The Journal of Pain, Vol 19, No 5 (May), 2018: pp 487-495

Available online at www.jpain.org and www.sciencedirect.com

The Involvement of the Endocannabinoid System in

the Peripheral Antinociceptive Action of Ketamine

Renata C. M. Ferreira,* Marina G. M. Castor,* Fabiana Piscitelli,† Vincenzo Di Marzo,†

Igor D. G. Duarte,* and Thiago R. L. Romero*
*Department of Pharmacology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Minas
Gerais, Brazil.
†
Endocannabinoid Research Group, Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Naples, Italy.

Abstract: Ketamine has been widely used as an analgesic and produces dissociative anesthetic
effects. The antinociceptive effects of ketamine have been studied, but the involvement of
endocannabinoids in these effects has not yet been investigated. In this study, we evaluated the
involvement of the endocannabinoid system in the peripheral antinociceptive effects induced by
ketamine. All drugs were administered via the intraplantar route. To induce hyperalgesia, rat paws
were injected with prostaglandin E2 (2 µg per paw). The nociceptive threshold for mechanical
stimulation was measured in the right hind paw of Wistar rats using the Randall–Selitto test. The
tissue levels of anandamide (AEA), 2-arachidonoylglycerol, palmitoylethanolamide, and
oleoylethanolamide were measured using liquid chromatography coupled to single quadrupole
mass spectrometry. The administration of the cannabinoid receptor type 1 (CB1) antagonist, N(piperidine-
1yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl 1 pyrazolcarboxamide (20, 40, and 80 µg per
paw), but not the cannabinoid receptor type 2 antagonist, 6-iodo-2-methyl-1-(2-morpholinoethyl)-
1H-indol-3-yl) (4-methoxyphenyl) methanone (100 µg per paw), antagonized the ketamine-induced
peripheral antinociception in a dose-dependent manner. Additionally, the administration of the
endocannabinoid metabolizing enzyme inhibitor (.5 µg per paw) or an AEA reuptake inhibitor,
(5Z,8Z,11Z,14Z)N(4Hydroxy2methylphenyl)5,8,11,14 eicosatetraenamide (2.5 µg per paw) signifi-
cantly enhanced low-dose ketamine-induced peripheral antinociception. AEA paw levels were increased
only after ketamine administration to prostaglandin E2-injected paws. These data suggest that
ketamine, in the presence of a nociceptive stimulus, induces a selective release of AEA levels and
subsequent CB1 cannabinoid activation at the peripheral level.
Perspective: This study suggests that ketamine antinociception depends at least in part on
AEA release and CB1 cannabinoid receptor activation in inflammatory conditions. This study
could potentially help clinicians in the use of ketamine as a peripheral analgesic for inflammatory
pain.
© 2017 by the American Pain Society
Key words: Ketamine, endocannabinoids, anandamide, cannabinoid receptors, peripheral
antinociception.

Received December 13, 2016; Revised August 11, 2017; Accepted Decem-
ber 3, 2017.
The research funding were provided by Fundação de Amparo a Pesquisa
de Minas Gerais PPMFAPEMIG 2015 Process number 0474-15 and Fundação
de Amparo a Pesquisa de Minas Gerais UNIVERSAL Process n° number
01307-14. Fellowship was provided by Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior.
The authors have no conflicts of interest to declare.
Address reprint requests to Thiago R. L. Romero, PhD, Department of
Pharmacology, Institute of Biological Sciences, Federal University of Minas
Gerais, Av. Antônio Carlos 6627, 31270-901, Belo Horizonte, MG, Brazil.
E-mail: thiromero@ufmg.br
1526-5900/$36.00
© 2017 by the American Pain Society
https://doi.org/10.1016/j.jpain.2017.12.002
K
etamine was first tested clinically by Domino and
co-authors in 196520 and, since then, has been ex-
tensively used for sedation, induction, and
maintenance of general anesthesia, as well as premedi-
cation and postoperative analgesic.59 In addition, there
are reports of its use for the treatment of acute pain,23
cancer, 45 neuropathic pain, 44 and burn, 65 as well as
phantom limb pain.3
In addition to the classic mechanism of action of
ketamine via the antagonism of the N-methyl-D-aspartate
receptor, the modulation of other signaling pathways by
ketamine has also been proposed, such as the inhibition

487
488 The Journal of Pain
66
of voltage-gated calcium channels and the activation
of cholinergic,1 serotonergic,41 and opioidergic5 systems.
Furthermore, Romero and Duarte54 showed the partici-
pation of the nitric oxide/cyclic guanosine monophosphate/
adenosine triphosphate-sensitive potassium channel
pathway in the peripheral antinociception that was
induced by ketamine treatment.
However, the relationship between ketamine and the
endocannabinoid system, consisting of 2 cannabinoid re-
ceptors (CBRs), type 1 (CB1) and type 2 (CB2), and of
their endogenous ligands, anandamide (AEA) and
2-arachidonoylglycerol (2-AG) as well as enzymes for AEA
and 2-AG biosynthesis and degradation, has not yet been
elucidated. Data that could be used as a link between
ketamine and the cannabinoid system were presented
in the study by Hesselink and Kopsky.32 These investigators
reported that the combination of palmitoylethanolamide
(PEA), an endocannabinoid-related molecule with no af-
finity for or direct activity at CBRs, and ketamine 10%
cream, reduced pain in more than 50% of patients with
chronic pain syndrome. This treatment also resulted in
reductions in skin swelling. However, the relationship
between PEA and the endogenous cannabinoid system
is not clear; PEA has been classified as a CB2 cannabinoid
agonist12,31,57 as well as a nuclear receptor peroxisome
proliferator-activated receptor alpha agonist.38
CBRs CB1 CB2 were cloned in 1990 and 1993, respec-
tively, as G protein-coupled receptors associated with Gi/0
subunits, 37 and activated by Cannabis psychotropic
principle, Δ 9 -tetrahydrocannabinol, as well as by
the “endocannabinoids” AEA 17 and 2-AG. 42 The
endocannabinoids are involved in several physiological
and pathological functions, such as analgesia, 26
inflammation,30 immunomodulation,11 inhibition of tumor
cell growth,46 regulation of food intake,7 and epilepsy.39
Several studies have shown the antinociceptive effect
of CB1 and CB2 agonists22 and ketamine.19 However, the
involvement of the endocannabinoid system in the
antinociceptive effect of this latter drug has not been in-
vestigated to date. Therefore, the aim of this study was
to determine whether or not activation of the
endocannabinoid system could be triggered by the pe-
ripheral administration of ketamine.
