European Journal of Pharmacology 755 (2015) 1–5

Contents lists available at ScienceDirect

European Journal of Pharmacology

journal homepage: www.elsevier.com/locate/ejphar

Neuropharmacology and analgesia

Tingenone, a pentacyclic triterpene, induces peripheral antinociception

due to NO/cGMP and ATP-sensitive K þ channels pathway activation
in mice
Clarice de Carvalho Veloso a, Vanessa Gregório Rodrigues b, Renata Cristina
Mendes Ferreira a, Lucienir Pains Duarte b, Andre Klein a, Igor Dimitri Duarte a,
Thiago Roberto Lima Romero a, Andrea de Castro Perez a,n
a
Department of Pharmacology, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG), Belo Horizonte, Minas Gerais, Brazil
b
Department of Chemistry, Institute of Exact Sciences, UFMG, Belo Horizonte, Minas Gerais, Brazil

art ic l e i nf o a b s t r a c t

Article history: Substances derived from plants play an important role in the development of new analgesic drugs, among
Received 27 August 2014 them, triterpenoids. The connection between the participation of L-arginine/NO/cGMP pathway and the
Received in revised form activation of ATP-sensitive K þ channels (KATP) has been established on the peripheral antinociception
19 February 2015
induced by various drugs. The study assessed the involvement of L-arginine/NO/cGMP/KATP pathway in the
Accepted 22 February 2015
antinociceptive effect induced by tingenone, from Maytenus imbricata, against the hyperalgesia evoked by
Available online 5 March 2015
prostaglandin E2 (PGE2) in peripheral pathway. The paw pressure test was used, with hyperalgesia induced
Keywords: by intraplantar injection of PGE2 (2 μg). Tingenone (200 mg/paw) administered into the right hind paw
Tingenone induced a local antinociceptive effect, that was antagonized by L-NOArg, nonselective nitric oxide synthase
Nitric oxide
(NOS) inhibitor and by L-NPA, selective neuronal NOS (nNOS) inhibitor. The L-NIO, selective inhibitor of
KATP
endothelial (eNOS), and the L-NIL, selective inhibitor of inducible (iNOS), did not alter the peripheral
Pain
Antinociception antinociceptive effect of the tingenone. The ODQ, selective soluble guanylyl cyclase inhibitor, prevented the
antinociceptive effect of tingenone, and zaprinast, inhibitor of the phosphodiesterase that is cyclic
guanosine monophosphate (cGMP) specific, intensified the peripheral antinociceptive effect of the smaller
dose of tingenone. Glibenclamide, ATP-sensitive K þ channels (KATP) blocker, but not tetraethylammonium
chloride, voltage-dependent K þ channel blocker; dequalinium dichloride, blocker of the small conductance
Ca2 þ -activated K þ channel, and paxilline, a potent blocker of high-conductance Ca2 þ -activated K þ
channels, respectively, prevented the peripheral antinociceptive effect of tingenone. The results demon-
strate that tingenone induced a peripheral antinociceptive effect by L-arginine/NO/cGMP/KATP pathway
activation, with potential for a new analgesic drug.
& 2015 Elsevier B.V. All rights reserved.

1. Introduction cyclase enzyme and increases cellular concentration of cyclic gua-

Initially, the function of nitric oxide (NO) was described as a
relaxing inductor of vascular smooth muscle and inhibitor of platelet
aggregation (Moncada et al., 1988). However, it has been reported
the involvement of the L-arginine/NO/cGMP pathway on peripheral
antinociception (Duarte et al., 1990). Nitric oxide is an endogenous
mediator, synthesized from L-arginine by the enzyme nitric oxide
synthase (NOS). Once it is formed, it stimulates the soluble guanylyl
n
Correspondence to: Laboratory of Pain and Analgesia, Department of Pharma-
cology, Institute of Biological Sciences, Federal University of Minas Gerais (UFMG),
Avenida Presidente Antônio Carlos, 6627, Pampulha, 31270-010 Belo Horizonte,
Minas Gerais, Brazil. Tel.: þ55 31 34092699.
E-mail address: acp@icb.ufmg.br (A. de Castro Perez).
nosine monophosphate (cGMP) (Moncada et al., 1988).
It has also been demonstrated the importance of K þ channels
opening on the antinociceptive effect induced by receptor agonists
coupled to the G protein, such as opioid and cannabinoid, NSAIDs
and natural products, such as resveratrol (Granados-Soto et al.,
2002; Ocaña et al., 2004; Ortiz et al., 2012). Several previous studies
established a connection between NO/cGMP and activation of K þ
ATP-sensitive (KATP) channels on peripheral antinociception. Soares
et al. (2000) showed that activation of channels for K þ sensitive to
ATP was the mechanism of the peripheral antinociceptive action of
sodium nitroprusside, a NO donor whose effect was prevented
by sulfonylureas, tolbutamide and glibenclamide, ATP-sensitive K þ
channels blockers (Nichols and Lederer, 1991; Edwards and Weston,
1993), with none effect on K þ voltage-dependent or Ca2 þ activated

http://dx.doi.org/10.1016/j.ejphar.2015.02.038
0014-2999/& 2015 Elsevier B.V. All rights reserved.
