2.4.6. NADPH oxidase activity
NADPH oxidase activity in intact aortic rings was performed by the lucigenin-enhanced chemiluminescence assay, as previously described (Zarzuelo et al., 2011). Briefly, aortic rings from all experimental groups were incubated for 30 minutes at 37°C in HEPES-containing physiological salt solution (pH 7.4) containing (in mM): NaCl 119, HEPES 20, KCl 4.6, MgSO4 1, Na2HPO4 0.15, KH2PO4 0.4, NaHCO3 1, CaCl2 1.2 and glucose 5.5. Aortic production of O2-was stimulated by addition of NADPH (100 μM). Rings were then placed in tubes containing physiological salt solution, with or without NADPH, and lucigenin was injected automatically at a final concentration of 5 μmol/L to withdraw known artefacts when used at higher concentrations. NADPH oxidase activity was determined by measuring luminescence over 200 s in a scintillation counter (Lumat LB 9507, Berthold, Germany) in 5-s intervals and was measured by subtracting the basal values from those in the presence of NADPH. Vessels were then dried, and their dry weight was determined. NADPH oxidase activity is shown as relative luminescence units (RLU)/min/mg dry aortic ring.