title: Naloxone inhibits immune cell function by suppressing superoxide production through a direct interaction with gp91phox subunit of NADPH oxidase.
abstract: 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 gp91phox-/- neutrophils and transfected COS-7 and HEK293 cells. The translocation of cytosolic subunit p47phox 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 gp91phox; in contrast, reduced 3H-naloxone binding was found in neutrophils deficient in gp91phox 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 gp91phox antibody, and 2) activation of NOX2 by PMA. Finally, western blot studies showed that naloxone suppressed translocation of the cytosolic subunit p47phox 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.
authors.affiliation: Neuropharmacology Section, Laboratory of Toxicology and Pharmacology, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA.
authors.forename: Qingshan
authors.initials: Q
authors.lastname: Wang
authors.forename: Hui
authors.initials: H
authors.lastname: Zhou
authors.forename: Huiming
authors.initials: H
authors.lastname: Gao
authors.forename: Shih-Heng
authors.initials: SH
authors.lastname: Chen
authors.forename: Chun-Hsien
authors.initials: CH
authors.lastname: Chu
authors.forename: Belinda
authors.initials: B
authors.lastname: Wilson
authors.forename: Jau-Shyong
authors.initials: JS
authors.lastname: Hong
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001713:Biphenyl Compounds / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D008070:Lipopolysaccharides / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000172:deficiency / Q000378:metabolism*; D008636:Mesencephalon / Q000166:cytology; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000172:deficiency / Q000378:metabolism*; D009270:Naloxone / Q000494:pharmacology*; D009457:Neuroglia / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D013482:Superoxide Dismutase / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate; D013778:Tetrazolium Salts / Q000494:pharmacology; D013960:Thyroid Function Tests / Q000379:methods; D014162:Transfection; D014316:Tritium / Q000493:pharmacokinetics; D014969:Xanthine Oxidase / Q000494:pharmacology
chemical_list: C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D001713:Biphenyl Compounds; D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D009861:Onium Compounds; D013778:Tetrazolium Salts; D014316:Tritium; C031291:diphenyliodonium; D013481:Superoxides; D009270:Naloxone; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate