doi: 10.1111/j.1472-8206.2009.00686.x

O R I G I N A L

A R T I C L E

Analgesic properties of S100A9 C-terminaldomain: a mechanism dependent on calciumchannel inhibition

Camila Squarzoni Dalea,b, Christophe Altierc, Nicolas Cenaca,b,Renata Giorgid, Maria Aparecida Julianoe, Luiz Julianoe,Gerald W. Zamponic, Nathalie Vergnollea,b,f*aINSERM, U563, Centre de Physiopathologie de Toulouse Purpan, Toulouse F-31300, FrancebUniversite Toulouse III Paul Sabatier, Toulouse F-31000, FrancecDepartment of Physiology and Biophysics Hotchkiss Brain Institute, University of Calgary, Calgary, AB T2N 4N1,

CanadadLaboratory of Pathophysiology, Butantan Institute, Sao Paulo, SP 05505-900, Brazil (RG)eDepartment of Biophysics, Pharmacology Institute, Federal University of Sao Paulo, Sao Paulo, SP 04044-020, BrazilfDepartment of Pharmacology and Therapeutics, University of Calgary, AB T2N4N1, Canada (CSD, NC, NV)

I N T R O D U C T I O N

The S100A9 protein belongs to the family of S100

proteins and is a calcium binding protein of the EF-hand

type [1,2]. Anti-inflammatory properties have been

described for S100A9, which inhibits leukocyte traf-

ficking [3], arachidonic acid metabolism [4] and is

able to de-activate activated peritoneal macrophages [5].

S100A9 protein also displays potent antinociceptive

properties in a mouse model of inflammatory pain [6].

S100A9, released by neutrophil, is responsible for the

inhibition of pain response in a model of neutrophilic

peritonitis induced by glycogen in mice [7,8]. Recent

reports suggest that the C-terminal domain of the

S100A9 protein (19 amino acids from the position

92–110 of murine S100A9) is responsible for most of

Keywords

antinociception,

dorsal root ganglia neurons,

inflammation,

N-type calcium channels,

S100A9,

voltage operated calcium

channels

Received 10 July 2008;

revised 7 November 2008;

accepted 16 December 2008

*Correspondence and reprints:

nathalie.vergnolle@inserm.fr

A B S T R A C T

Calcium-binding protein S100A9 and its C-terminus peptide (mS100A9p) are

anti-inflammatory and induce antinociception in rodents. We investigated the

mechanisms involved in this effect, and whether they depend or not on the anti-

inflammatory properties of mS100A9p. In mice, mS100A9p inhibited thermal and

mechanical hyperalgesia and allodynia induced by either carrageenan or formalin,

without interfering with paw edema. mS100A9p also inhibited myeloperoxidase

activity (MPO), a marker of granulocyte infiltration, induced by carrageenan, but

increased MPO after formalin intraplantar injection. The in vivo analgesic properties

of mS100A9p were independent of opioid receptor activation. Calcium flux into

dorsal root ganglia neurons induced by KCl was inhibited by mS100A9p, suggesting

that this protein is able to inhibit signaling, in sensory neurons. The inhibitory effects

of mS100A9p on primary afferent signaling were neither due to intracellular calcium

store inhibition nor to calcium chelating properties. However, mS100A9p was able to

inhibit calcium currents carried by transiently expressed N-type, but not L-type

calcium channels, as demonstrated both by gene transfection techniques and

electrophysiology. These data demonstrate that mS100A9p interferes with mecha-

nisms involved in nociception, hyperalgesia and calcium signaling in sensory

neurons, modulating primary afferent nociceptive signal by inhibiting activation of

N-type voltage operated calcium channels.

ª 2009 The Authors Journal compilation ª 2009 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 23 (2009) 427–438 427

the anti-inflammatory and anti-nociceptive properties

of S100A9. As a matter of fact, a synthetic peptide

corresponding to this part of the C-terminal domain

of the murine calcium-binding protein S100A9

(mS100A9p) inhibits spreading and phagocytic activity

of adherent peritoneal cells [9], inhibits hyperalgesia

induced by carrageenan in rats [10] and inhibits

hyperalgesia and edema induced by a metalloprotease

[11]. Most recently, we demonstrated that mS100A9p

also inhibits the hyperalgesia induced by an agonist

peptide of protease-activated receptor 2 (PAR2), modu-

lating PAR2 activation-induced nociceptive signal on

primary afferents [12].

The C-terminus of S100A9 protein is rich in histidine

residues and it has been reported to be involved with

zinc- and arachidonic acid-binding motif of S100A9

[13,14]. This portion of the molecule shows some

similarities with the sequence of the neutrophil inhibi-

tory factor that inhibits chemotaxia and neutrophil

migration in vitro [15,16], and with the contact domain

of high molecular weight kininogen [17]. However, no

clear mechanisms have been described to explain the

anti-inflammatory or anti-nociceptive properties of

S100A9 or its C-terminal domain. In this study, we

examined the mechanisms by which the C-terminal

domain of S100A9 exerts its analgesic properties. We

determined: (i) that mS100A9p exerts analgesic proper-

ties independently of its anti-inflammatory effects; (ii)

that the analgesic properties of mS100A9p are indepen-

dent of opioid receptor activation; (iii) that mS100A9p

can act directly on sensory neurons, inhibiting calcium

signaling in primary afferents and (iv) we defined that

mS100A9p acts as a voltage operated N-type calcium

channel inhibitor.

M A T E R I A L S A N D M E T H O D S

Animals

Male C57Bl6 mice were used throughout this study.

Animals were maintained under controlled light cycle

(12/12 h) and temperature (22 ± 2 �C), with free access

to food and water. The study was conducted in accor-

dance with the guidelines of the Institutional Animal

Care and Use Committee and was approved by the Ethics

Committee on Animal Experimentation of the University

of Calgary.

Reagents

A peptide identical to the C-terminus of murine S100A9

protein (H-E-K-L-H-E-N-N-P-R-G-H-G-H-S-H-G-K-G-NH2 –

mS100A9p) was synthesized in solid phase by fluor-

enylmethyloxycarbonyl (FMOC) technique. Characteri-

zation and purification were performed by high

performance liquid chromatography, and its mass

evaluated by matrix assisted laser desorption/ionization

time-of-flight (MALDI-TOF) spectrometry. The peptide

was diluted in saline at the final concentration of 1 mg/

mL. Formalin, 5% solution, diluted in saline, was injected

by the intraplantar (i pl.) route (50 lL/paw) of mice.

