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Involvement of transient receptor potential vanilloid 1 receptors in protease-activated receptor-2-induced joint inflammation and nociception
European Journal of Pain 14 (2010) 351–358
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Involvement of transient receptor potential vanilloid 1 receptors in protease-activated receptor-2-induced joint inflammation and nociception Zs. Helyes a,*,1, K. Sándor a,1, É. Borbély a, V. Tékus b, E. Pintér a, K. Elekes c, D.M. Tóth b, J. Szolcsányi a, J.J. McDougall d a
Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, H-7624 Pécs, Szigeti u. 12., Hungary Analgesic Research Laboratory, University of Pécs and Gedeon-Richter Plc., H-7624 Pécs, Szigeti u. 12., Hungary Institute of Pharmacognosy, Faculty of Medicine, University of Pécs, H-7624 Pécs, Rókus u. 2., Hungary d Department of Physiology and Pharmacology, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 Canada b c
a r t i c l e
i n f o
Article history: Received 25 February 2009 Received in revised form 23 June 2009 Accepted 21 July 2009 Available online 15 August 2009 Keywords: Capsaicin-sensitive sensory nerves Arthritis Mechanical hyperalgesia Allodynia Inflammatory cytokines
a b s t r a c t Protease-activated receptor-2 (PAR-2) is a G-protein-coupled receptor activated through proteolytic cleavage. It is localized on epithelial, endothelial and inflammatory cells, as well as on transient receptor potential vanilloid 1 (TRPV1) receptor-expressing neurones. It plays an important role in inflammatory/ nociceptive processes. Since there are few reports concerning PAR-2 function in joints, the effects of intraarticular PAR-2 activation on joint pain and inflammation were studied. Secondary hyperalgesia/allodynia, spontaneous weight distribution, swelling and inflammatory cytokine production were measured and the involvement of TRPV1 ion channels was investigated in rats and mice. Injection of the PAR-2 receptor agonist SLIGRL-NH2 into the knee decreased touch sensitivity and weight bearing of the ipsilateral hindlimb in both species. Secondary mechanical allodynia/hyperalgesia and impaired weight distribution were significantly reduced by the TRPV1 antagonist SB366791 in rats and by the genetic deletion of this receptor in mice. PAR-2 activation did not cause significant joint swelling, but increased IL-1b concentration which was not influenced by the lack of the TRPV1 channel. For comparison, intraplantar SLIGRL-NH2 evoked similar primary mechanical hyperalgesia and impaired weight distribution in both WT and TRPV1 deficient mice, but oedema was smaller in the knockouts. The inactive peptide, LRGILS-NH2, injected into either site did not induce any inflammatory or nociceptive changes. These data provide evidence for a significant role of TRPV1 receptors in secondary mechanical hyperalgesia/allodynia and spontaneous pain induced by PAR-2 receptor activation in the knee joint. Although intraplantar PAR-2 activation-induced oedema is also TRPV1 receptor-mediated, primary mechanical hyperalgesia, impaired weight distribution and IL-1b production are independent of this channel. Ó 2009 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.
1. Introduction The family of protease-activated receptors (PARs) consists of four members: three receptors for thrombin (PAR-1, PAR-3 and PAR-4) being involved in mainly coagulation processes and one for trypsin/mast cell tryptase (PAR-2). They are all heptahelical G-protein-coupled receptors activated by proteolytic cleavage
Abbreviation: TRPV1, transient receptor potential vanilloid 1; PAR-2 receptor, protease-activated receptor-2; SP, substance P; CGRP, calcitonin gene-related peptide. * Corresponding author. Address: Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, H-7624 Pécs, Szigeti u. 12., Hungary. Tel.: +36 72 536001/5591/5386; fax: +36 72 536218. E-mail address: [email protected] (Zs. Helyes). 1 Zs. Helyes and K. Sándor contributed equally to the present work.
through several proteases (Molino et al., 1997; Macfarlane et al., 2001; Hollenberg and Compton, 2002). PAR-2 has drawn great attention due to its different localization and function compared to the other three PARs. It is expressed on epithelial and endothelial cells in the skin (Steinhoff et al., 1999), joints (Ferrell et al., 2003; Boileau et al., 2007; Nakano et al., 2007), cardiovascular, gastrointestinal and respiratory systems (D’Andrea et al., 1998; Cocks et al., 1999; Kawabata et al., 2002), as well as on capsaicin-sensitive peripheral sensory nerves (Steinhoff et al., 2000; Vergnolle et al., 2001a,b; Vergnolle, 2005). Serine proteases play an important role in several inflammatory processes (Schmelz et al., 1999; Kanke et al., 2005; McIntosh et al., 2007) and pain (Vergnolle et al., 2001a,b; Coelho et al., 2003). The importance of PAR-2 in the induction and maintenance of inflammatory responses has been confirmed with PAR-2 deficient mice (Lindner et al., 2000). In the skin, PAR-2 agonists induce inflammatory
1090-3801/$36.00 Ó 2009 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpain.2009.07.005
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changes such as vasodilatation, oedema, leukocyte recruitment, adhesion and extravasation (Saifeddine et al., 1996; Vergnolle et al., 1998; Vergnolle, 1999; Seeliger et al., 2003). These depend on neurogenic mechanisms via the release of SP and CGRP from peripheral terminals of capsaicin-sensitive, transient receptor potential vanilloid 1 (TRPV1) receptor-expressing sensory nerves (Steinhoff et al., 2000; Coelho et al., 2003). In the innervated area, calcitonin gene-related peptide (CGRP) elicits arteriolar vasodilatation and potentiates the plasma protein extravasation-inducing effects of substance P (SP; Jancsó-Gábor and Szolcsányi, 1970; Szolcsányi, 1988; Holzer, 1992; Maggi, 1995; Szolcsányi, 1996; Helyes et al., 2003). Recently, the involvement of capsaicin-sensitive fibres in PAR-2 activation-induced inflammatory reactions and nociception has been suggested by the markedly reduced responses observed after capsaicin desensitization (Su et al., 2005; Gu and Lee, 2006; Shimizu et al., 2007; Paszcuk et al., 2008). Further studies with TRPV1 receptor antagonists revealed the role of this ion channel in PAR-2-evoked scratching behaviour (Costa et al., 2008), thermal hyperalgesia (Amadesi et al., 2004) and urinary bladder contractions (Shimizu et al., 2007). Data obtained with TRPV1 gene-deleted mice demonstrated that intraplantar PAR-2 activation-induced primary hyperalgesia is – at least partially – TRPV1 mediated (Amadesi et al., 2004; Dai et al., 2004). There are a limited number of in vivo experiments concerning PAR activation in joints (Russell and McDougall, 2009). PAR-2 stimulation in the murine knee promotes synovial hyperaemia and oedema indicating a role for PARs in inflammatory joint diseases (Ferrell et al., 2003; Busso et al., 2007). Thus, the aims of the present series of experiments were to analyse the inflammatory changes as well as secondary hyperalgesia/allodynia in response to intraarticular PAR-2 activation in rats and mice and to investigate the role of the TRPV1 ion channel in the observed actions with the help of a receptor antagonist and genetic deletion of the receptor. 2. Experimental procedures 2.1. Animals Experiments were performed on male Wistar rats (250–300 g) as well as on TRPV1 receptor gene-deficient mice (TRPV1 / ) backcrossed through 10 generations with the C57Bl/6 strain and their wildtype (WT) counterparts (20–25 g). The original breeding pairs of the rats and C57Bl/6 mice were purchased from Charles-River Ltd. (Hungary) and the TRPV1 / mice from Jackson Laboratories (USA). The animals were bred and kept in the Laboratory Animal House of the Department of Pharmacology and Pharmacotherapy of the University of Pécs at 24–25 °C and provided with standard rat chow and water ad libitum. 2.2. Ethics All experimental procedures were carried out according to the 1998/XXVIII Act of the Hungarian Parliament on Animal Protection and Consideration Decree of Scientific Procedures of Animal Experiments (243/1988) and complied with the recommendations of the International Association for the Study of Pain and the Helsinki Declaration. The studies were approved by the Ethics Committee on Animal Research of University of Pécs according to the Ethical Codex of Animal Experiments and licence was given (licence No.: BA 02/2000-11-2006).
