Cyanide-insensitive Oxygen Consumption Research Articles

Aminonaphthoquinone induces oxidative stress inStaphylococcus aureus

The biological activity of 5-amino-8-hydroxy-1,4-naphthoquinone (ANQ) on Staphylococcus aureus was investigated in comparison with the unsubstituted 1,4-naphthoquinone (NQ). Complete inhibition of microbial growth was observed with ANQ and NQ at 50 and 10 microg/mL, respectively. The antibacterial effect of naphthoquinones decreased in the presence of sodium ascorbate, but the superoxide scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid (Tiron) was able to protect S. aureus only from the harmful effect of ANQ. Naphthoquinones blocked oxygen uptake and induced cyanide-insensitive oxygen consumption. When combining rotenone or salicylhydroxamic acid with ANQ or NQ, a slight decrease in respiratory activity was observed. Assays in the presence of naphthoquinones induced an increase of lipid peroxidation in S. aureus, as determined by thiobarbituric acid reactive substances. These results showed that 1,4-naphthoquinones effectively act as electron acceptors and induce an increase in reactive oxygen species that are toxic to S. aureus cells.

Impact of pulmonary arterial endothelial cells on duroquinone redox status

The study objective was to use pulmonary arterial endothelial cells to examine kinetics and mechanisms contributing to the disposition of the quinone 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ) observed during passage through the pulmonary circulation. The approach was to add DQ, durohydroquinone (DQH 2), or DQ with the cell membrane-impermeant oxidizing agent, ferricyanide (Fe(CN) 6 3−), to the cell medium, and to measure the medium concentrations of substrates and products over time. Studies were carried out under control conditions and with dicumarol, to inhibit NAD(P)H:quinone oxidoreductase 1 (NQO1), or cyanide, to inhibit mitochondrial electron transport. In control cells, DQH 2 appears in the extracellular medium of cells incubated with DQ, and DQ appears when the cells are incubated with DQH 2. Dicumarol blocked the appearance of DQH 2 when DQ was added to the cell medium, and cyanide blocked the appearance of DQ when DQH 2 was added to the cell medium, suggesting that the two electron reductase NQO1 dominates DQ reduction and mitochondrial electron transport complex III is the predominant route of DQH 2 oxidation. In the presence of cyanide, the addition of DQ also resulted in an increased rate of appearance of DQH 2 and stimulation of cyanide-insensitive oxygen consumption. As DQH 2 does not autoxidize-comproportionate over the study time course, these observations suggest a cyanide-stimulated one-electron DQ reduction and durosemiquinone (DQ − ) autoxidation. The latter processes are apparently confined to the cell interior, as the cell membrane impermeant oxidant, ferricyanide, did not inhibit the DQ-stimulated cyanide-insensitive oxygen consumption. Thus, regardless of whether DQ is reduced via a one- or two-electron reduction pathway, the net effect in the extracellular medium is the appearance of DQH 2. These endothelial redox functions and their apposition to the vessel lumen are consistent with the pulmonary endothelium being an important site of DQ reduction to DQH 2 observed in the lungs.

Escherichia coli flavohaemoglobin (Hmp) with equistoichiometric FAD and haem contents has a low affinity for dioxygen in the absence or presence of nitric oxide

A purification procedure for flavohaemoglobin Hmp (NO oxygenase) is described that gives high yields of protein with equistoichiometric haem and FAD contents. H(2)O(2) accumulated on NADH oxidation by the purified protein and in cell extracts with elevated Hmp contents. H(2)O(2) probably arose by dismutation from superoxide, which was also detectable during oxygen reduction; water was not a product. In the absence of agents that scavenge superoxide and peroxide, the mean K(m) for oxygen was 80 microM; the addition of 15 microM FAD decreased the K(m) for oxygen to 15 microM without a change in V(max) but catalysed cyanide-insensitive oxygen consumption, attributed to electron transfer from flavins to O(2). Purified Hmp consumed NO in the absence of added FAD (approx. 1 O(2) per NO), which is consistent with NO oxygenation. However, half-maximal rates of NO-stimulated O(2) consumption required approx. 47 microM O(2); NO removal was ineffective at physiologically relevant O(2) concentrations (below approx. 30 microM O(2)). On exhaustion of O(2), NO was removed by a cyanide-sensitive process attributed to NO reduction, with a turnover number approx. 1% of that for oxygenase activity. These results suggest that the ability of Hmp to detoxify NO might be compromised in hypoxic environments.