Carrageenan, 2% solution, diluted in saline (carrageenan

lambda, type IV – Sigma, St Louis, MO, USA), was also

injected by the i.pl. route (50 lL/paw). Naloxone

(10 mg/kg – Sigma) was diluted in saline and injected

20 mins before carrageenan, by the subcutaneous route

[18]. Thapsigagin and calcium ionophore (A23187)

were purchased from Sigma.

Behavioral pain measures

Thermal nociception

Paw withdrawal latency to radiant heat stimulus was

measured using an Ugo Basile� Plantar test (Ugo Basile,

Comerio, Italy) essentially as preciously described [19].

The withdrawal latency was measured before and after

the i pl. injection of carrageenan (2%–50 lL), formalin

(5%–50 lL) or mS100A9p (1 lg – paw). Thermal

hyperalgesia was defined as a significant decrease in the

withdrawal latency compared with the basal measure-

ment at different times after treatment.

Mechanical hyperalgesia and allodynia

Mechanical hyperalgesia and allodynia of the hind paw

were assessed as described before [20]. Mice were placed

individually in a plastic cage. Von Frey filaments with

bending forces of 0.407 g (3.61 filament), 0.692 g (3.84

filament), and 1.202 g (4.08 filament) were pressed

perpendicularly against the plantar skin and held for 5 s.

The stimulation of the same intensity was applied three

times to each hind paw at intervals of several seconds.

The responses to these stimuli were ranked as follows: 0,

no response; 1, move away from von Frey filament; and

2, immediate flinching or licking of the hind paw.

Nociceptive score was calculated as follows:

Nociceptive score (%)

¼P

average score of each animalð Þ2� no. of animals tested

� 100

The nociceptive score was measured before and after

the i pl. injections of carrageenan (2%–50 lL), formalin

(5%–50 lL) or mS100A9p (1 lg/paw).

428 C.S. Dale et al.

ª 2009 The Authors Journal compilation ª 2009 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 23 (2009) 427–438

All the nociceptive tests were performed according to

the guidelines published in a Guest Editorial in Pain on

ethical standards, for investigations of experimental pain

in animals [21].

Paw inflammation

Paw volume was measured using an electronic caliper,

before and at different times after i pl. injection of

carrageenan (2%–50 lL), formalin (5%–50 lL) and/or

mS100A9p (1 lg/paw). After 4 h of treatments, the paw

tissues of mice were assayed for myeloperoxidase activity

(MPO), as an index of tissue infiltration by granulocytes

as previously described [22–24].

Neuronal culture and calcium imaging

Dorsal root ganglia (DRG) neurons isolated from mice

were rinsed in hanks balanced salt solution (HBSS), and

incubated in HBSS containing 1% Papain (Worthing-

ton, Cedarlane, Hornby, ON, Canada) for 10 mins at

37 �C as previously described [25]. After a wash with

filtered Leibovitz’s L-15 Medium solution [glutamine

(200 mM), glucose 20%, fetal bovine serum FBS 10%],

DRG neurons were incubated in HBSS containing

collagenase (1 mg/mL-1) and dispase (4 mg/mL-1) at

37 �C for 10 mins. After titration, cells were plated in

Poly-L-ornithine-laminine (Sigma) glass-bottom petri

dishes (35 mm diameter; MatTek Corporation, Ashland,

MA, USA) and recovered with the complete culture

media [Minimum Essential Medium (MEM), 2.5% FBS,

1% penicillin/streptomycin, glutamine (200 mM), 1%

Dextrose and cytosine B-D-Arabinofuranoside (ARAC),

5-fluoro-2-desoxi-uridine (FUDR), uridine 10 lM each].

Cells were cultured for a minimum of 72 h. The petri

dishes were then washed twice with a Hank’s buffered

salt solution, pH 7.4 and incubated for 60 mins at

37 �C with Hank’s buffered salt solution supplemented

with 0.1% bovine serum albumin in the same solution

to which 3–5 lM of Fluo3-AM (Molecular Probes,

Eugene, OR, USA) was added. After the incubation

period, the petri dishes were washed twice again with

the assay N-2-hydroxyethylpiperazine-N¢-2-ethanesu-

fonic acid (HEPES) buffer (containing in mM: NaCl, 150;

KCl, 3; CaCl, 1.5; Hepes, 20; glucose, 10; sulfinpirazone,

0.25) of which 2 mL were left in each petridish.

Cells were observed using a wide-field fluorescence

olympus IX-70 microscope and an LC Plan FL 40X

objective. A series of 30 images in 90 s was acquired; the

first five images were used to determine the baseline.

After the fifth image, neurons were treated by an acute

administration of KCl (50 mM), calcium ionophore

A23187 (2 lM) and/or mS100A9p (0.5, 5, 50 or

100 lM). The experiment was repeated three times per

group. Fluorescence measurements reflecting elevations

of intracellular calcium were conducted at 460–490 nm

excitation and 515 nm emission in individual cells using

the acquisition program OpenLab software (Improvision

Inc a PerkinElmer Company, Waltham, MA, USA).

Transient expression of recombinant

calcium channels

Human embryonic kidney (HEK) tsA-201 cells were

cultured as described previously [26], with the exception

that cells were not moved to 28 �C after transfection.

Cells were split and plated on glass coverslips. High

voltage activated (HVA) calcium channels Cav1.2 and

Cav2.2 cDNA constructs were transfected with calcium

phosphate method at a ratio of 1 : 1 : 1 for calcium a1,

a2-d1 and b1b (2 lg each). Cells were used 24–48 h

after transfection for recording.

Electrophysiology

Electrophysiological recordings of channels expressed in

tsA-cells were conducted as described in detail previously

[26], with 20 mM barium as the charge carrier. Cells

were held at )100 mV and current voltage-relations

were acquired by stepping to various test potentials. Peak

current amplitude was normalized to cell capacitance to

obtain current densities. In all recordings, series resis-

tance was compensated by 85%. For recordings of

calcium currents in DRG neurons, current-voltage rela-

tionships were obtained by stepping to various test

potentials. Peak current was usually elicited between

)20 and 0 mV and was again normalized to cell

capacitance to obtain current densities.

