Biochemical Pharmacology 198 (2022) 114965

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Biochemical Pharmacology
journal homepage: www.elsevier.com/locate/biochempharm

Bradykinin induces peripheral antinociception in PGE2-induced

hyperalgesia in mice
Renata Cristina Mendes Ferreira a, Flávia Cristina de Sousa Fonseca a,
Douglas Lamounier de Almeida a, Ana Cristina Nogueira Freitas b, Steve Peigneur c,
Thiago Roberto Lima Romero a, Flávio Almeida Amaral b, Igor Dimitri Gama Duarte a, *
a
Department of Pharmacology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil
b
Department of Biochemistry and Immunology, Institute of Biological Sciences, Federal University of Minas Gerais, Belo Horizonte, Brazil
c
Department of Toxicology and Pharmacology, KU Leuven, Leuven, Belgium

A R T I C L E I N F O A B S T R A C T

Keywords: Background: Bradykinin (BK) is an endogenous peptide involved in vascular permeability and inflammation. It
Bradykinin has opposite effects (inducing hyperalgesia or antinociception) when administered directly in the central nervous
Antinociception system. The aim of this study was to evaluate whether BK may also present this dual effect when injected
Opioid
peripherally in a PGE2-induced nociceptive pain model, as well as to investigate the possible mechanisms of
Cannabinoid
action involved in this event in mice.
Methods: Male Swiss and C57BL/6 knockout mice for B1 or B2 bradykinin receptors were submitted to a me­
chanical paw pressure test and hyperalgesia was induced by intraplantar prostaglandin E2 (2 µg/paw) injection.
Results: Bradykinin (20, 40 and 80 ng/paw) produced dose-dependent peripheral antinociception against PGE2-
induced hyperalgesia. This effect was antagonized by bradyzide (8, 16 and 32 μg/paw), naloxone (12.5, 25 and
50 μg/paw), nor-binaltorphimine (50, 100 and 200 μg/paw) and AM251 (20, 40 and 80 μg/paw). Bestatin (400
µg/paw), MAFP (0.5 µg/paw) and VDM11 (2.5 µg/paw) potentiated the antinociception of a lower 20 ng BK
dose. The knockout of B1 or B2 bradykinin receptors partially abolished the antinociceptive action of BK (80 ng/
paw), bremazocine (1 μg/paw) and anandamide (40 ng/paw) when compared with wild-type animals, which
show complete antinociception with the same dose of each drug.
Conclusion: The present study is the first to demonstrate BK-induced antinociception in peripheral tissues against
PGE2-induced nociception in mice and the involvement of κ-opioid and CB1 cannabinoid receptors in this effect.

1. Introduction
Apart from its well-characterized role in angioedema and vascular
permeability [1], bradykinin (BK) has been shown to induce nocifensive
responses through nociceptor activation. It is demonstrated that BK in­
jection into the human skin and muscle elicits pain perception [2,3].
Work from Kumazawa et al. (1991) [4] demonstrated how BK facilitates
heat-evoked nociceptive responses using in vitro canine testis-superior
spermatic nerve preparations and how they are reversed by blocking
the bradykinin B2 receptor.
Bradykinin exerts its effect by activation of bradykinin receptor B1
(B1) and bradykinin receptor B2 (B2). The B2 receptors are constitutively
expressed in a vast number of tissues (including sensory neurons). On
the other hand, the B1 receptor is known as an inducible GPCR which is
upregulated during inflammation or following tissue injury [5]. The
concept that B1 receptors are not constitutively expressed in animal
tissues is still controversy. Some literature data suggests basal expres­
sion levels of B1 receptors in central and peripheral nervous systems [6].
The main molecular mechanism by which BK exerts its effects in primary
afferent sensory neurons is the activation of Gq/11-mediated phospho­
lipase C (PLC) with subsequent production of DAG and IP3, culminating
with intracellular calcium increases. These pathways are responsible for

Abbreviations: BK, bradykinin; DOR, δ-opioid receptors; FAAH, fatty acid amide hydrolase; KOR, κ-opioid receptor; MOR, µ-opioid receptor; PLC, phospholipase C;
PGE2, prostaglandin E2.
* Corresponding author at: Department of Pharmacology, Institute of Biological Sciences, Federal University of Minas Gerais, Av. Antônio Carlos, 6627, Belo
Horizonte, MG CEP 31.270-100, Brazil.
E-mail address: dimitri@icb.ufmg.br (I.D.G. Duarte).

