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RESEARCH REPORT |
1 Department of Oral Anatomy and Cell Biology, and
2 Department of Fixed Prosthodontics, Graduate School of Dental Science, Kyushu University, Fukuoka 812-8582, Japan;
* corresponding author, kido{at}dent.kyushu-u.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: capsaicin • VR1 • tongue • oral epithelium • immunocytochemistry • rat
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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) nerve fibers (Holzer, 1991). As a general rule, capsaicin-sensitive sensory neurons contain peptides such as substance P or calcitonin gene-related peptide (CGRP), and they elicit an efferent function via a well-known "axon reflex" arrangement. Topical or intravenous application of capsaicin leads to plasma extravasation or increased blood flow in the oral mucosa of the rat, suggesting that capsaicin-sensitive afferents contribute to neurogenic inflammation in the oral cavity (Fazekás et al., 1990; Kondo et al., 1995). Furthermore, the ingestion of spicy food containing hot chili peppers is associated with an oral burning sensation (Lee, 1954). The application of capsaicin to the tongue or palate causes a reproducible burning sensation and salivation (Dunér-Engström et al., 1986). However, it is not known whether there are capsaicin receptors in the oral cavity, and little is known about the mechanisms underlying the irritant sensation. In 1997, vanilloid receptor 1 (VR1) was identified and characterized. This receptor is selective for capsaicin and participates in the detection of noxious heat (> 43°C) and acid (pH < 6) (Caterina et al., 1997; Tominaga et al., 1998). VR1 is considered a molecular integrator of noxious stimuli in the peripheral terminals of primary sensory neurons. Therefore, modulation of VR1 activity is now a pharmacological target for analgesics (Szallasi, 2001).
We provide direct evidence for the presence of VR1 in the rat oral cavity using RT-PCR and immunohistochemical techniques.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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RT-PCR
Three rats were killed with an overdose of pentobarbital sodium. The dorsal root ganglion (DRG), oral mucosa around the palatal rugae, and taste papillae were rapidly removed and frozen in liquid nitrogen. Total RNA was isolated by means of the SV Total RNA Isolation System (Promega, Madison, WI, USA), after which cDNA was synthesized by means of Rever TraAce
(Toyobo, Osaka, Japan), according to the manufacturers’ instructions. RNA was treated in parallel in the presence or absence of reverse transcriptase. PCR amplification was performed by means of a Gene Amp 9700 (PE Biosystems, Tokyo, Japan) as follows: 1 cycle at 94°C for 5 min, followed by 35 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min. The primer pairs were based on the cDNA sequence of rat VR1 (GenBank-AF029310) with G3PDH (GenBank-AB017801) as an internal control. The primer pairs were 5'-TGACCCTCTTGGTGGAGAATGG-3', and 5'-TAAGCGATCACCTCCAGAACCG-3' for VR1, and 5'-ATGGTGAAGGTCGGTGTGAAC-3', and 5'-GCTGACAA TCTTGAGGGAGT-3' for G3PDH. The amplification products were visualized on 2% agarose gels by the use of ethidium bromide and sequenced so that their identity could be confirmed.
Immunohistochemistry
Six rats were anesthetized by intraperitoneal injection of pentobarbital sodium (50 mg/kg) and perfused with heparinized saline, followed by a cold fixative containing 4% paraformaldehyde and 0.2% picric acid in phosphate buffer (PB). The palate and tongue, including the taste papillae, and dorsal root ganglia were dissected out and processed for immunohistochemistry by the avidin-biotin-peroxidase (ABC) method, as described previously (Kido et al., 2001). The sections were incubated with rabbit protein-gene-related product 9.5 (PGP 9.5) antiserum (Ultraclone, Wellow, UK), CGRP antiserum (Amersham, Piscataway, NJ, USA), or VR1 capsaicin receptor (Ab-1) antibody (Oncogene, Cambridge, MA, USA, 1:100), followed by goat biotinylated anti-rabbit IgG (1:200, Vector Laboratories, Burlingame, CA, USA) for 45 min and avidin-biotin-complex (1:50, Vector Laboratories) for 1 hr. The peroxidase reaction was developed for 5–10 min in 0.05 M Tris buffer, pH 7.6, containing 0.02% diaminobenzidine and 0.006% H2O2. Then, the specimens were counterstained with hematoxylin and photographed with an Axiocam on Axioskop2 plus Microscope (Carl Zeiss, Goettingen, Germany).
