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

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

Transformation of leukotriene A4 to lipoxins by rat kidney mesangial cell

Incubation of rat mesangial cells with leukotriene A4 in the presence of calcium ionophore A23187 led to a substrate dependent formation of lipoxin and its isomers. The major metabolite coeluted with authentic lipoxin A4 (LXA4) and lipoxin B4 (LXB4) in RP-HPLC system, and possessed a characteristic U.V. spectrum and C-value which were identical to authentic standards. GC/MS analysis on LXA4 further demonstrates that the mesangial cell derived LXA4 was identical to that reported by Serhan et al. (1) as LXA4 [5(S), 6,(R), 15(S)-trihydroxy7,9,13-trans-11-cis-eicosatetraenoic acid]. The formation of LXA4 was linear with substrate (LTA4) concentration. No similar products occurred in boiled controls. Incubation of mesangial cell with 15-HPETE failed to produce any lipoxin-like material. The absence of LX-like substance following incubation of 15-HPETE with mesangial cells suggested that 5-lipoxygenase activity is not expressed in mesangial cells under these conditions. The generation of LXA4 from LTA4 in mesangial cells suggested that there is an active 15- or 12- lipoxygenase activity in the kidney. The production of LX may play an important role in the regulation of renal function and the response to inflammatory stimuli.     

Novel pathway for the metabolism of 6-trans-leukotriene B4 by human polymorphonuclear leukocytes

Polymorphonuclear leukocytes convert arachidonic acid to leukotriene B4 as well as to two 6-trans isomers of this substance. Both leukotriene B4 and 6-trans-leukotriene B4 are metabolized by a hydroxylase in human polymorphonuclear leukocytes to 20-hydroxy metabolites. We have now found a second, previously unknown, metabolic pathway for 6-trans-leukotriene B4 involving reduction of either the 6- or the 10- double bond. One of the two major metabolites of 6-trans-leukotriene B4 in human polymorphonuclear leukocytes is formed by the action of this reductase, followed by hydroxylation by leukotriene B4 20-hydroxylase. On the basis of ultraviolet (maximum absorbance at 232 nm) and mass spectral evidence, this product is either 5,12,20-trihydroxy-6,8,14-eicosatrienoic acid or 5,12,20-trihydroxy-8,10,14-eicosatrienoic acid.     

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

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

Leukotriene A4: Enzymatic conversion to leukotriene C4

An unstable epoxide, leukotriene A₄ (5(S)-$\text{trans}$-5,6-oxido-7,9-$\text{trans}$-11,14-$\text{cis}$-eicosatetraenoic acid), was earlier proposed to be an intermediate in the conversion of arachidonic acid into the slow reacting substance (SRS), leukotriene C₄. In the present work synthetic leukotriene A₄ was incubated with human leukocytes or murine mastocytoma cells. A lipoxygenase inhibitor, BW755C, was added in order to prevent leukotriene formation from endogenous substrate. Leukotriene C₄ and 11-$\text{trans}$-leukotriene C₄ were the main products with SRS activity. It was not established whether the 11-$\text{trans}$-compound was formed by isomerization at the leukotriene A₄ or C₄ stage.     

Leukotriene A4, conversion to leukotriene B4 in human T-cell lines

Human T-cell lines (HSB, MOLT-4 and CCRF-CEM) produced leukotriene B4 when incubated with leukotriene A4. The product was characterized by chromatographic properties, UV-spectroscopy and gas chromatography mass spectrometry. About 10 pmol of leukotriene B4 was obtained per 10(6) cells. When incubated with arachidonic acid plus the calcium ionophore A23187 however, no leukotriene B4 was found, indicating that the T-cell lines lack 5-lipoxygenase yet contain LTA4 hydrolase.     

Carboxypeptidase A-catalyzed direct conversion of leukotriene C4 to leukotriene F4

Leukotrienes (LTs) are 5-lipoxygenase (5-LO)-derived arachidonic metabolites that constitute a potent set of lipid mediators produced by inflammatory cells. Leukotriene A(4), a labile allylic epoxide formed from arachidonic acid by dual 5-LO activity, is the precursor for LTB(4) and LTC(4) synthesis. LTC(4) is further transformed enzymatically by the sequential action of gamma-glutamyltranspeptidase and dipeptidase to LTD(4) and LTE(4), respectively. In this report, we present evidence that bovine pancreatic carboxypeptidase A (CPA), which shares significant sequence homology with CPA in mast cell granules, catalyzes the conversion of LTC(4) to LTF(4) via the hydrolysis of an amide bond. The identity of CPA-catalyzed LTC(4) hydrolysis product as LTF(4) was confirmed by several analytical criteria, including enzymatic conversion to conjugated tetraene by soybean LO, conversion to LTE(4) by gamma-glutamyltranspeptidase, cochromatography with the standard LTF(4) and positive-ion fast-atom bombardment mass spectral analysis. Thus, it appears that the physiological significance of this single-step transformation may point toward a major cellular homeostatic mechanism of metabolizing LTC(4), a potent bronco- and vasoconstrictor, to a less potent form of cysteinyl LTs.     

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.     

Enzymatic degradation of succinyl-coenzyme A by rat liver homogenates

When a dilute suspension of the mitochondrial fraction of rat liver homogenates was incubated with chemically synthesized succinyl-CoA, a product was rapidly formed which was retained at pH 3.9 on Dowex 50 (H⁺). Although its acid-base properties were indistinguishable from those of δ-aminolevulinic acid, the product did not form a pyrrole with acetylacetone, nor was its enzymatic formation dependent on added glycine. The enzyme which cleaved succinyl-CoA to the δ-aminolevulinic acid-like product was inhibited by phenylmethyl sulfonylfluoride. The first substance formed by the peptidase was the unstable thioester of succinic acid and cysteamine which underwent rearrangement to the more stable N-succinyl cysteamine above pH 4.0.It is apparent that the assay of δ-aminolevulinic acid synthetase (EC 2.3.1.37) by the ion-exchange method of Ebert et al. (Ebert, P.S., Tschudy, D.P., Choudhry, J.N. and Chirigos, M.A. (1970) Biochim. Biophys. Acta 208, 236–250) can yield erroneous results with succinyl-coenzyme A as substrate, especially when incubations are carried out for less than 25 min.     

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

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

Enzymatic degradation of succinyl-coenzyme A by rat liver homogenates.

When a dilute suspension of the mitochondrial fraction of rat liver homogenates was incubated with chemically synthesized succinyl-CoA, a product was rapidly formed which was retained at pH 3.9 on Dowex 50 (H+). Although its acid-base properties were indistinguishable from those of epsilon-aminolevulinic acid, the product did not form a pyrrole with acetylacetone, nor was its enzymatic formation dependent on added glycine. The enzyme which cleaved succinyl-CoA to the epsilon-aminolevulinic acid-like product was inhibited by phenylmethyl sulfonylfluoride. The first substance formed by the peptidase was the unstable thioester of succinic acid and cysteamine which underwent rearrangement to the more stable N-succinyl cysteamine above pH 4.0. It is apparent that the assay of epsilon-aminolevulinic acid synthetase (EC 2.3.1.37) by the ion-exchange method of Ebert et al. (Ebert, P.S., Tschudy, D.P., Choudhry, J.N. and Chirigos, M.A. (1970) Biochim. Biophys. Acta 208, 236--250) can yield erroneous results with succinyl-coenzyme A as substrate, especially when incubations are carried out for less than 25 min.     

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.     

Leukotriene A4: preparation and enzymatic conversion in a cell-free system to leukotriene B4

Treatment of leukotriene A4 (LTA4) methyl ester with sodium hydroxide in aqueous methanol at 4 degrees C afforded LTA4, the presence of which was inferred from the UV spectrum of the compound, its rate of reaction with water, and the identity of the hydration products obtained. The half-life of LTA4 in water (pH 7.4, room temperature) was increased from 14 to 500 s by 1 mg/ml of bovine serum albumin. This stabilized (chiral) LTA4 was converted to LTB4 by an epoxide hydrolase activity in the 100,000 x g supernatant fraction from sonified rat basophilic leukemia cells. Neither the ester of LTA4 nor the biologically incorrect enantiomer of LTA4 was metabolized to LTB4 under these conditions.     

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.     

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.     

Conversion of leukotriene B4 to dihydro and 19-hydroxy metabolites by rat polymorphonuclear leukocytes

Rat polymorphonuclear leukocytes metabolize leukotriene B4 (LTB4) by at least two major pathways. LTB4 is converted by a reductase in these cells to a dihydro metabolite in which one of the three conjugated double bonds has been reduced to give a conjugated diene with a UV absorption maximum at 230 nm. DihydroLTB4 appears to be a key intermediate in the metabolism of LTB4 by rat polymorphonuclear leukocytes, since a number of other metabolites, exhibiting UV absorbance at 235 nm, but not at 280 nm, have been detected by high pressure liquid chromatography. In addition, these cells contain a 19-hydroxylase, which converts LTB4 to 19-hydroxyLTB4, which has a typical leukotriene UV spectrum, exhibiting absorption maxima at 261, 270, and 282 nm.     

Specificity of the glutathione S-transferases in the conversion of leukotriene A4 to leukotriene C4

We have synthesized the 5,6-LTA4, 8,9-LTA4, and 14,15-LTA4 as methyl esters by an improved biomimetic method with yields as high as 70-80%. We have investigated the catalytic efficiency of the purified cytosolic glutathione S-transferase (GST) isozymes from rat liver in the conversion of these leukotriene epoxides to their corresponding LTC4 methyl esters. Among various rat liver GST isozymes, the anionic isozyme, a homodimer of Yb subunit, exhibited the highest specific activity. In general, the isozymes containing the Yb subunit showed better activity than the isozymes containing the Ya and/or Yc subunits. Interestingly, all three different LTA4 methyl esters gave comparable specific activities with a given GST isozyme indicating that regiospecificity of GSTs was not the factor in determining their ability to catalyze this reaction. Surprisingly, purified GSTs from sheep lung and seminal vesicles showed little activity toward these leukotriene epoxides, indicating a lack of the counterpart of rat liver anionic GST isozyme in these tissues.     

Metabolism of 6-trans isomers of leukotriene B4 to dihydro products by human polymorphonuclear leukocytes

The major dihydroxy metabolites of arachidonic acid formed by human polymorphonuclear leukocytes (PMNL) are leukotriene B4 (LTB4), 6-trans-LTB4, and 12-epi-6-trans-LTB4. LTB4, and to a lesser extent its 6-trans isomers, are metabolized to 20-hydroxy products by a hydroxylase in PMNL. We have recently reported the existence of a second pathway involving a reductase which, combined with the hydroxylase, results in the conversion of 6-trans-LTB4 to dihydro-6-trans-LTB4. We have now investigated some of the characteristics of this novel triene reductase pathway in human PMNL and have characterized some of the products and their mechanism of formation. At low substrate concentrations, the major pathway for the initial metabolism of both 6-trans-LTB4 and 12-epi-6-trans-LTB4 is reduction of the conjugated triene chromophore to give dihydro products with single absorption maxima at about 230 nm. Dihydro-6-trans-LTB4 is rapidly converted to its 20-hydroxy metabolite by LTB4 20-hydroxylase. However, 20-hydroxy-6-trans-LTB4 is not a substrate for the reductase. Neither 12-epi-6-trans-LTB4 nor its dihydro metabolite, 5,12-dihydroxy-7,9,14-eicosatrienoic acid, which was identified by gas chromatography-mass spectrometry, were very good substrates for the hydroxylase. The dihydro metabolites of 6-trans-LTB4 and 12-epi-6-trans-LTB4 were formed rapidly during the initial phase of the reaction, whereas the corresponding dihydro-20-hydroxy metabolites were formed only after a lag phase. Experiments utilizing deuterium-labeled 12-epi-6-trans-LTB4 indicated that a hydrogen atom is lost from the 5-position of the substrate, suggesting that the initial step in the formation of the dihydro products is the formation of a 5-oxo intermediate. LTB4 is metabolized very rapidly by LTB4 20-hydroxylase in PMNL, and we have not yet identified dihydro products derived from this substance. However, LTB4 strongly inhibits the conversion of 12-epi-6-trans-LTB4 to dihydro products, suggesting that it may also interact with the reductase.     

Leukotriene A: stereochemistry and enzymatic conversion to leukotriene B

Leukotriene A was assigned the structure 5(S)-$\text{trans}$-5,6-oxido-7,9-$\text{trans}\text{-11,14-}\text{cis}$-eicosatetraenoic acid by the enzymatic conversion of a synthetic product of known stereochemistry into the naturally occurring isomer of 5(S),12(R)-dihydroxy-6,8,10,14-eicosatetraenoic acid in human polymorphonuclear leukocytes.     

The antiinflammatory agent SC-41930 inhibits granulocyte infiltration of the rodent dermis induced by 6-trans-leukotriene B4.

Granulocyte diapedesis in response to the generation of defined chemotaxins such as leukotriene B4 (LTB4), 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE], C5a, platelet activating factor and others is a hallmark of the inflammatory process that is thought to contribute to the tissue pathology seen in a number of diseases. 6-trans-LTB4 arises through the myeloperoxidase (MPO)-dependent metabolism of sulfidopeptide leukotrienes and through the action of 5-lipoxygenase on 12(R)-HETE. The intradermal (i.d.) injection of 6-trans-LTB4 induces a dose and time dependent influx of granulocytes into the guinea-pig (Hartley) dermis. When various doses of the LTB4 receptor antagonist and antiinflammatory agent, SC-41930 (7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]-3,4-dihydro- 8-propyl-2H-1-benzopyran-2-carboxylic acid) given 30 min ahead of i.d. injection of 6-trans-LTB4 (10 micrograms/i.d. site), granulocyte infiltration, as assessed by dermal levels of the neutrophil marker enzyme MPO was inhibited with an ED50 value of 9.8 mg/kg in the guinea-pig. When various doses (10-25 micrograms) 6-trans-LTB4 were injected in the mouse (CD-1) dermis, there was a dose-related increase in granulocyte accumulation at 4 h. Furthermore when mice were pretreated (-30 min) with SC-41930 (1 mg/kg) orally, the trafficking of granulocytes was inhibited (p less than .01) as assessed by dermal MPO levels. SC-41930 orally inhibits 6-trans-LTB4-induced granulocyte accumulation in the guinea-pig more potently than against the response to 12(R)-HETE(ED50:13.4 mg/kg) but less potently than against LTB4 (ED50:0.6 mg/kg). These multiple activities may contribute to this compound's potential as an inflammatory agent.