Assessment of striatal D1 and D2 dopamine receptor-G protein coupling by agonist-induced [35S]GTPγS binding

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PII SOOZ4-3205(99)00412-9

Vol. 65, No. 16, pp. 1633-1645, 1999 Copyright 0 1999 Elsevier Science inc. Printedin the USA. All rights nsemd 0024-3205KWlsee hnt mattea

ASSESSMENT OF STRIATAL D 1 AND D2 DOPAMINE RECEPTOR-G COUPLING BY AGONIST-INDUCED [35S]GTPyS BINDING Muriel Geurts, Emmanuel Laboratoire

PROTEIN

Hermans] & Jean-Marie Maloteaux

de Pharmacologic, FARL 54.10, UniversitC catholique de Louvain, Avenue Hippocrate 54, B- 1200 Brussels, Belgium. (Receivedin final form June 18, 1999) Summary

The dopamine receptor-mediated modulation of guanosine 5’-0-(y[35S]thio)triphosphate ([35S]GTPyS) binding has been characterized in rat striatal membranes. In optimized experimental conditions, the potency of dopamine was 4.47 PM [3.02 - 6.61 PM] and a maximal response representing 54.8 f 4.5 % increase above basal level was observed. Data obtained with different agonists and antagonists clearly revealed that the most important fraction of this response was reflecting D2 receptor activation. Further analysis with specific antagonists also supported evidence for the involvement of Dl dopamine receptors. The potencies of compounds interacting with Dl and D2 receptors were deduced from [“S]GTPyS binding experiments and compared with their binding affinities for these receptors measured in similar experimental conditions. A good correlation between these parameters was observed, supporting the applicability of this technique for the study of dopamine receptors in the central nervous system. Key words [“S]GTPyS binding, spiperone, striatal membranes, G protein, Dl dopamine receptor, D2 dopamine receptor The use of high affinity and selective radioligands in binding experiments has been for many years the principal experimental approach for the characterization of dopamine receptors in the central nervous system. Meanwhile, dopamine receptors were shown to belong to the superfamily of seven-transmembrane-domain, G protein-coupled receptors (GPCRs). In this case, the first step following receptor activation is an exchange of GDP for GTP at the a-subunit of the interacting guanine nucleotide regulatory protein (G protein) (1). Based on this exchange, functional approaches for the study of GPCRs have been developed, including the measure of agoniststimulated GDP release or GTP hydrolysis (2). In the case of dopamine receptors, some authors have measured agonist-induced GTPase activities in rat striatal membranes (3-6). More recently, the discovery of high affinity, non-hydrolysable analogues of GTP has brought considerable interest to the study of agonist-induced increase in the specific binding of labeled nucleotides. Agonist-induced guanosine 5’-O-(y-[35S]thio)triphosphate ([35S]GTPyS) binding constitutes a valuable tool for the study of the functional properties of GPCRs by focusing on the first step of the signal transduction cascade. This method has been successfklly used to study the functional coupling of a large variety of receptors, such as muscarinic acetylcholine- (7) and AI adenosine(8) receptors. In addition, agonist-induced [3sS]GTPyS binding has also been applied to autoradiographic methods (for review, 9). In the case of dopamine receptors, agonist-induced ’ Corresponding author : Dr Emmanuel HERMANS, Laboratoire de Pharmacologic, UniversitC catholique de Louvain (54.10), Avenue Hippocrate 54, B-1200 Brussels, Belgium. Phone : 00 32 (02) 764 5317. Fax : 00 32 (02) 764 5460 Email : [email protected]

