Inhibition of human neutrophil arachidonate 5-lipoxygenase by 6, 9-deepoxy-6, 9-(phenylimino)- Δ 6, 8-prostaglandin I1 (U-60257)

6, 9-Deepoxy-6, 9-(phenylimino)-Δ 6, 8-prostaglandin I₁ (U-60257) and its methyl ester (U-56467) are selective inhibitors of leukotriene C and D biosynthesis both $\text{in}$$\text{vitro}$ and $\text{in vivo}$. In this study, we demonstrated that the principal site of inhibition may be arachidonate 5-lipoxygenase, the initial enzyme of leukotriene biosynthesis. U-60257 and its methyl ester block LTB₄ synthesis in human peripheral neutrophils with an ID₅₀ of 1.8 and 0.42 μM respectively. This inhibitory action of U-60257 on neutrophil 5-lipoxygenase can be reduced or reversed by a high concentration of exogenous arachidonic acid. U-60257 at 100 μM has no apparent effect on the following enzymes. 1) cyclooxygenase of sheep vesicular gland or human platelets; 2) 12-lipoxygenase of human platelets and 3) soybean 15-lipoxygenase. Thus, we conclude that U-60257 and its methyl ester potent and selective inhibitors of arachidonate 5-lipoxygenase.     

Formation of leukotrienes and other hydroxy acids during platelet-neutrophil interactions in vitro

Interactions of human platelets with neutrophils were studied in suspensions of [³H]arachidonate-labeled platelets and unlabeled neutrophils stimulated with ionophore A23187. Several radioactive arachidonate metabolites, not produced by platelets alone, were detected, including [³H]-labeled leukotriene B₄ (LTB₄), dihydroxyeicosatetraenoic acid (DHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). When [³H]12-HETE, a platelet product, was added to stimulated neutrophils, DHETE was formed. Similarly, when [³H]5-HETE, a neutrophil product, was added to stimulated platelets, DHETE was the major product. These results suggest that upon stimulation: 1) platelet-derived arachidonate may serve as precursor for the neutrophil-derived eicosanoids LTB₄ and 5-HETE, and 2) that platelet-derived 12-HETE can be converted to DHETE by human neutrophils. The present investigation documents cell-cell interactions via the lipoxygenase pathway, which may be important in hemostasis, thrombosis and inflammation.     

Inhibitory action of soluble elastin on thromboxane B2 formation in blood platelets

Soluble elastin, prepared from insoluble elastin by treatment with oxalic acid or elastase, was found to inhibit the formation of thromboxane B₂ both from [1-¹⁴C]arachidonic acid added to washed platelets and from [1-¹⁴C]arachidonic acid in prelabeled platelets on stimulation with thrombin. In both systems, the formation of 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) was accelerated. Oxalic acid-treated soluble elastin st 1 and 10 mg/ml inhibited the formation of thromboxane B₂ from exogenously supplied arachidonic acid 21 and 59%, respectively, and the formation of thromboxane B₂ in prelabeled platelets stimulated by thrombin 44 and 94%, respectively. These concentrations of elastin increased the formation of 12-HETE from exogenously supplied arachidonic acid about 3.4- and 7.3-times, respectively. Almost all the added arachidonic acid was converted to metabolites. In prelabeled platelets, soluble elastin at 1 and 10 mg/ml increased the formation of 12-HETE stimulated by thrombin about 1.3- and 2.8-times, respectively, and inhibited the thrombin-induced total productions of thromboxane B₂ (12-hydroxy-5,8,10-heptadecatrienoic acid (12-HETE) and free arachidonic acid by 26 and 25%, respectively. Elastase-treated digested elastin also inhibited the formation of thromboxane B₂ and stimulated the formation of 12-HETE in prelabeled platelets stimulated by thrombin. This inhibitory action of elastin was not replaced by desmosine. The level of cAMP in platelets was not affected by soluble elastin. Soluble elastin was also found to inhibit platelet aggregation induced by thrombin. However, the inhibitory action of soluble elastin on platelet aggregation cannot be explained by inhibition of thromboxane B₂ formation by the elastin.     

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, abd 5,6-duhydroxy-7,9,11,14-eicosatetraenoic acids). The enzyme was incubated with [10-D_R-³H]- or [10-L_S-³H]- labeled arachidonic acid, and 6-trans-LTB₄ and its 12-epimer were analyzed. More than 90% of 10-D_R-hydrogen was lost while about 100% of 10-L_S-hydrogen was retained, indicating a stereospecific hydrogen elimination from C-10 during the formation of leukotriene A₄.     

Deactivation of the effects of F-Met-Leu-Phe and leukotriene B4 on calcium mobilization in rabbit neutrophils

Preincubation of rabbit neutrophils with the synthetic chemotactic factor f-Met-Leu-Phe has been found to diminish the ability of these cells to mobilize calcium upon subsequent stimulation by f-Met-Leu-Phe or by leukotriene B₄. The preexposure of the neutrophils to leukotriene B₄ on the other hand results in a diminished subsequent response to itself but an unaltered response to f-Met-Leu-Phe. These results demonstrate that deactivation can be observed at the level of calcium mobilization, strengthen the postulated second messenger role of calcium in neutrophils and imply that neutrophil activation by chemotactic factors can bypass the arachidonic acid metabolic pathway.     

Formation of leukotrienes and hydroxy acids by human neutrophils and platelets exposed to monosodium urate

Monosodium urate (MSU) crystals stimulate the production of arachidonic acid metabolites by human neutrophils and platelets. Neutrophils exposed to MSU generated leukotriene B₄(LTB₄). 6- -LTB₄, 12- -6- -LTB₄, and 5S, 12S DHETE from endogenous sources of arachidonate. In addition to these metabolites both monohyroxyeicosatetraenoic acids (i.e., 5-HETE) and w-oxidation products (i.e., 20-COOH LTB₄) were formed by neutrophils exposed to MSU. Addition of exogenous arachidonic acid led to increased formation of each of these metabolites. When neutrophils were treated with colchicine (10 uM), LTB₄ but 5-HETE formation was impaired. (1-¹⁴C) Arachidonate-labeled platelets exposed to MSU released (1-¹⁴C)-arachidonate. (¹⁴C)-12 HETE, (¹⁴C)-HHT and (¹⁴C)-thromboxane B₂. Results indicate that MSU stimulates arachidonic acid metabolism in both human neutrophils and platelets. Moreover, they suggest not only that metabolites of arachidonate may be considered as possible candidates for mediators of inflammation in crystal-associated diseases, but that colchicine blocks the formation of LTB₄.     

A rapid miniature thin layer chromatography system for analysis of prostaglandins an lipoxygenase products

We have developed a miniature thin layer chromatography system for rapidly identifying the major arachidonate metabolites in a radiolabeled form elaborated by cells or tissues. This system separates the total spectrum of cyclooxygenase products and several of the most commonly found lipoxygenase pathway metabolites, while retaining the fine resolution of larger-scale and more time-consuming procedures. It requires less than one hour for extraction of metabolites, chromatography, and counting.     

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.     

On the mechanism of biosynthesis of leukotrienes and related compounds

[10D-³H; 3-¹⁴C]- and [10L-³H; 3-¹⁴C]arachidonic acids were incubated with human polymorphonuclear leukocytes and with human platelets. Leukotriene B₄ and 5(S),12(S)-dihydroxy-6trans,8cis,10trans,14-cis-eicosatetraenoic acid (5,12-DHETE) were isolated and the ³H/¹⁴C ratios determined. It could be concluded that the 10D (pro-R)-hydrogen is eliminated in the conversion of 5(S)-hydroperoxy-6trans,8cis,11cis,14cis-eicosatetraenoic acid into leukotriene A₄ whereas in the conversion of arachidonic acid into 5,12-DHETE the 10L (pro-S)-hydrogen is lost. Incubation of the doubly labeled arachidonic acids with human platelets confirmed and extended previous data on the stereochemistry of the hydrogen removal from C-10 during the conversion into 12(S)-hydroperoxy-5cis,8cis,10trans,14cis-eicosatetraenoic acid, i.e., the 10L (pro-S)-hydrogen is eliminated and the 10D (pro-R)-hydrogen retained.     

Evidence for mediation of acetyl glyceryl ether phosphorylcholine stimulation of adenosine 3′,5′-(cyclic)monophosphate levels in human polymorphonuclear leukocytes by leukotriene B4

Acetyl glyceryl ether phosphorylcholine induces human neutrophil aggregation. Incubation of neutrophils with either prostaglandin I₂, or the cyclic AMP-dependent phosphodiesterase inhibitor, RO 20-1724 before the addition of PAF-acether attenuates subsequent aggregation. Paradoxically, a small elevation in cyclic AMP is observed coincident with the initiation of PAF-acether-stimulated aggregation. The elevation in cyclic AMP in response to PAF-acether is amplified by RO 20-1724, and the magnitude of the response is dependent upon the concentration of PAF-acether. The elevation in cyclic AMP is not due to prostaglandins, because indomethacin actually enhances the elevation in cyclic AMP induced by PAF-acether. The involvement of the neutrophil 5-lipoxygenase, and subsequent leukotriene B₄ synthesis, is suggested by the observation that 5-lipoxygenase inhibitors limit both the elevation in cyclic AMP induced by PAF-acether, and the indomethacin enhancement. This indirect evidence is supported by the fact that leukotriene B₄ itself elevates neutrophil cyclic AMP levels in intact cells, and stimulates the adenylate cyclase in broken cell preparations. Although the elevation in cyclic AMP induced by either PAF-acether or leukotriene B₄ is coincident with the onset of neutrophil aggregation, it is not obligatory for aggregation. The adenylate cyclase inhibitor 2′,5′-dideoxyadenosine blocks the PAF-acether-stimulated increase in cyclic AMP, and actually enhances aggregation. It is suggested that the increase in cyclic AMP observed after the addition of PAF-acether is due to concomitant leukotriene B₄ synthesis, and is not obligatory for neutrophil aggregation, but is actually part of a feed-back regulatory system through which PAF-acether and leukotriene B₄ can limit their own activity in neutrophils.     

Facile syntheses for (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid through a common synthetic route

The delta 5,9 fatty acids (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid were synthesized for the first time in four steps (9-12% overall yield) starting from commercially available 2-(2-bromoethyl)-1,3-dioxolane. The synthetic approach provided enough material to corroborate the structure and stereochemistry of (5Z,9Z)-5,9-nonadecadienoic acid which was recently identified in the flowers of Malvaviscus arboreus (Malvaceae). The novel phospholipids 1-hexadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn-glycer o-3-phosphocholine and 1-octadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn- glycero-3-phosphocholine were also synthesized from commercially available L-alpha-phosphatidylcholine (egg yolk) and characterized by positive ion electrospray mass spectrometry. These are the first examples of unsymmetrical phospholipids with saturated fatty acids at the sn-1 position and delta 5,9 fatty acids at the sn-2 position.     

Metabolism of leukotriene B4 by guinea pig eosinophils

The metabolism of leukotriene B₄ (5(S),12(R)-dihydroxy-6-cis-8,10-trans-14-cis-eicosatetraenoic acid) by isolated guinea pig eosinophils was investigated. Incubation of guinea pig eosinophils with [³H]-leukotriene B₄ resulted in the rapid conversion of leukotriene B₄ to several more polar metabolites. Two of these metabolites were identified by ultraviolet spectroscopy and gas chromatography-mass spectrometry as the omega oxidation products 5(S),12(R),20-trihydroxy-6,8,10,14-eicosatetraenoic acid (20-hydroxy-leukotriene B₄) and 5(S),12(R),19-trihydroxy-6,8,10,14-eicosatetraenoic acid (19-hydroxy-leukotriene B₄). Two novel metabolites, 5(S),12(R),18,19-tetrahydroxy-6,8,10,14 eicosatetraenoic acid (18,19-dihydroxy-leukotriene B₄) and 5(S),12(R)-dihydroxy-1,18-dicarboxylic-6,8,10,14,16-octadecapentaenoic acid (Δ16,17–18-carboxy-19,20-dinor-leukotriene B₄) were tentatively identified. The identification of these compounds indicates that guinea pig eosinophils are capable of metabolizing leukotriene B₄ by both omega and beta oxidation. This catabolic activity may play a role in modulating inflammatory reactions by removing the chemoattractant leukotriene B₄ from inflammatory sites.     

Comparative activity of natural mono-, di- and tri-hydroxy derivatives of arachidonic acid on guinea-pig lungs: Myotropic effect and stimulation of cyclooxygenase activity

The activity of natural 5,6-Dihydroxy-eicosatetraenoic acid (5,6-DiHETE; 2 isomers), 5S,15S-DiHETE, 8S,15S-DiHETE, 5S,12S-DiHETE, Δ⁶-trans-leukotriene B₄, 12-epi-Δ⁶-leukotriene B₄, ω-hydroxy-leukotriene B₄, ω-carboxy-leukotriene B₄, 15S-hydroxy-eicosatetraenoic acid (15S-HETE), 12S-HETE, 5S-HETE and 12S-hydroxy-heptadecatrienoic acid was compared to TLB₄ on the guinea-pig lung parenchymal strip and on the release of prostaglandins and thromboxanes by the perfused guinea-pig lungs. The ω-hydroxy-LTB₄ appeared more potent than LTB₄ both for inducing a contraction and for releasing prostanoids whereas the ω-carboxy-LTB₄ was much less active on the parenchyma and did not release prostanoids at the dose used. All other hydroxy acids tested were either very weakly active or inactive in the two systems used with the exception of the 5,6-DiHETEs which showed significant activity. These di-hydroxy acids induced contractions of the lung parenchymal strip which could be blocked by PFL-55712 but were inactive on the guinea-pig ileum. The 5S-HETE, 12S-HETE and 15S-HETE were also tested for possible myotropic activity on selected smooth muscle preparations. Our results provide further informations on the structural requirements for LTB₄ (and other hydroxy acids) actions on the guinea-pig lungs.     

