Localization of neuropeptide Y Y1 receptor mRNA inhuman tooth pulp

Rolf Uddmana, *, Joji Katob, Leonor Cantera c, Lars Edvinsson c

aDepartment of Oto-rhino-laryngology, MalmoÈ General Hospital, MalmoÈbDepartment of Oral Anatomy and Developmental Biology, Osaka University Faculty of Dentistry, Osaka, Japan

cDivision of Experimental Vascular Research, Department of Internal Medicine, Lund University Hospital, Lund, Sweden

Accepted 29 October 1997

Abstract

With immunocytochemistry numerous nerve ®bres containing neuropeptide Y (NPY) were found in human molarpulp tissue, often around small blood vessels. Reverse transcriptase-polymerase chain reaction, using speci®cprimers, 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 andpain 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, neuropep-

tide 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 neuro-

peptide 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

blood pressure (Edvinsson et al., 1987; Zukowska-

Grojec et al., 1987). Neuropeptide Y exerts its biologi-

cal 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 recep-

tor 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 HaÊ kanson

1993). In the human dental pulp, neuropeptide Y-con-

taining 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 perivascu-

lar neuropeptide Y-containing ®bres in the human den-

tal pulp and demonstrated the presence of human

neuropeptide Y Y1 receptor mRNA.

Archives of Oral Biology 43 (1998) 389±394

0003-9969/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved.

PII: S0003-9969(97 )00117-9

ARCHIVESOFORALBIOLOGY

PERGAMON

* Corresponding author.

Abbreviations: RT-PCR, reverse transcriptase-polymerase

chain reaction.

2. Material and methods

2.1. Tissues

Pulp tissue from healthy human molars wasobtained from patients (age range 24±35 years, medianage 30 years) in conjunction with dental surgery. For

immunocytochemistry, tissue was obtained from sixpatients. For RNA experiments, tissue was collectedfrom another six patients, snap frozen in liquid nitro-

gen immediately after acquisition and stored at ÿ708Cuntil use. The project was approved by the EthicsCommittee of Lund University.

2.2. Immunocytochemistry

The specimens were ®xed by immersion in a mixtureof 2% formaldehyde and 0.2% picric acid in 0.1 mol/l

phosphate bu�er (pH 7.2) overnight and rinsedthoroughly in Tyrode bu�er containing 292 mmsucrose. They were frozen on dry ice and sectioned at10-mm thickness in a cryostat. For the immunocyto-

chemical demonstration of neuropeptide Y, indirectimmuno¯uorescence was used. The neuropeptide Yantigen was raised in a rabbit and used at a dilution of

1:320 (Eurodiagnostica, MalmoÈ , Sweden). The sectionswere exposed to the primary antiserum overnight at48C in a moist chamber. The site of the antigen±anti-

body reaction was revealed by application of ¯uor-escein isothiocyanate-labelled antibodies raised againstIgG (DAKO, Copenhagen, Denmark), in a dilution of1:80 for 1 hr at room temperature. Control sections

were exposed to antiserum that had been preabsorbedwith an excess amount of the antigen (10±100 mg ofneuropeptide Y per millilitre diluted antiserum).

Additionally, the antiserum was tested for cross-reac-tion with other peptides (10±100 mg of peptide permillilitre diluted antiserum). No such cross-reaction

was found. Cross-reactions with still other peptides orproteins containing amino acid sequences recognizedby the di�erent antisera cannot be excluded. It is

appropriate, therefore, to refer to the immunoreactivematerial as neuropeptide Y-like. For brevity, theshorter term neuropeptide Y is used.

2.3. Isolation of total RNA

Total cellular RNA was extracted using the TRIzolreagent (GIBCO BRL, Life Technologies, Sweden),following the manufacturer's instructions. Frozen tis-

sue was homogenized with 1 ml of TRIzol reagentuntil completely disrupted at room temperature, usingthe microprobe of a power homogenizer (Polytron

Kinematica AG, Model PT 1200, Labora; Sweden) for30 to 60 sec. The homogenates were mixed withchloroform and centrifuged at 12.000 x g for 15 min at

48C. The aqueous phase, containing RNA, was trans-ferred to a fresh tube and the RNA precipitated by the

addition of isopropanol. Samples were incubated atroom temperature for 10 min and centrifuged at 12.000x g for 10 min at 48C.The RNA pellet was ®nally washed with 70% ice-

cold ethanol, air-dried, dissolved in 20 ml of diethylpyr-ocarbonate-treated water and stored at ÿ208C until

use. The purity and yield of total RNA were deter-mined spectrophotometrically by measurement of theoptical density of a portion at 260 nm and 280 nm,

using a DU-65 spectrophotometer (BeckmanInstruments, Sweden). The ratio of absorption(260:280) was between 1.6 and 1.8. Finally, sampleswere subjected to gel electrophoresis and stained with

ethidium bromide to prove the integrity of the 18 and28 S ribosomal RNAs.

