scholarly journals The superoxide-generating NADPH oxidase of human neutrophils is electrogenic and associated with an H+ channel

1987 ◽  
Vol 246 (2) ◽  
pp. 325-329 ◽  
Author(s):  
L M Henderson ◽  
J B Chappell ◽  
O T G Jones
Keyword(s):  
Membrane Potential ◽  
Nadph Oxidase ◽  
Human Neutrophils ◽  
Superoxide Generation ◽  
Compensatory Movement ◽  
Diphenylene Iodonium

The membrane potential of cytoplasts, derived from human neutrophils, was depolarized by the activation of the superoxide-generating NADPH-dependent oxidase. The extent of the depolarization was inhibited by diphenylene iodonium and was therefore due directly to the activity of the oxidase, which must be electrogenic. The extent of the depolarization was influenced by alteration of the delta pH across the cytoplast membrane, indicating that the outward translocation of H+ eventually compensates for superoxide generation. The depolarization of the potential is enhanced by Cd2+, a blocker of H+ currents, suggesting that the compensatory movement is via an H+ channel.

scholarly journals A newly synthesized molecule derived from ruthenium cation, with antitumour activity, activates NADPH oxidase in human neutrophils

1997 ◽  
Vol 328 (2) ◽  
pp. 559-564 ◽  
Author(s):  
Modesto CARBALLO ◽  
Rosario VILAPLANA ◽  
Gracia MÁRQUEZ ◽  
Manuel CONDE ◽  
J. Francisco BEDOYA ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Antitumour Activity ◽  
Human Neutrophils ◽  
Phagocytic Cells ◽  
Diphenylene Iodonium ◽  
Ru Complexes ◽  
O2 Production ◽  
Cytosolic Factors ◽  
Oxygen Metabolites ◽  
Antitumour Effects

To determine the nature of the mechanism by which certain derived ruthenium (Ru) complexes induce regression in tumour growth, we have investigated the possibility that this mechanism was associated with an increase of superoxide anion (O2-•) production by phagocytic cells, which are usually found in tumour nodes. Here we present evidence that a newly synthesized complex, Ru3+-propylene-1,2-diaminotetra-acetic acid (Ru-PDTA), derived from Ru and the sequestering ligand (PDTA), specifically stimulates O2-• production. This increase was associated with the translocation of cytosolic factors p47phox and p67phox of NADPH oxidase to the plasma membrane. The Ru-PDTA-complex-dependent O2-• production was abrogated by staurosporine, partially inhibited by diphenylene iodonium, and it was insensitive to pertussis toxin or dibutyryl cyclic AMP pretreatment. An increase of cytosolic Ca2+ levels were also detected in neutrophils treated with the Ru-PDTA complex. Also, Ru-PDTA complex induced the phosphorylation of tyrosine residues of several proteins as assessed by Western blotting. Present data are consistent with the possibility that Ru-PDTA-dependent antitumour effects are due in part to the complex's ability to stimulate the release of toxic oxygen metabolites from phagocytic cells infiltrating tumour masses.

scholarly journals High rates of extracellular superoxide generation by cultured human fibroblasts: involvement of a lipid-metabolizing enzyme

1996 ◽  
Vol 318 (3) ◽  
pp. 805-812 ◽  
Author(s):  
Valerie B. O'DONNELL ◽  
Angelo AZZI
Keyword(s):  
Nadph Oxidase ◽  
Human Fibroblasts ◽  
Extracellular Enzyme ◽  
Competitive Inhibitor ◽  
Superoxide Generation ◽  
Diphenylene Iodonium ◽  
Cultured Human Fibroblasts ◽  
Prostaglandin H ◽  
Metabolizing Enzyme ◽  
Hydroperoxyeicosatetraenoic Acid