2 C. de Carvalho Veloso et al. / European Journal of Pharmacology 755 (2015) 1–5
channels. In another study, by Soares and Duarte (2001), it was
observed that dibutyryl-cGMP, an analog of cGMP, induced periph-
eral antinociception by activation of KATP channels.
The L-arginine/NO/cGMP pathway and activation of KATP was
described as the peripheral antinociceptive mechanism for opioid
agonists (Ferreira et al., 1991; Rodrigues and Duarte, 2000; Pacheco
et al., 2005), ketorolac (Lázaro-Ibáñez et al., 2001); diclofenac (Ortiz
et al., 2003; Alves et al., 2004), cannabinoids (Reis et al., 2011;
Romero and Duarte, 2012a), dipyrone and myrcene (terpene)
(Duarte et al., 1992), melatonin (Hernández-Pacheco et al., 2008),
among others. In a previous study accomplished by Veloso et al.
(2014b), it was demonstrated that tingenone administered into the
right hind paw induced a local antinociceptive effect that was
antagonized by naloxone, a non-selective antagonist to opioid
receptors, suggesting that the opioidergic system participates in
the peripheral antinociception induced by tingenone. Thus, the
purpose of this study was to investigate the L-arginine/NO/cGMP
pathway and activation of potassium channels in the peripheral
antinociceptive mechanism of locally applied tingenone, a penta-
cyclic triterpene, isolated from the roots of Maytenus imbricata
(Rodrigues et al., 2012). Other triterpenes that present antinocicep-
tive effects have been already studied (Gaertner et al., 1999; Ferreira
et al., 2000; Bortalanza et al., 2002). Our research group also demon-
strated the antinociceptive effect of tingenone in the second phase
of the formalin test (Veloso et al., 2014a).
2. Material and methods
2.1. Plant material
Roots of Maytenus imbricata (Celastraceae) were carefully col-
lected to prevent damage to the specimen. The collection area was
Ouro Preto municipality, Minas Gerais state, Brazil. The plant
material was identified by the botanists Rita M. de Carvalho Okano,
Botanic Department of the Federal University of Viçosa, and M.
Cristina Teixeira Braga Messias, Botanic Department of the Federal
University of Ouro Preto. A voucher specimen (number 27780) is
deposited in the collection of the Herbarium of the Botanic Depart-
ment of the Federal University of Viçosa, Brazil.
The roots of M. imbricata were dried at room temperature and
powdered in a mill. The powder (1.5 kg) was submitted to extraction
in a Soxhlet apparatus with the organic solvent hexane/ethyl ether
(1:1). The filtrate was removed in a rotator evaporator. The quantity
of filtrate obtained was 16.1 g for hexane/ethyl ether (1:1) extract.
From of this extract, 1.5 g of tingenone (Fig. 1) was isolated and
characterized (Rodrigues et al., 2012).
Fig. 1. Tingenone structure, a natural pentacyclic triterpene, isolated from roots of
Maytenus imbricata Mart. ex. Reissek.
2.2. Animals
Male Swiss mice (30–35 g), from the Bioterism Center of Federal
University of Minas Gerais (CEBIO/UFMG), were used in the experi-
ments. They were housed in standard cages and kept at a constant
temperature of 23 1C with a 12-h light–dark cycle and free access to
food and tap water. All testing procedures were in accordance with
the ethical guidelines of the International Association for the Study
of Pain (IASP) (Zimmermann, 1983) and approved by Ethics Com-
mittee in Animal Experimentation at the Federal University of Minas
Gerais (protocol 115/2012).
2.3. Measurement of hyperalgesia
Hyperalgesia was induced by subcutaneous injection of prosta-
glandin E2 (PGE2) (2 μg) into the plantar surface of the hind paw.
Hyperalgesia was measured according to the rat paw pressure test
described by Randall and Selitto (1957) and adapted to mice by
Kawabata et al. (1992). An analgesimeter was used (Ugo-Basile, 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
(g) required to elicit the nociceptive response of paw flexion was
determined as the nociceptive threshold. A cutoff value of 160 g was
used to reduce the possibility of damage to the paws. The nocicep-
tive threshold was measured in the right paw and determined as the
average of the three consecutive trials recorded before and 10 min
after 3 h after the PGE2 injection (time in which the maximum
antinociceptive effect of tingenone is observed). The threshold was
calculated as the difference between these averages (Δ of nocicep-
tive threshold) and is expressed in grams. Δ of nociceptive thresh-
old40 (zero) means hyperalgesia by PGE2 injection and decrease of
this value means antihyperalgesic effect by the tested drug.
