Metabolism of arachidonic acid by peripheral and elicited rat polymorphonuclear leukocytes. Formation of 18- and 19-oxygenated dihydro metabolites of leukotriene B4

Previous studies have shown that leukotriene B4 is metabolized by polymorphonuclear leukocytes (PMNL) by a 20-hydroxylase, a 19-hydroxylase, and a reductase. We have now identified for the first time LTB4 metabolites formed by a combination of the reductase and omega-oxidation pathways. We have also discovered that rat PMNL metabolize LTB4 by a novel pathway to 18-hydroxy products. Dihydro metabolites of LTB4 have formerly been reported only after incubation of exogenous LTB4 with PMNL, but we have now shown that they are formed to the same extent from endogenous arachidonic acid after stimulation of PMNL with the ionophore, A23187. The following metabolites have been identified after incubation of either LTB4 or arachidonic acid with rat PMNL: 10,11-dihydro-LTB4, 10,11-dihydro-12-epi-LTB4, 10,11-dihydro-12-oxo-LTB4, 19-hydroxy-LTB4, 19-hydroxy-10,11-dihydro-LTB4, 19-oxo-10,11-dihydro-LTB4, 18-hydroxy-LTB4, 18-hydroxy-10,11-dihydro-LTB4, and 18-hydroxy-10,11-dihydro-12-oxo-LTB4. Negligible amounts of 20-hydroxylated products were formed. Incubation of PMNL with 10,11-dihydro-LTB4 resulted in the formation of all of the above dihydro metabolites. However, none of the omega-oxidized metabolites of LTB4 was further metabolized to a significant extent when incubated with PMNL, possibly at least partially because they were not substrates for a specific LTB4 uptake mechanism. We found that the biosynthesis and metabolism of LTB4 is considerably enhanced in PMNL from an inflammatory site (carrageenan-induced pleurisy) compared with peripheral PMNL. When arachidonic acid was the substrate, the greatest increase was observed for products formed by the reductase pathway, which were about eight times higher in pleural PMNL. The rates of formation of both LTA hydrolase and omega-hydroxylase products were about three times higher, whereas the total amounts of 5-lipoxygenase products were about twice as high in pleural PMNL. The amounts of products formed by the above enzymatic pathways reached maximal levels about 4-6 h after injection of carrageenan and then declined.     

Lipopolysaccharide modulates receptors for leukotriene B4, C5a, and formyl-methionyl-leucyl-phenylalanine on rabbit polymorphonuclear leukocytes

Peripheral blood polymorphonuclear leukocytes (PMNL) isolated from rabbits after an i.v. injection of endotoxin exhibited decreased chemotactic migration in response to leukotriene B4 (LTB4) and C5a, but not N-formyl-methionyl-leucyl-phenylalanine (fMLP), after endotoxin treatment. The binding of radiolabeled LTB4, fMLP, and C5a to isolated PMNL was assessed in order to determine whether altered receptor expression could account for the observed functional changes. Control PMNL expressed binding sites for fMLP, LTB4, and C5a similar to those previously characterized from human PMNL. Control PMNL expressed a single class of 14,600 +/- 2700 receptors for fMLP with a mean dissociation constant (Kd) of 2.0 +/- 0.6 nM at 0 degrees C, whereas two subclasses of binding sites were expressed for LTB4: 10,300 +/- 6800 high-affinity and 85,600 +/- 53,000 low-affinity binding sites per PMNL with mean Kd for LTB4 of 0.75 +/- 0.43 nM and 70 +/- 58 nM (mean +/- SD, n = 5), respectively. Control PMNL bound [125I]-C5a in a dose-dependent and saturable manner at 24 degrees C. At saturating concentrations of C5a, PMNL obtained from control rabbits bound 270,000 +/- 50,000 molecules of [125I]-C5a with half-maximal binding occurring at [125I]-C5a concentrations of 5.5 +/- 1.9 nM. The binding of LTB4 and C5a to PMNL obtained 24 hr after an i.v. injection of endotoxin was markedly decreased compared with control PMNL. PMNL from endotoxin-treated rabbits exhibited 68% fewer high-affinity binding sites per PMNL for LTB4 and a 51% decrease in the amount of [125I]-C5a bound at saturating concentrations compared with control PMNL. There was no significant change in the Kd of the high-affinity binding sites for LTB4, no change in the Kd and number of the low-affinity binding sites for LTB4, and a small decrease in the apparent Kd for C5a to 3.3 +/- 1.1 nM. Even though the pretreatment with i.v. endotoxin did not alter chemotactic or degranulation responses elicited by fMLP, the endotoxin pretreatment induced an eightfold increase in the receptor density without altering the Kd for fMLP. Decreased receptor expression could account in large part for the decreased chemotactic responsiveness towards C5a and LTB4 induced by LPS. The finding that a substantial increase in receptors for fMLP need not be accompanied by a comparable functional change suggests that decreased efficiency in receptor coupling to intracellular biochemical events may also result from i.v. endotoxin.     

Ligand-induced formation of the leukotriene B4 receptor-G protein complex of human polymorphonuclear leukocytes.

The components of the polymorphonuclear leukocyte (PMNL) receptor for leukotriene B4 (LTB4) were examined by Sephacryl S-300 exclusion chromatography of PMNL membrane proteins, which were solubilized before and after the binding of [3H] LTB4. When the PMNL membranes were solubilized in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and filtered on Sephacryl S-300 prior to addition of [3H] LTB4, the binding activity was associated with a 65 kD protein. In contrast, the radioactivity of [3H] LTB4 bound to PMNL membranes prior to solubilization was recovered predominantly with a 140 kD protein. When PMNL membranes had been pretreated with pertussis toxin, but not cholera toxin, before the addition of LTB4 and subsequent solubilization, radioactivity was recovered predominantly with the 65 kD protein. The addition of guanylylimidodiphosphate (GMP-PNP), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMNL membrane receptors bearing [3H] LTB4 either prior to or after CHAPS solubilization reduced the yield of the 140 kD presumed LTB4 receptor protein-G protein complex. That the maximum specific binding of [35S] guanosine-5'-0-3-thiotriphosphate (GTP-gammaS) to LTB4-binding proteins in the Sephacryl S-300 effluent corresponded to the 140 kD protein supported the presence of a G protein in the LTB4 receptor complex.     

Affinity labeling of the membrane protein-binding component of human polymorphonuclear leukocyte receptors for leukotriene B4.

A radiolabeled N-(3-aminopropyl)-leukotriene B4 amide ([3H]LTB4-APA) analog of the potent leukocyte chemotactic factor leukotriene B4 (LTB4) binds to receptors for LTB4 in plasma membrane-enriched preparations from human blood polymorphonuclear leukocytes (PMNL) and intact PMNL with respective mean dissociation constants of 2.3 nM and 69 nM at 4 degrees C. The [3H]LTB4-APA bound to plasma membrane-enriched preparations from PMNL was covalently cross-linked to membrane proteins with disuccinimidyl suberate. Solubilization and resolution by SDS-PAGE of proteins from [3H]LTB4-APA-labeled PMNL membranes revealed predominant labeling of a 60-kDa protein. Labeling of the PMNL membrane protein was inhibited by LTB4 and its analogs at concentrations similar to those inhibiting the binding of [3H]LTB4 to its receptor, with an identical rank order of potency of LTB4 greater than 20-hydroxy-LTB4 greater than LTB4-APA = 5(S),12(R)-dihydroxy-eicosa-14-cis-6,8,10-trans-tetraenoic acid much greater than LTD4 = LTC4. GTP suppressed the labeling of the 60-kDa PMNL membrane protein to an extent consistent with the decrease in receptor affinity for LTB4 induced by GTP. The stereospecificity of the affinity cross-linking reaction and the regulation by GTP support the identification of an approximately 60-kDa protein as the binding component of the PMNL receptor for LTB4.     

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.     

Metabolism of arachidonic acid in neutrophils from alloxan-diabetic rabbits

The production of 5-lipoxygenase products from arachidonic acid was investigated in polymorphonuclear leukocytes (PMNL) isolated from non-diabetic and alloxan-induced diabetic rabbits: (i) production of 5-hydroxyeicosatetraenoic acid, leukotriene B4, and the two 6-trans-leukotriene B4 isomers were significantly decreased in the PMNL of diabetic rabbits when compared to non-diabetic rabbits; (ii) production of LTB4 and 5-HETE from diabetic PMNL required the addition of Ca2+ and A23187 to a greater degree than control incubations; and (iii) the availability of substrate in the PMNL of diabetics was not a limiting factor for 5-lipoxygenase product formation. Alternative pathways of arachidonic acid metabolism were also evaluated: the recovery of exogenous leukotriene B4 and 5-hydroxyeicosatetraenoic acid were identical using PMNL from control and diabetic rabbits and peptido-leukotrienes were not detected by radioimmunoassay. The data suggest that the activity of 5-lipoxygenase and the production of 5-hydroperoxyeicosatetraenoic acid in the diabetic PMNL may be limiting factors since the formation of leukotriene B4, leukotriene B4 isomers, and 5-hydroxyeicosatetraenoic acid are depressed in PMNL of diabetic rabbits. Alternative pathways do not account for the conversion of arachidonic acid to other products nor are the elimination pathways for LTB4 and 5-HETE different. Decreased formation of 5-hydroxyeicosatetraenoic acid and leukotriene B4 could predispose diabetic subjects to infection due to a decrease in mediators leading to the local accumulation of PMNL in the inflammatory response.     

Effects of dietary vitamin E on the biosynthesis of 5-lipoxygenase products by rat polymorphonuclear leukocytes (PMNL)

Activation of polymorphonuclear neutrophils (PMNL) leads to the release of arachidonate from cellular phospholipids via a phospholipase A2, and conversion of products of the 5-lipoxygenase pathway. Evidence to date indicates the dietary vitamin E ((R,R,R)-alpha-tocopherol) can influence both cyclooxygenase and phospholipase A2 activities and that the effect of this vitamin is cell/tissue specific. The present study was undertaken in order to examine the effects of varying dietary tocopherol on PMNL tocopherol content and 5-lipoxygenase product profile using the ionophore A23187 as stimulant in the presence and absence of exogenous arachidonate. Feeding semi-purified diets containing 0, 30 or 3000 ppm of (R,R,R)-alpha-tocopherol acetate to weanling rats for 17 weeks resulted in a dose-related enrichment of PMNL tocopherol. Stimulation of PMNL elicited a significant and rapid loss of tocopherol. When PMNL were stimulated with A23187 alone, the synthesis of 5-HETE, LTB4 and 19-hydroxy-LTB4 was decreased in proportion to increasing dietary tocopherol concentrations. However, when exogenous arachidonate was provided with A23187, intermediate amounts of dietary tocopherol (30 ppm) still suppressed the formation of 5-lipoxygenase products, but high doses (3000 ppm) did not have any additional inhibitory effect. This differential response to high concentrations of vitamin E in the presence and absence of exogenous arachidonate highly suggest that at these concentrations, tocopherol may act principally at the level of substrate release whereas at lower concentrations, 5-lipoxygenase is inhibited. Data from this study demonstrated that attenuation of the formation of 5-lipoxygenase products in PMNL can be achieved by dietary vitamin E enrichment.     

Metabolism of 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid and other 5(S)-hydroxyeicosanoids by a specific dehydrogenase in human polymorphonuclear leukocytes.

Human polymorphonuclear leukocytes (PMNL) convert 6-trans isomers of leukotriene B4 (LTB4) to dihydro metabolites (Powell, W.S., and Gravelle, F. (1988) J. Biol. Chem. 263, 2170-2177). In the present study we investigated the mechanism for the initial step in the formation of these products. We found that the 1,500 x g supernatant fraction from human PMNL converts 12-epi-6-trans-LTB4 to its 5-oxo metabolite which was identified by mass spectrometry and UV spectrophotometry. The latter compound was subsequently converted to the corresponding dihydro-oxo product, which was further metabolized to 6,11-dihydro-12-epi-6-trans-LTB4, which was the major product after longer incubation times. The 5-hydroxyeicosanoid dehydrogenase activity is localized in the microsomal fraction and requires NADP+ as a cofactor. These experiments therefore suggest that the initial step in the formation of dihydro metabolites of 6-trans isomers of LTB4 is oxidation of the 5-hydroxyl group by a microsomal dehydrogenase. Studies with a variety of substrates revealed that the microsomal dehydrogenase in human PMNL oxidizes the hydroxyl groups of a number of other eicosanoids which contain a 5(S)-hydroxyl group followed by a 6-trans double bond. There is little or no oxidation of hydroxyl groups in the 8-, 9-, 11-, 12-, or 15-positions of eicosanoids, or of the 5-hydroxyl group of LTB4, which has a 6-cis rather than a 6-trans double bond. The preferred substrate for this enzyme is 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5(S)-HETE) (Km, 0.2 microM), which is converted to 5-oxo-6,8,11,14-eicosatetraenoic acid. Unlike 5(S)-HETE, 5(R)-HETE is a poor substrate for the 5(S)-hydroxyeicosanoid dehydrogenase, indicating that in addition to exhibiting a high degree of positional specificity, this enzyme is also highly stereospecific. In addition to 5(S)-HETE and 6-trans isomers of LTB4, 5,15-diHETE is also a good substrate for this enzyme, being converted to 5-oxo-15-hydroxy-6,8,11,13-eicosatetraenoic acid (5-oxo-15-hydroxy-ETE). The oxidation of 5(S)-HETE to 5-oxo-ETE is reversible since human PMNL microsomes stereospecifically reduce 5-oxo-ETE to the 5(S)-hydroxy compound in the presence of NADPH. 5-Oxo-ETE is formed rapidly from 5(S)-HETE by intact human PMNL, but because of the reversibility of the reaction, its concentration only reaches about 25% that of 5(S)-HETE.     

Properties of leukotriene B4 20-hydroxylase from polymorphonuclear leukocytes

Human polymorphonuclear leukocytes (PMNL) convert arachidonic acid (20:4) to a number of dihydroxy metabolites, including leukotriene B4 (LTB4) 5S,12R-dihydroxy-6,8,10,14-EEEZ-icosatetraenoic acid (isomer-1), 5S,12S-dihydroxy-6,8,10,14-EEEZ-icosatetraenoic acid, 5S,12S-dihydroxy-6,8,10,14-EZEZ-icosatetraenoic acid (5S,12S-dh-20:4), 5,6-dihydroxy-7,9,11,14-icosatetraenoic acid, and 5,15-dihydroxy-6,8,11,13-icosatetraenoic acid. LTB4 was synthesized rapidly after stimulation of PMNL with the divalent cation ionophore, A23187, but its concentration rapidly declined after about 4 min, in contrast to the other dihydroxy metabolites of 20:4 whose concentrations remained stable for at least 20 min. The amounts of polar metabolites (identified primarily as 20-hydroxy-LTB4) increased steadily with time up to 20 min. These results suggest that LTB4 may be specifically converted to its 20-hydroxy metabolite by PMNL. We prepared 3H- and 14C-labeled analogs of the dihydroxyicosatetraenoic acid metabolites described above by incubation of labeled 20:4 with PMNL. Although all of these substances were metabolized to some extent by human PMNL, LTB4 (apparent Km, 1.0 microM) was metabolized the most rapidly, followed by 5S,12S-dh-20:4 (apparent Km, 2.4 microM) and isomer-1 (apparent Km, 4.8 microM). All three substrates were shown by mass spectrometry to be converted to their 20-hydroxy metabolites. LTB4 was also metabolized to its omega-carboxy derivative. Human mononuclear leukocytes and rabbit PMNL metabolized LTB4 very slowly, whereas rat PMNL metabolized this substrate at about one-sixth the rate of human PMNL. These results demonstrate that human PMNL contain an omega-hydroxylase that specifically converts LTB4 to its 20-hydroxy metabolite. This enzyme may be important for the regulation of LTB4 levels in vivo.     

Development of receptors for leukotriene B4 on HL-60 cells induced to differentiate by 1 alpha,25-dihydroxyvitamin D3