https://doi.org/10.1016/j.bcp.2022.114965
Received 7 December 2021; Received in revised form 10 February 2022; Accepted 11 February 2022
Available online 16 February 2022
0006-2952/© 2022 Published by Elsevier Inc.
R.C.M. Ferreira et al.
most of the depolarizing BK actions on peripheral neurons which are
associated with BK-mediated nociception [7].
Knockout mice lacking both B1 and B2 receptors showed a 70%
reduction of acute acetic acid-induced visceral nociception compared to
wild-type mice. Moreover, the same group of mice showed decreases in
heat hypersensitivity in carrageenan-induced paw inflammation, which
suggests that kinins are important for nociception associated with acute
short-lasting inflammation [8]. Furthermore, Gomez et al. (2011) [9]
shed light on the importance of the BK metabolism by metal­
loproteinases on BK-mediated nociception, showing how JA-2, a met­
alloendopeptidase EC3.4.24.15 (EP24.15) inhibitor, increased B2-
mediated bradykinin hyperalgesia in rats.
Parallel to the hyperalgesia previously mentioned, intrathecal BK
administration may inhibit spinal nociceptive sensory transmission and
produce analgesia by acting presynaptically on terminals of bulbospinal
noradrenaline-containing fibers [10]. This finding was later corrobo­
rated by Couto et al. (2006) [11]. They showed that microinjections of
bradykinin into the trigeminal nerve cause a long-lasting anti­
nociception that is antagonized by the damage of locus coeruleus-
noradrenergic neurons. In addition, treatment of rat trigeminal and
dorsal root ganglion cultures with BK caused robust trafficking of het­
erologously expressed δ-opioid receptors (DOR) to the plasma mem­
brane [12]. This data suggests that BK-induced membrane insertion of
DOR into the plasma membrane of peripheral sensory neurons may
underlie increased DOR analgesia in inflamed tissue.
In this context, the present study aimed to investigate, evaluate, and
characterize the newly demonstrated antinociceptive effect of nanogram
doses of bradykinin on peripheral tissues upon PGE2-induced hyper­
algesia. Furthermore, the participation of the opioid and cannabinoid
receptors in this analgesic mechanism is demonstrated.
2. Material and methods
2.1. Animals
The experiments were performed on 30–40 g male Swiss mice
(CEBIO-ICB/UFMG, Belo Horizonte, Brazil) and 15–25 g C57BL/6
knockout mice for BK receptors (Immunopharmacology laboratory of
ICB/UFMG), divided as follows: knockout mice for B1 (BKB1R− /− ) or B2
(BKB2R− /− ) bradykinin receptor, and C57BL/6 wild-type (WT). It was
used a total of 240 Swiss and 90 C57BL/6. The animals were maintained
in a temperature-controlled room (24 ± 2 ◦ C) on an automatic 12-hour
light/dark cycle (06:00–18:00). All tests were carried out during the
light phase and animals were randomly selected. Food and water were
available ad libitum. The animal experimental protocols were approved
by the UFMG Ethics Committee on the Use of Animals (protocol n◦ 247/
2019) and all animal care are in accordance with the recommendations
for the evaluation of experimental pain in animals [13]. Efforts were
made to minimize suffering and reduce the number of animals used for
the accomplishment of the experiments.
2.2. Measurement of nociceptive threshold
Hyperalgesia was induced by subcutaneous injection of prosta­
glandin E2 (PGE2; 2 µg/paw) into the mice’s plantar surface hind paw.
The mechanical nociceptive threshold was assessed by measuring the
response to a paw pressure test as described by Randall and Selitto
(1957) [14] and adapted to mice by Kawabata et al. (1992) [15]. An
analgesimeter (Ugo-Basile, Italy), which consists of a cone-shaped paw-
presser with a rounded tip, was used to apply a linearly increasing
pressure to the hind paw. The weight in grams required to elicit the
nociceptive response of the paw withdrawal was determined as the
nociceptive threshold. A cutoff value of 160 g was used to reduce
possible damage to the paws. The nociceptive threshold was measured
in the right hind paw and determined by the average of three consecu­
tive trials recorded before (baseline nociceptive threshold) and after
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Biochemical Pharmacology 198 (2022) 114965
180 min of PGE2 injection (peak of action). The results were calculated
by the difference between these two averages (Δ of nociceptive
threshold) and expressed in grams. To reduce stress, mice were habit­
uated to the apparatus two days prior to the experiments.
2.3. Drugs
Bradykinin acetate salt (Sigma-Aldrich, St. Louis, MO, USA) was
diluted in physiological saline. Prostaglandin E2 (Sigma-Aldrich, St.
Louis, MO, USA), the hyperalgesic agent, was diluted in ethanol 10% in
saline. Naloxone (Sigma-Aldrich, St. Louis, MO, USA), a nonselective
opioid antagonist; CTOP (Sigma-Aldrich, St. Louis, MO, USA), a selec­
tive µ-opioid receptor antagonist; naltrindole (Sigma-Aldrich, St. Louis,
MO, USA), a selective δ-opioid receptor antagonist; bestatin (Alfa Aesar,
MA, USA), an aminopeptidase-N inhibitor; bradyzide di(tri­
fluoroacetate) (Sigma-Aldrich, St. Louis, MO, USA), a selective B2 bra­
dykinin receptor antagonist; and bremazocine (Merck, Kenilworth, NJ,
USA), a κ-opioid receptor agonist, were diluted in physiological saline.
Anandamide (Tocris Bioscience; Bristol, UK), an agonist of CB1 receptor;
was diluted in tocrisolve 1%. Nor-Binaltorphimine dihydrochloride
(Sigma-Aldrich, St. Louis, MO, USA), a selective κ-opioid receptor
antagonist, was diluted in dimethyl sulfoxide (DMSO) 25%. AM251
(Sigma-Aldrich, St. Louis, MO, USA), a CB1 receptor antagonist, and
AM630 (Sigma-Aldrich, St. Louis, MO, USA), a CB2 receptor antagonist,
were diluted in DMSO 10%. MAFP (Tocris Bioscience; Bristol, UK), a
fatty acid amide hydrolase (FAAH) inhibitor was diluted in methyl ac­
etate 1%. VDM11 (Tocris Bioscience; Bristol, UK), an anandamide re­
uptake inhibitor; and JZL184 (Tocris Bioscience; Bristol, UK), an
inhibitor for monoacylglycerol lipase, were diluted in DMSO 1%. All the
drugs were injected into the plantar surface of the right paw in a volume
of 20 µL per paw.
2.4. Experimental protocol
In all experiments, each animal’s baseline nociceptive threshold was
first measured before the injection of any substance. To evaluate the
temporal development of the dose–response curve of bradykinin (BK),