2.4. Drugs administration
All drugs were administered using an injected volume of 20 μl/
paw. Tingenone was dissolved in 20% dimethylsulfoxide (DMSO)
and 1% Tween 20 in saline. Nonselective NO synthase (NOS)
inhibitor NG-nitro-L-arginine (L-NOarg; RBI, USA); selective neu-
ronal NO synthase inhibitor NW-propyl-L-arginine (L-NPA; Sigma);
selective endothelial NO synthase inhibitor N5-(1-iminoethyl)-L-
ornithine dihydrochloride (L-NIO; Sigma) and selective inducible
NO synthase inhibitor L-N6-(1-iminoethyl) lysine hydrochloride
(L-NIL; Sigma) were dissolved in isotonic saline. Specific soluble
guanylyl cyclase enzyme inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-
a]quinoxalin-1-one; RBI) and inhibitor of the phosphodiesterase
that is cGMP specific Zaprinast (1,4-dihydro-5-[2-propoxyphenyl]-
7H-1,2,3-triazolo[4,5-d]pyrimidine-7-one; Sigma) were dissolved
in 10% DMSO. Glibenclamide (TOCRIS), ATP-dependent K þ chan-
nels (KATP) blocker, was dissolved in 1% Tween 20; tetraethylam-
monium chloride (Sigma): voltage-dependent K þ channel blocker,
dequalinium dichloride (TOCRIS): a potent and selective non-
peptide blocker of the apamin-sensitive small conductance
Ca2 þ -activated K þ channel and paxilline (TOCRIS): potent blocker
of high-conductance Ca2 þ -activated K þ channels, were dissolved
in isotonic saline. The drug used as a hyperalgesic agent was PGE2
(Sigma, USA) dissolved in 2% ethanol in isotonic saline.
2.5. Experimental protocol
Tingenone was administered subcutaneously in the right hind
paw 3 h after local injection of PGE2. L-NOarg, L-NPA, L-NIO, L-NIL
and tetraethylammonium chloride were administered 30 min prior
to tingenone. Glibenclamide, dequalinium dichloride and paxilline
were administered 5 min prior to tingenone. ODQ and Zaprinast
were administered 10 min and 1 h prior to tingenone, respectively.
 C. de Carvalho Veloso et al. / European Journal of Pharmacology 755 (2015) 1–5
It should be noted that the protocols concerning dose and time of
administration of each drug used in this study were obtained
through literature data (Wagner et al., 2001; Reis and Duarte,
2006; Romero et al., 2012c; Costa et al., 2014; Galdino et al., 2014)
and pilot experiments.
2.6. Statistical analysis
The obtained results were analyzed using Graph Pad Prism
3.0 and expressed as mean7S.E.M. Statistically differences between
groups were calculated by one-way ANOVA followed by the Bon-
ferroni test. Statistically significance was set at Po0.05.
3. Results
The peripheral antinociceptive effect of tingenone (200 mg/paw)
was antagonized by intraplantar administration of L-NOArg (12, 18
and 24 mg/paw), NOS nonselective inhibitor, and L-NPA (12, 18
and 24 μg/paw), selective inhibitor of neuronal NOS, on a dose-
dependent manner (Fig. 2A and B, respectively). L-NIO, selective
inhibitor of eNOS, and L-NIL, selective inhibitor of iNOS, did not alter
the peripheral antinociceptive effect of the triterpene (Fig. 2C).
ODQ (25, 50 and 100 mg/paw), a specific soluble guanylyl cyc-
lase enzyme inhibitor, prevented the antinociceptive effect of
tingenone (200 mg/paw), on a dose-dependent manner (Fig. 3A).
Fig. 2. Effect of intraplantar administration of L-NOArg (A), L-NPA (B), L-NIO and
L-NIL (C) on the antinociceptive peripheral effect induced by tingenone. L-NOArg,
L-NPA, L-NIO and L-NIL were administrated 30 min before tingenone. Each column
represents the mean 7 S.E.M. of the Δ, measuring the nociceptive threshold
expressed in grams (g), concerning 4 animals. *indicates statistical significance
Po 0.05 when compared to the control group PGE2 2 mgþ V1 þ V2 and P o 0.05
when compared to the group PGE2 2 mgþ V1 þ Ting 200 mg. V1 (Saline); V2 (20%
DMSOþ 1% Tween 20 in saline) and Et (ethanol). Δ ¼difference of the nociceptive
threshold obtained in the beginning of the experiment, basal value, before any
injection (time zero), regarding the threshold measured 10 min after 3 h after the
PGE2 injection (time in which the maximum antinociceptive effect of tingenone is
observed).
