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Graduate Studies Department and 2Endodontics Department, School of Dentistry, Pontificia Universidad Javeriana, Bogota,
Colombia
Abstract
Caviedes-Bucheli J, Gutierrez-Guerra JE, Salazar F,
Pichardo D, Moreno GC, Munoz HR. Substance P receptor
expression in healthy and inflamed human pulp tissue. International Endodontic Journal, 40, 106111, 2007.
Introduction
Dental pulp inflammation is a complex process involving a variety of nervous and vascular reactions, which
are key components of the neurogenic phenomenon
that could lead to pulp necrosis (Byers et al. 1990, Kim
1990). Neuropeptides play an active role in homeostatic regulation under normal conditions and during
neurogenic inflammation of the pulp, controlling its
blood flow and regulating later stages of inflammation
106
and repairing processes (Olgart 1996). These neuropeptides include substance P (SP), calcitonin generelated peptide (CGRP), neurokinin A (NKA), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY)
amongst others (Casasco et al. 1990, Wakisaka 1990).
Substance P is produced in trigeminal cell bodies and
transported via axonal flow to nerve terminals in the
pulp, where it is co-stored with other sensory neuropeptides (Wakisaka & Akai 1989). These nerve
terminals are mainly C-type fibres, which closely follow
pulp microcirculation. When stimulated, neuropeptides
are released (Awawdeh et al. 2002).
Substance P interacts with a great variety of cells,
including mastocytes, macrophages, lymphocytes and
endothelial cells, inducing the release of inflammatory
mediators such as histamine, cytokines, prostaglandins
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Radioreceptor assay
Standard SP and 125I-SP were obtained from Phoenix
Peptide Pharmaceutical Laboratories (Ref. 061-05 and
T061-10, Belmont, CA, USA). Standard SP was reconstituted with distilled water and serially diluted with
HSA/TrisHCl buffer solution to obtain different reagent concentrations. 125I-SP was reconstituted with
distilled water and diluted until there were 10 000
counts per minute (cpm) in 100 lL reagent.
Each cell suspension sample was submitted to
competition assays with 125I-SP in the absence (total
binding) or presence (non-specific binding) of standard
SP and left incubated for 12 h at 4 C. The reaction
mixture was then passed through a 60 : 40 dibutylphthalatedioctylphthalate cushion (d 1.015 g mL)1)
to separate bound from free SP (Suarez et al. 2001).
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Statistical analysis
The values obtained are expressed in pmol of bound
peptide per 100 lg tissue protein. The mean and
standard deviation were calculated for each group. The
KruskalWallis test was performed to establish statistically significant differences in values obtained from
the three groups (P < 0.05). MannWhitneys post-hoc
tests were done for comparing differences between
different groups.
Results
Substance P receptor expression was found in all
human pulp tissue samples. Most receptors were found
in the group of pulps from teeth having a clinical
diagnosis of acute irreversible pulpitis, followed by the
group of pulps having induced inflammation. The least
number of receptors was expressed in the group of
healthy pulps.
Table 1 shows values obtained and mean receptor
expression for the three groups, with their respective
standard deviations.
The KruskalWallis test revealed statistically significant differences between the three groups (P < 0.01).
MannWhitneys post-hoc comparisons showed that the
greatest difference in the amount of receptors expressed
was between pulps having a diagnosis of acute
Healthy
pulpa
Acute irreversible
pulpitisa
1
2
3
4
5
Mean
SD
5.85
7.01
8.76
6.70
8.69
7.40
1.28
9.50
13.17
8.55
12.96
10.48
10.93
2.06
13.13
22.28
14.60
11.71
17.45
15.83
4.18
a
Values are given in pmol of bound peptide per 100 lg of tissue
protein.
Discussion
Neuropeptides and other biologically active peptides
exhibit a high degree of functional diversity within the
same group of peptides not only through the regulation
of peptide production but also through peptidereceptor
interaction. The mammalian tachykinin system represents a typical example; it comprises a group of multiple
peptides and demonstrates a functional diversity at the
levels of both peptide production and peptide reception
(Nakanishi 1991). SP induces inflammatory reactions
in peripheral tissues including the dental pulp, but its
regulatory effects in target tissues are dependent on
receptor signalling (Fristad et al. 1999). The objective
of this study was, therefore, to compare SP receptor
expression in pulp tissue having a clinical diagnosis of
acute irreversible pulpitis with pulp having induced
inflammation and healthy pulp.
Radioreceptor assay (RRA) measures a ligand interaction with its biological receptor by competition
binding assay in which receptors function as binding
proteins. RRA specificity completely depends on a
ligands affinity for its receptor (Chard 1990). This
technique has been widely used for determining the
presence of receptors, their affinity for their ligands and
determining changes in their expression in different
organs and pathological states (Caviedes-Bucheli et al.
2004b, 2005b).
Several studies have shown a significantly higher SP
expression in the inflamed pulp, suggesting that SP
plays an important role in the inflammatory process
(Byers et al. 1990, Kim 1990, Awawdeh et al. 2002,
Bowles et al. 2003, Caviedes-Bucheli et al. 2005a).
Results from the present study correlate with previous
statement, as there was a greater expression of SP
receptors in both inflammatory conditions, indicative of
SP active participation in pulpitis development.
Based on the results of a previous research (CaviedesBucheli et al. 2005b) where CGRP-receptor expression
was measured under the same conditions of the present
study, it was expected that SP receptor expression for
the group of acute irreversible pulpitis be higher than
the induced inflammation group. This could be
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Conclusion
Substance P receptor is expressed in human pulp tissue
with a significantly increased expression during clinical
inflammatory phenomena. Its presence has clinical
significance as it could play an important role in
controlling pulpal blood flow and regulating later
stages of inflammation and repair processes.
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