Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M511072200 on April 4, 2006

J. Biol. Chem., Vol. 281, Issue 25, 17304-17311, June 23, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/25/17304    most recent
M511072200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, C.-K.
Right arrow Articles by Oh, S. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, C.-K.
Right arrow Articles by Oh, S. B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Functional Expression of Thermo-transient Receptor Potential Channels in Dental Primary Afferent Neurons

IMPLICATION FOR TOOTH PAIN*

Chul-Kyu Park{ddagger}, Mi Sun Kim{ddagger}, Zhi Fang{ddagger}, Hai Ying Li{ddagger}, Sung Jun Jung§, Se-Young Choi{ddagger}, Sung Joong Lee{ddagger}, Kyungpyo Park{ddagger}, Joong Soo Kim{ddagger}, and Seog Bae Oh{ddagger}1

From the {ddagger}Department of Physiology and Molecular and Cellular Neuroscience Program, College of Dentistry and Dental Research Institute, Seoul National University, 28-2 Yeongeon-Dong Chongno-Ku, Seoul 110-749 and the §Department of Physiology, College of Medicine, Kangwon National University, Chunchon 200-710, Korea

Received for publication, October 12, 2005 , and in revised form, February 15, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Temperature signaling can be initiated by members of transient receptor potential family (thermo-TRP) channels. Hot and cold substances applied to teeth usually elicit pain sensation. This study investigated the expression of thermo-TRP channels in dental primary afferent neurons of the rat identified by retrograde labeling with a fluorescent dye in maxillary molars. Single cell reverse transcription-PCR and immunohistochemistry revealed expression of TRPV1, TRPM8, and TRPA1 in subsets of such neurons. Capsaicin (a TRPV1 agonist), menthol (a TRPM8 agonist), and icilin (a TRPM8 and TRPA1 agonist) increased intracellular calcium and evoked cationic currents in subsets of neurons, as did the appropriate temperature changes (>43 °, <25 °, and <17 °C, respectively). Some neurons expressed more than one TRP channel and responded to two or three corresponding stimuli (ligands or thermal stimuli). Immunohistochemistry and single cell reverse transcription-PCR following whole cell recordings provided direct evidence for the association between the responsiveness to thermo-TRP ligands and expression of thermo-TRP channels. The results suggest that activation of thermo-TRP channels expressed by dental afferent neurons contributes to tooth pain evoked by temperature stimuli. Accordingly, blockade of thermo-TRP channels will provide a novel therapeutic intervention for the treatment of tooth pain.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies have demonstrated that subfamilies of TRP2 channels play critical roles in the transduction of temperature and pain sensation (1). The temperature-activated TRP channels (thermo-TRP channels) include TRPV1, TRPM8, and TRPA1; they are activated by >43 °, <25 °, and <17 °C, respectively (1). They can also be activated by the exogenous ligands, capsaicin (TRPV1), menthol (TRPM8), and icilin (TRPM8 and TRPA1) (2-5). Given that the thermo-TRPs are involved in converting thermal information into electrical signals within the sensory nervous system (1), it is possible that thermo-TRPs expressed by dental primary afferents play a crucial role for transduction process of tooth pain, especially caused by noxious thermal stimulation.

The different forms of pain are produced by distinctive molecular and cellular mechanisms. It is now thought that elucidation of the main mechanism involved in a certain form of pain is key to the development of pain treatments, which specifically target underlying the cause rather than just symptoms (6). Tooth pain is commonly induced by the presence of hot or cold foods in the oral cavity (7-9). Tooth pain results from the exposure of dentin, which is caused by dissolution of the protective enamel covering the tooth crown by dental caries or gingival recession (7, 8). Since the proposition of Brännström, arguing that the fluid movement in dentinal tubules induced by diverse stimuli, including thermal stimuli, elicits tooth nerve firing to produce tooth pain (referred to as the hydrodynamic hypothesis) (10, 11), much evidence has been reported to support the hypothesis (12). However, it is not fully understood yet how such temperature changes are perceived as painful by the teeth, given that tooth afferents are generally considered to be purely nociceptive. We hypothesized that this is mediated by thermo-TRP channels (1, 13, 14).

In the present experiments we identified dental afferent neurons dissociated from the trigeminal ganglion of rats, by previously applying a fluorescent dye to the exposed dentin of molar teeth. The neurons were classified by size and expression of NaV1.8- and the isolectin B4 (IB4)-binding protein. We found evidence for expression of TRPV1, TRPM8, and TRPA1 in these neurons by measuring mRNA level through single cell reverse transcription (RT)-PCR, by immunohistochemistry, and by measuring inward currents or changes in intracellular calcium in response to the appropriate ligands or temperature changes. The results indicate that TRP channels could be responsible for the transduction of heat and cold stimuli into the sensation of pain.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
All surgical and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee in College of Dentistry, Seoul National University. Sprague-Dawley rats (Harlan, Indianapolis, IN), weighing ~160-180 g at the time of surgery, were housed at a temperature of 23 ± 2 °C with a 12-h light-dark cycle and fed food and water ad libitum. The animals were allowed to habituate to the housing facilities for 1 week before the experiments.