surface of the right paw (100 lg in 50 ll) or into the right knee joint of mice (100 lg in 50 ll) and rats (100 lg in 100 ll) under isoflurane anaesthesia. Measurements described below were performed at certain time points after the injections throughout the 6-h experimental period. In one group of rats, pre-treatment with the selective and in vivo active TRPV1 receptor antagonist SB366791 (500 lg/kg i.p.; Varga et al., 2005) was performed 15 min before intraarticular SLIGRL-NH2 injection and before each measurement. 2.4. Measurement of touch sensitivity of the paw Touch sensitivity of the plantar surface of the paw in both rats and mice were determined with dynamic plantar aesthesiometry (Ugo Basile 37400, Comerio, Italy) before and every hour after SLIGRL-NH2 (or in the control group LRGILS-NH2) injection. This device is a modified, electronic von Frey technique, which is used to assess touch sensitivity on the plantar surface of the paw. The animals move about freely in one of the two compartments of the enclosure positioned on the metal mesh surface. Following acclimation after cessation of exploratory behaviour, the operator places the touch stimulator unit under the animal’s paw, using the adjustable angled-mirror to position the filament below the target area of the paw. After pressing the ‘‘start” key an electrodynamic actuator of proprietary design advances a straight metal filament, which touches the plantar surface of the paw and begins to exert an increasing upward force at a preset rate of application until the animal removes its paw. The paw withdrawal threshold is numerically shown in grams on the digital screen. Changes of the touch sensitivity thresholds were expressed as % of control compared to the pre-injection values. Since this mechanical stimulus is basically non painful in rats, but slightly painful in mice, the drop of the threshold is considered to be mechanical allodynia in rats, but hyperalgesia in mice (Helyes et al., 2004; Szabó et al., 2005; Bölcskei et al., 2005). 2.5. Measurement of the mechanonociceptive threshold of the rat paw The mechanonociceptive threshold of the rat hindpaw was measured with the Randall-Sellitto test (Ugo Basile Analgesimeter 7210, Comerio, Italy) and hyperalgesia was expressed in % compared to the initial, pre-injection control values. In this measurement, the animal is gently restrained and the paw is placed under a plastic cone-shaped pusher, which exerts an increasing force/pressure. The force when the animal withdraws the paw is considered to be the mechanonociceptive threshold. Only rats can be measured with this instrument due to the size of the pusher and the force it exerts. Hyperalgesia was expressed as % drop of this threshold compared to the initial pre-injection values (Szolcsányi et al., 2004; Sándor et al., 2006). 2.6. Measurement of spontaneous weight distribution in rats and mice Spontaneous weight bearing on the two hindlimbs was determined by incapacitance tester (Linton Instrumentation, Norfolk, England) before the experiment and every hour during the 6-h experimental period. The percent weight distributed onto the right (treated) hindlimb was calculated by the following equation: [weight on the right hindlimb/(weight on the left + weight on the right)] 100. 2.7. Measurement of the paw volume
2.3. Intraarticular and intraplantar PAR-2 receptor activation The PAR-2-activating peptide, SLIGRL-NH2 (or in the control group the inactive LRGILS-NH2) was injected s.c. into the plantar
The volume of the paws was measured with plethysmometry (Ugo Basile Plethysmometer 7140, Comerio, Italy). This instrument consists of two vertical interconnected water-filled Perspex cells,
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the larger of which is used to measure volume displacement induced by immersion of the mouse paw. The water level in an interconnected smaller tube, which contains a force transducer, generates a proportional volume measurement of the mouse paw which is expressed in cm3 (Helyes et al., 2004; Szabó et al., 2005). The paw volumes were measured prior to intraplantar SLIGRL-NH2 (or in the control group the inactive LRGILS-NH2) injection, and every hour throughout 6 h after the treatment. Oedema was expressed in percentage compared to the initial volumes (Helyes et al., 2006). 2.8. Measurement of knee diameter in mice The medio-lateral and the anterio-posterior diameter of the knee joint was measured with a digital micrometer (Mitutoyo, Japan) under isoflurane anaesthesia before and every hour during a 6 h-period after the intraplantar injection of SLIGRL-NH2 or in the control group LRGILS-NH2. 2.9. Determination of inflammatory cytokines in tissue homogenates Mice were anaesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.m.), then sacrificed by cervical dislocation. The knee joints and the paws were excised, frozen in liquid nitrogen and kept at 80 °C until further processing. Each sample was homogenized in 1 ml buffer containing 990 ll RPMI 1640 medium and 10 ll PMSF (phenyl-methyl-sulphonyl-fluoride) protease inhibitor for 2 min at 21,000 rpm with Miccra D-9 Digitronic device (Art-moderne Laborteknik, Germany). Homogenates were centrifuged for 10 min at 5 °C and 12,500 rpm, the supernatants were then collected and kept at 20 °C. The concentrations of two inflammatory cytokines, IL-1b and TNF-a were measured with ELISA techniques. 2.10. Statistical analysis The number of animals in each group was 7–11, the data were checked for normality with the help of Origin 7.0 (Shapiro–Wilk test) and in each case normal distribution was found. Hyperalgesia/allodynia, spontaneous weight distribution, paw volume and joint diameter data obtained at several time points throughout the 6-h experimental period were evaluated by one-way analysis of variance (ANOVA) followed by Bonferroni’s modified t-test to compare the actions of the PAR-2 activating peptide to that of the inactive peptide, as well as to evaluate the effect of pre-treatment with the TRPV1 receptor antagonist in rats and the difference between WT and TRPV1 / mice. The cytokine results were analysed by Student’s t-test for unpaired comparison. In all cases P < 0.05 was considered to be significant. 2.11. Drugs and chemicals Thiopental was obtained from Sandoz GmbH (Austria), isoflurane from Abbott Ltd. (USA), ketamine from Richter Gedeon Plc. (Hungary), xylazine from Lavet Ltd. (Hungary), IL-1b OptEIA set from BD Biosciences (USA). SB366791 was synthesized at GlaxoSmithKline (UK) and was dissolved in dimethyl-sulphoxide to make a stock solution of 2 mg/kg and further diluted with saline. The ELISA kits for TNF-a were obtained from R&D System (USA). The PAR-2 receptor activating peptide, SLIGRL-NH2, was purchased from Sigma–Aldrich Ltd. (Hungary), the inactive peptide LRGILSNH2 was synthesized at the Department of Medical Biochemistry, Health Science Centre, University of Calgary, Alberta, Canada. Both compounds were dissolved in saline.
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3. Results 3.1. Role of the TRPV1 receptor in intraarticular SLIGRL-NH2-induced pain behaviour in rats Injection of the PAR-2 receptor activating peptide, SLIGRL-NH2 (100 lg in 100 ll) into the right knee joint induced a moderate, but significant drop of the touch sensitivity threshold of the plantar surface of the ipsilateral hindpaw. The force required to elicit a tactile response decreased from 48.5 ± 0.5 g to 40.0 ± 0.4 g. This 12– 17% secondary mechanical allodynia was markedly reduced by i.p. pre-treatment with the TRPV1 receptor antagonist SB366791 to 5–6% (control = 47.5 ± 0.7 g; treated = 44.5 ± 0.6 g) (Fig. 1A). The drop in the mechanonociceptive threshold of the paw determined with the Randall–Selitto test was much greater (about 35%; from 11.2 ± 0.7 g to 6.5 ± 0.6 g) in response to unilateral intraarticular injection of SLIGRL-NH2. Similarly to that observed for allodynia, the secondary mechanical hyperalgesia was abolished by TRPV1 receptor antagonist pre-treatment (Fig. 1B). In addition to mechanical allodynia and hyperalgesia, PAR-2 receptor activation in the knee joint decreased weight bearing on the affected side indicating the development of spontaneous pain. Although this symptom was also significantly diminished by TRPV1 receptor antagonism 1, 4 and 5 h after SLIGRL-NH2 administration, SB366791-evoked inhibition was not observed at the other time points (Fig. 1C). The inactive peptide, LRGILS-NH2 (100 lg in 100 ll), injected into the knee joint had no effect on either mechanonociception or spontaneous weight bearing (Figs. 1A–C). 3.2. Inflammatory cytokine concentrations in the rat joint The concentration of TNF-a in the rat joint homogenates were around the detection limit of the ELISA assay both in LRGILS- NH2 and SLIGRL-NH2-injected knees 6 h after the injection (data not shown). The level of IL-1b significantly increased in response to intraarticular PAR-2 receptor activation (792 ± 32.6 pg/g wet tissue in the SLIGRL-NH2-injected knees compared to the 687 ± 48.4 pg/g wet tissue measured in the LRGILS-NH2-treated joints; P < 0.05). However, IL-1b levels were not significantly altered by SB366791 pre-treatment (709 ± 45.5 pg/g wet tissue). 3.3. Role of the TRPV1 receptor in intraarticular SLIGRL-NH2-induced pain behaviour and inflammatory cytokine release in mice Injection of SLIGRL-NH2 (100 lg in 50 ll) into the right knee joint of wildtype mice induced about a 15% decrease of the mechanonociceptive threshold of the paw measured with aesthesiometry (from 8.5 ± 0.9 g to 7.0 ± 0.8 g), which is very similar to the extent of mechanical allodynia determined with the same technique. In the TRPV1 receptor gene-deficient (TRPV1 / ) group, this secondary hyperalgesia was almost absent at 3 and 4 h. Although some reduction was detected at later time points, the difference did not prove to be statistically significant compared to WT mice (Fig. 2A). Similar to that observed in rats, WT mice demonstrated reduced ipsilateral hindlimb weight bearing in response to intraarticular injection of SLIGRL-NH2 (Fig. 2B). TRPV1 / mice also showed a decrease in ipsilateral weight bearing during the first 4 h; however, hindlimb incapacitance subsequently returned to WT levels at 5 and 6 h post-injection of the PAR-2 agonist. Injection of the same volume of LRGILS-NH2 into the knee joint evoked no change in either mechanonociception or static weight bearing (Fig. 2A and B). In agreement with the rat experiments, PAR-2 receptor activation did not elevate joint TNF-a concentration, which was at the detection limit of the ELISA assay both in LRGILS-NH2 and SLIGRL-NH2-injected knees (data not shown). Intraarticular
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Change of touch sensitivity (%)
A
12
SLIGRL-NH2 SB366791 +SLIGRL-NH2
8
LRGILS-NH2 4 0
##
#
#
-4 -8
#
#
* **
*
*
-12
*
*
-16 -20 control
1
2
3
4
5
6
hours after i.a. administration
Change of mechanonociceptive threshold (%)
B
SLIGRL-NH2 SB366791+SLIGRL-NH2
10
LRGILS-NH2
5 0 -5 -10
### ###
-15
##
###
-20
*
-25
*
** ***
-30
#
-35
***
***
3
4
-40 -45 -50 1
2
5
6
hours after i.a. administration
Ipsilateral weight distribution (%)
C
SLIGRL-NH2
60
SB366791 +SLIGRL-NH2
58
LRGILS-NH2
56 54 52 #
50 48
**
*
46 44
#
#
*
*
*
42 40 control
1
2
3
4
5
6
hours after i.a. administration Fig. 1. Alterations of the (A) touch sensitivity thresholds and (B) mechanonociceptive thresholds of the paw as well as (C) spontaneous ipsilateral weight distribution in response to injecting the PAR-2 activating peptide SLIGRL-NH2 (100 lg in 100 ll) into the unilateral knee joint of rats. The inactive peptide, LRGILS-NH2, was injected the same way in control animals. In a separate groups rats pre-treatment with the TRPV1 receptor antagonist SB366791 (500 lg/kg i.p.) was performed 15 min before the intraarticular injections and before each measurement. Each data point represents the mean ± s.e.m. of n = 9–11 animals per group. *P < 0.05, **P < 0.01, ***P < 0.001 (SLIGRL-NH vs. LRGILS-NH ) and #P < 0.05, ##P < 0.01, ###P < 0.001 2 2 (SLIGRL-NH2 vs. SB366791 + SLIGRL-NH2) analysed by one-way ANOVA followed by Bonferroni’s post test.