2.4. Removal of genomic DNA from RNA samples

In order to eliminate eventual residual contami-

nating DNA that can produce a false-positive ampli®-cation signal in a RT-PCR, duplicate tubescontaining 1 mg of total RNA were pretreated with 1

unit of ampli®cation grade DNase I (GIBCO) inDNase I reaction bu�er, in the presence of 20 unitsof RNase inhibitor (Perkin Elmer AB, Sweden).

RNase-free water was added to each tube to a ®nalvolume of 10 ml and samples were incubated for15 min at room temperature. A volume (1 ml) of25 mM EDTA solution (pH 8.0) was added to each

tube and samples were heated for 10 min at 658C toinactivate the DNase I, and thereafter immediatelycooled on ice.

2.5. RT-PCR

Synthesis of ®rst-strand cDNA and subsequent PCRampli®cation were done with the GeneAmp RNAPCR kit reagents (Perkin-Elmer) in a PCR DNA ther-

mal cycler (Perkin-Elmer).DNase-treated RNA samples were reverse-tran-

scribed to cDNA in a 20 ml reaction volume in the pre-

sence of 1 � PCR bu�er (50 mM KCl, 10 mM Tris±HCl, pH 8.3), 5 mM MgCl2, 1 mM of each dNTP, 50pmol of oligo (dT) primers, 50 units of M-MLV(Moloney murine leukaemia virus) reverse transcrip-

tase. To determine if the ampli®cation product cameexclusively from the RNA, a reverse-transcriptasenegative reaction was run where the enzyme was

replaced by RNase-free water. The samples (20 ml)were incubated at room temperature for 10 min, at428C 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±394390

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. Theseprimers are based on published nucleotide sequences of

the human neuropeptide Y Y1 receptor (Larhammar etal., 1992; Herzog et al., 1992; 1993). The primers wereobtained 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 poly-merase (Perkin Elmer) was used as the thermostableenzyme. The PCR reaction was carried out by usingfour 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 electro-phoresed in a 1.5 % agarose gel (GIBCO), containing

0.5 mg/ml ethidium bromide (Sigma E 1510), in TBEbu�er (89 mM Tris±borate, 2 mM EDTA, pH 8.0) at5 V/cm for 1.5 hr. This analysis was performed in a

20 � 10 cm Midicell, Model EC 350 (E-C ApparatusCorporation; Techtum Lab AB, Sweden). A 100-bpDNA ladder (Promega , SDS, Sweden) was run ineach of the outside lanes to con®rm the molecular size

of the ampli®cation product.

3. Results

Numerous neuropeptide Y-containing nerve ®breswere seen in the apical and coronal part of the pulptissue. The majority of the immunoreactive nerve ®bres

were found in connection with blood vessels (Fig. 1).Occasional ®bres were seen in the stroma, withoutapparent association with blood vessels. Neuropeptide

Y-immunoreactive nerve ®bres were not seen in thesubodontoblastic region. Total RNA was successfullyextracted from the tooth pulp. By using one forwardand one backward primer in RT-PCR, the presence of

mRNA for the human neuropeptide Y Y1 receptorwas shown (Fig. 2). As a positive control the Y1 recep-tor expressed in the neuroblastoma cell line, SK-N-

MC, was used. The PCR products were of theexpected size (520 bp) in the pulp and in the SK-N-MC cell line, corresponding to mRNA encoding the

neuropeptide Y Y1 receptor. In addition, a small bandof about 100 bp was seen (Fig. 2), probably represent-ing a splice variant. In negative controls, when reverse-transcriptase enzyme was replaced by RNAse-free

water, no band was detected.

4. Discussion

Neuropeptide Y belongs to a family of homologouspeptides that include peptide YY and pancreatic poly-

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.

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).

