Expression of mRNA for four subtypes of the proteinase-activated receptor in rat dorsal root ganglia

Brain Research 1041 (2005) 205 – 211 www.elsevier.com/locate/brainres

Research report

Expression of mRNA for four subtypes of the proteinase-activated receptor in rat dorsal root ganglia Wan-Jun Zhu, Hiroki Yamanaka, Koichi Obata, Yi Dai, Kimiko Kobayashi, Toyoko Kozai, Atsushi Tokunaga, Koichi NoguchiT Department of Anatomy and Neuroscience, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan Accepted 4 February 2005 Available online 10 March 2005

Abstract Proteinase-activated receptors (PARs) are members of the superfamily of G-protein coupled receptors that initiate intracellular signaling by the proteolytic activity of extracellular serine proteases. Three member of this family (PAR-1, PAR-3, and PAR-4) are considered thrombin receptors, whereas PAR-2 is activated by trypsin and tryptase. Recently, activation of PAR-2 signal was identified as a pro-inflammatory factor that mediates peripheral sensitization of nociceptors. Activation of PAR-1 in the periphery is also considered to be a neurogenic mediator of inflammation that is involved in peptide release. Here, we investigated the expression of these four members of PARs in the adult rat dorsal root ganglia (DRG) using radioisotope-labeled in situ hybridization histochemistry. We detected mRNA for all subtypes of PARs in the DRG. Histological analysis revealed the specific expression patterns of the PARs. PAR-1, PAR-2, and PAR-3 mRNA was expressed in 29.0 F 4.0%, 16.0 F 3.2%, and 40.9 F 1.3% of DRG neurons, respectively. In contrast, PAR-4 mRNA was mainly observed in non-neuronal cells. A double-labeling study of PARs with NF-200 and alpha calcitonin gene-related peptide (CGRP) also revealed the distinctive expression of PARs mRNA in myelinated or nociceptive neurons. This study shows the precise expression pattern of PARs mRNA in the DRG and indicates that the cells in DRG can receive modulation with different types of proteinase-activated receptors. D 2005 Elsevier B.V. All rights reserved. Theme: Sensory systems Topic: Pain modulation: anatomy and physiology Keywords: Rat; Tryptase; Thrombin; In situ hybridization; DRG neuron

1. Introduction Proteinase-activated receptors (PARs) are members of the large family of seven transmembrane domain cell-surface receptors that mediate cellular signaling through heterotrimeric G proteins. Four different subtypes of PARs, PAR-1, PAR-2, PAR-3 and PAR-4, have been cloned. The pivotal step of activation of PARs by proteases is cleavage of the extracellular N-terminus of the receptors, unmasking a new N-terminus that acts as a tethered ligand and that binds intramolecularly to the receptor to induce signal trans* Corresponding author. Fax: +81 798 45 6417. E-mail address: [email protected] (K. Noguchi). 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.02.018

duction. According to their amino acid sequence, each subtype is specifically cleaved at a distinct site near its extracellular N-terminus leading to receptor activation. PAR-2 is activated by trypsin and mast cell tryptase, while PAR-1, PAR-3, and PAR-4 are considered to be thrombin receptors [4,17,21]. Extracellular serine proteases, which were originally identified outside nervous system, are also expressed in brain such as thrombin [8] and trypsin-like serine protease [10]. This indicates that extracellular protease activity is of physiological importance within the nervous system. For example, thrombin is known to be involved in the morphological alternation of neuronal and glia cells [2,3,11] and in regulating neuroprotective functions and degenerative mechanisms in a concentration

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dependent manner [20]. At least some of these effects are mediated by proteolytic activation of PARs. Recently, involvement of PAR-1 and PAR-2 in nociceptive signaling in the peripheral nervous system was reported [7,13,14,18]. Activation of PAR-2 signaling plays a role in neuronal sensitization and contributes to the pathogenesis of pain in inflammatory conditions mediating peptide release or modulation of the transient receptor potential vanilloid subfamily 1 (TRPV1) receptor [5,13]. It has not yet been shown whether PAR-1 activity in the peripheral nervous system is involved in nociceptive signal transduction or inhibition [1,7]. The physiological roles of PAR-3 and PAR4 in the nervous system are not known. Expression of PAR1 and PAR-2 mRNA in the dorsal root ganglia (DRG) was reported [7,13,18] and the locations of these four PARs proteins were reported in brain, cultured astrocytes, and bladder using immunohistochemistry [6,20,22]. However, the precise expression pattern of PARs for mRNA in the DRG has not been clarified. The purpose of this study was to clarify the expression of PARs mRNA in DRG to assess whether PARs are expressed in nociceptive neurons.

2. Materials and methods 2.1. Animal treatment and sample preparation Male Sprague–Dawley rats weighting 200–300 g were used as subjects. For immunohistochemistry and in situ hybridization (ISH), rats were deeply anesthetized with sodium pentobarbital (70–80 mg/kg body weight, i.p.) and perfused transcardially with 100 ml of 1% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, followed by 500 ml of 4% paraformaldehyde in 0.1 M PB. The L5 was dissected out and post-fixed in the same fixative for 4 h at 4 8C, followed by immersion in 20% sucrose in 0.1 M PB at 4 8C overnight. The tissue was frozen in powdered dry ice, cut on a cryostat at 10 Am thickness. Every effort was made to minimize animal suffering and reduce the number of animals used. All animal experimental procedures were approved by the Hyogo College of Medicine Committee on Animal Research and were carried out in accordance with the guidelines of the National Institutes of Health on animal care. 2.2. In situ hybridization To generate the template cDNA for ISH, we performed a reverse transcription-polymerase chain reaction (RT-PCR). Following ether anesthesia, L5 DRGs were removed and rapidly frozen with powdered dry ice and stored at 80 8C until use. Extraction of total RNA was done by a single step extraction method using ISOGEN (Nippon Gene, Tokyo, Japan) that was described in a previous paper [9]. PCR primers for PAR-1, PAR-2, PAR-3, PAR-4, and alpha calcitonin gene-related peptide (CGRP) cDNA were

designed corresponding to the coding regions of the genes. The primers sequences are shown in the list below. The amplified cDNA was cloned into p-GEM T-easy (Promega, MI, USA) and sequenced. These clones were used to generate the cRNA probes for ISH. Using the enzymedigested clones that contain partial cDNA of PAR-1, PAR-2, PAR-3, PAR-4 and CGRP, alpha 35S UTP- or digoxygenin (DIG)-labeled anti-sense and sense cRNA probes were synthesized using T7 and Sp6 RNA polymerase. Sections on slides were fixed in 4% formaldehyde in 0.1 M PB for 20 min. After washing with PB, the sections were treated with 10 Ag/ml protease K in 50 mM Tris–5 mM EDTA (pH 8.0) for 3 min at room temperature, post-fixed in the same fixative, acetylated with acetic anhydride in 0.1 M triethanolamine, rinsed with PB, and dehydrated through an ascending alcohol series. The 35S-labeled cRNA probes (anti-sense or sense) in hybridization buffer (50% deionized formamide, 0.3 M NaCl, 20 mM Tris–HCl, 5 mM EDTA, 10% dextran sulfate, 1 Denhardt’s solution, 0.2% sarcosyl, 250 Ag/ml yeast tRNA, 400 Ag/ml salmon testis DNA, and 20 mM dithiothreitol (DTT; pH 8.0)) were placed on the section and then incubated at 55 8C overnight. Then, sections were washed at 65 8C in 50% formamide, 2 SSC, and 5 mM DTT for 30 min. Sections were then treated with 1 Ag/ml RNase A in RNase buffer (0.5 M NaCl, 10 mM Tris–HCl, and 1 mM EDTA (pH 7.5)) for 30 min at 37 8C. Subsequently, sections were incubated at 65 8C in 50% formamide, 2 SSC, and 5 mM DTT for 30 min and then rinsed with 2 SSC and 0.1 SSC for 10 min at room temperature, dehydrated through an ascending alcohol series, and air-dried. Slides after the hybridization reaction were coated with NBT-3 emulsion (Kodak, Rochester, NY) and exposed for 5 weeks. Once developed in D-19 (Kodak, USA), the sections were stained with hematoxylin–eosin, dehydrated in graded ethanol series, cleared in xylene, and coverslipped. To confirm the specificity of ISH, sense probes for each of PARs mRNA were examined. Silver grain density in the sections with sense probe was very close to the background level of anti-sense probes. For the co-expression study, we used dual ISHH using DIG-labeled probes and 35S-labeled probes in the same sections. DIG-labeled RNA anti-sense probe for CGRP was prepared by in vitro transcription using the DIG RNA labeling kit (SP6/T7) (Roche). An 35S-labeled probe (2  106 cpm/ml per slide) and a DIG-labeled probe (1.0 mg/ml per slide) in hybridization buffer were placed on the cryostat sections. After incubation overnight at 55 8C, these sections were rinsed in the same condition with the single labeling ISH described above. These sections were rinsed in TBS (Tris buffered saline; 0.1 M Tris–HCl pH7.5, 150 mM NaCl) for 3 min followed by incubation in 30% methanol, 1% H2O2 in TBS for 20 min in order to inactivate the endogenous peroxidase. Then, the sections were rinsed 2 times each for 3 min in TBS and in TBS-T (0.1 M Tris–HCl pH7.5, 300 mM NaCl, 0.1% Tween-20) for 3 min. After blocking for 1 h in blocking buffer (TBS containing 1%

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blocking reagent, Roche), anti-DIG peroxydase–Fab fragments (Roche) were applied for 2 h at a concentration of 1.5 U/ml in blocking buffer. Sections were rinsed in TBS-T 4 times each for 5 min, visualized with diaminobenzidine (DAB) using a Gen Point System kit (DAKO, Osaka, Japan) in accordance with the instructions. Next, these slides were coated with NBT-3 emulsion (Eastman Kodak) and exposed for 5 weeks. 2.3. List of PCR primers for PAR-1, PAR-2, PAR-3, and PAR-4 PAR-1 (Accession number: X81642): sense; 5V-CCGTGQ CTCCCCTTCAAGATC-3V, anti-sense; 5V-GGCGGAGAAGGCGGAGAAAT-3V. PAR-2 (Accession number: u61373): sense; 5V-GGACGAGCACTCGGAGAAGA-3V, anti-sense; 5V-GAGCTGGAGGAGTAAGAGCTGG-3V. PAR-3 (Accession number: AF310076): sense; 5V-GCGA A C AT C G T G A C C C T G T G - 3 V, a n t i - s e n s e ; 5 VCCTGCTTGAGGATGGCCAAC-3V. PAR-4 (Accession number: AF310216): sense; 5V-GCACTGGCAGCC AATGGTCA-3V, anti-sense; 5V-GCCCGT ACCTTCTCCCTGAA-3V. CGRP (Accession number: M11597): sense; 5V-CTTGCTCCTGTACCAGGCAT-3V, anti-sense; GGCGGCGGCCGAAGGCTT-3V.

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olds of gray level density were set such that only silver grains were accurately discriminated from the background in the outlined cell or tissue profile and read by the computer pixel-by-pixel. Subsequently, the area of discriminated pixels was measured and divided by the area of the outlined profile, giving a grain density for each cell or tissue profile. To reduce the risk of biased sampling of the data because of varying emulsion thickness, we used a signal-tonoise (S/N) ratio for each cell in each tissue. The S/N ratio of an individual neuron and its cross-sectioned area, which was computed from the outlined profile, was plotted. Based on this scatter diagram, neurons with a grain density of tenfold the background level or higher (S/N ratio N 10) were considered positively labeled for PAR-1, PAR-2, PAR-3, and PAR-4 mRNA. To distinguish cell size-specific changes, we characterized the DRG neurons as small (b600 Am2)-, medium (600– 1200 Am2)-, and large (N1200 Am2)-size neurons, according to their cross-sectional area. Because a stereological approach was not used in this study, quantification of the data may represent a biased estimate of the actual numbers of neurons. At least 400 neurons from the L5 DRG of each rat were measured. The number of positively labeled DRG neurons was divided by the number of neuronal profiles counted in each DRG. For immunohistochemistry, only the signals that were clearly discriminative immunoreactive (ir) profiles were considered as the positive expressions.

2.4. Immunohistochemistry 3. Results The sections were pre-incubated in PBS containing 10% normal horse serum (NHS) and 0.3% Triton X-100 for 1 h then incubated in primary antibody in the same solution for 24 h at 4 8C. Mouse anti-NF200 (Clone N-52) monoclonal antiserum (1:10,000, Sigma, St. Louis, MO) and antineuronal nuclei (NeuN) monoclonal antibody (1:1000, Chemicon, CA, USA) were used. The sections were washed in PBS and then incubated in biotinylated anti-mouse IgG (1:400; Vector Laboratories, Burlingame, CA, USA) in PBS containing 5% NHS for 2 h at 4 8C followed by incubation in avidin–biotin–peroxidase complex (Elite ABC kit; Vector, CA, USA) for 1 h at room temperature. The horseradish peroxidase reaction was developed in 0.1 M Tris-buffered saline, pH 7.4, containing 0.05% 3,3-diaminobenzidine tetrahydrochloride (Sigma, MO, USA), 0.3% nickel sulfate, and 0.01% hydrogen peroxidase. Sections were then washed in PBS and used for ISH. 2.5. Quantitative analysis Measurements of the density of silver grains over randomly selected tissue profiles were performed using a computerized image analysis system (NIH Image, version 1.61), where only neuronal profiles that contained nuclei were used for quantification. At a magnification of 200 and with bright-field illumination, upper and lower thresh-

