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Expression of calcitonin gene-related peptide-1 receptor mRNA in human tooth pulp and trigeminal ganglion
ARCHIVES OF
PERGAMON
Archives of Oral Biology 44 (1999) 1±6
ORAL BIOLOGY
Expression of calcitonin gene-related peptide-1 receptor mRNA in human tooth pulp and trigeminal ganglion R. Uddman a, *, J. Kato b, P. Lindgren c, F. Sundler d, L. Edvinsson e a
Department of Oto-rhino-laryngology, MalmoÈ General Hospital, S-20502 MalmoÈ, Sweden Department of Oral Anatomy and Developmental Biology, Osaka University, Faculty of Dentristy, Osaka, Japan c Department of Maxillofacial Surgery, MalmoÈ General Hospital, MalmoÈ, Sweden d Department of Physiology and Neuroscience, Lund University Hospital, Lund, Sweden e Division of Experimental Vascular Research, Department of Internal Medicine, Lund University Hospital, Lund, Sweden b
Accepted 6 October 1998
Abstract Numerous nerve ®bres containing calcitonin gene-related peptide (CGRP) were found by immunocytochemistry in human molar pulp. These nerves were often seen around small blood vessels and as free endings without vascular contact. In the trigeminal ganglion a large number of CGRP-immunoreactive nerve-cell bodies, mostly of small to medium size, was encountered. Reverse transcriptase-polymerase chain reaction, using speci®c sense and antisense primers, detected mRNA expression of the human CGRP1 receptor in the pulp tissue and the trigeminal ganglion. Thus, both CGRP-containing nerve ®bres and CGRP1 receptor mRNA are present in human tooth pulp, where they may be involved in the regulation of vascular tone and other local reactions to injury. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Calcitonin gene-related peptide; Tooth pulp; Immunocytochemistry; Polymerase chain reaction
1. Introduction CGRP is a 37-amino acid peptide identi®ed from structural analysis of the products of calcitonin gene expression (Amara et al., 1982; Rosenfeld et al., 1983). The calcitonin gene can give rise to either calcitonin or CGRP. A second form of CGRP, b-CGRP, is the product of a dierent gene (Amara et al., 1985). Two human forms of CGRP, a and b, which dier in three out of 37 amino acids, are reported (Steenbergh et al., 1985). CGRP-immunoreactive nerve ®bres are abundant in the dorsal horns of the spinal cord and in sensory nuclei of the lower brainstem (Gibson et al., 1984; Sko®tsch and Jacobowitz, 1985).
Abbreviations: CGRP, calcitonin gene-related peptide, RT-PCR, reverse transcriptase-polymerase chain reaction. * Corresponding author.
In the tooth pulp there is a rich supply of peptide-containing nerve ®bres of which those containing substance P and CGRP are predominant (Olgart et al., 1977; Wakisaka et al., 1984, 1987; Uddman et al., 1986). CGRP is involved in peripheral sensory processing (Lundberg et al., 1985; Maggi, 1995). Sensory nerve reactions to dental injuries include sprouting of sensory ®bres and increased immunoreactivity for neuropeptides such as substance P and CGRP (Kimberley and Byers, 1988). Furthermore, CGRP is a potent vasodilator (Brain et al. 1985). In the guinea-pig ileum, fragments of CGRP (CGRP8±37 and CGRP12±37) act as competitive antagonists. However, the fragments do not antagonize the electrically stimulated, CGRP-induced inhibition of contraction of rat vas deferens. These ®ndings have led to the hypothesis that there are two subtypes of CGRP receptors of which CGRP1 is highly sensitive to CGRP8±37 while CGRP2 is not (Poyner, 1992;
0003-9969/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 9 8 ) 0 0 1 0 2 - 2
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R. Uddman et al. / Archives of Oral Biology 44 (1999) 1±6
Wimalawansa, 1996). One CGRP subreceptor has been cloned while the other is as yet only pharmacologically characterized (Aiyar et al., 1996). We have now investigated the distribution of CGRP-containing nerve ®bres in human pulp tissue and of CGRP-containing nerve-cell bodies in human trigeminal ganglia. In addition, we sought to demonstrate the presence of human CGRP1 receptor mRNA in these structures, showing a possible site of action.
active intestinal peptide, somatostatin or calcitonin (see also Grunditz et al., 1986). Additionally, the antiserum was tested for cross-reaction with other peptides (10±100 mg of peptide per ml diluted antiserum). No cross-reaction was found. Cross-reactions with still other peptides or proteins containing amino acid sequences recognized by the dierent antisera cannot be excluded. It is appropriate, therefore, to refer to the immunoreactive material as CGRP-like or CGRP-immunoreactive. For brevity, the shorter term CGRP is often used.
