Nadph

Grx1 activity was assayed as described previously^{73 –75 (link)}. Briefly, lung tissue was lysed in 137 mM Tris-HCl, pH 8.0, 130 mM NaCl, and 1% NP-40. Lysates were then cleared by centrifugation and 100 μg was incubated with reaction mixtures containing Na/K phosphate buffer (0.1 mM, pH 7.5), 0.5 mM GSH (Sigma-Aldrich, G4251), 2 units/mL GSSG reductase (Sigma-Aldrich, G3664), 0.1 mM L-CySSG (Cayman, 17582), 0.2 mM NADPH (Roche, 10107824001) and 1.5 mM EDTA (pH 8.0) or with reaction buffer consisting of 137 mM Tris-HCl buffer (pH 8.0), 0.5 mM GSH (Sigma-Aldrich, G4251), 1.2 units GSSG reductase (Roche, 10105678001), 2.5 mM 2-hydroxyethyl disulfide (Sigma-Aldrich, 380474), 0.35 mM NADPH (Roche, 10107824001), 1.5 mM EDTA (pH 8.0). The reaction was prepared in a 96-well plate (final volume of 200 µL) and proceeded at 30 °C. The consumption of NAPDH was followed spectrophotometrically at 340 nm. In each sample, the spontaneous consumption of NADPH was subtracted. Data are expressed in units (U)/mg protein for lung homogenates in which 1 U equals the oxidation of 1 μmol NADPH/min.

Tolbutamide was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). 4-hydroxyTolbutamide and 6-hydroxychlorzoxazone were obtained from Toronto Research Chemicals Inc. (North York, Canada). Dextromethorphan, dextrorphan and chlorzoxazone were supplied by Sigma-Aldrich Co. (St Louis, MO, USA). Testosterone was obtained from International Laboratory Limited (San Bruno, CA, USA). 6β-hydroxytestosterone was purchased from BD Biosciences Co. (Woburn, MA, USA). Phenacetin, cortisone acetate, EB and EE were from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). NADPH was obtained from Roche Diagnostics GmbH (Mannheim, Germany). All other reagents were of HPLC or analytical grade.