European Journal of Pharmacology 623 (2009) 30–36

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European Journal of Pharmacology

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Neuropharmacology and Analgesia

Spinal antinociception evoked by the triterpene 3β, 6β, 16β-trihydroxylup-20(29)-enein mice: Evidence for the involvement of the glutamatergic system via NMDA andmetabotropic glutamate receptors

Daniela Tagliari Longhi-Balbinot a, Evelise Fernandes Pietrovski b, Vinicius Maria Gadotti a,1,Daniel Fernandes Martins a, Valdir Alves Facundo c, Adair Roberto Soares Santos a,⁎a Departamento de Ciências Fisiológicas, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Trindade, Florianópolis 88040-900, SC, Brazilb Departamento de Farmacologia, Centro de Ciências Biológicas, Universidade Federal do Paraná, Curitiba 88015-420, PR, Brazilc Departamento de Química, Universidade Federal de Rondônia, Porto Velho 78900-500, RO, Brazil

⁎ Corresponding author. Departamento de Ciências FBiológicas, Universidade Federal de Santa Catarina, CaFlorianópolis 88040-900, SC, Brazil. Tel.: +55 48 3721 9

E-mail address: arssantos@ccb.ufsc.br (A.R.S. Santos1 Present address: Departamento de Fisiologia e Biof

University of Calgary, 3330 Hospital drive NW, Calgary,

0014-2999/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.ejphar.2009.09.004

a b s t r a c t

a r t i c l e i n f o

Article history:Received 5 March 2009Received in revised form 31 August 2009Accepted 8 September 2009Available online 15 September 2009

Keywords:TriterpeneAntinociceptionIonotropic glutamate receptorMetabotropic glutamate receptor

The present study investigated the possible involvement of the glutamatergic and neurokinin systems in theantinociception caused by triterpene 3β, 6β, 16β-trihydroxylup-20(29)-ene (TTHL) in mice. TTHL given byintraperitoneal (i.p., 2.1–65.5 µmol/kg), intraplantar (i.pl., 6.5–65.5 nmol/paw) or intrathecal (i.t., 21.8–655 nmol/site) routes, produced dose-dependent inhibition of glutamate-induced nociception with ID50

values of 12 µmol/kg; 34.2 nmol/paw; 233.8 nmol/site and inhibitions of 78±6; 82±4 and 77±8%,respectively. I.t. injection of TTHL (6.5–218 nmol/site, co-administered) also caused significant and dose-dependent reduction of nociceptive response induced by i.t. injection of glutamate (175 nmol/site), with ID50

value of 54.5 nmol/site and inhibition of 51±6%. Moreover, TTHL (65.5 nmol/site) co-injected by i.t. routewith agonist caused marked reduction of nociceptive responses induced by N-methyl-D-aspartate (NMDA,450 pmol/site), (±)-1-aminocyclopentane-trans-1,3 dicarboxylic acid (trans-ACPD, 10 nmol/site) andsubstance P (100 pmol/site), with inhibitions of 81±7; 79±7; 81±11%, respectively. Conversely, TTHLhad no effect on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, 135 pmol/site) and kainicacid (kainate, 110 pmol/site)-induced nociception. Moreover, the association of sub-effective doses of TTHL(6.5 nmol/site, i.t.) and MK-801(1 nmol/site, i.t.; non-competitive NMDA antagonist) or (RS)-MCPG(30 nmol/site, i.t.; non-selective group I/group II metabotropic glutamate receptor antagonist) produced asynergic antinociceptive effect in the nociception induced by NMDA or trans-ACPD, respectively. Together,these results provide experimental evidence for the involvement of the glutamatergic system (NMDA andmetabotropic glutamate receptors) in the antinociceptive action caused by TTHL in mice.

isiológicas, Centro de Ciênciasmpus Universitário Trindade,352; fax: +55 48 3721 9672.).ísica, Hotchkiss Brain Institute,Canada T2N 4N1.

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© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Combretum leprosumMart., a member of the Combretaceae family,is widely distributed in the North of Brazil and has been used in folkmedicine to heal wounds, to treat hemorrhages or as a sedative(Facundo et al., 1993, 2005). Phytochemical studies have revealed thepresence of flavonols 3-o-metilquercetina and 3-o-α-L-ramnopirano-silquercitrina and triterpenes mollic acid, arjunolic acid and 3β, 6β,16β-trihydroxylup-20(29)-ene in this plant (Facundo et al., 1993).

It is well established that the excitatory amino acid glutamateplays an important role on nociceptive pathways, either in central or

peripheral nervous system, due to the fact that glutamate is stored insensory C fibers which are responsible for the transmission ofnociceptive stimulus in the spinal cord (Bleakman et al., 2006;Fundytus, 2001; Millan, 1999). Glutamate exerts its effects throughboth ionotropic and metabotropic receptors.

Several studies have shown, by using glutamatergic-selectivereceptor agonists and antagonists, that all subtypes of ionotropic andmetabotropic glutamate receptors play significant roles in the estab-lishment of pain (Jesse et al., 2008; Neugebauer, 2001; Sawynok, 2003;Varney and Gereau, 2002). In this regard, it has been reported thatmodulation of glutamate receptors may have potential therapeuticactivity, since thismodulation exerts an antinociceptive effect in severalmammalian species including humans (Gadotti et al., 2006; Lutfy et al.,1997; Rosa et al., 2005;Wiech et al., 2004). Nevertheless, the clinical useof these drugs as analgesics is restricted due to the undesirable sideeffects (Millan, 1999).

Moreover, it was reported by Biasi and Rustioni (1988) that theactivation of nociceptors resulting from tissue damage, inflammation or

31D.T. Longhi-Balbinot et al. / European Journal of Pharmacology 623 (2009) 30–36

nerve injury evokes co-releasing of glutamate and substance P fromprimary afferents neurons either peripherally (Carlton et al.,1995;1998) or centrally (Dougherty and Willis, 1991; Randic et al.,1990) (for review see Chizh, 2002). It has also been demonstrated thatsubstance P increases glutamate-induced nociception and that theneurotransmitters glutamate and substance P can act synergistically inthe transmission of pain (Battaglia and Rustioni, 1988; Beirith et al.,2003; Malcangio et al., 1998; Teoh et al., 1996; Siebel et al., 2004).

In a previous study, we reported that the ethanolic extract obtainedfrom the flowers of C. leprosum caused dose-related antinociception inseveral models of chemical and thermal pain (Pietrovski et al., 2006).Additionally, it was demonstrated that 3β, 6β, 16β-trihydroxylup-20(29)-ene (TTHL, Fig. 1), given by oral route, produced a significant anddose-dependent inhibition of glutamate-induced nociception (Pietrovskiet al., 2006).

Regarding that glutamate plays important role in both peripheraland central pain transmission, this study aimed: i) to evaluate theantinociceptive effect of TTHL, administered either peripherically (byintraplantar route), systemically (by intraperitoneal route), or centrally(by intrathecal route), against nociception induced by intraplantarinjection of glutamate in the mouse hind paw, which causes a directstimulation of peripheral nociceptors; ii) we also investigated theinvolvement of the spinal glutamatergic systemby stimulation of spinalcord (central) neurons through intrathecal injection of glutamatereceptor agonists or antagonists and neurokinin substance P.

2. Material and methods

2.1. Animals

Experiments were conducted using Swiss mice of both sexes(25–35 g), housed at 22±2 °C under a 12-h light/12-h dark cycle (lightson at 06:00) andwith access to food andwater ad libitum. Animals (maleand female mice were equally distributed among groups) wereacclimatized to the laboratory for at least 1 h before testing and wereused only once throughout the experiments. The experiments wereperformed after protocol approval by the Institutional Ethics Committee(protocol number: PP0162) and were carried out in accordance with thecurrent guidelines for the care of laboratory animals and the ethicalguidelines for investigations of experimental pain in conscious animals(Zimmermann, 1983). The numbers of animals and intensities of noxiousstimuli usedwere theminimumnecessary to demonstrate the consistenteffect of the treatments.

2.2. Glutamate-induced nociception

In an attempt to evaluate the involvement of TTHL with theglutamatergic system, we investigated the effect of TTHL administeredby intraperitoneal (i.p.), intraplantar (i.pl.) or intrathecal (i.t.) routes, onglutamate-induced nociception. The procedure used was similar to thatpreviously described (Beirith et al., 2002). Mice were pre-treated with

Fig. 1.Molecular structure of triterpene 3β, 6β, 16β-trihydroxylup-20(29)-ene (TTHL).