3
Fig. 3. Effect of intraplantar administration of ODQ (A) and Zaprinast (B) on the
antinociceptive peripheral effect induced by tingenone. ODQ and Zaprinast were
administrated 10 min and 1 h, respectively, before tingenone. Each column repre-
sents the mean7 S.E.M. of the Δ, measuring the nociceptive threshold expressed in
grams (g), concerning 4 animals. *indicates statistical significance P o 0.05 when
compared to the control group PGE2 2 mgþ V1 þ V2 and P o 0.05 when compared to
the group PGE2 2 mg þV1 þ Ting 200 mg. V1 (10% DMSO in saline); V2 (20%
DMSOþ 1% Tween 20 in saline) and Et (ethanol). Δ¼ difference of the nociceptive
threshold obtained in the beginning of the experiment, basal value, before any
injection (time zero), regarding the threshold measured 10 min after 3 h after the
PGE2 injection (time in which the maximum antinociceptive effect of tingenone is
observed).
The administration of zaprinast (Zap, 50 mg/paw), inhibitor of the
phosphodiesterase that is cGMP specific, intensified the peripheral
antinociceptive effect of the smaller dose of tingenone (50 mg/paw)
(Fig. 3B). L-NOArg, L-NPA, ODQ and Zaprinast, when administered
at the highest dose and alone, did not induce antinociception or
hyperalgesia (Figs. 2A and B and 3A and B, respectively).
Intraplantar administration of glibenclamide (Glib; 20, 40 and
80 mg/paw), ATP-sensitive K þ channels (KATP) blocker, prevented
the peripheral antinociceptive effect of tingenone (200 mg/paw), in
a dose-dependent manner. The solo administration of the highest
dose did not induce antinociception or hyperalgesia (Fig. 4A).
Intraplantar injection of tetraethylammonium chloride (TEA;
60 mg/paw), a voltage-dependent K þ channel blocker, did not alter
the peripheral antinociceptive effect of tingenone (Fig. 4B). Admin-
istration of dequalinium dichloride (DQ; 50 mg/paw; Fig. 4B), blocker
of the small conductance Ca2 þ -activated K þ channel, and of paxil-
line (Pax; 20 mg/paw; Fig. 4B), a potent blocker of high-conductance
Ca2 þ -activated K þ channels, also did not alter the peripheral anti-
nociceptive effect of tingenone.
4. Discussion
The role of the L-arginine/NO/cGMP pathway on the peripheral
antinociception was observed for morphine μ opioid agonist (Ferreira
et al., 1991; Granados-Soto et al., 1997); dipyrone and myrcene
(terpene) (Duarte et al., 1992), for nimesulide (Islas-Cadena et al.,
1999); for meloxicam (Aguirre-Bañuelos and Granados-Soto, 2000);
for diclofenac (Ortiz et al., 2003), for SNC80 δ opioid receptor agonist
(Pacheco et al., 2005), melatonin (Hernández-Pacheco et al., 2008), for
endogenous agonist of the cannabinoid CB2 receptor, palmitoyletha-
nolamide (PEA) (Romero et al., 2012b), among others, whenever NO
synthesis and consequent stimulation of soluble guanylyl cyclase are
involved in peripheral antinociception.
4 C. de Carvalho Veloso et al. / European Journal of Pharmacology 755 (2015) 1–5
Fig. 4. Effect of intraplantar administration of glibenclamide (A), tetraethylammo-
nium chloride (B), dequalinium dichloride (B) and paxilline (B) on the antinoci-
ceptive peripheral effect induced by tingenone. Glibenclamide, dequalinium
dichloride and paxilline were administrated 5 min before tingenone. Tetraethy-
lammonium chloride was administrated 30 min before tingenone. Each column
represents the mean 7S.E.M. of the Δ, measuring the nociceptive threshold
expressed in grams (g), concerning 4 animals. *indicates statistical significance
Po 0.05 when compared to the control group PGE2 2 mgþ V1 þV2 and P o0.05
when compared to the group PGE2 2 mg þ V1 þTing 200 mg. Glibenclamide: V1 (1%
Tween 20 in saline); dequalinium dichloride, paxilline and tetraethylammonium
chloride: V1 (Saline); V2 (20% DMSOþ 1% Tween 20 in saline) and Et (ethanol).
Δ¼ difference of the nociceptive threshold obtained in the beginning of the
experiment, basal value, before any injection (time zero), regarding the threshold
measured 10 min after the 3 h after the PGE2 injection (time in which the
maximum antinociceptive effect of tingenone is observed).
With the purpose of verifying if tingenone leads to an increase of
NO synthesis, the nonselective NOS inhibitor was utilized, blocking the
synthesis of NO (Iacopucci et al., 2012). It was observed that L-NOARG
prevented the peripheral antinociceptive effect of this triterpene on a
dose-dependent manner, suggesting that NO is an important mediator.
In order to evaluate which isoform of NOS is involved in the
peripheral antinociception of tingenone, selective inhibitors for each
isoenzyme were utilized. NO is formed enzymatically from L-arginine,
by three isoenzymes, the constitutives (nNOS e eNOS) and the
inducible isoform (iNOS) (Reutov and Sorokina, 1998). The three
isoforms are located peripherally in neurons of the dorsal root
ganglion (Papadopolou et al., 2004). According to the results, we
suggest that tingenone administered peripherally activates nNOS,
and, hence, formation of NO. The eNOS and iNOS isoforms are not
involved in this process. Similar results have been described in the
peripheral antinociceptive mechanism of various kinds of analgesics,
including anandamide, acetylcholine, xylazine, dipyrone, diclofenac,
palmitoylethanolamide and opioid agonists, such as morphine and
SNC80 (Cunha et al., 2010; Romero et al., 2011, 2012b).
In a previous study by Veloso et al. (2014b), it was demonstrated
that tingenone administered into the right hind paw induced a local
antinociceptive effect that was antagonized by naloxone, a non-
selective antagonist to opioid receptors. Clocinnamox, naltrindole,
and nor-binaltorphimine are selective antagonists to m, δ and κ rece-
ptors, respectively, that prevented the peripheral antinociception
induced by tingenone. Bestatine acts as an inhibitor of aminopepti-
dase, an enzyme that degrades endogenous opioid peptides, and was
shown to intensify the antinociceptive effect of tingenone. The results
suggest that the opioidergic system participates in the peripheral
antinociception induced by tingenone. Cunha et al. (2010) demon-
strated that activation of the nitric oxide pathway by morphine was
dependent on an initial stimulation of PI3Kγ/AKT protein kinase B
(AKT) that in turn might cause the stimulation of nNOS and an
increase in NO production. As proposed by Moncada et al. (1988), NO,
once formed, promotes the stimulation of the soluble guanylyl
cyclase enzyme and thereby increases the concentration of cGMP.
The increase of this second messenger is associated with the peri-
pheral antinociceptive effect of various opioid analgesics (Duarte
et al., 1992; Ferreira et al., 1991; Pacheco et al., 2005) and cannabinoid
(Romero et al., 2012b). It was observed that the administration of
ODQ, an inhibitor of the soluble guanylyl cyclase enzyme prevented,
in a dose-dependent manner, the peripheral antinociceptive effect of
tingenone. Therefore, the results suggest that this triterpene in-
creases NO levels, with the consequent activation of the soluble
guanylyl cyclase enzyme and increase of cGMP levels.
cGMP possesses a limited action, due to the fact that it is
quickly degraded by phosphodiesterase-5 (Rybalkin et al., 2002).
Thus, substances that inhibit this enzyme, reducing the hydrolysis
of cGMP and increasing its concentration, increase analgesia
(Duarte et al., 1990). Administration of the phosphodiesterase
specific inhibitor, zaprinast, intensified the peripheral antinoci-
ceptive effect of tingenone on the lowest dose, demonstrating that
the concentration of cGMP is relevant for this purpose.
Several previous studies have established the link between the
participation of the NO/cGMP pathway and activation of the
potassium channels in peripheral antinociception (Soares et al.,
2000; Rodrigues and Duarte, 2000; Soares et al., 2001; Ortiz et al.,
2006; Hernández-Pacheco et al., 2008). Therefore, we verified the
involvement of channels for potassium, in the peripheral anti-
nociceptive mechanism of this triterpene.
In the present study, it was observed that the peripheral anti-
nociceptive effect of tingenone was prevented by local administration
of the sulfonylurea glibenclamide, ATP-sensitive K þ channels (KATP)
blocker (Nichols and Lederer, 1991). In order to verify the involvement
of other channels for K þ , we used tetraethylammonium chloride,
voltage-dependent K þ channel blocker, dequalinium dichloride, a
blocker of the small conductance Ca2 þ -activated K þ channel and
paxilline, potent blocker of high-conductance Ca2 þ -activated K þ
channels. As these blockers did not antagonize the antinociceptive
effect of tingenone, we suggest only the involvement of the KATP
channel.
The NO/cGMP pathway and the activation of the KATP channels
on the peripheral antinociception was demonstrated for several
analgesics: morphine (Rodrigues and Duarte, 2000; Cunha et al.,
2010), ketorolac (Lázaro-Ibáñez et al., 2001); dipyrone (Alves and
Duarte, 2002); diclofenac (Ortiz et al., 2003; Alves et al., 2004),
xylazine (Romero and Duarte, 2009a, 2009b), for the agonist of the
cannabinoid receptor CB1, anandamide (Reis et al., 2011), for the
agonist of the cannabinoid receptor CB2, palmitoylethanolamide
(PEA) (Romero and Duarte, 2012a), among others, and in the present
study, we observed that tingenone induced a peripheral antinoci-
ceptive effect by this pathway activation, with potential to be an
analgesic drug.