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[‘“S]GTPyS has been characterized in recombinant cells expressing the different subtypes of these receptors (1 O-l 5) and in reconstituted systems (16. 17). Further elegant distinction of the type of G-protein activated by striatal dopamine receptors has been achieved by combining [3”S]GTPyS binding and immunoprecipitation of G-protein subunits (18.19). Dopamine exerts its effects after binding to two types of receptors. known as the Dl and D2 receptors (20). Stimulation of adenylyl cyclasc viu Dl receptors and inhibition viu D2 receptors are mediated by distinct G proteins termed G, (stimulatory) and G, (inhibitory), respectively. In addition, Dl-like dopamine sites that couple to G,, have been proposed to mediate dopaminestimulated formation of inositol phosphates in the rat striatum (21). Molecular biological techniques have led to the identification of five subtypes of dopamine receptors (22) which can be classified as belonging to the D 1 (DI and Ds) or D2 (Dz. Di. D4) classes. The prevailing dopamine receptors in the striatum are the D, and D? subtypes (23-25). These receptors play a major role in the modulation of the locomotor activity and have been incriminated in movement disorders associated with chronic neuroleptic treatment or with neurological pathologies such as Parkinson’s disease and Huntington’s chorea (26). In the present study, the dopamine receptor-mediated [3’S]GTPyS binding has been characterized in rat striatal membranes. Different agonists and antagonists were used to assess the involvement of Dl and D2 receptors in the response to dopamine. Potencies of these drugs were deduced from [35S]GTPyS binding experiments and compared with their binding affinities for these receptors. A rather good correlation was found between these parameters, supporting the applicability of this technique for the study of dopamine receptors in the central nervous system.

Methods

The day of the binding assay. male Wistar rats here decapitated, the whole brain removed from the skull. The striata were dissected out on ice and immediately homogenized, by the use of a teflon/ glass homogenizer, in 50 volumes of ice-cold 50 mM Tris.HCl buffer (pH 7.4). The homogenate was centrifuged at 600 g for 10 min, and the supernatant obtained was centrifuged for 10 min at 49,000 g. The membranes were washed, resuspended in 100 volumes of ice-cold 50 mM Tris.HCl buffer (pH 7.4) and centrifuged for 10 min at 49,000 g twice. The pellet was resuspended in 20 volumes of ice-cold buffer containing 50 mM Tris.HCl (pH 7.5), 1 mM EDTA, 5 mM MgCll, and 150 mM NaCI. All procedures were carried out at 0-4°C until binding assay. Protein concentration was determined using Coomassie dye reagent (27). The [3”S]GTPyS binding assay was performed at 30 “C in plastic tubes containing 20 ug protein resuspended in a final volume of 1 ml. Binding buffer contained 50 mM TrisHCl (pH 7.5), 1 mM EDTA, 5 mM MgC12, 150 mM NaCI, 1 mM dithiothreitol and 0.1 % sodium metabisulfite. The binding was initiated by the addition of [35S]GTPyS (final concentration 0.1 nM). Non specific binding was measured in the presence of 0.1 mM 5’-guanylylimidodiphosphate (Gpp(NH)p). Unless indicated, incubation was performed for 60 min (dopamine. bromocriptine) or 15 min (other agonists) and in the presence of 10 uM GDP. It was terminated by addition of 3 ml ice-cold washing buffer (50 mM Tris.HCl (pH 7.5), 1 mM EDTA, 5 mM MgC12 , 150 mM NaCl). The suspension was immediately filtered through GFiB glass fiber filters and washed twice with the same buffer. Filters were immersed in 5 ml Aqualuma before determination of radioactivity by liquid scintillation counting.