TTHL i.p. (2.1–65.5 µmol/kg, 30 min before), i.pl. (6.5–65.5 nmol/paw,co-administered with glutamate) or i.t. (21.8–655 nmol/site, 15 minbefore). Control animals received an equal volume of vehicle by i.p.(10 ml/kg), i.pl. (20µl/paw) or i.t. (5µl/site) routes. A volume of 20 μlglutamate (10 μmol/paw prepared in saline) was injected i.pl. into theright hindpaw. Animals were observed individually for 15 minfollowing glutamate injection. The amount of time spent licking theinjected paw was recorded with a chronometer and was considered asindicative of nociception.

2.3. Algogen-induced overt nociception

2.3.1. Intrathecal (i.t.) injectionAs previously described by Hylden and Wilcox (1980), animals

were manually restrained, and a 30-gauge needle connected bypolyethylene tubing to a 25 μL Hamilton gas-tight syringe (Hamilton,Birmingham, UK), was inserted through the skin and between thevertebras into the subdural space of the L5–L6 spinal segments. Thetail reflex movement was considered as indicative of success ofadministration and the injections were given over a period of 5 s.

2.3.2. Spinal injection of excitatory aminoacids agonists and substance PIn parallel experiments, in order to investigate the spinal involve-

ment of TTHL with the central glutamatergic and neurokinin systems,TTHL was co-administered with ionotropic and metabotropic agonistsof glutamate receptors or substance P by i.t. route. In a first set ofexperiments, TTHL (dose range: 6.5–218 nmol/site) was co-adminis-tered via i.t. with glutamate (an excitatory amino acid, 175 nmol/site;Urca and Raigorodsky, 1988). In subsequent experiments, TTHL(65.5 nmol/site) was given by i.t. route and co-injected with N-methyl-D-aspartate (NMDA, a selective agonist of NMDA subtype ofionotropic glutamate receptors, 450 pmol/site; Urca and Raigorodsky,1988), (±)-1-aminocyclopentane-trans-1,3 dicarboxylic acid (trans-ACPD, a non-selective metabotropic glutamate receptor agonist,10 nmol/site; Boxall et al., 1998), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA, a selective agonist of AMPA subtype ofionotropic glutamate receptors, 135 pmol/site; Brambilla et al., 1996),kainic acid (kainate, a selective agonist of kainate subtype of ionotropicglutamate receptors, 110 pmol/site; Urca and Raigorodsky, 1988) orsubstance P (a neurokinin 1-receptor selective agonist, 100 pmol/site;Sakurada et al., 1990). In another group, vehicle (5 μl/site) wasadministered by i.t. route.

Immediately after i.t injection of the substances, mice wereindividually placed in observation chambers and the amount of time(seconds) the animal spent biting, licking or scratching the caudalregion (flanks, hindlimbs and tail) was taken as index of nociception.The ceiling for observation of nociceptive behavior was variable to thedifferents agonists: glutamate 3 min; NMDA and substance P 5 min;AMPA1 min; kainate 4 min; and trans-ACPD15 min (Boxall et al., 1998;Brambilla et al., 1996; Sakurada et al., 1990; Scheidt et al., 2002; Urcaand Raigorodsky, 1988).

2.3.3. I.t injection of NMDA and metabotropic glutamate receptorsantagonists

We also investigated if the commercially available antagonists ofNMDA and metabotropic receptors, such as MK-801 (selective non-competitive NMDA antagonist) and (RS)-MCPG (non-selective groupI/group II metabotropic glutamate receptor antagonist) are able toreverse the nociceptive response induced by i.t injection of glutamate,NMDA and trans-ACPD. Then, both antagonists MK-801 (3 nmol/site)and (RS)-MCPG (100 nmol/site) were intrathecally co-injected withglutamate (175 nmol/site), NMDA (450 pmol/site) or trans-ACPD(10 nmol/site). The nociceptive response (seconds) was quantified asdescribed above.

Fig. 2. Effect of TTHL given intraperitoneally (2.1–65.5 µmol/kg, i.p., Panel A),intraplantarly (6.5–65.5 nmol/paw, i.pl., Panel B) or intrathecally (21.8–655 nmol/site,i.t., Panel C) against glutamate (C; 10 μmol/paw)-induced licking in mice. Each columnrepresents the mean±S.E.M. of 6 to 9 animals. The symbols denote the levels ofsignificance: **P<0.01 and ***P<0.001 compared to their respective C group, #P<0.001from V group (one-way ANOVA followed by Newman–Keuls' test). Vehicle (V) andGlutamate=Control (C).

