746978.fig.002a
(a)
746978.fig.002b
(b)
Figure 2: Apocynin reverses glucose-induced NADPH oxidase activity. (a) Cultured bovine retinal capillary pericytes express active NADPH oxidase. The activity was determined by cytochrome c reduction. Lysate (25 μg protein) was added to 250 μM cytochrome c in a 96-well multiwell plate and incubated for 120 min, either in the presence or absence of NADPH (100 μM) or NADPH oxidase inhibitor or flavoprotein inhibitor, diphenyleneiodonium (DPI, 100 μM). The reduction of cytochrome c was measured by reading absorbance at 550 nm (SpectraMax 190). Superoxide (O2-) production in nmol/mg protein was calculated from absorbance of samples and the extinction coefficient for change of ferricytochrome c to ferrocytochrome c of 21 mmol/l/cm. Data are presented as mean±SEM of 4–6 separate experiments. P*<.001 versus lysate alone, P**<.01 versus lysate with NADPH. (b) Exposure to high glucose for 4 days increases NADPH oxidase which is significantly reversed by the addition of apocynin. Confluent cultures of BRP in 30 mm2 dishes were exposed to normal glucose (NG, 5.6 mM) and high glucose (HG, 25 mM) with and without 500 μM apocynin (HGA) for 4 days. After incubation, the cells were washed twice with ice-cold PBS; lysed, and NADPH-dependent O2- production was measured by DPI-inhibitable cytochrome c reduction assay. Data are presented as mean ± SEM of 4 separate experiments. P*<.05 versus HG. P**<.013 versus HG.