Suppression of 5-lipoxygenase activity by anionic cholesterol derivatives

5-Lipoxygenase (5-LO) is a key enzyme involved into biosynthesis of leukotrienes (LTs), mediating the host defense system, and acting simultaneously as inflammatory agents. In this work the effect of anionic cholesterol derivatives on 5-LO activity has been investigated. Cholesterol sulfate activates human polymorphonuclear leukocytes (PMNL) and stimulates their adhesion to endothelium and collagen. Cholesterol sulfate and cholesterol phosphate suppressed leukotriene production in PMNL and in rat basophil leukemia (RBL-1) cell line as well as in homogenates of these cells. Kinetic characteristics of the effect of anionic cholesterol derivatives on leukotriene synthesis have been obtained. In all experiments cholesterol phosphate (charge-2) was shown to be more potent inhibitor than cholesterol sulfate (charge-1). We believe that this fact highlights the importance of negatively charged ester groups for suppression of 5-LO activity.     

Induction of 5-lipoxygenase activation in polymorphonuclear leukocytes by 1-oleoyl-2-acetylglycerol

1,2-Diacylglycerols (DAGs) can prime polymorphonuclear leukocytes (PMNL) for enhanced release of arachidonic acid (AA) and generation of 5-lipoxygenase (5-LO) products upon subsequent agonist stimulation. Here, we demonstrate that in isolated human PMNL, 1-oleoyl-2-acetylglycerol (OAG) functions as a direct agonist stimulating 5-LO product formation (up to 42-fold). OAG caused no release of endogenous AA, but in the presence of exogenous AA, the magnitude of 5-LO product synthesis induced by OAG was comparable to that obtained with the Ca(2+)-ionophore A23187. Interestingly, OAG-induced 5-LO product synthesis was not connected with increased 5-LO nuclear membrane association. Examination of diverse glycerides revealed that the sn-2-acetyl-group is important, thus, also 1-O-hexadecyl-2-acetylglycerol (EAG) stimulated 5-LO product formation (up to 8-fold).Treatment of PMNL with OAG did not alter the mobilization of Ca(2+) but removal of intracellular Ca(2+) abolished the upregulatory OAG effects. Notably, the PKC activator phorbol-myristate-acetate hardly increased 5-LO product synthesis and PKC inhibitors failed to suppress the effects of OAG. Although OAG rapidly activated p38 MAPK and p42/44(MAPK), which can stimulate 5-LO for product synthesis, specific inhibitors of these kinases could not prevent 5-LO activation by OAG. Together, OAG acts as a direct agonist for 5-LO product synthesis in PMNL stimulating 5-LO by novel undefined mechanisms.     

Eugenol--the active principle from cloves inhibits 5-lipoxygenase activity and leukotriene-C4 in human PMNL cells

Polymorphonuclear leukocytes (PMNL) play an important role in the modulation of inflammatory conditions in humans. PMNL cells recruited at the site of inflammation, release inflammatory mediators such as leukotrienes, proteolytic enzymes and reactive oxygen species. Among these, leukotrienes are implicated in pathophysiology of allergic and inflammatory disorders like asthma, allergic rhinitis, arthritis, inflammatory bowel disease and psoriasis. 5-lipoxygenase (5-LO) is the key enzyme in biosynthetic pathway of leukotrienes. Our earlier studies showed that spice phenolic active principles significantly inhibit 5-LO enzyme in human PMNLs. In this study we have further characterized the inhibitory mechanism of eugenol, the active principle of spice-clove on 5-LO enzyme and also its effect on leukotriene C((4)) (LTC(4)). Substrate dependent enzyme kinetics showed that the inhibitory effect of eugenol on 5-LO was of a non-competitive nature. Further, eugenol was found to significantly inhibit the formation of LTC(4) in calcium ionophore A23187 and arachidonic acid (AA) stimulated PMNL cells. These data clearly suggest that eugenol inhibits 5-LO by non-competitive mechanism and also inhibits formation of LTC(4) in human PMNL cells and thus may have beneficial role in modulating 5-LO pathway in human PMNL cells.     

