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Page 8 of 15 Slattery et al. Neuroimmunol Neuroinflammation 2018;5:11 I http://dx.doi.org/10.20517/2347-8659.2018.05

150

Nitrite (% control) 100

50

0
0 2 20 50
P-aminophenol (µmol/L)

0 µmol/L SR144528 0.5 µmol/L SR144528
0.02 µmol/L SR144528 2 µmol/L SR144528

Figure 6. Effects of p-aminophenol alone or in combination with the selective CB2 receptor antagonist SR144528 on lipopolysaccharide
(LPS)-induced nitric oxide release. BV-2 cells were treated for 15 min with various concentrations of SR144528 (0.02, 0.5, 2 mmol/L),
then incubated with different concentrations (2, 20, 50 mmol/L) of p-aminophenol for a 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 four independent
experiments are normalized against nitrite concentration in samples stimulated in the absence of inhibitors. Nitrite concentration in
these samples was 19.6 ± 1.8 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 SR144528 (ANOVA
##
F = 4.17, P = 0.011); P < 0.01, significantly different from stimulated cells incubated in the absence of p aminophenol (ANOVA F = 232.2,
P < 0.0001). ANOVA interaction F = 0.79, P = 0.62

capacity, URB597 indirectly activates cannabinoid receptors by potentiating the effect of bioavailable
anandamide. Similarly, it has been identified that AM404 inhibits anandamide reuptake and degradation
through mechanisms which are currently unknown [51,52] . Accordingly, it has been reported that URB597
and AM404 have similar effects on LPS-induced inflammation in rats that are CB1 and CB2 receptor
dependent . These observations are consistent with our data showing that URB597 on its own [Figure 5],
[53]
similar to AM404 [Figure 2C], inhibits nitrite secretion by BV-2 microglia.

Next, we investigated the role of cannabinoid receptor signaling in mediating the effect of p-aminophenol
and AM404 on NO secretion by LPS-stimulated microglia. Controversy exists surrounding the ability of
AM404 to directly bind and activate CB1 receptors; however, it is widely agreed that AM404 attenuates
the reuptake and degradation of the endogenous cannabinoid, anandamide, thereby potentiating its
[23]
agonistic effect at CB1 and CB2 receptors . Moreover, it has been suggested that AM404-mediated
activation of TRPV1 is sufficient to induce the synthesis of additional anandamide, further increasing
[54]
signaling through both CB1 and CB2 receptors . It has been previously reported that signaling through
CB1 receptors has neurotoxic and psychoactive effects, while signaling through CB2 receptors reduces
[55]
the toxicity of the LPS-stimulated microglia . As BV-2 cells express functionally active CB2 receptors,
we determined whether the effect of p-aminophenol and AM404 on microglial activation was mediated
by CB2 receptor signaling [56-58] . Microglia were incubated with various concentrations of the selective CB2
receptor antagonist SR144528 (0.02, 0.5, 2 µmol/L) for 15 min prior to treatment with p-aminophenol or
AM404. Figures 6 and 7 indicate that CB2 receptor blockade did not reduce the secretion of NO from LPS-
stimulated BV-2 cells in the absence of p-aminophenol and AM404. SR144528 also did not attenuate the
inhibitory effects of p-aminophenol [Figure 6] or AM404 [Figure 7]; however, treatment with SR144528
did inhibit NO secretion in the presence of 2 µmol/L p-aminophenol [Figure 6]. These data indicate that
neither AM404 nor p-aminophenol exert their effect on NO production by BV-2 cells through engaging
the CB2 receptor and that CB2 receptor blockade may enhance the inhibitory effects of p-aminophenol on
microglial activation.