this drug was injected 180 min after PGE2 injection (peak of action) or at
time zero, without the PGE2-induced hyperalgesia. In the protocol used
to determine whether BK was acting only peripherally, PGE2 was
injected into both hind paws, whereas the highest dose of BK was given
only into the right paw while the contralateral paw received vehicle
(saline). Only for this experiment to verify the exclusion of the systemic
effect, the nociceptive threshold was measured in both hind paws.
BK was injected into the right hind paw 20 min prior to the mea­
surement of hyperalgesia (180 min). Naloxone, naltrindole, nor-
Binaltorphimine, and bestatin were intraplantarly given 30 min prior
to BK, while CTOP was injected 20 min before BK. Bradyzide, AM251,
AM630, MAFP, VDM11, and JZL184 were intraplantarly injected 10 min
before the BK intraplantar injection. Bremazocine and anandamide were
injected 5 min prior to the measurement of hyperalgesia (180 min). The
nociceptive threshold was measured in the right hind paw before any
injection (zero time) and at 180 min after PGE2 injection (peak action).
The difference between these values was expressed as Δ of the noci­
ceptive threshold. The protocols, concerning the dose and the injection
time of each drug used in this study, were obtained through literature
data and pilot experiments [16–19].
2.5. Data and Statistical analysis
Results are presented as the mean ± standard error of the mean (S.E.
M.). Statistical analysis was carried out using Graph Prism 8.0.2 soft­
ware and the data were analyzed by analysis of variance (ANOVA) fol­
lowed by Bonferroni test. Statistically, significance was set at p < 0.05.
R.C.M. Ferreira et al.
3. Results
3.1. Bradykinin algesic profile
Microgram doses of bradykinin (BK), injected into the right hind
paw, induced a hyperalgesic effect shown by a reduction in the noci­
ceptive threshold. The doses of 2 µg and 4 µg showed a peak of hyper­
algesic effect 60 min after injection. The dose of 8 µg induced the highest
reduction in the nociceptive threshold in 120 min and demonstrated a
sustained hyperalgesic effect until 300 min after injection (Fig. 1A). The
protocol used to determine whether microgram doses of BK were acting
outside the injection paw demonstrates that BK, at a dose of 8 µg, did not
produce hyperalgesia in the contralateral paw, indicating that, at this
dose, BK induced a peripheral hyperalgesic effect (Fig. 1B).
Nanogram doses of BK were able to produce antinociception against
2 μg PGE2-induced hyperalgesia. BK was injected 180 min after PGE2
and displayed a dose-dependent antinociceptive effect 20 min after its
injection. Therefore, this time point was used in the following experi­
ments for the evaluation of BK analgesic effects. The dose of 80 ng
completely reversed the PGE2-induced hyperalgesia, given that the
nociceptive threshold in this group was no different than the control (ET
2% + saline) group (Fig. 2A). BK, at the dose of 80 ng, did not produce
an antinociceptive effect in the contralateral paw, indicating that, at this
dose, it induced only a peripheral effect (Fig. 2B).
Bradyzide, a potent non-peptide B2 bradykinin receptor antagonist,
dose-dependently reversed the antinociception induced by 80 ng BK,
and the dose of 32 µg, completely reversed this antinociceptive effect
(Fig. 3). Bradyzide did not affect the nociceptive threshold of mice
injected only with PGE2.
3.2. Role of the opioid system on bradykinin antinociception
Non-selective antagonism of opioid receptors dose-dependently
reversed the antinociception induced by 80 ng BK. The higher dose of
naloxone (50 µg) completely reversed the antinociception produced by
80 ng BK (Fig. 4A). Naloxone did not have any effect on the nociceptive
threshold of mice injected only with PGE2.
Bestatin, an aminopeptidase inhibitor, at the dose of 400 µg/paw
could potentiate the lower antinociceptive effect of a 20 ng BK dose. The
potentiation effect completely abolished the hyperalgesia induced by 2
µg PGE2 (Fig. 4B). It is important to notice that, when given alone, 400
µg bestatin produced no alterations on the nociceptive threshold of PGE2
injected mice (Fig. 4B).
The selective κ-opioid receptor antagonist, nor-binaltorphimine,
Fig. 1. Nociceptive effect of intraplantar injection of bradykinin in mice. (A) Bradykinin (BK; 2, 4 and 8 μg/paw) was injected into the right hind paw and the
nociceptive thresholds were measured after 5 min of BK injection. (B) BK (8 μg/paw) was injected only into the right hind paw and its vehicle (saline) was given
solely into the left hind paw. The nociceptive thresholds were measured in both paws after 120 min of the injections. For both assays, each symbol/column represents
the mean ± S.E.M. (n = 5). * p < 0.05 compared with saline (p < 0.05), (A) two-way and (B) one-way ANOVA followed by the Bonferroni test.
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Biochemical Pharmacology 198 (2022) 114965
dose-dependently (50, 100, and 200 µg/paw) antagonized the anti­
nociceptive effect of 80 ng BK (Fig. 5A). When given alone, nor-
binaltrorphimine was not able to alter the nociceptive threshold of
PGE2 injected mice. On the other hand, neither CTOP (20 µg/paw) nor
naltrindole (60 µg/paw) were able to alter the antinociceptive effect of
80 ng BK (Fig. 5B and 5C, respectively).
The knockout of B1 or B2 bradykinin receptors did not abolish but
reduced BK-mediated antinociception at about 54% and 38%, respec­
tively. Note, in the wild type, the same dose of BK reduced 91% of
prostaglandin-induced hyperalgesia (Fig. 6A and B, respectively). Bre­
mazocine, 1 µg/paw, was used as a positive analgesic control as a
κ-opioid receptor-selective agonist in this experiment and produced
partial analgesia on BKB1R− /− and BKB2R− /− mice. Wild-type mice
treated with bremazocine showed a complete antinociceptive response
(Fig. 6A and B).
3.3. Role of the cannabinoid system on bradykinin antinociception
The selective CB1 cannabinoid receptor antagonist, AM251, dose-
dependently (20, 40, and 80 µg/paw) reversed the antinociceptive ef­
fect of 80 ng BK with the higher dose (80 µg/paw), showing a full block
of this effect. When injected alone, AM251 had no significant effect on
the nociceptive threshold of PGE2 injected mice (Fig. 7A). On the other
hand, the selective CB2 cannabinoid receptor antagonist, AM630, did
