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Localization of neuropeptide Y Y1 receptor mRNA in human tooth pulp
ARCHIVES OF
PERGAMON
Archives of Oral Biology 43 (1998) 389±394
ORAL BIOLOGY
Localization of neuropeptide Y Y1 receptor mRNA in human tooth pulp Rolf Uddman a, *, Joji Kato b, Leonor Cantera c, Lars Edvinsson c a
Department of Oto-rhino-laryngology, MalmoÈ General Hospital, MalmoÈ Department of Oral Anatomy and Developmental Biology, Osaka University Faculty of Dentistry, Osaka, Japan c Division of Experimental Vascular Research, Department of Internal Medicine, Lund University Hospital, Lund, Sweden b
Accepted 29 October 1997
Abstract With immunocytochemistry numerous nerve ®bres containing neuropeptide Y (NPY) were found in human molar pulp tissue, often around small blood vessels. Reverse transcriptase-polymerase chain reaction, using speci®c primers, detected mRNA of the human NPY Y1 receptor in the human pulp tissue. Thus, both NPY-containing nerve ®bres and NPY Y1 receptor mRNA are present in human tooth pulp, possibly regulating vascular tone and pain perception. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Neuropeptide Y; Tooth pulp; PCR; Innervation; Neuropeptide Y Y1 receptor mRNA
1. Introduction Neuropeptide Y is a 36 amino-acid peptide that has chemical similarities with the endocrine gut peptides pancreatic polypeptide and peptide YY. It is widely distributed in the central as well as in the peripheral nervous systems. In sympathetic neurones, neuropeptide Y is co-stored with noradrenaline and is released together with it upon sympathetic nerve activation (Fried et al., 1985; Pernow, 1988). Around blood vessels, arteries in particular, a rich supply of neuropeptide Y-containing nerve ®bres can be seen, often forming a dense plexus. Compared with noradrenaline, neuropeptide Y is a more potent vasoconstrictor and modulator of vascular tone, and in conscious rats, its systematic administration produces an increase in total peripheral resistance and a long-lasting increase in
Abbreviations: RT-PCR, reverse transcriptase-polymerase chain reaction. * Corresponding author.
blood pressure (Edvinsson et al., 1987; ZukowskaGrojec et al., 1987). Neuropeptide Y exerts its biological actions through several classes of receptors of which six types have been cloned (Larhammar et al., 1992; Herzog et al., 1992; 1993a,b; Bard et al., 1995; Gerald et al., 1995; 1996; Lundell et al., 1995; Rose et al., 1995; Ammar et al., 1996; Matsumoto et al., 1996; Hu et al., 1996). The human neuropeptide Y Y1 receptor is activated by neuropeptide Y, peptide YY and the Y1 agonist pro34 neuropeptide Y. The Y2 receptor is stimulated equally well by neuropeptide Y and by neuropeptide Y 13±36 (Grundemar and HaÊkanson 1993). In the human dental pulp, neuropeptide Y-containing nerve ®bres occur mainly around small blood vessels (Casasco et al., 1990; Ramieri et al., 1990; Luthman et al., 1992) but can also be seen without apparent connection to vessels. We have now followed the distribution of perivascular neuropeptide Y-containing ®bres in the human dental pulp and demonstrated the presence of human neuropeptide Y Y1 receptor mRNA.
