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

Qingshan Wang,Shih-Heng Chen,Hui Zhou,Belinda Wilson,Jau-Shyong Hong,Huiming Gao,Chun-Hsien Chu

doi:10.1186/1742-2094-9-32

Qingshan Wang, Shih-Heng Chen + Show 5 more

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https://doi.org/10.1186/1742-2094-9-32

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Abstract

BackgroundBoth (-) 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.MethodsInhibition 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.ResultsBoth (-) 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.ConclusionsStrong 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.

Highlights

Both (-) and (+)-naloxone attenuate inflammation-mediated neurodegeneration by inhibition of microglial activation through superoxide reduction in an opioid receptor-independent manner
These values are much higher than the IC50 for the binding of opioid receptors, but they are in similar ranges to those previously reported for inhibiting superoxide production in neutrophils [18]
To rule out the possibility that the reduction of LPSinduced superoxide release was attributed to the superoxide scavenging capacity of naloxone, we evaluated the effect of various concentrations of (-) and (+)-naloxone on superoxide production using the xanthine/xanthine oxidase superoxide-generating system

Summary

Introduction

Both (-) and (+)-naloxone attenuate inflammation-mediated neurodegeneration by inhibition of microglial activation through superoxide reduction in an opioid receptor-independent manner. It has been reported that naloxone displays neuroprotective effects in a two-hit seizure model by reducing both cytokine production and microglial activation [11]. Both (-) and (+)-naloxone have been found to be capable of reducing the severity of aortic atherosclerosis in apolipoprotein-E (apo-E)-deficient mice through inhibition of macrophage activation and superoxide release [12]. It is critical to elucidate the novel nonopioid binding site(s) and action mechanisms of naloxone

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