Activation of NADPH-oxidase and its associated whole-cell H+ current in human neutrophils by recombinant human tumor necrosis factor α and formyl-methionyl-leucyl-phenylalanine

Abstract

Proton accumulation and efflux associated specifically with NADPH oxidation in neutrophils remains to be elucidated. 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-leucyl-phenylalanine (fMLP) and recombinant human tumor necrosis factor α(rhTNFα). Intracellular pH changes were monitored utilizing the ratiometric imaging of the dual emission fluoroprobe, carboxyseminaphthorhodafluor-1, AM acetate. Bath application of 1000 units/ml rhTNFα or 0.1 μM fMLP changed the fluorescence of fluoroprobeloaded cells, indicating generation of cytosolic H+ ions. In the absence of Ca2+ in the pipette solution, exposure of cells to rhTNFα or fMLP for 10 s activated voltage-dependent H+ currents. From tail current analysis, the threshold voltage for H+ current activation was ≈ -50 mV. These fMLP- or rhTNFα-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 μM of apocynin, an NADPH oxidase inhibitor. These results indicate that rhTNFα- or fMLP-induced NADPH oxidase in human neutrophils gives rise to the activation of voltage-dependent H+ currents.