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C C Winterbourn

Publications and source records attributed to C C Winterbourn.

At least 127 records · Page 7Linked to original sources
PubMed1988

Superoxide modulates the activity of myeloperoxidase and optimizes the production of hypochlorous acid.

Myeloperoxidase catalyses the conversion of H2O2 and Cl- to hypochlorous acid (HOCl). It also reacts with O2- to form the oxy adduct (compound III). To determine how O2- affects the formation of HOCl, chlorination of monochlorodimedon by myeloperoxidase was investigated using xanthine oxidase and hypoxanthine as a source of O2- and H2O2. Myeloperoxidase was mostly converted to compound III, and H2O2 was essential for chlorination. At pH 5.4, superoxide dismutase (SOD) enhanced chlorination and prevented formation of compound III. However, at pH 7.8, SOD inhibited chlorination and promoted formation of the ferrous peroxide adduct (compound II) instead of compound III. We present spectral evidence for a direct reaction between compound III and H2O2 to form compound II, and for the reduction of compound II by O2- to regenerate native myeloperoxidase. These reactions enable compound III and compound II to participate in the chlorination reaction. Myeloperoxidase catalytically inhibited O2- -dependent reduction of Nitro Blue Tetrazolium. This inhibition is explained by myeloperoxidase undergoing a cycle of reactions with O2-, H2O2 and O2-, with compounds III and II as intermediates, i.e., by myeloperoxidase acting as a combined SOD/catalase enzyme. By preventing the accumulation of inactive compound II, O2- enhances the activity of myeloperoxidase. We propose that, under physiological conditions, this optimizes the production of HOCl and may potentiate oxidant damage by stimulated neutrophils.

Cyclohexanones↗
PubMed1988

Susceptibilities of lactoferrin and transferrin to myeloperoxidase-dependent loss of iron-binding capacity.

Apolactoferrin and apotransferrin lost their ability to subsequently bind iron when exposed to an excess of either HOCl or myeloperoxidase plus H2O2 and Cl-. Apolactoferrin, however, was more resistant than apotransferrin. By oxidizing a mixture of the two proteins, then separating them by immunoprecipitation, the difference in susceptibility was shown to be due to the greater reactivity of transferrin iron-binding groups, rather than protective groups on the lactoferrin molecule. The iron-saturated proteins were much more resistant to oxidative modification than the apoproteins. The greater resistance of apolactoferrin should be advantageous for maintaining its iron binding capacity when co-released with myeloperoxidase and reactive oxygen species from stimulated neutrophils.

Apoproteins↗
PubMed1988

Activation of human neutrophil gelatinase by endogenous serine proteinases.

The role of serine proteinases and oxidants in the activation of gelatinase released from human neutrophils was investigated. Gelatinase was measured by its ability to degrade both gelatin and native glomerular basement-membrane type IV collagen. When fMet-Leu-Phe or phorbol 12-myristate 13-acetate was used to stimulate the neutrophils, no gelatinase activity was measured in the absence of a mercurial activator, indicating that the enzyme was released entirely in latent form. However, when fMet-Leu-Phe-stimulated cells were treated with cytochalasin B, 50-70% of the maximal gelatinase activity was released. Activation was blocked by the serine-proteinase inhibitor phenylmethanesulphonyl fluoride and a specific inhibitor of neutrophil elastase, but was not affected by an inhibitor of cathepsin G. Addition of catalase or azide to prevent oxidative reactions did not affect activation of gelatinase under any conditions of stimulation, indicating that oxidants were not involved in activation. Our results imply that oxidative activation of gelatinase does not occur readily. However, neutrophil serine proteinases, particularly elastase, provide an alternative and apparently more efficient mechanism of activation.

Basement Membrane↗
PubMed1988

Microsomal reduction of low-molecular-weight Fe3+ chelates and ferritin: enhancement by adriamycin, paraquat, menadione, and anthraquinone 2-sulfonate and inhibition by oxygen.

