Naloxone inhibits immune cell function by suppressing superoxide production through a direct interaction with gp91phox subunit of NADPH oxidase

Journal of Neuroinflammation

Jau-Shyong Hong¹  Belinda Wilson¹  Chun-Hsien Chu¹  Shih-Heng Chen¹  Huiming Gao¹  Hui Zhou¹  Qingshan Wang¹ 

[1] Neuropharmacology Section (Q.S.W., H.Z., H.M.G., S.H.C., C.H.C., B.W., J.S.H.), Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA

关键词: Binding;    Opioid receptor;    NADPH oxidase;    Microglia;    Neuroinflammation;   

Others  :  1212804
DOI  :  10.1186/1742-2094-9-32

 received in 2011-09-30, accepted in 2012-02-16,  发布年份 2012

【 摘 要 】

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 (³H-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 ³H-naloxone with cold (-) and (+)-naloxone in microglia showed equal potency with an IC50 of 2.73 and 1.57 μM, respectively. ³H-Naloxone binding was elevated in COS-7 and HEK293 cells transfected with gp91^phox; in contrast, reduced ³H-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 ³H-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.

【 授权许可】

   
2012 Wang et al; licensee BioMed Central Ltd.

【 图 表 】

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