The incubation of HL-60 human promyelocytic leukemia cells for 7 days with 100 nM 1 alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] induced differentiation into monocyte-like cells, as assessed by morphologic and biochemical characteristics. Stereospecific receptors for leukotriene B4 (LTB4) developed on the surface of the HL-60 cell-derived monocytes that had the capacity to transduce LTB4 stimulation of a transient increase in the cytosolic concentration of calcium ([Ca+2]in). HL-60 cell-derived monocytes, but not undifferentiated HL-60 cells, expressed a high affinity subset of 6400 +/- 3700 receptors per cell with a dissociation constant (Kd) of 2.3 +/- 1 nM (mean +/- SD, n = 3) and a low affinity subset of approximately 2.2 X 10(6) receptors per cell with an apparent Kd of 680 +/- 410 nM. Derivatives of LTB4 inhibited the binding of [3H]LTB4 to HL-60 cell-derived monocytes with a rank order of potency of LTB4 greater than 20-OH-LTB4 greater than 3-aminopropyl amide-LTB4, which is similar to the order for LTB4 receptors of human blood PMNL. In contrast, leukotrienes C4 and D4 and formyl-methionyl chemotactic peptides did not inhibit the binding of [3H] LTB4, which demonstrates the specificity of these receptors for isomers of 5,12-dihydroxy-eicosatetraenoic acid. LTB4 stimulated an increase in [Ca+2]in in HL-60 cell-derived monocytes which reached 50% of the maximal level at an LTB4 concentration of 0.5 nM (EC50). Preincubation of HL-60 cell-derived monocytes with 10 nM LTB4 resulted in a selective loss of high affinity receptors, as assessed by binding of [3H]LTB4, and a 200-fold increase in the EC50 for stimulation by LTB4 of increases in [Ca+2]in, without alterations in either the low affinity receptors for LTB4 or the responsiveness of [Ca+2]in to formyl-methionyl chemotactic peptides. HL-60 cells that are induced to differentiate into monocytes thus develop stereospecific receptors for LTB4 with binding and transductional characteristics similar to those of human blood PMNL.     

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.     

Enhanced levels of leukotriene B(4) in synovial fluid in Lyme disease

The purpose of this study was to evaluate the potential role of LTB(4) and cysteinyl leukotrienes in Lyme disease (LD). Therefore, a total number of 34 patients divided into four groups was studied. The patients were classified as having Lyme arthritis (n = 7) or Lyme meningitis (n = 10), and as control groups patients with a noninflammatory arthropathy (NIA) (n = 7) and healthy subjects (n = 10). LTB(4) as well as LTC(4) secretion from stimulated polymorphonuclear leukocytes (PMNL) from all groups of patients showed no statistical differences. LTB(4) levels in synovial fluid were significantly increased in patients with Lyme arthritis (median 142 ng/ml, range 88-296) when compared to the control subjects with NIA (median 46 ng/ml, range 28-72) (p < 0.05). No statistical difference of urinary LTE(4) levels between all the different groups of patients was observed. These results show that cysteinyl leukotrienes do not play an important role in the pathogenesis of LD. In contrast to previous findings in rheumatoid arthritis, LTB(4) production from stimulated PMNL was not found to be increased in LD. However, the significantly elevated levels of LTB(4) in synovial fluid of patients with Lyme arthritis underline the involvement of LTB(4) in the pathogenesis of this disease.     

Natural forms of vitamin E and 13'-carboxychromanol, a long-chain vitamin E metabolite, inhibit leukotriene generation from stimulated neutrophils by blocking calcium influx and suppressing 5-lipoxygenase activity, respectively

Leukotrienes generated by 5-lipoxygenase (5-LOX)-catalyzed reaction are key regulators of inflammation. In ionophore-stimulated (A23187; 1-2.5 μM) human blood neutrophils or differentiated HL-60 cells, vitamin E forms differentially inhibited leukotriene B(4) (LTB(4)) with an IC(50) of 5-20 μM for γ-tocopherol, δ-tocopherol (δT), and γ-tocotrienol, but a much higher IC(50) for α-tocopherol. 13'-Carboxychromanol, a long-chain metabolite of δT, suppressed neutrophil- and HL-60 cell-generated LTB(4) with an IC(50) of 4-7 μM and potently inhibited human recombinant 5-LOX activity with an IC(50) of 0.5-1 μM. In contrast, vitamin E forms had no effect on human 5-LOX activity but impaired ionophore-induced intracellular calcium increase and calcium influx as well as the subsequent signaling including ERK1/2 phosphorylation and 5-LOX translocation from cytosol to the nucleus, a key event for 5-LOX activation. Further investigation showed that δT suppressed cytosolic Ca(2+) increase and/or LTB(4) formation triggered by ionophores, sphingosine 1-phosphate, and lysophosphatidic acid but not by fMLP or thapsigargin, whereas 13'-carboxychromanol decreased cellular production of LTB(4) regardless of different stimuli, consistent with its strong inhibition of the 5-LOX activity. These observations suggest that δT does not likely affect fMLP receptor-mediated signaling or store depletion-induced calcium entry. Instead, we found that δT prevented ionophore-caused cytoplasmic membrane disruption, which may account for its blocking of calcium influx. These activities by vitamin E forms and long-chain carboxychromanol provide potential molecular bases for the differential anti-inflammatory effects of vitamin E forms in vivo.     

