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

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.

The Quest for Selective Nox Inhibitors and Therapeutics: Challenges, Triumphs and Pitfalls

“…In fact, the IC 50 for both isomers was closed to 2 lM. It is interesting to note that naloxone was also effective at inhibiting the activity of preassembled enzyme (171). That said, no reports could be found that compare its binding or inhibition of other Nox subunits.…”

“…5), a commonly used antagonist of opioid receptors, was found to be highly effective in preventing dopaminergic degeneration in different models of rodent Parkinson's disease by inhibiting inflammatory responses (107). In search for the potential neuroprotective action of naloxone, it was found that it binds to Nox2 and blocks translocation of p47 phox to the plasma membrane, leading to inhibition of ROS production (171). In fact, the neuroprotective effect of this drug is dependent on Nox2 but independent of opioid receptors since ( + )-naloxone, the inactive isomer for the activation of opioid receptors, is as potent as ( -)-naloxone at inhibiting ROS production and binding to Nox2.…”

Discovery of GSK2795039, a Novel Small Molecule NADPH Oxidase 2 Inhibitor

“…Therefore, the assay may not have been optimal to detect its activity if the mode of action is via inhibition of subunit assembly. The reported pharmacology of naloxone at NOX2 is also complex as it is proposed to bind to the gp91 phox subunit of NOX2 and inhibit enzyme activity by modulation of p47 phox subunit translocation (65). The method for the cell-free assay used here differs from what was previously used to demonstrate naloxone activity because we used a fully recombinant enzyme activated with arachidonic acid, whereas the assay reported by Wang et al (65) used PMA-activated neutrophil membranes in a semirecombinant system.…”

“…The reported pharmacology of naloxone at NOX2 is also complex as it is proposed to bind to the gp91 phox subunit of NOX2 and inhibit enzyme activity by modulation of p47 phox subunit translocation (65). The method for the cell-free assay used here differs from what was previously used to demonstrate naloxone activity because we used a fully recombinant enzyme activated with arachidonic acid, whereas the assay reported by Wang et al (65) used PMA-activated neutrophil membranes in a semirecombinant system. In the case of Shionogi 2, however, we found that it is a potent PKC inhibitor, consistent with previous reports (21), and this likely explains its apparent inhibitory activity in HL60 cells.…”

NADPH-oxidase 2 activation promotes opioid-induced antinociceptive tolerance in mice

“…Blocking these pathways pharmacologically or using antiinflammatory strategies, such as anti-TNFα (i.e., Entanercept) (Hutchinson et al, 2008), direct recombinant IL10 (Lin et al, 2010) or indirect IL10 gene therapy (Johnston et al, 2004), protect morphine antinociceptive properties. Moreover, evidence suggests that subpicomolar morphine and micromolar naloxone, a mu-opioid antagonist, demonstrate antiinflammatory properties in TLR4-activated microglial cells independent of the opioid receptor through direct interactions with NOX2 lead to reductions in NOX2 activation (Qian et al, 2007, Wang et al, 2012a). This opioid-independent mechanism with morphine treatment is likely overshadowed by opioid-dependent events that lead to tolerance, however, it may contribute to the potentiation of morphine analgesia by naloxone (Qian et al, 2007, Wang et al, 2012a).…”