Leukotriene A synthase activity of purified mouse skin arachidonate 8-lipoxygenase expressed in Escherichia coli

Mouse skin 8-lipoxygenase was expressed in COS-7 cells by transient transfection of its cDNA in pEF-BOS carrying an elongation factor-1α promoter. When crude extract of the transfected COS-7 cells was incubated with arachidonic acid, 8-hydroxy-5,9,11,14-eicosatetraenoic acid was produced as assessed by reverse- and straight-phase high performance liquid chromatographies. The recombinant enzyme also reacted on α-linolenic and docosahexaenoic acids at almost the same rate as that with arachidonic acid. Eicosapentaenoic and γ-linolenic acids were also oxygenated at 43% and 56% reaction rates of arachidonic acid, respectively. In contrast, linoleic acid was a poor substrate for this enzyme. The 8-lipoxygenase reaction with these fatty acids proceeded almost linearly for 40 min. The 8-lipoxygenase was also expressed in an Escherichia coli system using pQE-32 carrying six histidine residues at N-terminal of the enzyme. The expressed enzyme was purified over 380-fold giving a specific activity of approximately 0.2 μmol/45 min per mg protein by nickel–nitrilotriacetate affinity chromatography. The enzymatic properties of the purified 8-lipoxygenase were essentially the same as those of the enzyme expressed in COS-7 cells. When the purified 8-lipoxygenase was incubated with 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid, two epimers of 6-trans-leukotriene B₄, degradation products of unstable leukotriene A₄, were observed upon high performance liquid chromatography. Thus, the 8-lipoxygenase catalyzed synthesis of leukotriene A₄ from 5-hydroperoxy fatty acid. Reaction rate of the leukotriene A synthase was approximately 7% of arachidonate 8-lipoxygenation. In contrast to the linear time course of 8-lipoxygenase reaction with arachidonic acid, leukotriene A synthase activity leveled off within 10 min, indicating suicide inactivation.     

Identification of specific binding sites for leukotriene C4 in human fetal lung

Specific leukotriene C₄ (LTC₄) binding sites were identified in membrane preparations from human fetal lung. Specific binding of [³H]-LTC₄ represented 95 percent of total binding, reached steadystate within 10 minutes and was rapidly reversible upon addition of excess unlabeled LTC₄. Binding assays were performed at 4°C under conditions which prevented metabolism of [³H]-LTC₄ (80 mM serineborate, 10 mM cysteine, 10 mM glycine). Under these conditions, greater than 95 percent of the membrane bound radioactivity, as analyzed by high performance liquid chromatography, co-eluted with the LTC₄ standard. Computer-assisted analyses of saturation binding data showed a single class of binding sites with a dissociation constant (K_d) of 26 + 6 nM and a density (B_max) of 84 ± 18 pmol/mg protein. Pharmacological specificity was demonstrated by competition studies in which specific binding of [³H]-LTC₄ was displaced by LTC₄ and its structural analogs with inhibition constants (K_j) of 10 to 30 nM, whereas LTD₄, diastereoisomers of LTD₁, LTE₄ and the end organ antagonist FPL 55712 were 150 to 700 fold less potent competitors than LTC₄. These results provide evidence for specific, reversible, saturable, high affinity binding sites for [³H]-LTC₄ in human fetal lung membranes.     

Phosphorylation mimicking mutations of ALOX5 orthologs of different vertebrates do not alter reaction specificities of the enzymes

5-Lipoxygenase (ALOX5) plays a key role in the biosynthesis of pro-inflammatory leukotrienes whereas 15-lipoxygenases (ALOX15) have been implicated in the formation of pro-resolving eicosanoids (lipoxins, resolvins). Recently, it has been suggested that a phosphorylation mimicking mutant (Ser663Asp) of a stabilized variant of human ALOX5 exhibits dominant arachidonic acid 15-lipoxygenase activity (> 95%). To test whether similar alterations in the reaction specificity can also be observed for ALOX5 orthologs of other species we expressed wildtype and phosphorylation mimicking mutants (Ser271Asp, Ser523Asp, Ser663Asp, Ser663Glu) of human, mouse and zebrafish ALOX5 in pro- and eukaryotic overexpression systems and characterized their reaction specificities. We found that neither of the phosphorylation mimicking mutants produced significant amounts of 15-hydroperoxyeicosatetraenoic acid and the 5-lipoxygenation/15-lipoxygenation ratio for all wildtype and mutant enzyme species was lower than 100:2. Taken together, this data suggest that phosphorylation of native ALOX5 orthologs of different vertebrates may not induce major alterations in the reaction specificity and thus may not inverse their biological activity.     

Rearrangement of 5S, 12S-dihydroxy-6,8,10,14-(E,Z,E,Z)-eicosatetraenoic acid during gas chromatography: formation of a cyclohexadiene derivative

The 5S, 12S-dihydroxy-6,8,10,14-(E,Z,E,Z,)-eicosatetraenoic acid, a product of double dioxygenation of arachidonic acid by lipoxygenases, undergoes severe decomposition during gas chromatography-mass spectrometric (GC-MS) analysis of the trimethylsilyl ether methyl ester derivative. The decomposition product was studied by GC-MS and identified as a cyclohexadiene derivative of the parent compound formed by ring closure at C6 and C11. Under identical GC conditions, two stereoisomers, i.e. 5S,12R-dihydroxy-6,8,10,14-(Z,E,E,Z)-eicosatetraenoic acid (leukotriene B4), and 6-trans-leukotriene B4 showed excellent chromatographic properties. These data indicated that the 5,12-dihydroxy derivative of arachidonic acid carrying the trans-cis-trans triene unit selectively undergoes cyclization during GC. These studies also provided an explanation to the controversial GC-MS data reported for this lipoxygenase product.     

Selective feed-back inhibition of the 5-lipoxygenation of arachidonic acid in human T-lymphocytes

Purified human T-lymphocytes exhibit 5-lipoxygenase activity as demonstrated by the conversion of arachidonic acid to 5-hydroxy-eicosatetraenoic acid (5-HETE), 5(S),12(R)-di-hydroxy-eicosa-6,14 cis-8,10 trans-tetraenoic acid (leukotriene B₄), and 5,12-di-HETE isomers of leukotriene B₄ that lack a 6-cis double bond. The concentrations of leukotriene B₄, 5-HETE, 11-HETE and 15-HETE in suspensions of T-lymphocytes were increased significantly by concanavalin A and by the calcium ionophore A23187. Preincubation of T-lymphocytes with 15-HETE at μM concentrations, characteristic of suspensions of stimulated lymphocytes, inhibited selectively the increases in the levels of 5-HETE and leukotriene B₄, but not of 11-HETE and prostaglandin E₂.     

Synthesis of [17,18-3H]trans-13-azaprostanoic acid. A labeled probe for the PGH2/TXA2 receptor

Because of its highly unstable nature, TXA₂, produced by platelet metabolism of arachidonic acid, does not lend itself to use as a receptor probe for its own receptor. As such, the stable TXA₂/PGH₂ antagonist, trans-13-azaprostanoic acid (trans-13-APA, 12b), was prepared as the [17,18 ³H] derivative ([³H] trans-13-APA, 12c) to study this receptor and to better evaluate the mechanism of action of these azaprostanoids. Tritiated trans-13-APA, 12c, was prepared in nearly theoretical specific activity (57 Ci/mmole) from (17z)-trans-13-azaprost-17-enoic acid (11b) by catalytic tritiation. The unsaturated 11b was prepared by condensation of cis-7-amino-3-heptene (8) with 2-(6-carboxyhexyl) cyclopentanone (9), NaBH₄ reduction, chromatography, and hydrolysis of the trans isomer so isolated. The olefins 11a and b were also of biochemical interest because of the unsaturation in the lower side chain. The presence of similar unsaturation in PGH₃ (4) and TXA₃ (3) renders these prostaglandins inactive as proaggregatory agents. Evaluation of the antiaggregatory activity of 11a and b indicated it to be about the same potency in inhibiting human platelet aggregation as the parent cis and trans-13-APAs, suggesting that introduction of a double bond at the 17 position in platelet prostaglandin antagonists is unlikely to result in enhanced antiplatelet activity.     

A novel leukotriene formed by transpeptidation of leukotriene E

A new leukotriene 5(S)-hydroxy-6(R)-$\text{S}$-γ-glutamylcysteine-7,9-$\text{trans}$-11,14-$\text{cis}$-eicosatetraenoic acid (leukotriene F₄) was isolated after incubating leukotriene E₄ with γ-glutamyltranspeptidase and glutathione. Leukotriene F₄ induced contractions of the isolated quinea pig ileum and was less potent in this respect than leukotriene E₄.