0003-9969/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 9 7 ) 0 0 1 1 7 - 9
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R. Uddman et al. / Archives of Oral Biology 43 (1998) 389±394
2. Material and methods 2.1. Tissues Pulp tissue from healthy human molars was obtained from patients (age range 24±35 years, median age 30 years) in conjunction with dental surgery. For immunocytochemistry, tissue was obtained from six patients. For RNA experiments, tissue was collected from another six patients, 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 292 mm sucrose. They were frozen on dry ice and sectioned at 10-mm thickness in a cryostat. For the immunocytochemical demonstration of neuropeptide Y, indirect immuno¯uorescence was used. The neuropeptide Y antigen was raised in a rabbit and used at a dilution of 1:320 (Eurodiagnostica, MalmoÈ, Sweden). The sections were exposed to the primary antiserum overnight at 48C in a moist chamber. The site of the antigen±antibody reaction was revealed by application of ¯uorescein isothiocyanate-labelled antibodies raised against IgG (DAKO, Copenhagen, Denmark), in a dilution of 1:80 for 1 hr at room temperature. Control sections were exposed to antiserum that had been preabsorbed with an excess amount of the antigen (10±100 mg of neuropeptide Y per millilitre diluted antiserum). Additionally, the antiserum was tested for cross-reaction with other peptides (10±100 mg of peptide per millilitre diluted antiserum). No such 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 neuropeptide Y-like. For brevity, the shorter term neuropeptide Y is used. 2.3. Isolation of total RNA Total cellular RNA was extracted using the TRIzol reagent (GIBCO BRL, Life Technologies, Sweden), following the manufacturer's instructions. Frozen tissue was homogenized with 1 ml of TRIzol reagent until completely disrupted at room temperature, using the microprobe of a power homogenizer (Polytron Kinematica AG, Model PT 1200, Labora; Sweden) for 30 to 60 sec. The homogenates were mixed with chloroform and centrifuged at 12.000 x g for 15 min at
48C. The aqueous phase, containing RNA, was transferred to a fresh tube and the RNA precipitated by the addition of isopropanol. Samples were incubated at room temperature for 10 min and centrifuged at 12.000 x g for 10 min at 48C. The RNA pellet was ®nally washed with 70% icecold ethanol, air-dried, dissolved in 20 ml of diethylpyrocarbonate-treated water and stored at ÿ208C until use. The purity and yield of total RNA were determined spectrophotometrically by measurement of the optical density of a portion at 260 nm and 280 nm, using 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. 2.4. Removal of genomic DNA from RNA samples In order to eliminate eventual residual contaminating DNA that can produce a false-positive ampli®cation signal in a 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). RNase-free water was added to each tube to a ®nal volume of 10 ml and samples were incubated for 15 min at room temperature. A volume (1 ml) of 25 mM EDTA solution (pH 8.0) was added to each tube and samples were heated for 10 min at 658C to inactivate the DNase I, and thereafter immediately cooled on ice. 2.5. RT-PCR Synthesis of ®rst-strand cDNA and subsequent PCR ampli®cation were done with the 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 1 PCR buer (50 mM KCl, 10 mM Tris± HCl, pH 8.3), 5 mM MgCl2, 1 mM of each dNTP, 50 pmol of oligo (dT) primers, 50 units of M-MLV (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 where the enzyme was replaced by 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. RT-PCR for neuropeptide Y Y1 receptor mRNA was performed with the following primers:
R. Uddman et al. / Archives of Oral Biology 43 (1998) 389±394
forward: 5'-TATGTAGGTATTGCTGTGATTTG-3', reverse 5'-CTGGAAGTTTTTGTTCAGGAACCCA-3', corresponding to regions in the vicinity of TM 4, and 7, respectively, of the human Y1 receptor. These primers are based on published nucleotide sequences of the human neuropeptide Y Y1 receptor (Larhammar et al., 1992; Herzog et al., 1992; 1993). The primers were obtained from Scandinavian Gene Synthesis AB, Sweden. Resultant cDNA was ampli®ed by PCR in a ®nal volume of 100 ml following the standard PCR protocol (GeneAmp RNA PCR kit), and AmpliTaq DNA polymerase (Perkin Elmer) was used as the thermostable enzyme. The PCR reaction was carried out by using four linked ®les: ®le 1, 2 min at 958C for 1 cycle; ®le 2, 1 min at 958C and 1 min at 608C for 35 cycles; ®le 3, 7 min at 728C for 1 cycle; ®le 4, incubation at 48C. 2.6. Electrophoretic analysis From each PCR-ampli®ed product 10 ml was electrophoresed in 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 hr. 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.
391
Fig. 2. Gel electrophoresis of RT-PCR reaction products after 35 cycles of ampli®cation of mRNA fragments corresponding to human neuropeptide Y Y1 receptor transcripts. Human dental pulp (lane 4). As a positive control a SK-N-MC cell line was used (lane 2). As a negative control, no ampli®cation product occurred when reverse transcriptase was omitted in the ®rst-strand cDNA reaction (lanes 1 and 3). A 100-bp DNA ladder (Promega) was run to con®rm the molecular size of the ampli®cation product (lane M).
3. Results Numerous neuropeptide Y-containing nerve ®bres were seen in the apical and coronal part of the pulp tissue. The majority of the immunoreactive nerve ®bres were found in connection with blood vessels (Fig. 1). Occasional ®bres were seen in the stroma, without apparent association with blood vessels. Neuropeptide Y-immunoreactive nerve ®bres were not seen in the subodontoblastic region. Total RNA was successfully extracted from the tooth pulp. By using one forward and one backward primer in RT-PCR, the presence of mRNA for the human neuropeptide Y Y1 receptor was shown (Fig. 2). As a positive control the Y1 receptor expressed in the neuroblastoma cell line, SK-NMC, was used. The PCR products were of the expected size (520 bp) in the pulp and in the SK-NMC cell line, corresponding to mRNA encoding the neuropeptide Y Y1 receptor. In addition, a small band of about 100 bp was seen (Fig. 2), probably representing a splice variant. In negative controls, when reversetranscriptase enzyme was replaced by RNAse-free water, no band was detected. 4. Discussion
Fig. 1. Human dental pulp (coronal part). Fine, beaded, neuropeptide Y-containing nerve ®bres are seen around small blood vessels and in the stroma. Bar = 40 mm.
Neuropeptide Y belongs to a family of homologous peptides that include peptide YY and pancreatic poly-
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peptide. It is highly conserved throughout evolution and is therefore thought to be an important neuropeptide which modulates numerous physiological processes including appetite, blood pressure, and circadian rhythms (Wahlestedt and Reis, 1993). Sympathetic ganglia harbour numerous neuropeptide Y-immunoreactive nerve-cell bodies and there is a rich supply of neuropeptide Y-immunoreactive nerve ®bres around peripheral blood vessels (Uddman et al., 1985). Double immunostaining has shown that in sympathetic nervecell bodies and around blood vessels, neuropeptide Y coexists with tyrosine hydroxylase and dopamine-b-hydroxylase, markers for adrenergic neurones (Lundberg et al., 1983). In human dental pulp tissue, early studies, using the Falck±Hillarp histo¯uorescence method, showed a plexus of adrenergic ®bres around blood vessels (Anneroth and Norberg, 1968). In the present study, a moderate supply of neuropeptide Y-containing nerve ®bres was observed in the human dental pulp. The majority of the ®bres were distributed around small blood vessels while some could also be seen in the pulpal stroma without apparent connection to vessels, con®rming previous studies (Casasco et al., 1990; Ramieri et al., 1990; Luthman et al., 1992). In pulp tissue of laboratory animals, numerous neuropeptide Y-containing nerve ®bres of sympathetic origin can be seen around blood vessels, arteries in particular (Uddman et al., 1984; Edwall et al., 1985; Wakisaka et al., 1996). Following combined chronic constriction injury of the alveolar nerve and sympathectomy and during regeneration of the inferior alveolar nerve a population of neuropeptide Y-immunoreactive nerve-cell bodies of mostly medium size appears in the trigeminal ganglion (Itotagawa et al., 1993; Fristad et al., 1996). In addition, neuropeptide Y-immunoreactive nerve ®bres can be seen in the odontoblast layer and dentine, an area normally innervated by aerent nerve ®bres (Fristad et al., 1995; 1996). Further, double immuno staining shows the coexistence of neuropeptide Y and calcitonin gene-related peptide in neurones of the trigeminal ganglion and in nerve ®bres in dental pulp tissue of rats in which the inferior alveolar nerve has been transected (Fristad et al., 1996). It could therefore be that some of the neuropeptide Y ®bres play a part in nociceptive transmission (Sasaki et al., 1994). Neuropeptide Y is a potent constrictor of arteries and veins. The neuropeptide Y-mediated vasoconstriction is long-lasting and resistant to a-adrenoceptor antagonists. In peripheral blood vessels, neuropeptide Y may act on vessel tone by direct constriction (Lundberg and Tatemoto, 1982), by potentiation of noradrenalineinduced contraction (Edvinsson et al., 1984) or by inhibition of presynaptic noradrenaline release (Lundberg and StjaÈrne 1984; Linton-DahloÈf, 1989). It has recently been demonstrated that the neuropeptide Y Y1 recep-