administration of SLIGRL-NH2 caused a significant increase in joint IL-1b levels in both WT and TRPV1 / mice, there was no difference
Fig. 2. Alterations of the (A) mechanonociceptive thresholds of the paw, (B) ipsilateral weight distribution and (C) IL-1b concentration measured in the joint homogenates in response to injecting the PAR-2 activating peptide SLIGRL-NH2 (100 lg in 50 ll) into the unilateral knee joint of C57Bl/6 wildtype (WT) and TRPV1 receptor gene-deleted TRPV1 / mice. The inactive peptide, LRGILS-NH2, was injected the same way in control animals. Each data point represents the mean ± s.e.m. of n = 9–11 animals per group. *P < 0.05, **P < 0.01, (SLIGRL-NH2 vs. LRGILS-NH2 in WT mice), #P < 0.05, ##P < 0.01 (SLIGRL-NH2-treated WT vs. TRPV1 / ), and +P < 0.05, ++P < 0.01 (SLIGRL-NH2 vs. LRGILS-NH2 in TRPV1 / mice) determined with one-way ANOVA followed by Bonferroni’s post test.
between these groups. LRGILS-NH2 had no effect on articular IL-1b release (Fig. 2C). 3.4. No effect of intraarticular SLIGRL-NH2 on knee diameter The control antero-posterior diameter of the mouse knee was 4.4 ± 0.1 mm, whereas the medio-lateral one was 4.3 ± 0.1 mm.
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Primary hyperalgesia induced by intraplantar injection of SLIGRL-NH2 (100 lg in 50 ll) was around 15–25% in WT mice throughout the 6-h period, it decreased from 8.7 ± 1.2 g to a maximum of 6.5 ± 0.5 g. Although it was smaller in the TRPV1 / group at certain time points, the two hyperalgesia curves referring to the total duration of the experiment did not prove to be statistically significant (Fig. 3A). Conversely, SLIGRL-NH2-evoked paw oedema was markedly smaller in TRPV1 / mice than in wildtypes throughout the whole 6 h-period. In WT animals, paw volume increased by about 22–26% (from 0.19 ± 0.05 cm3 to 0.24 ± 0.02 cm3) and in the animals by 10% (from 0.20 ± 0.03 cm3 to 0.22 ± TRPV1 / 0.01 cm3; Fig. 3B). Impaired weight distribution in response to the injection of the PAR-2-activating peptide into the paw was comparable between the TRPV1 / and wildtype groups without any statistically significant differences (Fig. 3C). Intraplantar administration of the inactive peptide, LRGILS-NH2 did not induce any changes in the mechanonociceptive thresholds, paw volumes or the spontaneous weight distribution in either group (Fig. 3A–C).
Change of mechanonociceptive threshold (%)
3.5. Involvement of the TRPV1 receptor in intraplantar SLIGRL-NH2induced pain behaviour and tissue oedema in mice
hours after i.pl. administration
A 1
2
3
4
5
6
10 5 0 -5
*
-10 +
-15
**
**
+
*
-20
**
++
-25 ++ WT (SLIGRL-NH2)
-30
-/-
TRPV1 (SLIGRL-NH2)
-35
WT (LRGILS-NH2) -/-
TRPV1 (LRGILS-NH2)
WT (SLIGRL-NH2)
B 35
-/-
TRPV1 (SLIGRL-NH2)
Oedema formation (%)
One hour following intraarticular SLIGRL-NH2 injection, only 5.1 ± 0.4% and 1.8 ± 0.1% increases were measured from these directions in wildtype mice, which was not considered as a significant swelling. This moderate oedema formation remained unchanged during the 6-h experimental period, the respective increases of these diameters were 4.9 ± 0.3% and 1.3 ± 0.2% at this time point. Similar minimal changes were observed in TRPV1 / mice as well as in response to LRGILS-NH2 injection in both WT and TRPV1 gene-deleted animals. Significant differences could not be determined between any groups.
WT (LRGILS-NH2)
30
-/-
TRPV1 (LRGILS-NH2)
25
*
20
***
***
***
**
***
15
## #
10
###
###
###
5
###
3.6. Inflammatory cytokine concentrations in the mouse paw 0
4. Discussion An appreciation of the precise nociceptive processes associated with inflammatory joint diseases is of great clinical importance, but for which we have a very poor understanding (McDougall, 2006). PAR activation is known to modulate inflammatory processes and there are some data concerning its significance in pain processing (Vergnolle et al., 2001a; Cenac et al., 2007), but the role of PARs in arthritis and related pain has not been clearly and thoroughly elucidated. In one of the few studies to address this issue, it has been shown that PAR-4 activation leads to joint inflammation and pain while local injection of a selective PAR-4 antagonist reduces arthritis severity and acute pain symptoms (McDougall et al., 2009). Whether this holds true for other PARs is currently unknown. The present results provide the first evidence that injection of the selective PAR-2 activating peptide SLIGRL-NH2 into the knee joint results in the development of a secondary mechanical hyperalgesia/allodynia in the paw as well as impaired weight distribution in both rats and mice. These pain-causing effects of PAR-2 activation are accompanied by an increase in IL-1b production. Conversely, the level of the other inflammatory cytokine tested, TNF-a, did not increase throughout the 6 h duration of the experiment. This particular time point was chosen based on the fact that
0
1
2
3
4
5
6
hours after i.pl. administration
C Ipsilateral weight distribution (%)
Similar to the results obtained in the joints, intraplantar injection of SLIGRL-NH2 did not induce an increased TNF-a production in the paw (data not shown). IL-1b, however, significantly increased in response to PAR-2 activation, but it was not altered by genetic deletion of the TRPV1 receptor (Fig. 4).
WT (SLIGRL-NH2) -/-
53
TRPV1 (SLIGRL-NH2)
52
WT (LRGILS-NH2) -/-
TRPV1 (LRGILS-NH2)
51 50 49 48 47 46 45 44 control
1
2
3
4
5
6
hours after i.pl. administration Fig. 3. Alterations of the (A) mechanonociceptive threshold and (B) volume of the paw as well as (C) ipsilateral weight distribution in response to injecting the PAR-2 activating peptide SLIGRL-NH2 (100 lg in 50 ll) intraplantarly in C57Bl/6 wildtype (WT) and TRPV1 receptor gene-deleted TRPV1 / mice. The inactive peptide, LRGILS-NH2, was injected the same way in control animals of both groups. Each data point represents the mean ± s.e.m. of n = 8–11 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (SLIGRL-NH vs. LRGILS-NH in WT mice), #P < 0.05, 2 2 ## P < 0.01, ###P < 0.001 (SLIGRL-NH2-treated WT vs. TRPV1 / ), and +P < 0.05, ++ P < 0.01 (SLIGRL-NH2 vs. LRGILS-NH2 in TRPV1 / mice) determined with oneway ANOVA followed by Bonferroni’s post test.
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3200 2800 2400 2000 1600 1200 800 400 0
Fig. 4. IL-1b concentration measured in the paw of C57Bl/6 wildtype (WT) and TRPV1 receptor gene-deleted TRPV1 / mice 6 h after intraplantar injection of the PAR-2 activating peptide SLIGRL-NH2 (100 lg in 50 ll). The inactive peptide, LRGILS-NH2, was injected the same way in control animals of both groups. Each column represents the mean ± s.e.m. of n = 6–8 samples per group. *P < 0.05, **P < 0.01 (SLIGRL-NH vs. LRGILS-NH in WT mice) and +P < 0.05, ++P < 0.01 2 2 (SLIGRL-NH2 vs. LRGILS-NH2 in TRPV1 / mice) determined with Student’s t-test for unpaired comparisons.
the PAR-2 receptor is rapidly desensitized after activation and it is generally considered to mediate acute inflammatory reactions over a short period of time, i.e. up to 6 h. There are several data available concerning the inflammatory and nociceptive actions of PAR-2 activation in the paw (Kawabata et al., 2001), the gastrointestinal tract (Cenac et al., 2002; Kawao et al., 2004) and the airways (Schmidlin et al., 2002; for revs. Vergnolle et al., 2001a; Fiorucci and Distrutti, 2002; Vergnolle, 2005; Cenac and Vergnolle, 2005; Kanke et al., 2005). The effects of articular PAR-2 stimulation was examined by Ferrell et al. who described swelling, hyperaemia and histological changes as well-defined signs of the development of an inflammatory reaction (Ferrell et al., 2003). We on the other hand did not find any change in knee joint diameter in response to a 100 lg dose of the PAR-2 activating peptide, although IL-1b concentration increased confirming the development of a distinctive inflammatory response in this organ. Furthermore, we have shown for the first time that PAR-2 receptor activation in joints leads to pro-nociceptive processes. Our experiments with rats pre-treated with the selective TRPV1 receptor antagonist SB366791 (Varga et al., 2005) as well as studies with TRPV1 receptor gene-deleted mice revealed that the secondary hyperalgesia/allodynia and the decreased spontaneous weight distribution on the affected side are predominantly mediated via TRPV1 receptor activation. While the PAR-2 activation-evoked changes were abolished by the TRPV1 antagonist in rats, these were significantly, but not completely inhibited by the genetic deletion of this ion channel in mice. This difference can be explained by a species difference regarding the importance of the TRPV1-mediated mechanisms in the joints, but compensatory mechanisms counteracting the lack of this receptor are also possible in knockout mice. The PAR-2-activation-induced significant mechanical hyperalgesia observed in TRPV1 gene-deleted mice compared to the inactive control at 2 h disappeared at later time points. The TRPV1 receptor therefore seems to be involved in PAR-2 activation-induced secondary mechanical hyperalgesia 3 h and in spontaneous pain 4 h after the induction of the inflammation. This is supported by the results obtained in rats with the TRPV1 receptor antagonist, although activation of this ion channel is likely to be a little faster in rats than in mice (2 and 3 h in cases of hyperalgesia and weight distribution, respectively).
