title: Intracellular shunting of O2(-) contributes to charge compensation and preservation of neutrophil respiratory burst in the absence of voltage-gated proton channel activity.
abstract: Proton efflux via voltage-gated proton channels (Hv1) is considered to mediate the charge compensation necessary to preserve NADPH oxidase activity during the respiratory burst. Using the Hv1 inhibitor Zn(2+), we found that the PMA-induced respiratory burst of human neutrophils is inhibited when assessed as extracellular production of O2(-) and H2O2, in accordance with literature studies, but, surprisingly, unaffected when measured as oxygen consumption or total (extracellular plus intracellular) H2O2 production. Furthermore, we show that inhibiting Hv1 with Zn(2+) results in an increased production of intracellular ROS. Similar results, i.e. decreased extracellular and increased intracellular ROS production, were obtained using a human granulocyte-like cell line with severely impaired Hv1 expression. Acidic extracellular pH, which dampens proton efflux, also augmented intracellular production of H2O2. Zinc caused an increase in the rate but not in the extent of depolarization and cytosolic acidification indicating that mechanisms other than proton efflux take part in charge compensation. Our results suggest a hitherto unpredicted mechanism of charge compensation whereby, in the absence of proton efflux, part of O2(-) generated within gp91(phox) in the plasma membrane is shunted intracellularly down electrochemical gradient to dampen excessive depolarization. This would preserve NADPH oxidase activity under conditions such as the inflammatory exudate in which the acidic pH hinders charge compensation by proton efflux.
authors.affiliation: Department of Life Sciences, University of Trieste, Trieste, Italy.
authors.forename: Eva
authors.initials: E
authors.lastname: Decleva
authors.affiliation: Department of Life Sciences, University of Trieste, Trieste, Italy. Electronic address: menegazz@units.it.
authors.forename: Renzo
authors.initials: R
authors.lastname: Menegazzi
authors.affiliation: Department of Life Sciences, University of Trieste, Trieste, Italy.
authors.forename: Alba
authors.initials: A
authors.lastname: Fasolo
authors.affiliation: Université Joseph Fourier, GREPI/AGIM CNRS FRE 3405, Grenoble, France.
authors.forename: Federica
authors.initials: F
authors.lastname: Defendi
authors.affiliation: Department of Life Sciences, University of Trieste, Trieste, Italy.
authors.forename: Michele
authors.initials: M
authors.lastname: Sebastianutto
authors.affiliation: Department of Life Sciences, University of Trieste, Trieste, Italy.
authors.forename: Pietro
authors.initials: P
authors.lastname: Dri
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000037:antagonists & inhibitors* / Q000378:metabolism; D017136:Ion Transport; D008564:Membrane Potentials; D019255:NADPH Oxidases / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism* / Q000502:physiology; D010100:Oxygen / Q000378:metabolism*; D011522:Protons; D016897:Respiratory Burst; D055672:Static Electricity; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
chemical_list: C510235:HVCN1 protein, human; D007473:Ion Channels; D011522:Protons; C016658:phorbolol myristate acetate; D006861:Hydrogen Peroxide; D019255:NADPH Oxidases; D015032:Zinc; D013755:Tetradecanoylphorbol Acetate; D010100:Oxygen