The external recording solution for DRGs contained of

CaCl2 (2 mM), HEPES (10 mM), tetraethylamonium

chloride (TEACl) (160 mM) and glucose (10 mM), pH

7.4. For tsA-201 cells, it had 20 mM BaCl2 (20 mM),

MgCl2 (1 mM), HEPES (10 mM), TEACl (40 mM), glucose

(10 mM), CsCl (HVA- 65 mM) or BaCl2 (2 mM), MgCl2

(1 mM), HEPES (10 mM), TEACl (40 mM), glucose

(10 mM) and CsCl (LVA – 105 mM). Borosilicate glass

pipettes (2–4 MW) were filled with internal solution

containing CsCl (110 mM), MgCl2 (3 mM), ethylene

glycol tetraacetic acid (EGTA) (10 mM), HEPES

(10 mM), MgATP (3 mM) and GTP (0.6 mM), pH 7.2

(for DRGs) or CsMeSO4 (108 mM), MgCl2 (4 mM), EGTA

(9 mM), HEPES (9 mM), MgATP (2.6 mM) and LiGTP

(0.6 mM), pH to 7.2 (for tsA-201 cells). Peptide

(mS100A9p) was prepared daily in external solution

S100A9 analgesic properties 429

ª 2009 The Authors Journal compilation ª 2009 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 23 (2009) 427–438

from 1 mM frozen stocks and was applied to cells by

microperfusion. Currents were elicited from a holding

potential of )90 mV (DRGs) or )100 mV (tsA-201 cells).

Presentation of data and statistical analysis

All data are presented as the mean ± SEM. Statistical

analysis of data was generated using GraphPAd Prism,

version 4.02 (GraphPad Software Inc., San Diego, CA,

USA). Statistical analysis between two samples was

performed using Student’s t-test. Statistical comparison

of more than two groups was performed using one way –

ANOVA with Tukey’s multiple comparisons post-test. In

all cases, a P value <0.05 was considered statistically

significant.

R E S U L T S

mS100A9p exerts antinociceptive effects

independently of anti-inflammatory properties

We investigated the anti-inflammatory and anti-nocicep-

tive effects of mS100A9p in experimental models of

inflammatory pain. Groups of mice injected by the i.pl.

route with carrageenan (2%) or formalin (5%) developed

thermal (decreased withdrawal latency) and mechanical

(increased nociceptive score) hyperalgesia (Figure 1a, b for

carrageenan and Figure 1e, f for formalin). Allodynia

(response to the innocuous stimulation of the 3.61 size

filament) was also observed after i.pl. injection of carra-

geenan (Figure 1b) or formalin (Figure 1f ). In addition,

mice injected with carrageenan or formalin showed

signs of inflammation such as edema (Figure 1c, g),

and granulocyte infiltration, as measured by MPO

activity (Figure 1d, h). Concomitant injection of 1 lg of

mS100A9p (final volume 50 lL), with carrageenan or

formalin, inhibited both thermal (Figure 1a and e) and

mechanical (Figure 1b and f ) hyperalgesia and allodynia

induced by either formalin or carrageenan. However, the

same dose of mS100A9p had no significant effect on

carrageenan- or formalin-induced edema (Figure 1c, 1g).

mS100A9p inhibited MPO activity induced by carra-

geenan injection in the mouse paw (Figure 1d), but

significantly increased MPO activity into the paw after

formalin injection (Figure 1h). We also evaluated the

effects of i pl. injections of mS100A9p alone, which did not

cause significant granulocyte infiltration compared to

vehicle-injected paws (Figure 1d) and caused a small

increase in paw volume (Figure 1c), which was not

significantly different from vehicle alone (not shown).

Overall, these data show analgesic properties for

i.pl. injection of mS100A9 peptide, which decreased

thermal and mechanical inflammatory allodynia and

hyperalgesia. The analgesic properties of mS100A9

were independent from the development of other

inflammatory parameters (edema and granulocyte

recruitment).

mS100A9p-induced analgesia is independent of

opioid receptor activation

Opioids are known to be important modulators of

nociception, and activation of opioid receptors on

primary afferents induces analgesia [27]. We investi-

gated whether the inhibitory effects of mS100A9p on

inflammatory hyperalgesia involved opioid receptor

activation. Naloxone treatment (10 mg/kg) antagonizes

opioid receptor activation non-selectively, inhibiting

mu-, delta- and kappa-opioid receptors [18]. The

analgesic effects of mS100A9p on carrageenan-induced

thermal (decreased withdrawal latency) or mechanical

(increased nociceptive score) hyperalgesia, and allo-

dynia were not affected by naloxone pre-treatment

(Figure 2a, b). Furthermore, naloxone treatment did not

modify carrageenan-induced edema and MPO activity

either (Figure 2c, d). These results suggest that opioid

receptor activation is not involved in the antinocicep-

tive effect of mS100A9p during carrageenan-induced

inflammation.

mS100A9p inhibits sensory neuron activity

We further investigated whether mS100A9p was able

to act directly on primary afferents, inhibiting signal-

ing in sensory neurons. Different concentrations of

mS100A9p (0.5, 5, 50 and 100 lM) did not induce

calcium mobilization in isolated DRG neurons, under

basal (unstimulated) conditions (not shown). However,

the same concentrations of mS100A9p, significantly

inhibited calcium mobilization induced by KCl (50 mM)

stimulation, in a dose-dependent manner (Figure 3a).