2. Materials and methods
2.3. Isolation of total RNA
2.1. Tissues
Total cellular RNA was extracted from frozen pulp tissue and trigeminal ganglia using the TRIzol Reagent according to the description provided (Gibco Brl, Life Technologies, Sweden). The RNA pellet was washed with 70% ice-cold ethanol, air-dried, dissolved in 20 ml of diethylpyrocarbonate-treated water and stored at ÿ208C until use. The purity and yield of total RNA was determined spectrophotometrically by measuring the optical density of a portion at 260 and 280 nm in a DU-65 spectrophotometer (Beckman Instruments, Sweden). The ratio of absorption (260:280) was between 1.6 and 1.8. Finally, samples were subjected to gel electrophoresis and stained with ethidium bromide to prove the integrity of the 18 and 28 S ribosomal RNAs.
For immunocytochemistry, pulp tissue, taken from extracted, partially erupted third molars, was obtained from six patients (age range 24±35 years). The teeth were split open and the tissue immediately placed in a ®xative. Trigeminal ganglia were obtained at autopsy within 8 h of death. None of the donors had suered from disease of the central nervous system. Average age at death was 74.8 years (range 51±85 years). For RNA experiments, pulp tissue obtained from six patients (third molars, age range 19±32 years) and trigeminal ganglia (autopsy specimens) collected from another six patients were snap frozen in liquid nitrogen immediately after acquisition and stored at ÿ708C until use. The project was approved by the Ethics Committee of Lund University. 2.2. Immunocytochemistry The specimens were ®xed by immersion in a mixture of 2% formaldehyde and 0.2% picric acid in 0.1 mol/l phosphate buer (pH 7.2) overnight and rinsed thoroughly in Tyrode buer containing 10% sucrose. They were frozen on dry ice and sectioned at 10 mm in a cryostat. The sections were thawmounted on to chrome±alum slides. For the immunocytochemical demonstration of CGRP, indirect immuno¯uorescence was used. The CGRP antiserum (Eurodiagnostica, MalmoÈ, Sweden) was raised in rabbit and used at a dilution of 1:1280. The sections were exposed to the primary antiserum overnight at 48C in a moist chamber. After rinsing for 10 min in phosphate-buered saline (three rinses), the sections were incubated for 90 min with a secondary antibody. The site of the antigen±antibody reaction was revealed by application of ¯uorescein isothiocyanatelabelled pig antirabbit immunoglobulin G (Dako, Copenhagen, Denmark), in a dilution of 1:320, for 1 h at room temperature. The antiserum does not cross-react with substance P, neurokinin A, vaso-
2.4. Removal of genomic DNA from RNA samples In order to eliminate any residual contaminating DNA that might produce a false-positive ampli®cation signal in RT-PCR, duplicate tubes containing 1 mg of total RNA were pretreated with 1 unit of ampli®cation grade DNase I (Gibco) in DNase I reaction buer, in the presence of 20 units of RNase inhibitor (Perkin Elmer AB, Sweden). 2.5. Reverse transcriptase-polymerase chain reaction Synthesis of ®rst-strand cDNA and subsequent PCR ampli®cation were done with GeneAmp RNA PCR kit reagents (Perkin-Elmer) in a PCR DNA thermal cycler (Perkin-Elmer). DNase-treated RNA samples were reverse-transcribed to cDNA in a 20-ml reaction volume in the presence of PCR buer (50 mM KCl, 10 mM Tris±HCl, pH 8.3), 5 mM MgCl2, 1 mM of each deoxyribonucleoside triphosphate, 50 pmol of oligo-(dT) primers, 50 units of Moloney murine Leukaemia virus reverse transcriptase. To determine if the ampli®cation product came exclusively from the RNA, a reverse-transcriptase negative reaction was run in which the enzyme was
R. Uddman et al. / Archives of Oral Biology 44 (1999) 1±6
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Fig. 1. (a) Human dental pulp (coronal part). A rich supply of ®ne beaded CGRP-containing nerve ®bres is seen in the stroma. (b) Human trigeminal ganglion. A moderate number of CGRP-immunoreactive nerve cell bodies mostly of small to medium size is seen. Numerous lipofuchsin granules scattered among the cells give non-speci®c staining. Bar = 40 mm.
replaced with RNase-free water. The samples (20 ml) were incubated at room temperature for 10 min, at 428C for 15 min, heated to 998C for 5 min and chilled to 58C for 5 min. The PCR reaction was done with speci®c oligonucleotide primers constructed according to published sequences of the human CGRP1 receptor and designed by computer using the OLIGO primer analysis software, version 4.0. The primers used to amplify a fragment of the human CGRP1 receptor generating a 339-bp product were as follows: sense primer, 5 0 -TCT GGT TCT CTT GCC TTT TTT TAT G-3 0 , corresponding to nucleotides 581±605; antisense primer, 5 0 -GTC CAT GTT CTG TTG CTT GCT G-3 0 complementary to nucleotides 898± 919. The PCR ampli®cation reaction for CGRP1 was composed of 2 ml of the ®rst-strand cDNA reaction mixture and 48 ml master mix containing PCR buer, 1.0 mM MgCl2, 0.2 mM of each sense and antisense primers, and 1.2 units of AmpliTaq Gold DNA polymerase. The PCR used for CGRP1 was: 9 min at 948C before undergoing 35 cycles of 1 min at 948C, 1 min at 638C, followed by a 7-min ®nal extension at 728C. 2.6. Electrophoretic analysis Portions (10 ml) from all PCR-ampli®ed products were electrophoretically separated on a 1.5% agarose gel (Gibco) containing 0.5 mg/ml ethidium bromide (Sigma E 1510), in TBE buer (89 mM Tris±borate, 2 mM EDTA, pH 8.0) at 5 V/cm for 1.5 h. This analysis was performed in a 20 10 cm Midicell,
Model EC 350 (E-C Apparatus Corporation; Techtum Lab AB, Sweden). A 100-bp DNA ladder (Promega, SDS, Sweden) was run in each of the outside lanes to con®rm the molecular size of the ampli®cation product. 3. Results Numerous ®ne-beaded CGRP-containing nerve ®bres were seen in the apical and coronal parts of the pulp tissue in all specimens examined. Control sections were negative. Some of the immunoreactive nerve ®bres were found in connection with blood vessels but the majority were in the stroma without apparent association with vessels (Fig. 1). Occasional ®bres were found close to the subodontoblastic layer. In the trigeminal ganglia, a large number of CGRP-immunoreactive nerve-cell bodies of mostly small to medium size was seen (Fig. 1). Auto¯uorescent lipofuscin granules characteristic of adult human nervous tissue were present within many perikarya. Total RNA was successfully extracted from the tooth pulp and the trigeminal ganglia. By using one forward and one backward primer in RT-PCR, the presence of mRNA for the human CGRP1 receptor was shown. Agarose gel electrophoresis of the RTPCR products from human pulp tissue and trigeminal ganglia demonstrated products of the expected sizes, corresponding to mRNA encoding the human CGRP1 receptor (339 bp) (Fig. 2). In negative controls, in which the reverse-transcriptase enzyme was replaced by RNAse-free water, no band was detected.
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Fig. 2. Gel electrophoresis of RT-PCR reaction products from human dental pulp and trigeminal ganglia after 35 cycles of ampli®cation of mRNA fragments corresponding to human CGRP1 receptor transcripts. Pulp (lane 3); trigeminal ganglion (lane 5). As negative controls, no ampli®cation products occurred when reverse transcriptase was omitted in the ®rst-strand cDNA reaction (lane 2 and 4). A 100-bp DNA ladder (Promega) con®rms the molecular size of the ampli®cation product (lane 1).
4. Discussion CGRP is a neuropeptide widely distributed in the central and peripheral nervous systems. Here we demonstrate numerous ®ne-beaded CGRP-containing nerve ®bres in the human dental pulp, consistent with previous ®ndings (Silverman and Kruger, 1987; Casasco et al., 1990; Luthman et al., 1992). The ®bres were located apically and coronally. Some ®bres were close to small blood vessels, but most were in the stroma with no apparent relation to vessels. The trigeminal ganglia, which contain the cell bodies of the CGRP ®bres, were rich in CGRP-containing cell bodies and nerve ®bres. CGRP appears to have two roles in sensory neurones, (a) to modulate sensory neurotransmission and (b) to relax blood vessels (Wimalawansa, 1996). In sensory neurones, CGRP may coexist with substance P and to a lesser degree with pituitary adenylate cyclaseactivating peptide (Mulder et al., 1994). After dental injuries there is sprouting of sensory nerves and an increase in neuropeptide immunoreactivity (Taylor
