Intact Polymorphonuclear Leukocytes Research Articles

Development of novel aminothiazole-comprising 5-LO inhibitors.

Leukotrienes are pivotal lipid mediators in various immune and inflammatory reactions. Herein, 5-LO is a validated target. 2-Aminothiazoles, as a privileged structure, implicate known 5-LO inhibitors like ST-1083 (IC50 [polymorphonuclear leukocytes (PMNL)] = 0.68 μM), yet deep structure-activity relationships (SAR) have not been established. Compounds were synthesized via Hantzsch thiazole synthesis. Inhibitory activities were evaluated using intact PMNL and purified 5-LO together with cytotoxicity measurements in U937 cells. We introduced novel functionalities at 2-, 3-, 4- and 5-position of the 2-aminothiazole scaffold and conducted bioisosteric replacement to optimize the parent scaffold. SARs of the 2-aminothiazole scaffold were deduced and extended primarily for inhibition of the 5-LO enzyme. SAR studies provided at least two optimized leads (ST-1853, ST-1906) with high potency (IC50 [polymorphonuclear leukocytes] = 0.05 μM), specificity and noncytotoxic behavior.

Structure–Activity Relationship and In Vitro Pharmacological Evaluation of Imidazo[1,2- a ] Pyridine-Based Inhibitors of 5-LO

5-LO is an important enzyme involved in the biosynthesis of leukotrienes, which are lipid mediators of immune and inflammation responses, with important roles in respiratory disease, cardiovascular disease, immune responses and certain types of cancer. Therefore, this enzyme has been investigated as a potential target for the treatment of these pathophysiological conditions. 5-LO inhibitory potential was investigated in intact polymorphonuclear leukocytes, a cell-free assay, in human whole blood and rodent cells to both elucidate structure-activity relationships and in vitro pharmacological evaluation. Chemical modifications for lead optimization via straight forward synthesis was used to combine small polar groups, which led to a suitable candidate (IC50 [polymorphonuclear leukocytes] = 1.15 µM, IC50 [S100] = 0.29 µM) with desired in vitro biopharmaceutical profiles in terms of solubility (451.9 µg/ml) and intrinsic clearance without demonstrating any cytotoxicity. Compound 9l is a novel, potent and selective 5-LO inhibitor with favorable preclinical drug-like properties.

Molecular pharmacological profile of a novel thiazolinone‐based direct and selective 5‐lipoxygenase inhibitor

The potency of many 5-lipoxygenase (5-LOX) inhibitors depends on the cellular peroxide tone and the mechanism of 5-LOX enzyme activation. Therefore, new inhibitors that act regardless of the mode of enzyme activation need to be developed. Recently, we identified a novel class of thiazolinone-based compounds as potent 5-LOX inhibitors. Here, we present the molecular pharmacological profile of (Z)-5-(4-methoxybenzylidene)-2-(p-tolyl)-5H-thiazol-4-one, compound C06. Inhibition of 5-LOX product formation was determined in intact cells [polymorphonuclear leukocytes (PMNL), rat basophilic leukaemia-1, RAW264.7] and in cell-free assays [homogenates, 100, 000×g supernatant (S100), partially purified 5-LOX] applying different stimuli for 5-LOX activation. Inhibition of peroxisome proliferator-activated receptor (PPAR), cytosolic phospholipase A(2) (cPLA(2) ), 12-LOX, 15-LOX-1 and 15-LOX-2 as well as cyclooxygenase-2 (COX-2) were measured in vitro. C06 induced non-cytotoxic, direct 5-LOX inhibition with IC(50) values about 0.66 µM (intact PMNL, PMNL homogenates) and approximately 0.3 µM (cell-free PMNL S100, partially purified 5-LOX). Action of C06 was independent of the stimulus used for 5-LOX activation and cellular redox tone and was selective for 5-LOX compared with other arachidonic acid binding proteins (PPAR, cPLA(2) , 12-LOX, 15-LOX-1, 15-LOX-2, COX-2). Experimental results suggest an allosteric binding distinct from the active site and the C2-like domain of 5-LOX. C06 was identified as a potent selective direct 5-LOX inhibitor exhibiting a novel and unique mode of action, different from other established 5-LOX inhibitors. This thiazolinone may possess potential for intervention with inflammatory and allergic diseases and certain types of cancer.

