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 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 mu M) 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 mu M) 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+.