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.     

Stereochemistry of leukotriene B4 metabolites formed by the reductase pathway in porcine polymorphonuclear leukocytes: inversion of stereochemistry of the 12-hydroxyl group

Leukotriene B4 (LTB4), a potent proinflammatory agent, is a major metabolite of arachidonic acid in polymorphonuclear leukocytes (PMNL). When porcine PMNL were incubated with LTB4 and the products purified by reversed-phase high-pressure liquid chromatography (HPLC), we previously identified two metabolites: 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 [Powell, W. S., & Gravelle, F. (1989) J. Biol. Chem. 264, 5364-5369]. Further analysis of the reaction products by normal-phase HPLC has now revealed the presence of a third major metabolite of LTB4. This product is not formed in detectable amounts in the first 5 min of the reaction but accounts for about 20-30% of the reaction products after 60 min, when LTB4 has been completely metabolized. The mass spectrum and gas chromatographic properties of the new metabolite are identical with those of 10,11-dihydro-LTB4, suggesting that it is a stereoisomer of this compound. This product was identified as 10,11-dihydro-12-epi-LTB4 [i.e., 5(S),12(R)-dihydroxy-6,8,14-eicosatrienoic acid] by comparison of its chromatographic properties with those of the authentic chemically synthesized compound. Both 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 were enzymatically converted to 10,11-dihydro-12-epi-LTB4 by porcine PMNL, the former compound being the better substrate. The reaction was reversible, since both 10,11-dihydro-12-epi-LTB4 and 10,11-dihydro-12-oxo-LTB4 could be converted to 10,11-dihydro-LTB4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism for the formation of dihydro metabolites of 12-hydroxyeicosanoids. Conversion of leukotriene B4 and 12-hydroxy-5,8,10,14-eicosatetraenoic acid to 12-oxo intermediates.

Eicosanoids containing a 12-hydroxyl group preceded by at least two conjugated double bonds are metabolized to 10,11-dihydro and 10,11-dihydro-12-oxo products by porcine polymorphonuclear leukocytes (PMNL) (Wainwright, S. L., Falck, J. R., Yadagiri, P., and Powell, W. S. (1990) Biochemistry 29, 10126-10135). These 10,11-dihydro metabolites could either have been formed by the direct reduction of the 10,11-double bond of the substrate, as previous evidence suggested, or via an initially formed 12-oxo intermediate. To gain some insight into the mechanism for the formation of dihydro products by this pathway, we investigated the metabolism of leukotriene B4 (LTB4), 12(S)-hydroxy-5,8,10,14-eicosatetraenoicacid(12(S)-HETE), and 12(R)-HETE by subcellular fractions from porcine PMNL. In the presence of NAD+ and a microsomal fraction from PMNL, each of the above 12-hydroxyeicosanoids was converted to a single product with a lambda max approximately 40 nm higher than that of the substrate, indicating that the conjugated diene or triene chromophore had been extended by one double bond, presumably by oxidation of the 12-hydroxyl group to an oxo group. In the case of LTB4, this was confirmed by mass spectrometry, which indicated that the product was identical to 12-oxo-LTB4. LTB4 was not converted to any products by a cytosolic fraction from PMNL, but was converted to both 10,11-dihydro-LTB4 and 10,11-dihydro-12-oxo-LTB4 by the 1500 x g supernatant in the presence of NAD+. Negligible amounts of dihydro products were formed in the presence of NADH or NADPH, suggesting that initial oxidation of the 12-hydroxyl group is a requirement for reduction of the 10,11-double bond. Consistent with this hypothesis, 12-oxo-LTB4 was rapidly metabolized to 10,11-dihydro-12-oxo-LTB4 by the cytosolic fraction in the presence of NADH. Only small amounts of this product, along with some LTB4, were formed by the microsomal fraction. These results indicate that the initial step in the formation of 10,11-dihydro products from 12-hydroxyeicosanoids is oxidation of the 12-hydroxyl group by a microsomal 12-hydroxyeicosanoid dehydrogenase in the presence of NAD+, which is followed by reduction of the olefinic double bond by a cytosolic delta 10-reductase in the presence of NADH.     

