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J Dent Res 85(11):1046-1049, 2006
© 2006 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Tissue pH and Temperature Regulate Pulpal Nociceptors

H.E. Goodis1,*, A. Poon2, and K.M. Hargreaves2,3

1 Division of Endodontics, Department of Preventive and Restorative Sciences, UCSF, 707 Parnasssus Ave., San Francisco, CA 94143, USA; and
2 Department of Endodontics and
3 Department of Pharmacology, UTHSCSA, San Antonio, TX, USA

* corresponding author, harold.goodis{at}ucsf.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The TRPV1 receptor acts as a sensor for environmental changes in pH and temperature. Since many nociceptors express TRPV1, it is possible that local tissue-cooling may inhibit nociceptor activity via reduction of TRPV1 activation. The present study used isolated superfused rat dental pulp to test the hypothesis that capsaicin receptors are activated in inflamed tissue, as measured by alterations in neuropeptide release. We tested the hypothesis that alterations in the tissue temperature and pH of isolated superfused rat dental pulp regulate capsaicin-induced release of calcitonin gene-related peptide (CGRP). Application of capsaicin with increased proton concentration (i.e., lowered pH) produced a nearly two-fold increase in peak immunoreactive CGRP release, as compared with capsaicin applied at a pH of 7.4. Reduction in tissue temperature from 37°C to 26°C completely blocked the capsaicin effect. The study indicates that environmental stimuli regulate the activity of capsaicin-sensitive neurons innervating dental pulp, and these factors may be significant clinically in the development and amelioration of dental pain.

KEY WORDS: TRPV1 • capsaicin • pH • temperature • CGRP


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although considerable research has determined that heat activates certain nociceptors, relatively little is known about the effects and mechanisms of cooling on primary afferent activity. Cloning of the capsaicin-sensitive vanilloid receptor (i.e., TRPV1, also known as VR1) has led to a more complete understanding of the mode of action of capsaicin, a noxious chemical that produces pain when administered to humans and nociceptive behavior when administered to animals. The TRPV1 receptor is a ligandgated non-selective cation channel expressed predominantly by a major class of sensory nociceptors (Caterina et al., 1997; Tominaga et al., 1998; Davis et al., 2000). Most studies of the TRPV1 receptor have utilized cell cultures and electrophysiologic measurements of inward current that occur across cell membranes when the cell is exposed to capsaicin, low extracellular pH, or noxious heat. These studies have demonstrated that TRPV1 acts as a cellular sensor for environmental changes in pH and heat (Bevan and Geppetti, 1994). These are important biophysical properties, since low extracellular pH (i.e., tissue acidosis) is frequently seen with inflamed tissue and results in activation of nociceptors (Kress and Reeh, 1996a,b), and noxious heat (≥45°C) also activates pain fibers (Cesare and McNaughton, 1996).

Calcitonin gene-related peptide (CGRP) is highly expressed in a subpopulation of afferent neurons, including nociceptors (Ju et al., 1987). Most of the CGRP-immunoreactivity has been found to be capsaicin-sensitive, suggesting an involvement in the mediation of pain (Franco-Cereceda et al., 1987), and denervation of the inferior alveolar nerve leads to rapid and significant reduction in pulpal levels of immunoreactive CGRP (Buck et al., 1999). Several past studies indicated that capsaicin receptors are present in the dental pulp of the incisor teeth and, when activated, release CGRP (Bowles et al., 2003). Increased numbers of CGRP-immunoreactive nerves were found in the coronal portion of rat molar and incisor pulp when teeth were subjected to experimental tooth movement (Kvinnsland and Kvinnsland, 1990). When antagonists to CGRP were used, involvement of CGRP in the mediation of antidiomic vasodilation was demonstrated (Kerezoudis et al., 1994). An anatomic study found CGRP-expressing nerve fibers located around blood vessels as well as in the odontoblast cell layer (Zhang et al., 1998). When dog and rat incisors were compared, the latter showed greater distribution of CGRP-immunoreactive pulpal nerves (Ngassapa et al., 1998). Two studies developed a model that used rat incisors to test the ability of irritants to cause release of pro-inflammatory mediators (Chidiac et al., 2002a,b). The resultant inflammatory response indicates that capsaicin-sensitive receptors are present in the incisor pulp of rats.

Collectively, these studies support the hypothesis that the TRPV1 receptor is activated in inflamed dental pulpal tissue, and provides a rationale for the hypothesis that inflammatory pain may be reduced or prevented by local tissue-cooling (Brandner et al., 1996). However, comparatively few studies have been conducted in native tissue; most research has been conducted in cell lines that may not produce a phenotype similar to that observed in native tissue, such as nociceptors innervating dental pulp. The present study tested the hypothesis that alterations in the tissue temperature and pH of isolated superfused rat dental pulp regulate capsaicin-induced release of calcitonin gene-related peptide (CGRP).


   MATERIALS & METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The protocol for the use of dental pulp tissue harvested from rat incisors was reviewed by the appropriate committees for animal research at the University of Texas Health Science Center, San Antonio.

The superfusion method used in this study was modified from a previously described procedure (Hargreaves et al., 1992; Goodis et al., 2000a) by the use of syringe pumps that permitted rapid changes to occur in buffer temperature or pH. Immunoreactive calcitonin gene-related peptide was selected as the evoked response in these experiments. Adult rats were decapitated, their maxillary and mandibular incisors were removed, and the pulp tissue was harvested. The tissues from 12 teeth (N = 3 animals) were pooled in a 1.5-mL chamber and superfused with Krebs buffer (36°C; pH 7.4) at a rate of 0.33 mL/min. The set-up allowed for the rapid transfer of the contents of the syringes through four-way stopcocks when solution pH was lowered. We controlled temperatures by placing the superfusion chambers in water baths previously set at the desired temperatures.

After a recovery period for tissue acclimation, fractions were collected every 7 min for the duration of the experiment. The effects of 30 µM capsaicin (CAP) (Hargreaves et al., 1992) stimulation of isolated dental pulp were studied at 37°C/pH 7.4 (n = 6), at 26°C/pH 7.4 (n = 7), at 37°C/pH 6.5 (n = 5), and at 37°C/pH 6.5 (n = 7) without capsaicin.

Superfusate levels of immunoreactive CGRP were measured by means of a previously validated radioimmunoassay (Hargreaves et al., 1992; Goodis et al., 2000a). Superfusate samples (2.1 mL) were pre-incubated for 48 hrs at 4°C with CGRP antisera (courtesy of Dr. Michael Iadarola, NIDCR, NIH, Bethesda, MD, USA). The radiolabeled tracer ([125Tyr-CGRP28–37 (approximately 20,000 cpm)] and 50 µL of goat anti-rabbit antisera, coupled to ferric beads (PerSeptive Diagnostics, Cambridge, MA, USA), were added and allowed to incubate for an additional 48 hrs at 4°C. Bound and free tracer was separated by immunomagnetic separation. The minimum detection of the assay is ~3 fmol/tube, with a 50% displacement of 20 fmol/tube. All drugs were tested for interference in the radioimmunoassay, and there was no cross-reactivity at the concentrations used in these studies.

Data are presented as means ± SEM. Statistical significance was determined by two-way ANOVA with repeated measures, followed by Duncan’s multiple-range post hoc analysis, with significance at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The effect of administration of capsaicin to dental pulp superfused at pH = 7.4 and pH = 6.5 was determined (Fig. 1Go). Capsaicin evoked a significant increase in immunoreactive CGRP over basal rates of release under both conditions (p < 0.05 for both). However, the application of capsaicin to dental pulp at a pH of 6.5 produced nearly a two-fold increase in peak immunoreactive CGRP release, as compared with the same concentration of capsaicin at a pH = 7.4 (p < 0.01).


Figure 1
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  		Figure 1. Effect of administration of capsaicin to the dental pulp at a pH of 7.4 and 6.5. Capsaicin evoked a significant increase in CGRP over basal rates of release: 9.5 fmmol/2.1 mL at a pH of 6.5 and 6.5 fmol/2.1 mL at a pH of 7.4. Recovery 1, 2, and 3 indicate successive fractions after capsaicin stimulation. While fractions continued to be collected, data (not shown) dictated that levels returned to baseline.

 
We determined the results of the temperature and pH studies by calculating the % increase in immunoreactive CGRP release over basal rates, with 100% defined as basal rates of release (Fig. 2Go). Reducing the pH of dental pulp from 7.4 to 6.5 was sufficient by itself to evoke significant immunoreactive CGRP release. In addition, reduction in tissue temperature from 37°C to 26°C completely blocked the excitatory effect of capsaicin administration. Analysis of superfusates at basal conditions of temperature and pH, prior to and after temperatures were lowered and pH increased, demonstrated levels of CGRP release consistent with those reported in our previous study (Goodis et al., 2000a; data not shown).


Figure 2
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  		Figure 2. Percent increase in immunoreactive CGRP (iCGRP) over basal rates at a pH of 7.4 and 6.5 and temperatures of 37°C and 26°C. There was a 140% increase in iCGRP release with CAP/pH 6.5.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The TRPV1 receptor is thought to serve as a neuronal receptor for noxious changes in environmental temperature and pH (Caterina et al., 1997). In the present study, we evaluated whether capsaicin-sensitive pulpal nociceptors possess these properties, and whether alterations in either tissue temperature or pH regulate activation of capsaicin-sensitive nociceptors innervating dental pulp. The results of the studies indicate that a reduction in tissue temperature (from 37°C to 26°C) completely abolished capsaicin-induced activation of these pulpal nociceptors. Further, elevations in tissue proton concentration (from pH 7.4 to 6.5) were sufficient to activate these neurons and significantly increased their responsiveness to capsaicin. Analysis of these data, collectively, supports the hypothesis that both tissue temperature and pH regulate the capsaicin-sensitive class of pulpal nociceptors through capsaicin receptor activation. Testing was carried out through cooling tissue and raising the local tissue pH.

Chaudhary et al.(2001), using retrograde transport of a fluorescent dye (DiIC18), found that labeled primary afferent pulpal neurons were excited by application of capsaicin, with the response appearing to be mediated by TRPV1. Studies indicate that TRPV1 immunoreactivity can be identical to pulp tissue used in other models in rat dental pulp. Stenholm et al.(2002) concluded that TRPV1 was down-regulated from normal values in dental pulp and gingiva when the interior alveolar nerve was injured. These authors reported that the results indicated that TRPV1 could play a significant role in rat pulpal and gingival transduction. Pan et al.(2003) used retrograde labeling of pulpal afferents and found that expression of CGRP was upgraded when teeth were injured. TRPV1 expression may therefore be evidence for nociceptors in the dental pulp. These studies indicate the presence of receptors that allow use of rat dental pulp as our model for this study and as seen in a more recent study (Wadachi and Hargreaves, 2006).

Since human dental pulp contains TRPV1 expressed on unmyelinated neurons, and since our model appears to have the same receptors (Renton et al., 2003), it is possible that the results of the present study have clinical significance. For example, stimuli that inhibit TRPV1 activity (e.g., desensitizing concentrations of capsaicin) act to reduce the responsiveness of 50% of slow A-delta fibers in pulp (Ikeda et al., 1997). Based on the present demonstration, that tissue-cooling reduces TRPV1 in dental pulp, this general mechanism may contribute to the understanding of prior observations reporting that tissue-cooling reduced responsiveness to electrical pulp testing (Goodis et al., 2000b), a response mediated, in part, by A-delta fibers. Thus, the effects of tissue-cooling on peripheral nociceptor activity, observed in this study, may mediate the clinical observation that tissue-cooling reduces pain due to severe pulpitis (e.g., drinking ice water) or following surgery (e.g., applying ice). This appears to be a general property of nociceptors derived from both trigeminal and dorsal root ganglia. For example, there was a significant reduction in morphine consumption in healthy patients undergoing lumbar spine surgery when the wound site was locally cooled by an externally applied cooling pad (Brandner et al., 1996). Other studies have reported similar effects following local cooling of inflamed or injured skin (Steen and Reeh, 1993; Kilo et al., 1995; Babes et al., 2002).

The reduction in tissue pH from 7.4 to 6.5 produced a significant increase in the rate of immunoreactive CGRP release when compared with basal rates of release, and potentiated the responsiveness to a simultaneous application of capsaicin. Thus, protons both activate and sensitize capsaicin-sensitive neurons that innervate dental pulp. These studies suggest that the local tissue acidosis that accompanies inflammation and reduction in pulpal blood flow might contribute to enhanced activity of pulpal nociceptors. Interestingly, the pH of normal extracellular tissue in rat dentin-predentin is 7.0 ± 0.2 (Lundgren et al., 1992). However, we can find no report of the pH of inflamed human dental pulp; this finding may have considerable significance in the pathophysiology of pain due to severe pulpitis.

While there is no direct evidence of a relationship between stimulation of both rat incisor pulp and human pulp, one must assume that the response seen in the rat should be the same as that seen in humans. The rat pulp is a convenient model for an evoked response of the tissue when stimulated. It is not unreasonable to believe, therefore, that the same response occurs in humans when patients present in pain.

The present study indicates that environmental stimuli, such as tissue temperature and pH, regulate the activity of capsaicin-sensitive neurons innervating dental pulp, and suggest that these factors may have clinical significance in the development and amelioration of dental pain.


   ACKNOWLEDGMENTS
 
The authors gratefully acknowledge a grant from the National Institutes of Health, #R37DE14228.

Received July 19, 2004; Last revision July 13, 2006; Accepted July 18, 2006


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS & METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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