Activation of PAR-2 receptors on the synovial cells, fibrocytes, inflammatory and immune cells, as well as sensory nerves results in the release of several pro-inflammatory mediators such as bradykinin, leukotriens, prostaglandins, cytokines and sensory neuropeptides (tachykinins, CGRP), which in turn activate/sensitize the TRPV1 channels. Direct sensitization of the TRPV1 receptor on the peripheral nerve terminal through protein kinases as described for thermal hyperalgesia (Amadesi et al., 2006) is not likely to be the primary mechanism for the secondary mechanical hyperalgesia and spontaneous pain. Intraplantar administration of SLIGRL-NH2 in mice produced paw oedema through a TRPV1-mediated mechanism, which is most likely to be the result of SP and CGRP release from the activated sensory nerve terminals as described by several previous investigations (Vergnolle et al., 2001b; Cottrell et al., 2003). In contrast, this ion channel was not involved in the development of PAR2-evoked primary hyperalgesia or impaired weight distribution. PAR-2 is known to cause hyperalgesia to thermal stimuli at the site of activation (Hoogerwerf et al., 2001; Vergnolle et al., 2001b) and the involvement of TRPV1 receptors on the peripheral nerve endings has been shown in this action (McLean et al., 2002). However, the ability of PAR-2 activation to induce mechanical hyperalgesia and allodynia is controversial. It has been suggested by two previous studies performed in TRPV1 receptor gene-deleted mice that thermal as well as mechanical hyperalgesia evoked by SLIGRLNH2 injection into the paw was absent or significantly smaller in the TRPV1 knockout group (Amadesi et al., 2004; Dai et al., 2004). In this latter study, mechanical hyperalgesia was moderate, but significantly smaller in TRPV1 / mice, which is in agreement with our findings obtained 2 h after intraplantar injection. However, at later time points this difference disappeared and the two primary hyperalgesia curves were not significantly different (Dai et al., 2004). In case of intraplantar administration of the PAR-2 receptor agonist the inflammation develops in the paw and since the nociceptive threshold is also measured there it is considered to be primary mechanical hyperalgesia. When the PAR-2 receptors are activated in the joints and the mechanonociceptive threshold is measured on the plantar surface of the paw, it is called secondary mechanical hyperalgesia/allodynia. Primary and secondary hyperalgesia develop by fundamentally different mechanisms. While primary hyperalgesia, especially to thermal stimuli, is predominantly mediated by peripheral sensitization of primary nociceptive nerve terminals, secondary hyperalgesia is attributed to altered processing in the central nervous system (Treede and Magerl, 2000). Altered neuronal response properties consistent with the characteristic of secondary hyperalgesia have been observed in the spinal cord, the thalamus, and the somatosensory cortex, but not in the peripheral endings of primary nociceptive afferents, which suggest that basic mechanisms of secondary hyperalgesia may consist of modulation of neural transmission in the spinal cord and in higher neural centres. Several mechanisms are triggered in the dorsal horn by noxious stimuli which lead to enhanced synaptic activation (central sensitization). However, intraarticular PAR-2 activation induced a weaker secondary hyperalgesia than the primary response after direct intraplantar administration of the agonist. There might be more constitutive endopeptidase activity in the synovial fluid which limits the PAR-2 induced responses. Besides the localization of the PAR-2 receptors on sensory nerve endings, in the joints they have also been described on other specific cell types such as synovial cells, immune- and inflammatory cells including mast cells. Pro-inflammatory mediators which are released from these specific cells in the joints might be involved in activation/ marked sensitization of the TRPV1 receptors (Amadesi et al., 2006; Kelso et al., 2006; Palmer et al., 2007).
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Although PAR-2 activation cannot generate action potentials itself, it has the ability to functionally interact with other receptors and ion channels (e.g. TRPV4; Grant et al., 2007) to induce depolarization due to Na+ influx as well as increased c-Fos expression and sensory neuropeptide release (e.g. SP and CGRP) in the spinal cord through increased intracellular Ca2+ concentration (Seeliger et al., 2003; Dai et al., 2004; Cenac and Vergnolle, 2005). These mechanisms result in an enhanced transmission of mechanonociceptive signals and the development of a secondary hyperalgesia/allodynia. Although peripheral sensitization of the TRPV1 receptor through protein kinase C-, protein kinase A- or phospholipase Cdependent processes has been described both in vitro (Dai et al., 2004) and in vivo (Amadesi et al., 2004) as an explanation of primary thermal hyperalgesia, these mechanisms are not likely to be involved in mechanical hyperalgesia and allodynia. Based on our results, it can be inferred that articular activation of PAR-2 receptors leads to joint pain and secondary allodynia. The activation of joint sensory nerve terminals themselves by the PAR-2 agonist is not TRPV1 receptor-mediated; however, TRPV1 does appear to be involved in PAR-2-mediated nociceptive processing involving higher pain centres. Acknowledgements The authors thank Mrs. Dóra Ömböli for her expert technical assistance in the experiments. This work was sponsored by Hungarian Grants: OTKA K73044, NK78059. Zs. Helyes is supported by János Bolyai Postdoctoral Research Fellowship. J.J. McDougall is an Alberta Heritage Foundation for Medical Research Senior Scholar and an Arthritis Society Investigator. References Amadesi S, Nie J, Vergnolle N, Cottrell GS, Grady EF, Trevisani M, et al. Proteaseactivated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia. J Neurosci 2004;24:4300–12. Amadesi S, Cottrell GS, Divino L, Chapman K, Grady EF, Bautista F, et al. Proteaseactivated receptor 2 sensitizes TRPV1 by protein kinase Ce- and A-dependent mechanisms in rats and mice. J Physiol 2006;575:555–71. Boileau C, Amiable N, Martel-Pelletier J, Fahmi H, Duval N, Pelletier J-P. Activation of protease-activated receptor 2 in human osteoarthritic cartilage upregulates catabolic and pro-inflammatory pathways capable of inducing cartilage degeneration: a basic science study. Arthritis Res Ther 2007;9:R121–30. } G, Elekes K, et al. Investigation of the Bölcskei K, Helyes Zs, Szabó Á, Sándor K, Petho role of TRPV1 receptors in acute and chronic nociceptive processes using genedeficient mice. Pain 2005;117:368–76. Busso N, Frasnelli M, Feifel R, Cenni B, Steinhoff M, Hamilton J, et al. Evaluation of protease-activated receptor 2 in murine models of arthritis. Arthritis Rheum 2007;56:101–7. Cenac N, Vergnolle N. Proteases and protease-activated receptors (PARs): novel signals for pain. Curr Top Med Chem 2005;5:569–76. Cenac N, Coelho AM, Nguyen C, Compton S, Andrade-Gordon P, McNaughton WK, et al. Induction of intestinal inflammation in mouse by activation of proteaseactivated receptor-2. Am J Pathol 2002;161:1903–15. Cenac N, Andrews CN, Holzhausen M, Chapman K, Cottrell G, Andrade-Gordon P, et al. Role of protease activity in visceral pain in irritable bowel syndrome. J Clin Invest 2007;117:636–47. Cocks TM, Fong B, Chow JM, Anderson GP, Frauman AG, Goldie RG, et al. A protective role for protease-activated receptors in the airways. Nature 1999;198:156–60. Coelho A-M, Ossovskaya V, Bunnett NW. Protease-activated receptor-2: physiological and pathophysiological roles. Curr Med Chem Cardiovasc Hematol Agents 2003;1:61–72. Costa R, Marotta DM, Manjavachi MN, Fernandes ES, Lima-Garcia JF, Paszcuk AF, et al. Evidence for the role of neurogenic inflammation components in trypsinelicited scratching behaviour in mice. Br J Pharmacol 2008;154:1094–103. Cottrell GS, Amadesi S, Schmidlin F, Bunnett NW. Protease-activated receptor 2: activation, signalling and function. Biochem Soc Trans 2003;31:1191–7. Dai Y, Moriyama T, Higashi T, Togashi K, Kobayashi K, Yamanaka H, et al. Proteaseactivated receptor-2-mediated potentiation of transient receptor potential vanilloid subfamily 1 activity reveals a mechanism for protease-induced inflammatory pain. J Neurosci 2004;24:4293–9. D’Andrea MR, Derian CK, Leturcq D, Baker SM, Brunmark A, Ling P, et al. Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues. J Histochem Cytochem 1998;46:157–64.