Reduction of iron is important in promoting xenobiotic-enhanced, microsomal lipid peroxidation, yet there is little evidence that Fe3+ chelates that promote lipid peroxidation can be reduced by the microsomal system. We have shown that rat liver microsomes catalyse NADPH-dependent reduction of Fe3+ without chelator, as well as Fe3+(ADP), Fe3+(ATP), Fe3+(citrate), Fe3+(EDTA), and ferrioxamine in N2. The NADPH oxidation that accompanied Fe3+ reduction was inhibited by CO for all chelates, except Fe3+ (EDTA). This implies that, except for Fe3+ (EDTA), cytochrome P450 was involved in reduction of the complexes. Adriamycin, paraquat, and anthraquinone 2-sulfonate (AQS) enhanced reduction of all the Fe3+ chelates, whereas menadione enhanced reduction only of Fe3+(ADP) and Fe3+(citrate). All the compounds enhanced oxidation of NADPH in the presence or absence of iron. This was not inhibited by CO, and the results are compatible with Fe3+ reduction occurring via the xenobiotic radicals produced by cytochrome P450 reductase. Microsomal reduction of the xenobiotics, except menadione, enabled the reduction and release of iron from ferritin. Fe3+ chelate reduction, both with and without xenobiotic, was inhibited by O2, although it still proceeded in air at 10-20% of the rate in N2. Iron-dependent lipid peroxidation was promoted by ADP and ATP, inhibited 50% by citrate, and completely inhibited by EDTA and desferrioxamine. Of the xenobiotics, only Adriamycin enhanced microsomal lipid peroxidation. These results indicate that the effects of chelators and xenobiotics on Fe3+ reduction do not correlate with lipid peroxidation and, although reduction is necessary, there must be other factors involved.

Anaerobiosis↗
PubMed1988

Gelatinase contributes to the degradation of glomerular basement membrane collagen by human neutrophils.

Neutrophils contain a number of proteinases active at neutral pH which are able to degrade extracellular matrices. We have determined the contribution of the major neutral proteinases to human neutrophil-mediated degradation of glomerular basement membrane type IV collagen in an in vitro model of immune complex-induced injury. Studies with proteinase inhibitors showed that with intact neutrophils stimulated by immune complexes trapped within the basement membrane, approximately 70% of the degradation was due to serine proteinases and 30% to metalloproteinases. Identical results were obtained with cell-free medium containing neutrophil granule contents. Elastase accounted for almost all the digestion by serine proteinases with a minimal contribution by cathepsin G. All the metalloproteinase activity was due to gelatinase rather than collagenase, and purified gelatinase was also shown to degrade basement membrane collagen. Hence, gelatinase has activity against type IV collagen and may be able to degrade collagens not cleaved by specific collagenases.

Basement Membrane↗
PubMed1988

Cleavage and inactivation of alpha 1-antitrypsin by metalloproteinases released from neutrophils.

Human neutrophils, when stimulated with phorbol myristate acetate or fMet-Leu-Phe in the presence or absence of cytochalasin B, released metalloproteinases that catalytically inactivated the plasma serine proteinase inhibitor, alpha 1-antitrypsin. Inactivation, measured as loss of elastase inhibitory capacity, was accompanied by cleavage of a Mr 4,000 peptide from the COOH-terminus. Cleavage of alpha 1-antitrypsin by cell supernatants was inhibited by EDTA, o-phenanthroline, and DTT, but not by inhibitors of serine or thiol proteinases. Gelatinase and collagenase were separated from the medium of stimulated neutrophils. Both preparations cleaved and inactivated alpha 1-antitrypsin, with cleavage occurring close to the reactive center, at the Phe-Leu bond between positions P7 and P6. Cleavage by purified gelatinase was very slow and could account for only a minor fraction of the activity of neutrophil supernatants. The collagenase preparation was more active. However, the unusual cleavage site, and the ability of fMet-Leu-Phe-stimulated neutrophils to cleave alpha 1-antitrypsin without releasing collagenase, suggests that collagenase is not responsible for cleavage by the cells, which, by implication, is due to an as yet uncharacterized metalloenzyme. Our results demonstrate that by releasing metalloproteinases, neutrophils could proteolytically inactivate alpha 1-antitrypsin at sites of inflammation. This provides an alternative to the previously documented mechanism of inactivation by neutrophil-derived oxidants.