not influence BK-induced antinociception (Fig. 7B).
The selective AEA hydrolysis inhibitor MAFP (0.5 µg/paw) and the
endocannabinoid membrane transporter inhibitor VDM11 (2.5 µg/paw)
were both able to potentiate the lower antinociceptive effect of a 20 ng
BK dose. The potentiation effect completely abolished the hyperalgesia
induced by 2 µg PGE2, (Fig. 8A and B). Notably, neither MAFP nor
VDM11 produced alterations on the nociceptive threshold of PGE2
injected mice. On the other hand, the selective 2-AG hydrolysis inhibitor
JZL184 was not able to further increase the antinociception of a lower
dose of 20 ng BK in mice injected with PGE2 (Fig. 8C).
AEA (40 ng/paw) elicited a less potent analgesic effect on BKB1R− /−
and BKB2R− /− animals compared with the Wild-type. It is interesting to
note that the impairment of the analgesic effect induced by BK was more
pronounced on BKB1R− /− mice (Fig. 9A and B).
4. Discussion
The role of BK in the development and maintenance of pain is well
described in the literature. Kinins, in general, might directly activate
sensory neurons and/or sensitize sensory fibers through increasing
R.C.M. Ferreira et al.
Fig. 2. Peripheral antinociceptive effect of bradykinin on PGE2-induced hyperalgesia in mice. (A) Bradykinin (BK; 20, 40 and 80 ng/paw) was injected into the right
hind paw 180 min after the intraplantar injection of prostaglandin E2 (PGE2; 2 μg/paw) and the nociceptive thresholds were measured after 5 min of BK injection. (B)
PGE2 (2 µg/paw) was injected into both right and left hind paws, followed by an injection of BK (80 ng/paw) only into the right paw, and vehicle into the left paw.
Both BK and vehicle were given at 160 min after local injection of PGE2. The nociceptive thresholds were measured in both paws at 180 min. For both assays, each
symbol/column represents the mean ± S.E.M. (n = 5). * p < 0.05 compared with PGE2 + Saline (p < 0.05), (A) two-way and (B) one-way ANOVA followed by the
Bonferroni test.
Fig. 3. Effect of the pretreatment with a selective bradykinin B2 receptor
antagonist on the BK-induced peripheral antinociception against the hyper­
algesia induced by PGE2. Bradyzide (BDZ; 8, 16 and 32 μg/paw) was injected
into the right hind paw 10 min prior to the intraplantar injection of bradykinin
(BK; 80 ng/paw). BK was given 160 min after the local injection of prosta­
glandin E2 (PGE2; 2 μg/paw). Measurements were made at 180 min. Each
column represents the mean ± S.E.M. (n = 5). Veh 1 = saline and Veh 2 =
ethanol 10%. * p < 0.05 compared to the PGE2 + Veh1 + Veh1 and # p < 0.05
compared to the PGE2 + BK + Veh1; one-way ANOVA followed by the Bon­
ferroni test.
neuronal excitability [20]. Interestingly, it has been shown that BK in­
jection directly in the central nervous system may induce opposite ef­
fects. Depending on the dose tested and the injection site, BK may
produce both, nociceptive and antinociceptive effects in the central
nervous system [21–23]. Many studies that investigate the pain
signaling and its components, used the peripheral injection of BK as a
model to induce hyperalgesia [9,24–27]. However, none has demon­
strated a possible peripheral antinociceptive effect of BK in well-
established models of pain. Therefore, the present study evaluated
whether BK may also induce, depending on the dose, a dual and opposite
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Biochemical Pharmacology 198 (2022) 114965
effect in the peripheral nervous system.
Our data demonstrated that the intraplantar injection of BK at
microgram doses had a hyperalgesic effect. On the other hand, at the
range of nanogram doses, BK induced peripheral antinociception on
PGE2-induced hyperalgesia. This is the first study demonstrating the
peripheral antinociceptive effect of BK. Likewise, previous studies from
our research group showed other well-known pharmacological media­
tors capable of inducing both nociception and antinociception depend­
ing on the dose, such as serotonin [28] and noradrenaline [29].
In the context of inflammation, besides bradykinin, prostaglandins
(PGs) are some of the main molecules responsible for sensitizing the
nociceptors in peripheral tissues, producing the increased sensation/
perception of pain. Upon activation of stimulatory G protein-coupled
receptors, prostaglandins induce sensitization of sodium and calcium
channels and suppression of outward potassium currents [30]. There­
fore, PGs-mediated activity causes an increase of primary sensory neu­
rons resting potential and, consequently, its sensitization. PGs are
considered as a prototype of potent direct sensitizers in animal models. A
single injection of PGE2 is capable of sensitizing nociceptors to me­
chanical and chemical stimuli [31]. Bradykinin acts through two G-
protein-coupled receptors, named B1 and B2 bradykinin receptors. These
receptors differ in their pharmacologic properties, desensitization abil­
ities and expression profiles. BK preferentially binds to the B2 receptor,
which is constitutively expressed, whilst the B1 receptor displays a high
affinity for kinin metabolites and is rarely expressed in normal tissue.
However, it might be upregulated in pathologic conditions [32–34]. In
2002, Sot and colleagues [23] showed that the antinociceptive effect of
intrathecal injection of BK was dependent on both B1 and B2 bradykinin
receptors. Nevertheless, the antinociception induced by BK in the central
nervous system may be resulting mostly from the B2 receptor activation
since HOE 140, a selective B2 receptor antagonist, abolished the anti­
nociceptive effect of BK against thermal noxious stimuli. Similarly, our
work showed that both B1 and B2 bradykinin receptors are involved in
the BK peripheral antinociception. Knockout mice for B1 or B2 brady­
kinin receptors presented a reduced antinociceptive effect induced by
BK when compared to wild-type (WT) mice. It is important to point out
that the antinociception produced by BK on B1 knockout mice might be
dependent on the B2 receptors, and on the other hand, the same possi­
bility might be true for the B2 knockout mice, where BK-mediated
antinociception might be dependent on B1 receptors. In addition,
pharmacologically blocking the B2 bradykinin receptor using a selective
antagonist (bradyzide), completely reverses the analgesic BK effect,
R.C.M. Ferreira et al. Biochemical Pharmacology 198 (2022) 114965