3.1. Expression of PARs in DRG To examine which sensory neurons had a modification of the extracellular protease, we performed ISH using adult rat DRG. First, we examined PAR-1 mRNA expression in the DRG. Both dense and weak hybridization signals of PAR-1 mRNA were observed in DRG neurons (Figs. 1A, B). Scatter plot analysis showed a distribution of signal intensity of PAR-1 mRNA in DRG neurons (Fig. 1C). Based on the signal/noise ratio analysis, PAR-1 mRNA expressing neurons (S/N N 10) were 29.0 F 4.0% of DRG neurons (n = 4, 2117 neurons.). In PAR-1 mRNA expressing neurons, 43.3 F 1.1% were small neurons (b600 Am2), 34.6 F 2.4% were medium (600–1200 Am2), and 21.8 F 3.0% were large neurons (N1200 Am2). Next, we demonstrated the expression of PAR-2 mRNA (Figs. 1D, F). In the naive DRG, PAR-2 mRNA expressing neurons (S/N N 10) were observed in 16.0 F 3.2% of DRG neurons (n = 4; 1884 neurons). PAR-2 mRNA was highly expressed in small DRG neurons and was visualized as an aggregation of silver grains in the cytoplasmic region. PAR2 positive neurons were mainly seen in small neurons using a scatter plot analysis (Figs. 1F, G). The quantification of the size–frequency data confirmed the specific expression pattern of PAR-2 in the DRG. We found that 62.5 F

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Fig. 1. Expression of PARs mRNA in adult rat DRG. (A, D, H, K) Low magnified dark-field images of the in situ hybridization (ISH) show PAR-1 (A), -2 (D), -3 (H), and -4 (K) mRNA expression, respectively. (B, F, I) Higher magnified bright-field images of the ISH show PAR-1 (B), -2 (F), and -3 (I) mRNA expression, respectively, in the control DRG neurons. (C, G, J, M) Scatter plot diagrams of PAR-1, -2, -3, and -4 mRNA expression, respectively, in the L4 and L5 DRG were made from sections obtained from 4 rats from each mRNA. Individual cell profiles are plotted according to their cross-sectional area (along the x axis) and signal/noise (S/N) ratio (along the y axis). The dashed line indicates the borderline between the negatively and positively labeled neurons (S/N ratio = 10). (L) Bright-field image of double labeling using a combination of ISH and immunohistochemistry shows PAR-4 mRNA and NeuN ir on the L5 DRG. Neurons with asterisks on the cell body indicate labeled neurons for NeuN ir. Arrowheads indicate PAR-4 mRNA expression on non-neuronal cells. Scale bars: 250 Am (A, D, H, K), 50 Am (B, F, I), 25 Am (L).

2.3% of PAR-2 mRNA labeled neurons were small and 33.8 F 1.7% neurons were medium in size. In large neurons, only a small proportion of PAR-2 mRNA expression was observed (3.4 F 0.9%). Expression of PAR-3 mRNA was also confirmed. In Figs. 1H and I, we present the hybridization signals of PAR3 mRNA in DRG neurons. Based on the scatter plot analysis (Fig. 1J), PAR-3 mRNA expressing neurons (S/N N 10) were distributed in 40.9 F 1.3% of DRG neurons (n = 4; 2246 neurons). Size–frequency analysis of the PAR-3 mRNA expressing neurons demonstrated that PAR-3 mRNA was distributed through all size neurons in the

DRG and showed no preferential expression based on the size of neurons. PAR-4 mRNA expression was also examined in the naive DRG. Under lower dark-field magnification, signals for PAR-4 mRNA were detected throughout the DRG (Fig. 1K). Higher bright-field magnification revealed that the PAR-4 mRNA labeling profiles were non-neuronal cells located around DRG neurons. To confirm the expression in non-neuronal cells, we performed double labeling ISH combined with the immunohistochemistry for NeuN that was thought as the neuronal marker. As shown in Fig. 1L, accumulation of silver grains was observed outside of the

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NeuN ir cell. Thus, most of the PAR-4 labeled profiles was likely non-neuronal cells, such as satellite cells. Scatter plot analysis performed on a total of 1876 neurons (n = 4) showed that there were no PAR-4 expressing neurons (S/N N 10) in the DRG. 3.2. Characterization of PAR labeled neuron In order to compare the expression of PAR mRNA between unmyelinated C-fiber neurons and myelinated Afiber neurons, we combined ISH for each PAR mRNA with immunohistochemistry for a 200 kDa neurofilament (NF200), which is a widely used monoclonal antibody that is considered to be a marker of myelinated A-fiber neurons (Fig. 2). NF200-ir neurons were 50.1 F 2.7% (n = 4, total 4187 neurons) of the total DRG neurons, and this value is consistent with that found in a previous report using the same antibody [15]. In PAR-1 mRNA expressing neurons, 60.2 F 2.0% were NF200 positive (n = 4, total 1289 neurons), suggesting that the PAR-1 mRNA was preferentially expressed by A-fiber neurons. As was expected from

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the size distribution, only 7.8 F 2.0% of the PAR-2 mRNA-expressing neurons were NF200 positive (n = 4; total 1517 neurons), suggesting that PAR-2 mRNA was preferentially expressed by C-fiber neurons. Co-localization of PAR-3 and NF200 was examined. About 50% (50.1 F 4.5%) of PAR-3 mRNA expressing neuron were colocalized with NF200 (n = 4; total 1381 neurons). Next, we examined the expression of mRNA for PARs in nociceptive DRG neurons using double labeling analysis of PARs mRNA and CGRP mRNA. CGRP is known to play a role in nociceptive transmission or peripheral neurogenic inflammation. In Fig. 3, ISH with non-RI labeled probe showed a typical expression of CGRP mRNA in DRG (46.7 F 1.9%), which is mainly observed in small to medium sized neurons (n = 4, total 6234 neurons). Coexpression of PAR-1 and CGRP mRNA profiles was shown in Figs. 3A–C. 77.3 F 3.8% of PAR-1 mRNA expressing neuron were double-labeled with CGRP mRNA (n = 4, total 2057 neurons). In CGRP mRNA labeled profiles, 51.1 F 3.8% were co-localized with PAR-1 mRNA. Co-localization of PAR-2 mRNA and CGRP mRNA was examined (Figs. 3D–F). In PAR-2 mRNA labeled neurons, many neurons (77.4 F 4.3%) expressed CGRP mRNA and 27.8 F 2.6% of CGRP mRNA labeled neurons showed co-localization with PAR-2 mRNA (n = 4, 2012 neurons). In PAR-3 mRNA labeled neurons, 84.1 F 4.3% neurons were CGRP mRNA positive, and 73.0 F 4.0% of CGRP mRNA expressing neurons were also labeled for PAR-3 mRNA (Figs. 3G–I).

4. Discussion

Fig. 2. Bright-field images of double labeling for PAR-1 (A), PAR-2 (B), and PAR-3 (C) mRNA and NF200 ir using a combination of ISH and immunohistochemistry on the L5 DRG. Arrows indicate co-localization of mRNA for PARs with NF200 ir. The sections were stained with hematoxylin. Scale bar: 25 Am.

In this study, we demonstrated the detailed expression of four distinct PARs, PAR-1, PAR-2, PAR-3, and PAR-4, in the adult rat DRG. The expression of diverse PAR subtypes in the DRG is important because only limited studies have reported the detection of PAR-1 and PAR-2 in the DRG. Particularly, de Garavilla et al. studied PAR-1 expression in rat DRG [7]. This study described a large population of DRG neurons that expressed PAR-1. In the present study, we precisely quantified PAR-1 mRNA in the adult rat DRG based on a signal/noise ratio analysis. We found that about 29% of DRG neurons were PAR-1 mRNA positive (S/N N 10). PAR-1 labeling profiles seemed to be expressed in more DRG neurons [7] than we have detected in this study. This discrepancy may have been because of variations in the sensitivity of the probes for ISH or the criterion of the quantification. Two recent studies on the activation of peripheral PAR-1 have shown the opposite results [1,7]. de Garavilla et al. reported that the activation of PAR-1 played pro-nociceptive roles, such as neurogenic inflammation in vivo or calcium mobilization in vitro [7]. The result of double labeling analysis with CGRP mRNA and PAR-1 showed that about 77% of PAR-1 mRNA expressing neurons are CGRP positive. This result suggests the pronociceptive role of PAR-1. In contrast, Asfaha et al. showed

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Fig. 3. Co-localization of PARs mRNA with CGRP mRNA in DRG neurons. Expression of mRNA of PAR-1 (A), -2 (D), and -3 (G) was shown in the darkfield images. DIG labeled probes for CGRP were visualized by immunohistochemistry for anti-DIG bright-field images (B, E, H). The dark-field and brightfield images were merged (C, F, I). Arrowheads indicate co-localization of mRNA for PARs and CGRP. The sections were stained with hematoxylin. Scale bar: 50 Am.

that activation of peripheral PAR-1 by the low dose administration of PAR-1 agonist peptide attenuated the nociceptive response to noxious stimuli [1]. In this present study, we showed that the PAR-1 mRNA was distributed not only in small and unmyelinated neurons, but also in large and myelinated neurons. We also observed various intensity of PAR-1 mRNA in each size population in the DRG, suggesting that there may be different expression levels of PAR-1 in peripheral terminals. Particularly, intense PAR-1 mRNA labeling was often seen in CGRP negative neurons. Dose dependent differences of the PAR-1 agonist on pain responses as reported by Asfaha et al. [1] may have been the result of this differential expression of PAR-1 based on size of neuron or expression levels in each neuron. Several studies have recently demonstrated the pronociceptive effect of the peripheral activation of PAR-2 [5,13,18,21]. These studies reported that PAR-2 was expressed in small DRG neurons. In this study, we also observed PAR-2 mRNA in small and unmyelinated neurons. Double labeling study showed many PAR-2 positive neurons co-expressed CGRP. These neurons were thought to include nociceptive neurons. Steinhoff et al., using immunohistochemistry, reported that 63% of DRG neurons expressed PAR-2 [18]. However, we detected PAR-2 mRNA expression in only 16% of DRG neurons (S/N N 10). This discrepancy may be because of the difference of the method (ISH versus immunohistochemistry) or the criterion used for histological quantification. Importantly, both our result and previous report strongly suggest that the PAR-2 can act exclusively in the nociceptive neurons in DRG.

Our study is the first to report the precise detection of PAR-3 mRNA expression by ISH in the DRG of the adult rat. So far, PAR-3 has been identified in platelets from mice and in a variety of human tissues. However, the role of PAR-3 in peripheral neurons is not clearly known. High percentage of co-localization with CGRP and PAR-3 mRNA was intriguing (~84%). It suggests that the activation of PAR-3 can be involved in the nociceptive mechanism in periphery. In platelets, PAR-3 is a cofactor of PAR-4 [16]. Thus, our data showing the lack of PAR-4 mRNA in DRG neurons may indicate that PAR-3 can act independently of PAR-4. An abundant expression of four PAR subtypes has been reported in the central nervous system [19]. In this study, none of the PAR-3 labeled area showed PAR-4 expression. Taken together with the expression of PAR-3 and 4 in the brain and our current observations, we hypothesize that PAR-3 activation in the nervous system must be different from that of platelets, where PAR-3 acts as the key factor in hemostasis and thrombosis. The abundant expression of the PAR subtypes in DRG neurons underscores the involvement of the proteinases in activating these receptors during distinct peripheral events, such as hemorrhage or inflammation and injury. Following peripheral injury, thrombin and tryptase may be expressed in damaged tissues. PARs have different sensitivities to these proteinases [12]. In this study, we showed the expression of the four subtypes of PAR mRNA expression in specific populations of DRG cells. This suggests that the expression pattern of PARs may be an important factor for the proteinase–PAR reaction that modulates neural activity, such as the neuro-

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peptide release or activation of channel proteins like TRPV1. In conclusion, histological diversity of PARs with different sensitivity to agonist proteinases in peripheral neurons may indicate that there is a variety of types of proteinase-mediated modulation in the peripheral nervous system.

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Acknowledgments [13]

This study was supported in part by Grants-in-Aid for Scientific Research and Grant for Open Research Center in Hyogo College of Medicine, from the Japanese Ministry of Education, Science and Culture. We also gratefully acknowledge the technical assistance from Nobumasa Ushio. We thank Dr. D.A. Thomas for correcting the English usage on the manuscript.

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