Verification and Classification of Dental Primary Afferent Neurons— Dental primary afferent neurons were identified by retrograde labeling with a fluorescent dye (DiI, D-282, Molecular Probes, Eugene, OR) (15-17). Three weeks after the dye was placed in maxillary molars, trigeminal ganglion neurons were prepared (18) and maintained in a humidified atmosphere of 95% O2/5% CO2 at 37 °C; they were used for calcium imaging and whole cell recording within 6 and 8 h. DiI-labeled neurons were identified by fluorescence and were further classified by treatment immediately after the experiment with IB4 conjugated to fluorescein isothiocyanate (10 µg/ml IB4-fluorescein isothiocyanate, Sigma) (19). IB4-positive neurons had an intense bright ring around the soma membrane (see Fig. 1A, panel f).

Intracellular Calcium Imaging—Neurons were loaded with fura-2 AM (acetoxymethyl, 2 µM; Molecular Probes) for 40 min at 37 °C in a balanced salt solution (in mM: 145 NaCl, 5 KCl, 2 CaCl2,1MgCl2, 10 HEPES, and 10 glucose), followed by rinsing and incubation for 30 min to de-esterify the dye. The cells plated onto poly-L-ornithine-coated coverslips were mounted onto the chamber, which then was placed onto the inverted microscope (Olympus IX70, Japan) and perfused continuously by balanced salt solution at 2 ml/min. All measurements were made at 36 °C (temperature controller PTC-20, ALA Scientific Instrument Inc., Westbury, NY). Cells were illuminated with a 175-watt xenon arc lamp, and excitation wavelengths (340/380 nm) were selected by a Lambda DG-4 monochromator wavelength changer (Shutter Instrument, Novato, CA). Intracellular free calcium concentration ([Ca2+]i) was measured by digital video microfluorometry with an intensified charge-coupled device camera (CasCade, Roper Scientific, Trenton, NJ) coupled to a microscope and software (Metafluor, Universal Imaging Corp., Downington, PA) on a computer with a Pentium 4 microprocessor chip.

Electrophysiology—Whole cell patch clamp recordings were performed to measure currents with Axopatch-1C amplifier (Axon Instruments, Union City, CA). The patch pipettes were pulled from borosilicate capillaries (Chase Scientific Glass Inc., Rockwood, TN). The resistance of pipette was 2-5 megohms, and series resistance was compensated (>80%). Data were low pass-filtered at 2 kHz and sampled at 10 kHz. The pClamp8 (Axon Instruments) software was used during experiments and analysis. Pipette solution was (in mM): 135 CsCl, 5 MgCl2, 10 HEPES, 5 Mg-ATP, 5 EGTA, 10 glucose, adjusted pH to 7.4 with CsOH, and 301 mosM. The bath was continuously perfused with extracellular solution containing (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, adjusted to pH 7.4 with NaOH, and 305 mosM. Experiments were performed at a holding potential of -60 mV at 28 °C.

Immunohistochemistry—Cells were identified after electrophysiological recording by marking the bottom of the cover glass surrounding the cells. To maintain the mark during staining, cover glasses were kept on parafilm during the immunohistochemical procedure (20, 21). Primary antibodies were rat anti-TRPM8 (1:500, a gift from Dr. Makoto Tominaga) and guinea pig anti-TRPV1 (1:500, Chemicon), incubated at 4 °C overnight. The cells were washed three times with phosphate-buffered saline and then incubated with Cy5-conjugated donkey anti-rat, fluorescein isothiocyanate-conjugated donkey anti-guinea pig (Jackson ImmunoResearch, West Grove, PA) at 1:200, respectively, for 1 h at room temperature. After washing with phosphate-buffered saline, the samples were covered with Vectashield mounting media (Vector Laboratories, Inc., Burlingame, CA) and visualized using a confocal microscope with the appropriate filter sets (LSM 5 PASCAL, Carl Zeiss, Germany). The primary antibodies were omitted as negative control (data not shown).

Single Cell RT-PCR—We adopted methods described by Silbert et al. (22). Briefly, following whole cell patch clamp recordings, we changed patch pipettes with a tip diameter of about 30 µm for cell collection. The intracellular content of a single cell was aspirated into a patch pipette under visual control and was gently put into a reaction tube containing reverse transcription agents. Optionally, to avoid genomic DNA contaminations, a DNase I (for 40 min at 37 °C) digest was performed before reverse transcription. After heat inactivation, RT was carried out for 50 min at 50 °C (superscript III, Invitrogen). Subsequently, the cDNA was divided into four or five 2-µl aliquots that were used in separate PCRs. All PCR amplifications were performed with nested primers (Table 1). The first round of PCR was preformed in 50 µl of PCR buffer containing 0.2 mM dNTPs, 0.2 µM "outer" primers, 5 µl of RT product, and 0.2 µlof platinum TaqDNA polymerase (Invitrogen). The protocol included 5 min of initial denaturation at 95 °C, followed by 35 cycles of 40 s of denaturation at 95 °C, 40 s of annealing at 55 °C, and then 40 s of elongation at 72 °C, and was completed with 7 min of final elongation. For the second round of amplification, the reaction buffer (20 µl) contained 0.2 mM dNTPs, 0.2 µM "inner" primers, 5 µl of the products from the first round, and 0.1 µl of platinum TaqDNA polymerase. The reaction was the same as the first round. The PCR products were then displayed on the ethidium bromide-stained 2% agarose gel. Gels were photographed using a digital camera.


