Identification of drug receptors in porcine dental pulp by the radioligand binding assay

Gen. Pharmac.Vol. 20, No. 4, pp. 525--528,1989 Printed in Great Britain. All fights reserved

0306-3623/89$3.00+ 0.00 Copyright © 1989PergamonPress pic

IDENTIFICATION OF D R U G RECEPTORS IN PORCINE DENTAL PULP BY THE RADIOLIGAND BINDING ASSAY MASAKAZUSANe, HAJIME MURAKAMIl' *, TAKASHITSUKIMURAand AKIRA YAMAZAKI Department of Oral Physiology, School of Dentistry, Showa University, Hatanodai, Shinagawa-Ku, Tokyo 142 and 1Department of Physiology and Anatomy, Nihon University, College of Pharmacy, Narashinodai, Funabashi, Chiba 274, Japan (Received 1 November 1988) Abstract--1. The acetylcholine (ACh), histamine and serotonin (5-HT) receptors in porcine dental pulp were characterized by the radioligand binding assay. 2. For [3H]nicotine binding site, Kd was 8.06 + 1.65 nM and Bm~ was 270.83 + 32.68 fmol/mg protein. 3. For [3H]QNB binding site, Kd was 1.04 + 0.14 nM and Bn~ was 24.83 + 3.09 fmol/mg protein. 4. For [3H]histamine binding site, Kd was 1.22 _+0.1 nM and Bm~ was 283.15 + 33.1 fmol/mg protein. 5. For [3H]5-HT binding site, Kd was 1.41 +0.1 nM and Bm~ was 53.1 + 3.4 fmol/mg protein. 6. These findings indicate that the specific receptors for ACh, histamine and 5-HT are present in the porcine dental pulp, and that the ACh receptor is predominantly nicotinic.

INTRODUCTION The topical applications of hypertonic NaC1 solution, KC1 solution, acetylcholine (ACh), histamine and serotonin (5-HT) to the exposed h u m a n dental pulp cause pain (Anderson and Naylor, 1962; Br/innstr6m, 1962). Furthermore, when applied topically to the dental pulp of cat and rat, these drugs cause the increase in the repetitive firing from the pulp and inferior alveolar nerves (Scott and Stewart, 1965; Horiguchi and Mathews, 1972; Nakajima, 1973; Sano et al., unpublished observation). These findings suggest that the ACh, histamine and 5-HT receptors are present in the m a m m a l i a n dental pulp. The present study, therefore, investigated the existence of neurotransmitter receptors in the porcine dental pulp with a direct method using radioligand binding technique, and characterized the kinetic parameters for each receptor. MATERIALS AND METHODS Preparation of crude synaptosomal fraction (P2fraction) The porcine dental pulp dissected from the teeth of pigs of either sex were collected, and washed with ice-cold buffer A (0.32M sucrose, 5raM HEPES and 10mM Tris/HCl, pH 7.4). The dental pulps were homogenized with a polytron homogenizer (Kinematica) in 10 vols of buffer A at 0°C with the reostat setting 9 for 5 sec. The homogenate was centrifuged at 1000g for 10 min. The supernatant was again centrifuged at 17,000g for 10min. After suspended in 50 mM Tris/HC1 buffer, the resultant pellet was centrifuged 3 times at 17,000g for 10rain. The final pellet was suspended in 50 mM Tris/HCl buffer as a P2 fraction of the porcine dental pulp. Aliquots of this suspension were diluted to a final protein concentration of 0.27-1.0mg/ml for binding assays. Thus prepared, the synaptosomes in the P2 fraction were structurally intact by electronmicroscopic analysis. *To whom all correspondence should be addressed. 525

