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J Dent Res 82(5): 398-401, 2003
© 2003 International and American Associations for Dental Research

RESEARCH REPORT
Biological

Intrinsic Regulation of CGRP Release by Dental Pulp Sympathetic Fibers

K.M. Hargreaves1,, W.R. Bowles2, and D.L. Jackson3

1 Department of Endodontics, UTHSCSA School of Dentistry, Mail Code 7892, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900;
2 Division of Endodontics, University of Minnesota School of Dentistry; and
3 Department of Oral Medicine, University of Washington School of Dentistry;

* corresponding author, Hargreaves{at}UTHSCSA.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neurotransmission from sympathetic and peptidergic afferent fibers participates in the regulation of pulpal blood flow (PBF) via opposing effects. In this study, we directly tested the hypothesis that activation of pulpal sympathetic terminals inhibits exocytosis of immunoreactive calcitonin gene-related peptide (iCGRP) from peptidergic afferents innervating bovine dental pulp. The results demonstrate that norepinephrine inhibits capsaicin-evoked iCGRP release. The application of {alpha}-adrenergic antagonists (phentolamine or phenoxybenzamine) increased spontaneous release of iCGRP. Moreover, administration of agents that evoke the release of sympathetic neurotransmitters (guanethidine or reserpine) inhibited capsaicin-evoked iCGRP release. Collectively, these results indicate that sympathetic neurotransmission inhibits exocytosis from pulpal peptidergic afferent fibers. Analysis of these data supports the hypothesis that peripheral sympathetic vasomotor control may operate by a direct mechanism (vasoconstriction) as well as by an indirect mechanism (e.g., inhibition of exocytosis from afferent fibers). Since capsaicin-sensitive neurons are nociceptors, it is possible that certain sympathetic neurotransmission may modulate pain.

KEY WORDS: dental pulp • sympathetic • capsaicin • CGRP


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Maintenance of pulpal tissue pressure, fluid exchange between vasculature and tissue, nutritionm and pulpal defense depend, to a large extent, on maintenance of vascular tone. Numerous factors are involved in the regulation of pulpal blood flow, including neurotransmitters released from peripheral terminals of sympathetic and trigeminal peptidergic afferent neurons (Suda and Ikeda, 2002). Electrical stimulation of sympathetic fibers reduces pulpal blood flow by the peripheral release of norepinephrine, neuropeptide Y, and other sympathetically derived transmitter substances (Tønder and Naess, 1978; Kim and Dörscher-Kim, 1990; Kim et al., 1996). In contrast, activation of capsaicin-sensitive peptidergic afferent neurons increases pulpal blood flow, and this effect is due primarily to peripheral release of calcitonin gene-related peptide (CGRP), substance P, and possibly other factors (Olgart, 1992; Andrew and Matthews, 1996; Berggreen and Heyeraas, 2000).

Several vascular physiology studies have suggested that activation of sympathetic fibers reduces pulpal blood flow in part via inhibition of capsaicin-sensitive peptidergic afferents (Kerezoudis et al., 1993a, b). Conversely, local application of capsaicin, which induces transmitter release from nociceptive axons, antagonizes sympathetically induced vasoconstriction (Takenaga and Kawasaki, 1999). These results have led to the hypothesis that sympathetically induced reduction in pulpal blood flow could be mediated by both direct (via constriction of arterioles) as well as indirect (via inhibition of CGRP or substance P release from afferent fibers) mechanisms. However, no study has tested directly whether endogenous sympathetic neurotransmitters inhibit exocytosis from pulpal peptidergic afferent neurons.

