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Intrinsic Regulation of CGRP Release by Dental Pulp Sympathetic Fibers
1 Department of Endodontics, UTHSCSA School of Dentistry, Mail Code 7892, 7703 Floyd Curl Drive, San Antonio, TX 78229-3900; Correspondence: * corresponding author, Hargreaves{at}UTHSCSA.edu
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  -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
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.
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.
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. 1  ). 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.
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 -adrenergic receptor antagonists and compared with vehicle treatment. Phentolamine and phenoxybenzamine (10 µM) were selected, since these antagonists block both the 1 and 2 subtypes of adrenergic receptors and act via different mechanisms of action (i.e., competitive and non-competitive antagonism, respectively). As seen in Fig. 2, 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.
The next experiment determined whether activation of sympathetic fibers could alter capsaicin-evoked release of iCGRP from peripheral terminals in dental pulp (Fig. 3 ). 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 -antagonist phentolamine (p < 0.01).
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 -antagonist (Fig. 4). 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).
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 -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 -adrenoceptor expressed on the afferent fiber) or by an indirect mechanism (i.e., activation of an -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 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.
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 for publication August 7, 2002. Revision received November 21, 2002. Accepted for publication January 16, 2003.
Journal of Dental Research, Vol. 82, No. 5,
398-401 (2003)
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