Marina A. Pushkareva

Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties

Caffeic acid phenethyl ester, an active component of propolis extract, inhibits 5‐lipoxygenase in the micromolar concentration range. The inhibition is of an uncompetitive type, i.e. the inhibitor binds to the enzyme‐substrate complex but not to the free enzyme. Caffeic acid phenethyl ester also exhibits antioxidant properties. At a concentration of 10 μM, it completely blocks production of reactive oxygen species in human neutrophils and the xanthine/xanthine oxidase system.

Sulphatides trigger polymorphonuclear granulocyte spreading on collagen-coated surfaces and inhibit subsequent activation of 5-lipoxygenase

Sulphatides are sulphate esters of galactocerebrosides that are present on the surfaces of many cell types and act as specific ligands to selectins. The present study was undertaken to investigate the effect of sulphatides on polymorphonuclear granulocyte (PMN) attachment, spreading and 5-lipoxygenase (5-LO) metabolism. Sulphatides, but not non-sulphated galactocerebrosides, dose-dependently enhanced attachment to collagen, as measured by the myeloperoxidase assay. Studies with blocking antibodies indicated that the increased attachment was mediated by CD11b/CD18 (Mac-1) beta 2 integrin. Scanning electron microscopy indicated that sulphatides also greatly enhanced the degree of cell spreading. In PMNs treated in suspension, sulphatides had no effect on the ionophore A23187-stimulated release of arachidonic acid and the synthesis of 5-LO metabolites. In contrast, in PMNs attached to collagen, the enzymic conversion of arachidonic acid by 5-LO was inhibited by sulphatides. Inhibition of 5-LO metabolism by sulphatides was observed even in the presence of exogenous substrate, suggesting that sulphatides directly inhibited 5-LO action. Consistent with this, sulphatides interfered with ionophore-induced translocation of the 5-LO to the nuclear envelope. Substances competing with sulphatide binding to cells, like dextran sulphate, or a strong inhibitor of cell spreading, like the actin-polymerizing agent jasplakinolide, prevented the effects of sulphatides on PMN attachment and spreading and leukotriene synthesis. We conclude that shape changes occurring in response to sulphatides specifically impair PMN leukotriene synthesis by inhibiting translocation of 5-LO.

Cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) selectivity of COX inhibitors

In vitro evaluations of the selectivity of COX inhibitors are based on a great variety of experimental protocols. As a result, data available on cyclooxygenase (COX)-1/COX-2/5- lipoxygenase (LOX) selectivity of COX inhibitors lack consistency. We, therefore, performed a systematic analysis of the COX-1/COX-2/5-LOX selectivity of 14 compounds with selective COX inhibitory activity (Coxibs). The compounds belonged to different structural classes and were analyzed employing the well-recognized whole-blood assay. 5-LOX activity was also tested on isolated human polymorphonuclear leukocytes. Among COX inhibitors, celecoxib and ML-3000 (licofelone) inhibited 5-LOX in human neutrophils at micromolar ranges. Surprisingly, ML-3000 had no effect on 5-LOX product synthesis in whole-blood assay. In addition, we could show that inhibition of COX pathways did not increase the transformation of arachidonic acid by the 5-LOX pathway.

Involvement of ecto‐ATPase and extracellular ATP in polymorphonuclear granulocyte‐endothelial interactions

We conclude that PMN‐endothelial adhesion is counteracted by an ecto‐ATPase or by ATP receptors with ATPase activity. Such interactions may play a role in PMN rolling and diapedesis as well as in the pathophysiology of PMN activation by an anergic endothelium.

Cholesterol and its anionic derivatives inhibit 5-lipoxygenase activation in polymorphonuclear leukocytes and MonoMac6 cells

5‐Lipoxygenase (5‐LO) is the key enzyme in the biosynthesis of leukotrienes (LTs), biological mediators of host defense reactions and of inflammatory diseases. While the role of membrane binding in the regulation of 5‐LO activity is well established, the effects of lipids on cellular activity when added to the medium has not been characterized. Here, we show such a novel function of the most abundant sulfated sterol in human blood, cholesterol sulfate (CS), to suppress LT production in human polymorphonuclear leukocytes (PMNL) and Mono Mac6 cells. We synthesized another anionic lipid, cholesterol phosphate, which demonstrated a similar capacity in suppression of LT synthesis in PMNL. Cholesteryl acetate was without effect. Cholesterol increased the effect of CS on 5‐LO product synthesis. CS and cholesterol also inhibited arachidonic acid (AA) release from PMNL. Addition of exogenous AA increased the threshold concentration of CS required to inhibit LT synthesis. The effect of cholesterol and its anionic derivatives can arise from remodeling of the cell membrane, which interferes with 5‐LO activation. The fact that cellular LT production is regulated by sulfated cholesterol highlights a possible regulatory role of sulfotransferases/sulfatases in 5‐LO product synthesis.