View this table:
[in this window]
[in a new window]
  		TABLE 1
DNA primers used for single cell RT-PCR

 
Thermal Stimulation and Drug Application—For calcium imaging experiment, temperature was controlled by PTC-20, a Peltier temperature control system (ALA Scientific Instrument Inc.), in which coverslips were cooled down at a rate of 0.13 °C/s from 36 °C to 10 °C and heated at a rate of 0.1 °C/s from 36 °C to 46 °C. For thermal stimulation for whole cell patch clamp recordings, we applied cold (4 °C) or heated (70 °C) extracellular solution for 1 min. Final temperatures of thermal stimuli, cold and hot, measured by a thermistor placed in the recording chamber, were 12 °C and 45 °C, respectively. Capsaicin, capsazepine, and menthol stock solutions were made in ethanol and stored at -20 °C. Icilin was dissolved in dimethyl sulfoxide (Me2SO) to make stock solution and kept at -20 °C. All drugs were purchased from Sigma. We confirmed that 0.01% Me2SO or ethanol did not affect [Ca2+]i and currents measured in the present study. Drugs were diluted to their final concentration using the extracellular solution and then applied by gravity through a bath perfusion system with a flow rate of 2 ml/min.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Distribution of Dental Primary Afferent Neurons—We considered fluorescent DiI-labeled neurons present in the dissociations of TG to be dental primary afferents (17, 23). DiI-labeled neurons were ~40 to 60 TG neurons per animal (n = 60), and they constituted ~13% of total neurons (Fig. 1A, panels a and b). In differential interference contrast and fluorescent images of the same field (Fig. 1A, panels c and d, respectively), we found DiI-labeled TG neurons with IB4-positive (Fig. 1A, panels e and f). IB4-positive neurons were 57% (229/403) of dental primary afferent neurons and almost exclusively below 40-µm diameter (Fig. 1B), consistent with previous reports (16, 19, 24). In the next experiments, we used IB4-positive dental primary afferent neurons (Fig. 1A, panel f). Interestingly, the proportion of IB4-positive neurons was relatively higher in dental primary afferents, compared with total TG neurons (Table 2).


View this table:
[in this window]
[in a new window]
  		TABLE 2
Numbers of trigeminal ganglion neurons and identified dental primary afferent neurons (Dil-labeled) responding to thermo-TRP ligands determined in single cell calcium imaging experiments The values are the percentages of cells in which calcium transients were observed; numbers in parentheses are numbers of cells tested.

 


Figure 1
View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 1.
Distribution of DiI-labeled dental primary afferent neurons and single cell RT-PCR analysis for thermo-TRP channels. A, DiI-labeled neurons in a slice of trigeminal ganglion (panels a and b), and after dissociation (c and d). IB4 labeling of DiI-labeled neurons (e and f). B,IB4 labels a subset of dental primary afferent neurons with a smaller average diameter. C, expression of thermo-TRP channels by single cell RT-PCR. a, typical gels from three individual neurons (lane 4 is the negative control obtained from control pipettes that did not harvest cell contents but were submerged in bath solution). Predicted product sizes are 245 bp (TRPV1), 244 bp (TRPM8), 189 bp (TRPA1), 215 bp (NaV1.8), and 233 bp (glyceraldehyde-3-phosphate dehydrogenase (GAPDH)), respectively. b, summary results from 55 neurons. The inset is a histogram of the soma sizes for NaV1.8.

 
Single Cell RT-PCR Analysis of Thermo-TRP Channel Expression by Dental Primary Afferent Neurons—We investigated using single cell RT-PCR whether dental primary afferent neurons would express TRPV1, TRPM8, and TRPA1, transmitting heat, cool, and cold sensation in sensory neurons (1, 13). Among the dental primary afferent neurons (n = 55) randomly collected irrespective of soma size, it was found that 45% of identified dental primary afferent neurons expressed TRPV1, 13% expressed TRPM8, and 11% expressed TRPA1 (Fig. 1C). Some neurons expressed more than one TRP channel subunit (Fig. 1C). When we examined the expression of TTX-resistant sodium channel, NaV1.8, which is known to be restricted to nociceptors (25), together with thermo-TRP channels, we found mRNA expression of NaV1.8 in 85% of these neurons, which were of small and medium sizes (Fig. 1C, panel b). These results suggested that dental primary afferent neurons with thermo-TRP channels might be associated with tooth pain.


