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Slattery et al. Neuroimmunol Neuroinflammation 2018;5:11 I http://dx.doi.org/10.20517/2347-8659.2018.05 Page 7 of 15

150

Nitrite (% control) 100

50

0
0 2 20 50
P-aminophenol (µmol/L)

0 µmol/L URB597 0.5 µmol/L URB597
0.02 µmol/L URB597 2 µmol/L URB597

Figure 5. Effects of p-aminophenol alone, or in combination with the fatty acid amide hydrolase inhibitor URB597, on the lipopolysaccharide
(LPS)-induced nitric oxide release. BV-2 cells were treated for 15 min with various concentrations of URB597 (0.02, 0.5, 2 mmol/L), then
incubated with different concentrations (2, 20, 50 mmol/L) of p-aminophenol for a further 15 min period, before being stimulated with LPS.
After 24 h, nitrite concentrations in the BV-2 cell-free supernatants were measured using the Griess assay. Data from nine independent
experiments are normalized against nitrite concentration in samples stimulated in the absence of inhibitors. Nitrite concentration in these
samples was 13.2 ± 1.0 mmol/L. The effect of treatments was assessed by the two-way analyses of variance (ANOVA), followed by Tukey’s
post hoc test. *P < 0.05 and **P < 0.01, significantly different from stimulated cells incubated in the absence of URB597 (ANOVA F = 7.87,
#
##
P < 0.0001); P < 0.05 and P < 0.01, significantly different from stimulated cells incubated in the absence of p-aminophenol (ANOVA F =
216.2, P < 0.0001). ANOVA interaction F = 1.83, P = 0.068
[27]
forming AM404 . In the CNS, AM404 activates TRPV1 but inhibits COX-1 and COX-2 enzymatic activity,
anandamide reuptake and metabolism, as well as the transcription factors NF-κB and nuclear factor of
activated T-cells (NFAT) [23,30] . Due to these various pharmacological activities, AM404 has been implicated
as the active substance responsible for the clinically beneficial effects of acetaminophen on nociception,
mechanical allodynia, thermal hyperalgesia, as well as inflammatory processes resulting from the gene
activation induced by the transcription factors NF-κB and NFAT. Specifically, the ability of AM404
to alleviate neuropathic pain in rodent models has been attributed to a reduction in NO and cytokine
[23]
production . In this study, we report that AM404 suppresses NO secretion by LPS-stimulated BV-2 cells
[Figure 2C].

Since both p-aminophenol and AM404 effectively reduced NO release by stimulated BV-2 microglia, we
hypothesized that FAAH present in BV-2 cells may allow p-aminophenol to exert its effect on microglial
activation through its metabolism to AM404 . To test this hypothesis, BV-2 cells were incubated with
[48]
various concentrations (0.02, 0.5, 2 µmol/L) of the specific FAAH inhibitor URB597 for 15 min prior to
treatment with p-aminophenol. We rationalized that the inhibitory effect of p-aminophenol would be
eliminated in the presence of URB597, if this metabolite were working through its conversion to AM404.
As illustrated in Figure 5, p-aminophenol attenuated NO secretion from stimulated microglia in the
presence of all concentrations of URB597 tested. Interestingly, URB597 itself showed an inhibitory effect
on NO release in the absence of p-aminophenol and in the presence of 2 µmol/L p-aminophenol. These
results indicate that, contrary to the initial hypothesis, p-aminophenol inhibits NO secretion through a
pathway independent of its conversion to AM404 by FAAH. Moreover, p-aminophenol and URB597 may
act synergistically to abate microglial activation.

It has been suggested that URB597 may function to attenuate microglial activation through the inhibition
[49]
of FAAH . In addition to catalyzing the conjugation of p-aminophenol and arachidonic acid, FAAH
[50]
functions in the degradation of fatty acid amides such as the endocannabinoid anandamide . In this