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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

Department of Biology, University of Copenhagen, Copenhagen, Denmark

Submitted 3 December 2007 ; accepted in final form 9 April 2008

Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H₂O₂ 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 µM) but is unaffected by the nitric oxide synthase inhibitor N-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 µM) 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⁺.

organic osmolytes; NOX4; lysophospholipids; arachidonic acid mobilization; adenosine triphosphate; calcium