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2.3.4. I.t injection of combined sub-effective doses of TTHL, NMDA andmetabotropic glutamate receptors antagonists

To clarify the involvement of NMDA and metabotropic glutamatereceptors in the antinociceptive effect of TTHL, we associated sub-effective doses [based on literature data (Roberts et al., 2005) or bypreliminar results obtained in our laboratory] of MK-801(1 nmol/site)plus TTHL (6.5 nmol/site) or (RS)-MCPG (30 nmol/site) plus TTHL(6.5 nmol/site) that were intrathecally administered just beforeNMDA (450 pmol/site) or trans-ACPD (10 nmol/site). For this exper-iment, the animals were injected twice (i.t. injections of 2.5 μl, totalvolume equal to 5 μl), as described above. Control group was injectedwith vehicle (2.5 μl/i.t.) before each agonist, NMDA or trans-ACPD(2.5 μl/site). The nociceptive response (seconds) was quantified asdescribed above.

2.4. Measurement of locomotor activity

To evaluate the effect of TTHL, MK-801 and (RS)-MCPG onspontaneous locomotor activity, mice were submitted to the openfield test (Rodrigues et al., 2002). The open-field apparatus consistedof a wooden boxmeasuring 40×60×50 cm. The floor of the arenawasdivided into 12 equal squares, and the number of squares crossed bythe animal with all paws was counted in a 6 min session. Mice weretreated with TTHL (65.5 and 218 nmol/site, i.t.), MK-801 (3 nmol/site), (RS)-MCPG (100 nmol/site) or vehicle (saline plus DMSO/Tween 80–5%; 5 µl/site, i.t.) 15 min beforehand.

2.5. Drugs

The following substances were employed: L-glutamic acid hydro-chloride (glutamate) from Sigma Chemical Co. (St. Louis, USA);dimethylsulfoxide (DMSO) from Merck A.G. (Darmstadt, Germany);kainic acid (kainate), (±)-1-aminocyclopentane-trans-1,3 dicarbox-ylic acid (trans-ACPD), α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), N-methyl-D-aspartic acid (NMDA),(5 S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohep-ten-5,10-imine maleate (MK-801 maleate), (RS)-α-methyl-4-carbox-yphenylglycine [(RS)-MGPG] and substance P from Tocris, CooksonInc. (Ellisville, USA). All drugs were dissolved in saline solution (0.9%),except TTHL which was dissolved in saline plus DMSO/Tween 80. Thefinal concentration of DMSO and Tween 80 did not exceed 5% and didnot cause any effect per se. The triterpene 3β, 6β, 16β-trihydroxylup-20(29)-ene was isolated from the flowers of C. leprosum at theDepartment of Organic Chemistry, Universidade Federal do Ceará,Brazil as previously described (Facundo et al., 1993).

2.6. Statistical analysis

The results are presented as means±standard error of the mean(S.E.M), except the ID50 values (i.e., the dose of TTHL that reduced theagonist-induced nociceptive response by 50% relative to the controlvalue), which are reported as geometric means accompanied by theirrespective 95% confidence limits. The statistical difference betweengroups was analyzed by one-way ANOVA followed by Newman–Keuls' test. P values less than 0.05 (P<0.05) were considered asindicative of significance. The ID50 value was determined by non-linear regression from individual experiments using Graph-Padsoftware (GraphPad software, San Diego, CA, USA).

3. Results

3.1. Glutamate-induced nociception

The results in Fig. 2 (A, B, and C) show that TTHL given systemicallyby i.p. route (2.1–65.5 µmol/kg), peripherally by i.pl. route (6.5–65.5 nmol/paw, co-administered with glutamate) or spinally, by i.t.

(21.8–655 nmol/site) route, caused significant and dose-dependentinhibition of glutamate-induced nociception. ID50 values were 12.0(8.0–17.0)µmol/kg; 34.2 (26.6–44.1)nmol/paw; and 233.8 (144.5–382.3)nmol/site, with inhibitions of 78±6%; 82±4% and 77±8%,respectively (Fig. 2A, B and C).

3.2. Effect of TTHL, MK-801 and (RS)-MCPG on algogen-induced overtnociception

3.2.1. Effect of TTHL on excitatory aminoacid and substance P-inducednociception

The results depicted in Fig. 3 show that TTHL administeredintrathecally (6.5–218 nmol/site, co-administered with glutamate)caused a significant and dose-dependent reduction of the nociceptiveresponse induced by glutamate injection. ID50 values were 54.5 (51.2–57.8)nmol/site and inhibition of 51±6% in the dose of 65.5 nmol/site.Thus, the dose of 65.5 nmol/site TTHLwas chosen for all further studies.

