Inhibition of leukotriene formation in human leukocytes by halothane

The effects of an inhalation anesthetic, halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) on the formation of 5-lipoxygenase metabolites such as leukotriene B4, 5(S)-hydroxyeicosatetraenoic acid (5-HETE), 6-trans-isomers of leukotriene B4 and leukotriene C4 were studied in human leukocytes stimulated with calcium ionophore A23187. Halothane inhibited the formation of all these metabolites dose dependently and the formation was restored by removal of the drug. The anesthetic also reversibly inhibited the release of [3H]arachidonic acid from neutrophils with a half-inhibition concentration of less than 0.19 mM. The formation of 5-lipoxygenase metabolites was not inhibited by the anesthetic when leukocytes were stimulated with the ionophore in the presence of exogenous arachidonic acid. These observations indicate that the inhibitory effect of halothane on the formation of 5-lipoxygenase metabolites in leukocytes is mainly due to the inhibition of arachidonic acid release.     

Guinea pig alveolar eosinophils and macrophages produce leukotriene B4 but no peptido-leukotriene

The metabolism of arachidonic acid (AA) was investigated in purified guinea pig alveolar eosinophils and macrophages. Alveolar eosinophils produced 12S-hydroxy-5,8,10-heptadecatraenoic acid (HHT) and small amounts only of 5-lipoxygenase products when stimulated by AA (10 microM) or ionophore A23187 (2 microM). However, when the cell suspensions were stimulated with both AA and A23187, the cells produced HHT, leukotriene (LT) B4, and 5S-hydroxy-6,8,11,14-eicosatetraenoic acid, whereas LTC4, D4, and E4 were undetectable. Similarly, alveolar macrophages stimulated with A23187 produced HHT, 5-hydroxy-6,8,11,14-eicosatetraenoic acid, and LTB4 but no peptido-leukotrienes. When LTA4 was added to suspensions of eosinophils and macrophages, only LTB4 was formed, whereas in parallel experiments, intact human platelets incubated with LTA4 produced LTC4. These data suggest that guinea pig alveolar eosinophils and macrophages contain both cyclooxygenase and 5-lipoxygenase, but do not produce peptido-leukotrienes, probably lacking LTA4 glutathione transferase activity. These studies demonstrate that guinea pig eosinophils differ from eosinophils of other animal species which have been shown to be major sources of leukotriene C4. The present data imply that eosinophils and macrophages are not the source of peptido-leukotrienes in anaphylactic guinea pig lungs.     

Activation of the Arachidonate 5-Lipoxygenase Pathway in the Canine Basilar Artery After Experimental Subarachnoidal Hemorrhage

Severe cerebral vasospasm as confirmed by angiography was induced in dogs by injection of autologous blood into the cisterna magna, and the resultant leukotriene formation in the isolated basilar artery was examined. When stimulated with calcium ionophore (A 23187), the arteries of the treated animals produced a significant amount of leukotrienes B4 (85 +/- 12 pmol/mg protein, n = 3) and C4 (72 +/- 14 pmol/mg), in addition to 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid. Structural elucidations of these metabolites were performed by radioimmunoassays or gas chromatography-mass spectrometry, following purification with HPLC. The artery of the untreated dog produced none of these compounds from either exogenous or endogenous arachidonic acid, under stimulation with the calcium ionophore. However, the homogenates from both animals converted exogenous leukotriene A4 to leukotrienes B4 and C4. These observations suggest that the normal basilar artery contains no detectable amount of 5-lipoxygenase, and that a prominent activation of this enzyme occurred (2.1 nmol 5-HETE/5 min/mg of protein) after subarachnoidal hemorrhage. The observation that fatty acid hydroperoxides stimulated the 5-lipoxygenase activity indicates a possible role of lipid peroxides in the development of vasospasm.     

Co-localization of leukotriene a4 hydrolase with 5-lipoxygenase in nuclei of alveolar macrophages and rat basophilic leukemia cells but not neutrophils.