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[3H]Spiperone binding experiments were performed in the conditions optimized for the [35S]GTPyS binding assay (incubation time and tern erature, buffer, GDP concentration). P Membrane proteins (100 ug) were incubated with [ Hlspiperone (0.3 nM in competition experiments, 25 pM to 1 nM in saturation experiments) in a final volume of 1 ml. Ketanserin (0.1 PM) was added in order to occlude the binding of the radiolabelled ligand to 5-NT2 receptors (28). Non specific binding was determined in the presence of domperidone (1 PM). [3’S]GTPyS (> 1,000 Ci/mmol) and [3H]spiperone (105 Ci/mmol) were purchased from Amersham. Bovine serum albumin, GDP, Gpp(NH)p, dopamine hydrochloride, clozapine, haloperidol, 2-bromo-a-ergocryptine (bromocriptine) methanesulfonate and pergolide mesylate were from Sigma. Ketanserin tartrate, R(+)-7-chloro-8-hydroxy-3-methyl-l-phenyl-2,3,4,5tetrahydro-lH-3-benzazepine hydrochloride (R(+)-SCH23390), R(+)-6-chloro-7,8-dihydroxy-lphenyl-2,3,4,5-tetrahydro-lH-3-benzazepine hydrobromide (R(+)-SKF8 1297), (-)-quinpirole hydrochloride, R(-)- 10,ll -dihydroxy-N-n-propylnorapomorphine hydrochloride (NPA), phentolamine mesylate and domperidone were obtained from RBI. Glass fiber filters were obtained from Whatman. Aqualuma was obtained from Lumac. All other materials were of the highest commercial purity available. Experimental protocols were approved by the local ethical committee and meet the guidelines of the responsible governmental agency (Administration de la Sante Animale et de la Qualite des Produits Animaux, Services Veterinaires du Ministere, Brussels). Data were analysed by non-linear regression and, where appropriate, statistical differences were determined by one-way ANOVA, followed by the Tukey’s test for multiple comparisons using the software Prism II (GraphPad Software, San Diego). In all statistical analyses, the null hypothesis was re_jected at the 0.05 level.

Results A preliminary step in this study was the optimization of the experimental conditions for the measure of [35S]GTPyS binding induced by dopamine in the model of rat striatal membranes. Both the basal and the dopamine-induced [35S]GTPyS specific binding were found to progressively increase with time (Figure 1A). Basal binding appeared to increase linearly up to 3 h whereas dopamine-stimulated binding had a half-time of association greater than 90 min. The ratio between dopamine-induced and basal [3SS]GTPyS specific binding remained relatively constant from 10 to 90 min. Similar observations were obtained with the agonist quinpirole (not shown). After 90 min, the dopamine-induced response tended to decrease. The proportion of non specific binding was very high at early points and progressively decreased with time, representing about 30 % at 15 min, 25 % at 60 min and 15 % at 3 h. Experimentally, dopamine- and bromocriptine-induced nucleotide binding were measured after 60 min, while a 15 min time was chosen for the other agonists. These two incubation times are a compromise between a substantial stimulation, stability of the preparation and sufficient specific-to-non specific binding ratio. The concentration of GDP in the incubation medium was found to dramatically influence the extent of basal and dopamine-stimulated [35S]GTPyS specific binding. Accordingly, when homogenates were incubated in the presence of low concentrations of GDP, the binding of [ 3”S]GTPyS was high, but was not significantly modified after addition of a high concentration of dopamine (Figure 1B). At high concentrations of GDP, the binding of [35S]GTPyS was very low and in these conditions, it was not possible to calculate the effect of the agonist. The optimal measurable effect of dopamine was detected in the presence of 10-5-10d M GDP. Similar results

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were obtained with other dopamine receptor agonists (bromocriptine, SKFS 1297 and NPA, data not shown) and therefore, all experiments were performed with a final concentration of 10 pM GDP. A 25Occ~

Incubation time (min)

log [GDP1 (MI

Fig. 1 (A) Time-course for basal- (open squares) and dopamine (100 PM)-induced (closed squares) [3’S]GTPyS specific binding to striatal membranes. GDP concentration was fixed at 10 &l. (B) Effect of increasing concentrations of GDP on basal-(main graph) or dopamine (100 PM)-induced (inset) [35S]GTPyS specific binding. Incubation time was fixed at 60 min. Data are mean + S.E.M. of 3 independent experiments performed in triplicate and are expressed in dpm of specifically bound [3”S]GTPyS (A and main graph of B) or as percentage of the basal binding (inset of B). DA, dopamine.

Using these optimized experimental conditions. the effect of increasing concentrations of different dopamine receptor agonists on [35S]GTPyS specific binding was measured. Analysis of the concentration-response curves (Figure 2) for specific D 1 (SKF8 1297) or D2 (bromocriptine, NPA. quinpirole and pergolide) agonists as well as for dopamine allowed to calculate their potency and efficacy in this assay. Pharmacological data from these curves - maximal stimulation (Emax) and concentration corresponding to half-maximal response (ECso) - are given in Table IA. As compared to a 55 % increase in the [3’S]GTPyS specific binding induced by high concentration of dopamine, the maximal responses to the putatively specific stimulation of Dl and D2 receptors were 11 % and 35 %, respectively. o

Dcpamlne

A NPA A Quinpirole .

-11

SKF81297

-10

-9

-6 log

-7

-6

-5

-4

-3

[agonist] (M)

Fig. 2 Effect of increasing concentrations of dopamine receptor agonists on [35S]GTPyS specific binding. Data are mean f S.E.M. of at least 4 independent experiments, performed in triplicate and are expressed as percentage stimulation above basal level.

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The dopamine-induced [“S]GTPyS specific binding was competitively, but not entirely, inhibited by potent and specific Dl and D2 receptor antagonists (SCH23390 and domperidone, respectively). The pKinh!b values for these drugs (Table IA) were calculated from experiments performed with a submaximal concentration of agonist (dopamine or NPA) and increasing concentrations of the antagonist (Figure 3), using the Cheng-Prusoff equation (29). An almost complete inhibition was obtained with the less specific antagonists clozapine (displaying similar affinity for Di and DZ subtypes) and haloperidol (which is -IO-fold specific for D2 versus Di). In the presence of increasing concentrations of clozapine (figure 4), or haloperidol (data not shown) the sigmoi’dal dose-response curve was progressively displaced to higher dopamine concentrations. The pKi”hib values for haloperidol and clozapine were derived from Schild plot analysis. At the highest concentrations tested, none of these antagonists were found to significantly change the basal level of [35S]GTPyS specific binding. A. _

B.

100

100

=t j&60 s

.Eu,E f

60

I

I

'Z g 60

60

40

40

g.g

5OG gx=:5iE -c

20 aE

0

SCH 23390

n

domperidone

-10

-9

log

-6

-7

20

domperidone

n

-6

[antagonist] (M)

-5

0h

-10

-9

-8

-7

. -6

-5

log [antagonist] (M)

Fig. 3 A) Effect of increasing concentrations of domperidone and SCH23390 on dopamine (100 PM)-induced [35S]GTPyS specific binding. B) Effect of increasing concentrations of domperidone on NPA (0.1 FM)-induced [35S]GTPyS specific binding. Data are expressed as percentage of the maximal response (measured in the absence of antagonist) and are mean 5 S.E.M. of 3 independent experiments performed in triplicate.

l CzPO.l)JM 0 CZPlpM

Fig. 4 Effect of clozapine (CZP) on the concentration-response curve for dopamine (DA)induced [35S]GTPyS specific binding. Results are expressed as percentage stimulation above basal binding. Data are mean k S.E.M. of triplicates from a typical experiment (n = 4). Inset : Schild plot analysis of the above-mentioned experiment. DR, EC50 ratio.

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As expected, domperidone (1 PM) completely inhibited the effect of the D2 receptor agonists NPA (0.01 PM). quinpirole (10 uM) and pergolide (0.1 PM) used at a concentration close to their ECso. Although bromocriptine (0.01 pM). known as a D2 dopamine receptor agonist. was significantly (p
A.