To investigate whether mS100A9p was able to block

calcium release from intracellular stores, DRG neurons

were treated with thapsigargin (250 nM), a specific

calcium inhibitor that promotes the discharge of

calcium from intracellular stores [28]. Pre-treatment

of DRG neurons with thapsigargin reduced KCl-

induced calcium signal, however, this treatment did

not modify the effect of mS100A9p on primary

afferents (Figure 3b). Moreover, mS100A9p had no

effect on DRG neuron signaling in response to 2 lM of

calcium ionophore, a carboxylic antibiotic that selec-

tively transfers calcium and other divalent cations

across biological membranes [29] (Figure 3c). Taken

430 C.S. Dale et al.

ª 2009 The Authors Journal compilation ª 2009 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 23 (2009) 427–438

(a)

Cg

Cg + mS100A9p

mS100A9p

Vehicle

** *

* *

***

10

8

6

4

2

00 1 2 3 4

Time (h)

Wit

hd

raw

al la

ten

cy (

s)

(b) Cg + vehicleCg + mS100A9p70

60

50

40

30

20

10

00 1 h 2 h 3 h 4 h 0 1 h 2 h 3 h 4 h 0 1 h 2 h 3 h 4 h

3.61 filament

No

cice

pti

ve s

core

(%

)

3.84 filament 4.08 filament

* * *

*

*

**

*

(c) Cg + vehicle

Vehicle

100

80

60

40

20

00 1 2 3 4

Time (h)

Δ P

aw v

olu

me

(%) Cg + mS100A9p

mS100A9p

(d)

Mye

lop

erox

idas

eac

tivi

ty (

U/m

g)

600

500

400

300

200

100

0Cg Cg

+mS100A9p

mS100A9p Vehicle

**

*

(h) 400

300

200

100

0Formalin

+mS100A9p

Formalin

Mye

lop

erox

idas

eac

tivi

ty (

U/m

g)

mS100A9p Vehicle

**

(e)

Vehicle20

15

10

5

00 30 60 90 120 150 180 210 240

***

*

Formalin + mS100A9pFormalin

Time (min)

Wit

hd

raw

al la

ten

cy (

s)

(f) Formalin + mS100A9p

70

60

50

40

30

20

10

00 0 0

10 m

in

10 m

in

10 m

in

120 m

in

120 m

in

120 m

in

240 m

in

240 m

in

240 m

in

180 m

in

180 m

in

180 m

in

60 m

in

60 m

in

60 m

in

30 m

in

30 m

in

30 m

in

20 m

in

20 m

in

20 m

in

Formalin

No

cice

pti

ve s

core

(%

)

**

*

**

*

** *

*

*

(g)

VehicleFormalin + mS100A9pFormalin

0

50

100

150

200

0 30 60 90 120 120 180 210 240

Time (min)

Δ P

aw v

olu

me

(%)

Figure 1 Mice were administered intraplantar (i.pl) with carrageenan (Cg; 2%) or formalin (5%) concomitantly to vehicle, or to 1 lg of

mS100A9p, or receive i.pl. injections of vehicle alone (vehicle) or mS100A9 alone. Withdrawal latency to a thermal stimulation (thermal

hyperalgesia) (a, e) nociceptive score in response to von Frey filament exposure (mechanical hyperalgesia and allodynia), (b, f) paw volume

(edema), (c, g) and myeloperoxidase activity (d, h) were evaluated hourly after i.pl. injections. Data represent mean ± SEM of 6–8 animals per

group. (*) denotes statistical differences compared to the control groups (Cg or Formalin) (P < 0.05, one-way ANOVA followed by Tukey’s

multiple comparison post-test).

S100A9 analgesic properties 431

ª 2009 The Authors Journal compilation ª 2009 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 23 (2009) 427–438

together, these results demonstrate that mS100A9p

inhibits calcium signaling in primary afferents, but by

a mechanism independent of intracellular calcium

store depletion and independent of calcium ion

chelation.

mS100A9p inhibits voltage-gated calcium

channel activity

We investigated the possibility that mS100A9 could act

as an inhibitor of calcium channels. Voltage-gated

calcium channels (VGCC) are present on sensory neu-

rons, where L-, and N-channels have been shown to be

involved in nociception [30,31]. We investigated the

effects of mS100A9 on calcium signaling in HEK cells

(tsA-201), which were transfected or not, with N-type or

L-type calcium channels. Different concentrations of

mS100A9p (0.5, 1, 5 or 50 lM) inhibited, in a dose-

dependent manner, the magnitude of calcium flux in

response to KCl (50 mM) in N-type transfected cells

(Figure 4a), but had no effect in non-transfected cells

(not shown). No effect of mS100A9p was observed on

L-type transfected cells, in response to KCl stimulation

(Figure 4b).

To confirm the inhibition by mS100A9p of KCl-

mediated calcium influx observed in native DRG neurons

and heterologous expression systems, we proceeded with

electrophysiological recordings on both DRG neurons

and transfected tsA-201 cells. DRG neurons express both

low voltage activated (LVA) and HVA calcium currents.

However, by using a holding potential of )60 mV and a

test pulse of 0, +10 mV, LVA channels could be

selectively eliminated. As shown in Figure 5a, the isolated

(a) Cg + vehicle

Cg + mS100A9p

Cg + NX + mS100A9p

* * * *

* * * *

10

8

6

4

2

0 0 1 2 3 4

Time (h)

Wit

hd

raw

al la

ten

cy (

s)

Cg + vehicle

Cg + mS100A9p

Cg + NX + mS100A9p

0 1 2 3 4 Time (h)

Δ P

aw v

olu

me

(%)

0

20

40

60

80

100

120 (c)

* *

Cg 0

200

400

Mye

lop

erox

idas

e ac

tivi

ty (

U/m

g) 600

800

+ + + +

+

–

– +

+ +

–

–

NX

S100

(d)

Cg100

80

60

40

20

No

cice

pti

ve s

core

(%

)

0 0 1 h 2 h 3 h 4 h 0 1 h 2 h 3 h 4 h 0 1 h 2 h 3 h 4 h

3.61 filament 3.84 filament 4.08 filament

Cg + mS100A9p

Cg + NLX + mS100A9p

* * *

*

* * * *

*

(b)

Figure 2 Mice were injected subcutaneously with naloxone (NX; 10 mg/kg) or its vehicle 20 mins before the intraplantar (i.pl) injection of

Carragenan (Cg; 2%), or before the concomitant treatment with Cg and mS100A9p. Withdrawal latency in response to a thermal stimulation

(thermal hyperalgesia) (a) nociceptive score in response to von Frey filament exposure (mechanical hyperalgesia and allodynia) (b) paw

volume (edema) (c) and myeloperoxidase activity (d) were evaluated after different times after i.pl. injections. Data represent mean ± SEM of

6–8 animals per group. (*) denotes statistical differences compared to the group injected with Cg + vehicle (P < 0.05, one-way ANOVA

followed by Tukey’s multiple comparison post-test).

