Activation of NADPH-oxidase and its associated whole-cell H+ current in human neutrophils by recombinant human tumor necrosis factor alpha and formyl-methionyl-leucyl-phenylalanine.
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.
Schumann
M A
MA
Division of Pulmonary and Critical Care Medicine, Stanford University School of Medicine, California 94305-5236, USA.
Leung
C C
CC
Raffin
T A
TA
D000595:Amino Acid Sequence; D001578:Benzopyrans; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D007473:Ion Channels / Q000378:metabolism*; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009284:Naphthols; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D011522:Protons; D011994:Recombinant Proteins / Q000494:pharmacology; D012235:Rhodamines; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D001578:Benzopyrans; D005456:Fluorescent Dyes; D007473:Ion Channels; D009284:Naphthols; D011522:Protons; D011994:Recombinant Proteins; D012235:Rhodamines; D014409:Tumor Necrosis Factor-alpha; C091666:seminaphthorhodaminefluoride; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate