scholarly journals The Antibacterial Activity of Human Neutrophils and Eosinophils Requires Proton Channels but Not BK Channels

2006 ◽  
Vol 127 (6) ◽  
pp. 659-672 ◽  
Author(s):  
Jon K. Femling ◽  
Vladimir V. Cherny ◽  
Deri Morgan ◽  
Balázs Rada ◽  
A. Paige Davis ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Respiratory Burst ◽  
Essential Role ◽  
Outward Current ◽  
Bk Channels ◽  
Bk Channel ◽  
Human Neutrophils ◽  
H2o2 Production ◽  
Proton Channels ◽  
Voltage Gated

Electrophysiological events are of central importance during the phagocyte respiratory burst, because NADPH oxidase is electrogenic and voltage sensitive. We investigated the recent suggestion that large-conductance, calcium-activated K+ (BK) channels, rather than proton channels, play an essential role in innate immunity (Ahluwalia, J., A. Tinker, L.H. Clapp, M.R. Duchen, A.Y. Abramov, S. Page, M. Nobles, and A.W. Segal. 2004. Nature. 427:853–858). In PMA-stimulated human neutrophils or eosinophils, we did not detect BK currents, and neither of the BK channel inhibitors iberiotoxin or paxilline nor DPI inhibited any component of outward current. BK inhibitors did not inhibit the killing of bacteria, nor did they affect NADPH oxidase-dependent degradation of bacterial phospholipids by extracellular gIIA-PLA2 or the production of superoxide anion (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2^{.}}^{{-}}\) \end{document}). Moreover, an antibody against the BK channel did not detect immunoreactive protein in human neutrophils. A required role for voltage-gated proton channels is demonstrated by Zn2+ inhibition of NADPH oxidase activity assessed by H2O2 production, thus validating previous studies showing that Zn2+ inhibited \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2^{.}}^{{-}}\) \end{document} production when assessed by cytochrome c reduction. In conclusion, BK channels were not detected in human neutrophils or eosinophils, and BK inhibitors did not impair antimicrobial activity. In contrast, we present additional evidence that voltage-gated proton channels serve the essential role of charge compensation during the respiratory burst.

scholarly journals Early signalling events implicated in leukotriene B4-induced activation of the NADPH oxidase in eosinophils: role of Ca2+, protein kinase C and phospholipases C and D

1995 ◽  
Vol 310 (3) ◽  
pp. 795-806 ◽  
Author(s):  
R S Perkins ◽  
M A Lindsay ◽  
P J Barnes ◽  
M A Giembycz
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Nadph Oxidase ◽  
Respiratory Burst ◽  
Leukotriene B4 ◽  
Human Neutrophils ◽  
H2o2 Production ◽  
Intact Cells ◽  
Kinase C ◽  
H2o2 Generation

The early signalling events that may ultimately contribute to the assembly and subsequent activation of the NADPH oxidase in guinea-pig peritoneal eosinophils were investigated in response to leukotriene B4 (LTB4). LTB4 promoted a rapid, transient and receptor-mediated increase in the rate of H2O2 generation that was potentiated by R 59 022, a diradylglycerol (DRG) kinase inhibitor, implicating protein kinase C (PKC) in the genesis of this response. This conclusion was supported by the finding that the PKC inhibitor, Ro 31-8220, attenuated (by about 30%) the peak rate of LTB4-induced H2O2 generation under conditions where the same response evoked by 4 beta-phorbol 12,13-dibutyrate (PDBu) was inhibited by more than 90%. Paradoxically, Ro 31-8220 doubled the amount of H2O2 produced by LTB4 which may relate to the ability of PKC to inhibit cell signalling through phospholipase C (PLC). Indeed, Ro 31-8220 significantly enhanced LTB4-induced Ins(1,4,5)P3 accumulation and the duration of the Ca2+ transient in eosinophils. Experiments designed to assess the relative importance of DRG-mobilizing phospholipases in LTB4-induced oxidase activation indicated that phospholipase D (PLD) did not play a major role. Thus, although H2O2 generation was abolished by butan-1-ol, this was apparently unrelated to the inhibition of PLD, as LTB4 failed to stimulate the formation of Ptd[3H]BuOH in [3H]butan-1-ol-treated eosinophils. Rather, the inhibition was probably due to the ability of butan-1-ol to increase the eosinophil cyclic AMP content. In contrast, Ca(2+)- and PLC-driven mechanisms were implicated in H2O2 generation, as LTB4 elevated the Ins(1,4,5)P3 content and intracellular free Ca2+ concentration in intact cells, and cochelation of extracellular and intracellular Ca2+ significantly attenuated LTB4-induced H2O2 generation. Pretreatment of eosinophils with wortmannin did not affect LTB4-induced H2O2 production at concentrations at which it abolished the respiratory burst evoked by formylmethionyl-leucylphenylalanine in human neutrophils. Collectively, these data suggest that LTB4 activates the NADPH oxidase in eosinophils by PLD- and PtdIns 3-kinase-independent mechanisms that involve Ca2+, PLC and PKC. Furthermore, the activation of additional pathways that do not require Ca2+ is also suggested by the finding that LTB4 evoked a significant respiratory burst in Ca(2+)-depleted cells.