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Involvement of transient receptor potential vanilloid 1 receptors in protease-activated receptor-2-induced joint inflammation and nociception Zs. Helyes a,*,1, K. Sándor a,1, É. Borbély a, V. Tékus b, E. Pintér a, K. Elekes c, D.M. Tóth b, J. Szolcsányi a, J.J. McDougall d a
Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, H-7624 Pécs, Szigeti u. 12., Hungary Analgesic Research Laboratory, University of Pécs and Gedeon-Richter Plc., H-7624 Pécs, Szigeti u. 12., Hungary Institute of Pharmacognosy, Faculty of Medicine, University of Pécs, H-7624 Pécs, Rókus u. 2., Hungary d Department of Physiology and Pharmacology, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, T2N 4N1 Canada b c
a r t i c l e
i n f o
Article history: Received 25 February 2009 Received in revised form 23 June 2009 Accepted 21 July 2009 Available online 15 August 2009 Keywords: Capsaicin-sensitive sensory nerves Arthritis Mechanical hyperalgesia Allodynia Inflammatory cytokines
a b s t r a c t Protease-activated receptor-2 (PAR-2) is a G-protein-coupled receptor activated through proteolytic cleavage. It is localized on epithelial, endothelial and inflammatory cells, as well as on transient receptor potential vanilloid 1 (TRPV1) receptor-expressing neurones. It plays an important role in inflammatory/ nociceptive processes. Since there are few reports concerning PAR-2 function in joints, the effects of intraarticular PAR-2 activation on joint pain and inflammation were studied. Secondary hyperalgesia/allodynia, spontaneous weight distribution, swelling and inflammatory cytokine production were measured and the involvement of TRPV1 ion channels was investigated in rats and mice. Injection of the PAR-2 receptor agonist SLIGRL-NH2 into the knee decreased touch sensitivity and weight bearing of the ipsilateral hindlimb in both species. Secondary mechanical allodynia/hyperalgesia and impaired weight distribution were significantly reduced by the TRPV1 antagonist SB366791 in rats and by the genetic deletion of this receptor in mice. PAR-2 activation did not cause significant joint swelling, but increased IL-1b concentration which was not influenced by the lack of the TRPV1 channel. For comparison, intraplantar SLIGRL-NH2 evoked similar primary mechanical hyperalgesia and impaired weight distribution in both WT and TRPV1 deficient mice, but oedema was smaller in the knockouts. The inactive peptide, LRGILS-NH2, injected into either site did not induce any inflammatory or nociceptive changes. These data provide evidence for a significant role of TRPV1 receptors in secondary mechanical hyperalgesia/allodynia and spontaneous pain induced by PAR-2 receptor activation in the knee joint. Although intraplantar PAR-2 activation-induced oedema is also TRPV1 receptor-mediated, primary mechanical hyperalgesia, impaired weight distribution and IL-1b production are independent of this channel. Ó 2009 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.
1. Introduction The family of protease-activated receptors (PARs) consists of four members: three receptors for thrombin (PAR-1, PAR-3 and PAR-4) being involved in mainly coagulation processes and one for trypsin/mast cell tryptase (PAR-2). They are all heptahelical G-protein-coupled receptors activated by proteolytic cleavage
Abbreviation: TRPV1, transient receptor potential vanilloid 1; PAR-2 receptor, protease-activated receptor-2; SP, substance P; CGRP, calcitonin gene-related peptide. * Corresponding author. Address: Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, H-7624 Pécs, Szigeti u. 12., Hungary. Tel.: +36 72 536001/5591/5386; fax: +36 72 536218. E-mail address: [email protected] (Zs. Helyes). 1 Zs. Helyes and K. Sándor contributed equally to the present work.
through several proteases (Molino et al., 1997; Macfarlane et al., 2001; Hollenberg and Compton, 2002). PAR-2 has drawn great attention due to its different localization and function compared to the other three PARs. It is expressed on epithelial and endothelial cells in the skin (Steinhoff et al., 1999), joints (Ferrell et al., 2003; Boileau et al., 2007; Nakano et al., 2007), cardiovascular, gastrointestinal and respiratory systems (D’Andrea et al., 1998; Cocks et al., 1999; Kawabata et al., 2002), as well as on capsaicin-sensitive peripheral sensory nerves (Steinhoff et al., 2000; Vergnolle et al., 2001a,b; Vergnolle, 2005). Serine proteases play an important role in several inflammatory processes (Schmelz et al., 1999; Kanke et al., 2005; McIntosh et al., 2007) and pain (Vergnolle et al., 2001a,b; Coelho et al., 2003). The importance of PAR-2 in the induction and maintenance of inflammatory responses has been confirmed with PAR-2 deficient mice (Lindner et al., 2000). In the skin, PAR-2 agonists induce inflammatory
1090-3801/$36.00 Ó 2009 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ejpain.2009.07.005
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changes such as vasodilatation, oedema, leukocyte recruitment, adhesion and extravasation (Saifeddine et al., 1996; Vergnolle et al., 1998; Vergnolle, 1999; Seeliger et al., 2003). These depend on neurogenic mechanisms via the release of SP and CGRP from peripheral terminals of capsaicin-sensitive, transient receptor potential vanilloid 1 (TRPV1) receptor-expressing sensory nerves (Steinhoff et al., 2000; Coelho et al., 2003). In the innervated area, calcitonin gene-related peptide (CGRP) elicits arteriolar vasodilatation and potentiates the plasma protein extravasation-inducing effects of substance P (SP; Jancsó-Gábor and Szolcsányi, 1970; Szolcsányi, 1988; Holzer, 1992; Maggi, 1995; Szolcsányi, 1996; Helyes et al., 2003). Recently, the involvement of capsaicin-sensitive fibres in PAR-2 activation-induced inflammatory reactions and nociception has been suggested by the markedly reduced responses observed after capsaicin desensitization (Su et al., 2005; Gu and Lee, 2006; Shimizu et al., 2007; Paszcuk et al., 2008). Further studies with TRPV1 receptor antagonists revealed the role of this ion channel in PAR-2-evoked scratching behaviour (Costa et al., 2008), thermal hyperalgesia (Amadesi et al., 2004) and urinary bladder contractions (Shimizu et al., 2007). Data obtained with TRPV1 gene-deleted mice demonstrated that intraplantar PAR-2 activation-induced primary hyperalgesia is – at least partially – TRPV1 mediated (Amadesi et al., 2004; Dai et al., 2004). There are a limited number of in vivo experiments concerning PAR activation in joints (Russell and McDougall, 2009). PAR-2 stimulation in the murine knee promotes synovial hyperaemia and oedema indicating a role for PARs in inflammatory joint diseases (Ferrell et al., 2003; Busso et al., 2007). Thus, the aims of the present series of experiments were to analyse the inflammatory changes as well as secondary hyperalgesia/allodynia in response to intraarticular PAR-2 activation in rats and mice and to investigate the role of the TRPV1 ion channel in the observed actions with the help of a receptor antagonist and genetic deletion of the receptor. 2. Experimental procedures 2.1. Animals Experiments were performed on male Wistar rats (250–300 g) as well as on TRPV1 receptor gene-deficient mice (TRPV1 / ) backcrossed through 10 generations with the C57Bl/6 strain and their wildtype (WT) counterparts (20–25 g). The original breeding pairs of the rats and C57Bl/6 mice were purchased from Charles-River Ltd. (Hungary) and the TRPV1 / mice from Jackson Laboratories (USA). The animals were bred and kept in the Laboratory Animal House of the Department of Pharmacology and Pharmacotherapy of the University of Pécs at 24–25 °C and provided with standard rat chow and water ad libitum. 2.2. Ethics All experimental procedures were carried out according to the 1998/XXVIII Act of the Hungarian Parliament on Animal Protection and Consideration Decree of Scientific Procedures of Animal Experiments (243/1988) and complied with the recommendations of the International Association for the Study of Pain and the Helsinki Declaration. The studies were approved by the Ethics Committee on Animal Research of University of Pécs according to the Ethical Codex of Animal Experiments and licence was given (licence No.: BA 02/2000-11-2006).