In vitro study of the aromatic hydroxylation of 1-methyltetrahydro-beta-carboline (methtryptoline) in rat

The incubation of 1-methyltetrahydro-beta-carboline (1-MeTHBC) with hepatocytes isolated from 3-methylcholanthrene-treated rats led to formation of the 5-, 6- and 7-hydroxylated products. The hydroxylating activity was associated with the microsomal fraction as indicated by testing different subcellular fractions. The highest activity for hydroxylating 1-MeTHBC was found in liver which was about ten times as active as lung. Only a trace amount of hydroxylating activity was present in brain and kidney tissue. Analysis using chiral gas chromatography revealed an unequal abundance of enantiomers in all three products. The formation of the 5-, 6- and 7-hydroxylated products was confirmed in vivo by analysis of 24 h urine samples after intraperitoneal administration of 1-MeTHBC to 3-methylcholanthrene-treated rats.     

Release of adenosine from human neutrophils stimulated by platelet activating factor, leukotriene B(4) and opsonized zymosan

Isolated human polymorphonuclear leukocytes (PMNL) stimulated by platelet activating factor (PAF), leukotriene B(4) (LTB(4)) or opsonized zymosan (OZ) released adenosine measured by thermospray high performance liquid chromatography mass spectrometry in the cell-free supernatants. Stimulation by PAF or LTB(4) resulted in a bellshaped concentration-effect curve; 5 x 10(-7) M PAF, 10(-8) M LTB(4) and 500 mug ml(-1) OZ induced peak adenosine release, thus cytotoxic concentrations did not elevate adenosine level in the supernatants. Therefore adenosine release was characteristic of viable cells. As calculated from concentration-effect curves, the rank order of potency for adenosine release was PAF > LTB > OZ. These resuits suggest that adenosine, when bound specifically to membrane receptor sites, may initiate signal transduction, and, in co-operation with other inflammatory mediators, may modulate phagocyte function, e.g. production of chemoluminescence (CL).     

Differential effects of 20-trifluoromethyl leukotriene B4 on human neutrophil functions

A leukotriene B4 (LTB4) analog, 20-trifluoromethyl LTB4 (20CF3-LTB4), has been synthesized and evaluated with human neutrophils for effects on chemotaxis and degranulation. 20CF3-LTB4 was equipotent to LTB4 as a chemoattractant (EC50, 3 nM), produced 50% of maximal activity of LTB4, and competed with [H] LTB4 for binding to intact human neutrophil LTB4 receptors. In contrast to chemotactic activity, 20CF3-LTB4 in nanomolar concentrations exhibited antagonist activity without agonist activity up to 10 microM on LTB4-induced degranulation. The analog had no significant effect on degranulation induced by the chemoattractant peptide, N-formyl-methionyl-leucyl-phenylalanine (fMLP). Like LTB4, 20CF3-LTB4 induced neutrophil desensitization to degranulation by LTB4. The results indicate that hydrogen atoms at C-20 of LTB4 are critical for its intrinsic chemotactic and degranulation activities. The fact that 20CF3-LTB4 is a partial agonist for chemotaxis and an antagonist for degranulation suggests that different LTB4 receptor subtypes are coupled to these neutrophil functions. Desensitization of the neutrophil degranulation response to LTB4 can result from receptor occupancy by an antagonist, and therefore, the desensitization is not specific for an agonist.     

Inhibitory effect of LY 255283 on the synthesis of leukotriene B(4) and thromboxane A(2) in human peripheral blood polymorphonuclear leukocytes and monocytes

LY 255283 [(1-(5-ethyl-2-hydroxy-4-(6-methyl-6-)1H-tetrazol-5-yl)-heptyloxy) phenyl)ethanone], a specific leukotriene B(4) (LTB(4)) receptor antagonist, inhibited the production of LTB(4) in human peripheral blood polymorphonuclear leukocytes (PMNL) and in monocytes activated by calcium ionophore A23187. In human monocytes activated by ionophore it inhibited also the production of thromboxane B(2) (TXB(2)). The effect of LY 255283 on 5-lipoxygenase (5-LO) and LTA(4) hydrolase activities which catalyse the production of LTB(4) and LTA(4) has not been studied yet. It is thought that LY 255283 may inhibit the production of LTB(4) and TXA(2) by antagonising the effect of ionophore-induced LTB(4) on 5-lipoxygenase and cyclooxygenase in human peripheral blood PMNL and monocytes.