Galina F. Sud'ina

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.

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.

Purification of prostaglandin H synthetase and a fluorometric assay for its activity

Prostaglandin H synthetase (PGH synthetase) has been purified to homogeneity from sheep vesicular glands. The pure enzyme has a specific activity of about 40 microM of arachidonic acid consumed per minute per milligram of protein, which corresponds to a turnover number of 2800 min-1 per subunit. The purified enzyme was obtained by one-stage chromatography on DEAE-Toyopearl 650 from Tween 20-solubilized microsomes. A sensitive fluorometric assay for PGH synthetase activity using homovanillic acid (HVA) as electron donor has been proposed. It has been shown that homovanillic acid may be used as the electron donor and that in the presence of HVA the enzyme has an activity of approximately 40 microM/min/mg.

Cell-cell contacts alter intracellular pH

Intracellular pH, an important regulatory factor for many cellular activities, was shown to be modulated by cell adhesion to the solid substratum. In the present work we have shown that cell-cell contacts also affect intracellular pH. pH(i) depends on how many contacts the cell has established with the substratum and the neighboring cells. pH(i) is low in single cells, not contacting each other. It increased with the increase of cell density. pH(i) is again decreased in confluent (topoinhibited) monolayers. pH(i)-shifts triggered by cell-cell contacts seem to be mediated by Na+/H(+)-antiporter. Dependence of pH(i) on cell density could be simulated by different concentration of Arg-Gly-Asp--which is part of the site of extracellular matrix proteins involved in integrin binding. The dependence of pH(i) on cell-cell contacts is discussed in relation to the phenomena of topoinhibition.

The role of adhesive interactions and extracellular matrix fibronectin from human polymorphonuclear leukocytes in the respiratory burst

The Arg-Gly-Asp (RGD) tripeptide and ajoene were used for studying the role of adhesive receptors in the respiratory burst. Activation of the respiratory burst was examined by using luminol-dependent and lucigenin-dependent chemiluminescence. Recently, it was shown that ajoene, (E, Z)-4,5,9-trithiadodeca-1,6,11-trien-9-oxide, a substance isolated from garlic extract, inhibits the binding of fibrinogen to activated platelets by direct interaction with fibrinogen receptor (Apitz-Castro, R., Lederma, E., Escalante, J. and Jain, M.K. (1986) Biochem. Biophys. Res. Commun. 141, 145-150). Taking into consideration the structural and functional similarity of integrins, it would be reasonable to assume that ajoene as well as RGD can inhibit adhesive interactions of human neutrophils. We have shown that the effect of various activators on the respiratory burst was abolished by ajoene or RGD treatment. The inhibitory effect of RGD and ajoene was dose-dependent. The treatment of neutrophils with antiserum against human plasma fibronectin inhibited the respiratory burst in response to formyl-methionyl-leucylphenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA). This effect is dose-dependent and reversible with the addition of fibronectin. These data indicate that the respiratory burst in human neutrophils is mediated by the integrin family of receptors and that interactions between the extracellular matrix fibronectin and cells are necessary for the respiratory burst.

Respiratory burst inhibition in human neutrophils by ultra-low doses of [D-Ala2]methionine enkephalinamide.

An ultra‐low dose (10−14 M) of opioid peptide [D‐Ala2]methionine enkephalinamide (DAMEA) is found to exert an inhibitory effect on the production of reactive oxygen species (respiratory burst) in human neutrophils. The validity of this phenomenon has been verified in a series of studies that comprised 30 experiments. The inhibition has proved to be statistically significant (P<0.001). The dose‐response dependence of the effect (10−15−10−9 M) followed a characteristic biphasic pattern (with the maximum effect at ultra‐low doses). An opioid antagonist, naloxone partially blocks the inhibitory effect, which indicates that the DAMEA action is at least partially mediated by opioid receptors.

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.

Regulation of intracellular pH by phospholipase A2 and protein kinase C upon neutrophil adhesion to solid substrata

Adhesion to solid substrata has been shown to increase intracellular pH (pH(i)) of fibroblasts and of other cells (FEBS Lett. (1988) 234, 449–450; Proc. Natl. Acad. Sci. USA (1989) 86, 4525–4529; J. Biol. Chem. (1990) 265, 1327–1332; Exp. Cell Res. (1992) 200, 211–214; FEBS Lett. (1995) 374,17–20). We have found that the inhibitors of PLA2, 4‐bromophenacyl bromide and manoalide, completely blocked the increase of pH(i) and spreading of neutrophils upon adhesion to solid substrata. Inhibition of phospholipase C with neomycin or removal of extracellular Ca2+ affects neither neutrophil spreading nor their pH(i). Inhibition of PKC with H‐7 or staurosporin increased pH(i). PMA, an activator of PKC, dramatically decreased pH(i) but did not impair the spreading of neutrophils. The effect of arachidonic acid, a product of PLA2 activity, on neutrophil pH(i) and spreading was similar to that of PMA. H‐7, an inhibitor of PKC, partially blocked the effect of arachidonic acid (AA) on pH(i). BW755C, an inhibitor of AA metabolism by cyclooxygenase or lipoxygenase, affected neither the pH(i) nor cell spreading. We propose that the increase of pH(i) upon neutrophil adhesion is mediated by PLA2 activity, while PKC decreased pH(i). AA produced by PLA2 activates PKC, thus forming a feedback regulation of pH(i).

Regulation of intracellular pH by cell-cell adhesive interactions

Inhibitors of phospholipase A2 (4‐bromophenacyl‐bromide), phospholipase C (neomycin) and protein kinase C (H‐7) dramatically change the way the pHi is modulated by local cell density. It is suggested that cell‐cell interactions regulate cell activities via modulation of pHi, which is under positive control from phospholipase A2 and under negative control from protein kinase C.