Figure 2
View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 2.
Calcium transients evoked by thermo-TRP ligands. A and B, concentration-response curves of menthol and icilin in dental primary afferent neurons obtained from Fura-2-based calcium-imaging experiments. Data are presented as mean ± S.E., p < 0.05. C-E, representative responses of individual neurons capsaicin (1µM), menthol (1 mM), and icilin (10µM). These cells each responded to only one agonist. F, a different neuron that responded to all three agonists. The numbers in parenthesis represent the number tested.

 
Ligands for Thermo-TRP Channels and Thermal Stimuli Increase Intracellular Calcium in Dental Primary Afferent Neurons—We examined the effects of capsaicin (a TRPV1 agonist), menthol (a TRPM8 agonist), or icilin (a TRPM8 and TRPA1 agonist) on [Ca2+]i as a screen for the presence of functional thermo-TRP channels in tooth pulp afferents, using ratiometric calcium imaging. The application of vehicle at the beginning of the experiment (i.e. 0.01% ethanol or Me2SOin 1 ml of balanced salt solution) did not alter [Ca2+]i (data not shown). However, as shown in Fig. 2 and Table 2, the subsequent application of thermo-TRP ligands clearly increased [Ca2+]i in subpopulations of neurons. To obtain the concentrations used in experiments, we confirmed the dose-response data (EC50, menthol, 85 ± 10.4 µM; icilin, 0.96 ± 0.2 µM, Fig. 2, A and B), which was similar to other previous reports (2, 5, 26). Measurement of [Ca2+]i in tooth pulp afferents showed increases evoked by capsaicin (1 µM: 60% of neurons, ratio changes 1.0 ± 0.2), menthol (1 mM: 20%, ratio changes 1.0 ± 0.2), and icilin (10 µM: 16%, ratio changes 0.9 ± 0.2) (Fig. 2, C-E, and Table 2).


Figure 3
View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 3.
Thermal stimuli evoke corresponding responses in individual neurons. A, in a capsaicin-responsive cell, a calcium transient was also induced by noxious heat (~43 °C). Cap, capsaicin (1 µM); CZP, capsazepine (20 µM). B, in a menthol- and icilin-responsive cell, a cold stimulus (below 25 °C) evoked a calcium transient. C, in a menthol-insensitive, icilin-sensitive cell, cold stimulation (below 17 °C) also evoked a calcium transient. Cell viability of neurons tested was confirmed by their response to high K+ (50 mM KCl solution) at the end of the experiments. D, temperature thresholds of thermo-TRPs in dental primary afferent neurons.

 
Several neurons responded to more than one ligand (capsaicin and menthol, 14% of neurons; menthol and icilin, 10%; capsaicin and icilin, 14%; capsaicin, menthol and icilin, 8%) (Fig. 2F and Table 2), indicating considerable co-localization of thermo-TRP channels in subpopulations of dental primary afferent neurons.

To confirm that neurons expressing thermo-TRP channel are responsive to natural thermal stimulation that might cause pain in teeth, we then examined the effects of heat or cold stimulation on [Ca2+]i. When heated above 42 ± 0.7 °C, capsaicin-sensitive neurons clearly showed increase of [Ca2+]i (0.3 ± 0.1, n = 10) (Fig. 3, A and D). Likewise, menthol- and icilin-sensitive neurons showed increase of [Ca2+]i (0.3 ± 0.1, n = 8, Fig. 3, B and D) below 25 ± 1.7 °C, and icilin-sensitive neurons responded below 17 ± 0.5 °C (0.2 ± 0.1, n = 7, Fig. 3, C and D). However, neurons that failed to respond to menthol and icilin also have no cold responses (n = 5). These results strongly suggest that thermo-TRP channels expressed by dental primary afferent neurons might act as specific detectors, which are activated at specific temperature point, for natural nociceptive thermal stimulation applied to teeth.