Binding assay P2 fraction was incubated at 37°C with varying concentrations of [3H]nicotine, [3H]QNB, [3H]histamine or [3H]5-HT in 300/zl of 50raM Tris/HCl, pHT.4. After 30 rain, the incubation mixture was diluted with I ml o f 50 mM Tris/HC1 (pH 7.4), and then immediately filtered through Whatman GF/C glass filters. The filters were washed three times with 15 ml of ice-cold 50 mM Tris]HCl (pH 7.4), and then dried. The radioactivity on filter was counted in a toluene base scintillator with the liquid scintillation spectrometer (Aloka LSC-900). A nonspecific binding was estimated as the radioactivity bound to P2 fraction which was not displaced by nicotine (3 x 10-s M), atropine (3 x 10-6 M), chlorpheniramine (10 -s M) or methyserglde (3 x 10-s M). Protein determination Protein concentration was determined according to the method of Lowry et al. (1951) using bovine serum albumin as a standard. Materials The radiolabelled compounds, [3H]nicotine (N-methyl[ 3H]-nicotine, 80 Ci/mmol); [3H]QNB (L-quinuclidinyl [phenyl-4-3H]bentilate, 60Ci/mmol); [3H]histamine ([2,5~H]histamine dihydrochloride, 31.7Ci/mmol) and [3H]serotonin (5-hydroxy-[G3H]tryptamine creatinine sulfate, 23 Ci/mmol) were obtained from New England Nuclear. L-Nicotine (nicotine bitartarate) and HEPES (N-2-hydroxyethylpiperazine-N'-2.ethanesulfonicacid were from Sigma. Chlorpheniramine (chlorpheniramine maleate) from Sankyo. Methysergide (methysergide dimaleate) from Sandoz. Other chemicals used were of analytical grade. RESULTS [ 3H]nicotine binding Figure IA shows the saturation curve of [ 3H]nicotine binding to the synaptosome (P2 fraction) from porcine dental pulp. The specific binding of [3 H]nicotine was calculated from a total binding by subtracting a non-specific binding which was not

526

MASAKAZU SANO e t

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[ 3H]histamine binding Figure 3A similarly shows the specific binding of [3H]histamine to the 1)2 fraction of porcine dental pulp with a plateau at the concentration of 5-10 nM [3H]histamine. The binding appeared to be a single population of specific binding site as shown by a single straight line on a Scatchard plot of the data (Fig. 3B). A Hill plot (Fig. 3C) showed a straight line with 0.98nH. The Kd of 1.22 _+ 0.1nM and Bmax of 283.15 _ 33.08 fmol/mg protein were derived from these figures.

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The [3 H]5-HT binding to the P2 fraction of porcine dental pulp was shown in Fig. 4A. The specific binding of [3H]5-HT reached a plateau at the

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Fig. 1. (A) Specific binding of [3H]nicotine to P2 fraction from porcine dental pulp. P2 fraction (about 0.3 mg protein) was incubated with 1.25-20 nM [3H]nicotine for 30min at 35°C in 300 #1 of 50 mM Tris/HCl, pH 7.4 in the absence or presence of 3 x 10-5 M L-nicotine. (B) A Scatchard plot of the specific binding of [3H]nicotine.

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1.0 displaced by 3 x 10-SM nicotine. The binding reached a plateau at the concentration of 10nM [3H]nicotine. A corresponding Scatchard plot (Fig. 1B) of the data provided a single straight line, indicating a single population of specific binding sites. A Hill plot (Fig. IC) gave a straight line with a Hill coefficient (nil) of 1.035. The dissociation constant (Kd) of [all]nicotine binding was calculated to be 8 . 0 6 _ 1.06 nM. The maximum binding sites (Bronx) were 270.83 _+ 32.68 fmol/mg protein.

Figure 2A shows the [aH]QNB binding to the P2 fraction of porcine dental pulp. The specific binding of [3H]QNB reached a plateau at the concentration of 2 nM [a H]QNB. A straight line on a corresponding Scatchard plot (Fig. 2B) suggested a single population of specific binding sites. A Hill plot (Fig. 2C) of the same data gave a straight line with 1.04 nil. Calculated from these figures, the Kd and Bm~

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Fig. 2. (A) Specific binding of [3H]QNB to P2 fraction from porcine dental pulp. P2 fraction (about 0.08 mg protein) was incubated with 0.2-4nM [3H]QNB in the absence or presence of 3 x 10-6 M atropine at the similar condition as Fig. 1A. The difference of bindings in the absence and presence of cold atropine was taken as a specific binding. (B) A Scatchard plot of the specific binding of [3H]QNB. (C) A Hill plot of the specific binding of [3H]QNB.

Dental pulp receptors

527 DISCUSSION

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The receptor for ACh, histamine and 5-HT were directly identified with radioligand binding technique. The analysis with Scatchard and Hill plot showed that they represented the specific binding site of a single population for each transmitter. Thus, these specific receptors appear to mediate the pain which is induced by these transmitters in the mammalian dental pulp. The maximum binding sites for [3H]nicotine in the dental pulp was 270 fmol/mg protein with the K~ of 8 nM. These findings indicated that the ACh receptors present in the dental pulp were predominantly the nicotinic ones.