Immunohistochemical controls involved immunostaining without the primary or secondary antibody or the avidin-biotin complex, or replacing the primary antibody with normal rabbit IgG or with antisera that were pre-adsorbed with the immunizing control peptide at 10-4-10-6 M.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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shows palatal and lingual views of the rat oral cavity. VR1 was localized in the intermolar rugae of the hard palate, the "Geschmacksstreifen" (taste stripe) of the soft palate, and the taste papillae of the tongue.
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). Two mRNA splice variants, VR1 and VR.5'sv, derived from the rat capsaicin receptor gene, have been identified so far. We used primer pairs specific to VR1. The VR1 PCR product was confirmed by sequencing.
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In the fungiform papillae (Figs. 3A–3C), numerous PGP-, CGRP- (Fig. 3C), or VR1-positive nerve fibers and bundles entered the base of each papilla, forming a subepithelial plexus. Nerve branches originating from the central stem were often observed, and ramifying branches penetrated the epithelium and taste buds. The VR1 staining pattern resembled that of CGRP. Small VR1-immunoreactive bundles surrounded taste buds, and some branches ran into the epithelium with small varicosities (Fig. 3B). Many fibers were distributed to the epithelium around taste buds. The density of VR1 nerves varied among the fungiform papillae.
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, 3E, 3F) and foliate (Figs. 3G, 3H, 3I, 3J) papillae, a dense network of nerve fibers was seen in the furrows of the papillae, at the subepithelial layer, and surrounding the taste bud area. The nerve fibers penetrated the basal lamina of the taste buds, and occasional varicose fibers reached the tips of the buds. Interestingly, the epithelium just around the taste buds received more VR1-immunoreactive nerve fibers than did the taste buds. Conspicuous VR1 nerves penetrated the boundary area of the epithelium just above the upper taste buds (Figs. 3F, 3I). Some fibers appeared to penetrate just underneath the stratum corneum and had terminal swellings. There were more nerves in the foliate papillae than in the circumvallate papillae.
The distribution patterns of PGP and VR1 immunoreactivity in the Geschmacksstreifen (Figs. 3K, 3L) resembled that in fungiform papilla.
VR1 Expression in the Oral Epithelium
VR1 was expressed in the epithelium facing the oral cavity. VR1 expression was seen in the tongue epithelium on top of the papillae (Figs. 3A, 3D, 3G, 3K). Taste cells seemed to be devoid of VR1 immunoreactivity, although the taste buds received a few VR1-positive nerves. Surprisingly, the strongest and most conspicuous VR1 expression was observed in the palatal epithelium, especially at the summit of the intermolar rugae (Figs. 4A, 4B), although they were not served by VR1-immunoreactive nerves. Immunoreactivity was observed from the stratum granulosum to the basal layer; the strongest staining was just beneath the spinosum corneum. PGP- or CGRP-immunoreactive nerves penetrated the rugal epithelium (Figs. 4C, 4D).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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It is interesting that epithelial VR1-immunoreactive nerves were more prominent than the nerves in the taste buds, which may allow for the simultaneous detection of various tastes with capsaicin or heat. This idea is in harmony with the observation that local capsaicin desensitization of the human tongue does not impair taste sensation (Szolcsanyi, 1977), while capsaicin changes the perception of a variety of tastes, despite acting mainly on nociceptors in humans (Karrer and Bartoshuk, 1995). These observations lead us to conjecture that VR1 is involved in the taste perception of special "hot" sense systems.
Recently, VR1 expression in epithelial cells has been demonstrated in the stomach (Nozawa et al., 2001). We showed that VR1 was localized in the epithelial cells of the dorsal surface of the tongue and the palate. The lingual epithelial expression of VR1 is partly in accord with the report of Liu and Simon (2001), who used RT-PCR to demonstrate the presence of VR1 in taste receptor cells and epithelial cells of the fungiform papillae. The application of capsaicin to the tongue or palate causes a burning sensation and salivation (Dunér-Engström et al., 1986). VR1 expression in the lingual epithelium and its unexpected strong expression at the summit of the palatal intermolar rugae support the idea that oral epithelial cells have specialized sensory and signaling properties. VR1 expression was stronger in the surface layer of the epithelium, suggesting that these cells can sense the chemical and physical environments. Furthermore, epithelial cells with the expression of VR1 may engage in reciprocal communication with neighboring nerves.
In conclusion, the capsaicin VR1 receptor-expressing nerves and epithelial cells in the oral cavity might provide defense mechanisms against toxic substances, and may be a future therapeutic target for the relief of oral pain.
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ACKNOWLEDGMENTS |
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Received June 24, 2002; Last revision January 23, 2003; Accepted January 31, 2003
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REFERENCES |
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