R. Uddman et al. / Archives of Oral Biology 43 (1998) 389±394 391

peptide. It is highly conserved throughout evolution

and is therefore thought to be an important neuropep-tide which modulates numerous physiological processesincluding appetite, blood pressure, and circadian

rhythms (Wahlestedt and Reis, 1993). Sympatheticganglia harbour numerous neuropeptide Y-immuno-reactive nerve-cell bodies and there is a rich supply of

neuropeptide Y-immunoreactive nerve ®bres aroundperipheral blood vessels (Uddman et al., 1985). Double

immunostaining has shown that in sympathetic nerve-cell bodies and around blood vessels, neuropeptide Ycoexists with tyrosine hydroxylase and dopamine-b-hy-droxylase, markers for adrenergic neurones (Lundberget al., 1983).

In human dental pulp tissue, early studies, using theFalck±Hillarp histo¯uorescence method, showed aplexus of adrenergic ®bres around blood vessels

(Anneroth and Norberg, 1968). In the present study, amoderate supply of neuropeptide Y-containing nerve®bres was observed in the human dental pulp. The ma-

jority of the ®bres were distributed around small bloodvessels while some could also be seen in the pulpal

stroma without apparent connection to vessels, con-®rming previous studies (Casasco et al., 1990; Ramieriet al., 1990; Luthman et al., 1992). In pulp tissue of

laboratory animals, numerous neuropeptide Y-contain-ing nerve ®bres of sympathetic origin can be seen

around blood vessels, arteries in particular (Uddmanet al., 1984; Edwall et al., 1985; Wakisaka et al., 1996).Following combined chronic constriction injury of the

alveolar nerve and sympathectomy and during regener-ation of the inferior alveolar nerve a population ofneuropeptide Y-immunoreactive nerve-cell bodies of

mostly medium size appears in the trigeminal ganglion(Itotagawa et al., 1993; Fristad et al., 1996). In ad-

dition, neuropeptide Y-immunoreactive nerve ®brescan be seen in the odontoblast layer and dentine, anarea normally innervated by a�erent nerve ®bres

(Fristad et al., 1995; 1996). Further, double immunostaining shows the coexistence of neuropeptide Y andcalcitonin gene-related peptide in neurones of the tri-

geminal ganglion and in nerve ®bres in dental pulp tis-sue of rats in which the inferior alveolar nerve has

been transected (Fristad et al., 1996). It could thereforebe that some of the neuropeptide Y ®bres play a partin nociceptive transmission (Sasaki et al., 1994).

Neuropeptide Y is a potent constrictor of arteries andveins. The neuropeptide Y-mediated vasoconstriction is

long-lasting and resistant to a-adrenoceptor antagon-ists. In peripheral blood vessels, neuropeptide Y mayact on vessel tone by direct constriction (Lundberg and

Tatemoto, 1982), by potentiation of noradrenaline-induced contraction (Edvinsson et al., 1984) or by inhi-bition of presynaptic noradrenaline release (Lundberg

and StjaÈ rne 1984; Linton-DahloÈ f, 1989). It has recentlybeen demonstrated that the neuropeptide Y Y1 recep-

tor is responsible for the potentiation of noradrena-line-induced contraction (Bergdahl et al., 1996). Thus,

neuropeptide Y seems to have several e�ects on thesympathetic neuroe�ector junction. In pulp tissue, elec-trical stimulation of sympathetic nerve ®bres causes

constriction of pulpal blood vessels and lowers the tis-sue-¯uid pressure (Ogilvie, 1967).Several receptor subtypes have been postulated for

the neuropeptide Y family of peptides, all of whichbelong to a superfamily that couples to G-proteins andcauses inhibition of cAMP accumulation. So far, six

human neuropeptide Y receptor subtypes have beencloned; the Y1 type (Larhammar et al., 1992; Herzoget 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 recep-

tors (Matsumoto et al., 1996) were discovered. Thepart played by the di�erent receptors has been di�cultto delineate due to the lack of selective receptor antag-

onists. The mRNA for the Y1 receptor has been shownin human arteries (Larhammar et al., 1992; Nilsson etal., 1996). The Y1 receptor is activated by neuropeptide

Y and the Y1 agonist pro34 neuropeptide Y. Y1

mRNA has widespread distribution in rat brain andhas been described in a subset of cells in dorsal rootganglia. The Y2 receptor is also abundant in brain

(Sheikh et al., 1989; Dumont et al., 1990; Gehlert etal., 1996). With the use of speci®c primers for theneuropeptide Y Y1 receptor the present study provides

evidence for the expression of the neuropeptide Y Y1

receptor mRNA in human pulp tissue. An additionalband about 100 bp longer was detected in the pulp tis-

sue, as has previously been shown in endothelial cellsand in the SK-N-MC cell line (Nilsson et al., 1996).The sequence of the human neuropeptide Y Y1 recep-tor contains a small intron (97 bp) with a stop codon

after the ®fth TM region (Herzog et al., 1993). Thisextra band is a splice variant whose functional role isunknown.