tor is responsible for the potentiation of noradrenaline-induced contraction (Bergdahl et al., 1996). Thus, neuropeptide Y seems to have several eects on the sympathetic neuroeector junction. In pulp tissue, electrical stimulation of sympathetic nerve ®bres causes constriction of pulpal blood vessels and lowers the tissue-¯uid pressure (Ogilvie, 1967). Several receptor subtypes have been postulated for the neuropeptide Y family of peptides, all of which belong to a superfamily that couples to G-proteins and causes inhibition of cAMP accumulation. So far, six human neuropeptide Y receptor subtypes have been cloned; the Y1 type (Larhammar et al., 1992; Herzog et al., 1992; 1993a), the Y2 type (Rose et al., 1995; Ammar et al., 1996), the Y3 type (Balasubramanian and Sheri, 1990; Michel, 1991; Herzog et al., 1993b), the Y4 type (Bard et al., 1995). Recently, the Y5 (Gerald et al., 1996; Hu et al., 1996) and the Y6 receptors (Matsumoto et al., 1996) were discovered. The part played by the dierent receptors has been dicult to delineate due to the lack of selective receptor antagonists. The mRNA for the Y1 receptor has been shown in human arteries (Larhammar et al., 1992; Nilsson et al., 1996). The Y1 receptor is activated by neuropeptide Y and the Y1 agonist pro34 neuropeptide Y. Y1 mRNA has widespread distribution in rat brain and has been described in a subset of cells in dorsal root ganglia. The Y2 receptor is also abundant in brain (Sheikh et al., 1989; Dumont et al., 1990; Gehlert et al., 1996). With the use of speci®c primers for the neuropeptide Y Y1 receptor the present study provides evidence for the expression of the neuropeptide Y Y1 receptor mRNA in human pulp tissue. An additional band about 100 bp longer was detected in the pulp tissue, as has previously been shown in endothelial cells and in the SK-N-MC cell line (Nilsson et al., 1996). The sequence of the human neuropeptide Y Y1 receptor contains a small intron (97 bp) with a stop codon after the ®fth TM region (Herzog et al., 1993). This extra band is a splice variant whose functional role is unknown. The ®nding of neuropeptide Y-containing nerve ®bres around blood vessels in human pulp tissue, the detection of neuropeptide Y Y1 receptor mRNA, and the known vasoconstrictor actions of neuropeptide Y and neuropeptide Y agonists makes it conceivable that a part of the vasoconstriction in dental pulp is mediated by the neuropeptide Y Y1 receptor.
Acknowledgements This study was supported by the Swedish Medical Research Council (no. 5958).
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PERGAMON
Archives of Oral Biology 43 (1998) 389±394
ORAL BIOLOGY
Localization of neuropeptide Y Y1 receptor mRNA in human tooth pulp Rolf Uddman a, *, Joji Kato b, Leonor Cantera c, Lars Edvinsson c a
Department of Oto-rhino-laryngology, MalmoÈ General Hospital, MalmoÈ Department of Oral Anatomy and Developmental Biology, Osaka University Faculty of Dentistry, Osaka, Japan c Division of Experimental Vascular Research, Department of Internal Medicine, Lund University Hospital, Lund, Sweden b
Accepted 29 October 1997
Abstract With immunocytochemistry numerous nerve ®bres containing neuropeptide Y (NPY) were found in human molar pulp tissue, often around small blood vessels. Reverse transcriptase-polymerase chain reaction, using speci®c primers, detected mRNA of the human NPY Y1 receptor in the human pulp tissue. Thus, both NPY-containing nerve ®bres and NPY Y1 receptor mRNA are present in human tooth pulp, possibly regulating vascular tone and pain perception. # 1998 Elsevier Science Ltd. All rights reserved. Keywords: Neuropeptide Y; Tooth pulp; PCR; Innervation; Neuropeptide Y Y1 receptor mRNA
1. Introduction Neuropeptide Y is a 36 amino-acid peptide that has chemical similarities with the endocrine gut peptides pancreatic polypeptide and peptide YY. It is widely distributed in the central as well as in the peripheral nervous systems. In sympathetic neurones, neuropeptide Y is co-stored with noradrenaline and is released together with it upon sympathetic nerve activation (Fried et al., 1985; Pernow, 1988). Around blood vessels, arteries in particular, a rich supply of neuropeptide Y-containing nerve ®bres can be seen, often forming a dense plexus. Compared with noradrenaline, neuropeptide Y is a more potent vasoconstrictor and modulator of vascular tone, and in conscious rats, its systematic administration produces an increase in total peripheral resistance and a long-lasting increase in
Abbreviations: RT-PCR, reverse transcriptase-polymerase chain reaction. * Corresponding author.
blood pressure (Edvinsson et al., 1987; ZukowskaGrojec et al., 1987). Neuropeptide Y exerts its biological actions through several classes of receptors of which six types have been cloned (Larhammar et al., 1992; Herzog et al., 1992; 1993a,b; Bard et al., 1995; Gerald et al., 1995; 1996; Lundell et al., 1995; Rose et al., 1995; Ammar et al., 1996; Matsumoto et al., 1996; Hu et al., 1996). The human neuropeptide Y Y1 receptor is activated by neuropeptide Y, peptide YY and the Y1 agonist pro34 neuropeptide Y. The Y2 receptor is stimulated equally well by neuropeptide Y and by neuropeptide Y 13±36 (Grundemar and HaÊkanson 1993). In the human dental pulp, neuropeptide Y-containing nerve ®bres occur mainly around small blood vessels (Casasco et al., 1990; Ramieri et al., 1990; Luthman et al., 1992) but can also be seen without apparent connection to vessels. We have now followed the distribution of perivascular neuropeptide Y-containing ®bres in the human dental pulp and demonstrated the presence of human neuropeptide Y Y1 receptor mRNA.
0003-9969/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved. PII: S 0 0 0 3 - 9 9 6 9 ( 9 7 ) 0 0 1 1 7 - 9
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2. Material and methods 2.1. Tissues Pulp tissue from healthy human molars was obtained from patients (age range 24±35 years, median age 30 years) in conjunction with dental surgery. For immunocytochemistry, tissue was obtained from six patients. For RNA experiments, tissue was collected from another six patients, 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 292 mm sucrose. They were frozen on dry ice and sectioned at 10-mm thickness in a cryostat. For the immunocytochemical demonstration of neuropeptide Y, indirect immuno¯uorescence was used. The neuropeptide Y antigen was raised in a rabbit and used at a dilution of 1:320 (Eurodiagnostica, MalmoÈ, Sweden). The sections were exposed to the primary antiserum overnight at 48C in a moist chamber. The site of the antigen±antibody reaction was revealed by application of ¯uorescein isothiocyanate-labelled antibodies raised against IgG (DAKO, Copenhagen, Denmark), in a dilution of 1:80 for 1 hr at room temperature. Control sections were exposed to antiserum that had been preabsorbed with an excess amount of the antigen (10±100 mg of neuropeptide Y per millilitre diluted antiserum). Additionally, the antiserum was tested for cross-reaction with other peptides (10±100 mg of peptide per millilitre diluted antiserum). No such 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 neuropeptide Y-like. For brevity, the shorter term neuropeptide Y is used. 2.3. Isolation of total RNA Total cellular RNA was extracted using the TRIzol reagent (GIBCO BRL, Life Technologies, Sweden), following the manufacturer's instructions. Frozen tissue was homogenized with 1 ml of TRIzol reagent until completely disrupted at room temperature, using the microprobe of a power homogenizer (Polytron Kinematica AG, Model PT 1200, Labora; Sweden) for 30 to 60 sec. The homogenates were mixed with chloroform and centrifuged at 12.000 x g for 15 min at
48C. The aqueous phase, containing RNA, was transferred to a fresh tube and the RNA precipitated by the addition of isopropanol. Samples were incubated at room temperature for 10 min and centrifuged at 12.000 x g for 10 min at 48C. The RNA pellet was ®nally washed with 70% icecold ethanol, air-dried, dissolved in 20 ml of diethylpyrocarbonate-treated water and stored at ÿ208C until use. The purity and yield of total RNA were determined spectrophotometrically by measurement of the optical density of a portion at 260 nm and 280 nm, using 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. 2.4. Removal of genomic DNA from RNA samples In order to eliminate eventual residual contaminating DNA that can produce a false-positive ampli®cation signal in a 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). RNase-free water was added to each tube to a ®nal volume of 10 ml and samples were incubated for 15 min at room temperature. A volume (1 ml) of 25 mM EDTA solution (pH 8.0) was added to each tube and samples were heated for 10 min at 658C to inactivate the DNase I, and thereafter immediately cooled on ice. 2.5. RT-PCR Synthesis of ®rst-strand cDNA and subsequent PCR ampli®cation were done with the 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 1 PCR buer (50 mM KCl, 10 mM Tris± HCl, pH 8.3), 5 mM MgCl2, 1 mM of each dNTP, 50 pmol of oligo (dT) primers, 50 units of M-MLV (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 where the enzyme was replaced by 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. RT-PCR for neuropeptide Y Y1 receptor mRNA was performed with the following primers:
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forward: 5'-TATGTAGGTATTGCTGTGATTTG-3', reverse 5'-CTGGAAGTTTTTGTTCAGGAACCCA-3', corresponding to regions in the vicinity of TM 4, and 7, respectively, of the human Y1 receptor. These primers are based on published nucleotide sequences of the human neuropeptide Y Y1 receptor (Larhammar et al., 1992; Herzog et al., 1992; 1993). The primers were obtained from Scandinavian Gene Synthesis AB, Sweden. Resultant cDNA was ampli®ed by PCR in a ®nal volume of 100 ml following the standard PCR protocol (GeneAmp RNA PCR kit), and AmpliTaq DNA polymerase (Perkin Elmer) was used as the thermostable enzyme. The PCR reaction was carried out by using four linked ®les: ®le 1, 2 min at 958C for 1 cycle; ®le 2, 1 min at 958C and 1 min at 608C for 35 cycles; ®le 3, 7 min at 728C for 1 cycle; ®le 4, incubation at 48C. 2.6. Electrophoretic analysis From each PCR-ampli®ed product 10 ml was electrophoresed in 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 hr. 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.
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Fig. 2. Gel electrophoresis of RT-PCR reaction products after 35 cycles of ampli®cation of mRNA fragments corresponding to human neuropeptide Y Y1 receptor transcripts. Human dental pulp (lane 4). As a positive control a SK-N-MC cell line was used (lane 2). As a negative control, no ampli®cation product occurred when reverse transcriptase was omitted in the ®rst-strand cDNA reaction (lanes 1 and 3). A 100-bp DNA ladder (Promega) was run to con®rm the molecular size of the ampli®cation product (lane M).
3. Results Numerous neuropeptide Y-containing nerve ®bres were seen in the apical and coronal part of the pulp tissue. The majority of the immunoreactive nerve ®bres were found in connection with blood vessels (Fig. 1). Occasional ®bres were seen in the stroma, without apparent association with blood vessels. Neuropeptide Y-immunoreactive nerve ®bres were not seen in the subodontoblastic region. Total RNA was successfully extracted from the tooth pulp. By using one forward and one backward primer in RT-PCR, the presence of mRNA for the human neuropeptide Y Y1 receptor was shown (Fig. 2). As a positive control the Y1 receptor expressed in the neuroblastoma cell line, SK-NMC, was used. The PCR products were of the expected size (520 bp) in the pulp and in the SK-NMC cell line, corresponding to mRNA encoding the neuropeptide Y Y1 receptor. In addition, a small band of about 100 bp was seen (Fig. 2), probably representing a splice variant. In negative controls, when reversetranscriptase enzyme was replaced by RNAse-free water, no band was detected. 4. Discussion
Fig. 1. Human dental pulp (coronal part). Fine, beaded, neuropeptide Y-containing nerve ®bres are seen around small blood vessels and in the stroma. Bar = 40 mm.
Neuropeptide Y belongs to a family of homologous peptides that include peptide YY and pancreatic poly-
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peptide. It is highly conserved throughout evolution and is therefore thought to be an important neuropeptide which modulates numerous physiological processes including appetite, blood pressure, and circadian rhythms (Wahlestedt and Reis, 1993). Sympathetic ganglia harbour numerous neuropeptide Y-immunoreactive nerve-cell bodies and there is a rich supply of neuropeptide Y-immunoreactive nerve ®bres around peripheral blood vessels (Uddman et al., 1985). Double immunostaining has shown that in sympathetic nervecell bodies and around blood vessels, neuropeptide Y coexists with tyrosine hydroxylase and dopamine-b-hydroxylase, markers for adrenergic neurones (Lundberg et al., 1983). In human dental pulp tissue, early studies, using the Falck±Hillarp histo¯uorescence method, showed a plexus of adrenergic ®bres around blood vessels (Anneroth and Norberg, 1968). In the present study, a moderate supply of neuropeptide Y-containing nerve ®bres was observed in the human dental pulp. The majority of the ®bres were distributed around small blood vessels while some could also be seen in the pulpal stroma without apparent connection to vessels, con®rming previous studies (Casasco et al., 1990; Ramieri et al., 1990; Luthman et al., 1992). In pulp tissue of laboratory animals, numerous neuropeptide Y-containing nerve ®bres of sympathetic origin can be seen around blood vessels, arteries in particular (Uddman et al., 1984; Edwall et al., 1985; Wakisaka et al., 1996). Following combined chronic constriction injury of the alveolar nerve and sympathectomy and during regeneration of the inferior alveolar nerve a population of neuropeptide Y-immunoreactive nerve-cell bodies of mostly medium size appears in the trigeminal ganglion (Itotagawa et al., 1993; Fristad et al., 1996). In addition, neuropeptide Y-immunoreactive nerve ®bres can be seen in the odontoblast layer and dentine, an area normally innervated by aerent nerve ®bres (Fristad et al., 1995; 1996). Further, double immuno staining shows the coexistence of neuropeptide Y and calcitonin gene-related peptide in neurones of the trigeminal ganglion and in nerve ®bres in dental pulp tissue of rats in which the inferior alveolar nerve has been transected (Fristad et al., 1996). It could therefore be that some of the neuropeptide Y ®bres play a part in nociceptive transmission (Sasaki et al., 1994). Neuropeptide Y is a potent constrictor of arteries and veins. The neuropeptide Y-mediated vasoconstriction is long-lasting and resistant to a-adrenoceptor antagonists. In peripheral blood vessels, neuropeptide Y may act on vessel tone by direct constriction (Lundberg and Tatemoto, 1982), by potentiation of noradrenalineinduced contraction (Edvinsson et al., 1984) or by inhibition of presynaptic noradrenaline release (Lundberg and StjaÈrne 1984; Linton-DahloÈf, 1989). It has recently been demonstrated that the neuropeptide Y Y1 recep-
tor is responsible for the potentiation of noradrenaline-induced contraction (Bergdahl et al., 1996). Thus, neuropeptide Y seems to have several eects on the sympathetic neuroeector junction. In pulp tissue, electrical stimulation of sympathetic nerve ®bres causes constriction of pulpal blood vessels and lowers the tissue-¯uid pressure (Ogilvie, 1967). Several receptor subtypes have been postulated for the neuropeptide Y family of peptides, all of which belong to a superfamily that couples to G-proteins and causes inhibition of cAMP accumulation. So far, six human neuropeptide Y receptor subtypes have been cloned; the Y1 type (Larhammar et al., 1992; Herzog et al., 1992; 1993a), the Y2 type (Rose et al., 1995; Ammar et al., 1996), the Y3 type (Balasubramanian and Sheri, 1990; Michel, 1991; Herzog et al., 1993b), the Y4 type (Bard et al., 1995). Recently, the Y5 (Gerald et al., 1996; Hu et al., 1996) and the Y6 receptors (Matsumoto et al., 1996) were discovered. The part played by the dierent receptors has been dicult to delineate due to the lack of selective receptor antagonists. The mRNA for the Y1 receptor has been shown in human arteries (Larhammar et al., 1992; Nilsson et al., 1996). The Y1 receptor is activated by neuropeptide Y and the Y1 agonist pro34 neuropeptide Y. Y1 mRNA has widespread distribution in rat brain and has been described in a subset of cells in dorsal root ganglia. The Y2 receptor is also abundant in brain (Sheikh et al., 1989; Dumont et al., 1990; Gehlert et al., 1996). With the use of speci®c primers for the neuropeptide Y Y1 receptor the present study provides evidence for the expression of the neuropeptide Y Y1 receptor mRNA in human pulp tissue. An additional band about 100 bp longer was detected in the pulp tissue, as has previously been shown in endothelial cells and in the SK-N-MC cell line (Nilsson et al., 1996). The sequence of the human neuropeptide Y Y1 receptor contains a small intron (97 bp) with a stop codon after the ®fth TM region (Herzog et al., 1993). This extra band is a splice variant whose functional role is unknown. The ®nding of neuropeptide Y-containing nerve ®bres around blood vessels in human pulp tissue, the detection of neuropeptide Y Y1 receptor mRNA, and the known vasoconstrictor actions of neuropeptide Y and neuropeptide Y agonists makes it conceivable that a part of the vasoconstriction in dental pulp is mediated by the neuropeptide Y Y1 receptor.
Acknowledgements This study was supported by the Swedish Medical Research Council (no. 5958).
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