Superoxide anion production was measured from frozen aortic slices using the lucigenin-enhanced chemiluminescence method as described previously [27,28]. Briefly, small slices from aorta were preincubated in Krebs-Hepes buffer (saturated with 95% O2 and 5% CO2, at room temperature) for 30 min and then transferred to a glass scintillation vial containing 5 mmol/L of lucigenin (200 mL/2 mL Krebs-Hepes) for the determination of basal O2 C- levels. The chemiluminescence was recorded every minute for 10 min at room temperature in a liquid scintillation counter (Wallac 1409, Turku, Finland). Lucigenin counts were expressed as cpm/mg of dry weight tissue. Moreover, 0.1 mM NADPH was added to the vials before counting to assess the activation of NADPH oxidase activity in the samples. Basal superoxide-induced luminescence was then subtracted from the luminescence value induced by NADPH [29]. To confirm the involvement of NADPH oxidase, diphenyleneiodonium (DPI, 10 mmol/L), a selective inhibitor of NADPH oxidase was used according to a previous study