukemia cell

Leukotriene A(4) (LTA(4)) is a chemically unstable triene epoxide product of 5-lipoxygenase metabolism of arachidonic acid. Despite this chemical reactivity and its synthesis at the perinuclear membrane, LTA(4) is enzymatically converted into the cysteinyl leukotrienes and leukotriene B(4). Furthermore, LTA(4) participates in transcellular biosynthesis and is thus transferred between cells as an intact molecule. A cytosolic fatty acid-binding protein present in the rat basophilic leukemia cells was identified using mass spectrometry. This protein was determined to be the stabilizing factor present in the cell cytosol responsible for increasing the effective chemical half-life of LTA(4). Rat epithelial fatty acid-binding protein (E-FABP) was isolated using partial protein purification and immunoprecipitation. In-gel digestion with trypsin followed by peptide fingerprint analysis using matrix-assisted laser desorption ionization mass spectrometry and sequencing the major tryptic peptide obtained from liquid chromatography/mass spectrometry/mass spectrometry analysis identified E-FABP in the active fraction. Semi-quantitative Western blot analysis indicated that E-FABP in the cytosolic fraction of RBL-1 cells was present at approximately 1-3 pmol/10(6) cells. E-FABP (9 microm) was tested for its ability to stabilize LTA(4), and at 37 degrees C E-FABP was able to increase the half-life of LTA(4) from the previously reported half-life less than 3 s to a half-life of approximately 7 min. These results present a novel function for the well studied fatty acid-binding protein as a participant in leukotriene biosynthesis that permits LTA(4) to be available for further enzymatic processing in various cellular regions.     

Immortalization of rat kidney glomerular mesangial cell and its coculture with glomerular epithelial cell

Mesangial cell has several key roles in the control of glomerular function: it participates in the regulation of glomerular filtration rate, macromolecular clearance, and as both a source and target of numerous hormones and autocrines. Many of these insights into mesangial cell function have been obtained by studying mesangial cells in culture. However, no suitable cell lines have been established yet. We here reported the immortalization of rat kidney glomerular mesangial cell by transfection of E6 and E7 genes of human papillomavirus type 16 (HPV-16) via electroporation and lipofection. The results showed that only electroporation could transfect the genes to mesangial cells and the transfected cells maintained the viability for longer than 6 months. Fluorescence microscopic observation showed that cellular contractility and phagocytosis, which are the two main phenotypes of mesangial cells, are well maintained after transfection. The coculture of transfected mesangial cells with rat glomerular epithelial cells showed that the growth of mesangial cells was suppressed by epithelial cell, but the growth of epithelial cells was enhanced by mesangial cells. Moreover, an enhancing effect on the phagocytosis of mesangial cell was also observed in coculture. Such results may imply that the glomerular cell-cell interaction plays an important role in the regulation of cell proliferation and differentiation.     

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.     

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.     

Human glomerular mesangial cells inactivate leukotriene B4 by reduction into dihydro-leukotriene B4 metabolites

Due to its potent chemotactic properties leukotriene B4 is an important mediator of inflammatory reactions. Cultured human kidney mesangial cells converted exogenously added leukotriene B4 efficiently into three different more lipophilic metabolites, two of them probably representing dihydro-leukotriene B4 isomers. This represents an alternative metabolic pathway, in contrast to leukotriene B4 omega-oxidation found in human polymorphonuclear leukocytes. Both dihydro-leukotriene B4 isomers had nearly completely lost their ability to induce leukocyte chemotaxis as compared to leukotriene B4.     

Formation of leukotriene B4-coenzyme A ester by rat liver microsomes

When leukotriene B4 (LTB4) was incubated with rat liver microsomal fraction in the presence of coenzyme A (CoA) and ATP, a more polar product (compound I) was detected on reverse-phase high-performance liquid chromatography (RP-HPLC). The product was identified as LTB4-CoA ester on the basis of ultraviolet spectrometry, alkaline hydrolysis followed by RP-HPLC, and fast atom bombardment mass spectrometry (FAB-MS). The activity forming LTB4-CoA ester was localized in the microsomal fraction. The reaction was proportional to the concentration of the microsomal protein with an optimal pH of 7.5-8.0 and completely dependent on CoA and ATP. Palmitic acid and myristic acid significantly inhibited the formation.     

Kinetics of leukotriene A4 synthesis by 5-lipoxygenase from rat polymorphonuclear leukocytes

When arachidonic acid is added to lysates of rat polymorphonuclear leukocytes, it is oxidized to (5S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid (5-HPETE). The 5-HPETE then partitions between reduction to the 5-hydroxyeicosanoid and conversion to leukotriene A4 (LTA4). Both steps in the formation of LTA4 are catalyzed by the enzyme 5-lipoxygenase. When [3H]arachidonic acid and unlabeled 5-HPETE were incubated together with 5-lipoxygenase, approximately 20% of the arachidonic acid oxidized at low enzyme concentrations was converted to LTA4 without reduction of the specific radioactivity of the LTA4 by the unlabeled 5-HPETE. A significant fraction of the [3H]-5-HPETE intermediate that is formed from arachidonic acid must therefore be converted directly to LTA4 without dissociation of the intermediate from the enzyme. This result predicts that even in the presence of high levels of peroxidase activity, which will trap any free 5-HPETE by reduction, the minimum efficiency of conversion of 5-HPETE to LTA4 will be approximately 20%, and this prediction was confirmed. 5-HPETE was found to be a competitive substrate relative to arachidonic acid, so that it is likely that the two substrates share a common active site.     

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

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

Bioconversion of leukotriene C4 by rat glomeruli and papilla

Since leukotriene C4 (LTC4) may be locally synthesized by bone marrow-derived cells infiltrating the kidney in inflammatory renal diseases we examined the in vitro metabolism of exogenously added [3H] LTC4 by rat glomeruli and papilla using radiometric HPLC. Homogenized as well as intact glomeruli converted [3H] LTC4 mainly into [3H] LTE4 (83%) and, at a smaller extent, into [3H] LTD4 (4%). Intact [3H] LTC4 represented 13% of the sum of radioactive leukotrienes. Addition of L-cysteine resulted in accumulation of LTD4. In contrast, there was nearly no conversion of [3H] LTC4 (87% intact) in the presence of homogenized papilla. The metabolism of [3H] LTC4 by the glomeruli was time- and temperature-dependent. The 10,000 g supernatant and pellet of homogenized glomeruli both retained the ability to metabolize [3H] LTC4. The papillary 10,000 g supernatant was inactive, as found for the total homogenate, whereas the papillary 10,000 g pellet separated from its supernatant could transform [3H] LTC4 into its metabolites, LTD4 and LTE4. Addition of increasing amounts of papillary 10,000 g supernatant to homogenized glomeruli progressively protected [3H] LTC4 from its bioconversion. These results demonstrate that both glomeruli and papilla possess the gamma-glutamyl transpeptidase and dipeptidase necessary to process LTC4. However, the enzyme activity of the papilla is unmasked only when the inhibitor present in the 10,000 g supernatant is separated from the enzyme present in the pellet.     

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.     

Stimulation of rat mesangial cell proliferation by macrophage interleukin 1

Conditioned media from LPS-activated rat peritoneal macrophages enhanced the proliferation rates of cultured rat glomerular mesangial cells. This macrophage-derived activity extensively co-purified with interleukin 1 (IL 1) activity through sequential ammonium sulfate precipitation, S-200 gel chromatography, DEAE-cellulose anion exchange chromatography, and phenyl-Sepharose chromatography. In addition, the macrophage-derived factor was heat-labile (80 degrees C) and inactivated by phenylglyoxal, thus allowing tentative identification as IL 1. Macrophage supernatants and purified IL 1 enhanced the proliferative rates of mesangial cells only in the presence of serum; the use of platelet-poor plasma or serum depleted of platelet-derived growth factor was without effect. IL 1 acted to increase the percentage of cycling cells, without a change in the length of the individual cell cycle times. These findings provide a potential mechanism whereby activated macrophages, in combination with platelet factors, enhance mesangial cell proliferation. Such processes may contribute to the mesangial hypercellularity frequently found in immune-mediated glomerulonephritis.     

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

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

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

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