Expression of NADPH oxidase and low superoxide generation (approx. 0.06 nmol/min per 106 cells) by cytokine- or ionophore-stimulated human fibroblasts is known. However, we here show that these cells also contain an ectoplasmic enzyme, distinct from NADPH oxidase, which can generate superoxide (2.19±0.14 nmol/min per 106 cells) at levels similar to phorbol ester-stimulated monocytes on exogenous NADH addition. Superoxide generation was temperature-dependent, insensitive to chelation (desferal), and had a Km(app)(NADH) of 11.5 µM. Inhibitor studies showed that there was no involvement of NADPH oxidase (diphenylene iodonium, diphenyl iodonium), prostaglandin H synthase (indomethacin), xanthine oxidase (allopurinol), cytochrome P-450 (metyrapone) or mitochondrial respiration (rotenone, antimycin A). NAD+ was a competitive inhibitor, whereas NADPH supported 40% of the rate seen with NADH. No luminescence was observed after the addition of lactate, malate, pyruvate, GSH or l-cysteine. NADH-stimulated superoxide generation was enhanced by the addition of (3–30 µM) arachidonic acid, linoleic acid or (5S)-hydroxyeicosatetraenoic acid [(5S)-HETE] but not palmitic acid, (15S)-hydroperoxyeicosatetraenoic acid [(15S)-HPETE], (15S)-HETE or (12S)-HETE. Several features suggest involvement of an enzyme related to 15-lipoxygenase, and, in support of this, we show superoxide generation and NADH oxidation by recombinant rabbit reticulocyte 15-lipoxygenase. The large amounts of superoxide measured suggest that the fibroblast extracellular enzyme could be a major source of reactive oxygen species after tissue damage.

scholarly journals Intracellular pH regulation during spreading of human neutrophils.

1996 ◽  
Vol 133 (6) ◽  
pp. 1391-1402 ◽  
Author(s):  
N Demaurex ◽  
G P Downey ◽  
T K Waddell ◽  
S Grinstein
Keyword(s):  
Nadph Oxidase ◽  
Acid Production ◽  
Ph Regulation ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Acid Generation ◽  
Diphenylene Iodonium ◽  
Physiological Values ◽  
Cytosolic Acidification ◽  
Intracellular Acid

The regulation of the intracelluar pH (pHi) during spreading of human neutrophils was studied by a combination of fluorescence imaging and video microscopy. Spreading on adhesive substrates caused a rapid and sustained cytosolic alkalinization. This pHi increase was prevented by the omission of external Na+, suggesting that it results from the activation of Na+/H+ exchange. Spreading-induced alkalinization was also precluded by the compound HOE 694 at concentrations that selectively block the NHE-1 isoform of the Na+H+ antiporter. Inhibition of Na+/H+ exchange by either procedure unmasked a sizable cytosolic acidification upon spreading, indicative of intracellular acid production. The excess acid generation was caused, at least in part, by the activation of the respiratory burst, since the acidification closely correlated with superoxide production, measured in single spreading neutrophils with dihydrorhodamine-123, and little acid production was observed in the presence of diphenylene iodonium, a blocker of the NADPH oxidase. Moreover, neutrophils from chronic granulomatous disease patients, which do not produce superoxide, failed to acidify. Comparable pHi changes were observed when beta 2 integrins were selectively activated during spreading on surfaces coated with anti-CD18 antibodies. When integrin engagement was precluded by pretreatment with soluble anti-CD18 antibody, the pHi changes associated with spreading on fibrinogen were markedly reduced. Inhibition of microfilament assembly with cytochalasin D precluded spreading and concomitantly abolished superoxide production and the associated pHi changes, indicating that cytoskeletal reorganization and/or an increase in the number of adherence receptors engaged are required for the responses. Neutrophils spread normally when the oxidase was blocked or when pHi was clamped near physiological values with nigericin. Spreading, however, was strongly inhibited when pHi was clamped at acidic values. Our results indicate that neutrophils release superoxide upon spreading, generating a burst of intracellular acid production. The concomitant activation of the Na+/H+ antiport not only prevents the deleterious effects of the acid released by the NADPH oxidase, but induces a net cytosolic alkalinization. Since several functions of neutrophils are inhibited at an acidic pHi, the coordinated activation of pHi regulatory mechanisms along with the oxidase is essential for sustained microbicidal activity.