scholarly journals Voltage-Gated Proton Channels Find Their Dream Job Managing the Respiratory Burst in Phagocytes

Physiology ◽  
2010 ◽  
Vol 25 (1) ◽  
pp. 27-40 ◽  
Author(s):  
Thomas E. DeCoursey
Keyword(s):  
Reactive Oxygen Species ◽  
Nadph Oxidase ◽  
Respiratory Burst ◽  
Proton Channel ◽  
Oxygen Species ◽  
Proton Extrusion ◽  
Proton Channels ◽  
Voltage Gated ◽  
Voltage Gated Ion Channels ◽  
Voltage Sensing Domain

The voltage-gated proton channel bears surprising resemblance to the voltage-sensing domain (S1–S4) of other voltage-gated ion channels but is a dimer with two conduction pathways. The proton channel seems designed for efficient proton extrusion from cells. In phagocytes, it facilitates the production of reactive oxygen species by NADPH oxidase.

scholarly journals Protein kinase C activity is not involved in N-formylmethionyl-leucyl-phenylalanine-induced phospholipase D activation in human neutrophils, but is essential for concomitant NADPH oxidase activation: studies with a staurosporine analogue with improved selectivity for protein kinase C

1993 ◽  
Vol 292 (3) ◽  
pp. 781-785 ◽  
Author(s):  
G C Kessels ◽  
K H Krause ◽  
A J Verhoeven
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cyclic Amp ◽  
Nadph Oxidase ◽  
Phospholipase D ◽  
Respiratory Burst ◽  
Feedback Inhibition ◽  
Human Neutrophils ◽  
Kinase C ◽  
Pkc Activation

Stimulation of human neutrophils by the receptor agonist N-formylmethionyl-leucyl-phenylalanine (fMLP) results in a respiratory burst, catalysed by an NADPH oxidase. Concomitantly, phospholipase D (PLD) is activated. To investigate the role of protein kinase C (PKC) in these neutrophil responses, we have compared the effects of staurosporine and a structural analogue of staurosporine (cgp41251), that reflects a higher selectivity towards PKC [Meyer, Regenass, Fabbro, Alteri, Rösel, Müller, Caravatti and Matter (1989) Int. J. Cancer 43, 851-856]. Both staurosporine and cgp41251 dose-dependently inhibited the production of superoxide induced by phorbol 12-myristate 13-acetate (PMA). Both compounds also caused inhibition of the fMLP-induced respiratory burst, but with a lower efficacy during the initiation phase of this response. This latter observation cannot be taken as evidence against PKC involvement in the activation of the respiratory burst, because pretreatment of neutrophils with ionomycin before PMA stimulation also results in a lower efficacy of inhibition. Activation of PLD by fMLP was enhanced in the presence of staurosporine, but not in the presence of cgp41251. Enhancement of PLD activation was also observed in the presence of H-89, an inhibitor of cyclic-AMP-dependent protein kinase (PKA). Both staurosporine and H-89 reversed the dibutyryl-cyclic-AMP-induced inhibition of PLD activation, whereas cgp41251 was without effect. These results indicate that the potentiating effect of staurosporine on PLD activation induced by fMLP does not reflect a feedback inhibition by PKC activation, but instead a feedback inhibition by PKC activation. Taken together, our results indicate that in human neutrophils: (i) PKC activity is not essential for fMLP-induced activation of PLD; (ii) PKC activity does play an essential role in the activation of the respiratory burst by fMLP, other than mediating or modulating PLD activation; (iii) there exists a negative-feedback mechanism on fMLP-induced PLD activation by concomitant activation of PKA.

scholarly journals Effect of Cleistanthin A on Voltage Gated Proton Channels of Human Neutrophils

2018 ◽  
Author(s):  
Rajasegaran Rajalakshmi ◽  
Soosai Manickam Amirtham ◽  
V Abirami ◽  
Sathya Subramani ◽  
Praghalathan Kanthakumar
Keyword(s):  
Human Neutrophils ◽  
Proton Channels ◽  
Voltage Gated

scholarly journals Relation of human neutrophil phorbol ester receptor occupancy and NADPH- oxidase activity

Blood ◽  
1982 ◽  
Vol 60 (2) ◽  
pp. 333-339 ◽  
Author(s):  
AI Tauber ◽  
DB Brettler ◽  
EA Kennington ◽  
PM Blumberg
Keyword(s):  
Nadph Oxidase ◽  
Phorbol Ester ◽  
Respiratory Burst ◽  
Human Neutrophil ◽  
Oxidase Activity ◽  
Receptor Occupancy ◽  
Human Neutrophils ◽  
Binding Studies ◽  
Nadph Oxidase Activity ◽  
Phorbol Ester Receptor

Abstract Phorbol esters are potent stimulants of the respiratory burst of the human neutrophil as assessed by superoxide (O2-) generation in whole cells and by NADPH-oxidase activity in a broken-cell 27,000-g particulate fraction. Phorbol 12-myristate, 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) stimulate production of O2- by human neutrophils with ED50 concentrations of 3.9 +/- 2.1 and 41.7 +/- 7.1 nM, respectively. The relation of biologic activity to receptor occupancy was assessed with binding studies of PMA and PDBu. Phorbol ester binding revealed a single high affinity phorbol ester receptor present at 7.6 x 10(5) sites/cell. The binding affinities for PMA and PDBu, 4.9 nM and 38.4 nM, respectively, agreed quantitatively with that of biologic potencies. Because of the high concentration of phorbol ester receptors (up to 125 nM) and the large amount of nonspecific binding at high cell density, apparent discrepancies between ED50′s for NADPH-oxidase and whole cell O2- generation were noted. With the use of low cell concentrations, quantitative agreement between intact cell production of O2-, NADPH-oxidase activity, and receptor binding was found. These results further support the identity of the NADPH-oxidase as the enzymatic source of respiratory burst O2- production in human neutrophils.

Neutrophil p29 Peroxiredoxin VI (Prdx VI) Enhances Bactericidal Activity and Chemotaxis in Myeloid Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1354-1354 ◽  
Author(s):  
Michael Ellison ◽  
Gail Thurman ◽  
Daniel R. Ambruso
Keyword(s):  
Nadph Oxidase ◽  
Bactericidal Activity ◽  
Respiratory Burst ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Human Neutrophils ◽  
Boyden Chamber ◽  
And Control ◽  
O2 Production ◽  
In Cells

Abstract Abstract 1354 Poster Board I-377 Introduction We have identified a 29 kDa protein from human neutrophils which binds to the NADPH oxidase component p67phox and enhances superoxide anion (O2−) production in a cell-free, reconstituted, NADPH oxidase system. The protein was identified as peroxiredoxin VI by sequence and the recombinant molecule was found to have both peroxiredoxin activity and calcium-independent PLA2 activity that is optimum at low pH (aiPLA2). Although p29 Prdx VI is found in many tissues, its role in myeloid cells is not well established. To explore other roles of p29, in addition to its effect on the respiratory burst, a PLB-985 cell line with shRNA mediated knockdown of p29 Prdx VI was established. Chemotaxis, as well as ingestion and killing S. aureus were determined in knockdown and control cells. Methods PLB-985 cells were transfected with a plasmid encoding a p29 Prdx VI targeting shRNA or a negative control plasmid and stable transfectants were selected in puromycin containing media. Knockdown of p29 Prdx VI was confirmed by Western blot with no changes in actin or other oxidase components. After maturation of the knockdown and control cells by DMSO for 4 days, each was combined with serum opsonized Staph. aureus in a 2 to1 human cell to bacterial cell ratio, bacterial cells remaining at various times were measured by plating aliquots of the cell mixtures and counting bacterial colonies which grew overnight. To evaluate ingestion, aliquots of the cell mixtures were transferred to slides by cytospin, stained, and examined under a light microscope to determine what proportion of PLB-985 cells had internalized bacteria. To evaluate chemotaxis, distances of migration toward chemo-attractant (opsonized zymosan) in a Boyden chamber were measured for differentiated p29 Prdx VI knockdown and control cells. Results Using stable expression of shRNA p29 protein was reduced to 31+/-18% (SD) of that in non-knockdown control cells. In two separate assays of bactericidal activity, cells without knockdown of p29 Prdx VI had 17 and 13% of initial bacteria surviving at 30 min; cells with p29 Prdx VI knockdown had 30 and 56% of bacteria surviving. This defect in bactericidal activity since ingestion was no different between the two types of cells at 0, 5, 10, and 15 min after addition of the bacteria. In response to zymosan activated serum, stimulated directed migration (distance of leading front in response to zymosan activated serum minus distance of leading front in response to buffer) was greater in cells without knockdown (23.9 ± 3.0 microns, mean ± SEM, n = 4 separate experiments) than movement by cells with knockdown of p29 Prdx VI (18.3 ± 5.3 microns). The difference was significant, p<0.05 by paired t test. Conclusion Optimal O2− production during the respiratory burst in intact myeloid cells is dependent on p29. Deficient bactericidal activity was demonstrated at 30 min; this decrease could not be associated with a difference in ingestion. In addition, directed cell migration was also decreased in cells with decreased amounts of p29 Prdx VI. These results indicate that in addition to its effect on the respiratory burst, p29 Prdx supports multiple functions in neutrophils. Disclosures No relevant conflicts of interest to declare.

scholarly journals C242TCYBAPolymorphism of the NADPH Oxidase Is Associated With Reduced Respiratory Burst in Human Neutrophils

Hypertension ◽  
2004 ◽  
Vol 43 (6) ◽  
pp. 1246-1251 ◽  
Author(s):  
Keith E. Wyche ◽  
Shaoshan S. Wang ◽  
Kathy K. Griendling ◽  
Sergey I. Dikalov ◽  
Harland Austin ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Respiratory Burst ◽  
Human Neutrophils
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