surface of the right paw (100 lg in 50 ll) or into the right knee joint of mice (100 lg in 50 ll) and rats (100 lg in 100 ll) under isoflurane anaesthesia. Measurements described below were performed at certain time points after the injections throughout the 6-h experimental period. In one group of rats, pre-treatment with the selective and in vivo active TRPV1 receptor antagonist SB366791 (500 lg/kg i.p.; Varga et al., 2005) was performed 15 min before intraarticular SLIGRL-NH2 injection and before each measurement. 2.4. Measurement of touch sensitivity of the paw Touch sensitivity of the plantar surface of the paw in both rats and mice were determined with dynamic plantar aesthesiometry (Ugo Basile 37400, Comerio, Italy) before and every hour after SLIGRL-NH2 (or in the control group LRGILS-NH2) injection. This device is a modified, electronic von Frey technique, which is used to assess touch sensitivity on the plantar surface of the paw. The animals move about freely in one of the two compartments of the enclosure positioned on the metal mesh surface. Following acclimation after cessation of exploratory behaviour, the operator places the touch stimulator unit under the animal’s paw, using the adjustable angled-mirror to position the filament below the target area of the paw. After pressing the ‘‘start” key an electrodynamic actuator of proprietary design advances a straight metal filament, which touches the plantar surface of the paw and begins to exert an increasing upward force at a preset rate of application until the animal removes its paw. The paw withdrawal threshold is numerically shown in grams on the digital screen. Changes of the touch sensitivity thresholds were expressed as % of control compared to the pre-injection values. Since this mechanical stimulus is basically non painful in rats, but slightly painful in mice, the drop of the threshold is considered to be mechanical allodynia in rats, but hyperalgesia in mice (Helyes et al., 2004; Szabó et al., 2005; Bölcskei et al., 2005). 2.5. Measurement of the mechanonociceptive threshold of the rat paw The mechanonociceptive threshold of the rat hindpaw was measured with the Randall-Sellitto test (Ugo Basile Analgesimeter 7210, Comerio, Italy) and hyperalgesia was expressed in % compared to the initial, pre-injection control values. In this measurement, the animal is gently restrained and the paw is placed under a plastic cone-shaped pusher, which exerts an increasing force/pressure. The force when the animal withdraws the paw is considered to be the mechanonociceptive threshold. Only rats can be measured with this instrument due to the size of the pusher and the force it exerts. Hyperalgesia was expressed as % drop of this threshold compared to the initial pre-injection values (Szolcsányi et al., 2004; Sándor et al., 2006). 2.6. Measurement of spontaneous weight distribution in rats and mice Spontaneous weight bearing on the two hindlimbs was determined by incapacitance tester (Linton Instrumentation, Norfolk, England) before the experiment and every hour during the 6-h experimental period. The percent weight distributed onto the right (treated) hindlimb was calculated by the following equation: [weight on the right hindlimb/(weight on the left + weight on the right)] 100. 2.7. Measurement of the paw volume
2.3. Intraarticular and intraplantar PAR-2 receptor activation The PAR-2-activating peptide, SLIGRL-NH2 (or in the control group the inactive LRGILS-NH2) was injected s.c. into the plantar
The volume of the paws was measured with plethysmometry (Ugo Basile Plethysmometer 7140, Comerio, Italy). This instrument consists of two vertical interconnected water-filled Perspex cells,
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the larger of which is used to measure volume displacement induced by immersion of the mouse paw. The water level in an interconnected smaller tube, which contains a force transducer, generates a proportional volume measurement of the mouse paw which is expressed in cm3 (Helyes et al., 2004; Szabó et al., 2005). The paw volumes were measured prior to intraplantar SLIGRL-NH2 (or in the control group the inactive LRGILS-NH2) injection, and every hour throughout 6 h after the treatment. Oedema was expressed in percentage compared to the initial volumes (Helyes et al., 2006). 2.8. Measurement of knee diameter in mice The medio-lateral and the anterio-posterior diameter of the knee joint was measured with a digital micrometer (Mitutoyo, Japan) under isoflurane anaesthesia before and every hour during a 6 h-period after the intraplantar injection of SLIGRL-NH2 or in the control group LRGILS-NH2. 2.9. Determination of inflammatory cytokines in tissue homogenates Mice were anaesthetized with ketamine (100 mg/kg, i.p.) and xylazine (10 mg/kg, i.m.), then sacrificed by cervical dislocation. The knee joints and the paws were excised, frozen in liquid nitrogen and kept at 80 °C until further processing. Each sample was homogenized in 1 ml buffer containing 990 ll RPMI 1640 medium and 10 ll PMSF (phenyl-methyl-sulphonyl-fluoride) protease inhibitor for 2 min at 21,000 rpm with Miccra D-9 Digitronic device (Art-moderne Laborteknik, Germany). Homogenates were centrifuged for 10 min at 5 °C and 12,500 rpm, the supernatants were then collected and kept at 20 °C. The concentrations of two inflammatory cytokines, IL-1b and TNF-a were measured with ELISA techniques. 2.10. Statistical analysis The number of animals in each group was 7–11, the data were checked for normality with the help of Origin 7.0 (Shapiro–Wilk test) and in each case normal distribution was found. Hyperalgesia/allodynia, spontaneous weight distribution, paw volume and joint diameter data obtained at several time points throughout the 6-h experimental period were evaluated by one-way analysis of variance (ANOVA) followed by Bonferroni’s modified t-test to compare the actions of the PAR-2 activating peptide to that of the inactive peptide, as well as to evaluate the effect of pre-treatment with the TRPV1 receptor antagonist in rats and the difference between WT and TRPV1 / mice. The cytokine results were analysed by Student’s t-test for unpaired comparison. In all cases P < 0.05 was considered to be significant. 2.11. Drugs and chemicals Thiopental was obtained from Sandoz GmbH (Austria), isoflurane from Abbott Ltd. (USA), ketamine from Richter Gedeon Plc. (Hungary), xylazine from Lavet Ltd. (Hungary), IL-1b OptEIA set from BD Biosciences (USA). SB366791 was synthesized at GlaxoSmithKline (UK) and was dissolved in dimethyl-sulphoxide to make a stock solution of 2 mg/kg and further diluted with saline. The ELISA kits for TNF-a were obtained from R&D System (USA). The PAR-2 receptor activating peptide, SLIGRL-NH2, was purchased from Sigma–Aldrich Ltd. (Hungary), the inactive peptide LRGILSNH2 was synthesized at the Department of Medical Biochemistry, Health Science Centre, University of Calgary, Alberta, Canada. Both compounds were dissolved in saline.
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3. Results 3.1. Role of the TRPV1 receptor in intraarticular SLIGRL-NH2-induced pain behaviour in rats Injection of the PAR-2 receptor activating peptide, SLIGRL-NH2 (100 lg in 100 ll) into the right knee joint induced a moderate, but significant drop of the touch sensitivity threshold of the plantar surface of the ipsilateral hindpaw. The force required to elicit a tactile response decreased from 48.5 ± 0.5 g to 40.0 ± 0.4 g. This 12– 17% secondary mechanical allodynia was markedly reduced by i.p. pre-treatment with the TRPV1 receptor antagonist SB366791 to 5–6% (control = 47.5 ± 0.7 g; treated = 44.5 ± 0.6 g) (Fig. 1A). The drop in the mechanonociceptive threshold of the paw determined with the Randall–Selitto test was much greater (about 35%; from 11.2 ± 0.7 g to 6.5 ± 0.6 g) in response to unilateral intraarticular injection of SLIGRL-NH2. Similarly to that observed for allodynia, the secondary mechanical hyperalgesia was abolished by TRPV1 receptor antagonist pre-treatment (Fig. 1B). In addition to mechanical allodynia and hyperalgesia, PAR-2 receptor activation in the knee joint decreased weight bearing on the affected side indicating the development of spontaneous pain. Although this symptom was also significantly diminished by TRPV1 receptor antagonism 1, 4 and 5 h after SLIGRL-NH2 administration, SB366791-evoked inhibition was not observed at the other time points (Fig. 1C). The inactive peptide, LRGILS-NH2 (100 lg in 100 ll), injected into the knee joint had no effect on either mechanonociception or spontaneous weight bearing (Figs. 1A–C). 3.2. Inflammatory cytokine concentrations in the rat joint The concentration of TNF-a in the rat joint homogenates were around the detection limit of the ELISA assay both in LRGILS- NH2 and SLIGRL-NH2-injected knees 6 h after the injection (data not shown). The level of IL-1b significantly increased in response to intraarticular PAR-2 receptor activation (792 ± 32.6 pg/g wet tissue in the SLIGRL-NH2-injected knees compared to the 687 ± 48.4 pg/g wet tissue measured in the LRGILS-NH2-treated joints; P < 0.05). However, IL-1b levels were not significantly altered by SB366791 pre-treatment (709 ± 45.5 pg/g wet tissue). 3.3. Role of the TRPV1 receptor in intraarticular SLIGRL-NH2-induced pain behaviour and inflammatory cytokine release in mice Injection of SLIGRL-NH2 (100 lg in 50 ll) into the right knee joint of wildtype mice induced about a 15% decrease of the mechanonociceptive threshold of the paw measured with aesthesiometry (from 8.5 ± 0.9 g to 7.0 ± 0.8 g), which is very similar to the extent of mechanical allodynia determined with the same technique. In the TRPV1 receptor gene-deficient (TRPV1 / ) group, this secondary hyperalgesia was almost absent at 3 and 4 h. Although some reduction was detected at later time points, the difference did not prove to be statistically significant compared to WT mice (Fig. 2A). Similar to that observed in rats, WT mice demonstrated reduced ipsilateral hindlimb weight bearing in response to intraarticular injection of SLIGRL-NH2 (Fig. 2B). TRPV1 / mice also showed a decrease in ipsilateral weight bearing during the first 4 h; however, hindlimb incapacitance subsequently returned to WT levels at 5 and 6 h post-injection of the PAR-2 agonist. Injection of the same volume of LRGILS-NH2 into the knee joint evoked no change in either mechanonociception or static weight bearing (Fig. 2A and B). In agreement with the rat experiments, PAR-2 receptor activation did not elevate joint TNF-a concentration, which was at the detection limit of the ELISA assay both in LRGILS-NH2 and SLIGRL-NH2-injected knees (data not shown). Intraarticular
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Change of touch sensitivity (%)
A
12
SLIGRL-NH2 SB366791 +SLIGRL-NH2
8
LRGILS-NH2 4 0
##
#
#
-4 -8
#
#
* **
*
*
-12
*
*
-16 -20 control
1
2
3
4
5
6
hours after i.a. administration
Change of mechanonociceptive threshold (%)
B
SLIGRL-NH2 SB366791+SLIGRL-NH2
10
LRGILS-NH2
5 0 -5 -10
### ###
-15
##
###
-20
*
-25
*
** ***
-30
#
-35
***
***
3
4
-40 -45 -50 1
2
5
6
hours after i.a. administration
Ipsilateral weight distribution (%)
C
SLIGRL-NH2
60
SB366791 +SLIGRL-NH2
58
LRGILS-NH2
56 54 52 #
50 48
**
*
46 44
#
#
*
*
*
42 40 control
1
2
3
4
5
6
hours after i.a. administration Fig. 1. Alterations of the (A) touch sensitivity thresholds and (B) mechanonociceptive thresholds of the paw as well as (C) spontaneous ipsilateral weight distribution in response to injecting the PAR-2 activating peptide SLIGRL-NH2 (100 lg in 100 ll) into the unilateral knee joint of rats. The inactive peptide, LRGILS-NH2, was injected the same way in control animals. In a separate groups rats pre-treatment with the TRPV1 receptor antagonist SB366791 (500 lg/kg i.p.) was performed 15 min before the intraarticular injections and before each measurement. Each data point represents the mean ± s.e.m. of n = 9–11 animals per group. *P < 0.05, **P < 0.01, ***P < 0.001 (SLIGRL-NH vs. LRGILS-NH ) and #P < 0.05, ##P < 0.01, ###P < 0.001 2 2 (SLIGRL-NH2 vs. SB366791 + SLIGRL-NH2) analysed by one-way ANOVA followed by Bonferroni’s post test.