Ligands for Thermo-TRP Channels and Thermal Stimuli Activate Inward Currents in Dental Primary Afferent Neurons—To further support our hypothesis, we next examined whether the activation of thermo-TRP channels would produce inward currents at the holding potential of -60 mV. Capsaicin, menthol, and icilin induced inward currents of 4.5 ± 1.8 nA (n = 30), 0.45 ± 0.15 nA (n = 12), and 0.32 ± 0.15 nA (n = 7), respectively (Fig. 4, A-C). The currents produced by these agonists reversed polarity at ~0 mV and showed outward rectification, indicating that these currents were produced by the opening of TRP-channels. This result agreed with other previous reports (2, 5, 26). Consistent with the results of calcium imaging, capsaicin-sensitive neurons also responded to heat (n = 9) and menthol-sensitive neurons were responsive to cold (n = 5, Fig. 4, D and E). To directly determine whether the responsiveness of dental primary afferent neurons to thermo-TRP ligands is associated with the expression of thermo-TRP channels, at the end of some electrophysiological recordings, the expression of TRP channels was determined by immunohistochemistry or single cell RT-PCR. In Fig. 5A, capsaicin- and menthol-sensitive dental primary afferent neuron (red) showed immunoreactivity of TRPV1 (green) and TRPM8 (blue). At single cell RT-PCR study, we confirmed the correlation between mRNA of each TRP channel and ligand-induced current. In Fig. 5B, capsaicin (panel a)-sensitive dental primary afferent expressed only TRPV1 mRNA, whereas icilin/capsaicin (panel b)- and menthol/capsaicin (panel c)-sensitive neurons expressed TRPA1/TRPV1 and TRPM8/TRPV1 mRNA, respectively. Taken together, this suggests that dental primary afferent neurons have TRP-channels as specific thermal detectors, which play an important role in the sensation of thermal stimuli.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrate that subfamilies of thermo-TRP channels such as TRPV1, TRPM8, and TRPA1 are expressed by dental primary afferent neurons and that responsiveness of individual tooth afferents to thermo-TRP ligands and temperature change is correlated with expression of thermo-TRP channels. Thus, we suggest that activation of thermo-TRP channels expressed by these neurons may initiate sensory signals that contribute to tooth pain by temperature stimuli.

It has been hypothesized that stimulation of tooth pulp by any type of stimulus results only in a painful sensation (15, 16, 27). This assumption leads many investigators to use dissociated trigeminal neurons, identified by retrograde labeling with DiI, as an in vitro model for the study of pain-sensing neurons (15, 16). However, evidence of non-nociceptive neuron in tooth pulp has challenged this assumption so that the "pure pain" sensation of teeth became controversial (28). Thus, we first determined the proportion of "nociceptive" neurons among dental primary afferents. On the basis of neurochemical properties primary afferent neurons fall into two main classes. The first binds IB4 and expresses P2X3, c-RET, and NaV1.8 sodium channels. The second group is IB4-negative, contains calcitonin gene-related peptide and/or substance P, and expresses TrkA (29). Our data obtained from IB4 staining revealed that a significant proportion of small primary afferent neurons were IB4-positive, whereas the large neurons were not (Fig. 1B). Single cell RT-PCR showed that most dental primary afferent neurons express NaV1.8 mRNA. These data strongly support the notion that the majority, but not all, of dental primary afferent neurons may be considered to be nociceptors.


Figure 4
View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 4.
A-C, representative currents evoked by capsaicin (1 µM), menthol (1 mM), and icilin (10 µM), respectively. Vertical deflections in the left panels indicate the voltage ramp (-100 to +100 mV in 200-ms intervals) used to determine current-voltage relations shown in the right panels. Vh = -60 mV. Note inward currents evoked by each thermo-TRP ligand (left panel) and reversal potential at 0 mV (right panel). D, heat-induced inward current in a dental primary afferent neuron, which was responsive to capsaicin but not responsive to menthol and icilin. E, cold-evoked inward currents in a menthol-sensitive neuron. In the same cell, capsaicin failed to evoke an inward current.

 
Stimuli that produce tooth pain include mechanical (toothbrush contact, instrument touch, and desiccation with air), chemical (citrus, sour, or sweet foods and beverages and acidic bacterial plaque by-products), and thermal (extremes of hot or cold food, liquids, or inhaled air). Thermal stimulation is often described as the most severe, and is widely used in dental clinic to test the vitality of the tooth pulp. Therefore, we focused in the present study on the molecular mechanisms that might underlie tooth pain caused by thermal stimulation.

TRPV1 is a Ca2+-permeable channel activated by capsaicin and temperatures higher than 42 °C, and it is highly expressed in a subset of peptidergic and IB4-binding nociceptive neurons (1, 2). By single cell RT-PCR analysis, we found that TRPV1 is highly expressed in dental primary afferent neurons (Fig. 1C), consistent with a previous report (23, 30). Ratiometric calcium imaging showed that [Ca2+]i was increased by capsaicin as well as by heat stimulation (Figs. 2C and 3A), indicating clear functional responses attributable to TRPV1. Consistent with these data, heat-evoked currents were found in capsaicin-sensitive neurons (Fig. 4, A and D), which were similar to the responses evoked by heterologous expression of TRPV1 (18). Furthermore, we observed expression of TRPV1 in the same neurons that gave capsaicin-evoked currents (Fig. 5). Thus, we propose that TRPV1 might be involved in nociceptive heat sensation in dental primary afferents.


Figure 5
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 5.
Confocal laser microscopy and single cell RT-PCR of thermo-TRPs following whole cell recordings. A, capsaicin (1 µM) and menthol (1 mM) evoked currents at -60 mV. Immunohisto-chemistry of the cell recorded A shows the presence of DiI-labeled (a, red), TRPV1 (b, green), TRPM8 (c, blue); d is the merged image. B, single cell RT-PCR analysis following whole cell recording in three different neurons. Lanes a-c show single cell RT-PCR products obtained from intracellular contents of the cells shown in panels a-c, respectively. Note the precise correspondence between mRNA expressed and responsiveness to ligands. The control is obtained from pipettes that did not harvest cell contents but were submerged in bath solution.