432 C.S. Dale et al.

ª 2009 The Authors Journal compilation ª 2009 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 23 (2009) 427–438

HVA currents were inhibited by �35% upon application

of 50 lM mS100A9 peptide, and a maximal inhibition

was observed �5 mins. after the beginning of the

response. The two major populations of HVA channels

in native DRG neurons are L-type and N-type, with

N-type channels pre-dominating [32,33]. After applica-

tion of x-conotoxin-GVIA, the selective N-type channels

blocker, effect of mS100A9p was significantly reduced

(Figure 5b, c). These results suggest that mS100A9-

(a) 1.5

1.4

1.3

1.2

1.1

1.00.5 5 50

mS100A9p (µM)

KCI (50 mM)

**

* **

**

Cal

ciu

m f

lux

inn

euro

ns

1.5

1.4

1.3

1.2

1.1

1.0

0.9

* *

* ** #

Cal

ciu

m f

lux

inn

euro

ns

KCIThapsig

mS100A9p

+ + + +

++

+ + ––

– –

(b)

2.5

2.0

1.5

1.05 50 100

mS100A9p (µM)Calcium ionophore (2 µM)

Cal

ciu

m f

lux

inn

euro

ns

(c)

Figure 3 Neurons were exposed to KCl (50 mM) concomitantly to

mS100A9p (0.5, 5, 50 or 100 lM – panel) (a) neurons treated only

with KCl were considered as control group. In panel (b), neurons

were incubated 5 mins with thapsigagrin (250 nM) and after were

exposed to KCl (50 mM) concomitantly to mS100A9p (50 lM).

Neurons treated only with KCl (control group) or KCl and

thapsigargin (Thapsg) were also evaluated. Data represent

mean ± SEM of 15–20 neurons per group. (*) denotes statistical

differences compared to KCl (P < 0.05), (***) denotes statistical

differences compared to KCl (P < 0.001), (#) denotes statistical

differences compared to KCl + thapsigargin (P < 0.05; one-way

ANOVA followed by Tukey’s multiple comparison post-test). In panel

(c), neurons were exposed to a calcium ionophore (2 lM) concom-

itantly to mS100A9p (0.5 5, 50 or 100 lM). Neurons treated only

with calcium ionophore were considered as the control group. The

calcium flux measurements were performed by fluorescence at

460–490 nm excitations and 515 nm emissions in individual cells

using a wide-field fluorescence microscope. A kinetic of 30 pictures

in 90 s was performed.

Figure 4 N-type transfected tsA-201 (a) or L-type transfected

tsA-201 (b) cells were exposed to KCl (50 mM) concomitantly to

mS100A9p (0.1, 0.5, 1, 5 or 50 lM). Data represent mean ± SEM

of 15–20 cells per group. (*) denotes statistical differences compared

to KCl (P < 0.05). The calcium flux measurements were performed

by fluorescence at 460–490 nm excitations and 515 nm emissions

in individual cells using a wide-field fluorescence microscope.

A kinetic of 30 pictures in 90 s was performed.

S100A9 analgesic properties 433

ª 2009 The Authors Journal compilation ª 2009 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 23 (2009) 427–438

peptide blocks N-type but not other HVA calcium

channels (L-type, P/Q-type) in DRG neurons. Then, we

recorded from tsA-201 cells transiently expressing either

L-type or N-type channels. N-type (Cav2.2+ b1b + a2- d1)

but not L-type (Cav1.2+ b1b + a2- d1) activity was

inhibited by �40% with 25 lM peptide (Figure 5d), and

this inhibition could be partially reversed upon washout,

as seen from the raw current traces (Figure 5d), and the

representative time course of mS100A9 effect (Figure 5e).

These data are consistent with the results observed with

Ca2+measurements from DRG neurons and tsA-201

cells. Superimposition of normalized current-voltage

relations in the absence and presence of the peptide

reveals that the voltage-dependence of activation was not

affected (Figure 5f). Taken together, our results demon-

strate that mS100A9p inhibits native and expressed

N-type calcium currents and this block does not affect

channel activation.

D I S C U S S I O N

Although different in vivo studies have reported anti-

nociceptive properties for the calcium-binding protein

S100A9 or its C-terminus domain [6–8,11,12], the

mechanism of this effect has never been identified. As

murine S100A9 C-terminal peptide (mS100A9p) also

showed anti-inflammatory properties, it is uncertain

whether the analgesic effects of mS100A9p are due to

Figure 5 Representative time course of calcium currents from dorsal root ganglia (DRG) neurons recorded during a + 10 mV test pulse

(50 ms) from a holding potential of )60 mV before and after application of 50 lM mS100A9-peptide (a) representative time course of calcium

current using the same protocol as in (a) but in the presence of x-conotoxin-GVIA (1 lM) (b) histogram of voltage gated calcium channels

block after application of 50 lM peptide in the absence or presence of x-conotoxin-GVIA (1 lM) (I/Icontrol = 0.67 ± 0.04, n = 9 and

I/Icontrol = 0.83 ± 0.05, n = 5 respectively. *P < 0.05) (c) mS100A9-peptide inhibits N-type current in tsA-201 cells transfected with the

Cav2.2 subunit (+b1b, a2- d1 – panel d). Note that the inhibition is partially reversible. Inset: histogram summarizing block of N-type but

not L-type channels expressed transciently in tsA-201 cells (I/Icontrol = 0.59 ± 0.03 for Cav2.2 and I/Icontrol = 0.94 ± 0.02 for Cav1.2).

Numbers in parentheses are the numbers of cells analyzed. Error bars reflect SEM. Asterisks denote statistical significance (***P < 0.001).

Time course of the block of N-type (Cav2.2 + b1b + a2- d1) current amplitude by 25 lM mS100A9p in tsA-201 cells (e). Current amplitude

was elicited by a + 10 mV test pulse (150 ms) from a holding potential of )90 mV. Normalized I-V curve from tsA-201 cell transfected

with Cav2.2 N-type channel, in the absence or presence of 25 lM mS100A9p (f) no shift in the I-V relation was observed.