Methods
Animals
All experiments used male Wistar rats weighing
between 180 and 200 g (from CEBIO, Federal University
of Minas Gerais, Minas Gerais, Brazil). The animals were
placed in plastic boxes with forage shavings, with free
access to water and food and kept in the trial room 2
days before the experiments, for habituation. They were
housed in a temperature-controlled room (23°C ± 1°C),
on an automatic 12-hour light/dark cycle. All tests were
conducted during the light phase (8:00 AM to 5:00 PM). All
animal procedures and protocols were approved by the
Ethics Committee on Animal Experimentation of the
Federal University of Minas Gerais and are in accor-
dance with the recommendations for evaluation of
Ketamine Induces Antinociception by CB1 Activation
experimental pain in animals.67 After the experimental
procedures, the animals were euthanized using an in-
traperitoneal injection of urethane 1.25 g/kg.
Measurement of Nociceptive Threshold
Hyperalgesia was induced using subcutaneous injec-
tion of prostaglandin E2 (PGE2; 2 µg) into the plantar
surface of the hind paw. Hyperalgesia was measured using
the mechanical paw pressure test described by Randall
and Selitto.49 An analgesiometer was used (Ugo-Basile SRL,
Varese, Italy) with a cone-shaped paw-presser with a
rounded tip, which applies a linearly increasing force to
the hind paw. The weight in grams required to elicit the
nociceptive response of paw withdrawal was deter-
mined as the nociceptive threshold. A cutoff value of
300 g was used to reduce the possibility of damage to
the paws.
The nociceptive threshold was expressed in grams and
it was determined in the right hind paw according to the
average of 3 consecutive trials recorded before (0 time)
and after PGE2 injection (3 hours). The time course of re-
sponses was computed. The time of injections and time
of nociceptive threshold measurements, as well as the
doses used, were on the basis of literature data and on
results of pilot experiments.57
Drug Administration
PGE2 (2 µg; Enzo Life Sciences, Farmingdale, NY)
was diluted in ethanol 2% whereas ketamine (10%;
Vetbrands; Sao Paulo, Brazil), was dissolved in physi-
ological saline. N(piperidine-1yl)-5-(4-iodophenyl)-1-(2,4-
dichlorophenyl)-4-methyl 1 pyrazolcarboxamide, a CB1
CBR antagonist (AM251; 20, 40, and 80 µg; Tocris, Pitts-
burgh, PA), 6-iodo-2-methyl-1-(2-morpholinoethyl)-1H-
indol-3-yl) (4-methoxyphenyl) methanone, a CB2 CBR
antagonist (AM630; 100 µg; Tocris); (5Z,8Z, 11Z,14Z)-
5,8,11,14-eicosatetraenyl-methylester phosphonofluoridic
acid, an irreversible nonselective fatty acid amide hydro-
lase (FAAH) inhibitor, (MAFP; .5 µg; Tocris) and
(5Z,8Z,11Z,14Z)N(4Hydroxy2methylphenyl)5,8,11,14
eicosatetraenamide, a selective inhibitor of AEA cellu-
lar reuptake, (VDM11, 2.5 µg, Tocris) were each diluted
in 10% dimethyl sulfoxide in sterile saline. All of the
aforementioned drugs were injected into the right plantar
surface of the paw in a volume of 50 µL per paw, except
PGE2 (100 µL per paw).
Endocannabinoid Extraction and
Quantification
Procedure of Extraction
The paw tissue from treated animals was extracted im-
mediately after sacrifice. The tissue was homogenized in
2 vol of chloroform/methanol/Tris-HCl (50 mM; 2:1:1) con-
taining 10 pmol of d8-AEA and d8-2-AG. Deuterated
standards were synthesized from d8-arachidonic acid and
ethanolamine or glycerol as described in Devane et al17
and Bisogno et al,9 respectively. Homogenates were
Ferreira et al
centrifuged at 13,000g for 16 minutes (4°C), and the
aqueous phase plus debris were collected and extracted
again twice with 1 vol of chloroform. The organic phases
from the 3 extractions were pooled, and the organic sol-
vents evaporated in a rotating evaporator. Lyophilized
samples were then stored frozen at −80°C in a nitrogen
atmosphere until analyzed.
Analysis of Endocannabinoid Contents
Lyophilized extracts were resuspended in chloroform/
methanol 99:1 by volume. The solutions were then
purified by open-bed chromatography on silica as de-
scribed in Bisogno et al.9 Fractions eluted with chloroform/
methanol 9:1 by volume (containing AEA and 2-AG) were
collected, and the excess solvent evaporated with a ro-
tating evaporator. In addition, aliquots were analyzed by
isotope dilution-liquid chromatography/atmospheric pres-
sure chemical ionization/mass spectrometry (MS), carried
out under conditions described previously40 and allow-
ing the separations of 2-AG and AEA. MS detection was
carried out in the selected ion-monitoring mode using
m/z values of 356 and 348 (molecular ions, 1 for deuter-
ated and undeuterated AEA) and 384.35 and 379.35
(molecular ions, 1 for deuterated and undeuterated 2-AG).
The area ratios between signals of deuterated and
undeuterated AEA varied linearly with varying amounts
of undeuterated AEA (30 fmol to 100 pmol). The same
applied to the area ratios between signals of deuter-
ated and undeuterated 2-AG in the 100-pmol to 20-
nmol interval. Therefore, AEA and 2-AG levels in unknown
samples were calculated on the basis of their area ratios
with the internal deuterated standard signal areas.
Experimental Protocol
In all protocols, ketamine was administered in the right
hind paw 2 hours and 55 minutes after local injection of
PGE2 and the nociceptive threshold was measured at the
third hour (peak of PGE2 nociception). To exclude sys-
temic effect, PGE2 was injected into both hind paws, and
ketamine into the right paw. The nociceptive threshold
was measured in both hind paws at the third hour.
AM251, AM630, MAFP, and VDM11 were adminis-
tered 10 minutes before ketamine injection.
To measure endocannabinoid (AEA and 2-AG) levels,
the paw tissues were collected always 3 hours after local
injection of PGE2, frozen in liquid nitrogen, and then
processed.
Statistical Analysis
The data were statistically analyzed using 1-way analy-
sis of variance followed by Bonferroni test for multiple
comparisons. Probabilities of <5% (P < .05) were consid-
ered to be statistically significant.
Results
Intraplantar injection of ketamine (80 µg per paw and
160 µg per paw) were made to exclude systemic effects
against hyperalgesia induced by PGE2 (2 µg per paw). The
The Journal of Pain 489
Figure 1. Exclusion of systemic antinociceptive effect of
ketamine (Ket) in hyperalgesic paws. PGE2 was injected into the
right hind paw (R-Paw) and left hind paw (L-Paw) and Ket 80
and 160 µg per paw was injected into the R-Paw. Ket 80 µg
showed a peripheral effect whereas Ket 160 µg induced a sys-
temic effect. Each column represents the mean ± standard error
of the mean (n = 4). *Indicates a significant difference com-
pared with PGE2 with saline (Veh; R-Paw and L-Paw; P < .05,
analysis of variance with Bonferroni post test).
dose of 80 µg per paw into the right paw did not produce
an antinociceptive effect in the left paw with vehicle,
however, the dose of 160 µg per paw produced
antinociception on the contralateral paw, which dis-
carded this dose as a peripheral effect (F5,18 = 71.84;
P < .0001; Fig 1).
To verify the possible involvement of CBRs in ketamine-
induced peripheral antinociceptive effect, the CB1 CBR
antagonist, AM251 (20, 40, and 80 µg per paw), or the
CB2 CBR antagonist, AM630 (100 µg per paw), were in-
jected before ketamine administration (80 µg per paw).
AM251 was able to antagonize the antinociceptive effect
of ketamine in a dose-dependent manner (F7,24 = 181.5;
P < .0001; Fig 2A), but did not induce hyperalgesia or
antinociception when used alone. The maximum dose of
AM251 (80 µg per paw) had no effect on the contralat-
eral paw (F5,18 = 142.4; P < .0001; Fig 2B). However, AM630
was not able to block the antinociceptive effect induced
by ketamine (F2,9 = 786.1; P < .0001; Fig 3).
To evaluate endocannabinoid involvement in this re-
sponse, the animals were pretreated with the nonselective
FAAH (the enzyme catalyzing AEA hydrolysis) MAFP (.5 µg
per paw; F5,18 = 106.3; P < .0001), at nonantinociceptive
dose. It potentiated the peripheral antinociceptive effect
of a low dose of ketamine (20 µg per paw; Fig 4A). The
exclusion of systemic effect of MAFP .5 µg per paw
(F5,20 = 29.01; P < .0001) against PGE2 (2 µg per paw)
showed that this drug had no effect on the contralat-
eral paw (Fig 4B). In addition, we evaluated whether
AM251 80 µg per paw is able to block the antinociceptive
effect induced by MAFP .5 µg per paw plus ketamine
80 µg per paw. Fig 4C shows that AM251 may antago-
nize this effect (F2,12 = 19.47; P = .0002).
To evaluate endocannabinoid involvement, the animals
were pretreated with the inhibitor of AEA cellular
reuptake VDM11 (2.5 µg per paw; F5,18 = 37.52; P < .0001)
at nonantinociceptive dose. This pretreatment potenti-
ated the peripheral antinociceptive effect of a low dose
of ketamine (20 µg per paw; Fig 5).
490 The Journal of Pain Ketamine Induces Antinociception by CB1 Activation

Figure 2. (A) Administration of a CB1 receptor antagonist blocks ketamine (Ket)-induced peripheral antinociception in hyperal-
gesic paws. AM251 (20, 40, and 80 µg) were injected 10 minutes before Ket (80 µg) and antagonized Ket-antinociceptive effect
against PGE2 injection. AM251 did not induce hyperalgesia or antinociception when used alone. Each column represents the mean ± stan-
dard error of the mean (n = 4). * and # indicate a significant difference compared with PGE2 with Veh 2 and Veh 1, and PGE2 with Ket
80 µg with Veh 1, respectively (P < .05, analysis of variance with Bonferroni post test). (B) Exclusion of systemic antinociceptive effect
of AM251 in hyperalgesic paws. PGE2 was injected into the right hind paw (R-Paw) and left hind paw (L-Paw), Ket 80 µg per paw
was injected into the R-Paw and AM251 80 µg per paw was injected into the L-Paw. AM251 did not show a systemic effect. Each
column represents the mean ± standard error of the mean. (n = 4). Veh 1 (L-Paw) = dimethyl sulfoxide 10% and Veh 2 (R-
Paw) = saline. *Indicates a significant difference compared with PGE2 with Veh 2 (P < .05, analysis of variance with Bonferroni post
test).