administration of SLIGRL-NH2 caused a significant increase in joint IL-1b levels in both WT and TRPV1 / mice, there was no difference
Fig. 2. Alterations of the (A) mechanonociceptive thresholds of the paw, (B) ipsilateral weight distribution and (C) IL-1b concentration measured in the joint homogenates in response to injecting the PAR-2 activating peptide SLIGRL-NH2 (100 lg in 50 ll) into the unilateral knee joint of C57Bl/6 wildtype (WT) and TRPV1 receptor gene-deleted TRPV1 / mice. The inactive peptide, LRGILS-NH2, was injected the same way in control animals. Each data point represents the mean ± s.e.m. of n = 9–11 animals per group. *P < 0.05, **P < 0.01, (SLIGRL-NH2 vs. LRGILS-NH2 in WT mice), #P < 0.05, ##P < 0.01 (SLIGRL-NH2-treated WT vs. TRPV1 / ), and +P < 0.05, ++P < 0.01 (SLIGRL-NH2 vs. LRGILS-NH2 in TRPV1 / mice) determined with one-way ANOVA followed by Bonferroni’s post test.
between these groups. LRGILS-NH2 had no effect on articular IL-1b release (Fig. 2C). 3.4. No effect of intraarticular SLIGRL-NH2 on knee diameter The control antero-posterior diameter of the mouse knee was 4.4 ± 0.1 mm, whereas the medio-lateral one was 4.3 ± 0.1 mm.
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Primary hyperalgesia induced by intraplantar injection of SLIGRL-NH2 (100 lg in 50 ll) was around 15–25% in WT mice throughout the 6-h period, it decreased from 8.7 ± 1.2 g to a maximum of 6.5 ± 0.5 g. Although it was smaller in the TRPV1 / group at certain time points, the two hyperalgesia curves referring to the total duration of the experiment did not prove to be statistically significant (Fig. 3A). Conversely, SLIGRL-NH2-evoked paw oedema was markedly smaller in TRPV1 / mice than in wildtypes throughout the whole 6 h-period. In WT animals, paw volume increased by about 22–26% (from 0.19 ± 0.05 cm3 to 0.24 ± 0.02 cm3) and in the animals by 10% (from 0.20 ± 0.03 cm3 to 0.22 ± TRPV1 / 0.01 cm3; Fig. 3B). Impaired weight distribution in response to the injection of the PAR-2-activating peptide into the paw was comparable between the TRPV1 / and wildtype groups without any statistically significant differences (Fig. 3C). Intraplantar administration of the inactive peptide, LRGILS-NH2 did not induce any changes in the mechanonociceptive thresholds, paw volumes or the spontaneous weight distribution in either group (Fig. 3A–C).
Change of mechanonociceptive threshold (%)
3.5. Involvement of the TRPV1 receptor in intraplantar SLIGRL-NH2induced pain behaviour and tissue oedema in mice
hours after i.pl. administration
A 1
2
3
4
5
6
10 5 0 -5
*
-10 +
-15
**
**
+
*
-20
**
++
-25 ++ WT (SLIGRL-NH2)
-30
-/-
TRPV1 (SLIGRL-NH2)
-35
WT (LRGILS-NH2) -/-
TRPV1 (LRGILS-NH2)
WT (SLIGRL-NH2)
B 35
-/-
TRPV1 (SLIGRL-NH2)
Oedema formation (%)
One hour following intraarticular SLIGRL-NH2 injection, only 5.1 ± 0.4% and 1.8 ± 0.1% increases were measured from these directions in wildtype mice, which was not considered as a significant swelling. This moderate oedema formation remained unchanged during the 6-h experimental period, the respective increases of these diameters were 4.9 ± 0.3% and 1.3 ± 0.2% at this time point. Similar minimal changes were observed in TRPV1 / mice as well as in response to LRGILS-NH2 injection in both WT and TRPV1 gene-deleted animals. Significant differences could not be determined between any groups.
WT (LRGILS-NH2)
30
-/-
TRPV1 (LRGILS-NH2)
25
*
20
***
***
***
**
***
15
## #
10
###
###
###
5
###
3.6. Inflammatory cytokine concentrations in the mouse paw 0
4. Discussion An appreciation of the precise nociceptive processes associated with inflammatory joint diseases is of great clinical importance, but for which we have a very poor understanding (McDougall, 2006). PAR activation is known to modulate inflammatory processes and there are some data concerning its significance in pain processing (Vergnolle et al., 2001a; Cenac et al., 2007), but the role of PARs in arthritis and related pain has not been clearly and thoroughly elucidated. In one of the few studies to address this issue, it has been shown that PAR-4 activation leads to joint inflammation and pain while local injection of a selective PAR-4 antagonist reduces arthritis severity and acute pain symptoms (McDougall et al., 2009). Whether this holds true for other PARs is currently unknown. The present results provide the first evidence that injection of the selective PAR-2 activating peptide SLIGRL-NH2 into the knee joint results in the development of a secondary mechanical hyperalgesia/allodynia in the paw as well as impaired weight distribution in both rats and mice. These pain-causing effects of PAR-2 activation are accompanied by an increase in IL-1b production. Conversely, the level of the other inflammatory cytokine tested, TNF-a, did not increase throughout the 6 h duration of the experiment. This particular time point was chosen based on the fact that
0
1
2
3
4
5
6
hours after i.pl. administration
C Ipsilateral weight distribution (%)
Similar to the results obtained in the joints, intraplantar injection of SLIGRL-NH2 did not induce an increased TNF-a production in the paw (data not shown). IL-1b, however, significantly increased in response to PAR-2 activation, but it was not altered by genetic deletion of the TRPV1 receptor (Fig. 4).
WT (SLIGRL-NH2) -/-
53
TRPV1 (SLIGRL-NH2)
52
WT (LRGILS-NH2) -/-
TRPV1 (LRGILS-NH2)
51 50 49 48 47 46 45 44 control
1
2
3
4
5
6
hours after i.pl. administration Fig. 3. Alterations of the (A) mechanonociceptive threshold and (B) volume of the paw as well as (C) ipsilateral weight distribution in response to injecting the PAR-2 activating peptide SLIGRL-NH2 (100 lg in 50 ll) intraplantarly in C57Bl/6 wildtype (WT) and TRPV1 receptor gene-deleted TRPV1 / mice. The inactive peptide, LRGILS-NH2, was injected the same way in control animals of both groups. Each data point represents the mean ± s.e.m. of n = 8–11 mice per group. *P < 0.05, **P < 0.01, ***P < 0.001 (SLIGRL-NH vs. LRGILS-NH in WT mice), #P < 0.05, 2 2 ## P < 0.01, ###P < 0.001 (SLIGRL-NH2-treated WT vs. TRPV1 / ), and +P < 0.05, ++ P < 0.01 (SLIGRL-NH2 vs. LRGILS-NH2 in TRPV1 / mice) determined with oneway ANOVA followed by Bonferroni’s post test.
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3200 2800 2400 2000 1600 1200 800 400 0
Fig. 4. IL-1b concentration measured in the paw of C57Bl/6 wildtype (WT) and TRPV1 receptor gene-deleted TRPV1 / mice 6 h after intraplantar injection of the PAR-2 activating peptide SLIGRL-NH2 (100 lg in 50 ll). The inactive peptide, LRGILS-NH2, was injected the same way in control animals of both groups. Each column represents the mean ± s.e.m. of n = 6–8 samples per group. *P < 0.05, **P < 0.01 (SLIGRL-NH vs. LRGILS-NH in WT mice) and +P < 0.05, ++P < 0.01 2 2 (SLIGRL-NH2 vs. LRGILS-NH2 in TRPV1 / mice) determined with Student’s t-test for unpaired comparisons.
the PAR-2 receptor is rapidly desensitized after activation and it is generally considered to mediate acute inflammatory reactions over a short period of time, i.e. up to 6 h. There are several data available concerning the inflammatory and nociceptive actions of PAR-2 activation in the paw (Kawabata et al., 2001), the gastrointestinal tract (Cenac et al., 2002; Kawao et al., 2004) and the airways (Schmidlin et al., 2002; for revs. Vergnolle et al., 2001a; Fiorucci and Distrutti, 2002; Vergnolle, 2005; Cenac and Vergnolle, 2005; Kanke et al., 2005). The effects of articular PAR-2 stimulation was examined by Ferrell et al. who described swelling, hyperaemia and histological changes as well-defined signs of the development of an inflammatory reaction (Ferrell et al., 2003). We on the other hand did not find any change in knee joint diameter in response to a 100 lg dose of the PAR-2 activating peptide, although IL-1b concentration increased confirming the development of a distinctive inflammatory response in this organ. Furthermore, we have shown for the first time that PAR-2 receptor activation in joints leads to pro-nociceptive processes. Our experiments with rats pre-treated with the selective TRPV1 receptor antagonist SB366791 (Varga et al., 2005) as well as studies with TRPV1 receptor gene-deleted mice revealed that the secondary hyperalgesia/allodynia and the decreased spontaneous weight distribution on the affected side are predominantly mediated via TRPV1 receptor activation. While the PAR-2 activation-evoked changes were abolished by the TRPV1 antagonist in rats, these were significantly, but not completely inhibited by the genetic deletion of this ion channel in mice. This difference can be explained by a species difference regarding the importance of the TRPV1-mediated mechanisms in the joints, but compensatory mechanisms counteracting the lack of this receptor are also possible in knockout mice. The PAR-2-activation-induced significant mechanical hyperalgesia observed in TRPV1 gene-deleted mice compared to the inactive control at 2 h disappeared at later time points. The TRPV1 receptor therefore seems to be involved in PAR-2 activation-induced secondary mechanical hyperalgesia 3 h and in spontaneous pain 4 h after the induction of the inflammation. This is supported by the results obtained in rats with the TRPV1 receptor antagonist, although activation of this ion channel is likely to be a little faster in rats than in mice (2 and 3 h in cases of hyperalgesia and weight distribution, respectively).
