Comparison of D2 and D3 dopamine receptor affinity of dopaminergic compounds in rat brain

PI1 SOO24-3205(98)00148-9

ELSEVIER

COMPARISON OF D2 AND D3 DOPAMINE DOPAMINERGIC COMPOUNDS

Life Sciences, Vol. 62, No. 20, pp. 182.5-1831, 1998 Copyright@ 1998 Ekvier Science Inc. Printed in the USA. All rights reserved 0024-3205/98 $19.00 t .cm

RECEPTOR AFFINITY IN RAT BRAIN

OF

Rebecca J. Flietstra and Beth Levant Department

of Pharmacology, Toxicology, and Therapeutics, University Center, Kansas City, KS 66160-7417, U.S.A.

of Kansas Medical

(Received in final form February 9, 1998) Summary This study used quantitative autoradiography to simultaneously evaluate the relative affinities of dopaminergic compounds for dopamine 9 and D3 receptors in rat brain. PD 152255, PD 128907, and I-nafadotride exhibited significantly higher affinity for cerebellar dopamine D3 sites than [3H]quinpirole-labeled sites in caudate/putamen (6.3-, 6.0-, and 2.3-fold, respectively). In contrast, chlorpromazine, risperidone, and domperidone were more potent at ‘striatal dopamine D2 receptors (3.8-, 3 l-, and 40-fold, respectively). Dopamine, quinelorane, (+)-UH 232, and RS-trans-7-OH-PIPAT exhibited relatively little D2/D3 selectivity. @ words D2 dopamine receptor, D3 dopaminereceptor, [3H]quinpirole,striatum, vestibulocerebellum

In 1990, Sokoloff et al. cloned the dopamine D3 receptor, a novel site with a structure and pharmacology similar to the D2 receptor. Because the D3 receptor is primarily expressed in limbic brain regions, rather than the striatum, it was proposed that the receptor may represent a target for antipsychotic drugs that might be free of extrapyramidal effects (1). A number of laboratories have sought to characterize the pharmacological profile of this site. D3 receptorexpressing cell lines have been widely used for this purpose resulting in the identification of a number of putatively Ds-selective compounds. In addition several putatively D3-selective radioligands have been synthesized and employed for binding studies in brain (for review see: 2). The degree of D@3 selectivity of certain compounds, however, has been highly variable and appears to be dependent on the use of rat or human receptors, the expression system, and the in vitro assay conditions used (3,4,5). Previously, we developed an autoradiographic method for the simultaneous comparison of D2/D3 receptor affinity in rat brain using the D2-D3 ligand [3H]quinpirole (6). This method is based on the observation that dopamine receptors in the molecular layer of the vestibulocerebellum, which appear to be colocalized exclusively with dopamine D3 receptor mRNA, exhibit a lack of guanyl Corresponding author: Beth Levant, Ph.D., Dept. of Pharmacology, Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7417, 7527; Fax: 913-588-7501; e-mail: [email protected].

University of Kansas U.S.A. Tel.: 913-58%

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nucleotide regulation and a pharmacological profile appropriate for the D3 site (4,6,7). As such, this brain area can binding site in brain tissue. In contrast, the caudate-putamen, which expresses substantially greater amounts of dopamine D2 receptor mRNA and exhibits relatively little dopamine D3 receptor binding, proves useful as a prototypical dopamine D2 tissue (4,6). Thus, by labeling these brain areas with a non-selective radioligand, such as [3H]quinpirole (3-S) it is possible to evaluate the D2/D3 selectivities of drugs in brain tissue under uniform in vitro assay conditions and in the same brain section.

In this study, we evaluate the dopamine D2/D3 selectivity of several novel putatively selective compounds. The D2/D3 selectivity of several clinically utilized antipsychotics other dopaminergic compounds is also examined.

DJand

Methods [3H]Quinpirole autoradiography. [3H]Quinpirole autoradiography was performed as previously described in detail (6). Slide-mounted sagittal brain sections (20 pm; lateral 1.O - 1.4 mm) from adult, male Sprague Dawley rats (200 - 300 g; Harlan Sprague-Dawley, Indianapolis, IN) were cut on a cryostat so that each section contained both caudatelputamen and vestibulocerebellum. Sections were incubated for 2 h at 23’ C with -10 nM [sH]quinpirole (40 Ci/mmol; DuPont NEN, Boston, MA) alone or in the presence of 5 concentrations of competing drug (10-s to 1O-5M) in assay buffer (50 mM Tris, 5 mM KCl, 2 mM MgCl2, and 2 mM CaC12, pH 7.4 @ 23” C). [sH]Quinpirole was prepared fresh for each experiment. The exact concentration was determined by scintillation counting for use in data analysis. Competition studies using dopamine were performed in the presence of 0.1% ascorbate. Nonspecific binding was defined in the presence of 1 PM spiperone. Duplicate sections from each animal were used for each data point. Sections from 4 individual animals were used for each drug. Following incubation, slides were dipped in ice-cold assay buffer, washed for 2 consecutive 5-min periods in ice-cold assay buffer, and dipped in ice-cold deionized H20. Radiolabeled sections were then dried and apposed to ‘H-Hypefilm (Amersham, Arlington Heights, IL) for a period of 8 weeks with [3H]methylmethacrylate standards. Data analysis. Autoradiographic images were digitized and quantified using the Macintoshbased video densitometry program NM “Image”. An optical density standard curve from [sH]methylmethacrylate standards was generated using a Rodbard plot. The density of [3H]quinpirole binding in caudate/putamen and the molecular layer of the vestibulocerebellum was sampled and expressed as fmol/mg tissue equivalent. Data from these brain regions were used to generate competition curves for each drug from which ICso values were derived by graphical analysis. Ki values were calculated using the Cheng and Prusoff equation: Ki = ICso/( 1 + [L]&). The previously determined KD value for [3H]quinpirole (2.3 nM) was used for this analysis (8). Kivalues for each drug in the caudate/putamen and vestibulocerebellum were compared using Student’s t-test (two -tailed). Significance was assumed at P < 0.05. Drugs. PD 128907, quinelorane, chlorpromazine, and domperidone were purchased from RBI, Inc. (Natick, MA); RS-trans-7-hydroxy-2-[N-propyl-N-(3’-iodo-2’-propenyl)amino]tetralin (OH-PIPAT), from Tocris Cookson (St. Louis, MO); and dopamine, from Sigma (St. Louis, MO). PD 152255 was donated by the Parke-Davis Co.; I-nafadotride, Bioprojet; (+)UH 232, the Upjohn Co.; and risperidone, Janssen Pharmaceuticals. Results Dense specific binding accumbens, olfactory

of [3H]quinpirole was observed in the caudate/putamen, nucleus tubercles, islands of Calleja and the molecular layer of the

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vestibulocerebellum as previously described (7) (Pig. 1). Caudate/putamen, a brain region associated primarily with dopamine D2 receptor mRNA, was used as a prototypical 9 tissue; vestibulocerebellum (D3 mRNA associated), was used as a dopamine D3 tissue for all further analyses. All drugs tested produced maximal inhibition of [Wjquinpirole binding similar to that produced by spiperone (1 p&I). Data from autoradiographic competition studies are shown in Fig. 2 and summarized in Table 1. Dopaminergic compounds inhibited [3H]quinpirole binding in caudatefputamen with the following rank order of potency: risperidone = domperidone > quinelorane = I-nafadotride = dopamine > RS-trans-7-OH-PIPAT 2 chlorpromazine > PD 128907 2: (+)-UH 232 2 PD 1552255. In the vestibulocerebellum, a somewhat different rank order was observed: I-nafadotride = quinelorane = dopamine > PD 128907 2 PD 152255 > RStrans-7-OH-PIPAT > risperidone = chlorpromazine = domperidone > (+)-WI 232. Under these assay conditions, PD 152255 and PD 128907 exhibited significantly higher affinity for cerebellar D3 sites (6.3- and 6.0-fold, respectively) followed by I-nafadotride (2.3-fold) than E3H]quinpirole-labeled sites in caudate/putamen. In contrast, chlorpromazine, risperidone, and domperidone were more potent at striatal DZ receptors (3.8-, 3 l-, and 40-fold, respectively). Dopamine, quinelorane, (+)-III-I 232, and RS-trans-7-OH-PIPAT exhibited relatively little selectivity for either dopamine receptor subtype.

Fig. 1 Autoradiographic localization of [3H]quinpirole-labeled sites in rat brain. Slidemounted sagiti brain sections (20 pm, lateral 1.0 - 1.4 mm) were incubated with -10 nM [%Ilquinpirole for 2h at 23’ C, washed, and apposed to sH-Hyperfilm for 8 weeks. A. Total binding. B. Nonspecific binding defined by 1 p.M spiperone. CPU - caudate/putamen; vestib. - vestibulocerebellum.

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Pharmacology

-9

-8

-7

-6

-9

-ii

-i

-6

PD 128907

I-Nafadotride

-tram-7-OH-PIPAT

-9

-8

-7

-6

-5

-9

-8

-7

-6

-5

-9

-8

Log Drug Concentration

Fig. 2 Competition curves of dopaminergic compounds on [5H]quinpirole binding in caudate/putamen (closed symbols) and vestibulocerebellum (open symbols). Slidemounted sagittal brain sections (lateral 1.O to 1.4 mm, in duplicate) were incubated for 2h at 23°C with -10 nM [3H]quinpirole in the presence or absence of 5 concentrations of competing drugs (10-g to 10-s M) and apposed to 3H-Hyperfilm. Autoradiographic images were digitized and quantified using the NIH “Image”. An optical density standard curve from [3H]methylmethacrylate standards was generated using a Rodbard plot, The density of [sH]quinpirole binding in caudate/putamen and the molecular layer of the vestibulocerebellum was sampled and expressed as fmol/mg tissue equivalent. These data were used to generate competition curves. Representative data are shown. Quantitative data are summarized in Table 1.

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TABLE 1 Pharmacological profiles of dopaminergic compounds in competition for [3H]quinpirole binding sites in rat caudate/putamen and vestibulocerebellum

Ki Cm) Compound

Caudate/putamen

PD 152255

175 f 65

PD 128907 I-Nafadotride

Vestibulocerebellum

Ki(caudate)/ Ki(vestib.j

28 If: 5.5*

6.3

109 I!I345

18 f 4.7*

6.0

7.0 f 1.2

3.0 + 0.4*

2.3

Dopamine

8.5 + 1.9

4.8 f 1.3

1.8

Quinelorane

5.7 f 0.9

3.4 f 1.1

1.7

(+)-UH 232

125 f 36

215 k 46

0.58

RS-truns-7-OH-PIPAT

20 f 7.7

47+ 18

0.42

Chlorpromazine

29+8.1

114f23*

0.26

Risperidone

3.3 f 0.8

103 f 24*

0.032

Domperidone

3.4 f 0.5

125 z!z48*

0.025

Slide-mounted sag&al brain sections (20 pm, lateral 1.0 - 1.4 mm) were incubated with -10 nM [3H]quinpirole alone or in the presence of 5 concentrations of competing ligand (10m9to 1O-5M) for 2h at 23’ C, washed, and apposed to 3H-Hyperfilm for 8 weeks. Nonspecific binding was defined with 1 p.M spiperone. Studies using dopamine were performed in the presence of 0.1% ascorbate. Data represent the mean I!I S.E.M. derived from competition analyses performed using duplicate sections from 4 individual animals. *P < 0.05 by Student’s t-test (two -tailed).

Discussion The dopamine D3 receptor has been proposed as a potential target for antipsychotic drugs. Messenger RNA for this novel Dz-related subtype, and presumably the receptors themselves, are present in much lower abundance than dopamine D1 and D2 receptors (2). Because of the low abundance of the receptors and the lack of highly selective pharmacological tools, most investigations of these novel subtypes have used cloned receptors expressed in cell lines. While this approach is of considerable merit, it has the inherent liability that the receptor expressed in a host cell may not exhibit the same binding characteristics as would be observed in brain tissue (9). Of note, certain drugs have exhibited dramatically different D2/D3 selectivities depending on the expression system used. For example, 7-OH-DPAT exhibited 98-fold selectivity between [3H]spiperone-labeled D3 and D2 receptors expressed in CHO-Kl cells, but only 3 l-fold selectivity when the receptors were expressed in CCL1.3 cells (10). The selectivities of other compounds, such as clozapine, (+)-butaclamol, and eticlopride were also shown to vary as much as 12-fold depending on the expression system used (5,lO). Hence, it is desirable to study the pharmacology of these receptors in brain as well as in transfected cells.

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In vitro assay conditions have also been shown to influence the affinity of compounds for dopamine receptors (3,lO,I 1). The autoradiographic method used in this study avoids the potentially confounding variable of using separate assays to measure dopamine D2 and D3 receptor affinity by using [%Ijquinpirole-labeled sites in striatum and vestibulocerebellum of the same brain section as prototypical D2 and D3 tissues. Because cerebellar dopamine sites are localized discretely in the molecular layer of the vestibulocerebellum, the autoradiographic method enables the detection of a signal adequate to allow pharmacological characterization using a tritiated radioligand (12). While this method does not allow the high degree of quantitation obtained in radioligand binding studies, the method is sufficiently quantitative to enable the determination of ICsc values and relative affinities in discrete brain regions. It should be noted, however, that because a small percentage of dopamine D3 receptors are expressed in the caudate/putamen, these data may somewhat underestimate the selectivity of D3-preferring ligands. The dopamine Dz/I$-selectivities of the compounds examined were generally in agreement with previous studies (for extensive review see: 2) in that antagonists PD 152255 and I-nafadotride and agonist PD 128927 exhibited modest, but significant selectivity for cerebellar D3 sites while domperidone and the antipsychotic risperidone exhibited significant D2-selectivity. However, these compounds exhibited greater relative preference for the D2 receptor than observed in the cloned receptors, perhaps due to the presence of low density D3 sites in the caudate/putamen (see above). Chlorpromazine exhibited significantly higher affinity for the D2 receptor in rat brain as reported in several studies using receptors expressed in various cell lines and antagonist radioligands (1,13). In agreement with Burris et al. (3) and Pugsley et al. (14) dopamine did not exhibit significant differences in afftnity between agonist-labeled D3 and D2 sites in rat brain although other studies have reported that the neurotransmitter exhibits substantially higher affinity for D3 receptors in some systems using antagonist radioligands. In contrast to the findings generated using transfected cell lines, quinelorane and RS-trans-7-OHPIPAT did not exhibit significant differences in affinity between the D3 and D2 sites in rat brain. Quinelorane has been reported to possess 43- or 95-fold higher affinity for the antagonist-labeled, cloned D3 receptor than the D2 in transfected cell lines (13,15). RS-trans-7-OH-PIPAT has been reported to exhibit 143-fold higher affinity for the D3 site in competition with [125I]R-(+)-trans7-OH-PIPAT (16). It must be noted, however, that the D2/D3 selectivity of compounds have been reported in only a few studies. As with the other compounds discussed, additional determinations by other laboratories using various methodologies may result is highly disparate results. Clearly, further study must resolve the issue of the D2/D3 selectivity of these compounds. In conclusion, a large body of highly variable data has been collected comparing the affinities of drugs at D2 and D3 sites. The present findings contribute additional information to this bank of data which may ultimately aid in the interpretation of the pharmacological effects of these compounds and to our understanding of the physiological roles of the D2 and D3 receptors. Acknowledgments We are grateful to Dr. Pierre Sokoloff of INSERM and Dr. Yves Joulin of Bioprojet for the generous donation of nafadotride; Dr. Montford Piercey of the Upjohn Co. for the donation of (+)-UH 232, Dr. Hyacinth Akunne of Parke-Davis for the donation of PD 152255, and Dr. Josee Leysen of Janssen Pharmaceuticals for the donation of risperidone. Supported by NIMH MH 52839.

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