Fig. 4. Effect of pretreatment with (A) a nonselective opioid receptor antagonist and (B) an aminopeptidase inhibitor on the BK-induced peripheral antinociception
against the hyperalgesia induced by PGE2. (A) Naloxone (NX; 12.5, 25 and 50 μg/paw) and (B) bestatin (Best; 400 μg/paw) were injected into the right hind paw 30
min prior to the intraplantar injection of bradykinin (BK; 80 or 20 ng/paw). BK was given (A) 160 min or (B) 165 min after the local injection of prostaglandin E2
(PGE2; 2 μg/paw). Measurements were made at 180 min. Each column represents the mean ± S.E.M. (n = 5). Veh 1 = saline and Veh 2 = ethanol 10%. * p < 0.05
compared to the PGE2 + Veh1 + Veh1 and # p < 0.05 compared to the PGE2 + BK + Veh1; one-way ANOVA followed by the Bonferroni test.

Fig. 5. Effect of the pretreatment with selective opioid receptors antagonists on the BK-induced peripheral antinociception against the hyperalgesia induced by PGE2.
(A) Nor-binaltorphimine (Nor-BNI; 50, 100 and 200 μg/paw) or (B) naltrindole (NTD; 60 μg/paw) were injected into the right hind paw 30 min prior to the
intraplantar injection of bradykinin (BK; 80 ng/paw) and (C) CTOP (20 μg/paw) was injected 20 min before the bradykinin. BK was given 160 min after the local
injection of prostaglandin E2 (PGE2; 2 μg/paw). Measurements were made at 180 min. Each column represents the mean ± S.E.M. (n = 5). Veh 1 = saline, Veh 2 =
DMSO 25% and Veh 3 = ethanol 10%. * p < 0.05 compared to the PGE2 + Veh1 + Veh and # p < 0.05 compared to the PGE2 + BK + Veh; one-way ANOVA followed
by the Bonferroni test.

