TY - JOUR
T1 - Naloxone inhibits immune cell function by suppressing superoxide production through a direct interaction with gp91phox subunit of NADPH oxidase
AU - Wang, Qingshan
AU - Zhou, Hui
AU - Gao, Huiming
AU - Chen, Shih Heng
AU - Chu, Chun Hsien
AU - Wilson, Belinda
AU - Hong, Jau Shyong
N1 - Funding Information:
This research was supported by the Intramural Research Program of the National Institutes of Health/National Institute of Environmental Health Sciences. We thank Dr. Saurabh Chatterjee and Baozhong Zhao at the National Institute of Environmental Health Sciences and Esteban Oyarzabal at University of North Carolina, Chapel Hill, for their helpful suggestions in editing this article.
PY - 2012/12/16
Y1 - 2012/12/16
N2 - 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.
AB - 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.
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U2 - 10.1186/1742-2094-9-32
DO - 10.1186/1742-2094-9-32
M3 - Article
C2 - 22340895
AN - SCOPUS:84858159054
SN - 1742-2094
VL - 9
JO - Journal of Neuroinflammation
JF - Journal of Neuroinflammation
M1 - 525
ER -