434 C.S. Dale et al.

ª 2009 The Authors Journal compilation ª 2009 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 23 (2009) 427–438

a reduced inflammatory response and thereby to a

reduced activation of primary afferents by inflammatory

mediators, or whether mS100A9p exerts antinociceptive

properties per se. One of the first aims of this study was to

identify whether the anti-nociceptive properties of

mS100A9p depend on its anti-inflammatory properties.

We used two different inflammatory models, which both

induce all inflammatory parameters (edema, granulocyte

recruitment, vasodilation, hyperalgesia, allodynia), but

with different profiles of mediators. The major mediators

involved in carrageenan-induced inflammation are eico-

sanoids and sympathomimetic amines, whereas the

major inflammatory mediators involved in formalin-

induced inflammatory pain are serotonin and histamine,

which directly activate sensitized nociceptors [34].

Despite the distinct mechanisms involved in the inflam-

matory pain induced by carrageenan and that induced

by formalin, mS100A9p was effective in both models, to

decrease inflammatory hyperalgesia and allodynia.

Although anti-inflammatory properties have been

described for S100A9, here in both models, the inflam-

matory edema was not reduced by mS100A9p. Edema

formation plays an important part in inducing hyper-

algesia, as compression of peripheral nerves causes

nociceptor activation [35]. The fact that mS100A9p

did not interfere with edema suggests that the mecha-

nism by which mS100A9p exerts antinociceptive prop-

erties could be a direct effect on nociceptive pathways,

and not an anti-inflammatory effect. This is further

suggested by the fact that in the formalin model, where

primary afferents are activated within seconds, and pain

appears in the first minutes after the i pl. injection when

this effect is not yet dependent on inflammatory infil-

trates, mS100A9p was effective at reducing mechanical

hyperalgesia as early as 10 mins after the i pl. injection

(Figure 1f). Finally, the dichotomy between antinocicep-

tive and anti-inflammatory properties of mS100A9p was

demonstrated by the effects of mS100A9p on granulo-

cyte infiltration. In the carrageenan model, mS100A9p

was able to inhibit MPO activity, as previously reported,

but in the formalin model, mS100A9p treatment

increased MPO activity. This last result definitively

demonstrates that the analgesic properties of mS100A9p

observed in both models are independent of anti-inflam-

matory (inhibition of granulocyte infiltration) properties

of mS100A9p. It is interesting to note that mS100A9p

showed better anti-inflammatory properties in the car-

rageenan model than in the formalin model. This could

be explained by the different mediators and pathways

involved in those two models.

As one of the major pathway of antinociception

involves the activation of opioid receptors and the

release of endogenous opioid receptor agonists, we

investigated the possibility that mS100A9p could acti-

vate opioid receptor pathways. This hypothesis was

supported by the fact that immune cells, which have

been shown to be targeted by mS100A9p [36] are a

major source of endogenous opioid agonists (endor-

phins). Treatment with naloxone, a non-selective mu-,

delta- and kappa-opioid receptor antagonist failed to

inhibit the antinociceptive effects of mS100A9p in the

carrageenan model, thereby demonstrating that activa-

tion of opioid receptor pathway was not involved in this

mechanism.

We have previously demonstrated that mS100A9p

can act directly on sensory neuron activation by

inhibiting calcium signaling on DRG neurons in response

to the activation of different receptors such as PAR2, B1

bradykinin receptor or Transient potential vanilloid

receptor 1 – TRPV1 [10]. Here, the dichotomy between

antinociceptive and anti-inflammatory properties of

mS100A9p suggests also that mS100A9p could act

directly on primary afferents. We observed here that

mS100A9p inhibits calcium flux on DRG neurons

induced by KCl. This demonstrates that the inhibitory

effects of mS100A9p on primary afferent signaling might

not be specific of some receptors such as PAR2 or BK

receptors, but that mS100A9 is able to interfere with

calcium signaling in general, in DRG neurons. We

further showed that this effect was not the result of

inhibition of intracellular calcium stores, as thapsigargin

treatment had no effect on mS100A9p inhibition of KCl-

induced calcium signal. The inhibitory effect of

mS100A9p was not due either to a chelating effect on

calcium ions, as mS100A9p was not able to inhibit the

effects of a calcium ionophore.

Calcium ions are widely recognized to play a funda-

mental role in the regulation of several biological

processes, such as modulation of neurotransmitter

release and modulation of cell membrane excitability

[30,37]. Evidence has accumulated for the involvement

of calcium ions also in nociception and anti-nociception

[30]. Particularly, the presence and functionality of L-,

N- and P/Q-type of VGCC were demonstrated in the

dorsal horn of the spinal cord [38] and to be involved

in nociceptive transmission. As in sensory neurons,

mS100A9p inhibited calcium flux induced by different

receptors [12] as well as by KCl (Figure 3); this suggests

that mS100A9p could act directly on calcium channels

present on primary afferents. Thus, we investigated

S100A9 analgesic properties 435

ª 2009 The Authors Journal compilation ª 2009 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 23 (2009) 427–438

whether mS100A9p could directly interfere with the

activation of L- or N-type voltage-gated calcium chan-

nels. Our results demonstrate that mS100A9p inhibits

N-type calcium channels expressed in tsA-201 cells,

whereas no effect was observed on L-type channels.

These data were confirmed by electrophysiology in

isolated sensory neurons, in which we observed that

S100A9-C-terminal peptide could specifically block

N-type calcium channels, while mS100A9p effect was

almost abolished in the presence of N-type channel

blocker x-conotoxin-GVIA. These results indicate that

S100A9-C-terminal peptide is able to inhibit native and

expressed N-type calcium currents without affecting

channel activation. The mechanism by which S100A9

inhibits N-type calcium channel will have to be further

investigated. One possibility is that parameters of

the gating of the channel are changed by S100A9.

We know that the current-voltage relation is not

modified by mS100A9p, but if the peptide blocks

the pore of the channel, the open probability or

conductance of the channel could be affected. In

addition, recent studies have shown that reducing

agents or endogenous compounds with metal chelator

properties (particularly Zn2+ions) are able to regulate

calcium channels [39]. Similar mechanism could be

hypothesized for S100A9.