To analyze the concentrations of the endocannabinoids Discussion

AEA and 2-AG, as well as of the AEA-related com-
pounds, PEA and oleoylethanolamide (OEA), in paws after
treatment with ketamine, isotope-dilution liquid
chromatography-MS analysis of the paw lipid extracts was
used. Treatment with ketamine or PGE2 alone was not
able to modulate endocannabinoid levels. However, the
co-injection of these 2 drugs induced a significant in-
crease in AEA concentrations (F4,14 = 3.652; P = .0307),
suggesting a synergistic effect between ketamine and
PGE2. However, the same response was not observed for
2-AG, PEA, and OEA. The levels of these compounds re-
mained unaltered in all groups (Fig 6).
Figure 3. Administration of a CB2 receptor antagonist did not
block ketamine (Ket)-induced peripheral antinociception in hy-
peralgesic paws. AM630 (100 µg) was injected 10 minutes before
Ket (80 µg) and did not antagonize Ket-antinociceptive effect
against PGE2 injection. Each column represents the mean ± stan-
dard error of the mean (n = 4). *Indicates a significant difference
compared with PGE2 with Veh 2 and Veh 1 (P < .05, analysis of
variance with Bonferroni post test). Veh 1 = DMSO 10%, Veh
2 = Saline.
In this study we investigated the hypothesis that the
endocannabinoid system might be involved in the pe-
ripheral analgesic actions of ketamine against the
nociceptive response to a key mediator of pain and in-
flammation (ie, PGE2). Previous studies show that ketamine
produces peripheral antinociceptive effects against
PGE2-induced hyperalgesia when this is evaluated using
the mechanical paw pressure test.56 Indeed, PGE2 is known
to sensitize primary afferent neurons through activa-
tion of Gs alpha subunit protein-coupled prostaglandin
E2 receptor type receptors in response to chemical,
thermal, and mechanical stimuli.8,48 The use of PGE2 is ad-
vantageous over other models of inflammatory
hyperalgesia, such as that induced by carrageenan,
because it allows the study of the peripheral effect of a
given drug directly and selectively on a major mediator
of pain, without the influence of the several other me-
diators that are normally produced during the
inflammatory process.64 However, the study of the pe-
ripheral mechanisms of pain, such as those triggered by
intrapaw injection of PGE2, allows in principle to develop
new analgesic drugs with fewer central side effects.
Previous work showed that ketamine might produce
analgesia through molecular mechanisms besides N-methyl-
D-aspartate antagonism, such as the peripheral activation
of the nitric oxide/cyclic guanosine monophosphate/ATP-
sensitive potassium channels pathway.54 In addition, other
studies have shown that the endocannabinoid AEA50,51
and its congener and non-CBR ligand, PEA,55 might act
through similar peripheral pathways, and that the latter
compound may potentiate the clinical efficacy of
ketamine.32 In the same context, several studies indicate
that noncannabinoid analgesics, such as nonsteroidal anti-
inflammatory drugs, exert their mechanism of action
through the activation of the endocannabinoid system,4,29
highlighting the possibility of interactions between this
system and ketamine. Therefore, to investigate this
Ferreira et al The Journal of Pain 491

Figure 4. (A) Potentiation by MAFP of a lower dose of ketamine (Ket)-induced peripheral antinociception in hyperalgesic paws.
MAFP (.5 µg per paw) was injected 10 minutes before Ket (20 µg) and potentiated Ket-antinociceptive effect against PGE2 injec-
tion. MAFP did not induce hyperalgesia or antinociception when used alone. Each column represents the mean ± standard error
of the mean (SEM; n = 4). * and # indicates a significant difference compared with PGE2 with Veh 2 with Veh 1 and PGE2 with Ket
20 µg with Veh 1, respectively (P < .05, analysis of variance [ANOVA] with Bonferroni post test). Veh 1 = Ethanol 3%, Veh 2 = Saline,
Veh 3 = Ethanol 2%. (B) Exclusion of systemic effect of MAFP in hyperalgesic paws. PGE2 was injected into the right hind paw (R-
Paw) and left hind paw (L-Paw), Ket 20 µg per paw was injected into the R-Paw and MAFP .5 µg was injected into the L-Paw. MAFP
did not show a systemic effect. Each column represents the mean ± SEM (n = 4). *Indicates a significant difference compared with
PGE2 with Veh 2 (P < .05, ANOVA with Bonferroni post test). Veh 1 (L-Paw) = ethanol 3% and Veh 2 (R-Paw) = saline. (C) AM251
blocks antinociceptive effect induced by MAFP with Ket in hyperalgesic paws. MAFP (.5 µg) and AM251 (80 µg) were injected 10 minutes
before Ket (20 µg). AM251 blocked the antinociceptive effect induced by MAFP with Ket. Each column represents the mean ± SEM
(n = 4). *Indicates a significant difference compared with PGE2 with Veh 2 and Veh 1 (P < .05, ANOVA with Bonferroni post test).
Veh 1 = DMSO 10%/ethanol 3% and Veh 2 = Saline.

hypothesis, we analyzed the involvement of CBRs (CB1
and CB2) in the ketamine-induced antinociceptive effect
against PGE2 by using the respective inverse agonists/
antagonists of the 2 receptors (ie, AM251 and AM630),57
which have been previously reported to antagonize the
analgesic actions of CBR agonists.22,57
Our results show that AM251 was able to antagonize, in
a dose-dependent manner, the peripheral antinociception
induced by ketamine. Indeed, CB1 receptors are densely
expressed in the dorsal root ganglia and in the peripheral
Figure 5. Potentiation by VDM11 of a lower dose of ketamine
(Ket)-induced peripheral antinociception in hyperalgesic paws.
