European Journal of Pharmacology 590 (2008) 127–135

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European Journal of Pharmacology

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Dual role of hydrogen sulfide in mechanical inflammatory hypernociception

Thiago M. Cunha a, Daniela Dal-Secco a, Waldiceu A. Verri Jr. a, Ana T. Guerrero a, Guilherme R. Souza a,Silvio M. Vieira a, Celina M. Lotufo a, Alberto F. Neto b, Sergio H. Ferreira a, Fernando Q. Cunha a,⁎a Department of Pharmacology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazilb Department of Pharmaceutical Science, Faculty of Pharmaceutical Sciences, University of São Paulo, Brazil

⁎ Corresponding author. Department of PharmacoRibeirão Preto, University of São Paulo, Brazil, Av. BandSP Brazil. 14049-900. Tel.: +55 16 3602 3204; fax: +55 1

E-mail address: fdqcunha@fmrp.usp.br (F.Q. Cunha).

0014-2999/$ – see front matter © 2008 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.05.048

A B S T R A C T

A R T I C L E I N F O

Article history:

Hydrogen sulfide (H2S) is a Received 21 December 2007Received in revised form 14 May 2008Accepted 31 May 2008Available online 7 June 2008

Keywords:Hydrogen sulfideInflammatory painHyperalgesiaNociceptionNeutrophilAntinociception

n endogenous gas involved in several biological functions, including modulationof nociception. However, the mechanisms involved in such modulation are not fully elucidated. The presentstudy demonstrated that the pretreatment of mice with PAG, a H2S synthesis inhibitor, reduced LPS-inducedmechanical paw hypernociception. This inhibition of hypernociception was associated with the prevention ofneutrophil recruitment to the plantar tissue. Conversely, PAG had no effect on LPS-induced production of thehypernociceptive cytokines, TNF-α, IL-1β and CXCL1/KC and on hypernociception induced by PGE2, a directlyacting hypernociceptive mediator. In contrast with the pro-nociceptive role of endogenous H2S, systemicadministration of NaHS, a H2S donor, reduced LPS-induced mechanical hypernociception in mice. Moreover,this treatment inhibited mechanical hypernociception induced by PGE2, suggesting a direct effect of H2S onnociceptive neurons. The antinociceptive mechanism of exogenous H2S depends on K(ATP)

+ channels since theinhibition of PGE2 hypernociception by NaHS was prevented by glibenclamide (K(ATP)

+ channel blocker).Finally, NaHS did not alter the thermal nociceptive threshold in the hot-plate test, confirming that its effect ismainly peripheral. Taken together, these results suggest that H2S has a dual role in inflammatoryhypernociception: 1. an endogenous pro-nociceptive effect due to up-regulation of neutrophil migration, and2. an antinociceptive effect by direct blockade of nociceptor sensitization modulating K(ATP)

+ channels.© 2008 Elsevier B.V. All rights reserved.

1. Introduction

Pain is one of the classic signs of the inflammatory process. It isnow accepted that the sensitization of the primary sensory neurons isan essential event in the genesis of inflammatory pain. In humans, thisnociceptor sensitization usually leads to clinical conditions known ashyperalgesia (an increased response to a stimulus that is normallypainful) or allodynia (pain due to a stimulus that does not normallyprovoke pain) better described as hypernociception in animal models(Cunha et al., 2007a). The mechanisms involved in the sensitization ofthe primary sensory neurons, and consequently in the establishmentof inflammatory hypernociception, may be divided into two phases.The first involves the non-neuronal events: the resident (macro-phages, mast cells, etc.) and migratory immune cells (neutrophils andlymphocytes) produce a vast number of hypernociceptive inflamma-tory mediators including pro-nociceptive cytokines (tumor necrosisfactor (TNF)-α, interleukin (IL)-1β and chemokines), leukotrienes(LTB4), nerve growth factor, endothelins and kinins, which trigger therelease of directly acting hypernociceptive mediators (Verri et al.,

logy, Faculty of Medicine ofeirantes 3900 Ribeirao Preto,6 3633 0021.

l rights reserved.

2006a). The most well-known, direct-acting hypernociceptive med-iators are the prostaglandins. These mediators are considered direct-acting because they activate their specific receptors present on themembrane of nociceptive neurons. The second phase includesneuronal events: activation of the receptors on primary nociceptiveneurons by direct-acting mediators, which triggers intracellularsignaling pathways dependent on cyclic AMP, protein kinase A andprotein kinase C (Aley and Levine, 1999; Khasar et al., 1999). Thesesignaling pathways result in the subsequent phosphorylation ofvoltage-dependent sodium channels (Gold et al., 1998) and inhibitionof voltage-dependent potassium channels (Evans et al., 1999; Nicolet al., 1997), leading to enhanced neuron excitability.

Hydrogen sulfide (H2S) is an endogenous gaseous substanceformed from the metabolism of L-cysteine by the action of cystathio-nine γ-lyase or cystathionine β-syntase (Szabo, 2007). Since thediscovery of the endogenous cysteine/H2S pathway, many efforts havebeen made to understand the physiologic and pathologic roles of thisgas (Li et al., 2005; Szabo, 2007). For instance, a potential role of H2S insystemic inflammatory and cardiovascular diseases such as septic andendotoxic shock and pulmonary hypertension has been suggested(Hui et al., 2003; Li et al., 2005; Szabo, 2007; Yanfei et al., 2006; Zhanget al., 2007b). Furthermore, there is evidence that H2S mediates localinflammation; for example, it is involved in neutrophil migration,edema formation and nociception (Bhatia et al., 2005; Cunha and

128 T.M. Cunha et al. / European Journal of Pharmacology 590 (2008) 127–135

Verri, 2007; Distrutti et al., 2006a; Kawabata et al., 2007; Li et al.,2006). Concerning the nociceptive process, in two seminal studies,Distrutti et al. (2006a,b) showed that systemic administration ofdifferent H2S donors inhibits visceral nociception by opening ATP-sensitive potassium channels (K(ATP)

+ channels) (Distrutti et al., 2006a;Distrutti et al., 2006b). On the other hand, a recent study using adifferent model demonstrated that H2S has a peripheral pro-nociceptive activity (Kawabata et al., 2007). This conclusion is mainlysubstantiated by the observation that intraplantar administration of aH2S donor induces a mechanical hypernociception of fast onset(25 min) and that the inhibition of endogenous H2S formation reduceslipopolysaccharide (LPS)-induced inflammatory hypernociception(Kawabata et al., 2007). The mechanisms of H2S mediation ofinflammatory hyperalgesia seem to be dependent on the directmodulation of T-type Ca2+ channel activity in nociceptors whileindependent of K(ATP)

+ channels (Kawabata et al., 2007). Thus, these studiesdetected discrepant nociceptive roles for the samemediator. Therefore, inthe present study we used two models of mechanical inflammatoryhypernociception to provide more evidence that could explain thesecontradictory findings concerning the nociceptive role of H2S.

2. Methods

2.1. Animals

Behavioral experiments were performed onmaleWistar rats (180–200 g) and male C57Bl6 mice (20–25 g) housed in the animal carefacility of the School of Medicine of Ribeirao Preto and taken to thetesting room at least 1 h before the experiments. Rats were preferablewhen the local effect of H2S was investigated, since it is easier toperform more than 2 injections into their paw. Food and water wereavailable ad libitum. All behavioral tests were performed between9:00 AM and 5:00 PM, and the animals were only used once. Animalcare and handling procedures were in accordance with the guidelinesof the International Association for the Study of Pain (IASP) on the useof animals in pain research. All efforts were made to minimize thenumber of animals used and their discomfort.

2.2. Drugs

The drugs used in this study were: diazoxide, glibenclamide,prostaglandin E2 (PGE2), DL-propargylglycine (PAG) and zymosan(from Sigma, St. Louis, MO). Bacterial endotoxin was from E. coli andreferred here as lipopolysaccharide (LPS-Difco Laboratories Ltd, WestMolsey, Surrey, U.K.). Sodium hydrosulfide (NaHS) was synthesized byProf. Dr. Alberto Federman Neto. A stock solution of PGE2 (1 μg/μl) wasprepared in 10% ethanol, and dilutions were made in 0.9% NaCl(saline); the final concentration of ethanol was 1%. The ATP-sensitivepotassium channel blockers (Alves and Duarte, 2002), glibenclamideand diazoxide, were dissolved in 1% DMSO and diluted in saline. Otherdrugs were diluted in sterile saline.

2.3. Evaluation of mechanical hypernociception

2.3.1. Paw testThe term hypernociception was used to define the decrease in

nociceptive withdrawal threshold (Verri et al., 2006a,b; Cunha et al.,2007b). Mechanical hypernociception was tested in mice and rats aspreviously reported (Cunha et al., 2004; Vivancos et al., 2004). Briefly,in a quiet room, rats or mice were placed in acrylic cages(12×20×17 cm) with wire grid floors, 15–30 min before the start ofthe test. The test consisted of evoking a hind paw flexion reflex with ahand-held force transducer adapted with a 0.7-mm2 or 0.5-mm2

polypropylene tip for rats and mice, respectively (Electronic von Frey;IITC Life Science, Woodland Hills, CA). A tilted mirror placed under thegrid provided a clear view of the rat hind paw. The investigator was

trained to apply the tip in between the five distal footpads with agradual increase in pressure. The stimulus was automaticallydiscontinued and its intensity recorded when the paw was with-drawn. The end-point was characterized by the removal of the paw ina clear flinch response after paw withdrawal. The animals were testedbefore and after treatments. A different investigator performed eachtest, as was the case in the preparation of solutions and treatment ofthe animals. The results are expressed by the Δ withdrawal threshold(in grams, g) which was calculated by subtracting the average of thelast three measurements after treatments from the average of threemeasurements before treatments.

2.3.2. Articular nociceptionArticular hypernociception was evaluated as previously described

(Guerrero et al., 2006). For thismodel, a polypropylene tip probewith anarea size of 4.2 mm2 that was adapted to a hand-held force transducer,instead of the standard tip probe (0.5 mm2), was applied on the plantarsurface of a hind paw to produce a tibio-tarsal flexion movement. Anincreasing perpendicular force was applied to the central area of theplantar surface of the hind paw to induce a dorsal flexion of the tibio-tarsal joint, followed by paw withdrawal. A tilted mirror below the gridprovided a clear view of the animal's hind paw. The electronic pressure-meter apparatus automatically recorded the intensity of the forceapplied when the paw was withdrawn. The test was repeated until 3subsequentlyconsistentmeasurementswereobtained (i.e., thevariationamong these measurements was less than 2 g). The flexion-elicitedwithdrawal threshold is expressed in grams (g).

2.4. Cytokine measurements

At 3 h after LPS intraplantar injection, animals were terminallyanesthetized, and the skin tissues of the plantar regionwere removedfrom the injected and control paws (saline). The samples were tri-turated and homogenized in 500 μl of the appropriate buffercontaining protease inhibitors followed by centrifugation for 10 minat 2000 ×g. The supernatants were used to determine the levels ofTNF-α, IL-1β and keratinocyte-derived chemokine (CXCL1/KC) asdescribed previously (Cunha et al., 2007b; Cunha et al., 2005; Gue-rrero et al., 2006) by enzyme-linked immunosorbent assay (ELISA).The results are expressed as picograms (pg) of each cytokine per paw.Control concentrations of these cytokines were determined in animalsinjected with saline.

2.5. Neutrophil migration to the plantar and joint tissue

Neutrophil migration to the hind paw plantar and tibio-tarsal jointtissues of mice was evaluated using a myeloperoxidase (MPO) kinetic-colorimetric assay, as previously described (Bradley et al., 1982;Guerrero et al., 2006; Valerio et al., 2007). Samples of subcutaneousplantar and joints tissue were collected in 50 mM K2HPO4 buffer (pH6.0) containing 0.5% hexadecyl trimethylammonium bromide (HTAB)and kept at −80 °C until use. Samples were homogenized using aPolytron (PT3100), centrifuged at 16,100 ×g for 4 min and the resultingsupernatant assayed for MPO activity spectrophotometrically at450 nm (Spectra max), with 3 readings in 1 min. MPO activity ofsampleswas compared to a standard curve of neutrophils. Briefly,10 μlof sample was mixed with 200 μl of 50 mM phosphate buffer, pH 6.0,containing 0.167 mg/ml O-dianisidine dihydrochloride and 0.0005%hydrogen peroxide. The results are presented asMPO activity (numberof neutrophils per mg of tissue).

2.6. Hot-plate test

Micewere placed in a 10-cmwide glass cylinder on a hot plate (IITCLife Science Inc. CA, USA) maintained at 55.0 °C. Two control latenciesat least 10 min apart were determined for each mouse. The normal

Fig. 1. The role of endogenous H2S in LPS- and zymosan-induced mechanicalinflammatory hypernociception. Panel A — Mice were pretreated with PAG (3–30 mg/kg; s.c.) 30 min before i.pl. injection of LPS (100 ng/paw). Panel B — Mice werepretreated with PAG (30 mg/kg; s.c.) 30 min before intrarticular injection of zymosan(zy; 30 mg/joint). Mechanical hypernociception was evaluated 3 h after LPS injectionand 1, 3 5 and 7 h after zymosan injection. ⁎For Pb0.05 when compared to saline-injected paws and joints, and #for Pb0.05 when compared to PAG vehicle-treatedgroup. One-way ANOVA followed by Tukey's post hoc test (n=5).

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latency (reaction time) was 5–9 s. The latency was also evaluated 15,30, 60, 180 min after drug administration. The reaction time wasscored when the animal jumped or licked its paws. A maximumlatency (cut-off) was set at 30 s to minimize tissue damage.

2.7. Statistical analysis

Results are presented asmeans±S.E.M. and are representative of twoexperiments. The letter n in the legends refers to the number of mice orrats used in the experimental group of each experiment. The differencesbetween the experimental groups were compared by ANOVA (one-way), and individual comparisons were subsequently made withTukey's post hoc test. The level of significance was set at Pb0.05.

3. Results

3.1. Endogenous H2S mediates mechanical inflammatory hypernociception

To address the involvement of endogenous H2S in the genesis ofmechanical inflammatory hypernociception, we analyzed the effect ofPAG, an irreversible inhibitor of H2S formation (Teague et al., 2002; Urenet al.,1978), onLPS-inducedmechanical inflammatoryhypernociceptionand in a model of zymosan-induced articular hypernociception in mice.The pretreatment of mice with PAG (3–30 mg/kg, 30 min before LPSinjection, s.c.) significantly reduced the mechanical hypernociceptioninduced by intraplantar injection of LPS (100 ng/paw) in a dose-dependentmanner (Fig.1A). Ahigherdoseof PAG (90mg/kg, s.c.) didnotproduce a further effect (data not shown). It is important tomention thatPAG treatment did not alter the baseline nociceptive threshold of theanimals (data not shown). The pretreatment of mice with PAG (30 mg/kg, s.c., 30 min before zymosan injection) also inhibited zymosan-induced articular hypernociception (Fig.1B), reinforcing the importanceof H2S in the genesis of inflammatory nociception.

3.2. Neutrophil migration seems to be involved in the pro-nociceptiverole of endogenous H2S

Our group and others have demonstrated that cytokines (TNF-α, IL-1βand CXC chemokine) (Cunha et al., 2005; Verri et al., 2007, 2006b) andneutrophils (Guerrero et al., 2008; Levine et al.,1985, Cunha et al., 2008a,b,Ting et al., 2008) play an important role in the genesis of mechanicalinflammatory hypernociception. Since endogenous H2S production hasbeen shown to be involved in the recruitment of neutrophils (Bhatia et al.,2005; Zhang et al., 2007b) and cytokine production during theinflammatory process (Li et al., 2005; Zhang et al., 2007a; Zhi et al.,2007), we tested the hypothesis that these mechanisms are involved inH2S mediation of inflammatory hypernociception. First, it was observedthat PAG (3–30 mg/kg, s.c.) pretreatment inhibited LPS-induced neutro-phil migration to mouse plantar tissue and zymosan-induced neutrophilmigration to the joint (Fig. 2A and B). However, pretreatment with PAG(30 mg/kg, s.c.) was not able to inhibit LPS-induced production of TNF-α,IL-1β and KC/CXCL1 in the mouse paw (Fig. 2C–E). Supporting thehypothesis that theantinociceptiveeffectof PAGdependson the inhibitionof H2S modulation of neutrophil migration, PAG (30 mg/kg, s.c.)pretreatment did not inhibit the mechanical hypernociception inducedby direct-acting hypernociceptive mediators such as PGE2 (Fig. 2F).Hypernociception induced by PGE2 does not depend on neutrophilmigration (Cunha et al., 2008a).

3.3. Exogenous H2S inhibits mechanical inflammatory hypernociception

Contrary to the pro-nociceptive role of endogenous H2S mentionedabove, systemic pretreatment ofmicewith aH2S donor (NaHS; 3–90 μmol/kg, s.c., 15 min before LPS injection) inhibited LPS-induced mechanicalhypernociception in a dose-dependentmanner (Fig. 3A). In further supportof the antinociceptive action of exogenous H2S, pretreatment with NaHS

(90 μmol/kg,15min before zymosan injection, s.c.) also inhibited zymosan-induced articular hypernociception (Fig. 3B). In contrast with thisantinociceptive activity of exogenous H2S, pretreatment with NaHS (3–90 μmol/kg, s.c., 15min before LPS injection and 90 μmol/kg,15min beforezymosan injection, s.c.) enhanced LPS-induced neutrophil migration to thepaw (Fig. 3C). The results showed only a tendency toward increasedneutrophil migration (MPO activity) in the zymosan group treated withNaHS when compared with the vehicle-treated group (Fig. 3D). Theenhancementwasnot statisticallydifferentprobablybecause themigrationobserved in the control group was near maximal due to the use of a highdose of zymosan.

It was also observed that the treatment of mice with NaHS(90 μmol/kg, s.c., 15 min before LPS injection) did not alter either theproduction of cytokines (TNF-α, KC and IL-1β induced by LPS) or thebaseline production of these cytokines (Fig. 4).

3.4. H2S promotes antinociception by direct blockade of hypernociception

Next, we investigated the effect of H2S on mechanical hypernoci-ception induced by a direct-acting hypernociceptive mediator, PGE2.This type of hypernociceptive mediator acts directly on its receptor

Fig. 2. Role of neutrophils and cytokines in the pro-nociceptive action of endogenous H2S. Panel A—Mice were pretreated with PAG (3–30 mg/kg; s.c.) 30 min before i.pl. injection ofLPS (100 ng/paw). Panel B — Mice were pretreated with PAG (30 mg/kg; s.c.) 30 min before intrarticular injection of zymosan (zy; 30 mg/joint). After the evaluation of mechanicalhypernociception (Fig. 1A and B), subcutaneous plantar hind paw tissue or tibio-tarsal joint tissue was collected for MPO analysis. (Panels A and B, respectively) Panels C–E — Micewere pretreated with PAG (30 mg/kg; s.c.) 30 min before i.pl. injection of LPS (100 ng/paw). At 3 h after LPS injection, the subcutaneous plantar hind paw tissues were collected,homogenized and the levels of cytokines (TNF-α, IL-1β and CXCL1/KC) weremeasured by ELISA. Panel F—Micewere pretreatedwith PAG (30mg/kg; s.c.) 30min before i.pl. injectionof PGE2 (100 ng/paw). ⁎For Pb0.05 when compared to saline-injected paws and joints, and #for Pb0.05 when compared to PAG vehicle-treated group. One-way ANOVA followed byTukey's post hoc test (n=5).

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present on the membrane of nociceptive fibers triggering theirsensitization, which is detected as hypernociception (Bley et al.,1998). Systemic pretreatment of mice with NaHS (90 μmol/kg, s.c.,15min before PGE2 injection) also inhibited PGE2-inducedmechanicalhypernociception, suggesting that H2S directly blocks mechanicalhypernociception (Fig. 5A).

In examining the possible local site for the antinociceptive actionof exogenous H2S, local post-treatment (hind paw) of rats with NaHS(0.1–100 nmol/paw, 2 h after PGE2 injection) reversed the ongoinghypernociception induced by PGE2 (100 ng/paw; Fig. 5B). Moreover,the injection of NaHS (100 nmol/paw) into the rat hind paw did notalter the baseline nociceptive threshold (Fig. 5B). When injected intothe contralateral paw of rats, NaHS did not produce antinociception,confirming that NaHS at the dose that inhibited PGE2-inducedhypernociception acts only locally (Fig. 5B).

In contrast to the peripheral H2S site of antinociceptive action, thesystemic administration of NaHS (90 μmol/kg, s.c.,) in mice, at a dosethat inhibited PGE2-induced mechanical hypernociception, modifiedneither the baseline thermal nociceptive threshold in the hot-platetest (Fig. 5C) nor the baseline mechanical nociceptive threshold (datanot shown). The antinociceptive effect of morphine in the hot-platetest was used as control (Fig. 5C).

3.5. Role ofK(ATP)+ channel activation in antinociceptive actionof exogenousH2S

It has been demonstrated that the activation of K(ATP)+ channels in the

periphery by different substances is able to reverse ongoing hyperno-ciception induced by PGE2 (Sachs et al., 2004; Soares et al., 2000).Taking into account that the vasodilator effect of H2S depends on theactivation of this K+ channel population (Zhao et al., 2001), we testedthe hypothesis that direct blockade of mechanical hypernociceptionproduced by exogenous H2S could also be dependent on the opening ofK(ATP)+ channels in the periphery. In agreement with this hypothesis, it

was observed that the treatment of mice (40 μmol/kg, s.c.) or rats(160 μg/paw) with glibenclamide prevented the antinociceptive effectof exogenous H2S (NaHS) on PGE2-induced mechanical hypernocicep-tion (Fig. 6A and B, respectively). It is important to mention that neithersystemic nor local treatment with only glibenclamide changed PGE2-induced hypernociception in mice and rats, respectively (Fig. 6A and B).It was also seen that dizoxide (K(ATP)

+ channel opener, 300 μmol/kg, s.c.)had no effect in the hot-plate test (Fig. 5C), similar to the finding withNaHS, supporting the notion of a peripheral H2S/K(ATP)

+ channelantinociceptive pathway.

It was also investigated whether nitric oxide mediates the peri-pheral antinociceptive effect of H2S. Local treatment of rat paws with a

Fig. 4. H2S donor did not modulate LPS-induced cytokines production. Mice were pretreated with NaHS (3–90 μmol/kg; s.c.) 15 min before i.pl. injection of LPS (100 ng/paw). At 3 hafter LPS injection, the subcutaneous plantar hind paw tissues were collected, homogenized and the levels of: Panel A (TNF-α), Panel B (CXCL1/KC) and Panel C (IL-1β) weremeasuredby ELISA. ⁎For Pb0.05 when compared to saline-injected paws. One-way ANOVA followed by Tukey's post hoc test (n=5).

Fig. 3. Antinociceptive effect of exogenous H2S on inflammatory hypernociception. Panel A—Mice were pretreated with NaHS (3–90 μmol/kg; s.c.) 15 min before i.pl. injection of LPS(100 ng/paw). Panel B — Mice were pretreated with NaHS (90 μmol/kg; s.c.) 15 min before intrarticular injection of zymosan (zy; 30 mg/joint). Mechanical hypernociception wasevaluated 3 h after LPS injection and 1, 3 5 and 7 h after zymosan injection. Panels C and D — After the evaluation of mechanical hypernociception, subcutaneous plantar hind pawtissue or tibio-tarsal joint tissue was collected for MPO analysis. ⁎For Pb0.05 when compared to saline-injected paws and joints, and #for Pb0.05 when compared to NaHS vehicle-treated group. One-way ANOVA followed by Tukey's post hoc test (n=5).

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Fig. 5. Effect of exogenous H2S on PGE2-induced mechanical hypernociception: peripheral effect. Panel A — Mice were pretreated with NaHS (90 μmol/kg; s.c.) 15 min before i.pl.injection of PGE2 (100 ng/paw). Mechanical hypernociception was evaluated 1 h after PGE2 injection. Panel B — Rats received an i.pl. injection of PGE2 followed 2 h later by an i.pl.injection of NaHS (0.1–100 nmol/paw). NaHS (100 nmol/paw) was also injected into the contralateral (CL) paws. Mechanical hypernociceptionwas evaluated 3 h after PGE2 injection.Panel C— The nociceptive thermal threshold of micewas tested before and 15, 30, 60 and 180min after s.c. injection of NaHS (90 μmol/kg; s.c.), morphine (8mg/kg, s.c.) and diazoxide(300 μmol/kg) in the hot-plate test. ⁎For Pb0.05 when compared to saline-injected animals, and #for Pb0.05 when compared to NaHS vehicle-treated group. One-way ANOVAfollowed by Tukey's post hoc test (n=5).

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non-selective inhibitor of nitric oxide synthase (L-NMMA, 50 μg/paw,30 min before NaHS administration, Napimoga et al., 2008) did notprevent the antinociceptive action of NaHS (Fig. 5C).

4. Discussion

In the present study, H2S was shown to play a dual role ininflammatory hypernociception. On the one hand, production ofendogenous H2S during LPS-induced paw and zymosan-induced jointinflammation mediates the induction of mechanical hypernocicep-tion. The pro-nociceptive role of H2S seems to be closely associatedwith up-regulation of neutrophil migration to the inflammatory site.On the other hand, in contrast to the action of H2S on neutrophilmigration, the direct action of H2S on peripheral nociceptive neuronsproduces antinociception by blockade of nociceptor sensitization. This

Fig. 6. Role of K(ATP)+ channel activation and nitric oxide in the antinociceptive effect of exogen

(DMSO 2%) 15 min after they were treated with NaHS (30 μmol/kg, s.c.), and 15 min later micof PGE2 (100 ng/paw) followed 2 h later by an i.pl. injection of NaHS (100 nmol/paw). Oneinjection of NaHS. Panel C — Rats received an i.pl. injection of PGE2 (100 ng/paw) followedpretreated with L-NMMA (50 μg/paw) 30 min before injection of NaHS. Mechanical hypernoinjected paw group; #for Pb0.05 when compared to NaHS vehicle-treated group; and ⁎⁎

followed by Tukey's post hoc test (n=5).

antinociceptive effect seems to be dependent on the activation ofperipheral K(ATP)

+ channels. A dual nociceptive role for an endogen-ously formed gas/mediator is not novel, since nitric oxide (NO) andcarbon monoxide (CO) also display ambiguous actions in nociception,depending on many factors such as the nociceptive model used andnociceptive system level (Meller et al., 1994; Prado et al., 2002; Steineret al., 2001; Vivancos et al., 2003).

It has been demonstrated that inflammatory hypernociception ismediated by a cascade of cytokines, which includes the release of TNF-α, IL-1β and CXCL1/KC chemokine (Cunha et al., 1992, 2005; Ferreiraet al., 1988; Verri et al., 2006a). Although H2S is able to induce theproduction of several cytokines such as TNF-α (Zhang et al., 2007a;Zhi et al., 2007), this effect was not observed with endogenous orexogenous H2S on our experimental conditions. Actually, neitherinhibition of H2S production by PAG nor H2S donor alters LPS-induced

ous H2S. Panel A—Mice were pretreated with glibenclamide (40 μmol/kg, i.p) or vehiclee received an i.pl. injection of LPS (100 ng/paw). Panel B— Rats received an i.pl. injectiongroup of the animals was pretreated with glibenclamide (160 μg/paw) 20 min before2 h later by an i.pl. injection of NaHS (100 nmol/paw). One group of the animals was

ciception was evaluated 3 h after PGE2 injection. ⁎For Pb0.05 when compared to saline-for Pb0.05 when compared to glibenclamide vehicle-treated group. One-way ANOVA

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cytokine production in the mouse paw. On the other hand, themediation of inflammatory hypernociception by endogenous H2S,observed in the present study, was closely associated with its capacityto drive neutrophil migration toward the inflammatory site. The role ofneutrophils in the development of inflammatory hypernociception hasbeen investigated in different inflammatory models, such as carragee-nin-, LPS- and zymosan-induced inflammation (Cunha et al., 2008a,b;Ting et al., 2008; Guerrero et al., 2008). In agreement with our presentfindings, we recently demonstrated that emigrating neutrophils are notresponsible for theproduction of hypernociceptive cytokines (TNF-α, IL-1β and CXCL1/CINC-1) during carrageenin-induced inflammation in ratpaw, but they could be involved in the production of direct-actinghypernociceptive mediators such as PGE2 (Cunha et al., 2008a).Furthermore, during zymosan-induced arthritis, neutrophils alsomediate the establishment of mechanical hypernociception by promot-ing PGE2 release (Guerrero et al., 2008). H2S triggering neutrophilmigration as its pro-nociceptive role is substantiated by the fact that theinhibition of H2S formation did not reduce hypernociception induced byPGE2. In fact, PGE2 acts directly on receptors present on nociceptormembranes, triggering their sensitization (Cunhaet al.,1992). Therefore,hypernociceptionproduced by PGE2 is independent of the production ofother inflammatorymediators or cells such as neutrophils (Cunha et al.,2008a,b). The role of endogenous H2S in the recruitment of neutrophilshas been demonstrated in many inflammatory models (Bhatia et al.,2005; Zhang et al., 2007b; Dal-Secco et al., unpublished observation). Itseems that increased production of H2S during inflammationmodulatesneutrophil rolling and adhesion aswell as their locomotion. Indeed, H2Smediates the increase in the expression of ICAM-1 in the endothelialcells of mesenteric vessels induced by LPS challenge in the peritonealcavity (Dal-Secco et al., unpublished observation). Furthermore, it alsopromotes more availability of CXCR2 receptors on the neutrophilmembrane, which can explain the enhancement of MIP-2-inducedneutrophil chemotaxis by H2S (Dal-Secco et al., unpublished observa-tion; Zhang et al., 2007b). It is noteworthy that the up-modulation effectof H2S on the events responsible for neutrophil migration, as describedabove, was dependent on the activation of K(ATP)

+ channel (Secco et al.,unpublished observation).

Although we did not determine the source of H2S productionduring LPS-inducedmouse paw inflammation, there is evidence in theliterature that the production of H2S increases in the inflamed site, forexample, during carrageenin-induced rat paw inflammation (Bhatia etal., 2005). Moreover, we have shown that the activation of neutrophilswith MIP-2/(CXCL2), a CXCR1/2 ligand, induces H2S production,suggesting that H2S could be produced in the inflammatory focus bymigrating leukocytes (Secco et al., unpublished observation).

In contrast to the pro-nociceptive role of endogenous H2S in LPS-and zymosan-induced inflammatory hypernociception, exogenousH2S displays antinociceptive activity. The antinociceptive action ofexogenous H2Swas first described by Distrutti et al. (2006a,b). In thesestudies the systemic administration of different H2S donors inhibitedcolorectal distension-induced nociception in rats independent ofsmooth muscle contractility (Distrutti et al., 2006a,b). With regard tothe possible site of the antinociceptive action of H2S, a possible centraleffect was excluded because systemic administration of a H2S donor ata dose that inhibited inflammatory hypernociception, did not produceany change in the thermal or mechanical baseline nociceptivethreshold of mice. Therefore, H2S could act on the peripheralnociceptive system to produce antinociception. Interestingly, althoughexogenous H2S has an antinociceptive effect, it enhanced neutrophilmigration. This result is apparently in contrast to the pro-nociceptiverole of neutrophils. However, it was observed that H2S inhibitedhypernociception induced by a direct-acting mediator, PGE2, both inmice and rats, thereby suggesting that H2S is able to directly inhibitongoing hypernociception. This fact also supports the finding thateven while inducing neutrophil migration, H2S still has an antinoci-ceptive effect.

The difference in H2S concentration reached by endogenousformation or exogenous administration could also explain thesecontradictory results. At low doses, endogenously produced H2S actspredominantly on neutrophil/endothelium adhesion, enhancing thisprocess, and is consequently involved in the cascade of events leadingto neutrophil migration and inflammatory hypernociception. How-ever, it is unlikely that endogenous H2S is sufficient to causeantinociception, since antinociception was observed when H2Sproduction was inhibited during LPS-induced inflammation, insteadof enhancement of hypernociception. On the other hand, only highamounts of H2S (exogenous) would reach nociceptive neurons, whereit also activates K(ATP)

+ channels restoring the threshold of nociceptorsthat counteract the pro-nociceptive action of migrating neutrophils.

Experimentally, the peripheral pharmacologic control of inflam-matory pain is based on two main strategies. The first is the use ofdrugs that prevent nociceptor sensitization, such as non-steroidalanti-inflammatory drugs (NSAIDs) (aspirin and aspirin-like drugs),which inhibit prostaglandin synthesis (Ferreira, 1972), therebypreventing the development of hypernociception. The second strategyis the direct blockade of ongoing nociceptor sensitization, which canbe achieved by the use of peripheral morphine (opioids), dipyrone,diclofenac and other substances that activate the L-arginine/NOS/NO/cGMP pathway in sensitive neurons (Duarte et al.,1992; Ferreira,1993;Ferreira et al., 1991). In fact, these drugs reverse the alreadyestablished hypernociception induced by prostaglandin E2 (PGE2).Thus, the results obtained with exogenous H2S suggest that it belongsin the second class of peripheral antinociceptive drugs.

The activation of K(ATP)+ channels in the peripheral nociceptive system

has been shown to be involved in the modulation of nociception (Soaresand Duarte, 2001; Soares et al., 2000). For instance, peripheralantinociceptive drugs that directly block ongoing hypernociceptioninduced by PGE2, such as morphine and dipyrone, exert their effects byopening K(ATP)

+ channels stimulated by the L-arginine/NOS/NO/cGMPantinociceptive pathway (Rodrigues andDuarte, 2000; Sachs et al., 2004;Soares andDuarte, 2001; Soares et al., 2000). Concerning theH2S system,several biological effects produced by this gas have been attributed to theregulation of K(ATP)

+ channel activity (Johansen et al., 2006; Yang et al.,2005; Zhao et al., 2001). For instance, the vasodilator action of H2S isprevented by the inhibition of this K+ channel with glibenclamide,suggesting that K(ATP)

+ channel activation could be involved (Zhao et al.,2001). Taking into account this information,we tested thehypothesis thatthe antinociceptive effect of H2S on direct hypernociception induced byPGE2 is dependent on K(ATP)

+ channels in the periphery. Supporting thishypothesis, glibenclamide prevented the antinociceptive effect ofexogenous H2S in mice and in rats. A possible direct hypernociceptiveeffect of glibenclamide was excluded, as glibenclamide administrationalone in the rat paw did not produce mechanical hypernociception(Rodrigues and Duarte, 2000). Further supporting these findings, localadministration of a K(ATP)

+ channel opener also directly blocks hyperno-ciception induced by PGE2 (Sachs et al., 2004). Moreover, it was shownelectrophysiologically that K(ATP)

+ channel activation reduces theenhanced excitability of rat nociceptive sensory neurons induced byPGE2 (Chi et al., 2007). Although we did not investigate the mechanisminvolved in themodulation of K(ATP)

+ channels inprimary sensory neuronsby H2S, there is evidence using whole-cell and single-channel patch-clamp technique that H2S directly modulates K(ATP)

+ channels in vascularsmooth muscle cells (Tang et al., 2005). Actually, H2S was found toenhance the amplitude of whole-cell K(ATP)

+ currents and to increase theopen probability of single K(ATP)

+ channel (Tang et al., 2005). On the otherhand, the action of H2S on K(ATP)

+ channels could be indirect. For instance,it could bemediated byNO. This suggestion is supported by the followingfindings: a) NO has an antinociceptive role in the peripheral nociceptivesystem, and this effect is mediated by the activation of K(ATP)

+ channels(Soares et al., 2000); b) previous studies have shown that some actions ofH2S may involve interactions with NO (Hosoki et al., 1997); and c) non-selective inhibition of nitric oxide synthase prevents the antinociceptive

134 T.M. Cunha et al. / European Journal of Pharmacology 590 (2008) 127–135

effect of H2S (Distrutti et al., 2006a). In contrast to this hypothesis, ourpresent data showed that the treatment of rat paw with a non-selectiveinhibitor of nitric oxide synthase did not prevent the peripheralantinociceptive action of H2S.

There is evidence in the literature that activation of K(ATP)+ channels in

the central nervous system, by icv injection of diazoxide, producesantinociception (Lohmann and Welch, 1999). However, as mentionedabove, H2S did not showa central effect as evaluated in the hot-plate test.In further support of this notion, systemic administration of diazoxidealso did not change the thermal threshold in the hot-plate test. Thesedrugs administered systemically probably did not reach a sufficientconcentration in the central nervous system to produce antinociception.Webelieve that further studies, for exampledeterminingwhether centraladministration of H2S produces antinociception, are necessary toabsolutely exclude any central effect.

In conclusion, our results boost the importance of H2S system in themodulation of the nociceptive process mainly during the inflammatoryprocess. Furthermore, they suggest that H2S may play a dual role ininflammatory hypernociception. Endogenously produced H2S actspredominantly on neutrophil/endothelium adhesion, enhancing thisprocess, and is consequently involved in the cascade of events leading toneutrophil migration and inflammatory hypernociception. On the otherhand, exogenously administered H2S acts on sensitive neurons promot-ing the opening of K(ATP)

+ channels and consequently antinociception.

Acknowledgments

The authors wish to express their appreciation to Ieda Regina dosSantos Schivo, Sérgio Roberto Rosa and Giuliana Bertozi Francisco forexcellent technical assistance. This work was supported by grants fromFAPESP and CNPq. T.M. Cunha is a recipient of a PhD studentship fromFAPESP (Brazil). Dr. A. Leyva provided English editing of themanuscript.

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