et al., 1988; Byers et al., 1990). CGRP in dorsal-root ganglia may be upregulated in response to peripheral in¯ammation (Walsh et al., 1992) which further suggests that CGRP is involved in the response to in¯ammation and nociception. In addition, CGRP, released from the peripheral aerent nerve-®bre endings of sensory neurones, induces vasodilatation and increases vascular permeability in the dental pulp (Heyeraas and Kvinnsland 1992; Olgart, 1992; Heyeraas et al., 1994). It has been demonstrated that activation of the CGRP1 receptor results in dilatation of human cerebral arteries (Jansen-Olesen et al., 1996). A possible role for sensory peptides in transendothelial migration of immunocompetent cells has been suggested. Following electrical stimulation of the crown an increased number of immunocompetent cells can be seen in the pulp of rats (Fristad et al., 1997). Recently, the cloning of a cDNA encoding a human CGRP1 receptor was reported (Aiyar et al., 1996). This product shares signi®cant sequence homology with the human calcitonin receptor, a member of the G-protein-coupled receptor superfamily. Highly speci®c CGRP receptors have been identi®ed both in the intima and the media of peripheral blood vessels such as the superior mesenteric, the femoral and the coronary arteries (Sigrist et al., 1986; Wimalawansa et al., 1987). CGRP causes marked stimulation of adenylate cyclase in cultured aortic smooth muscle and endothelial cells. These binding sites are linked to the generation of cyclic AMP via a non-endothelium-dependent mechanism (Edvinsson et al., 1985). The C-terminal fragment of human a-CGRP, hCGRP [8±37], has an antagonistic eect on CGRP-induced adenylate cyclase activity in liver-cell membranes and acts as a competitive a-hCGRP receptor antagonist in the cardiovascular system of the rat, in the guinea-pig atrium and the guinea-pig ileum (Dennis et al., 1990; Donoso et al., 1990, Jansen, 1991; Jansen-Olesen et al., 1996). hCGRP [8±37] has a weak antagonistic eect in rat vas deferens and is ineective against a-hCGRPinduced hyperthermia. Consequently, the existence of at least two classes of CGRP receptors has been suggested (Dennis et al., 1990), one being sensitive and the other being insensitive to hCGRP [8±37] (Poyner, 1992). The CGRP1 receptor is highly sensitive to the antagonistic properties of CGRP [8±37]. Receptors that do not respond to CGRP [8±37] are at present grouped together as CGRP2 (Dennis et al., 1990). Thus, the ®nding of CGRP1 receptor mRNA in the tooth pulp is compatible with a role for CGRP in modulating neuronal activities and agrees well with previous studies which have suggested that CGRP may, like substance P, have a role in nociception (Olgart et al., 1977; Andersen et al., 1978; Henry et al., 1980; Wakisaka et al., 1987).
R. Uddman et al. / Archives of Oral Biology 44 (1999) 1±6
The mRNA for the CGRP1 receptor has been shown in the human trigeminal ganglion and cerebral arteries (Edvinsson et al., 1997). With the use of speci®c primers for the CGRP1 receptor we here provide evidence for the expression of the CGRP1 receptor mRNA in human pulp tissue. This suggests a target role of these receptors for CGRP released from pulpal nerve ®bres. However, we cannot with RT-PCR demonstrate the exact localization of the receptors; this will have to await the development of suitable in situ PCR for this tissue. In conclusion, the presence of CGRP-containing nerve ®bres around blood vessels in the human pulp tissue together with the detection of CGRP1 receptor mRNA in the same area, and the known eects of CGRP, make it conceivable that the CGRP1 receptor plays a part in the regulation of pulpal sensitivity. The known vasodilatory eect of CGRP suggests that part of the vasodilatation is mediated by a CGRP1 receptor.
Acknowledgements This study was supported by the Swedish Medical Research Council (nos. 5958 and 11238).
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Luthman, J., Luthman, D., HoÈkfelt, T., 1992. Occurrence and distribution of dierent neurochemical markers in the human dental pulp. Archs. oral Biol. 37, 193±208. Maggi, C.A., 1995. Tachykinins and calcitonin gene-related peptide (CGRP) as co-transmitters released from peripheral endings of sensory nerves. Progr. Neurobiol. 45, 1±98. Mulder, H., Uddman, R., Moller, K., Zhang, Y.Z., Ekblad, E., Alumets, J., Sundler, F., 1994. Pituitary adenylate cyclase activating polypeptide expression in sensory neurons. Neuroscience 63, 307±312. Olgart, L., HoÈkfelt, T., Nilsson, G., Pernow, B., 1977. Localization of substance P-like immunoreactivity in nerves in the tooth pulp. Pain 4, 153±159. Olgart, L.M., 1992. Involvement of sensory nerves in hemodynamic reactions. Proc. Finn. Dent. Soc. 88 (Suppl 1), 403± 410. Poyner, D.R., 1992. Calcitonin gene-related peptide: multiple actions, multiple receptors. Pharmac. Ther. 56, 23±51. Rosenfeld, M.G., Mermod, J.J., Amara, S.G., Swanson, L.W., Sawchenko, P.E., Rivier, J., Vale, W.W., Evans, R.M., 1983. Production of a novel neuropeptide encoded by the calcitonin gene via tissue-speci®c RNA processing. Nature 304, 129±135. Sigrist, S., Franco-Cereceda, A., Mu, R., Henke, H., Lundberg, J.M., Fisher, J.A., 1986. Speci®c receptor and cardiovascular eects of calcitonin gene-related peptide. Endocrinology 119, 381±389. Silverman, J.D., Kruger, L., 1987. An interpretation of dental innervation based upon the pattern of calcitonin gene-related peptide (CGRP)-immunoreactive thin sensory axons. Somatosensory Res. 5, 157±175. Sko®tsch, G., Jacobowitz, D.M., 1985. Calcitonin gene-related peptide: detailed immunohistochemical distribution in the central nervous system. Peptides 6, 721±745.