VDM11 (2.5 µg) was injected 10 minutes before Ket (20 µg) and
potentiated Ket-antinociceptive effect against PGE2 injection.
MAFP did not induce hyperalgesia or antinociception when used
alone. Each column represents the mean ± standard error of the
mean (n = 4). * and # indicates a significant difference com-
pared with PGE2 with Veh 2 with Veh 1 and PGE2 with Ket
20 µg and Veh 1, respectively (P < .05, analysis of variance
with Bonferroni post test). Veh 1 = tocrisolve 10%, Veh 2 = Saline,
Veh 3 = ethanol 2%.
terminals of primary afferent neurons,2,10,35 which anatomi-
cally supports the finding that antinociception induced by
the local administration of ketamine occurs partly through
these receptors. However, treatment with the CB2 receptor
antagonist (AM630) was unable to reverse the peripheral
antinociceptive effect of ketamine at the maximum dose
previously reported to reverse the effect of a CB2 CBR
agonist.57 In their study, Hohmann and Herkenham34 were
not able to identify the presence of CB2 in the dorsal
root ganglion, although other studies have reported
such evidence6,24 as well as the occurrence of CB2 in periph-
eral terminals.21,61 Such presence would explain, for
example, why the peripheral analgesic effects of PEA13,31,57
are attenuated by CB2 antagonists. Furthermore, previous
studies have described several agents that induce periph-
eral antinociception through the activation of CB1 as well
as CB2 receptors.16,52 Nevertheless, our data indicate that
ketamine acts via activation of CB1 selectively over CB2
receptors.
When we observed the involvement of CB1 receptors
in the antinociceptive effect induced by ketamine, we
then evaluated the role of endocannabinoids in this
mechanism. FAAH and monoacylglycerol lipase are known
to be responsible for the degradation of AEA and 2-AG,
respectively. 47 Therefore, we studied the effect on
ketamine of an easily available compound (ie, MAFP),
which is able to inhibit these enzymes and significantly
increase endocannabinoid levels in tissues. Further-
more, we also studied the effect of VDM11, an
endocannabinoid reuptake inhibitor,28 also a substrate
for FAAH, capable of reducing the metabolism of FAAH
hydrolysis products (eg, AEA and PEA)33,63 inducing the
inhibition of this enzyme, consequently, evaluating
the participation of endocannabinoids release on the
ketamine-antinociceptive effect.
We found that MAFP and VDM11 were both able to
potentiate the ketamine-induced antinociceptive effects.
492 The Journal of Pain
Figure 6. Increase of AEA levels, but not 2-AG, PEA, and OEA in rat hind paws. Each column represents the mean ± standard error
of the mean (n = 4) of the measured levels expressed in pmol/g by AEA and pmol/mg by others. # and * and & indicates a signifi-
cant difference compared with saline (Sal), ketamine (Ket), and PGE2 with saline (PGE2 + Sal), respectively (P < .05, analysis of variance
with Bonferroni post test).
da Fonseca Pacheco and coauthors14 used MAFP in the
peripheral pathway and showed that it was able to
intensify the antinociception that was generated by ex-
posure to low doses of morphine. This finding suggests
the participation of the endocannabinoid system when
the opioidergic system is activated. In addition, VDM11
was shown to prolong the antinociceptive effect that was
evoked by exercise27 and caused a potentiation of the
antinociception that was induced by stress.36 This finding
suggests that the endocannabinoid system is also in these
conditions. Besides, the use of AM251 and MAFP to-
gether with ketamine, was not able to reverse the entire
potentiated analgesic effect, suggesting that this effect
could be through a non-CB1 mechanisms when this re-
ceptor is antagonized.
To further evaluate this hypothesis, we measured the
levels of endocannabinoids, PEA and OEA, in rat paw
tissues after ketamine-induced counteraction of the no-
ciceptive response to PGE2. Our liquid chromatography-
MS data indicated that, in the presence of PGE2, ketamine
selectively increases AEA levels in the treated paw. The
fact that AEA, unlike 2-AG, preferentially activates CB1
over CB2 receptors37 may explain why the effects of
ketamine were shown in this study to be antagonized
by a CB1, and not a CB2, receptor antagonist. Our data,
however, do not explain how ketamine induces this in-
crease in paw AEA levels, although some hypotheses could
be made.
On the basis of previous research, endocannabinoid bio-
synthesis could be triggered by neuronal depolarization
and intracellular calcium elevation.18 However, although
PGE2 may cause intracellular calcium elevation,25 the clas-
sical mechanism of ketamine involving blockade of
N-methyl-D-aspartate receptor would reduce Ca2+ influx.
Ketamine Induces Antinociception by CB1 Activation
Therefore, mechanisms alternative to intracellular calcium
elevation are likely to underlie the effect of ketamine
on AEA levels. For example, the analgesic effect of
ketamine may be associated with the opioid system at
the peripheral level. Accordingly, administration of the
nonselective opioid antagonist naloxone was able to
reverse the antinociceptive effect induced by ketamine.5
The same response was observed using the formalin test
in rats,62 suggesting a relationship between this drug and
the opioid system. Indeed, ketamine may even directly
bind to opioid receptors.60 However, the activation of
the µ-opioid receptor was suggested to lead to
endocannabinoid release in central15 and peripheral14
systems, and CB1 antagonists were reported to reduce
morphine-induced analgesia.43 Additionally, CB1 and µ-
and δ-opioid receptors may form heteromers, as sug-
gested by Rios and co-authors53 and Rozenfeld and
co-authors,58 reinforcing the idea of an interaction between
opioid and CBRs. Therefore, it is possible that ketamine
activates the opioid system, which in turn may activate
CB 1 receptors either directly or via increased AEA
biosynthesis.