B. 0

Non spmflc

,

I---,

0

’

-----I

250

500

[‘H]spiperone

150

(PM)

1000

-11

-10

-9

-8

-7

log [competiior]

-5

-5

-4

(M)

Fig. 5 (A) [‘H]Spiperone saturation experiment. Striatal membranes were incubated in the presence of increasing concentrations of [3H]spiperone, as described under Methods. Data are mean + S.E.M. of quadruplicates from a typical experiment, performed 12 times independently. (B) Competition experiments. Striatal membranes were incubated with 0.3 nM [3H]spiperone and varying concentrations of dopamine, bromocriptine, clozapine, domperidone. or haloperidol. Data are mean & S.E.M. of triplicates from a typical experiment, performed 2 to 5 times independently.

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(mean + S.E.M., n = 12). Competition experiments were performed with dopamine, bromocriptine, clozapine, haloperidol, and dom eridone (figure 5B), and the inhibition constants (pK,), calculated from I& values and the Ko of [PHlspiperone, are indicated in Table IB. The relationship between the dissociation constants for dopamine receptors (pKi) and the functional constants measured in [3%]GTPyS binding assay (pE&o for agonists and pK,nhib for antagonists) is presented in Fig. 6. Concerning the agonists, dissociation constants for D2 dopamine receptors were compared to pE& values from Table IA. When dopamine-, NPA- or quinpirole- induced [35S]GTPyS binding were determined in the presence of 20 nM SCH23390, in order to maximally antagonise Dl- without affecting D2- receptors, only a modest and nonsignificant rightward shift of the concentration-response curves was observed (data not shown). Therefore, potencies of these agonists measured in the [35S]GTPyS binding assay were considered to reflect their potencies at D2 dopamine receptors. For pharmacological accuracy, data obtained with agonists and antagonists were considered separately. In these conditions, although a rather good correlation was obvious for both classes of drugs, the total number of substances in each group was not sufficient to perform a statistical analysis of the relationship between the functional and radioligand binding data. However, when the entire set of compounds used in the study was considered, a highly significant correlation between these parameters was observed (p
7

8

9

5

10

PKi

6

7

8

9

PKi

Fig. 6 Relationship between dissociation constants and functional constants for dopamine receptor antagonists (A) and agonists (B). pECs0 and pKi”hib were derived from [3SS]GTPyS binding experiments (Table IA.). Dissociation constants for dopamine, haloperidol and domperidone were determined in bromocriptine, clozapine, [3H]spiperone competition experiments. The dissociation constant of SCH23390 for Dl dopamine receptors was from Ref. 30. The pK, values for NPA, pergolide and quinpirole were from previous reports in which experimental conditions shifted D2 dopamine receptors towards the low-affinity state (30, 3 1).

Discussion In the present study, we have analyzed the functional response to dopamine in rat striatal membranes by measuring agonist-induced [3sS]GTPyS binding. This extends previous reports on the dopamine receptor-G protein coupling assessed using this technique in transfected cells or

