ACTIVATION OF NADPH-OXIDASE AND ITS ASSOCIATED WHOLE-CELL H-NECROSIS-FACTOR-ALPHA AND FORMYL-METHIONYL-LEUCYL-PHENYLALANINE( CURRENT IN HUMAN NEUTROPHILS BY RECOMBINANT HUMAN TUMOR)

Proton accumulation and efflux associated specifically with NADPH oxid ation in neutrophils remains to be elucidated. Using confocal fluoresc ence and patch-clamp recordings from single human neutrophils, in the presence of protein kinase C inhibitors, we studied the transient cyto solic acidification and whole cell H+ current induced by N-formyl-meth ionyl-leucyl-phenylalanine (fMLP) and recombinant human tumor necrosis factor alpha (rhTNF alpha). Intracellular pH changes mere monitored u tilizing the ratiometric imaging of the dual emission fluoroprobe, car boxyseminaphthorhodafluor-1, AM acetate. Bath application of 1000 unit s/ml rhTNF alpha or 0.1 mu M fMLP changed the fluorescence of fluoropr obe-loaded cells, indicating generation of cytosolic H+ ions. In the a bsence of Ca2+ in the pipette solution, exposure of cells to rhTNF alp ha or fMLP for 10 s activated voltage dependent HC currents. From tail current analysis, the threshold voltage for H+ current activation was approximate to -50 mV. These fMLP- or rhTNF alpha-activated voltage-d ependent H+ currents were augmented further in the presence of 0.1 mM of NADPH in the pipette solution, and they were inhibited by bath appl ication of 50 mu M of apocynin, an NADPH oxidase inhibitor. These resu lts indicate that rhTNF alpha- or fMLP-induced NADPH oxidase in human neutrophils gives rise to the activation of voltage-dependent H+ curre nts.