Chemical Fractionation↗
PubMed1987

Inhibition of neutrophil degranulation and superoxide production by sulfasalazine. Comparison with 5-aminosalicylic acid, sulfapyridine and olsalazine.

Sulfasalazine is a potent inhibitor of superoxide production and granule enzyme release by stimulated neutrophils, and modulation of these responses may contribute to its anti-inflammatory properties. It is a composite drug consisting of 5-aminosalicylic acid and sulfapyridine joined through an azo linkage. To investigate which functional groups on the molecule are active against neutrophil responses, 5-aminosalicylic acid, sulfapyridine and olsalazine were added to cells stimulated with fMet-Leu-Phe or immune complexes. The inhibitory effects of sulfasalazine on superoxide production, degranulation and neutrophil-mediated collagen degradation were closely mimicked by olsalazine, with the other two compounds having little effect on either function. Thus the azo link appears to be the important structural feature of sulfasalazine that affects neutrophil responses. This suggests that sulfasalazine could be anti-inflammatory in its own right rather than just acting as a source of 5-aminosalicylic acid. Our findings are also a favourable indication for olsalazine (Dipentum), which is currently under trial as an anti-inflammatory agent.

Aminosalicylic Acids↗
PubMed1987

Radical driven Fenton reactions--evidence from paraquat radical studies for production of tetravalent iron in the presence and absence of ethylenediaminetetraacetic acid.

Micromolar concentrations of nonchelated ferrous sulfate catalyze a reaction between H2O2 and radiolytically generated paraquat radicals, causing the concurrent oxidation of deoxyribose to thiobarbituric acid reactive products. The oxidation yield per paraquat radical increases with increasing concentration of deoxyribose, and decreases as the instantaneous or steady-state concentration of paraquat radicals is increased, thus explaining previous anomalies in which oxidation was not observed at high paraquat radical concentrations. The process is not mediated by OH. (which gives different products) but is attributed to an oxidizing intermediate resulting from the two electron oxidation of Fe2+ to a peroxo complex, or a derivative of tetravalent iron. Similar but less pronounced concentration dependences occur in the corresponding oxidation of formate or of deoxyribose catalyzed by Fe(EDTA), where at pH 7.3 90% of the pathway is attributed to one electron oxidation of the Fe2+(EDTA) by H2O2, producing OH., while two electron oxidation accounts for the remaining 10%.

Chemical Phenomena↗
PubMed1987

Inhibition by nonsteroidal anti-inflammatory drugs of superoxide production and granule enzyme release by polymorphonuclear leukocytes stimulated with immune complexes or formyl-methionyl-leucyl-phenylalanine.

The effects of nonsteroidal anti-inflammatory agents on superoxide production and granule enzyme release by human polymorphonuclear leukocytes stimulated with either formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe] or immune complexes were investigated. Cytochrome c reduction and the release of lysozyme, beta-glucuronidase, myeloperoxidase and gelatinase were measured. Auranofin, phenylbutazone, sulfasalazine and the phospholipase A2 inhibitor, 4-bromophenacyl bromide, strongly inhibited these responses in fMet-Leu-Phe stimulated cells, at concentrations below 50 microM. Indomethacin, piroxicam, mefenamic acid, primaquine and quinacrine at 50-250 microM were inhibitory. Up to 1 mM ibuprofen and chloroquine inhibited superoxide production but had little effect on degranulation. With cells stimulated by IgG aggregates (immune complexes), up to 1 mM ibuprofen, mefenamic acid and piroxicam did not inhibit either response. Indomethacin, phenylbutazone, sulfasalazine and primaquine inhibited, but considerably higher concentrations were required than with fMet-Leu-Phe. Quinacrine inhibited superoxide production equally well with both stimuli but inhibited enzyme release only with fMet-Leu-Phe. Only auranofin, 4-bromophenacyl bromide, and the weakly effective chloroquine exerted approximately the same effect with both stimuli. D-Penicillamine did not affect enzyme release with either stimulus and interfered in the superoxide assay. Gelatinase release induced by fMet-Leu-Phe was affected to the same extent, or slightly more, than release of the other granule enzymes. With immune complexes, there was only modest inhibition of gelatinase release by any of the drugs at 250-1000 microM. Our results reinforce previous observations that many anti-inflammatory drugs affect neutrophil functions, but their effects vary with stimulus. The relative insensitivity of immune complex-induced responses to most of the drugs must be taken into account when considering their mode of action.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1987

Myeloperoxidase-dependent oxidative inactivation of neutrophil neutral proteinases and microbicidal enzymes.

The susceptibility of a number of human neutrophil granule enzymes to oxidative inactivation was investigated. Addition of H2O2 to the cell-free medium from stimulated neutrophils resulted in inactivation of all enzymes tested. This was inhibited by azide and methionine, indicating that inactivation was due to myeloperoxidase-derived oxidants. Lysozyme was more than 50% inactivated by one addition of 100 nmol of H2O2/ml, whereas myeloperoxidase, beta-glucuronidase, gelatinase and collagenase were almost completely inactivated by three additions. Cathepsin G was slightly less susceptible, whereas elastase was extremely resistant to oxidative attack. Myeloperoxidase-dependent enzyme inactivation may be a means whereby the neutrophil can terminate the activity of its granule enzymes and control the release of degradative enzymes into the tissues.

Glycoside Hydrolases↗
PubMed1987

Iron binding to microsomes and liposomes in relation to lipid peroxidation.

The effects of ADP, ATP, citrate and EDTA on iron-dependent microsomal and liposomal lipid peroxidation, and on 59FeCl3 binding to the lipid membranes were measured. The aim was to test if initiation of lipid peroxidation is a site-specific mechanism requiring bound iron. In the absence of chelator, iron was bound to both membranes. EDTA and citrate removed the iron and inhibited peroxidation. ATP and ADP stimulated peroxidation, but whereas ADP allowed only half of the iron to remain bound, all was removed by ATP. Chelators, therefore, cannot be simply influencing a site-specific mechanism. Their effects must relate to the reactivities of the different iron chelates as initiators of lipid peroxidation.

Adenosine Diphosphate↗
PubMed1987

Inhibition of neutrophil-mediated degradation of isolated basement membrane collagen by nonsteroidal antiinflammatory drugs that inhibit degranulation.

The ability of nonsteroidal antiinflammatory drugs to inhibit neutrophil-mediated degradation of type IV collagen in an in vitro tissue injury model using glomerular basement membrane (GBM) containing immune complexes was investigated. Auranofin (2.5-10 microM), phenylbutazone (50-250 microM), sulfasalazine (250-1,000 microM), and 4-bromophenacyl bromide (5-20 microM) each inhibited up to 70% of the collagen degradation, in parallel with almost complete inhibition of the release of azurophil and specific granule enzymes. These drugs had much less an effect on gelatinase release. Indomethacin and the antimalarials, which inhibited the neutrophil oxidative burst but not degranulation, had little effect on GBM collagen degradation. Our results do not necessarily imply that inhibition of neutrophil-mediated degradation of connective tissue is relevant to the action of nonsteroidal antiinflammatory drugs in vivo; however, using the GBM model system, we have shown that when a drug inhibits granule enzyme release, there is an associated decrease in collagen degradation, whereas inhibition of the oxidative burst has relatively little effect.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed1987

Radical-driven Fenton reactions: studies with paraquat, adriamycin, and anthraquinone 6-sulfonate and citrate, ATP, ADP, and pyrophosphate iron chelates.