Biosynthesis of leukotriene B4 in human polymorphonuclear leukocytes: regulation by cholesterol and other lipids

Leukotriene B4 (LTB4) is one of the most potent chemotactic compounds produced in macrophages and neutrophils. LTB4 is a product of arachidonic acid oxygenation by 5-lipoxygenase pathway. We present here the data on regulation of LT synthesis in human polymorphonuclear leukocytes by cholesterol, cholesterol sulfate and cholesterol phosphate. The addition of Pseudomonas aeruginosa lipopolysaccharides (LPS) with lipid vesicles containing phosphatidylcholine or phosphatidylcholine/cholesterol (70:30) showed that omitting cholesterol abolished the effect of LPS on LT synthesis. We show here the capacity of cholesterol sulfate, the most abundant sulfated sterol in human blood, to suppress LT production in human neutrophils and to neutralize the effect of P. aeruginosa LPS on LT synthesis. We suggest that sulfated lipids serve as specific endogenous regulators of LT synthesis in neutrophils, and anti-inflammatory therapy may be based on modification of cholesterol level and its conversion to anionic derivatives.

Competitive inhibition of the 5-lipoxygenase-catalysed linoleate oxidation by arachidonic and 5-hydroperoxy-eicosatetraenoic acids.

Linoleic and arachidonic acids are competing substrates for 5–1ipoxygenase from barley. When these two substrates are added simultaneously, arachidonic acid acts as a competitive inhibitor of linoleic acid oxidation with K i of 20 μM, the same value as the Michaelis constant for arachidonate oxygenation by this enzyme (22 ± 3 μM). Linoleic acid hydroperoxide accumulated in the reaction mixture does not inhibit the enzymatic process, while arachidonic acid hydroperoxy product (5–hydroperoxy–6,8,11,14–eicosatetraenoic acid) inhibits it with very low K i equal to 0.5 μM.

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.

Quasi-collinear AOTF with improved resolution

A number of possible configurations of acousto-optical tunable filter (AOTF) with a quasi-collinear geometry of interaction in the paratellurite crystal are observed. A theoretical examination of the AOTF resolution improving problem in the case of quasi-collinear geometry of acousto-optical interaction is presented. The analysis of interaction of light and sound in different planes of the paratellurite crystal is carried out. Devices with spherical angles of sound equal to 85.5° and 88° in the plane (110) are calculated. Some new and original configurations of AOTF are described. Possible angle apertures of sound and light in all these kinds of devices are observed and the frequency dependence, figure of merit dependencies are presented.

Regulation of leukotriene synthesis by arachidonic acid in human polymorphonuclear leukocyte adhesive interactions is dependent on the presence of albumin.

We have previously demonstrated that the pretreatment of polymorphonuclear leukocytes (PMNs) with the chemotherapeutic drug, Suramin, increases both cell attachment and inhibits calcium ionophore A23187‐stimulated leukotriene (LT) synthesis. Here, we examined the effects of extracellular arachidonic acid (AA) and albumin on attachment and LT synthesis in the interaction of PMNs with both collagen‐coated surfaces and human umbilical vein endothelial cell (HUVEC) monolayers. Suramin decreased the release of radiolabelled AA and 5‐lipoxygenase metabolites by [14C‐AA]‐prelabelled PMNs stimulated with A23187, with and without human serum albumin (HSA) in the culture medium. Addition of 1μM AA together with calcium ionophore stimulated the release of endogenous AA to the same level as control and Suramin‐pretreated cells, but attachment was unaffected and LT synthesis was still inhibited with Suramin treatment. Using 24μM AA, regulation of LT synthesis was dependent on the presence of HSA in the medium. Without HSA, 24μM AA induced detachment of PMNs and increased LT synthesis in Suramin‐treated cells above the control level. In the presence of HSA, 24μM AA did not influence PMN attachment or abolish Suramin‐induced inhibition of LT synthesis. These results suggest that tight attachment of PMNs to a solid surface leads to decreased LT synthesis during subsequent stimulation of the cells by A23187 in the presence or absence of exogenous substrate.