Naloxone inhibits superoxide production through inhibition of NOX2. A. (-) and (+)-Naloxone isomers are equipotent in inhibiting LPS-induced superoxide production. Neuron/glia cultures were pretreated with (-) or (+)-naloxone for 30 minutes before the addition of LPS (15 ng/ml), the production of superoxide was immediately assessed after LPS challenge by measuring SOD-inhibitable reduction of the WST-1. B. Both (-) and (+)-naloxone lack superoxide scavenging capacity. Briefly (-) or (+)-naloxone, xanthine oxidase (10 mU), and WST-1 (250 μM) were mixed in potassium phosphate buffer (PBS, 50 mM, pH 7.6). Xanthine (50 μM, final concentration) was added to initiate the reaction (final volume, 1 ml). Absorbance at 450 nm was continuously monitored. C. (-) and (+)-Naloxone (10 μM) inhibited NADPH-dependent superoxide production by PMA-stimulated neutrophil membranes in a cell-free system, as measured by the reduction of ferricytochrome c. Results are expressed as a percentage of the control and are mean ± SEM of three independent experiments performed in duplicate. SOD was used as a control to demonstrate its effectiveness to remove superoxide from the system. **P < 0.01 compared with corresponding control group. NOX2, NADPH oxidase; SOD, superoxide dismutase; WST-1, water soluble tetrazolium salt 1; PMA, phorbol myristate acetate.
Wang et al. Journal of Neuroinflammation 2012 9:32 doi:10.1186/1742-2094-9-32