Voltage gated calcium channels as a family repre-

sent one of the most important regulators of Ca2+

concentration in neurons, as they do in many cell

types, and hence have an important role in neuronal

functions, including nociception and chronic/neuro-

pathic pain [40]. Particularly, the N-type VGCCs have

been demonstrated to be involved in short lasting

nociception, hyperexcitability, inflammation and neuro-

pathic pain [38,41–44]. Our results demonstrating the

inhibitory effects of mS100A9p on N-type VGCCs are

consistent with such a role and suggest for mS100A9p

an important role as therapeutic tool for the control

of nociception. Furthermore, our results reinforce the

concept that S100A9 protein is an important actor

of the modulation of nociceptive responses. Whether

native S100A9 plays such an endogenous role as a pain

modulator via N-type VGCC inhibition still has to be

demonstrated. However, these studies depend on the

availability of more specific tools for the study of S100A9

(knock-out mice, blocking antibodies, large supplies of

pure S100A9 native protein).

In conclusion, our study demonstrates, for the first

time the mechanisms involved in the antinociception

induced by the C-terminus of murine S100A9. Our

findings highlight mS100A9p as a new important tool

for the inhibition of N-type VGCC, and for the inhibition

of inflammatory pain.

A C K N O W L E D G E M E N T S

This work was supported by Coordenacao de Aperfeicoa-

mento de Pessoal de Nıvel Superior-CAPES, the Canadian

Institute for Health Research, and the Institut UPSA de la

douleur. CSD holds a Canadian Association of Gastroen-

terology – CAG/SHERING fellowship. NV is supported by

an AVENIR-INSERM program, by the ‘Fondation Betten-

court-Schueller’ and the ‘Fondation Schlumberger’, and

was an Alberta Heritage Foundation for Medical

Research (AHFMR) scholar, and a Canadian Institute

for Health Research Investigator. GWZ is a Canada

Research Chair and AHFMR Scientist. CA was supported

by an AHFMR Fellowship Award.

We thank Dr. Pina Colarusso from the Department of

Physiology & Biophysics of the University of Calgary for

the use of the fluorescence microscope.

R E F E R E N C E S

1 Hessian P.A., Edgeworth J., Hogg N. MRP-8 and MRP-14, two

abundant Ca(2+)-binding proteins of neutrophils and mono-

cytes. J. Leukoc. Biol. (1993) 53 197–204.

2 Roth J., Vogl T., Sunderkotter C., Sorg C. Chemotactic activity of

S100A8 and S100A9. J. Immunol. (2003) 171 5651.

3 Raftery M.J., Harrison C.A., Alewood P., Jones A., Geczy C.L.

Isolation of the murine S100 protein MRP14 (14 kDa migra-

tion-inhibitory-factor-related protein) from activated spleen

cells: characterization of post-translational modifications and

zinc binding. Biochem. J. (1996) 316 285–293.

4 Kerkhoff C., Klempt M., Kaever V., Sorg C. The two calcium-

binding proteins, S100A8 and S100A9, are involved in the

metabolism of arachidonic acid in human neutrophils. J. Biol.

Chem. (1999) 274 32672–32679.

5 Kerkhoff C., Klempt M., Sorg C. Novel insights into structure

and function of MRP8 (S100A8) and MRP14 (S100A9).

Biochim. Biophys. Acta. (1998) 1448 200–211.

6 Giorgi R., Pagano R.L., Dias M.A., Aguiar-Passeti T., Sorg C.,

Mariano M. Antinociceptive effect of the calcium-binding

protein MRP-14 and the role played by neutrophils on the

control of inflammatory pain. J. Leukoc. Biol. (1998) 64 214–

220.

7 Pagano R.L., Dias M.A., Dale C.S., Giorgi R. Neutrophils and the

calcium-binding protein MRP-14 mediate carrageenan-induced

antinociception in mice. Mediators Inflamm. (2002) 11 203–

210.

8 Pagano R.L., Mariano M., Giorgi R. Neutrophilic cell-free

exudate induces antinociception mediate by the protein

S100A9. Mediators Inflamm. (2006) 2006, 36765.

436 C.S. Dale et al.

ª 2009 The Authors Journal compilation ª 2009 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 23 (2009) 427–438

9 Pagano R.L., Sampaio S.C., Juliano L., Juliano M.A., Giorgi R.

The C-terminus of murine S100A9 inhibits spreading and

phagocytic activity of adherent peritoneal cells. Inflamm. Res.

(2005) 54 204–210.

10 Dale C.S., Pagano R.L., Paccola C.C. et al. Effect of the

C-terminus of murine S100A9 protein on experimental

nociception. Peptides (2006) 27 2794–2802.

11 Dale C.S., Goncalves L.R.C., Juliano L., Juliano M.A., da Silva

A.M., Giorgi R. The C-terminus of murine S100A9 inhibits

hyperalgesia and edema induced by jararhagin. Peptides (2004)

25 81–89.

12 Dale C.S., Cenac N., Britto L.R. et al. The C-terminus of murine

S100A9 protein inhibits hyperalgesia induced by the agonist

peptide of protease-activated receptor 2 (PAR2). Br. J. Phar-

macol. (2006) 149 374–384.

13 Loomans H.J., Hahn B.L., Li Q.Q., Phadnis S.H., Sohnle P.G.

Histidine-based zinc-binding sequences and the anti-

microbial activity of calprotectin. J. Infect. Dis. (1998)

177 812–814.

14 Sopalla C., Leukert N., Sorg C., Kerkhoff C. Evidence for the

involvement of the unique C-tail of S100A9 in the binding of

arachidonic acid to the heterocomplex S100A8/A9. Biol. Chem.

(2002) 383 1895–1905.

15 Watt K.W., Brightman I.L., Goetzl E.J. Isolation of two

polypeptides comprising the neutrophil-immobilizing factor of

human leucocytes. Immunology (1983) 48 79–86.

16 Freemont P., Hogg N., Edgeworth J. Sequence identity. Nature

(1989) 339 516.

17 Hessian P.A., Wilkinson L., Hogg N. The S100 family protein

MRP-14 (S100A9) has homology with the contact domain of

high molecular weight kininogen. FEBS Lett. (1995) 371 271–

275.

18 Juni A., Klein G., Kest B. Morphine hyperalgesia in mice is

unrelated to opioid activity, analgesia, or tolerance: evidence for

multiple diverse hyperalgesic systems. Brain Res. (2006) 1070

35–44.

19 Hargreaves K., Dubner R., Brown F., Flores C., Joris J. A new

and sensitive method for measuring thermal nociception in

cutaneous hyperalgesia. Pain (1988) 32 77–88.

20 Takasaki I., Andoh T., Nojima H., Shiraki K., Kuraishi Y.

Gabapentin antinociception in mice with acute herpetic pain

induced by herpes simplex virus infection. J. Pharmacol. Exp.

Ther. (2001) 296 270–275.

21 Zimmermann M. Ethical guidelines for investigations of exper-

imental pain in conscious animals. Pain (1983) 6 109–110.

22 Bradley P.P., Priebat D.A., Christensen R.D., Rothstein G.

Measurement of cutaneous inflammation: estimation of neu-

trophil content with an enzyme marker. J. Invest. Dermatol.

(1982) 78 206–209.

23 Vergnolle N., Hollenberg M.D., Sharkey K.A., Wallace J.L.

Characterization of the inflammatory response to proteinase-

activated receptor-2 (PAR2)-activating peptides in the rat paw.

Br. J. Pharmacol. (1999) 127 1083–1090.

24 Asfaha S., Cenac N., Houle S. et al. Protease-activated receptor-

4: a novel mechanism of inflammatory pain modulation. Br. J.

Pharmacol. (2007) 150 176–185.

25 Cenac N., Andrews C., Holzhausen M. et al. Proteases are

released in Irritable Bowel Syndrome: a role in visceral pain.

J. Clin. Invest. (2007) 117, 636–647.

26 Beedle A.M., McRory J.E., Poirot O. et al. Agonist-independent

modulation of N-type calcium channels by ORL1 receptors. Nat.

Neurosci. (2004) 7 118–125.

27 Vanderah T.W. Pathophysiology of pain. Med. Clin. North Am.

(2007) 91 1–12.

28 Thastrup O., Cullen P.J., Drobak B.K., Hanley M.R., Dawson

A.P. Thapsigargin, a tumor promoter, discharges intracellular

Ca2+ stores by specific inhibition of the endoplasmic reticulum

Ca2(+)-ATPase. Proc. Natl Acad. Sci. USA (1990) 87 2466–

2470.

29 Luckasen J.R., White J.G., Kersey J.H. Mitogenic properties of a

calcium ionophore, A23187. Proc. Natl. Acad. Sci. USA (1974)

71 5088–5090.

30 Prado W.A. Involvement of calcium in pain and antinocicep-

tion. Braz. J. Med. Biol. Res. (2001) 34 449–461.

31 Lee Y., Lee C.H., Oh U. Painful channels in sensory neurons.

Mol. Cells. (2005) 20 315–324.

32 Sutton K.G., Martin D.J., Pinnock R.D., Lee K., Scott R.H.

Gabapentin inhibits high-threshold calcium channel currents in

cultured rat dorsal root ganglion neurons. Br. J. Pharmacol.

(2002) 135 257–265.

33 Altier C., Khosravani H., Evans R.M. et al. ORL1 receptor-

mediated internalization of N-type calcium channels. Nat.

Neurosci. (2006) 9 31–40.

34 Parada C.A., Tambeli C.H., Cunha F.Q., Ferreira S.H. The major

role of peripheral release of histamine and 5-hydroxytryptamine

in formalin-induced nociception. Neuroscience (2001) 102

937–944.

35 Dray A., Bevan S. Inflammation and hyperalgesia: highlighting

the team effort. Trends Pharmacol. Sci. (1993) 14 287–290.

36 Striz I., Trebichavsky I. Calprotectin – a pleiotropic molecule in

acute and chronic inflammation. Physiol. Res. (2004) 53 245–

253.

37 Zamponi G.W., Snutch T.P. Modulation of voltage-dependent

calcium channels by G proteins. Curr. Opin. Neurobiol. (1998)

8 351–356.

38 Vanegas H., Schaible H. Effects of antagonists to high-threshold

calcium channels upon spinal mechanisms of pain, hyperalge-

sia and allodynia. Pain (2000) 85 9–18.

39 Nelson M.T., Joksovic P.M., Su P. et al. Molecular mechanisms

of subtype-specific inhibition of neuronal T-type calcium

channels by ascorbate. J. Neurosci. (2007) 27 12577–

12583.

40 Gribkoff V.K. The role of voltage-gated calcium channels in

pain and nociception. Semin. Cell Dev. Biol. (2006) 17 555–

564.

41 Maggi C.A., Tramontana M., Cecconi R., Santicioli P. Neuro-

chemical evidence for the involvement of N-type calcium chan-

nels in transmitter secretion from peripheral endings of sensory

nerves in guinea pigs. Neurosci. Lett. (1990) 114 203–206.

42 Santicioli P., Del Bianco E., Tramontana M., Geppetti P., Maggi

C.A. Release of calcitonin gene-related peptide like-immuno-

reactivity induced by electrical field stimulation from rat spinal

S100A9 analgesic properties 437

ª 2009 The Authors Journal compilation ª 2009 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 23 (2009) 427–438

afferents is mediated by conotoxin-sensitive calcium channels.

Neurosci. Lett. (1992) 136 161–164.

43 Smith M.T., Cabot P.J., Ross F.B., Robertson A.D., Lewis R.J. The

novel N-type calcium channel blocker, AM336, produces potent

dose-dependent antinociception after intrathecal dosing in rats

and inhibits substance P release in rat spinal cord slices. Pain

(2002) 96 119–127.

44 Altier C., Dale C.S., Kisilevsky A.E. et al. Differential role of N-

type calcium channel splice isoforms in pain. J. Neurosci.

(2007) 27 6363–6373.

438 C.S. Dale et al.

ª 2009 The Authors Journal compilation ª 2009 Societe Francaise de Pharmacologie et de TherapeutiqueFundamental & Clinical Pharmacology 23 (2009) 427–438