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reconstituted systems (1 O-l 7). Although the involvement of D2 receptor activation was clearly demonstrated and pharmacologically characterized. the possible role for Dl receptors was less obvious and only measurable wit!1 dopamine after antagonizing D2 receptors. According to previous studies performed with other GPCRs (for review. 32), optimization of binding conditions, such as the guanine nucleotide concentration, was found critical for the determination of agonist-stimulated [35S]GTPyS binding. Indeed, in physiological conditions, GDP and GTP are in competition for binding to the a-subunit of the G protein and agonists incline the competition in favor of GTP. Although Gardner and colleagues (10) reported that the optimal concentration of GDP to measure the dopamine agonist-induced [3’S]GTPyS binding was 1 uM, in the present study, the highest signal-to-noise ratio was obtained with lo- 100 uM of this nucleotide. This discrepancy could be explained by differences in the models used. Indeed, it was shown by these authors that the stimulation depended on the cell type, and not on the level of receptor expression. In brain membranes, Lorenzen et al. (8) found that 10 uM was the optimal GDP concentration in order to measure a sufficient response to A1 adenosine receptor agonists. It is well known that experimental conditions can modulate the affinity of agonists for GPCRs. Thus, in rat striatal membranes (28), as well as in the anterior pituitary gland (33) D2 dopamine receptors were shown to exist in two inter-convertible states with equal antagonist affinities but with differing agonist affinities. IJnder low temperature (22°C) sodium- and guanine nucleotidesfree conditions [3H]dopamine labels a high-affinity state of D2 dopamine receptors. In more physiological conditions (presence of Na’, guanine nucleotides, higher temperature), a proportion of high- and low- affinity states co-exist (28). On this basis, the affinity of some agonists and antagonists used in the present work was determined in the experimental conditions optimized for the [35S]GTPyS assay. As expected on the basis of their antagonist profile, saturation of [3H]spiperone binding as well as competition with domperidone, haloperidol and clozapine revealed affinities similar to those previously reported (28,30,34,3.5), indicating their insensitivity to experimental conditions known to affect agonist binding. By contrast, competition with dopamine revealed an affinity for D2 receptors that was intermediate between the values generally obtained when measured in conditions favoring either the high- or the low- affinity states. Contrarily to most agonists of GPCRs. bromocriptine does not discriminate between high- and low- affinity states of dopamine receptors and therefore, a single affinity constant is generally measured (30, 36) which is consistent with the present findings. On the basis of the relatively low affinity of dopamine for D2 dopamine receptors measured in our experimental conditions. it is likely that a high proportion of these receptors are in the low-affinity state. Agonist potencies, found in the functional assay, were in good agreement with the affinity values measured in similar conditions. In a previous study, McDonald et al. (37) reported ECjo values revealing a very high potency for the dopaminergic agonists tested in inhibiting adenylyl cyclase activity. Indeed, in this case, experimental conditions were such that a high proportion of dopamine receptors was in a high-affinity state. By contrast, rather low potencies of dopamine agonists, consistent with those found in the present study, were reported by other groups when measuring agonist-induced GTPase activity or [3’S]GTPyS binding in striatal membranes or cultured cells (4,5,11,12). It is indeed well documented that the presence of guanine nucleotides and Na’ is generally required in order to study G-protein activation by these techniques. In a similar fashion, Adapa and Toll (38) showed that when using experimental conditions favouring the low-affinity state of opioid receptors, functional responses were observed such as CAMP accumulation and stimulation of [35S]GTPyS binding with potencies corresponding to the lowaffinity state.

Dopamine-induced [3JS]GTPySBinding

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TABLE I Pharmacological Profile of Dopamine Agonists and Antagonists in [35S]GTPyS and [3H]Spiperone Binding Assays. A. Potencies in [35S]GTPyS assay pE& or pKi”hib (K, r&f,)

Agonist efficacies in [35S]GTPyS assay n E mau(% above basal binding

5.35 7.49 8.08 6.53 5.82 6.52

f 0.17 * 0.25 + 0.11 ?I 0.25 f 0.16 + 0.40

(4,500) (32) (8.3) (290) (1,500) (302)

54.83 + 27.90 + 31.74 f 34.81 + 29.48 k 11.50f

9.38 7.30 9.15 8.84 8.34

f f f + +

(0.42)

Agonists

Dopamine Bromocriptine NPA Pergolide Quinpirole SKF 81297 Antagonists SCH23390

Clozapine Haloperidol Domperidone

O.Oga 0.09a 0.22& O.Oga 0. 13b

4.50 2.48 1.19 2.45 2.51 1.95

0-O) (0.71) (1.4) (4.61

B.