Escherichia coli flavohaemoglobin (Hmp) with equistoichiometric FAD and haem contents has a low affinity for dioxygen in the absence or presence of nitric oxide

A purification procedure for flavohaemoglobin Hmp (NO oxygenase) is described that gives high yields of protein with equistoichiometric haem and FAD contents. H2O2 accumulated on NADH oxidation by the purified protein and in cell extracts with elevated Hmp contents. H2O2 probably arose by dismutation from superoxide, which was also detectable during oxygen reduction; water was not a product. In the absence of agents that scavenge superoxide and peroxide, the mean Km for oxygen was 80µM; the addition of 15µM FAD decreased the Km for oxygen to 15µM without a change in Vmax but catalysed cyanide-insensitive oxygen consumption, attributed to electron transfer from flavins to O2. Purified Hmp consumed NO in the absence of added FAD (approx. 1 O2 per NO), which is consistent with NO oxygenation. However, half-maximal rates of NO-stimulated O2 consumption required approx. 47µM O2; NO removal was ineffective at physiologically relevant O2 concentrations (below approx. 30µM O2). On exhaustion of O2, NO was removed by a cyanide-sensitive process attributed to NO reduction, with a turnover number approx. 1% of that for oxygenase activity. These results suggest that the ability of Hmp to detoxify NO might be compromised in hypoxic environments.

Respiratory stimulation and generation of superoxide radicals in Pseudomonas aeruginosa by fungal naphthoquinones.

The mechanism of action of antimicrobial naphthoquinones from the fungus Fusarium was studied by using Pseudomonas aeruginosa. Bostricoidin, methyl ether fusarubin, and fusarubin stimulated the oxygen consumption of bacterial cells and induced cyanide-insensitive oxygen consumption. These activities of the tested compounds were also observed in bacterial membrane preparations in a dose-dependent manner. Naphthoquinones stimulated the generation of superoxide anion and hydrogen peroxide. The naphthoquinone effectively acted as the electron acceptors for bacterial diaphorase, which could explain the antibacterial activity of Fusarium naphthoquinones since electron acceptors lead to the stimulation of respiratory activity and the generation of oxygen radical species.

7] Measurement of nitric oxide and hydrogen peroxide production from human neutrophils

Publisher Summary This chapter discusses the measurement of nitric oxide (NO) and hydrogen peroxide production from human neutrophils. Human neutrophils, polymorphonucleocytes (PMNs) exposed to soluble or particulate stimuli largely increase cyanide-insensitive oxygen consumption with the production of reactive oxygen species in a process termed the respiratory burst, which seems responsible for the microbicidal activity of PMNs. Circulating human neutrophils release NO when they are exposed to appropriate stimuli by activation of a constitutive nitric oxide synthase (NOS), while inflammatory neutrophils appear to express an inducible NOS. The NO and superoxide anion (O 2 – ) are able to interact to form peroxynitrite 1° and other reactive species that apparently contribute to host defense against infections. NO production is measured in a double beam-double wavelength 356 Perkin–Elmer spectrophotometer. A direct measurement of released NO can be made with Clark-type nitric oxide selective electrodes. Neutrophils release NO after stimulation either with phorbol esters such as phorbol myristate acetate (PMA) or with formylmethionylleucylphenylalanine (fMLP); typical traces showing the spectrophotometric changes in performing the oxymyoglobin assay. The detection of nitrite (and nitrate) is useful for high cell concentrations such as those in culture.

Effects of growth conditions on mitochondrial morphology in Saccharomyces cerevisiae.

