Binding of [3H]sulpiride to purified rat striatal synaptic membranes

0306-4522/81/030407WSO202.00/0 PergamonPressLtd 0 1981IBRO

NewxctenccVol. 6, No. 3, PP. 407to 410,1981 Printedin GreatBritain

BINDING OF [3H]SULPIRIDE STRIATAL SYNAPTIC

TO PURIFIED MEMBRANES

G. N. WOODNJFFand S. B.

RAT

FREEDMAN

Department of Physiology and Pharmacology, Medical & Biological Sciences Building, Southampton University, Southampton, U.K. Aluttract-Previous results showing that sulpiride, unlike classical neuroleptics, does not block the effects of dopamine on the dopamine-sensitive adenylate cyclase have led to the concept of dopaminergic D1 and Dr receptors. It has been suggested that sulpiride is a specific antagonist of D2 receptors and [“Hjsulpiride has been used as a specific ligand for these. This report examines the binding of C3H]sulpiride to purified synaptic membranes from rat striata. There appears to be a single saturable binding component with an affinity constant of 7 nM and a maximum binding capacity of 240 fmol/mg protein. This binding site appears to be dopaminergic in origin since it is present in high concentration in the striatum and nucleus accumbens but in low concentration in non-dopaminergic regions of the brain. Our results show that sulpiride binding is potently inhibited by classical neuroleptics and henzamides, whilst dopamine agonists have much weaker activity. The binding site shows stereochemical specificity for cis-flupenthixol, cis-clopenthixol, (+ )-butaclamol and E( -)sulpiride. The rank order of potency of the dopamine antagonists is not consistent with a specific benzamide binding site since the classical neuroleptics were very potent inhibitors of binding. In particular, the thioxanthines, reputedly good inhibitors of the dopamine-sensitive adenylate cyclase, were amongst the most potent inhibitors of C3H]sulpiride binding. Thus it is clear that these results are not in accord with the present concept and classification of dopaminergic Dr and D2 receptors.

SULPIRIDE,which is a substituted benzamide has, amongst its spectrum of activity, properties which resemble those of classical neuroleptics and those which do not. Thus it is an antiemetic (LAVILLE& MARGARIT, 1968), increases serum prolactin levels in man (MANCINI, GUITELMAN,VARGAS, DEBEL~UK& APARICIO, 1976) and is a potent antagonist of vascular dopamine receptors (GOLDBERG,MUSGRAVE& KOHLI, 1978). It is also active in certain behavioural models of neuroleptic activity. It inhibits apomorphine-induced circling behaviour (ELLIOT JENNER,HUIZING, MARSDEN & MILLER, 1977) and is as potent as fluphenazine in inhibiting locomotor activity induced by 2-amino6,7_dihydroxy-1,2,3,4_tetrahydronaphthalene (ADTN) (WOODRUFF& ANDREWS,1978). However, it does not induce catalepsy (LAVILLE,1972) is weak in displacing [‘Hlhaloperidol binding (SPANO,G~VONI & Tnmuccm, 1978) and is inactive upon the dopamine-sensitive adenylate cyclase model (TRABUCCHI, LONGON, Fnnsu & SPANO,1975). These findings have been interpreted as evidence for two sorts of dopamine receptor, one linked to the adenylate cyclase and a second type which is cyclase: independent and inhibited by sulpiride (KEZBABIAN & CALNE, 1979). It is possible that the use of [3H]sulpiride as the ligand in binding studies might be expected to label Abbreviations: ADTN, 2-amino-6,7-dihydroxy-1,2,3,4tetrahydronaphthalene; 5-HT, S-hydroxytryptamine.

407

preferentially the second type of receptor. This possibility has been investigated. EXPERIMENTAL PROCEDURES Purified striatal synaptic membranes were prepared from 200-300 g Wistar rats by density gradient centrifugation of the crude synaptosomal pellet as described by DAVE, WOODRUFF, POAT & FREEDMAN (1980). The synaptosome layer was collected, washed and lysed by homogenisation in 5 mM tris-HCl buffer, pH 8.0. The pellet was resuspended in 50mM tris-Krebs’, pH 7.4 containing 0.1% ascorbic acid and 10 PM nialamide. The membranes were incubated for 10 min at 37°C to inactivate monoamine oxidase and stored at -20°C for a period not exceeding one month. Membranes (K&200 pg protein) were preincubated for 2 min at 37°C in the presence and absence of 1 PM s-( - )sulpiride, which was used to define specific binding. Drugs or vehicle were added at this stage. The binding reaction was initiated by the addition of C3H]sulpiride (specific activity 26.2 Ci/mmol), at a concentration of 1520 nM, and terminated, after a 10 min incubation period by placing the tubes on ice. Free and bound ligand were separated by millipore filtration using HAWP 02400 filters presoaked in 50m~ trisKrebs’ buffer, pH 7.4. The filters were washed with 10 ml ice-cold buffer and dissolved in l-2 ml methyl cellosolve. Bound radioactivity was determined by liquid scintillation spectrometry, all counts being corrected for background, quenching and machine efficiency, and the results expressed as fmol [sH]sulpiride bound per mg protein. Under these assay conditions, specific binding rep resented approximately 55% total binding. Preliminary ex-

408

G. N. WOODRUFFand S. Et. FREEDMAN

perimcnts showed that specific binding to Nil&ore filters occurred. The blank rate, in the absence of tissue, was 1.4 f 0.3 fmoi/fiiter compared with a typical value of 12.9 f 1.4 (16)fmol/filter in the presence of tissue. Binding to the filter was independent of ligand concentration and unafTected by the drugs used at their pharmacologically active concentrations. In view of this observation, blanks were routinely incorporated into each experiment. Protein was estimated according to LOWRY,ROSEROUGH, FARR &

TABLE 1. THE BINDING OF [‘H]SULPIJUDE

RANDALL (1951).

Medulla Hip~ampus

Materi&

REGIONS

TO DIFFERENT

OF THE RAT BRAIN ____

---_

Region

Specific binding fmol bound/mg protein

Striatum Nucleus accumbens

154 f 7 103 f 5

Occipital cortex Cerebellum

8?5 17 f 12 19 + 4 11 &2

Cis-flupenthixol, trans-flupcnthixol, cis-clopenthixol and transclopenthixol were gifts from Lundbeck, Copenhagen; s-(-)-sulpiride and R-( +)-sulpiridc from Ravixxa SpA.; tiapride, ( f )-sultopride and [3H~sulpi~de from Chemitechna and fluphenazine from E. R. Squibb & Co.

Binding was determined in synaptic membranes prepared from the above regions using 20 no [3H]sulpiride in the presence and absenoe of 1 I(M (f~sulpiride to de& specihc binding. Results are expressed as mean f S.E.M. of 6 observations.

RESULTS

of the brain gave the results shown in Table 1. Binding was highest in the striatal and nucleus accumbens fractions, and very low in other regions examined.

of binding

Characteristics

[3H]sulpiride bound specifically to rat striatal synaptic membranes. This specific binding was saturable in the &and concentration range 1-4On~. At these concentrations non-specific binding was linear (Fig la). Scatchard analysis of the data revealed a single binding component with an afhnity constant 7.40~ and a m~imum binding capacity of 24ofmol/mg protein (Fig. lb). The specific binding increased linearly with protein concentration (W-300 ~cgprotein) in contrast to non-specific binding which was protein-independent. The specific binding of the ligand was rapid, occurring with a tj of approx 4min at 0°C whilst binding of the non-specific component was virtually instantaneous. Re~jo~~ dis~~ut~~ of [~iijsdpiride

binds

An examination of the specific binding of [3HJ~ulpiride to membrane preparations from different areas

1X0-

E

TI

The rank order of potency of dopaminergic agonists and antagonists in displacing [3H$ulpiride binding in striatal synaptic membranes is shown in Table 2, expressed as K&, values. The most potent displacers of binding are the neuroleptic drugs. The phenothiazine, !Iuphenazine, the thioxanthine cis-cfopenthixol and ( + kbutaclamol were the most active of the drugs tested. Slightly weaker, but still fairly potent inhibitors of binding were haloperidoi and the atypicaf neuroleptics of the benxam3e group, ( f )_SttitOpride and s-(-)-sulpiride. The table shows also that C3H]sulpiride binding shows a marked stereospecificity towards neurokptics. Of the two enantiomers of sulpiride s-( -)-sulpiride is 20 times more active in displacing the binding than a-(+)-sulpiride and ( + )-butaclamol is more than lO,Wl times more active

200

fo)

i

0,

.+ J/

Eflect of drugs on [“H)sulpiride binding

woo-

*

.

I’

0

0

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.

-1 l

OC

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BcunQ / 20

30

40

nM sulpwde

FtO. la. Non spa&e (0) and spec& (e) binding of [“H’jsulpii O&xam: [*Hfautpkide m (d.p.m.). Abscissa: [sH-Jaulpiri& amcaoaatiOn (aw).

tree

FIG. lb. Scatchard analysis of speci6c binding Of [‘H~~uIpiride. Ordinate: Bound (fmol . mg- ‘) Abscissa: Bound,Free(

fmolz-

y)

Binding of [3Hjsulpiri& to rat strirtal synaptic membranes as a function of concentration of &and. Specific binding was defined using 1 FM s-( - )-sulpiride.