As presented in Fig. 4, i.t. administration of NMDA, trans-ACPD,AMPA, kainate and substance P promoted significant nociceptiveresponse in mice. The results were: 156.0±7.3 for NMDA; 209.6±15.1 for trans-ACPD; 37.1±2.6 for AMPA; 163.3±28.3 for kainate and95.4±9.6 for substance P. I.t. injection of vehicle induced nociceptiveresponse thatwas not significant (data not shown). In all groups, exceptAMPA and kainate groups, TTHL co-injection (65.5 nmol/site, i.t.)significantly reduced the nociceptive response when compared with

Fig. 3. Effect of TTHLgiven i.t. (6.5–218 nmol/site, co-administered)on glutamate-inducednociceptive response (C, 175 nmol/site, i.t). Each column represents themean±S.E.M. of 6to 12 animals. The symbols denote the levels of significance: ***P<0.001 compared to Cgroup, # P<0.001 from V group (one-way ANOVA followed by Newman–Keuls' test).Vehicle (V) and Glutamate=Control (C).

Fig. 5. Effect of MK-801 (3 nmol/site) or (RS)-MCPG (100 nmol/site) given i.t. (co--administered) on nociceptive response induced by i.t. injection of glutamate (175 nmol/site) in mice. Each column represents the mean±S.E.M. of 6 to 11 animals. The symbolsdenote the levels of significance: **P<0.01 and ***P<0.001 compared to C group (one-way ANOVA followed by Newman–Keuls' test). Glutamate=Control (C).

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the respective control group (Fig. 4). The inhibition observed was 81±7%; 79±7% and 81±11% for NMDA, trans-ACPD and substance P,respectively.

3.2.2. Effect of MK-801 and (RS)-MCPG on spinal glutamate, NMDA ortrans-ACPD- induced nociceptive response

First,we evaluated the effect of i.t. co-injection ofMK-801(a selectivenon-competitive NMDA antagonist, 3 nmol/site) or (RS)-MCPG (a non-selective group I/group II mGlu receptor antagonist, 100 nmol/site) inglutamate-induced nociceptive response. We observed that the noci-ceptive behavior induced by glutamate (175 nmol/site, i.t) wassignificantly reduced by both antagonists, with inhibitions of 78±10%for MK-801 and 63±15% for (RS)-MCPG (Fig. 5).

Fig. 6 illustrates the effects of i.t. co-injection of MK-801 or (RS)-MCPGwithNMDA(450 pmol/site, Fig. 6A) or trans-ACPD (10 nmol/site,Fig. 6B). The results showed that NMDA-induced nociceptive responsewas inhibited by i.t. co-injection ofMK-801 (inhibition of 77±11%), butit was unaffected by (RS)-MCPG (Fig. 6A). Conversely, trans-ACPD-induced nociception was inhibited by either antagonists (RS)-MCPG orMK-801. The inhibition observed was 77±9% and 37±9% for (RS)-MCPG andMK-801, respectively (Fig. 6B). Furthermore, the injection ofMK-801 or (RS)-MCPG per se did not induce nociceptive behavior whencompared to mice treated with vehicle (5 μl/site) (results not shown).

3.2.3. Effect of combined i.t. sub-effective doses of TTHL,MK-801 and (RS)-MCPGon spinal NMDA or trans-ACPD-induced nociceptive response

The results presented in Fig. 7 show the effect of combinedadministration of sub-effective doses of MK-801 (1 nmol/i.t.) withTTHL (6.5 nmol/i.t.) or (RS)-MCPG (30 nmol/i.t.) against NMDA(450 pmol/i.t.) or trans-ACPD (10 nmol/i.t.)-induced nociceptive re-sponse (Fig. 7A and B).When administered alone at sub-effective doses

Fig. 4. Effect of TTHL given i.t. (65.5 nmol/site, co-administered) on nociceptivebehavior induced by i.t injection of NMDA (450 pmol/site), trans-ACPD (10 nmol/site),AMPA (135 pmol/site), kainate (110 pmol/site) and SP (100 pmol/site) in mice. Eachcolumn represents the mean±S.E.M. of 6 to 11 animals. The symbols denote the levelsof significance: **P<0.01 and ***P<0.001 compared to their respective C group (one-way ANOVA followed by Newman–Keuls' test). NMDA, trans-ACPD, AMPA, Kainate,SP=Control (C).