Activation of PAR-2 receptors on the synovial cells, fibrocytes, inflammatory and immune cells, as well as sensory nerves results in the release of several pro-inflammatory mediators such as bradykinin, leukotriens, prostaglandins, cytokines and sensory neuropeptides (tachykinins, CGRP), which in turn activate/sensitize the TRPV1 channels. Direct sensitization of the TRPV1 receptor on the peripheral nerve terminal through protein kinases as described for thermal hyperalgesia (Amadesi et al., 2006) is not likely to be the primary mechanism for the secondary mechanical hyperalgesia and spontaneous pain. Intraplantar administration of SLIGRL-NH2 in mice produced paw oedema through a TRPV1-mediated mechanism, which is most likely to be the result of SP and CGRP release from the activated sensory nerve terminals as described by several previous investigations (Vergnolle et al., 2001b; Cottrell et al., 2003). In contrast, this ion channel was not involved in the development of PAR2-evoked primary hyperalgesia or impaired weight distribution. PAR-2 is known to cause hyperalgesia to thermal stimuli at the site of activation (Hoogerwerf et al., 2001; Vergnolle et al., 2001b) and the involvement of TRPV1 receptors on the peripheral nerve endings has been shown in this action (McLean et al., 2002). However, the ability of PAR-2 activation to induce mechanical hyperalgesia and allodynia is controversial. It has been suggested by two previous studies performed in TRPV1 receptor gene-deleted mice that thermal as well as mechanical hyperalgesia evoked by SLIGRLNH2 injection into the paw was absent or significantly smaller in the TRPV1 knockout group (Amadesi et al., 2004; Dai et al., 2004). In this latter study, mechanical hyperalgesia was moderate, but significantly smaller in TRPV1 / mice, which is in agreement with our findings obtained 2 h after intraplantar injection. However, at later time points this difference disappeared and the two primary hyperalgesia curves were not significantly different (Dai et al., 2004). In case of intraplantar administration of the PAR-2 receptor agonist the inflammation develops in the paw and since the nociceptive threshold is also measured there it is considered to be primary mechanical hyperalgesia. When the PAR-2 receptors are activated in the joints and the mechanonociceptive threshold is measured on the plantar surface of the paw, it is called secondary mechanical hyperalgesia/allodynia. Primary and secondary hyperalgesia develop by fundamentally different mechanisms. While primary hyperalgesia, especially to thermal stimuli, is predominantly mediated by peripheral sensitization of primary nociceptive nerve terminals, secondary hyperalgesia is attributed to altered processing in the central nervous system (Treede and Magerl, 2000). Altered neuronal response properties consistent with the characteristic of secondary hyperalgesia have been observed in the spinal cord, the thalamus, and the somatosensory cortex, but not in the peripheral endings of primary nociceptive afferents, which suggest that basic mechanisms of secondary hyperalgesia may consist of modulation of neural transmission in the spinal cord and in higher neural centres. Several mechanisms are triggered in the dorsal horn by noxious stimuli which lead to enhanced synaptic activation (central sensitization). However, intraarticular PAR-2 activation induced a weaker secondary hyperalgesia than the primary response after direct intraplantar administration of the agonist. There might be more constitutive endopeptidase activity in the synovial fluid which limits the PAR-2 induced responses. Besides the localization of the PAR-2 receptors on sensory nerve endings, in the joints they have also been described on other specific cell types such as synovial cells, immune- and inflammatory cells including mast cells. Pro-inflammatory mediators which are released from these specific cells in the joints might be involved in activation/ marked sensitization of the TRPV1 receptors (Amadesi et al., 2006; Kelso et al., 2006; Palmer et al., 2007).
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Although PAR-2 activation cannot generate action potentials itself, it has the ability to functionally interact with other receptors and ion channels (e.g. TRPV4; Grant et al., 2007) to induce depolarization due to Na+ influx as well as increased c-Fos expression and sensory neuropeptide release (e.g. SP and CGRP) in the spinal cord through increased intracellular Ca2+ concentration (Seeliger et al., 2003; Dai et al., 2004; Cenac and Vergnolle, 2005). These mechanisms result in an enhanced transmission of mechanonociceptive signals and the development of a secondary hyperalgesia/allodynia. Although peripheral sensitization of the TRPV1 receptor through protein kinase C-, protein kinase A- or phospholipase Cdependent processes has been described both in vitro (Dai et al., 2004) and in vivo (Amadesi et al., 2004) as an explanation of primary thermal hyperalgesia, these mechanisms are not likely to be involved in mechanical hyperalgesia and allodynia. Based on our results, it can be inferred that articular activation of PAR-2 receptors leads to joint pain and secondary allodynia. The activation of joint sensory nerve terminals themselves by the PAR-2 agonist is not TRPV1 receptor-mediated; however, TRPV1 does appear to be involved in PAR-2-mediated nociceptive processing involving higher pain centres. Acknowledgements The authors thank Mrs. Dóra Ömböli for her expert technical assistance in the experiments. This work was sponsored by Hungarian Grants: OTKA K73044, NK78059. Zs. Helyes is supported by János Bolyai Postdoctoral Research Fellowship. J.J. McDougall is an Alberta Heritage Foundation for Medical Research Senior Scholar and an Arthritis Society Investigator. References Amadesi S, Nie J, Vergnolle N, Cottrell GS, Grady EF, Trevisani M, et al. Proteaseactivated receptor 2 sensitizes the capsaicin receptor transient receptor potential vanilloid receptor 1 to induce hyperalgesia. J Neurosci 2004;24:4300–12. Amadesi S, Cottrell GS, Divino L, Chapman K, Grady EF, Bautista F, et al. Proteaseactivated receptor 2 sensitizes TRPV1 by protein kinase Ce- and A-dependent mechanisms in rats and mice. J Physiol 2006;575:555–71. Boileau C, Amiable N, Martel-Pelletier J, Fahmi H, Duval N, Pelletier J-P. Activation of protease-activated receptor 2 in human osteoarthritic cartilage upregulates catabolic and pro-inflammatory pathways capable of inducing cartilage degeneration: a basic science study. Arthritis Res Ther 2007;9:R121–30. } G, Elekes K, et al. Investigation of the Bölcskei K, Helyes Zs, Szabó Á, Sándor K, Petho role of TRPV1 receptors in acute and chronic nociceptive processes using genedeficient mice. Pain 2005;117:368–76. Busso N, Frasnelli M, Feifel R, Cenni B, Steinhoff M, Hamilton J, et al. Evaluation of protease-activated receptor 2 in murine models of arthritis. Arthritis Rheum 2007;56:101–7. Cenac N, Vergnolle N. Proteases and protease-activated receptors (PARs): novel signals for pain. Curr Top Med Chem 2005;5:569–76. Cenac N, Coelho AM, Nguyen C, Compton S, Andrade-Gordon P, McNaughton WK, et al. Induction of intestinal inflammation in mouse by activation of proteaseactivated receptor-2. Am J Pathol 2002;161:1903–15. Cenac N, Andrews CN, Holzhausen M, Chapman K, Cottrell G, Andrade-Gordon P, et al. Role of protease activity in visceral pain in irritable bowel syndrome. J Clin Invest 2007;117:636–47. Cocks TM, Fong B, Chow JM, Anderson GP, Frauman AG, Goldie RG, et al. A protective role for protease-activated receptors in the airways. Nature 1999;198:156–60. Coelho A-M, Ossovskaya V, Bunnett NW. Protease-activated receptor-2: physiological and pathophysiological roles. Curr Med Chem Cardiovasc Hematol Agents 2003;1:61–72. Costa R, Marotta DM, Manjavachi MN, Fernandes ES, Lima-Garcia JF, Paszcuk AF, et al. Evidence for the role of neurogenic inflammation components in trypsinelicited scratching behaviour in mice. Br J Pharmacol 2008;154:1094–103. Cottrell GS, Amadesi S, Schmidlin F, Bunnett NW. Protease-activated receptor 2: activation, signalling and function. Biochem Soc Trans 2003;31:1191–7. Dai Y, Moriyama T, Higashi T, Togashi K, Kobayashi K, Yamanaka H, et al. Proteaseactivated receptor-2-mediated potentiation of transient receptor potential vanilloid subfamily 1 activity reveals a mechanism for protease-induced inflammatory pain. J Neurosci 2004;24:4293–9. D’Andrea MR, Derian CK, Leturcq D, Baker SM, Brunmark A, Ling P, et al. Characterization of protease-activated receptor-2 immunoreactivity in normal human tissues. J Histochem Cytochem 1998;46:157–64.
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