Inhibitors of actin polymerisation stimulate arachidonic acid release and 5-lipoxygenase activation by upregulation of Ca2+ mobilisation in polymorphonuclear leukocytes involving Src family kinases

Here, we show that actin polymerisation inhibitors such as latrunculin B (LB), and to a minor extent also cytochalasin D (Cyt D), enhance the release of arachidonic acid (AA) as well as nuclear translocation of 5-lipoxygenase (5-LO) and 5-LO product synthesis in human polymorphonuclear leukocytes (PMNL), challenged with thapsigargin (TG) or N-formyl-methionyl-leucyl-phenylalanine. The concentration-dependent effects of LB (EC50 approximately 200 nM) declined with prolonged preincubation (>3 min) prior TG and were barely detectable when PMNL were stimulated with Ca2+-ionophores. Investigation of the stimulatory mechanisms revealed that LB (or Cyt D) elicits Ca2+ mobilisation and potentiates stimulus-induced elevation of intracellular Ca2+, regardless of the nature of the stimulus. LB caused rapid but only moderate activation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK)2. The selective Src family kinase inhibitors PP2 and SU6656 blocked LB- or Cyt D-mediated Ca2+ mobilisation and suppressed the upregulatory effects on AA release and 5-LO product synthesis, without affecting AA metabolism evoked by ionophore alone. We conclude that in PMNL, inhibitors of actin polymerisation cause enhancement of intracellular Ca2+ levels through Src family kinase signaling, thereby facilitating stimulus-induced release of AA and 5-LO product formation.     

An experimental cell-based model for studying the cell biology and molecular pharmacology of 5-lipoxygenase-activating protein in leukotriene biosynthesis

BackgroundSubcellular distribution of 5-lipoxygenase (5-LO) to the perinuclear region and interaction with the 5-LO-activating protein (FLAP) are assumed as key steps in leukotriene biosynthesis and are prone to FLAP antagonists.MethodsFLAP and/or 5-LO were stably expressed in HEK293 cells, 5-LO products were analyzed by HPLC, and 5-LO and FLAP subcellular localization was visualized by immunofluorescence microscopy.Results5-LO and FLAP were stably expressed in HEK293 cells, and upon Ca^2 +-ionophore A23187 stimulation exogenous AA was efficiently transformed into the 5-LO products 5-hydro(pero)xyeicosatetraenoic acid (5-H(p)ETE) and the trans-isomers of LTB₄. A23187 stimulation caused 5-LO accumulation at the nuclear membrane only when FLAP was co-expressed. Unexpectedly, A23187 stimulation of HEK cells expressing 5-LO and FLAP without exogenous AA failed in 5-LO product synthesis. HEK cells liberated AA in response to A23187, and transfected HEK cells expressing 12-LO generated 12-HETE after A23187 challenge from endogenous AA. FLAP co-expression increased 5-LO product formation in A23187-stimulated cells at low AA concentrations. Only in cells expressing FLAP and 5-LO, the FLAP antagonist MK886 blocked FLAP-mediated increase in 5-LO product formation, and prevented 5-LO nuclear membrane translocation and co-localization with FLAP.ConclusionThe cellular biosynthesis of 5-LO products from endogenously derived substrate requires not only functional 5-LO/FLAP co-localization but also additional prerequisites which are dispensable when exogenous AA is supplied; identification of these determinants is challenging.General significanceWe present a cell model to study the role of FLAP as 5-LO interacting protein in LT biosynthesis in intact cells and for characterization of putative FLAP antagonists.     

Caffeic acid phenethyl ester and its amide analogue are potent inhibitors of leukotriene biosynthesis in human polymorphonuclear leukocytes

Background

5-lipoxygenase (5-LO) catalyses the transformation of arachidonic acid (AA) into leukotrienes (LTs), which are important lipid mediators of inflammation. LTs have been directly implicated in inflammatory diseases like asthma, atherosclerosis and rheumatoid arthritis; therefore inhibition of LT biosynthesis is a strategy for the treatment of these chronic diseases.

