Naloxone inhibits immune cell function by suppressing superoxide production through a direct interaction with gp91(phox )subunit of NADPH oxidase

BACKGROUND: Both (-) and (+)-naloxone attenuate inflammation-mediated neurodegeneration by inhibition of microglial activation through superoxide reduction in an opioid receptor-independent manner. Multiple lines of evidence have documented a pivotal role of overactivated NADPH oxidase (NOX2) in inflammation-mediated neurodegeneration. We hypothesized that NOX2 might be a novel action site of naloxone to mediate its anti-inflammatory actions. METHODS: Inhibition of NOX-2-derived superoxide by (-) and (+)-naloxone was measured in lipopolysaccharide (LPS)-treated midbrain neuron-glia cultures and phorbol myristate acetate (PMA)-stimulated neutrophil membranes by measuring the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt (WST-1) or ferricytochrome c. Further, various ligand ((3)H-naloxone) binding assays were performed in wild type and gp91(phox-/- )neutrophils and transfected COS-7 and HEK293 cells. The translocation of cytosolic subunit p47(phox )to plasma membrane was assessed by western blot. RESULTS: Both (-) and (+)-naloxone equally inhibited LPS- and PMA-induced superoxide production with an IC50 of 1.96 and 2.52 μM, respectively. Competitive binding of (3)H-naloxone with cold (-) and (+)-naloxone in microglia showed equal potency with an IC50 of 2.73 and 1.57 μM, respectively. (3)H-Naloxone binding was elevated in COS-7 and HEK293 cells transfected with gp91(phox); in contrast, reduced (3)H-naloxone binding was found in neutrophils deficient in gp91(phox )or in the presence of a NOX2 inhibitor. The specificity and an increase in binding capacity of (3)H-naloxone were further demonstrated by 1) an immunoprecipitation study using gp91(phox )antibody, and 2) activation of NOX2 by PMA. Finally, western blot studies showed that naloxone suppressed translocation of the cytosolic subunit p47(phox )to the membrane, leading to NOX2 inactivation. CONCLUSIONS: Strong evidence is provided indicating that NOX2 is a non-opioid novel binding site for naloxone, which is critical in mediating its inhibitory effect on microglia overactivation and superoxide production.