Comparison of Effects of Tiapride and Sulpiride on D-1, D-2, D-3 and D-4 Subtypes of Dopamine Receptors in Rat Striatal and Bovine Caudate Nucleus Membranes
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Comparison of Effects of Tiapride and Sulpiride on D-1, D-2, D-3 and D-4 Subtypes of Dopamine Receptors in Rat Striatal and Bovine Caudate Nucleus Membranes Takashi ARI MA, Naomi SAM U RA, Yasuyuki NOM U RA* and Tomio SEGAWA** Department of Pharmacology, Institute of Pharmaceutical Sciences, Hiroshima University School of Medicine, Kasumi 1-2-3, Minami-ku, Hiroshima 734, Japan *Department of Pharmacology , Research Institute for Wakan-Yaku, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-01, Japan Accepted May 5, 1986 Abstract-To determine the affinity of tiapride to D-1, D-2, D-3 and D-4 subtypes of dopamine (DA) receptors, inhibitory effects of tiapride on [3H]-cis-flupenthixol, [3H]spiperone and [3H]N-propylapomorphine binding were examined in the rat striatum and bovine caudate nucleus membranes and compared to those of sulpiride and haloperidol. The IC50 values of tiapride, sulpiride and haloperidol were estimated as follows: 1440, 132 and 0.295 /LM for D-1 ; 45.8, 8.8 and 0.004 /tM for D-2; greater than 100, greater than 100 and 0.64 ,uM for D-3; 11.7, 2.88 and 0.0044 PM for D-4, respectively. It is suggested that the affinity of tiapride is high to D-2 and D-4, but is not high to D-1 and D-3. The affinity pattern of tiapride to each DA receptor subtype is similar to but lower than those of sulpiride and haloperidol. In the D-2 receptor assay, the IC50 values of tiapride and sulpiride were 1/22.7 and 1/19.1 of those in the presence of 100 mM NaCI, respectively, suggesting that benzamide drug binds to the D-2 subtype with higher affinity in the presence of Na+ than in the control. Tiapride (N-[2-(diethyl amino)ethyl] -5 (FPT), [3H]spiperone and [3H]N-propyl (methylsulfonyl) -o-anisamide) has been apomorphine (NPA) were examined in the demonstrated as a dopamine (DA) receptor membranes of rat striatum and bovine antagonist which selectively suppresses caudate nucleus. dyskinesia (1). DA receptors are classified Male adult rats of the Wistar strain were as D-1, D-2, D-3 and D-4 subtypes (2). used for D-1 and D-2 receptor binding assay. D-1 and D-2 subtypes cause a stimulation Since specific binding of [3H]NPA to rat and inhibition in adenylate cyclase activity, striatal membranes was too low in both D-3 respectively (3, 4). D-3 receptors seem to be and D-4 receptor assays of preliminary presynaptically located to regulate DA release experiments, bovine brain obtained from a (2). In addition, it is also suggested that D-4 local slaughterhouse was used for D-3 and subtypes may be an interconvertible state of D-4 receptor assay. The striatum from rats or D-2 subtypes (5), although the details of the caudate nucleus from bovine brain were functional significances cannot be clarified. rapidly dissected and homogenized in 50 vol. To identify the affinity of tiapride to D-1, of ice-cold 50 mM Tris-HCI buffer (pH 7.7) D-2, D-3 and D-4 subtypes as well as those using a Polytron® (setting at No. 6 for 30 sec) of sulpiride and haloperidol, the inhibitory and then centrifuged at 50,000 x g for 1 5 min, effects of each drug on [3H]cis-flupenthixol followed twice by resuspension in fresh buffer and centrifugation. D-1 DA receptor assay was carried out by the modified method of Murrin (6). The by 0.1 W speiperone, respectively. The pellets from rat striatum were resuspended in specific bindings of D-3 and D-4 were assay buffer (50 mM Tris-HCI buffer con estimated as 57.0 and 71.0%, respectively. taining 120 mM NaCI, 5 mM KCI, 2 mM CaCl2 Protein content was determined by the and 1 MM MgCl2, pH 7.4). Aliquots (0.8 ml) method of Lowry et al. (8). of the suspension (0.13-0.2 mg/ml), 0.1 ml Statistical significances were analyzed by of drug or buffer and 0.1 ml of [3H] FPT Student's t-test. (final concentra ion, 1.0 nM) were incubated [3H]cis-Flupenthixol (10.4 Ci/mmol, New at 37°C for 5 min. Whatman GF/C filters were England Nuclear), [3H]spieprone (16.0 Ci/ soaked in the assay buffer containing Tween mmol, Amersham) and [3H] N-propyl 80 (polyoxyethylene sorbitan monooleate) at apomorphine (53.1 Ci/mmol, New England room temperature for at least 1 5 min prior to Nuclear) were used. Tiapride hydrochloride, use. The incubation was terminated by rapid sulpiride and haloperidol were purchased vacuum filtration over treated GF/C filters from Fujisawa Pharmaceutical Co., Ltd.; followed by four 5 ml rinses with ice-cold ketanserin from Kyowa Hakkoh Kogyo Co., assay buffer. The radioactivity on the filters Ltd.; spiperone from Eisai Pharmaceutical was assayed by liquid scintillation counting. Co., Ltd.; (+)-butaclamol hydrochloride from Blank values were generated by inclusion of Ayerst Res. Lab. (Canada); dopamine hydro 20 /iM (+)-butaclamol. Specific binding chloride from Nakarai Chemicals Co., Ltd. was 60.0% of the total binding. Sulpiride was dissolved in 0.1 N HCI and D-2 DA receptor assay was carried out by diluted with buffer. Haloperidol and the modified method of Usdin et al. (7). The ketanserin were dissolved in ethylalcohol, rat striatal membrane pellets described above followed by dilution with buffer. Ethyl were resuspended in 50 mM Tris-HCI buffer alcohol did not affect the binding. Tiapride with 0.1% ascorbic acid (pH 7.1). The and dopamine were dissolved in buffer. membrane suspension (0.3-0.4 mg/tube) Preliminary experiments showed that neither was incubated with 0.4-0.5 nM [3H] 0.1% (D-2 assay) nor 0.02% (D-3 and D-4 spiperone, 0.1 uM ketanserin and test drug assay) ascorbic acid significantly affected the at 37°C for 15 min. Membranes were binding. recovered by rapid filtration (Whatman GF-B Effects of tiapride, sulpiride, haloperidol filters) under vacuum with three 5 ml rinses and DA on D-1 sites were investigated by of ice-cold buffer. Specific binding was examining effects of each drug on [3H] FPT measured as the excess over blanks taken in binding to rat striatal membranes. From con the presence of 1 itM (+)-butaclamol and centration-inhibition curves, the IC50 values was estimated as 69.0% of the total binding. of tiapride, sulpiride, haloperidol and DA were D-3 and D-4 DA receptor assays were 1440+370, 132±44, 0.295±0.052 and performed by the modified method of Seeman 2.53±0.37 ,_cM, respectively (Table 1). To (2). Membrane pellets from bovine caudate avoid [3H]spiperone binding to S-2 serotonin nucleus were resuspended in 15 mM Tris binding sites, [3H]spiperone binding to D-2 HCI buffer (pH 7.4) containing 5 mM EDTA sites in rat straital membranes was carried out Na2, 0.02% ascorbic acid and 12.5 ; iM in the presence of 0.1 /iM ketanserin. As nialamide. In the D-3 receptor assay, mem shown in Table 1, the IC50 of tiapride, brane suspension (0.3-0.4 mg protein/tube) sulpiride, haloperidol and DA were estimated containing 0.1 /iM spiperone was incubated as 45.8+4.3, 8.8±1.24, 0.004±0.0006 and with 0.29 nM [3H]NPA and each test drug 1.41 ±0.32 ,uM, respectively. In the presence at 22°C for 30 min. To assay D-4 receptors, of 100 mM NaCI, inhibitory effects of tiapride membrane suspension (0.3-0.4 mg protein/ and sulpiride were enhanced, and the IC50 tube) containing 25 nM DA was incubated values of these drugs become 1/22.7 and with 0.29 nM [3H]NPA and each test drug 1/19.1, respectively. Previously reported at 22°C for 30 min. Specific bindings of D-3 results (9) showed that the addition of 0.1 1'M and D-4 receptor assays were defined as unlabeled spiperone into the binding medium the binding displaceable by 25 nM DA and was enough to avoid [3H]NPA binding to Table 1. The IC50 values of tiapride, sulpiride, haloperidol and dopamine on D-1 , D-2, D-3 and D-4 sites D-4 binding sites. Inhibitory effects of test were examined by the modified method of drugs on [3H]NPA binding were examined Seeman (2). Based on the very close in the presence of 0.1 1W spiperone, and the relationship between inhibitory effects of IC50 values are shown in Table 1. In com several neuroleptics on [3H] FPT binding and parison to the potent inhibitory action of DA on adenylate cyclase (14, 15), [3H] FPT (IC50=0.002±0.0009 itM) and to the binding seems to bind to D-1 sites. Although relatively potent action of haloperidol (IC50 FPT binds to D-2 DA sites with higher =0 .640±0.050 iiM), neither tiapride nor affinitythan to D-1 sites (6, 16), the maximum sulpiride caused 50% inhibition at 100 p M. number of D-1 binding sites is much higher Inhibitory effects of test drugs on [3H]NPA (over 2,000 fmol/mg protein) than that of D-2 binding to D-4 sites of bovine caudate nucleus sites (334 fmol/mg protein) in the rat striatum membranes were determined in the presence (2). In fact, several authors have suggested of 25 nM unlabeled DA to exclude [3H]NPA that 80% of[ 3H]FPT binding seems to be binding to D-3 sites as described in the D-1 sites (10, 15, 17).