Acknowledgments
This work was supported by Coordenação de Aperfeiçoamento
de Pessoal de Nível Superior (CAPES-Brazil) and Conselho Nacional
de Pesquisa (CNPq-Brazil) (Grant no. 3536130).
References
Aguirre-Bañuelos, P., Granados-Soto, V., 2000. Evidence for the participation of the
nitric oxide-cyclic GMP pathway in the antinociceptive action of meloxicam in
the formalin test. Eur. J. Pharmacol. 395, 9–13.
Alves, D.P., Duarte, I.D.G., 2002. Involvement of ATP-sensitive K þ channels in the
peripheral antinociceptive effect induced by dipyrone. Eur. J. Pharmacol. 444,
47–52.
 C. de Carvalho Veloso et al. / European Journal of Pharmacology 755 (2015) 1–5
Alves, D.P., Tatsuo, M.A.F., Leite, R., Duarte, I.D.G., 2004. Diclofenac-induced
peripheral antinociception is associated with ATP-sensitive K þ channels
activation. Life Sci. 74, 2577–2591.
Bortalanza, L.B., Ferreira, J., Hess, S.C., Monache, F.D., Yunes, R.A., Calixto, J.B., 2002.
Anti-allodynic action of the tormentic acid, a triterpene isolated from plant,
against neuropathic and inflammatory persistent pain in mice. Eur. J. Pharma-
col. 453, 203–208.
Costa, A., Galdino, G., Romero, T., Silva, G., Cortes, S., Santos, R., Duarte, I., 2014. Ang-
(1–7) activates the NO/cGMP and ATP-sensitive K þ channels pathway to induce
peripheral antinociception in rats. Nitric Oxide 37, 11–16.
Cunha, T.M., Roman-Campos, D., Lotufo, C.M., Duarte, H.L., Souza, G.R., Verri Jr., W.A.,
Funez, M.I., Dias, Q.M., Schivo, I.R., Domingues, A.C., Sachs, D., Chiavegatto, S., Teixeira,
M.M., Hothersall, J.S., Cruz, J.S., Cunha, F.Q., Ferreira, S.H., 2010. Morphine peripheral
analgesia depends on activation of the PI3Kγ/AKT/nNOS/KATP signaling pathway.
Proc. Natl. Acad. Sci. USA 107, 4442–4447.
Duarte, I.D.G., Lorenzetti, B.B., Ferreira, S.H., 1990. Peripheral analgesia and activa-
tion of the oxide-cyclic GMP pathway. Eur. J. Pharmacol. 186, 289–293.
Duarte, I.D.G., dos Santos, I.R., Lorenzetti, B.B., Ferreira, S.H., 1992. Analgesia by
direct antagonism of nociceptor sensitization involves the arginine-nitric
oxide-cGMP pathway. Eur. J. Pharmacol. 217, 225–227.
Edwards, G., Weston, A.H., 1993. The pharmacology of ATP-sensitive potassium
channels. Annu. Rev. Pharmacol. Toxicol. 33, 597–637.
Ferreira, S.H., Duarte, I.D.G., Lorenzetti, B.B., 1991. The molecular mechanism of
action peripheral morphine analgesia: stimulation of the cGMP system via
nitric oxide release. Eur. J. Pharmacol. 201, 121–122.
Ferreira, J., Floriani, A.E.O., Filho, V.C., Monache, F.D., Yunes, R.A., Calixto, J.B., Santos,
A.R.S., 2000. Antinociceptive properties of the methanolic extract and two
triterpenes isolated from Epidendrum monsenii stems (Orchidaceae). Life Sci. 66,
791–802.
Gaertner, M., Muller, L., Roos, J.F., Cani, G., Santos, A.R.S., Niero, R., Calixto, J.B.,
Yunes, R.A., Monache, F.D., Cechinel-Filho, V., 1999. Analgesic triterpenes from
Sebastiania schottiana roots. Phytomedicine 1, 41–44.
Galdino, G.S., Xavier, C.H., Almeida, R., Silva, G., Fontes, M.A., Menezes, G., Duarte, I.D.,
Perez, A.C., 2015. The Nitric oxide/CGMP/KATP pathway mediates systemic and
central antinociception induced by resistance exercise in rats. Int.
J. Neurosci..
Granados-Soto, V., Rufino, M.O., Gomes Lopes, L.D., Ferreira, S.H., 1997. Evidence for
the involvement of the nitric oxide-cGMP pathway in the antinociception of
morphine in the formalin test. Eur. J. Pharmacol. 340, 177–180.