suggesting that the B2 receptors may play a major role in the BK anti­
nociceptive effect. It is known from the literature, that bradyzide is a
potent B2 receptor competitive antagonist, with Ki values ranging from
0.18 to 0.89 nM depending on the cell type used [35]. Moreover, bra­
dyzide has been shown to display high selectivity for the rodent B2 re­
ceptors over other species, including humans, which validates the use of
this drug in the present study [35].
The apparent contradiction between the data obtained by genetic
deletion and pharmacological antagonism, in which there was, respec­
tively, a partial and total reversal of BK-induced antinociception, can be
explained by reciprocal upregulation of B2 and B1 receptors, probably
compensate for the loss of its action in knockout mice. In fact, the se­
lective P2X7 antagonist treatment completely prevented the thermal
hyperalgesia in the hind paws induced by NTG in wild-type, but not in
knockout mice, compatible with this phenomenon [36]. Specifically in
relation to knockout animals for B2 receptors, the lack of effect in the
formalin test has been reported [37]. It is in contrast to a significant
attenuation of responding by the selective B2 antagonist, HOE140 [38].
The authors conclude that the subtle phenotypic changes exhibited by
B2 KO mice can be regarded as evidence of the important compensatory
role of B1 receptors in the maintenance of inflammation and
hyperalgesia.
A previous work showed that in a neuropathic pain model, the pe­
ripheral injection of µ and δ-opioid agonists in knockout mice lacking
both B1 and B2 receptors presented a significantly reduced anti­
nociception when compared to WT animals [8]. This study showed that
kinin receptors contribute to the peripheral antinociception induced by
the activation of µ and δ-opioid receptors, suggesting an interaction