Dopamine

pKi from [3H]spiperone binding assay (K, nM) 6.24 f 0.07 (n = 3) (5 70)

pK, for D2 dopamine receptors from previous reports (K,, nM) 5.37’ (low-affinity state) (4,300) 8.12’ (high-afftnity state) (7.5)

Bromocriptine Clozapine Haloperidol Domperidone

8.39 + 0.09 (n = 2) (4.03) 7.21 + 0.11 (n = 5)

8.32’

(62) 8.50 + 0.01 (n = 3) (3.2) 8.79 + 0.13 (n = 3) (1.6)

(230) 8.92’ (1.2) 9.60d (0.25)

(low- /high- affinity state)

(4.8) 6.64’

The [35S]GTPyS and [3H]spiperone binding assays were carried out in the same conditions (time, temperature, buffer, membranes preparation). Data are mean + S.E.M. of n independent experiments, performed in triplicate. (A) Potencies of agonists in inducing [35S]GTPyS binding were expressed as (p)ECso values. @)Kinhib values of antagonists were determined via inhibition of dopamine- (“) or NPA- (b) induced [35S]GTPyS binding. (B) Afftnity of agonists and antagonists for D2 dopamine receptors were determined using [3H]spiperone competition experiments or were from previous reports (’ Ref. 30, d Ref. 28).

24 18 6 5 4 7

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pK,“h,b of the selective DI (SCtE3390) and D2 (domperidonej dopamine receptor antagonists obtained from competition experiments are in good correlation with dissociation constants for the corresponding receptors. This is zonsistcnt with the fact that the dopamine response is mediated via either Dl or D2 receptors in these experimental conditions. It must be pointed out that the analysis of the effect of SCH23390 and to a lesser degree of domperidone revealed inhibitions spreading over 3 orders of magnitude. Indeed. the amplitude of the inhibition by SCH23390 is rather small and precludes any definitive conclusion about this observation. Nevertheless. it clearly reveals the complexity of the response mediated by dopamine and its inhibition by these antagonists. Indeed, a heterogeneous population of dopamine receptors exists in striatal membranes. Complexity may also result fi-om the diversity of G proteins associated with Dl (G,. G,lr, G,) and D? (G,. G,,) dopamine receptors in the striatum (for review. see Ref. 39). Interestmglq. no significant inhibition of the SK}:8 1397-mediated response could be observed with the Dl receptor antagonist SCH23390. .The rather limited amplitude of the response to the agonist SKF81297 (as well as SKF38393. data not sho\\n) hampered the accurate analysis of experimental data. In fact. such compounds have been proposed to act as partial agonists at Dl receptors (4042). In order to analyse the putative imolvement of Dl receptors in the dopamine-induced [“S]GTPyS binding. we have characterized the response mediated by dopamine when measured in the presence of a saturating concentration ofthr highly selective D2 dopamine receptor antagonist. domperidone. It is noteworthy that the extent of the response to dopamine that was specifical]) inhibited by the D2 antagonist \cas consistent \vith the maximal effect obtained using specific D2 receptor agonists. The remaining stimulation \\as found to be mainly due to Dl dopamine receptors as indicated by the inhibition obtained \vith SCH23390. Surprisingly, a significant inhibition of the non-D2 response \cas also obtained using the a-adrenergic antagonist. phentolamine. Previous reports have shown that dopamine could act as an u-adrenoceptor agonist at high concentrations (43) and therefore. the invol\,ement of cx-adrenergic receptors in our results can not be excluded. Another interesting feature of this study was the unusual effect observed with bromocriptine. Indeed, contrarily with the results obtained Lvith the other D2 agonists (NPA, quinpirole and pergolide). the bromocriptine-evoked effect on guanine nucleotide binding was not entirely antagonized by domperidone. Similar observations \vere previously reported by Yue and co]]. when measuring agonist-induced ~‘I‘hc acti\,it> in rat striatum (3). Bromocriptine has been shown to bind to 5HT- (44). opiate- (45). and (I. adrenergic receptors (46). however, in our experimental conditions. specific blockade of thcsc receptors does not significantly inhibit the bromocriptine (10 PM)-evoked [ “SJGTPyS binding (Geurts and Maloteaux, unpublished results). Although the effect of bromocriptine on GTPase activity was found to be more efficiently antagonized by a combination of Dl and D2 antagonists (3). the bromocriptine induced [35S]GTPyS binding was not further inhibited b!, the combination of SCH23390 and domperidone or by less specific dopamine receptor antagonists such as clozapine and haloperidol. In accordance with these observations. bromocriptine does not show any agonist activity at Dl receptors (47). Unusual binding properties have been reported for bromocriptine. As mentioned above, its binding is unaffected by changes in incubation temperature or in Na+ and GDP concentrations (30). Moreover, it was suggested that bromocriptine interact with dopamine receptors in a nearly irreversible manner, perhaps because of its cyclic peptide chain undergoing additional binding reactions with adjacent receptor sites (for review. 36). This proposed model could explain the poor efficacy of D2 receptor antagonists to inhibit bromocriptine-mediated G-protein activation. As mentioned above, other strategies have been developed protein coupling, such as agonist-stimulated GTP hydrolysis.