scholarly journals Internal pH changes associated with the activity of NADPH oxidase of human neutrophils. Further evidence for the presence of an H+ conducting channel

1988 ◽  
Vol 251 (2) ◽  
pp. 563-567 ◽  
Author(s):  
L M Henderson ◽  
J B Chappell ◽  
O T G Jones
Keyword(s):  
Plasma Membrane ◽  
Membrane Potential ◽  
Nadph Oxidase ◽  
Initial Rate ◽  
External Medium ◽  
Human Neutrophils ◽  
Conducting Channel ◽  
Proton Conducting ◽  
Ph Changes ◽  
Internal Ph

The internal pH (pHi) of cytoplasts, derived from human neutrophils, falls 0.05 pH units upon activation of the superoxide-generating NADPH oxidase. The decrease in pHi is absent in diphenyleneiodonium-treated cytoplasts and therefore it is likely to arise directly from the activity of the oxidase. The addition of amiloride, to diminish the Na+/H+ exchanger, enhanced the extent of the internal acidification but not the initial rate. However the electroneutral Na+/H+ exchanger cannot be a contributor to H+ efflux to compensate for charge translocated by the oxidase. In the presence of Cd ions or valinomycin, phorbol-induced acidification of the cytosol was greatly increased, suggesting an inability to translocate the cytosolic H+ generated by an electrogenic oxidase. In the presence of both Cd and valinomycin the cytoplasts retained 0.8 H+ per O2-. generated. The rate of acidification of the external medium by stimulated cytoplasts is greatly reduced in the presence of Zn and valinomycin. Our results support the view that the plasma membrane of neutrophils contains Zn2+- or Cd2+-sensitive proton-conducting channels which maintain a stable membrane potential and pHi during the activity of the electrogenic NADPH oxidase.

Dual role of phagocytic NADPH oxidase in bacterial killing

Blood ◽  
2004 ◽  
Vol 104 (9) ◽  
pp. 2947-2953 ◽  
Author(s):  
Balázs K. Rada ◽  
Miklós Geiszt ◽  
Krisztina Káldi ◽  
Csaba Timár ◽  
Erzsébet Ligeti
Keyword(s):  
Membrane Potential ◽  
Nadph Oxidase ◽  
Classical Model ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Membrane Depolarization ◽  
Chemical Effect ◽  
Bacterial Survival ◽  
Phagocytic Cells ◽  
Bacterial Killing ◽  
Diphenylene Iodonium

Abstract The classical model of bacterial killing by phagocytic cells has been recently challenged by questioning the toxic effect of oxygen products and attributing the fundamental role to K+ ions in releasing antimicrobial proteins within the phagosome. In the present study we followed \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\cdot}-}\) \end{document} production, changes of membrane potential, K+ efflux, and bacterial killing in the presence of increasing concentrations of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor diphenylene iodonium. Efficiency of bacterial killing was assessed on the basis of bacterial survival measured by a new semiautomated method. Very low rates of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\cdot}-}\) \end{document} production were accompanied by significant membrane depolarization and K+ release and parallel improvement of bacterial killing. When \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\cdot}-}\) \end{document} production exceeded 20% of its maximal capacity, no further change was detected in the membrane potential and only minimal further K+ efflux occurred, yet bacterial survival decreased parallel to the increase of \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\cdot}-}\) \end{document} production. The presented results indicate that both electrophysiological changes (depolarization and consequent ion movements) and the chemical effect of reactive oxygen species play a significant role in the killing of certain pathogens. The observation that an increase of membrane depolarization can compensate for decreased \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\cdot}-}\) \end{document} production may be important for potential therapeutic applications.