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Fig. 3. (A) Specific binding of [3H]histamine to I)2 fraction from porcine dental pulp. Similary as in Fig. IA, 1)2 fraction (about 0.15mg protein) was incubated with 0.65-10nM [3H]histamine in the absence or presence of 10-SM chioropheniramine. A specific binding was obtained from the difference. (B) A Scatchard plot of the specific binding of [3H]histamine. (C) A Hill plot of the specific binding of [ 3H]histamine.

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concentration of 4 nM [3H]5-HT. A straight line on a Scatchard plot (Fig. 4B) of the data suggested a single population of specific binding sites for [3H]5HT. A Hill coefficient was 0.99 nH (Fig. 4C). Calculated from these figures, the Kd and Bm,x were 1.41 +0.1 nM and 53.11 + 3 . 4 4 f m o l / m g protein respectively. Table 1. Binding parameters for [3H]nicotine, [3H]QNB, [3H]histamine and [3H]5-HT Kd (nM) B,,,x (fmol/mg protein) [3H]Ni¢otine 8.06 -t- 1.06 270.83 4- 32.68 [~H]QNB 1.04 4- 0.14 24.83 4- 3.09 [3H]Histamine 1.22 4- 0.10 283.15 4- 33.08 [3H]Serotonin 1.41 4- 0.10 53.11 + 3.44 The values are mean 4- SE of at least 3 experiments for each radioactive ligand.

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Fig. 4. (A) Specific binding of [3H]5-HT to P2 fraction from porcine dental pulp. P2 fraction (about 0.3 mg protein) was incubated with 0.94--5nM [3I-I]5-HT in the absence or presence of 3 x 10-SM methyserside at the similar condition as Fig. IA. The difference was taken as a spe~fic binding of [3H]5-HT. (B) A Scatchard plot of the specific binding of [3H]5-HT. ((2) A Hill plot of the specific binding of [3I-I]5-HT.

MASAKAZUSANOet al.

528

The specific receptor for [3H]5-HT was present in the porcine dental pulp at the maximum binding sites of 53 fmol/mg protein. Presently, the multiple subtypes for 5-HT receptor have been identified in the central and peripheral neuron systems (Bradley et aL, 1986; Conn and Sanders-Bush, 1987). The peripheral efferent and afferent neurones have been found to be 5-HT 3 subtype (Fozard, 1984). The 5-HT 3 subtype appears to be involved in inducing pain (Donatsch et al., 1984). A similar further analysis of 5-HT receptor in the dental pulp remains to be shown. REFERENCES

Anderson D. J. and Naylor N. (1962) Chemical excitants of pain in human dentine and dental pulp. Arch. oral Biol. 7, 412-415. Bradley P. B., Engel G., Feniuk W., Fozard J. R., Humphrey P. P. A., Middlemiss D. N., Mylecharance E. J., Richardson B. P. and Saxena P. R. (1986) Proposal for the classification and nomenclature of functional receptors for 5-hydroxytriptamine. Neuropharmacology 25, 563-576.

Br~innstr6m M. (1962) The elicitation of pain in human dentine and pulp by chemical stimuli. Arch. oral Biol. 7, 59-62. Conn and Sanders-Bush E. (1987) Central serotonin receptors: effector systems, physiological role and regulations. Psychopharmacology 92, 267-277. Donatsch P., Engel G., Richardson B. P. and Stadler P. (1984) The inhibitory effect of neuronal 5-hydroxytryptamine (5-HT) receptor antagonists on experimental pain in humans. Br. J. Pharmac. 81, 33P. Fozard J. R. (1984) Neuronal 5-HT receptors in the periphery. Neuropharmacology 23, 1473-1486. Horiuchi H. and Matthews B. (1972) Evidence that nerve impulsescan be recorded from dentine, in Oral Physiology (Edited by N. Emmeline and Y. Zotterman) pp. 297-302. Pergamon Press, Oxford. Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Nakajima T. (1973). The effects of pain producing substances on dental pulp of cats. Jpn J. Conserv. Dent. 16, 235-247. (in Japanese). Scott D. Jr. and Stewart G. G. (1965) Excitation of dentinal receptor of the cat by heat and chemical agents. Oral Surg. 20, 784-794.