Intracellular shunting of O2− contributes to charge compensation and preservation of neutrophil respiratory burst in the absence of voltage-gated proton channel activity
| Autor: | Pietro Dri, Alba Fasolo, Renzo Menegazzi, F. Defendi, Eva Decleva, Michele Sebastianutto |
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| Přispěvatelé: | Decleva, Eva, Menegazzi, Renzo, Fasolo, Alba, Defendi, Federica, Michele, Sebastianutto, Dri, Pietro |
| Rok vydání: | 2013 |
| Předmět: |
neutrophils
NADPH oxidase zinc respiratory burst Neutrophils DMSO dimethyl sulfoxide HBS-BSA HEPES-buffered saline solution containing 0.2% BSA Ion Channels Membrane Potentials HBS HEPES-buffered saline solution biology NHA 5-N N-hexamethylene amiloride neutrophil HRP horseradish peroxidase Depolarization Hydrogen-Ion Concentration Respiratory burst Voltage-gated proton channels Zinc Biochemistry Tetradecanoylphorbol Acetate Efflux Protons Intracellular Research Article Static Electricity pHo extracellular pH HEPES N-2-hydroxyethylpiperazine-N′-2-ethanesulphonic acid RB respiratory burst ROS reactive oxygen species PMA phorbol-12-myristate-13-acetate Cell Line Tumor SOD superoxide dismutase Extracellular Humans Electrochemical gradient Ion Transport Voltage-gated proton channel activity Cell Membrane NADPH Oxidases Hydrogen Peroxide Cell Biology pHi intracellular pH Oxygen di-O-C5(3) 3-3′dipentyloxacarbocianine biology.protein Biophysics BCECF-AM 2′ 7′-bis(2carboxyethil)-5 6-carboxy-fluoresceine acetoxymethyl ester BSA bovine serum albumin DHR dihydrorhodamine 123 |
| Zdroj: | Experimental Cell Research |
| ISSN: | 0014-4827 |
| Popis: | 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 Zn2+, 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 Zn2+ 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 gp91phox 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. Highlights • Neutrophils’ respiratory burst is not inhibited by the H+ channel inhibitor Zn2+. • Intracellular production of O2− and H2O2 is increased in the presence of Zn2+. • Intracellular H2O2 production is increased in H+ channels knock-down cells. • Zn2+ increases the rate but not the extent of depolarization and pHi decrease. • Intracellular shunting of O2− contributes to charge compensation in neutrophils. |
| Databáze: | OpenAIRE |
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