Inhibition of the neutrophil NADPH oxidase and associated H+ channel by diethyl pyrocarbonate (DEPC), a histidine-modifying agent: evidence for at least two target sites

2001 ◽  
Vol 358 (2) ◽  
pp. 315-324 ◽  
Author(s):  
Tosti J. MANKELOW ◽  
Lydia M. HENDERSON
Keyword(s):  
Nadph Oxidase ◽  
Human Neutrophils ◽  
Closed Channel ◽  
Superoxide Generation ◽  
Free System ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate ◽  
Target Sites ◽  
A Cell ◽  
Membrane Components

Diethyl pyrocarbonate (DEPC), a histidine-modifying reagent, has been utilized to demonstrate the importance of histidine residues in the functioning of proteins. In previous studies of the NADPH oxidase, histidine residues have been determined to be important in the ability of gp91phox to function as an H+ pathway and in the binding of haem and FAD. We have investigated the ability of DEPC to inhibit H+ flux and superoxide generation by human neutrophils. Proton flux through the NADPH oxidase-associated H+ channel was inhibited by DEPC only if applied simultaneously with an activator of the channel. This suggested that the site modified by DEPC is not accessible in the closed channel. Superoxide generation by the NADPH oxidase was also inhibited by DEPC when applied after or simultaneously with the activator. Translocation of the NADPH oxidase cytosolic components, p67phox and p47phox, to the membrane was unaffected by DEPC. In a cell-free system, DEPC-treated membranes failed to support superoxide generation or the reduction of Iodonitrotetrazolium Violet and showed a loss of the characteristic cytochrome b558 spectrum. Superoxide generation by DEPC-treated cytosol was inhibited slightly. Therefore it can be concluded that there are two sites within the NADPH oxidase that interact with DEPC, one in the H+ pathway, only accessible in the activated oxidase, and a second accessible prior to activation of the NADPH oxidase. The latter non-proton pathway DEPC site is located within the membrane components of the NADPH oxidase and is associated with the binding of haem in the enzyme complex.

scholarly journals Sex-dependent dysregulation of human neutrophil responses by bisphenol A

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Wioletta Ratajczak-Wrona ◽  
Marzena Garley ◽  
Malgorzata Rusak ◽  
Karolina Nowak ◽  
Jan Czerniecki ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Bisphenol A ◽  
Total Concentration ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Human Neutrophils ◽  
Boyden Chamber ◽  
No Production ◽  
Pathogen Elimination ◽  
Latex Beads

Abstract Background In the present study, we aimed to investigate selected functions of human neutrophils exposed to bisphenol A (BPA) under in vitro conditions. As BPA is classified among xenoestrogens, we compared its action and effects with those of 17β-estradiol (E2). Methods Chemotaxis of neutrophils was examined using the Boyden chamber. Their phagocytosis and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase activity were assessed via Park’s method with latex beads and Park’s test with nitroblue tetrazolium. To assess the total concentration of nitric oxide (NO), the Griess reaction was utilized. Flow cytometry was used to assess the expression of cluster of differentiation (CD) antigens. The formation of neutrophil extracellular traps (NETs) was analyzed using a microscope (IN Cell Analyzer 2200 system). Expression of the investigated proteins was determined using Western blot. Results The analysis of results obtained for both sexes demonstrated that after exposure to BPA, the chemotactic capacity of neutrophils was reduced. In the presence of BPA, the phagocytic activity was found to be elevated in the cells obtained from women and reduced in the cells from men. Following exposure to BPA, the percentage of neutrophils with CD14 and CD284 (TLR4) expression, as well as the percentage of cells forming NETs, was increased in the cells from both sexes. The stimulatory role of BPA and E2 in the activation of NADPH oxidase was observed only in female cells. On the other hand, no influence of E2 on the expression of CD14 and CD284, chemotaxis, phagocytosis, and the amount of NET-positive neutrophils was found for both sexes. The study further showed that BPA intensified NO production and iNOS expression in the cells of both sexes. In addition, intensified expression of all tested PI3K-Akt pathway proteins was observed in male neutrophils. Conclusions The study demonstrated the influence of BPA on neutrophil functions associated with locomotion and pathogen elimination, which in turn may disturb the immune response of these cells in both women and men. Analysis of the obtained data showed that the effect of this xenoestrogen on the human neutrophils was more pronounced than E2.

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