Effects of growth conditions on mitochondrial morphology were studied in living Saccharomyces cerevisiae cells by vital staining with the fluorescent dye dimethyl-aminostyryl-methylpyridinium iodine (DASPMI), fluorescence microscopy, and confocal-scanning laser microscopy. Cells from respiratory, ethanol-grown batch cultures contained a large number of small mitochondria. Conversely, cells from glucose-grown batch cultures, in which metabolism was respiro-fermentative, contained small numbers of large, branched mitochondria. These changes did not significantly affect the fraction of the cellular volume occupied by the mitochondria. Similar differences in mitochondrial morphology were observed in glucose-limited chemostat cultures. In aerobic chemostat cultures, glucose metabolism was strictly respiratory and cells contained a large number of small mitochondria. Anaerobic, fermentative chemostat cultivation resulted in the large, branched mitochondrial structures also seen in glucose-grown batch cultures. Upon aeration of a previously anaerobic chemostat culture, the maximum respiratory capacity increased from 10 to 70 mumole.min-1.g dry weight-1 within 10 h. This transition resulted in drastic changes of mitochondrial number, morphology and, consequently, mitochondrial surface area. These changes continued for several hours after the respiratory capacity had reached its maximum. Cyanide-insensitive oxygen consumption contributed ca. 50% of the total respiratory capacity in anaerobic cultures, but was virtually absent in aerobic cultures. The response of aerobic cultures to oxygen deprivation was qualitatively the reverse of the response of anaerobic cultures to aeration. The results indicate that mitochondrial morphology in S. cerevisiae is closely linked to the metabolic activity of this yeast: conditions that result in repression of respiratory enzymes generally lead to the mitochondrial morphology observed in anaerobically grown, fermenting cells.

Hemocytes release phenoloxidase upon contact reaction, an allogeneic interaction, in the ascidian Halocynthia roretzi.

Contact reaction is the morphological expression of allogeneic recognition by hemocytes in the ascidian, Halocynthia roretzi; namely they undergo an exocytotic burst upon contact with non-self (allogeneic) hemocytes in vitro. We have found that contact reaction is accompanied by a burst of cyanide-insensitive oxygen consumption that, unlike exocytotic events of mammalian phagocytes, is Ca(2+)-dependent and does not culminate in the production of superoxide anions or H2O2. Instead, the burst is due to the release of phenoloxidase from hemocytes as known for the self-defense systems in insects and crustaceans. The activity of phenoloxidase released from hemocytes corresponds well to the degree of contact reaction observed under the microscope. Therefore, it is possible to quantify the contact reaction simply by measuring the activity of phenoloxidase released from hemocytes into the medium.

Enhancement of daunomycin toxicity by the differentiation inducer hexamethylene bisacetamide in erythroleukemia cells

Cytotoxic effects of daunomycin were investigated upon differentiation of Friend erythroleukemia cells induced with hexamethylene bisacetamide, a process during which a 20-fold increase in the hemoglobin content occurred. Daunomycin proved to be more toxic to differentiated Friend cells than to their undifferentiated counterparts. No changes in the daunomycin uptake rates of the two cell types were detectable. Externally added catalase and desferrioxamine mesylate protected against the additional cytotoxicity of daunomycin in differentiated cells, pointing to hydrogen peroxide and iron ions as mediators of the toxic effect. Daunomycin-dependent, cyanide-insensitive oxygen consumption of control and induced cells did not differ significantly, and the rate of formation of the daunomycin semiquinone radical electron paramagnetic resonance signal was similar in both cell types, indicating that the difference in toxicity was not due to increased drug activation by plasma membrane enzymes. Differentiated cells had a lowered catalase content; the cellular iron content was shown to increase by 2.8-fold upon cell differentiation, with hemoglobin-bound iron being about 50% of the total. Altogether, the results suggest increased intracellular hydrogen peroxide generation mediated by hemoglobin, combined with a decrease in catalase activity and an increase in accessible iron, as responsible for the higher sensitivity to daunomycin shown by differentiated Friend cells. This represents the first experimental system where the increase in anthracycline cytotoxicity upon cell differentiation can be attributed to redox activation and the formation of reactive oxygen species.

Role of Xanthine Oxidase in the Potentiation of Doxorubicin-Induced Cardiotoxicity by Mitomycin C

Clinical evidence has suggested that mitomycin C (MMC) potentiates doxorubicin (DOX) induced cardiotoxicity. In this study a mouse model was used to examine the effect of DOX on the ability of cardiac tissue to bioactivate MMC to generate oxygen radicals. Cardiac damage was assessed by measuring serum CPK-MB isoenzyme levels and thiobarbituric acid reactive substances (TBARS) in the cardiac tissue. The exposure of animals to DOX or DOX and MMC over a three week period led to an increase in serum CPK-MB isoenzyme levels as well as TBARS. Treatment with DOX led to an increase in MMC-dependent, NADH-dependent, cyanide insensitive oxygen consumption, compared to control animals, thereby suggesting increased MMC-dependent oxygen radical generation. Levels of xanthine oxidase (XO; EC 1.1.3.22) and NADPH:cytochrome C reductase, two enzymes known to bioactivate MMC with subsequent oxygen radical generation, were measured in cardiac tissue with a 4.5 x increase in XO activity seen in DOX treated animals vs controls and no change in NADPH:cytochrome C reductase activity. Cardiac levels of xanthine dehydrogenase (XDH; EC 1.1.1.204) activity in DOX treated animals decreased while the XO/XDH ratio increased, suggesting a conversion of XDH to XO following DOX treatment.

Multifunctional analysis of the interaction of anthralin and its metabolites anthraquinone and anthralin dimer with the inner mitochondrial membrane

We studied the interaction of the antipsoriatic compound anthralin (1.8-dihydroxy-9-anthrone), and its metabolites anthraquinone (1.8-dihydroxy-9.10-anthraquinone) and anthralin dimer (1.8.1'.8'.-tetrahydroxy-10.10'-bis-9[10]-dianthrone) with the inner mitochondrial membrane. Mitochondrial membrane functions such as ubiquinone redox equilibria, redox status of iron sulfur clusters, cyanide-sensitive and cyanide-insensitive oxygen consumption, adenosine triphosphate (ATP) synthesis, ATP hydrolysis, and adenine nucleotide content of mitochondria were analyzed. Anthralin is an inhibitor of mitochondrial oxygen uptake in the presence of ADP and substrate (cyanide-sensitive respiration), inhibits ATP synthesis without affecting ATP hydrolysis, and depletes mitochondria of ATP. Anthralin dimer is a much weaker inhibitor of mitochondrial functions and anthraquinone is almost inactive. Anthralin, but not anthraquinone and anthralin dimer, reverses uncoupler stimulated oxygen consumption, stimulates cyanide-insensitive respiration, reduces mitochondrial ubiquinone-9 and -10 to the corresponding ubiquinols and reduces mitochondrial iron sulfur clusters. Anthralin may induce formation of reactive oxygen species by enhancing autoxidation of mitochondrial components and/or by catalyzed oxidation of anthralin. Taken together, anthralin acts as an electron donor to inner mitochondrial membrane associated redox components, inhibits the electron transport chain, and has an oligomycin-like effect. Anthralin dimer and anthraquinone do not function as electron donors and act by a different reaction mechanism. Respiratory measurements in human keratinocytes revealed similar results as obtained with isolated mitochondria. We suggest that modulation of membrane redox status may be a common concept of anthralin action in target cells such as keratinocytes and neutrophils.