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R.C.M. Ferreira et al. Biochemical Pharmacology 198 (2022) 114965

Fig. 6. Effect of the treatment with bradykinin and bremazocine in WT, (A) BKB1R− /− and (B) BKB2R− /− mice in PGE2-induced hyperalgesia. Bradykinin (BK; 80 ng/
paw) or bremazocine (Bre; 100 μg/paw) was injected into the right hind paw of WT and knockout mice 160 and 175 min, respectively, after the intraplantar injection
of prostaglandin E2 (PGE2; 2 μg/paw). Measurements were made at 180 min. Each column represents the mean ± S.E.M. (n = 5). Veh = saline. * p < 0.05 compared
to the PGE2 + Veh, & p < 0.05 compared to the PGE2 + BK in WT mice and # p < 0.05 compared to the PGE2 + Bre in WT mice; one-way ANOVA followed by the
Bonferroni test.

Fig. 7. Effect of the pretreatment with selective cannabinoid receptors antagonists on the BK-induced peripheral antinociception against the hyperalgesia induced by
PGE2. (A) AM251 (20, 40 and 80 μg/paw) or (B) AM630 (100 μg/paw) were injected into the right hind paw 10 min prior to the intraplantar injection of bradykinin
(BK; 80 ng/paw). BK was given 160 min after the local injection of prostaglandin E2 (PGE2; 2 μg/paw). Measurements were made at 180 min. Each column represents
the mean ± S.E.M. (n = 5). Veh 1 = DMSO 10%, Veh 2 = saline and Veh 3 = ethanol 10%. * p < 0.05 compared to the PGE2 + Veh2 + Veh1 and # p < 0.05 compared
to the PGE2 + BK + Veh1; one-way ANOVA followed by the Bonferroni test.

between kinins and the opioid system. Our results support this interac­
tion hypothesis since the complete antinociception of bremazocine in
WT mice was partially abolished in both knockout mice for B1 or B2
receptors. In addition, it was demonstrated that naloxone, a non-
selective opioid receptor antagonist, also abolished the BK-induced pe­
ripheral antinociception. Interestingly, our study suggests that the
κ-opioid receptor (KOR), but not µ- (MOR) and δ (DOR)-opioid re­
ceptors, might be modulating the opioid-bradykinin system interaction.
Data from the literature show a possible interaction between bra­
dykinin receptors and the opioid system. According to Lee et al. [39]
dynorphin A interacts with opioid receptors producing antinociception.
However, under chronic pain conditions, up-regulated spinal dynorphin
A may also interact with bradykinin receptors to promote hyperalgesia
through a neuroexcitatory effect. This interaction was also demon­
strated during neuropathic-induced plasticity events in rat spinal cord
neurons [40]. Strikingly, a yet novel interaction possibility has been
demonstrated on a molecular level by Ji et. al. 2017 [41] where they
found the formation of κ-opioid receptors/bradykinin B2 receptors het­
eromers in human embryonic kidney 293 (HEK293) cells and how this
heteromer could be activated by dynorphin A.
This collection of data from the literature associated with the find­
ings from the present work with the selective κ-opioid receptor agonist
bremazocine producing partial analgesia on BK knockout mice further
reinforces the interaction of bradykinin and opioid receptors and their
effect on neuronal excitability.
Furthermore, we suggest the participation of endogenous opioids on
BK-induced antinociception since the inhibition of opioid degradation
potentiated its peripheral antinociceptive effect. The κ-opioid receptor is
selectively activated by the endogenous opioid dynorphin [42], sug­
gesting that this endogenous opioid might be involved in the BK-induced

6
R.C.M. Ferreira et al. Biochemical Pharmacology 198 (2022) 114965

Fig. 8. Effect of pretreatment with (A) MAFP, (B) VDM11 or (C) JZL184 on the BK-induced peripheral antinociception against the hyperalgesia induced by PGE2. (A)
MAFP (0.5 μg/paw), (B) VDM11 (2.5 μg/paw) or (C) JZL184 (4 μg/paw) were injected into the right hind paw 10 min prior to the intraplantar injection of bradykinin
(BK; 20 ng/paw). BK was given 165 min after the local injection of prostaglandin E2 (PGE2; 2 μg/paw). Measurements were made at 180 min. Each column represents
the mean ± S.E.M. (n = 5). Veh 1 = methyl acetate 1%, Veh 2 = saline, Veh 3 = ethanol 10% and Veh4 = DMSO 1%. * p < 0.05 compared to the PGE2 + Veh2 + Veh
and # p < 0.05 compared to the PGE2 + BK + Veh; one-way ANOVA followed by the Bonferroni test.

Fig. 9. Effect of the treatment with bradykinin and anandamide in WT, (A) BKB1R− /− and (B) BKB2R− /− mice in PGE2-induced hyperalgesia. Bradykinin (BK; 80 ng/
paw) or anandamide (AEA; 40 ng/paw) was injected into the right hind paw of WT and knockout mice 160 and 175 min, respectively, after the intraplantar injection
of prostaglandin E2 (PGE2; 2 μg/paw). Measurements were made at 180 min. Each column represents the mean ± S.E.M. (n = 5). Veh = saline. * p < 0.05 compared
to the PGE2 + Veh, & p < 0.05 compared to the PGE2 + BK in WT mice and # p < 0.05 compared to the PGE2 + AEA in WT mice; one-way ANOVA followed by the
Bonferroni test.