Molecular characterization of EP6—A novel imidazo[1,2- a]pyridine based direct 5-lipoxygenase inhibitor

5-Lipoxygenase (5-LO) is a crucial enzyme of the arachidonic acid (AA) cascade and catalyzes the formation of bioactive leukotrienes (LTs) which are involved in inflammatory diseases and allergic reactions. The pathophysiological effects of LTs are considered to be prevented by 5-LO inhibitors. In this study we present cyclohexyl-[6-methyl-2-(4-morpholin-4-yl-phenyl)-imidazo[1,2- a]pyridin-3-yl]-amine ( EP6), a novel imidazo[1,2- a]pyridine based compound and its characterization in several in vitro assays. EP6 suppresses 5-LO activity in intact polymorphonuclear leukocytes with an IC 50 value of 0.16 μM and exhibits full inhibitory potency in cell free assays (IC 50 value of 0.05 μM for purified 5-LO). The efficacy of EP6 was not affected by the redox tone or the concentration of exogenous AA, characteristic drawbacks known for the class of nonredox-type 5-LO inhibitors. Furthermore, EP6 suppressed 5-LO activity independently of the cell stimulus or the activation pathway of 5-LO contrary to what is known for some nonredox-type inhibitors. Using molecular modeling and site-directed mutagenesis studies, we were able to derive a feasible binding region within the C2-like domain of 5-LO that can serve as a new starting point for optimization and development of new 5-LO inhibitors targeting this site. EP6 has promising effects on cell viability of tumor cells without mutagenic activity. Hence the drug may possess potential for intervention with inflammatory and allergic diseases and certain types of cancer including leukemia.

A Class of 5-Benzylidene-2-phenylthiazolinones with High Potency as Direct 5-Lipoxygenase Inhibitors

A novel class of potent direct 5-lipoxygenase (5-LO) inhibitors bearing a thiazolinone-scaffold identified by virtual screening is presented. A range of substitutions and the importance of the 2-phenyl moiety were evaluated. This series is characterized by high potency in intact polymorphonuclear leukocytes and a cell-free system, exemplified by (Z)-2-(4-chlorophenyl)-5-(4-methoxybenzylidene)-5H-thiazol-4-one (18, IC(50) = 0.28 and 0.09 μM). These disubstituted thiazolinones may possess potential for intervention with inflammatory and allergic diseases and certain cancer types.

ChemInform Abstract: 4-Hydroxythiazole Inhibitors of 5-Lipoxygenase.

4-Hydroxythiazoles have been identified as potent inhibitors of 5-lipoxygenase in vitro exhibiting IC50's of less than 1 microM. An investigation of structure-activity relationships showed that the most potent inhibitors of this series are the 5-phenyl derivatives. The corresponding thiazolidin-4-one analogues were found to be relatively inactive. The 4-hydroxythiazoles were active inhibitors against 5-lipoxygenase in both intact rat polymorphonuclear leukocytes and human whole blood. The compounds were also selective inhibitors of 5-lipoxygenase, displaying only weak activity against other related enzymes, cyclooxygenase and 12- and 15-lipoxygenase.

Carnosic acid and carnosol potently inhibit human 5-lipoxygenase and suppress pro-inflammatory responses of stimulated human polymorphonuclear leukocytes

Carnosic acid (CA) and carnosol (CS) are phenolic diterpenes present in several labiate herbs like Rosmarinus officinalis (Rosemary) and Salvia officinalis (Sage). Extracts of these plants exhibit anti-inflammatory properties, but the underlying mechanisms are largely undefined. Recently, we found that CA and CS activate the peroxisome proliferator-activated receptor gamma, implying an anti-inflammatory potential on the level of gene regulation. Here we address short-term effects of CA and CS on typical functions of human polymorphonuclear leukocytes (PMNL). We found that (I), CA and CS inhibit the formation of pro-inflammatory leukotrienes in intact PMNL (IC 50 = 15–20 μM [CA] and 7 μM [CS], respectively) as well as purified recombinant 5-lipoxygenase (EC number 1.13.11.34, IC 50 = 1 μM [CA] and 0.1 μM [CS], respectively), (II) both CA and CS potently antagonise intracellular Ca 2+ mobilisation induced by a chemotactic stimulus, and (III) CA and CS attenuate formation of reactive oxygen species and the secretion of human leukocyte elastase (EC number 3.4.21.37). Together, our findings provide a pharmacological basis for the anti-inflammatory properties reported for CS- and CA-containing extracts.

Borrelia burgdorferi are susceptible to killing by a variety of human polymorphonuclear leukocyte components.

The killing of Borrelia burgdorferi by intact human polymorphonuclear leukocytes (PMNL) and by individual PMNL components was compared. Intact PMNL killed B. burgdorferi 6.5-fold more efficiently and 5-fold more completely when spirochetes were opsonized with specific antibodies. U-cytoplasts, which have activatable oxidase, killed opsonized B. burgdorferi with an efficiency similar to that of intact PMNL in killing unopsonized B. burgdorferi. Although B. burgdorferi were susceptible to H(2)O(2) and nitric oxide, PMNL lysates killed B. burgdorferi nearly as well as intact PMNL killed opsonized B. burgdorferi, suggesting a critical role for granule contents. B. burgdorferi were killed by the PMNL antimicrobial components elastase, LL-37, bactericidal/permeability-increasing protein, and human neutrophil peptide-1. B. burgdorferi had limited susceptibility to killing by lysozyme and were not killed by azurocidin, proteinase 3, or lactoferrin. The efficient killing of B. burgdorferi by a variety of PMNL mechanisms highlights the paradoxical persistence of spirochetes in vivo.

The Polymorphonuclear Leukocyte – A New Target for Erythropoietin

A previous study from our laboratory has shown that erythropoietin (EPO), beside its traditional role in erythropoiesis, acts as an alleviator of oxidative stress and inflammation in chronic hemodialysis (HD) patients, conferred in part by activated polymorphonuclear leukocytes (PMNLs). To substantiate this phenomenon, the existence of EPO receptors (EPO-Rs) on PMNL membrane was examined at the transcriptional and translational levels. mRNA for EPO-R was detected in PMNLs using specific primers directed towards the extracellular region of human EPO-R cDNA. The predicted 300-bp fragment was amplified by reverse transcriptase-polymerase chain reaction. Subcloning and sequence analysis revealed 100% homology of this fragment with human EPO-R. The receptor protein was detected on the surface of intact PMNLs using ¹²⁵I-EPO. The protein was further demonstrated by flow cytometric analysis using a fluorescent monoclonal anti-EPO-R. The percentage of PMNLs expressing EPO-R showed a strong correlation with the level of EPO in the serum, suggesting an upregulation of the receptor by the hormone. Taken together with our recent findings that EPO attenuates the oxidative stress and inflammation contributed by PMNLs in HD patients, the detection of functional EPO-R expression in PMNLs places these cells among the nonerythroid, EPO-responsive target populations.