The ®nding of neuropeptide Y-containing nerve®bres around blood vessels in human pulp tissue, thedetection of neuropeptide Y Y1 receptor mRNA, and

the known vasoconstrictor actions of neuropeptide Yand neuropeptide Y agonists makes it conceivable thata part of the vasoconstriction in dental pulp is

mediated by the neuropeptide Y Y1 receptor.

Acknowledgements

This study was supported by the Swedish MedicalResearch Council (no. 5958).

R. Uddman et al. / Archives of Oral Biology 43 (1998) 389±394392

References

Ammar, D.A., Eadie, D.M., Wong, D.J., Ma, Y-Y.,

Kolakowski, L.F., Yang-Feng, T.L., Thompson, D.A.,

1996. Characterization of the human type 2 neuropeptide Y

receptor gene (neuropeptide Y2R) and localization to the

chromosome 4q region containing the type 1 neuropeptide

Y receptor gene. Genomics 38, 392±398.

Anneroth, G., Norberg, K-A., 1968. Adrenergic vasoconstric-

tor innervation in the human dental pulp. Acta Odontol.

Scand. 26, 89±93.

Balasubramaniam, A., Sheri�, S., 1990. Neuropeptide Y (18-

36) is a competitive antagonist of neuropeptide Y in rat

cardiac membranes. J. Biol. Chem. 265, 14724±14727.

Bard, J.A., Walker, M.W., Branchek, T.A., Weinshank, R.L.,

1995. Cloning and functional expression of a human Y4

subtype receptor for pancreatic polypeptide, neuropeptide

Y, and peptide YY. J. Biol. Chem. 270, 26762±26765.

Bergdahl, A., Nilsson, T., Cantera, L., Nilsson, L., Sun, X-

Y., Hedner, T., Erlinge, D., Valdermarsson, S., Edvinsson,

L., 1996. Neuropeptide Y potentiates noradrenaline-induced

contraction through the neuropeptide Y Y1 receptor. Eur.

J. Pharmacol. 316, 59±64.

Casasco, A., Calligaro, A., Casasco, M., Springall, D.R.,

Polak, J.M., Poggi, P., Marchetti, C., 1990. Peptidergic

nerves in human dental pulp. Histochemistry 95, 115±121.

Dumont, Y., Fournier, A., St-Pierre, S., Schwartz, T.W.,

Quirion, R., 1990. Di�erential distribution of neuropeptide

Y1 and Y2 receptors in the rat brain. Eur. J. Pharmacol.

191, 501±503.

Edvinsson, L., Ekblad, E., HaÊ kanson, R., Wahlestedt, C.,

1984. Neuropeptide Y potentiates the e�ect of various vaso-

constrictor agents on rabbit blood vessels. Br. J.

Pharmacol. 83, 519±525.

Edvinsson, L., HaÊ kanson, R., Wahlestedt, C., Uddman, R.,

1987. E�ects of neuropeptide Y on the cardiovascular sys-

tem. Trends Pharmacol. Sci. 8, 231±235.

Edwall, B., Gazelius, B., Fazekas, A., Theodorsson-Norheim,

E., Lundberg, J.M., 1985. Neuropeptide Y (NPY) and sym-

pathetic control of blood ¯ow in oral mucosa and dental

pulp in the cat. Acta Physiol. Scand. 125, 253±264.

Fried, G., Terenius, L., HoÈ kfelt, T., Goldstein, M., 1985.

Evidence for di�erential localization of noradrenaline and

neuropeptide Y in neuronal storage vesicles isolated from

rat vas deferens. J. Neurosci. 5, 450±458.

Fristad, I., Heyeraas, K.J., Kvinnsland, I.H., 1996.

Neuropeptide Y expression in the trigeminal ganglion and

mandibular division of the trigeminal nerve after inferior

alveolar nerve axotomy in young rats. Exp. Neurol. 142,

276±286.

