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.

Membrane depolarization and NADPH oxidase activation in aortic endothelium during ischemia reflect altered mechanotransduction

2005 ◽  
Vol 288 (1) ◽  
pp. H336-H343 ◽  
Author(s):  
Ikuo Matsuzaki ◽  
Shampa Chatterjee ◽  
Kris DeBolt ◽  
Yefim Manevich ◽  
Qunwei Zhang ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Membrane Potential ◽  
Nadph Oxidase ◽  
Membrane Depolarization ◽  
Ros Generation ◽  
Similar Response ◽  
Aortic Endothelial Cells ◽  
Aortic Endothelium ◽  
Aortic Endothelial

We previously showed that “ischemia” (abrupt cessation of flow) leads to rapid membrane depolarization and increased generation of reactive oxygen species (ROS) in lung microvascular endothelial cells. This response is not associated with anoxia but, rather, reflects loss of normal shear stress. This study evaluated whether a similar response occurs in aortic endothelium. Plasma membrane potential and production of ROS were determined by fluorescence microscopy and cytochrome c reduction in flow-adapted rat or mouse aorta or monolayer cultures of rat aortic endothelial cells. Within 30 s after flow cessation, endothelial cells that had been flow adapted showed plasma membrane depolarization that was inhibited by pretreatment with cromakalim, an ATP-sensitive K+ (KATP) channel agonist. Flow cessation also led to ROS generation, which was inhibited by cromakalim and the flavoprotein inhibitor diphenyleneiodonium. Aortic endothelium from mice with “knockout” of the KATP channel (KIR6.2) showed a markedly attenuated change in membrane potential and ROS generation with flow cessation. In aortic endothelium from mice with knockout of NADPH oxidase (gp91phox), membrane depolarization was similar to that in wild-type mice but ROS generation was absent. Thus rat and mouse aortic endothelial cells respond to abrupt flow cessation by KATP channel-mediated membrane depolarization followed by NADPH oxidase-mediated ROS generation, possibly representing a cell-signaling response to altered mechanotransduction.

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 Relationship between phosphorylation and translocation to the plasma membrane of p47phox and p67phox and activation of the NADPH oxidase in normal and Ca2+-depleted human neutrophils

1993 ◽  
Vol 290 (1) ◽  
pp. 173-178 ◽  
Author(s):  
S Dusi ◽  
V Della Bianca ◽  
M Grzeskowiak ◽  
F Rossi
Keyword(s):  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Phorbol Esters ◽  
Second Messengers ◽  
Human Neutrophils ◽  
Phosphorylated Proteins ◽  
Kinase C ◽  
Activated State ◽  
Phagocyte Oxidase ◽  
Hydrolysis Of

Stimulation of neutrophils with different agonists activates a latent multicomponent NADPH oxidase that reduces molecular oxygen to superoxide anion. Evidence has accumulated that phosphorylation of p47phox (the 47 kDa cytosolic phagocyte oxidase factor) and translocation of the two cytosolic components p47phox and p67phox are essential steps in the activation of NADPH oxidase in response to phorbol esters. We analysed the relationships between activation of the NADPH oxidase and phosphorylation and translocation of p47phox and p67phox in normal and Ca(2+)-depleted neutrophils stimulated by the receptor-mediated agonists formyl-methionyl-leucyl-phenylalanine and concanavalin A. The results produced the following conclusions: (1) Translocation of p47phox and p67phox is an essential mechanism for activation of the NADPH oxidase. (2) A continuous translocation of p47phox and p67phox is necessary to maintain the NADPH oxidase in an activated state. (3) Only a fraction of p47phox and p67phox translocated to the plasma membrane is functional for the activation of the oxidase. (4) Translocation is independent of protein kinase C, and is linked to transmembrane signalling involving Ca2+ transients and production of lipidic second messengers. However, under some conditions, such as in Ca(2+)-depleted neutrophils, translocation can also occur independently of signalling pathways involving production of second messengers from hydrolysis of phospholipids and Ca2+ transients. (5) Phosphorylation of p47phox and p67phox can be quantitatively dissociated from translocation, as staurosporine markedly inhibits phosphorylation but not translocation. (6) The activity of NADPH oxidase is not correlated with the amounts of the phosphorylated proteins present in the plasma membrane.

scholarly journals In vitro and in vivo modulation of NADPH-oxidase activity and reactive oxygen species production in human neutrophils by alpha-1 antitrypsin

2021 ◽  
pp. 00234-2021
Author(s):  
Padraig Hawkins ◽  
Thomas McEnery ◽  
Claudie Gabillard-Lefort ◽  
David A Bergin ◽  
Bader Alfawaz ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Ex Vivo ◽  
Human Neutrophils ◽  
Driving Mechanism ◽  
Increased Risk ◽  
Alpha 1 Antitrypsin

