NADPH Oxidase versus Mitochondria-Derived ROS in Glucose-Induced Apoptosis of Pericytes in Early Diabetic Retinopathy

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 $\mu$g protein) was added to 250 $\mu$M cytochrome c in a 96-well multiwell plate and incubated for 120 min, either in the presence or absence of NADPH (100 $\mu$M) or NADPH oxidase inhibitor or flavoprotein inhibitor, diphenyleneiodonium (DPI, 100 $\mu$M). The reduction of cytochrome c was measured by reading absorbance at 550 nm (SpectraMax 190). Superoxide (${{\text{O}}_2}^{-}$) 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 $\text{mean}\pm \text{SEM}$ 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 $\mu$M apocynin (HGA) for 4 days. After incubation, the cells were washed twice with ice-cold PBS; lysed, and NADPH-dependent ${{\text{O}}_2}^{-}$ production was measured by DPI-inhibitable cytochrome c reduction assay. Data are presented as mean $\pm$ SEM of 4 separate experiments. ${}_{}{}^{*}P<.05$ versus HG. ${}_{}{}^{**}P<.013$ versus HG.

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