Fristad, I., Heyeraas, K.J., Jonsson, R., Kvinnsland, I.H.,

1995. E�ect of inferior alveolar nerve axotomy on immune

cells and nerve ®bres in young rat molars. Archs oral Biol.

40, 1053±1062.

Gehlert, D.R., Beavers, L., Johnson, D., Gackenheimer, S.L.,

Schober, D.A., Gadski, R.A., 1996. Expression cloning of a

human brain neuropeptide Y Y2 receptor. Mol. Pharmacol.

49, 224±228.

Gerald, C., Walker, M.W., Vaysse, P.J-J., He, C., Branchek,

T.A., Weinshank, R.L., 1995. Expression cloning and phar-

amcological characterization of a human hippocampal

neuropeptide Y/peptide YY Y2 receptor subtype. J. Biol.

Chem. 270, 26758±26761.

Gerald, C., Walker, M.W., Criscione, L., Gustafson, E.L.,

Batzl-Hartmann, C., Smith, K.E., Vaysse, P., Durkin,

M.M., Laz, T.M., Linemeyer, D.L., Scha�hauser, A.O.,

Whitebread, S., Hofbauer, K.G., Taber, R.I., Branchek,

T.A., Weinshank, R.L., 1996. A receptor subtype involved

in neuropeptide-Y-induced food intake. Nature 382, 168±

171.

Grundemar, L., HaÊ kanson, R., 1993. Neuropeptide Y e�ector

systems: perspective for drug development. Trends

Pharmacol. Sci. 15, 153±159.

Herzog, H., Hort, Y.J., Ball, H.J., Hayers, G., Shine, J.,

Selbie, L.A., 1992. Cloned human neuropeptide Y receptor

couples to two di�erent second messenger systems. Proc.

Natl. Acad. Sci. USA 89, 5794±5798.

Herzog, H., Baumgartner, M., Vivero, C., Selbie, L.A., Auer,

B., Shine, J., 1993a. Genomic organization, localization and

allelic di�erences in the gene for the human neuropeptide Y

Y1 receptor. J. Biol. Chem. 268, 6073±6707.

Herzog, H., Hort, Y.J., Shine, J., Selbie, L.A., 1993b.

Molecular cloning, characterization, and localization of the

human homolog to the reported bovine NPY Y3 receptor:

Lack of neuropeptide Y binding and activation. DNA Cell

Biol. 12, 465±471.

Hu, Y., Bloomquist, B.T., Corn®eld, L.J., DeCarr, L.B.,

Flores-Riveros, J.R., Friedman, L., Jiang, P., Lewis-

Higgins, L., Sadlowski, Y., Schaefer, J., Velazquez, N.,

McCaleb, M.L., 1996. Identi®cation of a novel hypothala-

mic neuropeptide Y receptor associated with feeding beha-

vior. J. Biol. Chem. 271, 26315±26319.

Itotagawa, T., Yamanaka, H., Wakisaka, S., Sasaki, Y.,

Kato, J., Kuriso, K., Tsuchitani, Y., 1993. Appearance of

neuropeptide Y-like immunoreactive cells in the rat trigem-

inal ganglion following dental injuries. Archs oral Biol. 38,

725±728.

Larhammar, D., Blomqvist, A.G., Yee, F., Jazin, E., Yoo,

H., Wahlestedt, C., 1992. Cloning and functional expression

of a human neuropeptide Y/peptide YY receptor of the Y1

type. J. Biol. Chem. 267, 10935±10938.

Linton-DahloÈ f, P., 1989. Modulatory interactions of neuro-

peptide Y (NPY) on sympathetic neurotransmission. Acta

Physiol. Scand. 137 (suppl 586), 1±85.

Lundberg, J.M., Tatemoto, K., 1982. Pancreatic polypeptide

family (APP, BPP, NPY and PYY) in relation to sympath-

etic vasoconstriction resistant to a-adrenoceptor blockade.

Acta Physiol. Scand. 116, 393±402.

Lundberg, J.M., Terenius, L., HoÈ kfelt, T., Goldstein, M.,

1983. High levels of neuropeptide Y in peripheral noradren-

ergic neurons in various mammals including man. Neurosci.

Lett. 42, 167±172.