neitherMK-801, (RS)-MCPG nor TTHL affected the nociception inducedby NMDA or trans-ACPD (Fig. 7A and B). However, the combinedadministration of sub-effective doses ofMK-801 (1 nmol/i.t.) with TTHL(6.5 nmol/i.t.) produced a synergistic antinociceptive effect (inhibitionof 73±5%) onNMDA-induced nociceptive response inmice (Fig. 7A). Inaddition, the combined administration of sub-effective doses ofMK-801(1 nmol/i.t.) with (RS)-MCPG (30 nmol/i.t.) or RS-MCPG with TTHLreduced in 33±3 and 38±4%, but not significantly, the nociceptivebehavior caused by NMDA, respectively (Fig. 7A).

The results depicted in Fig. 7B show that the combined administra-tion of sub-effective doses of (RS)-MCPG (30 nmol/i.t.) with TTHL(6.5 nmol/i.t.) produced a synergistic antinociceptive effect (inhibitionof 78±7%) on trans-ACPD-induced nociceptive response in mice.However, the combined administration of sub-effective doses of MK-801 (1 nmol/i.t.) with (RS)-MCPG (30 nmol/i.t.) or MK-801 with TTHLdid not significantly affect the nociceptive behavior caused by trans-ACPD (Fig. 7B).

Fig. 6. Effect of MK-801 (3 nmol/site) or (RS)-MCPG (100 nmol/site) given i.t. (co--administered) on nociceptive response induced by i.t. injection of NMDA (450 pmol/site, Panel A) or trans-ACPD (10 nmol/site, Panel B) inmice. Each column represents themean±S.E.M. of 6 to 11 animals. The symbols denote the levels of significance:**P<0.01 and ***P<0.001 compared to their respective C group (one-way ANOVAfollowed by Newman–Keuls' test). NMDA, trans-ACPD=Control (C).

Fig. 7. Effect of sub-effective doses of MK-801 (1 nmol/site), (RS)-MCPG (30 nmol/site),TTHL (6.5 nmol/site), MK-801 (1 nmol/site) plus TTHL (6.5 nmol/site), (RS)-MCPG(30 nmol/site) plus TTHL (6.5 nmol/site) or MK-801 (1 nmol/site) plus (RS)-MCPG(30 nmol/site) given i.t. (co-administered) on nociceptive response induced by i.tinjection of NMDA (450 pmol/site, Panel A) or trans-ACPD (10 nmol/site, Panel B) inmice. Each column represents the mean±S.E.M. of 6 to 11 animals. The symbols denotethe levels of significance: **P<0.01, ***P<0.001 and non-significant (NS) compared totheir respective C group (one-way ANOVA followed by Newman–Keuls' test). NMDA,trans-ACPD=Control (C).

34 D.T. Longhi-Balbinot et al. / European Journal of Pharmacology 623 (2009) 30–36

3.3. Effect of TTHL, MK-801 and (RS)-MCPG on the locomotor activity(open-field test)

Intrathecal injection of TTHL (65.5 and 218 nmol/site), MK-801(3 nmol/site) or (RS)-MCPG (100 nmol/site), given 15 min beforehand,did not affect the locomotor activity in the open-field test whencompared to mice that received vehicle. The means±S.E.M. of crossingnumbers were 103.4±3.8 for vehicle, 104.2±11.9 for 65.5 nmol/siteTTHL, 94.5±6.5 for 218 nmol/site TTHL; 117.6±11.9 for MK-801 and130.5±5.6 for (RS)-MCPG.

4. Discussion

The present study extended the previous data and demonstratedthat the glutamatergic system plays a significant role in the systemic(intraperitoneal), peripheral (intraplantar) and central (intrathecal)antinociceptive effect produced by TTHL in mice. It has beendemonstrated that glutamate promotes depolarization of primaryafferent C fibers and consequently it causes a direct activation ofnociceptive neurons. Once activated, nociceptive neurons releaseseveral inflammatory mediators and neuropeptides that are involvedin nociceptive transmission in both central and peripheral nervoussystem (Fundytus, 2001; Millan, 1999). Furthermore, Beirith et al.(2002) have found that nociceptive response induced by intraplantarglutamate appears to involve peripheral, spinal and supraspinal sites ofaction and is significantly mediated by both ionotropic and metabo-tropic glutamate receptors.