Conclusions
This study demonstrated at least part of the
mechanism of action underlying the peripheral
antinociceptive effect induced by ketamine. This
mechanism could be possibly through release of the
endogenous cannabinoid, AEA, and subsequent CB1 re-
ceptor activation at the peripheral level, leading
to counteract PGE2-induced analgesia. Because prosta-
glandin E2 type receptor activation by PGE2 produces
its effects through elevation of adenosine 3’,5’-cyclic
Ferreira et al
monophosphate levels, it is possible that the
antinociceptive action of ketamine is due to CB1-mediated
inhibition of adenylyl cyclase.37 Further pharmacologi-
cal and biochemical studies are required to investigate
References
1. Abelson KS, Goldkuhl RR, Nylund A, Höglund AU: The
effect of ketamine on intraspinal acetylcholine release: In-
volvement of spinal nicotinic receptors. Eur J Pharmacol 534:
122-128, 2006
2. Ahluwalia J, Urban L, Capogna M, Bevan S, Nagy I: Can-
nabinoid 1 receptors are expressed in nociceptive primary
sensory neurons. Neuroscience 100:685-688, 2000
3. Alviar MJ, Hale T, Dungca M: Pharmacological interven-
tions for treating phantom limb pain. Cochrane Database
Syst Rev (12):06380, 2011
4. Ates M, Hamza M, Seidel K, Kotalla CE, Ledent C, Gühring
H: Intrathecally applied flurbiprofen produces an
endocannabinoid-dependent antinociception in the rat for-
malin test. Eur J Neurosci 17:597-604, 2003
5. Baumeister A, Advokat C: Evidence for a supraspinal
mechanism in the opioid-mediated antinociceptive effect of
ketamine. Brain Res 566:351-353, 1991
6. Beltramo M, Bernardini N, Bertorelli R, Campanella M,
Nicolussi E, Fredduzzi S, Reggiani A: CB2 receptor-mediated
antihyperalgesia: Possible direct involvement of neural mecha-
nisms. Eur J Neurosci 23:1530-1538, 2006
7. Berry EM, Mechoulam R: Tetrahydrocannabinol and
endocannabinoids in feeding and appetite. Pharmacol Ther
95:185-190, 2002
8. Bevan S: Nociceptive peripheral neurons: Cellular prop-
erties, in Melzack R, Wall D (eds): Textbook of Pain, 4th ed.
London, Churchill Livingstone, 1999, pp 85-103
9. Bisogno T, Maurelli S, Melck D, De Petrocellis L, Di Marzo
V: Biosynthesis, uptake, and degradation of anandamide and
palmitoylethanolamide in leukocytes. J Biol Chem 272:3315-
3323, 1997
10. Bridges D, Rice AS, Egertová M, Elphick MR, Winter J,
Michael GJ: Localisation of cannabinoid receptor 1 in rat
dorsal root ganglion using in situ hybridisation and immu-
nohistochemistry. Neuroscience 119:803-812, 2003
11. Buckley NE, McCoy KL, Mezey E, Bonner T, Zimmer A,
Felder CC, Glass M, Zimmer A: Immunomodulation by can-
nabinoids is absent in mice deficient for the cannabinoid CB(2)
receptor. Eur J Pharmacol 396:141-149, 2000
12. Calignano A, La Rana G, Giuffrida A, Piomelli D: Control
of pain initiation by endogenous cannabinoids. Nature 394:
277-281, 1998
13. Calignano A, La Rana G, Piomelli D: Antinociceptive
activity of the endogenous fatty acid amide,
palmitylethanolamide. Eur J Pharmacol 419:191-198, 2001
14. da Fonseca Pacheco D, Klein A, de Castro Perez A, da
Fonseca Pacheco CM, de Francischi JN, Duarte ID: The µ-opioid
receptor agonist morphine, but not agonists at δ- or κ-opioid
receptors, induces peripheral antinociception mediated by
cannabinoid receptors. Br J Pharmacol 154:1143-1149, 2008
The Journal of Pain 493
this hypothesis and elucidate whether ketamine may
act as an exogenous agonist of CBRs and what its affin-
ity for those receptors is, besides the mechanisms through
which ketamine may release AEA in the periphery.
15. da Fonseca Pacheco D, Klein A, Perez AC, da Fonseca
Pacheco CM, de Francischi JN, Reis GM, Duarte ID: Central
antinociception induced by µ-opioid receptor agonist mor-
phine, but not δ- or k-, is mediated by cannabinoid CB1
receptor. Br J Pharmacol 158:225-231, 2009
16. dos Santos GG, Dias EV, Teixeira JM, Athie MC, Bonet
IJ, Tambeli CH, Parada CA: The analgesic effect of dipyrone
in peripheral tissue involves two different mechanisms: Neu-
ronal K(ATP) channel opening and CB(1) receptor activation.
Eur J Pharmacol 741:124-131, 2014
17. Devane WA, Hanus L, Breuer A, Pertwee RG, Steven-
son LA, Griffin G, Gibson D, Mandelbaum A, Etinger A,
Mechoulam R: Isolation and structure of a brain constitu-
ent that binds to the cannabinoid receptor. Science 258:
1946-1949, 1992
18. Di Marzo V: Endocannabinoid signaling in the brain: Bio-
synthetic mechanisms in the limelight. Nat Neurosci 4:9-15,
2011
19. Domino EF: Taming the ketamine tiger. 1965. Anesthe-
siology 113:678-684, 2010
20. Domino EF, Chodoff P, Corssen G: Pharmacologic effects
of CI-581, a new dissociative anesthetic, in man. Clin
Pharmacol Ther 6:279-291, 1965
21. Duncan M, Mouihate A, Mackie K, Keenan CM, Buckley
NE, Davison JS, Patel KD, Pittman QJ, Sharkey KA: Canna-
binoid CB2 receptors in the enteric nervous system modulate
gastrointestinal contractility in lipopolysaccharide-treated rats.