The Antipsoriatic Compound Anthralin Influences Bioenergetic Parameters and Redox Properties of Energy Transducing Membranes

Bioenergetic parameters and redox properties of energy transducing membranes in rat liver mitochondria and cyanobacteria were investigated in the presence of the antipsoriatic compound anthralin (1,8-dihydroxy-9-anthrone). Transmembrane pH and electrical gradients were determined using electron paramagnetic resonance spectroscopy. In mitochondria, ubiquinones 9,10 and other redox components of the electron transport chain are reduced by anthralin; the proton motive force is increased. In the absence of ADP, anthralin slightly stimulates mitochondrial cyanide-insensitive oxygen consumption. It is suggested that increased cyanide-insensitive respiration is due to enhanced autoxidation of mitochondrial components and/or catalyzed oxidation of anthralin. In the presence of ADP mitochondrial respiration is decreased, and ATP synthesis is inhibited. Uncoupler-induced mitochondrial respiration is also decreased by anthralin, indicating inhibition of the electron transport chain. In the cyanobacterium Synechococcus PCC 6311 anthralin increases the pH gradient and decreases ATP levels. Thus, anthralin acts as an electron donor to membrane associated redox components and inhibits ATP synthesis in two different biologic systems. In human keratinocytes oxygen metabolism is influenced by anthralin in a similar pattern as in isolated mitochondria, and ATP content is decreased. Because anthralin reacts with redox components in different biologic membranes, alterations of subcellular/cellular redox status and energy metabolism might contribute significantly to its antiproliferative activity.

2-Bromohydroquinone-induced toxicity to rabbit renal proximal tubules: Evidence against oxidative stress

2-Bromohydroquinone (BHQ) plays an important role in bromobenzene-induced nephrotoxicity and is a model toxic hydroquinone. Since BHQ has a quinone nucleus and various quinones have been shown to produce cytotoxicity via oxidative stress, the goal of this study was to determine whether BHQ produced cytotoxicity in a suspension of rabbit renal proximal tubules via oxidative stress. t-Butyl hydroperoxide (TBHP), an agent known to produce cytotoxicity via oxidative stress in this preparation, was used as a positive control. BHQ decreased tubular glutathione disulfide content whether glutathione reductase was inhibited or not. Inhibition of glutathione reductase did not result in the potentiation of BHQ-induced mitochondrial dysfunction or cell death. In contrast, TBHP increased tubular glutathione disulfide content. TBHP-induced increases in glutathione disulfide content, mitochondrial dysfunction, and cell death were potentiated when glutathione reductase was inhibited. Unlike TBHP, BHQ did not initiate lipid peroxidation nor was the antioxidant butylated hydroxytoluene protective. However, BHQ and TBHP both increased sodium cyanide-insensitive oxygen consumption. These results suggest that BHQ may undergo “redox cycling,” but BHQ-induced mitochondrial dysfunction and cell death are not due to oxidative stress.

Nitrofurantoin-stimulated superoxide production by channel catfish ( Ictalurus punctatus) hepatic microsomal and soluble fractions

Nitroaromatic compounds, which frequently contaminate the environment, are known to be reduced to corresponding aromatic amines by fish as well as mammals under anaerobic conditions. Although amine products are not generally formed aerobically, “nitroreducase”-mediated redox cycling of nitroaromatics may occur under these conditions, leading to enhanced production of a potentially toxic oxygen species, superoxide (O 2 −). In this study, we have investigated the ability of channel catfish ( Ictalurus punctatus) hepatic microsomal and soluble fractions to stimulate O 2 −) production upon exposure to a model redox cycling nitroaromatic compound, nitrofurantoin (NF). Two assays for O 2 − production, cytochrome c reduction and cyanide-insensitive oxygen consumption, were stimulated by NF exposure to both hepatic fractions. These reactions were partially inhibited by superoxide dismutase (SOD), and by SOD and catalase in the oxygen consumption assay, providing specific evidence for the involvement of O 2 − in the stimulatory effect by NF. Furthermore, results of cofactor requirement and inhibition studies suggest that NF enhancement of O 2 − production was mediated by NADPH-cytochrome P-450 ( c) reductase in the microsomal fraction and xanthine oxidase in the soluble fraction. These findings comprehensively suggest that the in vitro stimulation of O 2 − production by nitroaromatics as indicated in mammals may also occur in fish and, therefore, suggests a similar potential for oxyradical-mediated toxicities in these species.

Iron-induced Ca 2+ release from rat liver mitochondria via H 2O 2 metabolism

The addition of iron(III)-gluconate complex to isolated rat liver mitochondria induced a net efflux of Ca 2+ not inhibitable by ruthenium red, which resulted in enhancement of Ca 2+ cycling and a simultaneous membrane potential drop. Iron caused extensive oxidation of mitochondrial pyridine nucleotides. However, it failed to induce appreciable changes in either the oxidation level of pyridine nucleotides or the membrane potential in mitochondria isolated from rats fed with a selenium-deficient diet. Iron also induced a transient increase in the mitochondria cyanide-insensitive oxygen consumption. This effect was largely prevented by the addition of the hydrogen peroxide scavenger catalase. It was concluded that iron induced the activation of a specific Ca 2+ efflux pathway via the oxidation of pyridine nucleotides due to the hydrogen peroxide metabolism by the glutathione enzyme system.

Hypertonic glycerol induces a respiratory burst in leukocytes

Exposure to hypertonic glycerl induced cyanide-insensitive oxygen consumption and formation of superoxide anion (O 2 −) in leukocytes such as porcine blood polymorphonuclear leukocytes, guinea pig peritoneal leukocytes and guinea pig alveolar macrophages. Generation of O 2 − occurred after a short lag time, remained maximal for a certain and then stopped. Its termination was not due to cell damage, since cells exposed to glycerol did not release cytosolic enzymes such as lactate dehydrogenase and exhibited a subsequent respiratory burst upon addition of other stimulators such as myristic acid and phorbol myristate acetate. The period of O 2 − generation increased linearly as a function of the glycerol concentrations; cells exposed to 20% (v/v) glycerol produced O 2 − for 10 min. The maximal velocity of O 2 − generation also increased with the concentration of glycerol, reaching a plateau at 10% glycerol. Membrane vesicles isolated from the cells exposed to 20% glycerol showed high activity of NADPH-dependent O 2 − generation as compared to those of unexposed cells. Activation of leukocytes by glycerol was not accompanied by degranulation, unlike stimulation by phagocytosis. A marked change in shape of the cell membrane of glycerol-treated cells was observed by light and scanning electron microscopies.

Oxidative metabolism associated with phagocytosis in Acanthamoeba castellanii.

When amebae were incubated with latex beads, cyanide-insensitive oxygen consumption increased nearly two-fold. This cyanide-insensitive respiration was inhibited by salicylhydroxamate. Furthermore, cell fractionation studies revealed a localization for a portion of the NAD(P)H oxidase activity in phagolysosomes. The presence of low concentrations of divalent metal during fractionation resulted in an increased yield of oxidative activity in the phagolysosome fraction. In addition, the phagolysosome membrane was enriched about two-fold in a b-type cytochrome. These results show that oxidative metabolism in amebae has some striking similarities to the respiratory burst oxidase of neutrophils.

Ca2+ and Mg2+-enhanced reduction of arsenazo III to its anion free radical metabolite and generation of superoxide anion by an outer mitochondrial membrane azoreductase.

At the concentrations usually employed as a Ca2+ indicator, arsenazo III underwent a one-electron reduction by rat liver mitochondria to produce an azo anion radical as demonstrated by electron-spin resonance spectroscopy. Either NADH or NADPH could serve as a source of reducing equivalents for the production of this free radical by intact rat liver mitochondria. Under aerobic conditions, addition of arsenazo III to rat liver mitochondria produced an increase in electron flow from NAD(P)H to molecular oxygen, generating superoxide anion. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H azoreductase reaction unless the mitochondria were solubilized by detergent or anaerobiosis. In addition, NAD(P)H azoreductase activity was higher in the crude outer mitochondrial membrane fraction than in mitoplasts and intact mitochondria. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated cyanide-insensitive oxygen consumption were enhanced by calcium and magnesium, suggesting that, in addition to an enhanced azo anion radical-stabilization by complexation with the metal ions, enhanced reduction of arsenazo III also occurred. Accordingly, addition of cations to crude outer mitochondrial membrane preparations increased arsenazo III-stimulated cyanide-insensitive O2 consumption, H2O2 formation, and NAD(P)H oxidation. Antipyrylazo III was much less effective than arsenazo III in increasing superoxide anion formation by rat liver mitochondria and gave a much weaker electron spin resonance spectrum of an azo anion radical. These results provide direct evidence of an azoreductase activity associated with the outer mitochondrial membrane and of a stimulation of arsenazo III reduction by cations.

Superoxide enhances photobleaching during cellular immune attack against fluorescent lipid monolayer membranes

Lipid hapten-containing monolayer membranes with bound, anti-hapten antibody molecules serve as model immunological target membranes. Targets with bound-IgG trigger guinea pig macrophages to (a) adhere, (b) spread, (c) release lysosomal enzymes, and (d) increase cyanide-insensitive oxygen consumption. When the target membranes are derivatized with fluorescein, there is a 2–3-fold enhancement in the rate of fluorescein photobleaching in regions of cell-monolayer contact. This effect is due to release of O 2 − from macrophages, as shown by inhibition with superoxide dismutase and by the fact that enhanced photobleaching is not observed with cells of the RAW264 macrophage line, which undergo responses (a)–(d), but do not release O 2 − extracellularly. The O 2 −-dependent photobleaching reaction appears to be relatively specific for fluorescein, as it did not occur with two other fluorophores, 4-nitrobenz-2-oxa-1,3-diazole and tetramethylrhodamine. Because stimulated neutrophils release large quantities of O 2 −, the photobleaching of fluorescein-labeled target membranes in response to neutrophils was examined. Monolayer membranes with specifically bound IgG caused neutrophils to adhere and become markedly motile during incubation at 37°C. Like macrophages, neutrophils induced O 2 −-dependent photobleaching of fluorescein-labeled IgG in regions of cell-monolayer contact. In addition, neutrophils gave rise to a slower, nonphotochemical loss of fluorescence in the same contact regions. The latter effect is apparently due to cleavage of target-bound fluorescent IgG by proteolytic enzymes secreted by the neutrophils in response to the target surface.

Opsonized‐zymosan induces a respiratory burst in human blood platelets

Opsonized-zymosan-stimulated polymorphonuclear cells show a cyanide-insensitive oxygen consumption. We have investigated whether opsonized-zymosan could induce similar metabolic change in human blood platelets. Preparation of intact human blood platelets, obtained by separation through a Ficoll layer (23% w/v) were challenged with opsonized-zymosan. The polymorphonuclear cell contamination was less than 1/10(8) platelets. The opsonized-zymosan-stimulated platelets showed an increase of oxygen consumption. The mean of oxygen burst measured by a polarographic method with a Clark electrode was 11 nmole/10(9) platelets/min (S.E.M. 4; n = 15). The duration of the burst was 2 min. Unstimulated platelets did not show the oxygen burst. The inhibitors of respiratory chain and prostaglandin synthesis completely abolished the oxygen consumption by opsonized-zymosan-stimulated platelets. The simultaneous addition of NADH (1 mM) and opsonized-zymosan induced a burst of oxygen consumption, which occurred after a variable lag phase (10-12 min) from the stimulation, also in the presence of inhibitors. This burst, which lasted about 1 min, amounted to 10 nmole/10(9) platelets/min (S.E.M. 2; n = 15) and it was higher in the presence of NAN3, a catalase inhibitor. Zymosan treated with hydrazine or heated plasma (56 degrees C) did not cause increased oxygen consumption. Inulin or inulin-treated serum did not stimulate platelets. In these experimental conditions some NADH disappeared, as shown by isotachophoresis. The results demonstrated that an immunological stimulus may activate a membrane-linked cyanide-insensitive oxygen metabolizing system.