Steenbergh, P.H., Hoppener, J.W.M., Zandberg, J., Lips, C.J.M., Jansz, H.S., 1985. A second human calcitonin/ CGRP gene. FEBS Lett. 183, 403±407. Taylor, P.E., Byers, M.R., Redd, P.E., 1988. Sprouting of CGRP nerve ®bers in response to dentin injury in rat molars. Brain Res 461, 371±376. Uddman, R., Grunditz, T., Sundler, F., 1986. Calcitonin gene related peptide: a sensory transmitter in dental pulps?. Scand. J. Dent. Res. 94, 219±224. Wakisaka, S., Ichikawa, H., Nishimoto, S., Matsuo, S., Yamamoto, K., Nakata, T., Akai, M., 1984. Substance Plike immunoreactivity in the pulp-dentine zone of human molar teeth demonstrated by indirect immuno¯uorescence. Archs. oral Biol. 29, 73±75. Wakisaka, S., Ichikawa, H., Nishikawa, S., Matsuo, S., Takano, Y., Akai, M., 1987. The distribution and origin of calcitonin gene-related peptide-containing nerve ®bres in feline dental pulp. Relationship with substance P-containing ®bres. Histochemistry 86, 585±589. Walsh, D.A., Wharton, J., Blake, D.R., Polak, J.M., 1992. Neural and endothelial regulatory peptides, their possible involvement in in¯ammation. Int. J. Tiss. React. 14, 101± 111. Wimalawansa, S.J., 1996. Calcitonin gene-related peptide and its receptors: molecular genetics, physiology, pathophysiology, and therapeutic potentials. Endocrine Rev. 17, 533± 585. Wimalawansa, S.J., Emson, P.C., MacIntyre, I., 1987. Regional distribution of calcitonin gene-related peptide and its speci®c binding sites in rats with particular reference to the nervous system. Neuroendocrinology 46, 131± 136.
PERGAMON
Archives of Oral Biology 44 (1999) 1±6
ORAL BIOLOGY
Expression of calcitonin gene-related peptide-1 receptor mRNA in human tooth pulp and trigeminal ganglion R. Uddman a, *, J. Kato b, P. Lindgren c, F. Sundler d, L. Edvinsson e a
Department of Oto-rhino-laryngology, MalmoÈ General Hospital, S-20502 MalmoÈ, Sweden Department of Oral Anatomy and Developmental Biology, Osaka University, Faculty of Dentristy, Osaka, Japan c Department of Maxillofacial Surgery, MalmoÈ General Hospital, MalmoÈ, Sweden d Department of Physiology and Neuroscience, Lund University Hospital, Lund, Sweden e Division of Experimental Vascular Research, Department of Internal Medicine, Lund University Hospital, Lund, Sweden b
Accepted 6 October 1998
Abstract Numerous nerve ®bres containing calcitonin gene-related peptide (CGRP) were found by immunocytochemistry in human molar pulp. These nerves were often seen around small blood vessels and as free endings without vascular contact. In the trigeminal ganglion a large number of CGRP-immunoreactive nerve-cell bodies, mostly of small to medium size, was encountered. Reverse transcriptase-polymerase chain reaction, using speci®c sense and antisense primers, detected mRNA expression of the human CGRP1 receptor in the pulp tissue and the trigeminal ganglion. Thus, both CGRP-containing nerve ®bres and CGRP1 receptor mRNA are present in human tooth pulp, where they may be involved in the regulation of vascular tone and other local reactions to injury. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Calcitonin gene-related peptide; Tooth pulp; Immunocytochemistry; Polymerase chain reaction
1. Introduction CGRP is a 37-amino acid peptide identi®ed from structural analysis of the products of calcitonin gene expression (Amara et al., 1982; Rosenfeld et al., 1983). The calcitonin gene can give rise to either calcitonin or CGRP. A second form of CGRP, b-CGRP, is the product of a dierent gene (Amara et al., 1985). Two human forms of CGRP, a and b, which dier in three out of 37 amino acids, are reported (Steenbergh et al., 1985). CGRP-immunoreactive nerve ®bres are abundant in the dorsal horns of the spinal cord and in sensory nuclei of the lower brainstem (Gibson et al., 1984; Sko®tsch and Jacobowitz, 1985).
Abbreviations: CGRP, calcitonin gene-related peptide, RT-PCR, reverse transcriptase-polymerase chain reaction. * Corresponding author.
In the tooth pulp there is a rich supply of peptide-containing nerve ®bres of which those containing substance P and CGRP are predominant (Olgart et al., 1977; Wakisaka et al., 1984, 1987; Uddman et al., 1986). CGRP is involved in peripheral sensory processing (Lundberg et al., 1985; Maggi, 1995). Sensory nerve reactions to dental injuries include sprouting of sensory ®bres and increased immunoreactivity for neuropeptides such as substance P and CGRP (Kimberley and Byers, 1988). Furthermore, CGRP is a potent vasodilator (Brain et al. 1985). In the guinea-pig ileum, fragments of CGRP (CGRP8±37 and CGRP12±37) act as competitive antagonists. However, the fragments do not antagonize the electrically stimulated, CGRP-induced inhibition of contraction of rat vas deferens. These ®ndings have led to the hypothesis that there are two subtypes of CGRP receptors of which CGRP1 is highly sensitive to CGRP8±37 while CGRP2 is not (Poyner, 1992;
0003-9969/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 9 8 ) 0 0 1 0 2 - 2
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R. Uddman et al. / Archives of Oral Biology 44 (1999) 1±6
Wimalawansa, 1996). One CGRP subreceptor has been cloned while the other is as yet only pharmacologically characterized (Aiyar et al., 1996). We have now investigated the distribution of CGRP-containing nerve ®bres in human pulp tissue and of CGRP-containing nerve-cell bodies in human trigeminal ganglia. In addition, we sought to demonstrate the presence of human CGRP1 receptor mRNA in these structures, showing a possible site of action.
active intestinal peptide, somatostatin or calcitonin (see also Grunditz et al., 1986). Additionally, the antiserum was tested for cross-reaction with other peptides (10±100 mg of peptide per ml diluted antiserum). No cross-reaction was found. Cross-reactions with still other peptides or proteins containing amino acid sequences recognized by the dierent antisera cannot be excluded. It is appropriate, therefore, to refer to the immunoreactive material as CGRP-like or CGRP-immunoreactive. For brevity, the shorter term CGRP is often used.
2. Materials and methods
2.3. Isolation of total RNA
2.1. Tissues
Total cellular RNA was extracted from frozen pulp tissue and trigeminal ganglia using the TRIzol Reagent according to the description provided (Gibco Brl, Life Technologies, Sweden). The RNA pellet was washed with 70% ice-cold ethanol, air-dried, dissolved in 20 ml of diethylpyrocarbonate-treated water and stored at ÿ208C until use. The purity and yield of total RNA was determined spectrophotometrically by measuring the optical density of a portion at 260 and 280 nm in a DU-65 spectrophotometer (Beckman Instruments, Sweden). The ratio of absorption (260:280) was between 1.6 and 1.8. Finally, samples were subjected to gel electrophoresis and stained with ethidium bromide to prove the integrity of the 18 and 28 S ribosomal RNAs.
For immunocytochemistry, pulp tissue, taken from extracted, partially erupted third molars, was obtained from six patients (age range 24±35 years). The teeth were split open and the tissue immediately placed in a ®xative. Trigeminal ganglia were obtained at autopsy within 8 h of death. None of the donors had suered from disease of the central nervous system. Average age at death was 74.8 years (range 51±85 years). For RNA experiments, pulp tissue obtained from six patients (third molars, age range 19±32 years) and trigeminal ganglia (autopsy specimens) collected from another six patients were snap frozen in liquid nitrogen immediately after acquisition and stored at ÿ708C until use. The project was approved by the Ethics Committee of Lund University. 2.2. Immunocytochemistry The specimens were ®xed by immersion in a mixture of 2% formaldehyde and 0.2% picric acid in 0.1 mol/l phosphate buer (pH 7.2) overnight and rinsed thoroughly in Tyrode buer containing 10% sucrose. They were frozen on dry ice and sectioned at 10 mm in a cryostat. The sections were thawmounted on to chrome±alum slides. For the immunocytochemical demonstration of CGRP, indirect immuno¯uorescence was used. The CGRP antiserum (Eurodiagnostica, MalmoÈ, Sweden) was raised in rabbit and used at a dilution of 1:1280. The sections were exposed to the primary antiserum overnight at 48C in a moist chamber. After rinsing for 10 min in phosphate-buered saline (three rinses), the sections were incubated for 90 min with a secondary antibody. The site of the antigen±antibody reaction was revealed by application of ¯uorescein isothiocyanatelabelled pig antirabbit immunoglobulin G (Dako, Copenhagen, Denmark), in a dilution of 1:320, for 1 h at room temperature. The antiserum does not cross-react with substance P, neurokinin A, vaso-
2.4. Removal of genomic DNA from RNA samples In order to eliminate any residual contaminating DNA that might produce a false-positive ampli®cation signal in RT-PCR, duplicate tubes containing 1 mg of total RNA were pretreated with 1 unit of ampli®cation grade DNase I (Gibco) in DNase I reaction buer, in the presence of 20 units of RNase inhibitor (Perkin Elmer AB, Sweden). 2.5. Reverse transcriptase-polymerase chain reaction Synthesis of ®rst-strand cDNA and subsequent PCR ampli®cation were done with GeneAmp RNA PCR kit reagents (Perkin-Elmer) in a PCR DNA thermal cycler (Perkin-Elmer). DNase-treated RNA samples were reverse-transcribed to cDNA in a 20-ml reaction volume in the presence of PCR buer (50 mM KCl, 10 mM Tris±HCl, pH 8.3), 5 mM MgCl2, 1 mM of each deoxyribonucleoside triphosphate, 50 pmol of oligo-(dT) primers, 50 units of Moloney murine Leukaemia virus reverse transcriptase. To determine if the ampli®cation product came exclusively from the RNA, a reverse-transcriptase negative reaction was run in which the enzyme was
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Fig. 1. (a) Human dental pulp (coronal part). A rich supply of ®ne beaded CGRP-containing nerve ®bres is seen in the stroma. (b) Human trigeminal ganglion. A moderate number of CGRP-immunoreactive nerve cell bodies mostly of small to medium size is seen. Numerous lipofuchsin granules scattered among the cells give non-speci®c staining. Bar = 40 mm.