Am J Physiol Gastrointest Liver Physiol 295:78-G87, 2008
22. Ebrahimzadeh M, Haghparast A: Analgesic effects of can-
nabinoid receptor agonist WIN55,212-2 in the nucleus
cuneiformis in animal models of acute and inflammatory pain
in rats. Brain Res 1420:19-28, 2011
23. Elia N, Tramer MR: Ketamine and postoperative pain—a
quantitative systematic review of randomised trials. Pain 113:
61-70, 2005
24. Elmes SJ, Jhaveri MD, Smart D, Kendall DA, Chapman
V: Cannabinoid CB2 receptor activation inhibits mechani-
cally evoked responses of wide dynamic range dorsal horn
neurons in naive rats and in rat models of inflammatory and
neuropathic pain. Eur J Neurosci 20:2311-2320, 2004
25. Ferreira SH, Nakamura M: I—Prostaglandin hyperalge-
sia, a cAMP/Ca2+ dependent process. Prostaglandins 18:179-
190, 1979
26. Fox A, Bevan S: Therapeutic potential of cannabinoid
receptor agonists as analgesic agents. Expert Opin Investig
Drugs 14:695-703, 2005
27. Galdino G, Romero TR, Silva JF, Aguiar DC, de Paula AM,
Cruz JS, Parrella C, Piscitelli F, Duarte ID, Di Marzo V, Perez
AC: The endocannabinoid system mediates aerobic exercise-
induced antinociception in rats. Neuropharmacology 77:
313-324, 2014
28. Gamaleddin I, Guranda M, Goldberg SR, Le Foll B: The
selective anandamide transport inhibitor VDM11 attenuates
494 The Journal of Pain
reinstatement of nicotine seeking behaviour, but does not
affect nicotine intake. Br J Pharmacol 164:1652-1660, 2011
29. Gühring H, Hamza M, Sergejeva M, Ates M, Kotalla CE,
Ledent C, Brune K: A role for endocannabinoids in
indomethacin-induced spinal antinociception. Eur J Pharmacol
454:153-163, 2002
30. Guindon J, De Lean A, Beaulieu P: Local interactions
between anandamide, an endocannabinoid, and ibupro-
fen, a nonsteroidal anti-inflammatory drug, in acute and
inflammatory pain. Pain 121:85-93, 2006
31. Helyes Z, Németh J, Thán M, Bölcskei K, Pintér E,
Szolcsányi J: Inhibitory effect of anandamide on
resiniferatoxin-induced sensory neuropeptide release in vivo
and neuropathic hyperalgesia in the rat. Life Sci 73:2345-
2353, 2003
32. Hesselink JM, Kopsky DJ: Treatment of chronic re-
gional pain syndrome type 1 with palmitoylethanolamide
and topical ketamine cream: Modulation of nonneuronal cells.
J Pain Res 6:239-245, 2013
33. Hillard CJ, Shi L, Tuniki VR, Falck JR, Campbell WB: Studies
of anandamide accumulation inhibitors in cerebellar granule
neurons: Comparison to inhibition of fatty acid amide hy-
drolase. J Mol Neurosci 33:18-24, 2007
34. Hohmann AG, Herkenham M: Cannabinoid receptors
undergo axonal flow in sensory nerves. Neuroscience 92:
1171-1175, 1999
35. Hohmann AG, Herkenham M: Localization of central can-
nabinoid CB1 receptor messenger RNA in neuronal
subpopulations of rat dorsal root ganglia: A double label
in situ hybridization study. Neuroscience 90:923-931, 1999
36. Hohmann AG, Suplita RL, Bolton NM, Neely MH, Fegley
D, Mangieri R, Krey JF, Walker JM, Holmes PV, Crystal JD,
Duranti A, Tontini A, Mor M, Tarzia G, Piomelli D: An
endocannabinoid mechanism for stress-induced analgesia.
Nature 435:1108-1112, 2005
37. Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P,
Devane WA, Felder CC, Herkenham M, Mackie K, Martin BR,
Mechoulam R, Pertwee RG: International Union of Pharma-
cology. XXII. Classification of cannabinoid receptors.
Pharmacol Rev 54:161-202, 2002
38. Lo Verme J, Russo R, La Rana G, Fu J, Farthing J, Mattace-
Raso G, Meli R, Hohmann A, Calignano A, Piomelli D: Rapid
broad- spectrum analgesia through activation of peroxi-
some proliferator- activated receptor-alpha. J Pharmacol Exp
Ther 319:1051-1061, 2006
39. Lutz B: On-demand activation of the endocannabinoid
system in the control of neuronal excitability and epilepti-
form seizures. Biochem Pharmacol 68:1691-1698, 2004
40. Marsicano G, Goodenough S, Monory K, Hermann H, Eder
M, Cannich A, Azad SC, Cascio MG, Gutiérrez SO, van der
Stelt M, López-Rodriguez ML, Casanova E, Schütz G,
Zieglgänsberger W, Di Marzo V, Behl C, Lutz B: CB1 canna-
binoid receptors and on-demand defense against
excitotoxicity. Science 302:84-88, 2003
41. Martin LL, Smith DJ: Ketamine inhibits serotonin uptake
in vivo. Neuropharmacology 21:113-118, 1982
42. Mechoulam R, Ben-Shabat S, Hanus L, Ligumsky M,
Kaminski NE, Schatz AR, Gopher A, Almog S, Martin BR,
Compton DR, Pertwee RG, Griffin G, Bayewitch M, Barg J,
Ketamine Induces Antinociception by CB1 Activation
Vogel Z: Identification of an endogenous 2-monoglyceride,
present in canine gut, that binds to cannabinoid receptors.
Biochem Pharmacol 50:83-90, 1995
43. Miller LL, Picker MJ, Schmidt KT, Dykstra LA: Effects of
morphine on pain-elicited and pain-suppressed behavior in
CB1 knockout and wildtype mice. Psychopharmacology (Berl)
215:455-465, 2011
44. Niesters M, Martini C, Dahan A: Ketamine for chronic
pain: Risks and benefits. Br J Clin Pharmacol 77:357-367, 2014
45. Okon T: Ketamine: An introduction for the pain and pal-
liative medicine physician. Pain Physician 10:493-500, 2007
46. Patsos HA, Hicks DJ, Greenhough A, Williams AC,
Paraskeva C: Cannabinoids and cancer: Potential for colorectal
cancer therapy. Biochem Soc Trans 33:712-714, 2005
47. Petrosino S, Di Marzo V: FAAH and MAGL inhibitors:
Therapeutic opportunities from regulating endocannabinoid
levels. Curr Opin Investig Drugs 11:51-62, 2010
48. Raja SN, Meyer RA, Ringkamp M, Campbell JN: Periph-
eral neural mechanisms of nociception, in Melzack R, Wall
D (eds): Textbook of Pain, 4th ed. London, Churchill Living-
stone., 1999
49. Randall LO, Selitto JJ: A method for measurement of an-
algesic activity on inflamed tissues. Arch Int Pharmacodyn
Ther 111:409-419, 1957
50. Reis GM, Pacheco D, Perez AC, Klein A, Ramos MA, Duarte
ID: Opioid receptor and NO/cGMP pathway as a mecha-
nism of peripheral antinociceptive action of the cannabinoid
receptor agonist anandamide. Life Sci 85:351-356, 2009
51. Reis GM, Ramos MA, da Fonseca Pacheco D, Klein A, Perez
AC, Duarte ID: Endogenous cannabinoid receptor agonist
anandamide induces peripheral antinociception by activa-
tion of ATP-sensitive K+ channels. Life Sci 88:653-657, 2011
52. Rezende RM, Paiva-Lima P, Dos Reis WG, Camêlo VM,
Faraco A, Bakhle YS, Francischi JN: Endogenous opioid and
cannabinoid mechanisms are involved in the analgesic effects
of celecoxib in the central nervous system. Pharmacology 89:
127-136, 2012
53. Rios C, Gomes I, Devi LA: mu opioid and CB1 cannabi-
noid receptor interactions: Reciprocal inhibition of receptor
signaling and neuritogenesis. Br J Pharmacol 148:387-395,
2006
54. Romero TR, Duarte ID: Involvement of ATP-sensitive K+
channels in the peripheral antinociceptive effect induced by
ketamine. Vet Anaesth Analg 40:419-424, 2013
55. Romero TR, Galdino GS, Silva GC, Resende LC, Perez AC,
Cortes SF, Duarte ID: Involvement of the L-arginine/nitric
oxide/cyclic guanosine monophosphate pathway in periph-
eral antinociception induced by N-palmitoyl-ethanolamine
in rats. J Neurosci Res 90:1474-1479, 2012
56. Romero TR, Galdino GS, Silva GC, Resende LC, Perez AC,
Côrtes SF, Duarte ID: Ketamine activates the L-arginine/
nitric oxide/cyclic guanosine monophosphate pathway to
induce peripheral antinociception in rats. Anesth Analg 113:
1254-1259, 2011
57. Romero TR, Resende LC, Guzzo LS, Duarte ID: CB1 and
CB2 cannabinoid receptor agonists induce peripheral
antinociception by activation of the endogenous noradren-
ergic system. Anesth Analg 116:463-472, 2013
Ferreira et al
58. Rozenfeld R, Bushlin I, Gomes I, Tzavaras N, Gupta A,
Neves S, Battini L, Gusella GL, Lachmann A, Ma’ayan A, Blitzer
RD, Devi LA: Receptor heteromerization expands the rep-
ertoire of cannabinoid signaling in rodent neurons. PLoS One
7:29239, 2012
59. Sinner B, Graf BM: Ketamine. Handb Exp Pharmacol 182:
313-333, 2008
60. Smith DJ, Pekoe GM, Martin LL, Coalgate B: The inter-
action of ketamine with the opiate receptor. Life Sci 26:
789-795, 1980
61. Stander S, Schmelz M, Metze D, Luger T, Rukwied R: Dis-
tribution of cannabinoid receptor 1 (CB1) and 2 (CB2) on
sensory nerve fibers and adnexal structures in human skin.
J Dermatol Sci 38:177-188, 2005
62. Tandon OM, Mehta AK, Halder S, Khanna N, Sharma KK:
Peripheral interaction of opioid and NMDA receptors in in-
The Journal of Pain 495
flammatory pain in rats. Indian J Physiol Pharmacol 54:21-
31, 2010
63. Vandevoorde S, Fowler CJ: Inhibition of fatty acid amide
hydrolase and monoacylglycerol lipase by the anandamide
uptake inhibitor VDM11: Evidence that VDM11 acts as an
FAAH substrate. Br J Pharmacol 145:885-893, 2005
64. Vinegar R, Truax JF, Selph JL, Johnston PR, Veneable AL,
McKenzie KK: Pathway to carrageenan-induced inflamma-
tion in the hind limb of the rat. Fed Proc 46:118-126, 1987
65. White PF, Way WL, Trevor AJ: Ketamine—its pharma-
cology and therapeutic uses. Anesthesiology 56:119-136, 1982
66. Yamakage M, Hirshman CA, Croxton TL: Inhibitory effects
of thiopental, ketamine, and propofol on voltage-dependent
Ca2+ channels in porcine tracheal smooth muscle cells. An-
esthesiology 83:1274-1282, 1995
67. Zimmermann M: Ethical guidelines for investigation of
experimental pain in conscious animals. Pain 16:109-110, 1983