Granados-Soto, V., Argüelles, C.F., Ortiz, M.I., 2002. The peripheral antinociceptive
effect of resveratrol is associated with activation of potassium channels.
Neuropharmacology 43, 917–923.
Hernández-Pacheco, A., Araiza-Saldaña, C.I., Granados-Soto, V., Mixcoatl-Zecuatl, T.,
2008. Possible participation of the nitric oxide-cyclic GMP-protein kinase G-K þ
channels pathway in the peripheral antinociception of melatonin. Eur.
J. Pharmacol. 596, 70–76.
Iacopucci, A.P., Mello, R.O., Barbosa-Silva, R., Melo-Thomas, L., 2012. L-NOARG-
induced catalepsy can be influenced by glutamatergic neurotransmission
mediated by NMDA receptors in the inferior colliculus. Behav. Brain Res. 234,
149–154.
Islas-Cadena, M., Aguirre-Bañuelos, P., Granados-Soto, V., 1999. Evidence for the
participation of the nitric oxide-cyclic GMP pathway in the antinociceptive
effect of nimesulide. J. Pharmacol. Toxicol. Methods 42, 87–92.
Kawabata, A., Nishimura, Y., Takagi, H., 1992. L-Leucyl-L-arginine, naltrindole and D-
arginine block antinociception elicited by L-arginine in mice with carrageenin-
induced hyperalgesia. Br. J. Pharmacol. 107, 1096–1101.
Lázaro-Ibáñez, G.G., Torres-López, J.E., Granados-Soto, V., 2001. Participation of the
nitric oxide-cyclic GMP-ATP-sensitive K(þ ) channel pathway in the antinoci-
ceptive action of ketorolac. Eur. J. Pharmacol. 426, 39–44.
Moncada, S., Radomski, M.W., Palmer, R.M.J., 1988. Endothelium-derived relaxing
factor. Identification as nitric oxide and role in the control of vascular tone and
platelet function. Biochem. Pharmacol. 37, 2495–2501.
Nichols, C.G., Lederer, W.J., 1991. Adenosine triphosphate-sensitive potassium
channels in the cardiovascular system. Am. J. Physiol. 261, H1675–H1690.
Ocaña, M., Cendán, C.M., Cobos, E.J., Entrena, J.M., Baeyens, J.M., 2004. Potassium
channels and pain: present realities and future opportunities. Eur. J. Pharmacol.
500, 203–219.
Ortiz, M.I., Granados-Soto, V., Castañeda-Hernández, G., 2003. The NO-cGMP-K þ
channel pathway participates in the antinociceptive effect of diclofenac, but not
of indomethacin. Pharmacol. Biochem. Behav. 76, 187–195.
Ortiz, M.I., Medina-Tato, D.A., Sarmiento-Heredia, D., Palma-Martínez, J., Granados-
Soto, V., 2006. Possible activation of the NO-cyclic GMP-protein kinase G-K þ
channels pathway by gabapentin on the formalin test. Pharmacol. Biochem.
Behav. 83, 420–427.
5
Ortiz, M.I., Castañeda-Hernández, G., Izquierdo-Vega, J.A., Sánchez-Gutiérrez, M.,
Ponce-Monter, H.A., Granados-Soto, V., 2012. Role of ATP-sensitive K þ channels
in the antinociception induced by non-steroidal anti-inflammatory drugs in
streptozotocin-diabetic and non-diabetic rats. Pharmacol. Biochem. Behav. 102,
163–169.
Pacheco, D.F., Reis, G.M.L., Francischi, J.N., Castro, M.S.A., Perez, A.C., Duarte, I.D.G.,
2005. δ-Opioid receptor agonist SNC80 elicits peripheral antinociception via δ1
and δ2 receptors and activation of the L-arginine/nitric oxide/cyclic GMP
pathway. Life Sci. 78, 54–60.
Papadopolou, S., Hartmann, P., Lips, K.S., Kummer, W., Haberberger, R.V., 2004.
Nicotinic receptor mediated stimulation of NO-generation in neurons of rat
thoracic dorsal root ganglia. Neurosci. Lett. 361, 32–35.
Randall, L.O., Selitto, J.J., 1957. A method for measurement of analgesic activity on
inflamed tissues. Arch. Int. Pharmacodyn. Ther. 111, 409–419.
Reis, G.M.L., Duarte, I.D.G., 2006. Baclofen, an agonist at peripheral GABAB
receptors, induces antinociception via activation of TEA-sensitive potassium
channels. Br. J. Pharmacol. 149, 733–739.
Reis, G.M., Ramos, M.A., Pacheco, D.F., Klein, A., Perez, A.C., Duarte, I.D., 2011.