Lundberg, J.M., StjaÈ rne, L., 1984. Neuropeptide Y (NPY)

depresses the secretion of 3H-noradrenaline and the con-

tractile response evoked by ®eld stimulation in the rat vas

deferens. Acta Physiol. Scand. 120, 477±479.

Lundell, I., Blomqvist, A.G., Berglund, M.M., Schober, D.A.,

Johnson, D., Statnick, M.A., Gadski, R.A., Gehlert, D.R.,

Larhammar, D., 1995. Cloning of a human receptor of the

NPY receptor family with high a�nity for pancreatic poly-

peptide and peptide YY. J. Biol. Chem. 270, 29123±29128.

R. Uddman et al. / Archives of Oral Biology 43 (1998) 389±394 393

Luthman, J., Luthman, D., HoÈ kfelt, T., 1992. Occurrence and

distribution of di�erent neurochemical markers in the

human dental pulp. Archs oral Biol. 37, 193±208.

Matsumoto, M., Nomura, T., Momose, K., Ikeda, Y.,

Kondou, Y., Akiho, H., Togami, J., Kimura, Y., Okada,

M., Yamaguchi, T., 1996. Inactivation of a novel neuropep-

tide Y/peptide YY receptor gene in primate species. J. Biol.

Chem. 271, 27217±27220.

Michel, M.C., 1991. Receptors for neuropeptide Y: Multiple

subtypes and multiple second messengers. Trends

Pharmacol. Sci. 12, 389±394.

Nilsson, T., Cantera, L., Edvinsson, L., 1996. Presence of

neuropeptide Y Y1 receptor mediating vasoconstriction in

human cerebral arteries. Neurosci. Lett. 204, 145±148.

Ogilvie, R.W., 1967. Direct observation of the cat dental pulp

microvascular response to electrical and drug stimuli. Anat.

Rec. 159, 379.

Pernow, J., 1988. Co-release and functional interactions of

neuropeptide Y and noradrenaline in peripheral sympath-

etic vascular control. Acta Physiol. Scand. 133 (suppl. 568),

1±56.

Ramieri, G., Anselmetti, G.C., Baracchi, F., Panzica, G.C.,

Viglietti-Panzica, C., Modica, R., Polak, J.M., 1990. The

innervation of human teeth and gingival epithelium as

revealed by means of an antiserum for protein gene product

9.5 (PGP 9.5). Am. J. Anatomy 189, 146±154.

Rose, P.M., Fernandes, P., Lynch, J.S., Frazier, S.T., Fisher,

S.M., Kodukula, K., Kienzle, B., Seethala, R., 1995.

Cloning and functional expression of a cDNA encoding a

human type 2 neuropeptide Y receptor. J. Biol. Chem. 270,

22661±22664.

Sasaki, Y., Wakisaka, S., Kurisu, K., 1994. E�ects of periph-

eral axotomy of the inferior alveolar nerve on the levels of

neuropeptide Y in rat trigeminal primary a�erent neurons.

Brain Res. 664, 108±114.

Sheikh, S.P., HaÊ kanson, R., Schwartz, T.W., 1989. Y1 and

Y2 receptors for neuropeptide Y. FEBS Lett. 245, 209±214.

Uddman, R., Grunditz, T., Sundler, F., 1984. Neuropeptide

Y: Occurrence and distribution in dental pulps. Acta

Odontol. Scand. 42, 361±365.

Uddman, R., Ekblad, E., Edvinsson, L., HaÊ kanson, R.,

Sundler, F., 1985. Neuropeptide Y-like immunoreactivity in

perivascular nerve ®bres of the guinea-pig. Reg. Pept. 10,

243±257.

Wahlestedt, C., Reis, D.J., 1993. Neuropeptide Y-related pep-

tides and their receptors-are the receptors potential thera-

peutic drug targets?. Annu. Rev. Pharmacol. Toxicol. 32,

309±352.

Wakisaka, S., Youn, S.H., Kato, J., Takemura, M., Kurisu,

K., 1996. Neuropeptide Y-immunoreactive primary a�er-

ents in the dental pulp and periodontal ligament following

nerve injury to the inferior alveolar nerve in the rat. Brain

Res. 712, 11±18.

Zukowska-Grojec, Z., Marks, E.S., Haass, M., 1987.

Neuropeptide Y is a potent vasoconstrictor and cardiode-

pressant in rats. Am. J. Physiol. 253, H12334±H12339.

R. Uddman et al. / Archives of Oral Biology 43 (1998) 389±394394