16( R)-hydroxy-5,8,11,14-eicosatetraenoic acid, a new arachidonate metabolite in human polymorphonuclear leukocytes

Intact human polymorphonuclear leukocytes (PMNL) incubated with substimulatory amounts of arachidonic acid in the absence of a calcium ionophore formed four metabolites that were isolated by reverse-phase HPLC and characterized structurally by GC/MS. A major metabolite eluting as the most abundant peak of radioactivity lacked UV chromophores above 215 nm, and its formation was sensitive to 2-diethylaminoethyl-2,2-diphenylvalerate hydrochloride (SKF525A) but not 3-amino-1-[ m(trifluoromethyl)phenyl]-2-pyrazoline (BW755C), suggesting that it was likely to be a product of cytochrome P450. The GC/MS analysis revealed the presence of two components: 20-hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) and 16-hydroxy-5,8,11,14-eicosatetraenoic acid (16-HETE) in an approximate ratio of 4:1. The minor metabolites were identified as 15-HETE and 5-HETE. Although 20-HETE has been observed previously as a product of arachidonic acid metabolism in PMNL, the occurrence of 16-HETE was a novel finding. The stereochemistry of the hydroxyl group in PMNL-derived 16-HETE was established by analysis of 1-pentafluorobenzyl-16-naphthoyl derivatives on a chiral-phase chromatographic column and comparison with authentic synthetic stereoisomers. The PMNL-derived radioactive metabolite co-eluted with the synthetic 16( R)-HETE stereoisomer. Analysis of the total lipid extracts from intact PMNL followed by mild alkaline hydrolysis resulted in detectable amounts of 16-HETE (108 ± 26 pg/10 8 cells) and 20-HETE (341 ± 69 pg/10 8 cells), which suggested that these HETEs were formed from endogenous arachidonic acid and esterified within PMNL lipids. Thus, in contrast to calcium ionophore-stimulated neutrophils that generate large amounts of 5-lipoxygenase products, the intact PMNL generate 20-HETE and 16( R)-HETE via a cytochrome P450 ω- and ω-4 oxygenase(s).

Distinct granule populations in human neutrophils and lysosomal organelles identified by immuno-electron microscopy

In this paper, we illustrate the fine structural localization of distinct marker proteins in the organelles of human neutrophils and outline our preferred methods for processing ultrathin cryosections for use with immunoelectron microscopy. Previous work has determined the subcellular localization of certain marker proteins within intact polymorphonuclear neutrophilic leukocytes (PMN) and PMN fractions. These are as follows: myeloperoxidase (MPO) for azurophilic granules, lactoferrin for specific/secondary granules, gelatinase for gelatinase/tertiary granules, albumin for the secretory vesicles, and HLA class I and L-selectin for the plasma membrane. In addition to analyzing the heterogeneity of the PMN granule populations, new information on the lysosomal system of this cell is reviewed and extended by the localization of the lysosome-associated membrane proteins (LAMPs) and the cation-independent mannose 6-phosphate receptor (CI-M6PR). LAMPs were absent in all identified granule populations, but were found in the membranes of vesicles, multivesicular bodies (MVB), and multilaminar compartments (MLC). We show here that MVB contain CI-M6PR whereas MLC do not. Furthermore, since MLC contain LAMPs but not the receptor, they probably correspond to the late endosome. By current criteria, the true lysosomes of the resting PMN are MVB and MLC. Finally, although azurophil granules contain acid hydrolases their membranes do not contain LAMPs and they cannot be classified as lysosomes, but rather are more similar to regulated secretory granules.

Inhibition of arachidonate lipoxygenase activities by 2-(3,4-dihydroxyphenyl)ethanol, a phenolic compound from olives.

The effects of olive fruit extract on arachidonic acid lipoxygenase activities were investigated using rat platelets and rat polymorphonuclear leukocytes (PMNL). Olive extract strongly inhibited both 12-lipoxygenase (12-LO) and 5-lipoxygenase (5-LO) activities. One of the compounds responsible for this inhibition was purified and identified as 2-(3,4-dihydroxyphenyl)ethanol (DPE). DPE inhibited platelet 12-LO activity (IC50, 4.2 microM) and PMNL 5-LO activity (IC50, 13 microM) but not cyclooxygenase activity in cell-free conditions. It also inhibited 12-LO activity in intact platelets (IC50, 50 microM) and reduced leukotriene B4 production in intact PMNL stimulated by A23187 (IC50, 26 microM). The inhibition by DPE of both lipoxygenase activities was stronger than that by oleuropein, caffeic acid, or 7 other related phenolic compounds, especially in intact cells. These results suggest that DPE is a potent specific inhibitor of lipoxygenase activities.

Lipoxin A4 and B4 are potent stimuli for human monocyte migration and adhesion: selective inactivation by dehydrogenation and reduction.

Monocyte recruitment and adherence are important events in inflammatory and vascular diseases. Here, we evaluated the actions of lipoxin A4 (LXA4) and LXB4, a series of lipoxygenase products from arachidonic acid generated by cell-cell interactions, on human monocytes. LXA4 and LXB4 (10(-7) M) each increased monocyte migration in chamber chemotaxis assays and, in migration under agarose, exhibited chemotactic indices similar to those of the chemotactic peptide formyl-methionyl-leucyl-phenylalanine at 10(-10)-10(-8) M and to the chemokine macrophage inflammatory protein-1 alpha (MIP-1 alpha) at 10(-8)-10(-7) M with a rank order of potency: Monocyte chemotactic protein-1 alpha > LXA4 approximately LXB4 approximately MIP-1 alpha. Lipoxins also stimulated monocyte adherence to laminin. In addition, human monocytes rapidly transformed LXA4 and LXB4 to several metabolites. LXB4 (> 80%) was converted within 30 s to new products, in a trend similar to that of LXA4. The novel monocyte-derived LXB4 products were identified as 5-oxo-6,7-dihydro-LXB4 and 6,7-dihydro-LXB4, indicating a role for site-selective dehydrogenation and reduction. Unlike monocytes, intact polymorphonuclear leukocytes (PMN) did not metabolize LXA4 in significant quantities, and only approximately 12% of exogenous LXB4 was omega-oxidized to 20-OH-LXB4 and 20-COOH-LXB4 by PMN. To determine if lipoxin conversion altered bioactivity, we evaluated the actions of these metabolites on monocytes. Each of the novel products of LXA4 and LXB4 from monocytes, namely oxo- and dihydrolipoxins, were essentially inactive in stimulating monocyte adherence. In contrast, the omega-oxidation products of LXB4 isolated from PMN were equipotent with LXB4 for monocyte adherence. Dehydrogenation of LXA4 in monocytes appears to be carried out by a 15-hydroxyprostaglandin dehydrogenase, which is present in human monocytes as determined by reverse transcription PCR and Western blots. Together, these results provide the first evidence that LXA4 and LXB4 are both potent stimulants for migration and adherence of human monocytes. Moreover, they underscore the importance of the major route of lipoxin metabolism in leukocytes, namely, the rapid dehydrogenation and inactivation carried out by monocytes.

Inversely-correlated inhibition of human 5-lipoxygenase activity by bay X1005 and other quinoline derivatives in intact cells and a cell-free system—implications for the function of 5-lipoxygenase activating protein

A series of quinoline derivatives were analysed for the influence on leukotriene synthesis as a parameter for 5-LOX (EC 1.13.11.34) activity in a cell-free system of the 10,000 g supernatant of human PMNL (polymorphonuclear leukocytes). The ratios of the ic 50 values for leukotriene synthesis inhibition in this cell-free system and in A23187-stimulated intact PMNL ranged from 1–1100. Consequently, plotting of the two values resulted in a random distribution ( r = −0.281, N = 18) suggesting that no relationship between the inhibition of leukotriene synthesis in the cell-free system and in intact cells exists. At first sight this finding was not surprising since we have shown earlier that in intact cells this class of quinoline derivatives shares the same mode of action as MK-886, i.e. an indirect inhibition of 5-LOX activity by binding to FLAP. However, we found that the potency of these compounds in intact cells is strongly influenced by the K value (partition coefficient) which is a parameter for the ability of a substance to accumulate in a lipid (membrane) phase compared to the water phase. Therefore, the ic 50 values for leukotriene synthesis inhibition in intact PMNL were corrected for the corresponding K value of the compounds and the resulting values again plotted against the ic 50 values for inhibition of leukotriene synthesis in the cell-free system. As a result, a significant correlation ( r = −0.878, N = 18) was obtained. In order to simplify this relationship the influence of the partition coefficient was eliminated by comparing compounds with about the same K value ( K = 7243 ± 1646, N = 7). As a result, the ic 50 values for inhibition of leukotriene synthesis in the 10,000 g supernatant fraction (indicative for the affinity of the compounds to 5-LOX) and in intact cells (indicative for the affinity of the compounds to FLAP) were highly, but inversely correlated ( r = −0.992). That means that a compound with a high affinity to 5-LOX will have a low affinity to FLAP and vice versa. We hypothesized that this pharmacologically obtained relationship could be indicative of a physiologically occurring equivalent. We therefore propose a model in which FLAP binds arachidonic acid as its physiological substrate with low affinity and allows 5-LOX to get access to its substrate (assuming a higher affinity of 5-LOX to arachidonic acid) after 5-LOX translocation from the cytosol to the membrane. In support of this model we provide evidence that arachidonic acid and other cis-unsaturated fatty acids, but neither a trans-unsaturated nor a saturated fatty acid, inhibit BAY X1005 binding to FLAP in intact human PMNL.

Effects of phorbol myristate acetate on the synthesis of 5-oxo-6,8,11,14-eicosatetraenoic acid by human polymorphonuclear leukocytes.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a newly discovered chemotactic agent for human polymorphonuclear leukocytes (PMNL) which has potent stimulatory effects on cytosolic calcium levels in these cells. Although we have shown that it is synthesized from 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE) by a highly specific microsomal dehydrogenase, little is known about the synthesis of this substance by intact PMNL. In the present study we found that in contrast to PMNL microsomes, intact, unstimulated PMNL produced relatively small amounts of 5-oxo-ETE from 5-HETE, but instead converted 5-HETE primarily to its omega-oxidation product, 5,20-diHETE. However, preincubation of PMNL with phorbol myristate acetate (PMA; EC50, ca. 4 nM) dramatically increased the ratio of 5-oxo-ETE to 5,20-diHETE from 0.07 in its absence to 1.85 in the presence of 100 nM PMA. Both effects were completely reversed by staurosporine, indicated that they were mediated by a protein kinase. PMA also stimulated the formation of 5-oxo-ETE, 5-HETE, and leukotriene B4 (LTB4) from exogenous arachidonic acid. The greatest enhancement was observed for 5-oxo-ETE, which, under all conditions, was produced in greater quantities than LTB4. PMA stimulated the formation of 5-oxo-ETE by PMNL stimulated with either A23187 or zymosan. A23187-stimulated PMNL initially produced more LTB4 than 5-oxo-ETE, but at longer time points, 5-oxo-ETE predominated. These results demonstrate that PMA-activated human PMNL can synthesize substantial amounts of 5-oxo-ETE and raise the possibility that this substance may be an important inflammatory mediator.

Mode of action of the new selective leukotriene synthesis inhibitor BAY X 1005 {( R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid} and structurally related compounds

BAY X 1005 ( R)-2-[4-(quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid has been demonstrated to be a potent inhibitor of leukotriene B 4 (LTB 4) and 5-hydroxyeicosatetraenoic acid (5-HETE) synthesis in various in vitro systems. Using mainly human polymorphonuclear leukocytes (PMNL) this study elucidates the mechanism of inhibition of 5-lipoxygenase (5-LOX, EC 1.13.11.34)-derived arachidonic acid metabolites by BAY X 1005. At concentrations of BAY X 1005 which almost totally inhibited the formation of 5-LOX-derived metabolites, both arachidonic acid release and platelet-activating factor synthesis were only modestly affected. This suggests that the inhibitory effect of BAY X 1005 is not due to a limitation of substrate availability for 5-LOX. Compared to the inhibition of leukotriene synthesis in intact human PMNL about 800-fold higher concentrations of BAY X 1005 were required to inhibit leukotriene formation in a cell-free system suggesting that the inhibitory effect of BAY X 1005 cannot be explained by a direct effect on 5-LOX. In an attempt to identify possible target proteins of BAY X 1005, [ 14C]BAY X 1005 was used in binding studies under equilibrium conditions. The quantitative analysis of specific binding in intact human PMNL revealed two binding sites for BAY X 1005. Upon subcellular fractionation of these cells the BAY X 1005 high affinity binding site was localized in the microsomal fraction whereas the low affinity binding site was localized in the granule fraction. The K d for BAY X 1005 binding to the high affinity binding site (0.165 μmol L ) was almost identical to the ic 50value for inhibition of LTB 4 synthesis (0.22 μmol L ). Furthermore, the ic 50 values for competition of BAY X 1005 binding at the high affinity binding site were almost identical to the ic 50 values for inhibition of LTB 4 synthesis in the case of BAY X 1005, 12 other structurally related quinoline derivatives and the reference compounds REV-5901, WY-50,295 and MK-886, but not in the case of the direct 5-LOX inhibitors A-64077 and AA-861. The analysis of BAY X 1005 binding in rat PMNL also revealed two binding sites. Whereas the low affinity binding site in rat PMNL exhibited a K d similar to the human, the rat high affinity binding site showed a 5.5-fold higher affinity for BAY X 1005 compared to the human. This correlates well with the 8.5-fold higher sensitivity of rat versus human PMNL concerning inhibition of LTB 4 synthesis. Competition experiments verified that this relationship holds true also for 12 other quinoline derivatives as well as for REV-5901, WY-50,295 and MK-886. Taken together, these results indicate a causal relationship between the binding of BAY X 1005 to the high affinity binding site and the inhibition of leukotriene synthesis in human and rat PMNL. In addition, the localization of this binding site in the microsomal fraction and the competition of BAY X 1005 binding by MK-886 suggest that the target protein of BAY X 1005 mediating leukotriene synthesis inhibition is identical to five lipoxygenase activating protein.

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.

Effects of oxygen radicals on the conformation of sulfhydryl groups on human polymorphonuclear leukocyte membranes

The healthy intact polymorphonuclear leukocytes(PMNs) were labeled with 4-maleimide-TEMPO spin labeling compound (MAL) to study the effects of oxygen radicals produced by phorbol myristate acetate (PMA)-stimulated PMNs on the conformation of sulfhydryl (SH) groups of PMN membrane proteins. The lipid peroxidation induced by PMA-stimulated PMNs was detected by evaluating the formation of malonaldehyde (MDA) with the thiobarbituric acid (TBA) test. From the experiments of luminol-dependent chemiluminescence (CL) and fluorometry, it was found that Chinese herbs schizandrin B (Sin B) and quercetin (Q) possessed scavenging properties for oxygen radicals produced during the PMN respiratory burst. These two herbs can also inhibit the conformation changes in SH binding sites on the PMN membrane proteins caused by oxygen radicals produced by the PMNs themselves. They also decrease the amount of MDA, which was a final product formed during lipid peroxidation.

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.

Differential effect of extracellular calcium on the Na + -K + pump activity in intact polymorphonuclear leucocytes and erythrocytes

The effect of extracellular calcium on the Na + -K + pump activity in human polymorphonuclear leucocytes and erythrocytes was studied and compared with the activity in mixed peritoneal leucocytes from rats. While there was maximal decrease in the pump activity (25–30%) of leucocytes from both rat and human by calcium 0.6 mM, a concentration of 0.1 mM caused a substantial decrease indicating a high sensitivity for extracellular calcium. In contrast, calcium had no effect on the pump activity in crythrocytes. The effect of calcium on the pump activity in leucocytes may be due to regulation of the influx of sodium across the plasma membrane, since in human leucocytes calcium had no effect on the pump activity if the cells were loaded with sodium.