Endogenous cannabinoid receptor agonist anandamide induces peripheral
antinociception by activation of ATP-sensitive K þ channels. Life Sci. 88,
653–657.
Reutov, V.P., Sorokina, E.G., 1998. NO-synthase and nitrite-reductase components of
nitric oxide cycle. Biochemistry 63, 874–884.
Rodrigues, A.R.A., Duarte, I.D.G., 2000. The peripheral antinociceptive effect
induced by morphine is associated with ATP-sensitive K þ channels. Br.
J. Pharmacol. 129, 110–114.
Rodrigues, V.G., Duarte, L.P., Silva, G.D.F., Silva, F.C., Góes, J.F., Takahashi, J.A.,
Pimenta, L.P.S., 2012. Evaluation of antimicrobial activity and toxic potential
of extracts and triterpenes isolated from Maytenus imbricata. Quím. Nova 35,
1375–1380.
Romero, T.R.L., Duarte, I.D.G., 2009a. α2-Adrenoceptor agonist xylazine induces
peripheral antinociceptive effect by activation of the L-arginine/ nitric oxide/
cyclic GMP pathway in rat. Eur. J. Pharmacol. 613, 64–67.
Romero, T.R.L., Duarte, I.D.G., 2009b. Involvement of ATP-sensitive K þ channels in
the peripheral antinociceptive effect induced by the α2-adrenoceptor agonist
xylazine. J. Pharmacol. Sci. 111, 323–327.
Romero, T.R., Resende, L.C., Duarte, I.D., 2011. The neuronal NO synthase participa-
tion in the peripheral antinociception mechanism induced by several analgesic
drugs. Nitric Oxide 25, 431–435.
Romero, T.R.L., Duarte, I.D., 2012a. N-palmitoyl-ethanolamine (PEA) induces per-
ipheral antinociceptive effect by ATP-sensitive K þ -channel activation. J. Phar-
macol. Sci. 118, 156–160.
Romero, T.R.L., Galdino, G.S., Silva, G.C., Resende, L.C., Perez, A.C., Cortes, S.F., Duarte, I.D.,
2012b. Involvement of the L-arginine/nitric oxide/cyclic guanosine monophosphate
pathway in peripheral antinociception induced by N-palmitoyl–ethanolamine in
rats. J. Neurosci. Res. 90, 1474–1479.
Romero, T.R.L., Guzzo, L.S., Perez, A.C., Klein, A., Duarte, I.D.G., 2012c. Noradrenaline
activates the NO/cGMP/ATP-sensitive K þ channels pathway to induce periph-
eral antinociception in rats. Nitric Oxide 26, 157–161.
Rybalkin, S.D., Rybalkina, I.G., Feil, R., Hofmann, F., Beavo, J.A., 2002. Regulation of
cGMP-specific phosphodiesterase (PDE5) phosphorylation in smooth muscle
cells. J. Biol. Chem. 277, 3310–3317.
Soares, A.C., Leite, R., Tatsuo, M.A.K.F., Duarte, I.D.G., 2000. Activation of ATP-
sensitive K þ channels: mechanism of peripheral antinociceptive of the nitric
oxide donor, sodium nitroprusside. Eur. J. Pharmacol. 400, 67–71.
Soares, A.C., Duarte, I.D.G., 2001. Dibutyryl-cyclic GMP induces peripheral anti-
nociception via activation of ATP-sensitive K þ channels in the rat PGE2-induced
hyperalgesic paw. Br. J. Pharmacol. 134, 127–131.
Veloso, C.C., Rodrigues, V.G., Azevedo, A.O., Oliveira, C.C., Gomides, L.F., Duarte, L.P.,
Duarte, I.D., Klein, A., Perez, A.C., 2014a. Antinociceptive effects of Maytenus
imbricata Mart. ex. Reissek (Celastraceae) root extract and its tingenone
constituent. J. Med. Plants Res. 8, 68–76.
Veloso, C.C., Rodrigues, V.G., Ferreira, R.C.M., Duarte, L.P., Klein, A., Duarte, I.D.,
Romero, T.R.L., Perez, A.C., 2014b. Tingenone, a pentacyclic triterpene, induces
peripheral antinociception due to opioidergic activation. Planta Med. 80,
1615–1621.
Wagner, E.J., Ronnekleiv, O.K., Kelly, M.J., 2001. The noradrenergic inhibition of an
apamin-sensitive, small-conductance Ca2 þ -activated K þ channel in hypotha-
lamic gamma-aminobutyric acid neurons: pharmacology, estrogen sensitivity,
and relevance to the control of the reproductive axis. J. Pharmacol. Exp. Ther.
299, 21–30.
Zimmermann, M., 1983. Ethical guidelines for investigations of experimental pain
in conscious animals. Pain 16, 109–110.