 
TRPM8 was originally identified as a cold and menthol receptor (CMR1) (3, 5); it is activated by menthol and cooling (8-25 °C). Because the temperature ranges that activate TRPM8 are similar to those that activate native innocuous cold-sensitive fibers, and because pain markers such as calcitonin gene-related peptide, substance P, TRPV1, or IB4 do not co-localize with TRPM8, it has been suggested that TRPM8 has physiological function for innocuous thermosensation (5). However, our findings, that TRPM8 is expressed by a subset of pain and temperature-sensing neurons such as TrkA-positive small diameter neurons and activated by the noxious cold range of temperatures (3, 31, 32), led us to re-examine the possibility of involvement of TRPM8 in cold sensation. A subset of neurons that expressed TRPM8 were found (Fig. 1C), and menthol and cold stimulation not only increased [Ca2+]i (Figs. 2D and 3B) but also evoked inward currents (Fig. 4B). The percentage of menthol-sensitive neurons in the trigeminal ganglion was similar to that previously reported (5, 33, 34). Although it was originally suggested that TRPM8 does not co-exist with TRPV1 (3), other reports indicated colocalization of TRPM8 and TRPV1 (33, 35, 36). In agreement with these latter reports, we also found TRPM8 to be co-localized with TRPV1 in some primary afferent neurons (Figs. 1C and 5B). Moreover, some neurons were responsive to both menthol and capsaicin (Fig. 5, A and C), and sometimes to icilin as well (Fig. 2F). Taken together, our data suggested the potential role of TRPM8 channels in thermosensation of dental afferents.

TRPA1 is a more distantly related TRP channel, homologous to Drosophila Painless (37, 38) and activated by noxious cold temperature (<~17 °C): this temperature is reported as being painfully cold by humans. TRPA1 is also activated by icilin, but not by menthol. Unlike TRPM8, TRPA1 is expressed in a subset of sensory neurons that express the nociceptive markers such as calcitonin gene-related peptide and substance P (4). We also found the expression of TRPA1 in a subset of dental primary afferents (Fig. 1C), suggesting a potential action as a transducer of cold sensation in teeth. The percentage of TRPA1-expressing cells was similar to that of total trigeminal ganglion previously reported (4). It is interesting to find that some neurons responded to icilin alone (Figs. 2E and 3C), whereas other neurons responded to both icilin and menthol (Figs. 2D, 2F, and 3B). Based on the specificity of the ligands, it is likely that individual neurons can express either TRPA1 or TRPM8, respectively. TRPA1 is also known to be expressed in a subset of sensory neurons that express the noxious-heat receptor, TRPV1 (4). We also found that TRPA1 and TRPV1 were expressed together in a subset of dental primary afferent neurons (Figs. 1C and 5C); these might act as polymodal nociceptive neurons that respond to both noxious heat and cold. The co-expression of subtypes of thermo-TRP channels in dental primary afferent neurons provide a likely explanation why pain is the sensation mostly perceived in teeth, irrespective of the sub-modality of thermal stimuli.

Given that the sensation perceived from teeth by cold and hot stimuli is not always identical, the cellular and molecular mechanisms underlying thermal and pain sensations of teeth is likely to be more complex, rather than simply induced by the activation of molecules such as thermo-TRPs in dental primary afferents, which are highly specialized to detect specific ranges of temperature (1). Odontoblasts, which have a process extending into the dentin, can also contribute to nociceptive and non-nociceptive sensory processing in teeth (39). Non-TRP channels such as TREK-1 (a member of a family of mammalian two-pore domain K+channel) might also be involved in thermosensation (1) and have been demonstrated to be expressed by odontoblasts (39). Odontoblasts also express subfamilies of thermo-TRP channels.3 From the observation that various stimuli, including cold, heat, acids, pressure, chemicals, and high osmotic solutions, create the movement of fluid within the dentin tubules, Brännström proposed that any kinds of stimulus that cause the fluid within the tubules to flow inward or outward create a mechanical disturbance or cellular perturbation, thereby exciting the nerves in the tooth (10, 11). If this is the case, dental primary afferents are also likely to express low threshold mechanoreceptors with high sensitivity. Our ongoing work on these two possibilities will also help us further understand the detailed cellular and molecular mechanisms underlying tooth pain.

In conclusion, our results support a potential role of thermo-TRP channels in the transduction of tooth pain. Thermo-TRP channels are expressed by dental primary afferent neurons, and the neurons are activated by the appropriate thermal stimuli. Given that rational treatment of pain requires identification of pain mechanisms as a target of drug therapy, thermo-TRP channels might constitute a new distinctive target for the development of therapeutic intervention for tooth pain.