Considering that TTHL administered either systemically or centrallyevoked antinociceptive effect against the nociception induced byperipheral injection of glutamate, we suggest that this effect could beby a direct action on glutamatergic neurons presents in the spinal cord.Then, we demonstrated that the intrathecal injection of TTHLsignificantly inhibited nociception caused by spinal administration ofglutamate. Glutamate, exerts its effects through both ionotropic and

metabotropic glutamate receptors. Ionotropic glutamate receptors areknown to activate cation-specific ion channels that are classified in threedifferent groups: AMPA, kainate and NMDA (Nakanishi, 1992).Differently,metabotropic glutamate receptors are coupled toG-proteinsand modulate the activation of second messengers, mainly inositol1,4,5,-trisphosphate, diacyl–glicerol and cyclic nucleotides (for a reviewsee Bleakman et al., 2006; Hudspith, 1997; Ozawa et al, 1998). To date,eight subtypes of metabotropic glutamate receptors have been clonedand they are termed metabotropic glutamate receptors 1–8. Thesereceptors are classified into I (metabotropic glutamate receptors 1 and5), II (metabotropic glutamate receptors 2 and 3) and III (metabotropicglutamate receptors 4, 6, 7 and 8) groups. The classification is based ontheir sequence homologies, pharmacology and coupling to intracellulareffector systems (for a review see Bleakman et al., 2006; Conn and Pin,1997).

In this study we showed that the intrathecal injection of65.5 nmol/site TTHL, also described to reduce significantly thenociception caused by spinal administration of glutamate, signifi-cantly attenuated the nociceptive response induced by i.t. injection ofthe ionotropic and metabotropic receptor agonist, NMDA and trans-ACPD. However, TTHL did not inhibit the nociceptive response causedby i.t. injection of the ionotropic receptor agonists AMPA and kainate.Although AMPA and kainate receptors were not involved in theantinociceptive response by spinal injection of TTHL in our scheme oftreatment, the activation of different kind of receptors seems to play acrucial role in pain transmission in spinal cord (Sahara et al., 1997;Willis, 2001). According to our earlier hypothesis, there is aninvolvement of spinal glutamatergic system on the central antinoci-ceptive effect of TTHL, possibly through interaction with NMDA andmetabotropic glutamate receptors.

Based on the results above, we investigated whether NMDA andmetabotropic receptor antagonists injected i.t. were able to producethe same effect as TTHL to inhibit the nociceptive response induced byspinal injection of glutamate, NMDA and trans-ACPD. The resultsshowed that like TTHL, MK-801 (a non-competitive NMDA receptorantagonist) and (RS)-MCPG (non-selective metabotropic glutamatereceptor antagonist) significantly inhibited the nociception inducedby i.t. injection of glutamate. We also demonstrated that MK-801, in adose that reduces by 77±11% the nociceptive response caused byNMDA, partially reduced (37±9%) the nociceptive behavior inducedby i.t. trans-ACPD, a non-selective metabotropic glutamate receptoragonist. Furthermore, our data showed that (RS)-MCPG, was notcapable to significantly inhibit the nociceptive behavior induced byNMDA. However, it inhibited the nociceptive behavior induced by i.t.trans-ACPD in mice. Noteworthy, the present study showed that TTHLand MK-801, were effective to inhibit the nociceptive responseinduced by i.t. injection of NMDA and trans-ACPD. Therefore, themetabotropic glutamate receptor antagonist (RS)-MCPG had no effecton NMDA-induced nociception, but the NMDA receptor antagonist(MK-801) attenuated trans-ACPD-induced nociception, which sug-gests an interaction between NMDA and metabotropic glutamatereceptors to produce nociception. Taking this into account, the resultsobtained with TTHL are interesting for demonstrating that it caninhibit the activation of both NMDA and metabotropic glutamatereceptors.

It has been described a positive postsynaptic interaction betweenNMDA and group I metabotropic glutamate receptors in dorsal hornneurons (Bleakman et al., 1992; Bond and Lodge, 1995; Cerne andRandic, 1992; Jones and Headley, 1995; Zhong et al., 2000). Inagreement with these data, several studies have provided evidencethat spinal group I metabotropic glutamate receptors (1 and 5) maypotentiate NMDA responses and are involved on nociceptive transmis-sion (Fisher and Coderre, 1996; Fundytus, 2001; Neugbauer et al., 1994;Zanchet et al., 2004). In addition, spinal metabotropic glutamatereceptors 1 and 5 contribute to the regulation of the strength ofexcitatory amino acid mediated primary afferent neurotransmission.