replaced with RNase-free water. The samples (20 ml) were incubated at room temperature for 10 min, at 428C for 15 min, heated to 998C for 5 min and chilled to 58C for 5 min. The PCR reaction was done with speci®c oligonucleotide primers constructed according to published sequences of the human CGRP1 receptor and designed by computer using the OLIGO primer analysis software, version 4.0. The primers used to amplify a fragment of the human CGRP1 receptor generating a 339-bp product were as follows: sense primer, 5 0 -TCT GGT TCT CTT GCC TTT TTT TAT G-3 0 , corresponding to nucleotides 581±605; antisense primer, 5 0 -GTC CAT GTT CTG TTG CTT GCT G-3 0 complementary to nucleotides 898± 919. The PCR ampli®cation reaction for CGRP1 was composed of 2 ml of the ®rst-strand cDNA reaction mixture and 48 ml master mix containing PCR buer, 1.0 mM MgCl2, 0.2 mM of each sense and antisense primers, and 1.2 units of AmpliTaq Gold DNA polymerase. The PCR used for CGRP1 was: 9 min at 948C before undergoing 35 cycles of 1 min at 948C, 1 min at 638C, followed by a 7-min ®nal extension at 728C. 2.6. Electrophoretic analysis Portions (10 ml) from all PCR-ampli®ed products were electrophoretically separated on a 1.5% agarose gel (Gibco) containing 0.5 mg/ml ethidium bromide (Sigma E 1510), in TBE buer (89 mM Tris±borate, 2 mM EDTA, pH 8.0) at 5 V/cm for 1.5 h. This analysis was performed in a 20 10 cm Midicell,
Model EC 350 (E-C Apparatus Corporation; Techtum Lab AB, Sweden). A 100-bp DNA ladder (Promega, SDS, Sweden) was run in each of the outside lanes to con®rm the molecular size of the ampli®cation product. 3. Results Numerous ®ne-beaded CGRP-containing nerve ®bres were seen in the apical and coronal parts of the pulp tissue in all specimens examined. Control sections were negative. Some of the immunoreactive nerve ®bres were found in connection with blood vessels but the majority were in the stroma without apparent association with vessels (Fig. 1). Occasional ®bres were found close to the subodontoblastic layer. In the trigeminal ganglia, a large number of CGRP-immunoreactive nerve-cell bodies of mostly small to medium size was seen (Fig. 1). Auto¯uorescent lipofuscin granules characteristic of adult human nervous tissue were present within many perikarya. Total RNA was successfully extracted from the tooth pulp and the trigeminal ganglia. By using one forward and one backward primer in RT-PCR, the presence of mRNA for the human CGRP1 receptor was shown. Agarose gel electrophoresis of the RTPCR products from human pulp tissue and trigeminal ganglia demonstrated products of the expected sizes, corresponding to mRNA encoding the human CGRP1 receptor (339 bp) (Fig. 2). In negative controls, in which the reverse-transcriptase enzyme was replaced by RNAse-free water, no band was detected.
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Fig. 2. Gel electrophoresis of RT-PCR reaction products from human dental pulp and trigeminal ganglia after 35 cycles of ampli®cation of mRNA fragments corresponding to human CGRP1 receptor transcripts. Pulp (lane 3); trigeminal ganglion (lane 5). As negative controls, no ampli®cation products occurred when reverse transcriptase was omitted in the ®rst-strand cDNA reaction (lane 2 and 4). A 100-bp DNA ladder (Promega) con®rms the molecular size of the ampli®cation product (lane 1).