antinociception. Moreover, Ji et al. (2017) [41] demonstrated that KOR
and B2 receptors possibly form a heterodimer that may trigger the
dynorphin A (1–13)-induced Gαs/protein kinase A signaling pathway
activity as part of the molecular mechanism for cell proliferation. This
signaling pathway is known to be triggered by BK as well [7]. Therefore,
the KOR/B2 heterodimer might be the link between the KOR and B2
peripheral analgesia, mediated by BK as observed in the present data set.
It is important to notice that the KOR/B2 heterodimer was found only in
human embryonic kidney 293 (HEK293) cells co-expressing both re­
ceptors, which does not mean that the same is found in peripheral
tissues.
Several studies suggest that the opioid system and the cannabinoid
system have synergistic activities on pain modulation mechanisms.
Studies performed by our research group showed the participation of
endocannabinoids and the CB1 cannabinoid receptor in the central [43]
and peripheral [44] antinociception of morphine. In addition, Desroches
et al. (2014) [45] demonstrated that intraplantar injection of morphine
in knockout mice for cannabinoid receptors induced a less pronounced
antinociceptive effect in the formalin test when compared to WT.
Therefore, taking into account the extensive evidence of participation of
the cannabinoid system in analgesics processes [46], its interaction with
the opioid system and the involvement of the latter in the BK peripheral
antinociception, we decided to evaluate also the involvement of the
cannabinoid system in the peripheral antinociceptive effect of BK.
Our work indicates that the endocannabinoid system plays an
important role in BK-mediated antinociception. The complete and dose-
dependent reversion of the BK antinociceptive effect by the CB1
cannabinoid receptor antagonist suggests that BK might modulate the
cannabinoid receptor activation to induce analgesia. The same effect
was not observed when mice were treated with a selective antagonist of
CB2 receptors, suggesting that, between the cannabinoid receptors, CB1
might play a major role in BK-mediated antinociception in peripheral
tissues. Moreover, inhibition of endogenous AEA hydrolysis and reup­
take by MAFP and VDM11, respectively, potentiated the antinociceptive
effect of a lower BK dose, inducing complete analgesia. The same was
not observed when inhibition of 2-AG hydrolysis by JZL184 was tested
since the nociceptive threshold remained seemingly unaltered compared
with the control group.
To further reinforce the role of the endocannabinoid system in BK-
mediated analgesia, the present work also showed that AEA was not
able to induce the same antinociceptive response in BKB1R− /− and
BKB2R− /− against PGE2-induced hyperalgesia compared with their WT

7
R.C.M. Ferreira et al.
counterparts that received the same treatment. This data set strongly
suggests that BK-induced analgesia might involve the activation of the
endocannabinoid system and indicates that the AEA-induced anti­
nociception in peripheral tissues might also involve the participation of
BK receptors. The literature is very scarce regarding the interactions of
endocannabinoids and BK receptors in promoting analgesia. Therefore,
the present work brings another novelty in the field of peripheral
analgesia mediated by bradykinin and the endocannabinoid system.
It is also important to highlight that cannabinoid and opioid re­
ceptors share several molecular features, such as the interaction be­
tween their intracellular pathways, the reciprocal release of opioid
peptides by cannabinoids or endocannabinoids by opioids, and the ex­
istence of a direct receptor-receptor interaction when they are co-
expressed in the same cells [47]. Knowing that this interaction is
possible in peripheral and central tissues and given that the opioid re­
ceptors have been shown to form heterodimers with the B2 bradykinin
receptors [38], it is possible to suggest that cannabinoid and BK re­
ceptors might also form heterodimers to modulate pain and other
cellular processes.
In conclusion, the present work highlighted the dual effect of bra­
dykinin on nociception and demonstrated, for the first time, the pe­
ripheral antinociceptive effect of BK. In addition, this study
demonstrated part of the mechanism of action underlying the peripheral
antinociceptive effect induced by BK, which is mediated by both B1 and
B2 receptors, besides KOR and the CB1 receptor. Furthermore, this work
provides insights into the involvement of endogenous dynorphin and
anandamide in this antinociceptive mechanism in peripheral tissues.
The present data highlights a new role of bradykinin as a peripheral
analgesic agent and suggests a possible interaction between the canna­
binoid and opioid systems with bradykinin receptors.
Author contributions
Renata C M Ferreira; Flávia C. de S. Fonseca; Douglas L. de Almeida
and Ana C. N. Freitas performed experiments, analyzed the data, and
wrote the manuscript. Steve Peigneur helped to discuss and correct the
manuscript. Thiago R. L. Romero; Flávio A. Amaral and Igor D. G. Duarte
supervised the study, provided the infrastructure and edited the manu­
script. All authors read and approved the final manuscript.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors thank the Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES) and Fundação de Amparo à Pesquisa
do Estado de Minas Gerais (FAPEMIG) for financial support. TRLR
would like to thank the Conselho Nacional de Desenvolvimento Científico e
Tecnológico - CNPq - Brasil for the Productivity Fellowship, level 2
number 301496/2018-8. SP was supported by KU Leuven funding
(PDM/19/164) and F.W.O. Vlaanderen grant 12W7822N.
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