Methodology/Principal Findings

Analogues of caffeic acid, including the naturally-occurring caffeic acid phenethyl ester (CAPE), were synthesized and evaluated for their capacity to inhibit 5-LO and LTs biosynthesis in human polymorphonuclear leukocytes (PMNL) and whole blood. Anti-free radical and anti-oxidant activities of the compounds were also measured. Caffeic acid did not inhibit 5-LO activity or LT biosynthesis at concentrations up to 10 µM. CAPE inhibited 5-LO activity (IC₅₀ 0.13 µM, 95% CI 0.08–0.23 µM) more effectively than the clinically-approved 5-LO inhibitor zileuton (IC₅₀ 3.5 µM, 95% CI 2.3–5.4 µM). CAPE was also more effective than zileuton for the inhibition of LT biosynthesis in PMNL but the compounds were equipotent in whole blood. The activity of the amide analogue of CAPE was similar to that of zileuton. Inhibition of LT biosynthesis by CAPE was the result of the inhibition of 5-LO and of AA release. Caffeic acid, CAPE and its amide analog were free radical scavengers and antioxidants with IC₅₀ values in the low µM range; however, the phenethyl moiety of CAPE was required for effective inhibition of 5-LO and LT biosynthesis.

Conclusions

CAPE is a potent LT biosynthesis inhibitor that blocks 5-LO activity and AA release. The CAPE structure can be used as a framework for the rational design of stable and potent inhibitors of LT biosynthesis.     

Prolonged exposure to lipopolysaccharide inhibits macrophage 5-lipoxygenase metabolism via induction of nitric oxide synthesis

LPS from bacteria can result in the development of sepsis syndrome and acute lung injury. Although acute exposure to endotoxin primes leukocytes for enhanced synthesis of leukotrienes (LT), little is known about the effect of chronic exposure. Therefore, we determined the effect of prolonged LPS treatment on 5-lipoxygenase (5-LO) metabolism of arachidonic acid in alveolar macrophages (AM) and in peripheral blood monocytes. Pretreatment of AM with LPS caused time- and dose-dependent suppression of LT synthetic capacity. LPS pretreatment failed to inhibit arachidonic acid (AA) release. The fact that LPS inhibited LT synthesis from endogenous AA more than from exogenous AA suggested an effect on 5-LO-activating protein (FLAP). In addition, an inhibitory effect of LPS treatment on AM 5-LO activity was suggested by cell-free 5-LO enzyme assay. No effect on the expression of either 5-LO or FLAP proteins was observed. New protein synthesis was necessary for LPS-induced reduction of 5-LO metabolism in AM, and immunoblotting demonstrated marked induction of NO synthase (NOS). Inhibition by LPS was reproduced by an NO donor and was abrogated by inhibitors of constitutive and inducible NOS. Compared with AM, peripheral blood monocytes exhibited no suppression by LPS of 5-LO metabolism and no induction of inducible NOS. We conclude that prolonged exposure to LPS impairs AM 5-LO metabolism by NO-mediated suppression of both 5-LO and FLAP function. Because LT contribute to antimicrobial defense, this down-regulation of 5-LO metabolism may contribute to the increased susceptibility to pneumonia in patients following sepsis.     

Protein kinase A inhibits leukotriene synthesis by phosphorylation of 5-lipoxygenase on serine 523

Leukotrienes (LTs) are lipid messengers generated by leukocytes that drive inflammation and modulate neighboring cell function. The synthesis of LTs from arachidonic acid is initiated by the enzyme 5-lipoxygenase (5-LO). We report for the first time that LT synthesis is inhibited by the direct action of protein kinase A (PKA) on 5-LO. The catalytic subunit of PKA directly phosphorylated 5-LO in vivo and in vitro and inhibited activity in intact cells and in vitro. Mutation of Ser-523 on human 5-LO prevented phosphorylation by PKA and restored LT synthesis. Treatment with PKA activators inhibited LTB(4) synthesis in 3T3 cells expressing wild type 5-LO but not in cells expressing the S523A mutant of 5-LO. The mechanism of inhibition of LTB(4) synthesis did not involve either reduced membrane association of activated 5-LO or redistribution of 5-LO from the nucleus to the cytoplasm. Instead, PKA phosphorylation of recombinant 5-LO inhibited in vitro activity, as did co-transfection of cells with 5-LO plus the catalytic subunit of PKA. Also, substitution of Ser-523 with glutamic acid, mimicking phosphorylation, resulted in the total loss of 5-LO activity. These results indicate that PKA phosphorylates 5-LO on Ser-523, which inhibits the catalytic activity of 5-LO and reduces cellular LT generation. Thus, PKA activation, as can occur in response to adenosine, prostaglandin E(2), beta-adrenergic agonists, and other mediators, is a means of directly reducing 5-LO activity and LT synthesis that may be important in limiting inflammation and maintaining homeostasis.     

Nitric oxide mediates distinct effects of various LPS chemotypes on phagocytosis and leukotriene synthesis in human neutrophils

We investigated the effect of lipopolysaccharide (LPS) chemotypes differing in their carbohydrate chain length on phagocytosis of serum-opsonized zymosan (OZ) particles and related functions of human polymorphonuclear leukocyte (PMNL, neutrophils). LPS from deep core mutant (Re), complete core (Ra) and smooth (S) phenotypes of Salmonella typhimurium was studied. Priming of neutrophils with various LPSs caused prominent enhancement of OZ phagocytosis, superoxide production and leukotriene (LT) synthesis in neutrophils, with LPS effects increasing as Re < S < Ra. The LPS forms were less potent to activate OZ uptake in the presence of MK-886, 5-lipoxygenase activating protein inhibitor, suggesting the regulatory function of 5-lipoxygenase (5-LO)-derived LTs. Direct measurement of nitrite release from OZ-stimulated neutrophils revealed that the effects of LPS on NO synthesis increased in the range of Ra < S < Re. Nitric oxide synthase (NOS) inhibitor l-NAME increased phagocytosis, LT and superoxide formation by neutrophils, and abolished the difference in the action of the LPSs forms. Further mechanistic studies revealed that NO modulates cellular 5-LO activity in a guanylyl cyclase and protein kinase G dependent manner, as well as interplay between NO and superoxide, and peroxynitrite generation contribute to distinct effects of LPS chemotypes on phagocytosis and LT synthesis in human neutrophils. Our investigation of the three LPS species demonstrates that the LPS polysaccharide core is mostly essential for the PMNL activation and is able to suppress lipid A-induced increase in NOS activity in phagocyting cells by triggering specific signaling cascades.     

Sulfatides inhibit leukotriene synthesis in human polymorphonuclear granulocytes by a mechanism involving lipid rearrangement in intracellular membranes

Sulfatides - sulfated derivatives of galactocerebroside - are endogenous ligands for P- and L-selectins and are able to induce intracellular signaling in neutrophils through a L-selectin dependent pathway. Sulfatides are implicated in a variety of physiological functions and have been found to suppress the synthesis of 5-lipoxygenase (5-LO) metabolites and impede 5-LO translocation to the nuclear envelope in adherent human polymorphonuclear leukocytes (PMNs) [Sud'ina, G. F., Brock, T. G., Pushkareva, M. A., Galkina, S. I., Turutin, D. V., Peters-Golden, M., et al. (2001). Sulphatides trigger polymorphonuclear granulocyte spreading on collagen-coated surfaces and inhibit subsequent activation of 5-lipoxygenase. The Biochemical Journal, 359, 621-629]. In this study we investigated the mechanism of the leukotriene (LT) synthesis inhibition by sulfatides. Sulfatides neither attenuated the ionophore-induced rise in [Ca(2+)](i) nor promoted PKA activation. We demonstrated that sulfatides directly inhibited 5-LO enzyme activity in a cell-free assay. BODIPY-labeled sulfatides were able to rapidly penetrate into the cells. Sulfatides induced rearrangement and redistribution of cytoskeletal components in adherent PMNs. The lipid incorporation as well as sulfatide-induced inhibition of LT synthesis were abolished by cytochalasin D, an inhibitor of actin polymerization and endocytosis. Importantly, sulfatides caused a prominent intracellular cholesterol redistribution, increasing its abundance at the uropod region. On the basis of these data, we suggest that increased cholesterol accumulation in cell compartments represents a novel mechanism by which sulfatides abrogate 5-LO translocation and activation.     