4. Discussion CGRP is a neuropeptide widely distributed in the central and peripheral nervous systems. Here we demonstrate numerous ®ne-beaded CGRP-containing nerve ®bres in the human dental pulp, consistent with previous ®ndings (Silverman and Kruger, 1987; Casasco et al., 1990; Luthman et al., 1992). The ®bres were located apically and coronally. Some ®bres were close to small blood vessels, but most were in the stroma with no apparent relation to vessels. The trigeminal ganglia, which contain the cell bodies of the CGRP ®bres, were rich in CGRP-containing cell bodies and nerve ®bres. CGRP appears to have two roles in sensory neurones, (a) to modulate sensory neurotransmission and (b) to relax blood vessels (Wimalawansa, 1996). In sensory neurones, CGRP may coexist with substance P and to a lesser degree with pituitary adenylate cyclaseactivating peptide (Mulder et al., 1994). After dental injuries there is sprouting of sensory nerves and an increase in neuropeptide immunoreactivity (Taylor
et al., 1988; Byers et al., 1990). CGRP in dorsal-root ganglia may be upregulated in response to peripheral in¯ammation (Walsh et al., 1992) which further suggests that CGRP is involved in the response to in¯ammation and nociception. In addition, CGRP, released from the peripheral aerent nerve-®bre endings of sensory neurones, induces vasodilatation and increases vascular permeability in the dental pulp (Heyeraas and Kvinnsland 1992; Olgart, 1992; Heyeraas et al., 1994). It has been demonstrated that activation of the CGRP1 receptor results in dilatation of human cerebral arteries (Jansen-Olesen et al., 1996). A possible role for sensory peptides in transendothelial migration of immunocompetent cells has been suggested. Following electrical stimulation of the crown an increased number of immunocompetent cells can be seen in the pulp of rats (Fristad et al., 1997). Recently, the cloning of a cDNA encoding a human CGRP1 receptor was reported (Aiyar et al., 1996). This product shares signi®cant sequence homology with the human calcitonin receptor, a member of the G-protein-coupled receptor superfamily. Highly speci®c CGRP receptors have been identi®ed both in the intima and the media of peripheral blood vessels such as the superior mesenteric, the femoral and the coronary arteries (Sigrist et al., 1986; Wimalawansa et al., 1987). CGRP causes marked stimulation of adenylate cyclase in cultured aortic smooth muscle and endothelial cells. These binding sites are linked to the generation of cyclic AMP via a non-endothelium-dependent mechanism (Edvinsson et al., 1985). The C-terminal fragment of human a-CGRP, hCGRP [8±37], has an antagonistic eect on CGRP-induced adenylate cyclase activity in liver-cell membranes and acts as a competitive a-hCGRP receptor antagonist in the cardiovascular system of the rat, in the guinea-pig atrium and the guinea-pig ileum (Dennis et al., 1990; Donoso et al., 1990, Jansen, 1991; Jansen-Olesen et al., 1996). hCGRP [8±37] has a weak antagonistic eect in rat vas deferens and is ineective against a-hCGRPinduced hyperthermia. Consequently, the existence of at least two classes of CGRP receptors has been suggested (Dennis et al., 1990), one being sensitive and the other being insensitive to hCGRP [8±37] (Poyner, 1992). The CGRP1 receptor is highly sensitive to the antagonistic properties of CGRP [8±37]. Receptors that do not respond to CGRP [8±37] are at present grouped together as CGRP2 (Dennis et al., 1990). Thus, the ®nding of CGRP1 receptor mRNA in the tooth pulp is compatible with a role for CGRP in modulating neuronal activities and agrees well with previous studies which have suggested that CGRP may, like substance P, have a role in nociception (Olgart et al., 1977; Andersen et al., 1978; Henry et al., 1980; Wakisaka et al., 1987).
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The mRNA for the CGRP1 receptor has been shown in the human trigeminal ganglion and cerebral arteries (Edvinsson et al., 1997). With the use of speci®c primers for the CGRP1 receptor we here provide evidence for the expression of the CGRP1 receptor mRNA in human pulp tissue. This suggests a target role of these receptors for CGRP released from pulpal nerve ®bres. However, we cannot with RT-PCR demonstrate the exact localization of the receptors; this will have to await the development of suitable in situ PCR for this tissue. In conclusion, the presence of CGRP-containing nerve ®bres around blood vessels in the human pulp tissue together with the detection of CGRP1 receptor mRNA in the same area, and the known eects of CGRP, make it conceivable that the CGRP1 receptor plays a part in the regulation of pulpal sensitivity. The known vasodilatory eect of CGRP suggests that part of the vasodilatation is mediated by a CGRP1 receptor.
Acknowledgements This study was supported by the Swedish Medical Research Council (nos. 5958 and 11238).
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