Using paraquat, adriamycin, and anthraquinone 6-sulfonate, we have investigated the ability of radical-driven Fenton reactions to oxidize formate or deoxyribose when catalyzed by iron complexed with citrate, ADP, ATP, or pyrophosphate. Radicals were generated either radiolytically or enzymatically with xanthine oxidase or ferredoxin reductase. With each radical source, the citrate, ADP, and ATP complexes were at least 50% as active as Fe(EDTA) at catalyzing deoxyribose oxidation, and slightly less active as catalysts of CO2 formation from formate. Fe(pyrophosphate) was less efficient and in some cases inactive. Although it is not possible to definitively identify the oxidant involved, it behaved more like the hydroxyl radical than the proposed ferryl or peroxoferrous species formed in equivalent reactions catalyzed by nonchelated iron, which can oxidize deoxyribose but not formate. Chelator concentrations of 1-2 mM were required for maximum effect, which implies that the major effect of the chelators is on the reactivity of Fe2+ in the Fenton reaction with H2O2. This also suggests that any iron available physiologically could participate in the Fenton reaction in a nonchelated form, and produce a ferryl species rather than the hydroxyl radical. Reactions of the organic radicals contrast with the equivalent reactions of superoxide (Haber-Weiss reaction) for which the same iron chelates are all very inefficient catalysts. Fenton reactions driven by organic reducing radicals may therefore contribute more to the toxicity of redox cycling compounds than equivalent reactions of superoxide.

Adenine Nucleotides↗
PubMed1987

The ability of scavengers to distinguish OH. production in the iron-catalyzed Haber-Weiss reaction: comparison of four assays for OH.

Kinetic analysis has been used to access how well scavenger inhibition can characterize the reactivity of oxidants produced in the iron-catalyzed reaction of H2O2 with xanthine oxidase-derived O2-.. Formate oxidation to CO2, deoxyribose oxidation, benzoate hydroxylation, and ethylene production from alpha-keto-gamma-methiolbutyric acid (KMB) were measured. With Fe(EDTA) as catalyst, inhibition by most scavengers was quantitatively as expected for OH. involvement. Exceptions were urate and thiourea, which inhibited excessively and appeared to scavenge intermediates of the detection reactions. With nonchelated iron, there was minimal formate oxidation, but benzoate, KMB, and deoxyribose gave, respectively, 17%, 25%, and approximately the same product yield as with Fe(EDTA). Deoxyribose oxidation was not inhibited by some scavengers and excessively inhibited by others. However, scavengers that did not inhibit deoxyribose oxidation did inhibit with KMB and benzoate, and differences in scavenger effects in the presence and absence of EDTA in these assays were relatively minor. The results with formate and deoxyribose, but not KMB and benzoate, can therefore exclude free OH. as a significant oxidant product of the nonchelated iron-catalyzed Haber-Weiss reaction. It is proposed that the different patterns of scavenger inhibition arise in the different assays because scavengers can react with intermediates in the detection reactions, all of which are multistep chains. Thus, inhibition may not signify OH. involvement, and similarities with inhibition expected for OH. my be fortuitous.

Benzoates↗
PubMed1987

Human red cells scavenge extracellular hydrogen peroxide and inhibit formation of hypochlorous acid and hydroxyl radical.

The ability of intact human red cells to scavenge extracellularly generated H2O2 and O2-, and to prevent formation of hydroxyl radicals and hypochlorous acid has been examined. Red cells inhibited oxidation of ferrocytochrome c by H2O2. Cells treated with aminotriazole no longer inhibited, indicating that protection was almost entirely due to intracellular catalase. Contribution by the GSH system was slight, and apparent only with low H2O2 concentrations when catalase was inhibited by aminotriazole. The cells were about a quarter as efficient at inhibiting cytochrome c oxidation as an equivalent concentration of purified catalase. No inhibition of O2(-)-dependent reduction of ferricytochrome c or nitroblue tetrazolium was observed, although extracted red cell superoxide dismutase inhibited nitroblue tetrazolium reduction at one fortieth the concentration of that in the cells. Red cells efficiently inhibited deoxyribose oxidation by hydroxyl radicals generated from H2O2, O2- and Fe(EDTA), and myeloperoxidase-dependent oxidation of methionine to methionine sulfoxide by stimulated neutrophils. Most of the red cell inhibition of hydroxyl radical production, and all the inhibition of methionine oxidation, was prevented by blocking intracellular catalase with aminotriazole. Thus red cells are able to efficiently scavenge H2O2, but not O2-, produced in their environment, and to inhibit formation of hydroxyl radicals and hypochlorous acid. They may therefore have an important role in extracellular antioxidant defense.

Amitrole↗