Monocyte-derived soluble protein confers 5-lipoxygenase activity Ca2+-dependent

5-Lipoxygenase (5-LO) is a Ca2+-stimulated enzyme that initializes the formation of proinflammatory leukotrienes from arachidonic acid (AA). In this report, we demonstrate that a soluble protein of the monocytic cell line Mono Mac 6 confers 5-LO activity Ca2+-dependent in vitro. Thus, in broken cell preparations of human polymorphonuclear leukocytes (PMNL) and rat basophilic leukemia (RBL)-1 cells, 5-LO converted AA (>20 microM) in the absence of Ca2+, whereas Ca2+ was absolutely required for 5-LO activity in broken cell preparations of MM6 cells. 5-LO partially purified from MM6 cells was substantially active in the absence of Ca2+. Recombination experiments revealed that the cytosolic fraction of MM6 cells contains a factor that suppresses the activity of partially purified 5-LO from PMNL, RBL-1, and MM6 cells in the absence but not in the presence of Ca2+. Further characterization showed that this factor is a 80-100 kDa heat-sensitive protein.     

1-Oleoyl-2-acetylglycerol stimulates 5-lipoxygenase activity via a putative (phospho)lipid binding site within the N-terminal C2-like domain

5-Lipoxygenase (5-LO) catalysis is positively regulated by Ca2+ ions and phospholipids that both act via the N-terminal C2-like domain of 5-LO. Previously, we have shown that 1-oleoyl-2-acetylglycerol (OAG) functions as an agonist for human polymorphonuclear leukocytes (PMNL) in stimulating 5-LO product formation. Here we have demonstrated that OAG directly stimulates 5-LO catalysis in vitro. In the absence of Ca2+ (chelated using EDTA), OAG strongly and concentration-dependently stimulated crude 5-LO in 100,000 x g supernatants as well as purified 5-LO enzyme from PMNL. Also, the monoglyceride 1-O-oleyl-rac-glycerol and 1,2-dioctanoyl-sn-glycerol were effective, whereas various phospholipids did not stimulate 5-LO. However, in the presence of Ca2+, OAG caused no stimulation of 5-LO. Also, phospholipids or cellular membranes abolished the effects of OAG. As found previously for Ca2+, OAG renders 5-LO activity resistant against inhibition by glutathione peroxidase activity, and this effect of OAG is reversed by phospholipids. Intriguingly, a 5-LO mutant lacking tryptophan residues (Trp-13, -75, and -102) important for the binding of the 5-LO C2-like domain to phospholipids was not stimulated by OAG. We conclude that OAG directly stimulates 5-LO by acting at a phospholipid binding site located within the C2-like domain.     

Interaction between nitric oxide,reactive oxygen intermediates,and peroxynitrite in the regulation of 5-lipoxygenase metabolism

We have shown that overnight lipopolysaccharide (LPS) suppresses alveolar macrophage (AM) leukotriene (LT) synthesis mediated in part by induction of inducible nitric oxide synthase (iNOS) and NO production. Here we examined the possibility that reactive oxygen intermediates (ROI) generated by LPS pretreatment contribute to the suppression of 5-lipoxygenase (5-LO) metabolism. Pretreatment of AM with xanthine/xanthine oxidase, which generates high concentrations of ROI, resulted in suppression of LT synthetic capacity. Since NO and ROI reactive species are known to react and form peroxynitrite (ONOO(-)), we examined the effect of ONOO(-) on 5-LO metabolism. Exogenous ONOO(-) caused a dose-dependent suppression of recombinant 5-LO cell-free activity. ONOO(-) also suppressed LT synthesis in intact AM, which was reversed by the ONOO(-) scavenger tetrakis(4-benzoic acid)porphyrin. ONOO(-) treatment also resulted in dose-dependent nitrotyrosination and S-nitrosylation of the recombinant 5-LO enzyme. Since the direct 5-LO inhibitor zileuton prevents the LPS-induced suppression of LT synthesis, we examined if 5-LO itself was the source of ROI. Zileuton reduced ROI generation in LPS-treated cells. These studies identify an important role for ROI and ONOO(-) in the suppression of 5-LO metabolism by LPS.     

Arachidonic acid regulates the translocation of 5-lipoxygenase to the nuclear membranes in human neutrophils

Elevation of the intracellular cAMP concentration in agonist-activated human neutrophils (PMN) leads to the concomitant inhibitions of arachidonic acid (AA) release, 5-lipoxygenase (5-LO) translocation, and leukotriene (LT) biosynthesis. We report herein that exogenous AA completely prevents cAMP-dependent inhibition of 5-LO translocation and LT biosynthesis in agonist-activated PMN. Moreover, the group IVA phospholipase A2 inhibitor pyrrophenone and the MEK inhibitor U-0126 inhibited AA release and 5-LO translocation in activated PMN, and these effects were also prevented by exogenous AA, demonstrating a functional link between AA release and 5-LO translocation. Polyunsaturated fatty acids of the C18 and C20 series containing at least three double bonds located from carbon 9 (or closer to the carboxyl group) were equally effective as AA in restoring 5-LO translocation in pyrrophenone-treated agonist-activated PMN. Importantly, experiments with the 5-LO-activating protein inhibitor MK-0591 and the intracellular Ca2+ chelator BAPTA-AM demonstrated that the AA-regulated 5-LO translocation is FLAP- and Ca2+-dependent. Finally, the redox and competitive 5-LO inhibitors L-685,015, L-739,010, and L-702,539 (but not cyclooxygenase inhibitors) efficiently substituted for AA to reverse the pyrrophenone inhibition of 5-LO translocation, indicating that the site of regulation of 5-LO translocation by AA is at or in the vicinity of the catalytic site. This report demonstrates that AA regulates the translocation of 5-LO in human PMN and unravels a novel mechanism of the cAMP-mediated inhibition of LT biosynthesis.     

Guanylyl cyclase and protein kinase G mediate nitric oxide suppression of 5-lipoxygenase metabolism in rat alveolar macrophages

We have previously demonstrated that exogenous nitric oxide (NO) directly inhibits alveolar macrophage (AM) cell-free activity of the enzyme 5-lipoxygenase (5-LO), thereby inhibiting metabolism of arachidonic acid to the important proinflammatory lipid mediators, leukotrienes (LT). Here, we explored the possibility that NO indirectly inhibited AM LT synthesis via activation of soluble guanylyl cyclase (sGC) in rat AM. The selective sGC inhibitor, LY83583, abrogated the suppression of cellular LT synthesis elicited by either exogenous or endogenous NO. A non-NO-dependent activator of sGC, YC-1, also inhibited macrophage LT synthesis. We next determined if sGC-mediated suppression of AM LT synthesis was dependent on protein kinase G (cGK). The selective cGK inhibitor, KT5823, reversed the suppression of cellular 5-LO metabolism following treatment with exogenous NO and YC-1. cGK1 activation resulted in phosphorylation of 5-LO. In contrast to peritoneal macrophages, AM exhibited localization of sGC, cGK1 and cGKII to the cell nucleus. In summary, in addition to its direct effects, NO-induced suppression of 5-LO action can be mediated indirectly through activation of the sGC and cGK pathways in AM. The nuclear localization of enzymes sGC, CGK1 and cGKII in the AM, which also demonstrates preferential nuclear 5-LO expression, may confer tighter regulation of LT synthesis.     

Studies of the inhibitory activity of MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-indol- 2-yl]-2,2-dimethyl propanoic acid) on arachidonic acid metabolism in human phagocytes.

We have investigated the inhibitory activity of compound MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)-i ndol-2- yl]-2,2-dimethyl propanoic acid) on 5-lipoxygenase (5-LO) product synthesis in various human phagocytes stimulated with either the ionophore A23187, opsonized zymosan (OPZ), platelet-activating factor (PAF), or formyl-methionyl-leucyl-phenylalanine (fMLP). The lipoxygenase products were analyzed by reversed-phase HPLC. MK-0591 inhibited the formation of 5-hydroxyeicosatetraenoic acid, leukotriene (LT) B4, its omega-oxidation products, and 6-trans-isomers with IC50 values of 2.8-4.8 nM in A23187-stimulated neutrophils. In these conditions, arachidonic acid at a concentration of 10 microM had no effect on MK-0591 inhibitory activity. In neutrophils stimulated with OPZ, the synthesis of LTB4, its omega-oxidation products, and 6-trans-isomers was inhibited with IC50 values of 9.5-11.0 nM. MK-0591 inhibited 5-LO product synthesis in A23187-stimulated blood monocytes, eosinophils, and alveolar macrophages with IC50 values of 0.3-0.9, 3.7-5.3, and 8.5-17.3 nM, respectively. In neutrophils primed with granulocyte--macrophage colony-stimulating factor and stimulated with PAF, lipoxygenase product synthesis was inhibited with IC50 values of 7.7-8.7 nM. At the concentration of 1 microM, MK-0591 had no inhibitory effect on 15-lipoxygenase activity in human polymorphonuclear leukocytes, nor on human platelet 12-lipoxygenase and cyclooxygenase. In conclusion, MK-0591 is a very potent and specific inhibitor of 5-LO product synthesis in various types of human phagocytes.     

Interaction of ceruloplasmin and 5-lipoxygenase

The interaction between ceruloplasmin (CP), the multicopper oxidase of human plasma, and 5-lipoxygenase (5-LO), the key enzyme of leukotriene synthesis, is shown for the first time. By Western-blotting and mass spectrometry of tryptic fragments, it is shown that 5-LO from protein extract of human leukocytes binds with immobilized CP. Dose-dependent influence of intact CP on leukotrienes synthesis is found: CP reduced leukotrienes synthesis in leukocytes in a dose above 50 μg/ml (normal CP concentration in plasma is about 300–400 μg/ml). Proteolyzed CP and apo-form of CP is unable to inhibit activity of 5-LO. CP increased activity of 5-LO at low doses (5–10 μg/ml). On the whole, the influence of CP on phagocytosis index of leukocytes coordinates with influence on activity of 5-LO: the index increased in the range of 2–10 μg/ml CP and decreased at doses of CP above 40 μg/ml. The dual role of CP in regulation of cellular response of leukocytes is discussed.     

Modulation of human 5-lipoxygenase activity by membrane lipids

Mammalian 5-lipoxygenase (5-LO) catalyzes the conversion of arachidonic acid (AA) to leukotrienes, potent inflammatory mediators. 5-LO is activated by a Ca(2+)-mediated translocation to membranes, and demonstrates the characteristic features of interfacially activated enzymes, yet the mechanism of membrane binding of 5-LO is not well understood. In an attempt to understand the mechanism of lipid-mediated activation of 5-LO, we have studied the effects of a large set of lipids on human recombinant 5-LO activity, as well as mutual structural effects of 5-LO and membranes. In the presence of 0.35 mM phosphatidylcholine (PC) and 0.2 mM Ca(2+), there was substrate inhibition at >100 microM AA. Data analysis at low AA concentrations yielded the following: K(m) approximately 103 microM and k(cat) approximately 56 s(-1). 5-LO activity was supported by PC more than by any other lipid tested except for a cationic lipid, which was more stimulatory than PC. Binding of 5-LO to zwitterionic and acidic membranes was relatively weak; the extent of binding increased 4-8 times in the presence of Ca(2+), whereas binding to cationic membranes was stronger and essentially Ca(2+)-independent. Polarized attenuated total reflection infrared experiments implied that 5-LO binds to membranes at a defined orientation with the symmetry axis of the putative N-terminal beta-barrel tilted approximately 45 degrees from the membrane normal. Furthermore, membrane binding of 5-LO resulted in dehydration of the membrane surface and was paralleled with stabilization of the structures of both 5-LO and the membrane. Our results provide insight into the understanding of the effects of membrane surface properties on 5-LO-membrane interactions and the interfacial activation of 5-LO.     

Effects of Suramin on PMN Interactions with Different Surfaces

Human polymorphonuclear leukocytes (PMN) were found to tightly adhere on endothelial (lines EAhy926 and ECV304) and collagen surfaces under the influence of the chemotherapeutic drug suramin. This was observed by scanning electron microscopy and quantitated by myeloperoxidase assays. Suramin also inhibited Ca²⁺ ionophore A23187-stimulated leukotriene (LT) synthesis in PMN interaction with endothelial cells or with collagen surface. Suramin decreased the release of radiolabeled arachidonic acid (AA) and 5-lip-oxygenase (5-LO) metabolites by prelabeled PMN stimulated with A23187. Using agents releasing the suramin-stimulated adhesion namely jasplakonolide and dextran sulfate, we observed a reversal of the suramin effect on leukotriene synthesis. Jasplakonolide released the adhesion of PMN on endothelial and collagen-coated surfaces and restored 5-LO activity. Dextran-sulfate released adhesion on collagen-coated surfaces and abolished suramin inhibition. Arachidonate could also overcome adhesion and inhibition of 5-LO. We conclude that suramin-induced tight attachment of PMN on to solid surfaces lead to decreased leukotriene synthesis during subsequent A23187 stimulation in the absence of exogenous substrates.