Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts.
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+.
Friis
Martin Barfred
MB
Department of Biology, University of Copenhagen, Copenhagen, Denmark.
Vorum
Katrine Gribel
KG
Lambert
Ian Henry
IH
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
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