Oxidative stress from innate immune cells is a driving mechanism that underlies COPD pathogenesis. Individuals with alpha-1 antitrypsin (AAT) deficiency (AATD) have a dramatically increased risk of developing COPD. To understand this further, the aim of this study was to investigate whether AATD presents with altered neutrophil NADPH-oxidase activation, due to the specific lack of plasma AAT. Experiments were performed using circulating neutrophils isolated from healthy controls and individuals with AATD. Superoxide anion (O2−) production was determined from the rate of reduction of cytochrome c. Quantification of membrane NADPH-oxidase subunits was performed by mass spectrometry and western blot analysis. The clinical significance of our in vitro findings were assessed in patients with AATD and severe COPD receiving intravenous AAT replacement therapy. In vitro, AAT significantly inhibited O2− production by stimulated neutrophils and suppressed receptor stimulation of cyclic adenosimonophosphate (cAMP) and extracellular-signal regulated kinase (ERK)1/2 phosphorylation. In addition, AAT reduced plasma membrane translocation of cytosolic phox components of the NADPH-oxidase. Ex vivo, AATD neutrophils demonstrated increased plasma membrane associated p67phox and p47phox and significantly increased O2− production. The described variance in phox protein membrane assembly was resolved post AAT augmentation therapy in vivo, the effects of which significantly reduced AATD neutrophil O2− production to that of healthy control cells. These results expand our knowledge on the mechanism of neutrophil driven airways disease associated with AATD. Therapeutic AAT augmentation modified neutrophil NADPH-oxidase assembly and ROS production, with implications for clinical use in conditions in which oxidative stress plays a pathogenic role.

scholarly journals Membrane Dynamics and Organization of the Phagocyte NADPH Oxidase in PLB-985 Cells

2020 ◽  
Vol 8 ◽  
Author(s):  
Jérémy Joly ◽  
Elodie Hudik ◽  
Sandrine Lecart ◽  
Dirk Roos ◽  
Paul Verkuijlen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Proteolytic Enzymes ◽  
Super Resolution ◽  
Membrane Dynamics ◽  
Small Gtpase ◽  
Human Neutrophils ◽  
Number Of Clusters ◽  
Phagocyte Nadph Oxidase ◽  
Phagosomal Membrane

Neutrophils are the first cells recruited at the site of infections, where they phagocytose the pathogens. Inside the phagosome, pathogens are killed by proteolytic enzymes that are delivered to the phagosome following granule fusion, and by reactive oxygen species (ROS) produced by the NADPH oxidase. The NADPH oxidase complex comprises membrane proteins (NOX2 and p22phox), cytoplasmic subunits (p67phox, p47phox, and p40phox) and the small GTPase Rac. These subunits assemble at the phagosomal membrane upon phagocytosis. In resting neutrophils the catalytic subunit NOX2 is mainly present at the plasma membrane and in the specific granules. We show here that NOX2 is also present in early and recycling endosomes in human neutrophils and in the neutrophil-like cell line PLB-985 expressing GFP-NOX2. In the latter cells, an increase in NOX2 at the phagosomal membrane was detected by live-imaging after phagosome closure, probably due to fusion of endosomes with the phagosome. Using super-resolution microscopy in PLB-985 WT cells, we observed that NOX2 forms discrete clusters in the plasma membrane. The number of clusters increased during frustrated phagocytosis. In PLB-985NCF1ΔGT cells that lack p47phox and do not assemble a functional NADPH oxidase, the number of clusters remained stable during phagocytosis. Our data suggest a role for p47phox and possibly ROS production in NOX2 recruitment at the phagosome.

scholarly journals p21-activated kinase (Pak) regulates NADPH oxidase activation in human neutrophils

Blood ◽  
2005 ◽  
Vol 106 (12) ◽  
pp. 3962-3969 ◽  
Author(s):  
Kendra D. Martyn ◽  
Moon-Ju Kim ◽  
Mark T. Quinn ◽  
Mary C. Dinauer ◽  
Ulla G. Knaus
Keyword(s):  
Plasma Membrane ◽  
Nadph Oxidase ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Human Neutrophils ◽  
C Terminus ◽  
Nadph Oxidase Complex ◽  
Direct Binding ◽  
Membrane Target ◽  
P21 Activated Kinase ◽  
Flavocytochrome B

The phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase plays an instrumental role in host defense and contributes to microbicial killing by releasing highly reactive oxygen species. This multicomponent enzyme is composed of membrane and cytosolic components that assemble in the plasma membrane or phagolysosome. While the guanosine S′-triphosphatase (GTPase) Rac2 has been shown to be a critical regulator of NADPH oxidase activity and assembly, the role of its effector, p21-activated kinase (Pak), in oxidase function has not been well defined. Using HIV-1 Tat-mediated protein transduction of Pak inhibitory domain, we show here that Pak activity is indeed required for efficient superoxide generation in intact neutrophils. Furthermore, we show that Pak translocates to the plasma membrane upon N-formyl-methionyl-leucyl-phenylalanine (fMLF) stimulation and colocalizes with translocated p47phox and with p22phox, a subunit of flavocytochrome b558. Although activated Pak phosphorylated several essential serine residues in the C-terminus of p47phox, direct binding to p47phox was not observed. In contrast, active Pak bound directly to p22phox, suggesting flavocytochrome b was the oxidase-associated membrane target of this kinase and this association may facilitate further phosphorylation of p47phox in the assembling NADPH oxidase complex.

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