The synthesis of leukotriene B(4) from arachidonic acid requires the sequential action of two enzymes: 5-lipoxygenase and leukotriene A(4) hydrolase. 5-Lipoxygenase is known to be present in the cytoplasm of some leukocytes and able to accumulate in the nucleoplasm of others. In this study, we asked if leukotriene A(4) hydrolase co-localizes with 5-lipoxygenase in different types of leukocytes. Examination of rat basophilic leukemia cells by both immunocytochemistry and immunofluorescence revealed that leukotriene A(4) hydrolase, like 5-lipoxygenase, was most abundant in the nucleus, with only minor occurrences in the cytoplasm. The finding of abundant leukotriene A(4) hydrolase in the soluble nuclear fraction was substantiated by two different cell fractionation techniques. Leukotriene A(4) hydrolase was also found to accumulate together with 5-lipoxygenase in the nucleus of alveolar macrophages. This result was obtained using both in situ and ex vivo techniques. In contrast to these results, peripheral blood neutrophils contained both leukotriene A(4) hydrolase and 5-lipoxygenase exclusively in the cytoplasm. After adherence of neutrophils, 5-lipoxygenase was rapidly imported into the nucleus, whereas leukotriene A(4) hydrolase remained cytosolic. Similarly, 5-lipoxygenase was localized in the nucleus of neutrophils recruited into inflamed appendix tissue, whereas leukotriene A(4) hydrolase remained cytosolic. These results demonstrate for the first time that leukotriene A(4) hydrolase can be accumulated in the nucleus, where it co-localizes with 5-lipoxygenase. As with 5-lipoxygenase, the subcellular distribution of leukotriene A(4) hydrolase is cell-specific and dynamic, but differences in the mechanisms regulating nuclear import must exist. The degree to which these two enzymes are co-localized may influence their metabolic coupling in the conversion of arachidonic acid to leukotriene B(4).     

Inhibition of leukotriene A4 hydrolase/aminopeptidase by captopril

Captopril ((2S)-1-(3-mercapto-2-methyl-propionyl)-L-proline) inhibited the bifunctional, Zn(2+)-containing enzyme leukotriene A4 hydrolase/aminopeptidase reversibly and competitively with Ki = 6.0 microM for leukotriene B4 formation and Ki = 60 nM for L-lysine-p-nitroanilide hydrolysis at pH 8. Inhibition was independent of pH between pH 7 and 8, the optimum range for each catalytic activity. Half-maximal inhibition of leukotriene B4 formation by intact erythrocytes and neutrophils required 50 and 88 microM captopril, respectively. In neutrophils and platelets neither 5(S)-hydroxyeicosatetraenoic acid, 12(S)-hydroxyeicosatetraenoic acid, nor leukotriene C4 formation were reduced, indicating selective inhibition of leukotriene A4 hydrolase/aminopeptidase, not 5-lipoxygenase, 12-lipoxygenase, or leukotriene C4 synthase. In whole blood, captopril inhibited leukotriene B4 formation with an accompanying redistribution of substrate toward formation of cysteinyl leukotrienes. The decrease in leukotriene B4 was more substantial than the corresponding increase in cysteinyl leukotrienes suggesting that nonenzymatic hydration predominates over transcellular metabolism of leukotriene A4 by platelets during selective inhibition of leukotriene A4 hydrolase. Enalapril dicarboxylic acid and Glu-Trp-Pro-Arg-ProGln-Ile-Pro-Pro which inhibit angiotensin-converting enzyme: angiotensin I, bradykinin, and N-[3-(2-furyl)acryloyl]Phe-Gly-Gly which are substrates; and chloride ions which activate angiotensin-converting enzyme did not modulate leukotriene A4 hydrolase/aminopeptidase activity. The results indicate that: (i) the sulfhydryl group of captopril is an important determinant for inhibition of leukotriene A4 hydrolase/aminopeptidase, probably by binding to an active site Zn2+; (ii) aminopeptidase and leukotriene A4 hydrolase display differential susceptibility to inhibition; (iii) there is minimal functional similarity between angiotensin-converting enzyme (peptidyl dipeptidase) and leukotriene A4 hydrolase/aminopeptidase; (iv) captopril may be a useful prototype to identify more potent and selective leukotriene A4 hydrolase inhibitors.     

Metabolism of arachidonic acid by guinea pig Clara cells.

A method for the isolation of non-ciliated bronchiolar epithelial (Clara) cells from the guinea pig is described. Following digestion of the lung tissue with Type XXIV protease, the isolated lung cells showed a viability greater than 90% and contained 3% of Clara cells. Several cell populations were then separated on the basis of size using 2 centrifugal elutriations. The macrophages and endothelial cells were removed from the Clara cells enriched fractions by differential adherence on Petri dishes. The Clara cell-rich suspension was then further purified by centrifugation on Percoll non-continuous density gradients consisting of 48-52-55% Percoll solution. The lower interface and the pellet of the non-continuous gradient consisted of approximately 80% Clara cells. Identification of isolated Clara cells was confirmed by light microscopic observations after nitroblue tetrazolium staining and by ultrastructural characteristic features as observed by electron microscopy. The metabolism of arachidonic acid into prostaglandins and TxB2 by purified Clara cells was examined by enzyme immunoassay (EIA) and leukotriene formation was investigated by reverse phase high performance liquid chromatography (RP-HPLC). Enriched guinea pig Clara cells incubated with arachidonic acid released TxB2, PGE2 and 6-keto PGF1 alpha, but did not produce leukotrienes. These cells could however transform exogenous leukotriene A4 into leukotriene B4. These results suggest that guinea pig Clara cells possess the enzymes of the cyclooxygenase pathway required for TxB2, PGE2 and 6-keto-PGF1 alpha synthesis. Clara cells do not possess the 5-lipoxygenase enzyme but show some leukotriene A4 hydrolase activity since they can produce leukotriene B4 upon incubation with leukotriene A4.     

On the mechanism of transcellular lipoxin formation in human platelets and granulocytes

Endogenous arachidonic acid was converted to lipoxins A4, B4 and (6S)-lipoxin A4, in ionophore-A23187-stimulated mixtures of human platelets and granulocytes, while no lipoxins were formed when these cells were incubated separately. However, pure platelet suspensions transformed exogenous leukotriene A4 to lipoxins, including lipoxin A4 and (6S)-lipoxin A4, but not lipoxin B4. This compound was produced exclusively in the presence of granulocytes. A common unstable tetraene intermediate in lipoxin formation, 15-hydroxy-leukotriene A4 [5(6)-epoxy-15-hydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid], was indicated by trapping experiments with methanol. Thus, identical profiles of less polar tetraene-containing derivatives were formed from leukotriene A4 in platelet suspensions, from exogenous 15-hydroxyeicosatetraenoic acid in granulocyte suspensions and from endogenous substrate in mixed platelet/granulocyte suspensions. Evidence for the involvement of 12-lipoxygenase in platelet-dependent lipoxin formation was obtained. Thus, lipoxin synthesis from leukotriene A4 and 12-hydroxyeicosatetraenoic acid production from arachidonic acid by human platelets was equally inhibited by 15-hydroxyeicosatetraenoic acid with 50% inhibition obtained at 7.0 microM and 8.2 microM, respectively. In experiments with subcellular preparations from platelets, lipoxin synthesis was observed in both the particulate and soluble fraction and was paralleled by the 12-lipoxygenase activity. Furthermore, lipoxin formation from leukotriene A4 in platelet sonicates was dose-dependently inhibited by exogenous arachidonic acid. Finally, 12-lipoxygenase-deficient platelets from a patient with chronic myelogenous leukemia were totally unable to produce lipoxins from exogenous or granulocyte-derived leukotriene A4. It is concluded that the transcellular lipoxin synthesis is dependent on the platelet 12-lipoxygenase and proceeds via the unstable intermediate, 15-hydroxy-leukotriene A4. This tetraene epoxide is transformed to lipoxin B4 by a granulocyte epoxide hydrolase activity or to lipoxin A4 and lipoxins A4/B4 isomers by enzymatic or nonenzymatic hydrolysis.     

Stereoselective hydrogen abstraction in leukotriene A4 synthesis by purified 5-lipoxygenase of porcine leukocytes

Arachidonate 5-lipoxygenase purified from porcine leukocytes transformed arachidonic acid to 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid. By the leukotriene A synthase activity of the same enzyme the product was further metabolized to leukotriene A4 (actually detected as 6-trans-leukotriene B4, 12-epi-6-trans-leukotriene B4, and 5,6-dihydroxy-7,9,11,14-eicosatetraenoic acids). The enzyme was incubated with [10-DR-3H]- or [10-LS-3H]-labeled arachidonic acid, and 6-trans-LTB4 and its 12-epimer were analyzed. More than 90% of 10-DR-hydrogen was lost while about 100% of 10-LS-hydrogen was retained, indicating a stereospecific hydrogen elimination from C-10 during the formation of leukotriene A4.     

Kinetic resolution of racemic leukotriene A4 by mammalian cytosolic epoxide hydrolases

Cytosols of rat and guinea pig liver and of human placenta were screened for their capacity to catalyze the conversion of racemic leukotriene A4 into 5S, 12R-dihydroxy-(Z,E,E,Z)-6,8,10,14-eicosatetraenoic acid (leukotriene B4). The epoxide hydrolase activities showed some specificity for the 5S,6S-oxido-(E,E,Z,Z)-7,9,11,14-eicosatetraenoic acid (LTA4) and produced mixtures of leukotriene B4 and its enantiomer containing up to 78-87% of leukotriene B4.     

Evidence for inhibition of leukotriene A4 synthesis by 5,8,11,14-eicosatetraynoic acid in guinea pig polymorphonuclear leukocytes

The sensitivity of the 5-lipoxygenase to inhibition by 5,8,11,14-eicosatetraynoic acid (ETYA) is species- and/or tissue-dependent. Guinea pig peritoneal polymorphonuclear leukocytes prelabeled with [3H]arachidonic acid and stimulated with ionophore A23187 formed 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), as well as several dihydroxy fatty acids, including 5(S),12(R)-dihydroxy-6,8,10-(cis/trans/trans)-14-(cis)-eicosatetraenoic acid. ETYA (40 microM) did not inhibit, but, rather, increased the incorporation of 3H label into 5-HETE. In contrast, ETYA markedly inhibited the formation of radiolabeled dihydroxy acid metabolites by the A23187-stimulated cells. Assay of products from polymorphonuclear leukocytes incubated with exogenous arachidonic acid plus A23187, by reverse phase high performance liquid chromatography combined with ultraviolet absorption, showed a concentration-dependent inhibition of the formation of dihydroxy acid metabolite by ETYA (1-50 microM) and an increase in 5-HETE levels (maximum of 2- to 3-fold). The latter finding was verified by stable isotope dilution assay with deuterated 5-HETE as the internal standard. Another lipoxygenase inhibitor, nordihydroguaiaretic acid, potently inhibited the formation of both 5-HETE and dihydroxy acids, with an IC50 of 2 microM. The data suggest that ETYA can inhibit the enzymatic step whereby 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid is converted to leukotriene A4 in guinea pig polymorphonuclear leukocytes.     

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.     

Effect of various lipoxygenase metabolites of arachidonic acid on degranulation of polymorphonuclear leukocytes

Lipoxygenase metabolites of guinea pig peritoneal polymorphonuclear leukocytes stimulated with 10 microM A23187 plus arachidonic acid were isolated and identified. These metabolites were compared with each other and to chemically synthesized arachidonate metabolites for their ability to stimulate leukocyte degranulation. 5(S),12(R)-Dihydroxy-6,8,10-(cis/trans/trans)14-cis-eicosatetraenoic acid (leukotriene B4) produced a significant release of lysozyme, but not beta-glucuronidase or beta-N-acetylglucosaminidase at low concentrations (EC50 = 6.5 x 10(-9) M), while the leukocyte nonenzymatically generated 5,12-or 5,6-dihydroxyeicosatetraenoic acids had no effect at these concentrations. Higher concentrations (1--10 microM) of all the dihydroxyeicosatetraenoic acids, 5-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) and its hydroperoxy precursor stimulated significant lysozyme release which was greater than that produced by 15-hydroxy-5,8,11-13-eicosatetraenoic acid, arachidonic acid, or its acetylene analogue, 5,8,11,14-eicosatetraynoic acid. Micromolar concentrations of leukotriene B4 and 5-HETE also stimulated significant release of beta-N-acetylglucosaminidase above controls, but not beta-glucuronidase. These results suggest that leukotriene B4 may play a role in regulating the release of certain granule-bound enzymes from polymorphonuclear leukocytes.     

Purification of natural killer-like Kurloff cells and arachidonic acid metabolism.

Pulmonary and splenic Kurloff cells have been purified from estrogen-treated guinea pig. Enzymatic digestion of lung tissue and mechanical dispersion of cells yielded about 650 x 10(6) viable cells. After centrifugal elutriation and centrifugation on continuous Percoll gradient, a population of high-density (1,100 g/ml) pulmonary Kurloff cells were obtained with high viability (approximately 99%) and purity (approximately 99%). Splenic Kurloff cells have been isolated by disruption of spleen tissue and centrifugation on continuous Percoll gradient. High-density splenic Kurloff cells (150 x 10(6) cells per spleen) were also obtained with high purity (approximately 99%) and viability (approximately 99%). Pulmonary and splenic Kurloff cells were incubated with various concentrations of arachidonic acid (10, 30 and 100 microM) in the absence or presence of 2 microM ionophore A23187. With 10 microM arachidonic acid the relative production of cyclooxygenase products was the following: TxB2 greater than PGE2 approximately PGI2. For an arachidonic acid concentration superior to 10 microM, the profile of release was PGE2 much greater than TxB2 greater than PGI2. Arachidonic acid metabolism through the 5-lipoxygenase pathway was also studied by incubating pulmonary or splenic Kurloff cells with 10 microM arachidonic acid in the absence or presence of 2 microM ionophore A23187, or in some experiments, with 2.5 microM leukotriene A4. Reverse phase HPLC profiles clearly indicated that high-density Kurloff cells did not express 5-lipoxygenase activity. However, these cells showed the ability to convert exogenous leukotriene A4 into leukotriene B4 suggesting the presence of LTA4 hydrolase activity. These data have been confirmed by a sensitive RIA method. This study constitutes the first report on the purification of pulmonary Kurloff cells and on arachidonic acid metabolism by these cells. The possible implications of Kurloff cells in various biological events are discussed.     

Identification of leukotriene D4 receptor binding sites in guinea pig lung homogenates using [3H]leukotriene D4

We have characterized [3H]leukotriene D4 binding to guinea pig lung homogenates. Both biphasic dissociation kinetics and curvilinear Scatchard plots indicated the presence of [3H]leukotriene high and low affinity states of the binding sites. The rank order of potency for the competition study was leukotriene C4 = leukotriene D4 greater than leukotriene E4 much greater than arachidonic acid, and for their contractile effect on lung strips was leukotriene C4 = leukotriene D4 = leukotriene E4 much greater than arachidonic acid. FPL-55712 was the only other agent tested that inhibited binding. These results suggest that binding of [3H]leukotriene D4 to the homogenate is consistent with its binding to specific leukotriene D4 receptor sites.     

Na+/H+ exchange modulates the production of leukotriene B4 by human neutrophils.

Human neutrophils produce various compounds of the 5-lipoxygenase pathway, including (5S)-hydroxyeicosatetraenoic acid, leukotriene B4, its 6-trans isomers and omega-oxidation metabolites of LTB4, when the cells are stimulated with the Ca2+ ionophore A23187. The elevation in the extracellular pH (pHo) facilitated the cytoplasmic alkalinization induced by the ionophore as determined fluorometrically using 2',7'-bis(carboxyethyl)carboxyfluorescein and enhanced the production of all the 5-lipoxygenase metabolites. The production decreased when the alkalinization was blocked by the decrease in the pHo, the removal of the extracellular Na+ or the addition of specific inhibitors of the Na+/H+ exchange, such as 5-(NN-hexamethylene)amiloride, 5-(N-methyl-N-isobutyl)amiloride and 5-(N-ethyl-N-isopropyl)amiloride. The alkalinization of the cytoplasm with methylamine completely restored the production suppressed by the removal of Na+ from the medium. These findings suggest that the change in the cytoplasmic pH (pHi) mediated by the Na+/H+ exchange regulates the production of the lipoxygenase metabolites. The site of the metabolism controlled by the pHi change seemed to be the 5-lipoxygenase, because the production of all the metabolites decreased in parallel and the release of [3H]arachidonic acid from the neutrophils in response to the ionophore was not affected by the pHi change. Furthermore, the production of the 5-lipoxygenase metabolites stimulated by A23187 with or without exogenous arachidonic acid showed a similar pHo-dependence and the production induced by N-formylmethionyl-leucylphenylalanine (chemotactic peptide) with exogenous arachidonic acid also decreased when the cytoplasmic alkalinization was inhibited.     

Biosynthesis of peptidyl leukotrienes and other lipoxygenase products by rat pancreatic islets. Comparison with macrophages and neutrophils

Biochemical evidence in support of a role for arachidonic acid 5-lipoxygenase activity in pancreatic islet insulin secretion has been obtained. Peptidyl leukotriene metabolism was studied in rat islets using a dual-labeling technique in extended culture, with analysis of arachidonic acid metabolites by reverse-phase high-performance liquid chromatography. The production of [3H]arachidonoyl/[35S]cysteinyl leukotrienes C4 and E4 by islets was compared with that by mouse resident peritoneal macrophages and with the lipoxygenase metabolism of rabbit polymorphonuclear leukocytes. The stimulus-specific nature of leukotriene biosynthesis was characterized by low basal biosynthesis in unstimulated islet cells with a calcium-mediated activation of 5-lipoxygenase product formation.     

Structural requirements in hydroxyeicosanoids for the activation of the 5-lipoxygenase in PT-18 mast/basophil cells

We have previously reported that 15-hydroxyeicosatetraenoic acid (15-HETE) stimulated the 5-lipoxygenase in the murine PT-18 mast/basophil cell line to produce leukotriene B4 and 5-HETE from exogenously added arachidonic acid. In order to determine the structural requirements in the HETE molecule that are necessary for the activation of this 5-lipoxygenase, various isomeric HETEs, derivatives and analogs were prepared, purified and tested. The order of stimulatory potencies was: 15-HETE acetate greater than 15-HETE = 15-hydroperoxyeicosatetraenoic acid (15-HPETE) greater than 5-HPETE = 12-HPETE greater than 5-HETE. 15-HETE methyl ester, 12-HETE and prostaglandin E2 were ineffective over the concentration range tested. Several diHETEs were also tested. 5S,15S-DiHETE was somewhat less potent than 15-HETE, whereas both 8S,15S-diHETE and leukotriene B4 were inactive. The calcium ionophore A23187 was much less effective than 15-HETE. These structure-activity studies indicate the importance of the nature, position and location of the various functional groups in the HETE molecule and suggest that a specific recognition site is involved in the activation of the 5-lipoxygenase in PT-18 cells.     

Phorbol myristate acetate and the calcium ionophore A23187 synergistically induce release of LTB4 by human neutrophils: involvement of protein kinase C activation in regulation of the 5-lipoxygenase pathway

Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, and the calcium ionophore A23187 synergistically induced the noncytotoxic release of leukotriene B4 (LTB4) and other 5-lipoxygenase products of arachidonic acid metabolism from human neutrophils. Whereas neutrophils incubated with either A23187 (0.4 microM) or PMA (1.6 microM) alone failed to release any 5-lipoxygenase arachidonate products, neutrophils incubated with both stimuli together for 5 min at 37 degrees C released LTB4 as well as 20-COOH-LTB4, 20-OH-LTB4, 5-(S),12-(R)-6-trans-LTB4, 5-(S),12-(S)-6-trans-LTB4, and 5-hydroxyeicosatetraenoic acid, as determined by high pressure liquid chromatography. This synergistic response exhibited concentration dependence on both PMA and A23187. PMA induced 5-lipoxygenase product release at a concentration causing a half-maximal effect of approximately 5 nM in the presence of A23187 (0.4 microM). Competition binding experiments showed that PMA inhibited the specific binding of [3H]phorbol dibutyrate ([3H]PDBu) to intact neutrophils with a 50% inhibitory concentration (IC50) of approximately 8 nM. 1-oleoyl-2-acetyl-glycerol (OAG) also acted synergistically with A23187 to induce the release of 5-lipoxygenase products. 4 alpha-phorbol didecanoate (PDD), an inactive phorbol ester, did not affect the amount of lipoxygenase products released in response to A23187 or compete for specific [3H]PDBu binding. PMA and A23187 acted synergistically to increase arachidonate release from neutrophils prelabeled with [3H]arachidonic acid but did not affect the release of the cyclooxygenase product prostaglandin E2. Both PMA and OAG, but not PDD, induced the redistribution of protein kinase C activity from the cytosol to the membrane fraction of neutrophils, a characteristic of protein kinase C activation. Thus, activation of protein kinase C may play a physiologic role in releasing free arachidonate substrate from membrane phospholipids and/or in modulating 5-lipoxygenase activity in stimulated human neutrophils.     