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When activated they lead to the release of glutamate which may,consequently, enhance NMDA receptor activity in the dorsal horn(Cerne and Randic, 1992; Neugbauer et al., 1994; Sevostianova andDanysz, 2006;Ugolini et al., 1997;Urban et al., 1994). Evidences suggestthat activation of metabotropic glutamate receptors 1 and 5 stimulatephospholipase Cβ through Gq/11 proteins to convert phosphatidilino-sitol biphosphate into diacyl-glycerol (DAG) and IP3. Hence, DAGactivates Ca2+-dependent and independent PKC isoforms, which canphosphorylate serine/threonine sites on NMDA receptor (Sánchez-Pérez and Felipo, 2005). Thus, one functional consequence of metabo-tropic glutamate receptor activation is to sensitizeNMDA receptors for afurther hyperexcitability (Gabra et al., 2007; Guo et al., 2004).

With this in mind, we investigated the possible involvement ofNMDA and metabotropic glutamate receptors (or an interactionbetween them) on the antinociceptive effect of TTHL. We associatedsub-effective doses of (RS)-MCPG and TTHL or MK-801 and TTHLagainst nociception induced by i.t. injection of NMDA or trans-ACPD.Interestingly, the combined administration of sub-effective doses of(RS)-MCPG plus TTHL or MK-801 plus TTHL produced a synergicantinociceptive effect on trans-ACPD- andNMDA-induced nociceptiveresponse, respectively. The inhibitionswere similar to those caused bythe individual injections of the antagonists or TTHL. Conversely, sub-effective doses of (RS)-MCPG plus TTHL or MK-801 plus TTHL did notaffect the nociceptive response induced by NMDA and trans-ACPD,respectively. Together, these results suggest that the antinociceptiveeffect of TTHL could be due to an interaction with commonintracellular glutamatergic pathways e.g. protein kinase C, intracellu-lar increase of calcium, mitogen-activated kinases (MAPKs/ERK)(Meller et al., 1996; Wei et al., 2006; Yang et al., 2004) rather thanto a direct interaction with NMDA and metabotropic glutamatereceptors. However, other experiments are necessary to furthersubstantiate this assertion.

It has been suggested that i.t. injection of substance P activatesneurokinin 1 receptors, triggers the release of glutamate in the dorsalhorn and transmits nociceptive signal to higher areas of the centralnervous system (Battaglia and Rustioni, 1988; Biasi and Rustioni,1988; Lima-Júnior et al., 2007; Millan, 1999). In line with this, the co-administration of TTHL significantly inhibited nociceptive responseinduced by substance P. This result, associated with the inhibition ofspinal glutamate-, NMDA- and trans-ACPD-induced nociceptiveresponse reinforce a possible inhibitory effect of TTHL on glutama-tergic transmission.

Of interest, the results of the present study also showed that theantinociception caused by TTHL was not associated with non-specificeffects such as locomotor impairment. In this regard, it has beenreported that several drugs that antagonize NMDA receptors (Parsons,2001) or group I metabotropic glutamate receptors (Pietraszek et al.,2007; Takahashi and Afford, 2002) produce significant disturbances inlocomotor activity. Nonetheless, the used doses of NMDA, metabo-tropic glutamate antagonists, MK-801 and (RS)-MCPG did notsignificantly affect the locomotor activity of mice. Thus, these dataclearly indicate that TTHL produces significant and specific antinoci-ceptive action against glutamate-, NMDA- and trans-ACPD-inducednociception without causing any detectable locomotor impairment.

Collectively, the results of this present study showed that theantinociceptive effect of TTHL is closely related to an effect on theglutamatergic system, mainly on NMDA and metabotropic glutamatereceptors or their intracellular signaling pathways.

Acknowledgements

This work was supported by grants from Conselho Nacional deDesenvolvimento Científico e Tecnológico (CNPq), Coordenação deAperfeiçoamento de Pessoal de Nível superior (CAPES), Fundação deApoio à Pesquisa Científica Tecnológica do Estado de Santa Catarina(FAPESC) and Financiadora de Estudos e Projetos [FINEP, Rede

Instituto Brasileiro de Neurociência (IBN-Net)], Brazil. DT Longhi-Balbinot and DF Martins are PhD students in Neuroscience; VMGadotti and EF Pietrovski are PhD in Neuroscience and Pharmacology,respectively. They thank CNPq and CAPES for financial support. Dr.ARS Santos is a CNPq recognized researcher (1C category) and has anadditional financial support from CNPq.

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