for the assessment of receptor-G In the case of dopamine receptors

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studied in the rat striatum, some authors have measured agonist-induced modulation of GTPase activities (3-6). Overall, our results are consistent with these studies, also showing some limitations in characterizing the Dl component. This observation could reflect differences in the densities in the rat striatum. However, previous of dopamine receptor subtypes imunohistochemical studies have shown that D1 and D2 dopamine receptors (the prevailing subtypes in the striatum) are approximately expressed at the same level in this structure (23). In addition, the relationship between the amplitude of the agonist-induced [35S]GTPyS binding and the receptor density remains unclear (10). Odagaki and colleagues (6) have reported that functional receptor activation, as measured by agonist-induced GTPase activity, is more easily detected for G,-type G proteins as compared with other G protein subfamilies. In addition, it is proposed that agonist-induced guanine nucleotide binding assays more efficiently detect pertussis toxin-sensitive (G,) G-proteins (48). Therefore both the nature and the densities of G proteins activated by dopamine receptors need to be considered. It has been proposed that in most cell types, G,- are higher than G, levels (39). In addition, when agonist-induced stimulation of each G protein subtype was distinguished by combining agonist-induced [35S]GTPyS binding and immunoprecipitation methods, a robust stimulation was measured for both Gi and G, proteins (18, 19). Altogether, these results tend to prove that in native tissues, the detection of G, activation is masked by the activity of Gi. Surprisingly however, when low concentrations of GTP were added to striatal membranes, stimulation, rather than inhibition of adenylyl cyclase activity was observed (35, 49). Moreover, dopamine was found to induce an intense stimulation of adenylyl cyclase activity in striatal homogenates (35, 49, 50). Another group (5 1) also measured a high stimulation of striatal adenylyl cyclase after addition of the Dl agonist, SKF82526. Taken together, these observations indicate that when dopamine receptor-stimulated G protein activities are measured in a direct manner, the Dl component is less detectable than its D2 counterpart, for a reason which is not yet fully understood. When low stimulations are measured (< lOoh) and the signal-to-noise ratio is particularly high, as observed in GTPase activities assays performed in native membranes (2), the Dl component may become impossible to detect. Using the [35S]GTPyS binding assay, we have studied the primary step occurring in the signalling pathway of striatal dopamine receptors after binding of dopamine agonists. More than just measuring the density of receptors, which could correspond in some situations to spare receptors, this assay allows to assess the functional state of dopamine receptors in striatal tissue. This represents a useful tool to evaluate the functional coupling of dopamine receptors with G proteins in physiological and pathological conditions. Finally, in a pharmacological context, this method allows the functional study of the interaction of clinically used drugs at their presumed site of action.

Acknowledgments The authors wish to thank A. Lebbe and H. Lenaer-t for their excellent technical assistance. This work was supported by the National Fund for Scientific Research (F.N.R.S., Belgium, Convention FRSM 3.453.997F), by a Grant of Ministry of Scientific Policy (Belgium) (ARC 95/00-ISS), and by the Belgian Queen Elisabeth Medical Foundation. M. Geurts and E. Hermans are Research Assistant and Research Associate of the National Fund for Scientific Research, respectively.

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