Abstract
Proton accumulation and efflux associated specifically with NADPH oxidation in neutrophils remains to be elucidated. Using confocal fluorescence and patch-clamp recordings from single human neutrophils, in the presence of protein kinase C inhibitors, we studied the transient cytosolic acidification and whole-cell H+ current induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) and recombinant human tumor necrosis factor alpha (rhTNF alpha). Intracellular pH changes were monitored utilizing the ratiometric imaging of the dual emission fluoroprobe, carboxyseminaphthorhodafluor-1, AM acetate. Bath application of 1000 units/ml rhTNF alpha or 0.1 microM fMLP changed the fluorescence of fluoroprobe-loaded cells, indicating generation of cytosolic H+ ions. In the absence of Ca2+ in the pipette solution, exposure of cells to rhTNF alpha or fMLP for 10 s activated voltage-dependent H+ currents. From tail current analysis, the threshold voltage for H+ current activation was approximately -50 mV. These fMLP- or rhTNF alpha-activated voltage-dependent H+ currents were augmented further in the presence of 0.1 mM of NADPH in the pipette solution, and they were inhibited by bath application of 50 microM of apocynin, an NADPH oxidase inhibitor. These results indicate that rhTNF alpha- or fMLP-induced NADPH oxidase in human neutrophils gives rise to the activation of voltage-dependent H+ currents.
Highlights
From the Division of Pulmonary and Critical Care Medicine, Stanford University School of Medicine, Stanford, California 94305-5236
Using confocal fluorescence and patchclamp recordings from single human neutrophils, in the presence of protein kinase C inhibitors, we studied the transient cytosolic acidification and whole-cell H+ current induced by N.formyl-methionyl-Ieucyl-phenylalanine and recombinant human tumor necrosis factor 0:
Chemiluminescence could have been used as an indirect sensitive measurement of NADPH oxidase activity in human neutrophils, we used a more quantitative method utilizing a cytochrome reduction to investigate the production of O2 by neutrophils exposed to phorbol 12-myristate 13-acetate (PMA), tMLP, and recombinant human tumor necrosis factor 0: (rhTNFa)
Summary
From the Division of Pulmonary and Critical Care Medicine, Stanford University School of Medicine, Stanford, California 94305-5236. The evidence is based on the following observations in PMA-stimulated cells: 1) the mode of action of NADPH oxidase has been shown to be electrogenic; 2) the subsequent effiux of produced H+ ions, through a proposed Zn 2+ - and Cd 2+ -inhibitable channel (known as "H+ -channel"), provides the necessary charge compensation; and 3) the effiux of H+ ions (repolarization) initially lagging behind the generation of O2 (depolarization) may explain the membrane depolarization-repolarization sequence In these studies, correlative changes in pH to membrane potential have been determined using the cytosolic pH indicator, 2',7'-bis(2-carboxyethyl)-5 (and -6)·carboxyfluorescein and a membrane potential-sensitive probe.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