[3H]sulpiride binding to striatal membranes

409

The distribution of binding sites is consistent with the distribution of dopamine in the brain as binding DRUGS was greatest in the striatum and nucleus accumbens. Binding to regions low in dopaminergic activity was Drug Go Oh4 low being barely above blank levels. Furthermore, the Fluphenazine 0.5 ability of dopamine and its agonists ADTN and Cis-clopenthixol apomorphine to displace binding, in contrast to the :: ( + )-Butaclamol inactivity of noradrenaline, 5-HT ~-~nobuty~~ and 1:s Cis-flu~thixol gfutamic acids in the system, suggests that the binding 4.5 Haloperidoi ( + )-Suhopride 7.0 component may be connected with central dopaminChlorpromazine 9.0 erg& sites. It is interesting to note, however, that the s-(- )-sulpiride 11.0 S-HT antagonist cyproheptadine, was a weak disTram-clopenthixof 40.0 placer of binding. Cyproheptadine 100.0 The rank order of potency of the neuroleptics in 110.0 Tiapride 120.0 ADTN displacing [3H]sulpiride binding is of particular interTram-flupenthixol 140.0 est. Classical neuroleptics such as fluphenazine, Aponorphine 220.0 (+)-butaclamol and cis-flupenthixol are extremely R-(+ )-sulpiride 240.0 potent in this respect. Cis-flupenthixol was only very 3ooo.o Dopamine weakly active in the assay of THEODoROU et af. (1979), ( - tButa&mol, 5-HT, ( - )-Noradrenawho interpreted this observation as su~~tive of a line, (~~~opr~oIo1, glutantic acid, non-cyclase linked component of E3H]sulpiride bindatropine and GABA were relatively inacing. The reasons for this major discrepancy are tive (ICso > 10,000nhf). unclear and we cannot offer any reasonable explanaAssays were performed using 1.5nht C3H]sulpiride and 1 pm s-f- )-sulpiride to tion, since in our system the related thioxanthine clodefine maximum specific binding. All penthixol was also a potent inhibitor of binding. The drugs were tested at least at 4 different use of crude homogenates, as employed by THEOconcentrations in triplicate in 24 experiDOROU et al. (1979), can lead to false binding informents and IClo values determined mation but we are not sure that this could totally graphically. account for this important discrepancy. The benzamides are active in displacing C3H]sulpiride binding although not as active as the classical neuroleptics than the pharmacologically inactive ( - )-enantiomer. and furthermore they do not appear to fall into a Cis-flupenthixol and cis-olopentbixol are approxiwell-define group, such as would be expected if they mately 70 times more active than their clinically inactive transisomers. The dop~inergic agonists tested in were specificity labelling a non-cyclase linked dopamine receptor. this system appeared to be generally weaker in inhiThe results demonstrate that the inhibition of biting C3HJsulpiride binding than the antagonists. The most potent of these was ADTN, which holds the [3H]sulpiride by neuroleptics of the various classes is stereospecific. Thus, S( - )-sulpiride, for example is dopamine molecule fixed in its fl-rotameric form and which is a potent dopamine receptor agonist (WOOD- more potent than R-(+)-sulpiride, a finding which is consistent with S-(- )-sulpiride being more active in RUFF,1978). ADTN is as potent in displacing binding as the weak benzamide tiapride and is about 30 times inhibiting locomotor activity (ANDREWS& Wormmore active than dopamine. The table shows that the RUFF, 1978).This difference in activity of the sulpiride binding of the ligand appears to be displaced by stereoisomers has been utilised in an attempt to increase the sensitivity of the binding assay by using dopaminergic agents specifically, since noradrenaline, s-(-)-sulpiride as the displacing ligand in the stanpropr~olol, S-HT, glutamic and ~-aminobutyric dard assay procedure. acids were inactive. The dopamine antagonists are generally more potent in displacing [3H]sulpiride binding than DISCUSSION agonists, the rigid dopamine analogue ADTN being the most potent of the agonists tested. This is consistThe results presented show that [3H]sulpiride binds to purified striatal membranes in a specific ent with its activity in many dopaminergic systems saturable manner. There appears to be a single com- (WOODRUFF,1978). This observation is in contrast to the situation found in studying C3H]ADTN binding ponent with an affinity constant of 7 nM and a BmaX of 24Ofmol/mg protein. These values are similar to in a similar preparation, here dopamine antagonists those described by THEODOROU, C~ocm, JAR & and agonists had similar potency as displacing agents (DAVIS,POAT & WOODRUFF, 1980). MARSDEN(1979) in their recent study of f3H]sulpiride binding using crude membranes and different assay CONCLUSIONS conditions. The higher umax reported in the present The results presented show that there is a specific study is probably a reflection of the differences in sulpiride binding site in the central nervous system purity of the preparations. TABLET 2. INHIBITION OF [3H]~~~~~~~ BINDINGTURATSTRIATALMEMBRANESBY

410

G. N. WOODRUFFand S. B. FREEDMAN

which is probably closely linked with dopamine binding sites. There does not appear to he any difference in the activity of classical neuroleptics and benzamides upon this site and thus the study has not produced supportive evidence for the two types of

preparation

dopaminereceptortheory.It slm.N however,be

Acknowledgement-S.B.F. is in receipt of an s.R.C. (CASE) studentship award in conjunction with Chemitechna.

noted

that

even in a purified

synaptic

membrane

it is possible

that

binding

could

presynaptic and ~stsynaptic sites, at least possibility is at present under investigation.

be to This

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