Lipoxygenase products of arachidonic acid modulate biosynthesis of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human neutrophils via phospholipase A2

When human neutrophils, previously labeled in their phospholipids with [14C]arachidonate, were stimulated with the Ca2+-ionophore, A23187, plus Ca2+ in the presence of [3H]acetate, these cells released [14C]arachidonate from membrane phospholipids, produced 5-hydroxy-6,8,11,14-[14C]eicosatetraenoic acid (5-HETE) and 14C-labeled 5S,12R-dihydroxy-6-cis,8,10-trans, 14-cis-eicosatetraenoic acid ([14C]leukotriene B4), and incorporated [3H]acetate into platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine). Ionophore A23187-induced formation of these radiolabeled products was greatly augmented by submicromolar concentrations of exogenous 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE), 5-HETE, and leukotriene B4. In the absence of ionophore A23187, these arachidonic acid metabolites were virtually ineffective. Nordihydroguaiaretic acid (NDGA) and several other lipoxygenase/cyclooxygenase inhibitors (butylated hydroxyanisole, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 1-phenyl-2-pyrazolidinone) caused parallel inhibition of [14C]arachidonate release and [3H]PAF formation in a dose-dependent manner. Specific cyclooxygenase inhibitors, such as indomethacin and naproxen, did not inhibit but rather slightly augmented the formation of these products. Furthermore, addition of 5-HPETE, 5-HETE, or leukotriene B4 (but not 8-HETE or 15-HETE) to neutrophils caused substantial relief of NDGA inhibition of [3H]PAF formation and [14C]arachidonate release. As opposed to [3H]acetate incorporation into PAF, [3H]lyso-PAF incorporation into PAF by activated neutrophils was little affected by NDGA. In addition, NDGA had no effect on lyso-PAF:acetyl-CoA acetyltransferase as measured in neutrophil homogenate preparations. It is concluded that in activated human neutrophils 5-lipoxygenase products can modulate PAF formation by enhancing the expression of phospholipase A2.     

Cloning and characterization of a bifunctional leukotriene A(4) hydrolase from Saccharomyces cerevisiae

In mammals, leukotriene A(4) hydrolase is a bifunctional zinc metalloenzyme that catalyzes hydrolysis of leukotriene A(4) into the proinflammatory leukotriene B(4) and also possesses an arginyl aminopeptidase activity. We have cloned, expressed, and characterized a protein from Saccharomyces cerevisiae that is 42% identical to human leukotriene A(4) hydrolase. The purified protein is an anion-activated leucyl aminopeptidase, as assessed by p-nitroanilide substrates, and does not hydrolyze leukotriene A(4) into detectable amounts of leukotriene B(4). However, the S. cerevisiae enzyme can utilize leukotriene A(4) as substrate to produce a compound identified as 5S,6S-dihydroxy-7,9-trans-11, 14-cis-eicosatetraenoic acid. Both catalytic activities are inhibited by 3-(4-benzyloxyphenyl)-2-(R)-amino-1-propanethiol (thioamine), a competitive inhibitor of human leukotriene A(4) hydrolase. Furthermore, the peptide cleaving activity of the S. cerevisiae enzyme was stimulated approximately 10-fold by leukotriene A(4) with kinetics indicating the presence of a lipid binding site. Nonenzymatic hydrolysis products of leukotriene A(4), leukotriene B(4), arachidonic acid, or phosphatidylcholine were without effect. Moreover, leukotriene A(4) could displace the inhibitor thioamine and restore maximal aminopeptidase activity, indicating that the leukotriene A(4) binding site is located at the active center of the enzyme. Hence, the S. cerevisiae leukotriene A(4) hydrolase is a bifunctional enzyme and appears to be an early ancestor to mammalian leukotriene A(4) hydrolases.