   FOOTNOTES
 
* This work was supported by the Basic Research Program of the Korea Science & Engineering Foundation (Grant R01-2004-000-10384-0) and by the Brain Research Center of the 21st Century Frontier Research Program (Grant M103KV010009-04K2201-00930) funded by the Ministry of Science and Technology, Republic of Korea. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed. Tel.: 82-2-740-8656; Fax: 82-2-762-5107; E-mail: odolbae{at}snu.ac.kr.

2 The abbreviations used are: TRP, transient receptor potential; thermo-TRPs, temperature-activated transient receptor potential channels; IB4, isolectin B4; RT, reverse transcription; TG, trigeminal ganglion; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate. Back

3 C.-K. Park, M. S. Kim, Z. Fang, H. Y. Li, S. J. Jung, S.-Y. Choi, S. J. Lee, K. Park, J. S. Kim, and S. B. Oh, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Makoto Tominaga of the National Institutes of Natural Sciences, Japan, for his kind gift of TRPM8 antibody.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Patapoutian, A., Peier, A. M., Story, G. M., and Viswanath, V. (2003) Nat. Rev. Neurosci. 4, 529-539[CrossRef][Medline] [Order article via Infotrieve]
  2. Caterina, M. J., Schumacher, M. A., Tominaga, M., Rosen, T. A., Levine, J. D., and Julius, D. (1997) Nature 389, 816-824[CrossRef][Medline] [Order article via Infotrieve]
  3. Peier, A. M., Moqrich, A., Hergarden, A. C., Reeve, A. J., Andersson, D. A., Story, G. M., Earley, T. J., Dragoni, I., McIntyre, P., Bevan, S., and Patapoutian, A. (2002) Cell 108, 705-715[CrossRef][Medline] [Order article via Infotrieve]
  4. Story, G. M., Peier, A. M., Reeve, A. J., Eid, S. R., Mosbacher, J., Hricik, T. R., Earley, T. J., Hergarden, A. C., Andersson, D. A., Hwang, S. W., McIntyre, P., Jegla, T., Bevan, S., and Patapoutian, A. (2003) Cell 112, 819-829[CrossRef][Medline] [Order article via Infotrieve]
  5. McKemy, D. D., Neuhausser, W. M., and Julius, D. (2002) Nature 416, 52-58[CrossRef][Medline] [Order article via Infotrieve]
  6. Scholz, J., and Woolf, C. J. (2002) Nat. Neurosci. 5, 1062-1067[Medline] [Order article via Infotrieve]
  7. Pashley, D. H. (1996) Crit. Rev. Oral Biol. Med. 7, 104-133[Abstract/Free Full Text]
  8. Nair, P. N. (1995) Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 80, 710-719[Medline] [Order article via Infotrieve]
  9. Mengel, M. K., Stiefenhofer, A. E., Jyvasjarvi, E., and Kniffki, K. D. (1993) Pain 55, 159-169[CrossRef][Medline] [Order article via Infotrieve]
  10. Brannstrom, M., Linden, L. A., and Astrom, A. (1967) Caries Res. 1, 310-317[Medline] [Order article via Infotrieve]
  11. Brannstrom, M., and Astrom, A. (1972) Int. Dent. J. 22, 219-227[Medline] [Order article via Infotrieve]
  12. Andrew, D., and Matthews, B. (2000) J. Physiol. 529, 791-802[Abstract/Free Full Text]
  13. Clapham, D. E. (2003) Nature 426, 517-524[CrossRef][Medline] [Order article via Infotrieve]
  14. Moran, M. M., Xu, H., and Clapham, D. E. (2004) Curr. Opin. Neurobiol. 14, 362-369[CrossRef][Medline] [Order article via Infotrieve]
  15. Taddese, A., Nah, S. Y., and McCleskey, E. W. (1995) Science 270, 1366-1369[Abstract/Free Full Text]
  16. Cook, S. P., Vulchanova, L., Hargreaves, K. M., Elde, R., and McCleskey, E. W. (1997) Nature 387, 505-508[CrossRef][Medline] [Order article via Infotrieve]
  17. Eckert, S. P., Taddese, A., and McCleskey, E. W. (1997) J. Neurosci. Methods 77, 183-190[CrossRef][Medline] [Order article via Infotrieve]
  18. Yang, B. H., Piao, Z. G., Kim, Y. B., Lee, C. H., Lee, J. K., Park, K., Kim, J. S., and Oh, S. B. (2003) J. Dent. Res. 82, 781-785[Abstract/Free Full Text]
  19. Stucky, C. L., and Lewin, G. R. (1999) J. Neurosci. 19, 6497-6505[Abstract/Free Full Text]
  20. Petruska, J. C., Napaporn, J., Johnson, R. D., Gu, J. G., and Cooper, B. Y. (2000) J. Neurophysiol. 84, 2365-2379[Abstract/Free Full Text]
  21. Galoyan, S. M., Petruska, J. C., and Mendell, L. M. (2003) Eur. J. Neurosci. 18, 535-541[CrossRef][Medline] [Order article via Infotrieve]
  22. Silbert, S. C., Beacham, D. W., and McCleskey, E. W. (2003) J. Neurosci. 23, 34-42[Abstract/Free Full Text]
  23. Chaudhary, P., Martenson, M. E., and Baumann, T. K. (2001) J. Dent. Res. 80, 1518-1523[Abstract/Free Full Text]
  24. Dirajlal, S., Pauers, L. E., and Stucky, C. L. (2003) J. Neurophysiol. 89, 513-524[Abstract/Free Full Text]
  25. Lai, J., Porreca, F., Hunter, J. C., and Gold, M. S. (2004) Annu. Rev. Pharmacol. Toxicol. 44, 371-397[CrossRef][Medline] [Order article via Infotrieve]
  26. Chuang, H. H., Neuhausser, W. M., and Julius, D. (2004) Neuron 43, 859-869[CrossRef][Medline] [Order article via Infotrieve]
  27. Fors, U., Ahlquist, M. L., Skagerwall, R., Edwall, L. G., and Haegerstam, G. A. (1984) Pain 18, 397-408[Medline] [Order article via Infotrieve]
  28. Dong, W. K., Chudler, E. H., and Martin, R. F. (1985) Brain Res. 334, 389-395[CrossRef][Medline] [Order article via Infotrieve]
  29. Hunt, S. P., and Mantyh, P. W. (2001) Nat. Rev. Neurosci. 2, 83-91[Medline] [Order article via Infotrieve]
  30. Morgan, C. R., Rodd, H. D., Clayton, N., Davis, J. B., and Boissonade, F. M. (2005) J. Orofac. Pain 19, 248-260[Medline] [Order article via Infotrieve]
  31. Tominaga, M., and Caterina, M. J. (2004) J. Neurobiol. 61, 3-12[CrossRef][Medline] [Order article via Infotrieve]
  32. McKemy, D. D. (2005) Mol. Pain 1, 16[CrossRef][Medline] [Order article via Infotrieve]
  33. Abe, J., Hosokawa, H., Okazawa, M., Kandachi, M., Sawada, Y., Yamanaka, K., Matsumura, K., and Kobayashi, S. (2005) Brain Res. Mol. Brain Res. 136, 91-98[Medline] [Order article via Infotrieve]
  34. Nealen, M. L., Gold, M. S., Thut, P. D., and Caterina, M. J. (2003) J. Neurophysiol. 90, 515-520[Abstract/Free Full Text]
  35. Babes, A., Zorzon, D., and Reid, G. (2004) Eur. J. Neurosci. 20, 2276-2282[CrossRef][Medline] [Order article via Infotrieve]
  36. Okazawa, M., Inoue, W., Hori, A., Hosokawa, H., Matsumura, K., and Kobayashi, S. (2004) Neurosci. Lett. 359, 33-36[CrossRef][Medline] [Order article via Infotrieve]
  37. Goodman, M. B. (2003) Trends Neurosci. 26, 643-645[Medline] [Order article via Infotrieve]
  38. Tracey, W. D., Jr., Wilson, R. I., Laurent, G., and Benzer, S. (2003) Cell 113, 261-273[CrossRef][Medline] [Order article via Infotrieve]
  39. Magloire, H., Lesage, F., Couble, M. L., Lazdunski, M., and Bleicher, F. (2003) J. Dent. Res. 82, 542-545[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 JDRHome page
K.Y. Yeon, G. Chung, M.S. Shin, S.J. Jung, J.S. Kim, and S.B. Oh
Adult Rat Odontoblasts Lack Noxious Thermal Sensitivity
Journal of Dental Research, April 1, 2009; 88(4): 328 - 332.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
Y. Takashima, R. L. Daniels, W. Knowlton, J. Teng, E. R. Liman, and D. D. McKemy
Diversity in the Neural Circuitry of Cold Sensing Revealed by Genetic Axonal Labeling of Transient Receptor Potential Melastatin 8 Neurons
J. Neurosci., December 19, 2007; 27(51): 14147 - 14157.
[Abstract] [Full Text] [PDF]

Home page
 JDRHome page
A. Diogenes, A.N. Akopian, and K.M. Hargreaves
NGF Up-regulates TRPA1: Implications for Orofacial Pain
Journal of Dental Research, June 1, 2007; 86(6): 550 - 555.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. Rev.Home page
B. Nilius, G. Owsianik, T. Voets, and J. A. Peters
Transient Receptor Potential Cation Channels in Disease
Physiol Rev, January 1, 2007; 87(1): 165 - 217.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
H. Katsura, K. Obata, T. Mizushima, J. Sakurai, K. Kobayashi, H. Yamanaka, Y. Dai, T. Fukuoka, M. Sakagami, and K. Noguchi
Activation of SRC-family kinases in spinal microglia contributes to mechanical hypersensitivity after nerve injury.
J. Neurosci., August 23, 2006; 26(34): 8680 - 8690.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
281/25/17304    most recent
M511072200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Park, C.-K.
Right arrow Articles by Oh, S. B.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Park, C.-K.
Right arrow Articles by Oh, S. B.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2006 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement