title: Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts.
abstract: Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H(2)O(2) potentiates the concomitant release of the organic osmolyte taurine (Lambert IH. J Membr Biol 192: 19-32, 2003). The increase in ROS production [5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein diacetate fluorescence] is detectable after a reduction in the extracellular osmolarity from 335 mosM (isotonic) to 300 mosM and reaches a maximal value after a reduction to 260 mosM. The swelling-induced ROS production is reduced by the flavoprotein inhibitor diphenylene iodonium chloride (25 microM) but is unaffected by the nitric oxide synthase inhibitor N omega-nitro-l-arginine methyl ester, indicating that the volume-sensitive ROS production is NADPH oxidase dependent. NIH3T3 cells express the NADPH oxidase components: p22 phox, a NOX4 isotype; p47 phox; and p67 phox (real-time PCR). Exposure to the Ca2+-mobilizing agonist ATP (10 microM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid (LPA, 10 nM) potentiates the swelling-induced taurine release as well as the ROS production. Overexpression of Rac1 or p47 phox or p47 phox knockdown [small interfering (si)RNA] had no effect on the swelling-induced ROS production or taurine release. NOX4 knockdown (siRNA) impairs the increase in the ROS production and the concomitant taurine release following osmotic exposure. It is suggested that a NOX4 isotype plus p22 phox account for the swelling-induced increase in the ROS production in NIH3T3 cells and that the oxidase activity is potentiated by PKC and LPA but not by Ca2+.
authors.affiliation: Department of Biology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Martin Barfred
authors.initials: MB
authors.lastname: Friis
authors.forename: Katrine Gribel
authors.initials: KG
authors.lastname: Vorum
authors.forename: Ian Henry
authors.initials: IH
authors.lastname: Lambert
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D053119:Benzophenanthridines / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology; D048429:Cell Size; D003573:Cytochrome b Group / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D007038:Hypotonic Solutions; D008246:Lysophospholipids / Q000378:metabolism; D051379:Mice; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D041681:NIH 3T3 Cells; D009861:Onium Compounds / Q000494:pharmacology; D009997:Osmotic Pressure; D010750:Phosphoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D034622:RNA Interference; D017382:Reactive Oxygen Species / Q000378:metabolism; D013654:Taurine / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: D053119:Benzophenanthridines; D002227:Carbazoles; D003573:Cytochrome b Group; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D007038:Hypotonic Solutions; D008246:Lysophospholipids; D009861:Onium Compounds; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; C079789:neutrophil cytosol factor 67K; C081021:Go 6976; D013654:Taurine; D016718:Arachidonic Acid; C007517:diphenyleneiodonium; D000255:Adenosine Triphosphate; C016299:chelerythrine; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C422943:Nox4 protein, mouse; C515614:Cyba protein, mouse; C060505:neutrophil cytosolic factor 1; D011493:Protein Kinase C; D020830:rac1 GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate; C032881:lysophosphatidic acid; D002118:Calcium