Accordingly, this study evaluated the hypothesis that endogenous sympathetically derived neurotransmitters inhibit exocytosis of iCGRP from trigeminal peptidergic afferent neurons innervating bovine dental. CGRP in the dental pulp is found only in sensory axons (Wakisaka et al., 1987), and thus the release of this neuropeptide into superfusates from dental pulp represents a selective marker for activation of certain trigeminal peptidergic afferent fibers.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
The superfusion method was used as previously described (Hargreaves et al., 1992). In brief, we collected mandibular incisors from Holstein cows (2–4 yrs old) at a local slaughterhouse, and the pulp tissue was removed, sectioned, and then chopped into 200-µm2 slices (McIlwain tissue chopper, Mickle Lab Eng Co. Ltd., Gomshall, UK). The tissue was superfused with oxygenated Krebs’ buffer (420 µL/min) at 37°C. After a 60-minute wash-out period, the samples were collected over seven-minute periods. The Krebs’ buffer (pH 7.4) was made fresh daily (NaCl [135 mM], KCl [3.5 mM], MgCl [1.1 mM], NaH2PO4 [1 mM], CaCl2 [2.5 mM], dextrose [3.3 mM], bovine serum albumin [0.1%], bacitracin [3 mg%], and 0.1 mM ascorbic acid). All chemicals and test drugs were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

Levels of iCGRP were measured by radioimmunoassay (RIA) as previously described (Richardson et al., 1998). Each incisor provided sufficient tissue for one chamber (the sample sizes are listed in the legend to each Fig.), and pulp tissue was exposed to only one experimental condition. Each experiment (i.e., all data generated for each Fig.) was assayed in a separate RIA. Separate standard curves were prepared with aliquots of Krebs’ buffer containing relevant drug concentrations to facilitate respective comparison with experimental treatments.

The data were analyzed by one-way ANOVA with repeated measures followed by Duncan’s multiple-range test to determine differences between groups. A Student’s t test was conducted when two groups were compared. Release data were normalized by calculation of the % increase over baseline rates of iCGRP release according to the formula 100 x (peak release - baseline)/(baseline). This reduced intra-experimental variability due to differences among animals. A difference was accepted as significant if the probability that it occurred due to chance alone was less than 5% (p < 0.05). Data are presented as mean ± SEM.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Capsaicin (10 µM) evoked a significant (p < 0.01) two- to three-fold increase in the release in iCGRP from the peripheral terminals of peptidergic neurons innervating dental pulp (Fig. 1Go). Pre-treatment with a physiologically relevant concentration of norepinephrine (3 nM) significantly (p < 0.01) inhibited capsaicin-evoked iCGRP exocytosis. Analysis of these data indicates that exogenous norepinephrine is capable of inhibiting efferent neurotransmission from peripheral peptidergic fibers that innervate dental pulp.



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  		Figure 1. Effect of norepinephrine administration on capsaicin-evoked release of immunoreactive calcitonin gene-related peptide (iCGRP) from in vitro-superfused dental pulp. Bovine dental pulp was collected, sliced into 200-µm2 slices, and superfused with oxygenated Krebs’ buffer. Levels of iCGRP released from dental pulp were collected over a seven-minute fraction and measured by radioimmunoassay. Norepinephrine (3 nM) or vehicle was administered from fractions 0–28 min, and capsaicin (10 µM) was administered to all chambers over a 21- to 28-minute period. Data are calculated as % increase over baseline 100 x (peak release - baseline)/baseline. Error bars are SEM. **p < 0.01 vs. vehicle. N = 6/group.

 
We next evaluated whether the spontaneous release of endogenous catecholamines from isolated slices of dental pulp was able to modulate basal rates of exocytosis of iCGRP. To test this hypothesis, we treated dental pulp with one of two {alpha}-adrenergic receptor antagonists and compared with vehicle treatment. Phentolamine and phenoxybenzamine (10 µM) were selected, since these antagonists block both the {alpha}1 and {alpha}2 subtypes of adrenergic receptors and act via different mechanisms of action (i.e., competitive and non-competitive antagonism, respectively). As seen in Fig. 2Go, treatment with either antagonist significantly (p < 0.01) increased spontaneous release of iCGRP from superfused dental pulp. Analysis of these data indicates that endogenous catecholamines provide an intrinsic inhibitory tone to peripheral terminals of peptidergic afferent fibers.



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  		Figure 2. Effect of pre-treatment with {alpha}-adrenoceptor antagonists (phentolamine and phenoxybenzamine, 10 µM) or vehicle on spontaneous release of iCGRP from superfused dental pulp. Levels of iCGRP were measured as described in the legend to Fig. 1Go. Error bars are SEM. **p < 0.01 vs. vehicle. N = 8–16/group.

 
The next experiment determined whether activation of sympathetic fibers could alter capsaicin-evoked release of iCGRP from peripheral terminals in dental pulp (Fig. 3Go). To evaluate this hypothesis, we pre-treated separate groups of chambers with either guanethidine (100 µM) or vehicle. Chronic guanethidine administration produces a chemical sympathectomy (Demas and Bartness, 2001; Lipnicki and Drummond 2001), whereas an acute exposure to guanethidine induces a massive release of sympathetically derived neurotransmitter substances (Lipnicki and Drummond, 2001). In this study, guanethidine was acutely administered prior to the application with capsaicin. Guanethidine treatment significantly inhibited capsaicin-evoked release of iCGRP (p < 0.01). Approximately 80% of this effect was reduced in the group pre-treated with the {alpha}-antagonist phentolamine (p < 0.01).



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  		Figure 3. Effect of guanethidine treatment on capsaicin-evoked release of iCGRP from superfused dental pulp. Pulpal tissue was treated with vehicle or phentolamine 10 µM for 7 min, and then either vehicle or guanethidine, 100 µM, was administered for an additional 21 min before stimulation with capsaicin (10 µM). Levels of iCGRP were measured as described in the legend to Fig. 1Go. Error bars are SEM. **p < 0.01 vs. both other groups. N = 4–5/group.

 
To confirm these findings, we repeated this experiment using reserpine as a stimulant for sympathetic fibers (Kong et al., 1990; Vizi et al., 1992) and phenoxybenzamine as the {alpha}-antagonist (Fig. 4Go). Pre-treatment with reserpine significantly (p < 0.01) reduced the iCGRP release evoked by capsaicin (10 µM). This effect was attenuated by pre-treatment with phenoxybenzamine (p < 0.01).



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  		Figure 4. Effect of reserpine treatment on capsaicin-evoked release of iCGRP from superfused dental pulp. Pulpal tissue was treated with vehicle or phenoxybenzamine, 10 µM, for 7 min, and then either vehicle or reserpine, 100 µM, was administered for an additional 21 min before stimulation with capsaicin (10 µM). Levels of iCGRP were measured as described in the legend to Fig. 1Go. Error bars are SEM. **p < 0.01 vs. both other groups. N = 5–6/group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study evaluated the hypothesis that sympathetically derived neurotransmitters inhibit the exocytotic activity of trigeminal peptidergic afferent neurons terminating in isolated dental pulp. The results indicate that exogenous norepinephrine reduces exocytotic activity from the capsaicin-sensitive subpopulation of peptidergic nociceptors. (Capsaicin activates the VR1 receptor expressed on a large proportion of nociceptors [Caterina et al., 1997; Caterina and Julius, 2001]). The present results strongly support the hypothesis that the endogenous release of sympathetically derived neurotransmitters inhibits efferent exocytotic activity of peptidergic afferent neurons innervating dental pulp, since administration of {alpha}-adrenergic antagonists increases the spontaneous release of iCGRP, and activation of sympathetic terminals (with guanethidine or reserpine) reduces capsaicin-evoked release of iCGRP. These effects must occur via interaction with the peripheral terminals of the peptidergic afferent fibers, since this isolated superfused in vitro preparation of dental pulp precludes any possible systemic effects. It is possible that sympathetic neurotransmitters inhibit capsaicin-sensitive afferent fibers by either a direct mechanism (i.e., activation of an {alpha}-adrenoceptor expressed on the afferent fiber) or by an indirect mechanism (i.e., activation of an {alpha}-adrenoceptor expressed on an intermediate cell type).

Prior physiologic studies measuring various parameters of pulpal blood flow have suggested that sympathetic neurotransmitters inhibit the efferent release of neuropeptides from certain trigeminal sensory neurons innervating dental pulp (Kerezoudis et al., 1992, 1993a,Kerezoudis et al., b). However, to our knowledge, this is the first study to demonstrate that activation of pulpal sympathetic fibers inhibits the exocytotic activity of the capsaicin-sensitive class of pulpal sensory neurons. This constitutes direct biochemical evidence in support of sympathetic regulation of capsaicin-sensitive primary afferent fibers. Moreover, these findings support the hypothesis that sympathetic regulation of pulpal blood flow may be mediated by both direct and indirect mechanisms.

Treatment with guanethidine and reserpine acutely releases sympathetic neurotransmitters (Kong et al., 1990; Vizi et al., 1992; Demas and Bartness, 2001; Lipnicki and Drummond, 2001). In the present study, acute treatment with either agent reduced capsaicin-evoked iCGRP release by approximately 80%. Pre-treatment with {alpha}-adrenoceptor antagonists (phentolamine or phenoxybenzamine) significantly, though incompletely, reduced this effect. This incomplete blockade could be due to several possibilities. First, the concentration of the {alpha}-adrenoceptor antagonists may not have blocked all {alpha}-adrenergic receptors. Although possible, we view this hypothesis as unlikely, since the concentration of both antagonists was sufficient to increase basal rates of iCGRP release. Moreover, phenoxybenzamine is a non-competitive antagonist whose inhibitory effect is independent of pulpal catecholamine concentrations. Second, it is possible that several sympathetically derived neurotransmitters, in addition to norepinephrine, inhibit exocytosis from capsaicin-sensitive neurons. For example, neuropeptide Y (NPY) is found in pulpal sympathetic neurons and regulates pulpal blood flow (Kim et al., 1996; Zhang et al., 1998). Previously, we have demonstrated that VR1-immunoreactive sensory neurons express the Y1 receptor for NPY, and that administration of Y1 agonists inhibits capsaicin-evoked exocytosis from central neuronal terminals (in the spinal cord), from neuronal somata (trigeminal ganglion), and from peripheral terminals (in dental pulp) (Gibbs et al., unpublished observations). Third, it is possible that norepinephrine may activate ß-adrenoceptors. Although we have evidence that exogenous catecholamines can suppress capsaicin-evoked iCGRP release by a ß-adrenoceptor mechanism (Bowles et al., unpublished observations), we do not know whether endogenous catecholamines can activate these receptors. Thus, it is possible that the incomplete reversal of the inhibitory effects of guanethidine and reserpine by the {alpha}-adrenoceptor antagonists may be due to the concurrent release of multiple neurotransmitters or activation of multiple receptors that inhibit exocytosis from capsaicin-sensitive fibers.

The studies presented here provide direct biochemical support for the hypothesis that sympathetic neurotransmitters inhibit basal and stimulated neuropeptide release from capsaicin-sensitive neurons. Although iCGRP was measured, it is possible that other neuropeptides co-expressed in these neurons (e.g., substance P) may also be regulated by this mechanism. This may constitute a significant physiologic regulatory system for the control of pulpal blood flow and the initiation of neurogenic inflammation. In addition, certain persistent pain conditions that occur after injury are thought to be due to an interaction between sympathetic fibers and peripheral nociceptors (Sato and Perl, 1991; Drummond, 2001; Raja and Grabow, 2002). It is possible, therefore, that these persistent pain conditions derive in part from a pathologic alteration of this pre-existing vascular regulatory system. Increased understanding of the mechanisms mediating this change may reveal novel therapeutic approaches for managing these pain conditions.


   ACKNOWLEDGMENTS
 
This research was supported by NIDCR/NIH grants DE12888 and DE00270. We thank Dr. Christopher Flores for his advice and comments on this manuscript.

Received August 7, 2002; Last revision November 21, 2002; Accepted January 16, 2003


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