Functional dopamine receptors in the brain

.~~urophornrrrcol~~~, Vol. 23. No. 28, pp. 243-246. I984 PrInted in Great Britain. All rights reserved

FUNCTIONAL

DOPAMINE

Copyright c

RECEPTORS

0028.3908:‘84 S3.00 + 0.00 1984 Pergamon Press Ltd

IN THE BRAIN

G. N. WOODRUFF*, L. HOLDEN-DYE, K. SENIOR and J. A. POAT Department of Physiology and Pharmacology, University of Southampton, England

The neurotransmitter dopamine is implicated in numerous neurological and psychiatric disorders. Over the last decade there has been an increasing flurry of research directed towards the identification, localisation, characterisation and isolation of brain dopamine receptors. By analogy with other neurotransmitter systems, it is likely that multiple dopamine receptors will prove to have functional and pharmacological significance. However, a newcomer to the field of dopamine receptor research would probably conclude that the literature is in a confused state. Thus, there have been several schemes which propose the existence of multiple receptors for dopamine (Costall and Naylor, 1981) and it has been variously suggested that there are D,, D,, D,, D,, dopamine receptors (Seeman, 1980; Seeman and List, 1982) and that there is only one (Laduron, 1982). The most widely quoted classification of dopamine receptors is the D,. and Dz, scheme. Kebabian, Petzold and Greengard (1972) showed that dopamine-rich areas of the brain contain a dopamine-sensitive adenylate cyclase that is stimulated by micromolar concentrations of dopamine. The structure-activity requirements for dopamine-like activity on the dopamine-stimulated adenylate cyclase were similar to those which had been previously reported using peripheral and invertebrate models (Woodruff, 1978): this added weight to the suggestion that this adenylate cyclase was an important link between receptor activation and physiological response to dopamine. It soon became apparent however, that there were numerous inconsistencies with the theory. For example, the substituted benzamide. sulpiride, was found to be inactive as an antagonist of the dopamine-sensitive adenylate cyclase (Trabucchi, Longoni, Fresia and Spano, 1975). This led to the suggestion that there might be multiple receptors for dopamine, a D, receptor linked to the adenylate cyclase and a D? receptor, non-cyclase linked (Kebabian and Calne, 1979). To date however, there is still no evidence for a physiological role for the so-called D, receptor and it may be that the I& receptor is linked to adenylate cyclase, but linked to inhibition of the cyclase rather than activation (Woodruff. 1982). On the other hand the dopamine *Present address: Neuroscience Research Centre, Merck, Sharp & Dohme. Hertford Road. Hoddesdon, Hertfordshire,

England.

receptor identified by sulpiride is strongly implicated in mediating the electrophysiological, neurochemical and biochemical actions of dopamine. In the present paper the use of sulpiride and the dopamine receptor agonist ADTN in the study of brain dopamine receptors will be discussed. ADTN

ADTN (2 amino-6, 7-dihydroxy-1,2,3,4-tetrahydronaphthalene) is one of the most potent dopamine receptor agonists in a variety of test systems (Woodruff, 1982). This molecule is a rigid analogue of dopamine in its extended form, corresponding to the b-rotamer. ADTN has been used extensively in receptor binding studies, not only as the radioactive ligand but also as the displacing agent for other ligands (Seeman, 1980). ADTN

binding

[)H]-ADTN binds to striatal synaptic membranes of the rat in a saturable fashion over the concentration range I-20 nM (Kd l6nM from Scatchard analysis and B,,, 130 fmol/mg protein). The ability of some dopamine agonists and antagonists to displace binding is shown in Table 1. It can be seen that the Table I. InhibItion of binding of [‘HI-ADTN to striatal membranes from rat brain Drug N.N dl-n-propyl iso-ADTN ( + )-Butaclamol ( + )-ADTN Domperidone Sulpiride ( + )-ADTN Apomorphme Dopamine Spiroperidol ( - )-ADTN Cb-flupenthixol Iso-ADTN Ergometrine Bromocrlptine SKF 38393 Haloperidol Pergolide Truns-tlupenthlxol ( - )-Butaclamol

IC,,, (aM) 0.4 1.3 1.7 2.1 2.2 3.6 7.1 12.6 18.2 23.0 23.0 36.0 50.0 56.0 149.0 468.0 1645.0 2143.0 I1800.0

Inactive (IC,, > 10,000 nM) were ( - )-noradrenalme, ( - )-adrenaline and 5-hydroxytryptamine. The IC,, is the concentration of drug required to displace 50% of [‘HI-ADTN (5 nM) specific binding. The specific binding was defined by I p M ( i )-ADTN. Each value represents results from at least 3 experiments, using at least 4 concentrations with each estimation in triplicate. Taken from Templeton and Woodruff (1982).

243

binding of ADTN is readily and specifically displaced by dopamine receptor agonists and antagonists, including sulpiride.

Table 3. Displacement Drug

Sulpiride

1,) rat .,,I.LLI~

/I 1 II s 0 ‘1 Ih 1i ,x ,I II 0 I(1 0 ‘5 0 -. 2x 0 12 0 10 0 50 0 750 0 720 II ix0 0 > lo.t1l~(l 0

jAssays were performed using IS nM [‘H]aulpir~dc and I I’M S-I sulpiride to define specific bmding Drug\ wre tertcd ~1 ,i minimum of 4 concentrations m rrlphcate 1112 4 c*perlmct,tx For a further list of compounds tested on [‘Hjrulplrldc hlndlnF see Woodrut? and Freedman (IW3).

hiding

[3H]Sulpiride is a useful ligand for labelling dopamine receptors. It binds to striatal membranes of the rat in a saturable reversible stereospecific manner. [3H]Sulpiride also binds to striatal membranes prepared from other species with similar binding characteristics. Table 2 shows the kinetic properties of [3H]sulpiride binding sites from pig and rat striatal tissue. The value for the equilibrium dissociation constants (Kc,) are very similar in both species although the pig tissue appears to have a lower density of binding sites suggested from the significantly reduced B,,_. The distribution of [3H]sulpiride binding sites is similar to that of dopamine in the CNS and the effect of drugs in displacing the ligand from its binding site (Table 3) suggests that [‘Hlsulpiride is labelling striatal dopamine receptors. One interesting and unusual characteristic of specific [‘Hlsulpiride binding is that it is completely dependent upon the presence of Na’ in the incubating buffers (Theodorou, Hall, Jenner and Marsden, 1980: Freedman and Woodruff, 1982). In the absence of Na ’ . specific binding is reduced to almost zero. Although other neuroleptic binding sites, for example those identified by [‘Hlspiroperidol are Na’-dependent, omission of Nat reduces binding rather than abolishes it (Usdin, Creese and Snyder, 1980). It has previously been shown that binding of [‘Hlsulpiride to striatal membranes was affected by guanine nucleotides (Freedman, Poat and Woodruff, I98 I). The ability of agonists to displace [‘Hlsulpiride

Table 2. Kinetic orouertles

K, (nM) B rn‘ll

hding I(‘,,, tnhl,

Spwoperidol Domperidone Fluphenarmc CkHupenth~xol Bromocriptinc Zetidolinc I ~ )-Sulpirlde R-C + J-ADTN l’ergolidt: ( i )mADTN Apomorphme Trunvflupenthixol Iso-ADTN ( ~ )-ADTN Ergomotrmc D0paIlI1ne SKF 3839

SL’LPIRIDE

Sulpiride is a clinically effective atypical neuroleptic drug (Edwards. Alexander. Alexander, Gordon and Zutchi. 1980). which has been used as a dopamine receptor antagonist routinely in these laboratories. It offers distinct advantages over most classical neuroleptic hgands in that it is relatively specific for dopamine receptors and in binding studies its low lipid solubility ensures that the non-specific binding component is relatively small. Although sulpiride penetrates the blood-brain barrier weakly, it is a potent dopamine antagonist either in behavioural or electrophysiological tests, when it is applied directly into the brain (Woodruff and Freedman, 1983).

of \peutic [‘Hj\ulpmdc membrancr by drug\

of I’Hlsultxr~de

bmdme

utes

Pig

Rat

6.8 t 0.8 231 & 3s

x.1 rt I 433 f 27

[‘H]Sulpiride specific bindmg as defined by the additmn of I HIM S-( - )-sulpiride wa, saturable over the concentratmn range LdOnM. Values are the mean of at least 6 detsrmmations

from striatal membranes is signiticantly reduced in the presence of GTP, GDP and the non-hydrolysable GTP analogue Gpp(NH)p. In contrast. the ability of antagonists to displace the binding is unaffected by the addition of guanine nucleotides. Protein

tndfj~ing

ugents

These studies have concentrated on the uxc 01. dithiothreitol (DTT), a compound which reduces disulphide bonds. and N-ethylmaleamide (NEM ) ;I compound which forms covalent bonds with SH groups, to study the active site of the dopamine receptor in the brain. Although NEM is used as a thiol group reagent, the possibility cannot he ruled out that it reacts with alternative groups. DITHlOTHRElTOl.

(DTT)

Dithiothreitol can distinguish between the btnding of [‘HI-ADTN and the binding of [‘Hlsulpiride. Thus. it was previously shown that binding of [‘HI-ADTN is inhibited by small concentrations of DTT. concentrations which have no elfect on binding 01 [iH]sulpiride (Freedman. Templeton and Woodrulf. 1982). The tact that the pattern of displacement 01 [‘HI-ADTN binding by drugs shows similarities to the displacement of [‘Hlsulpiride binding by the jame drugs and the fact that various hrain lesions have similar effects on both [‘HI-ADTN and [‘H]xulpiridc binding suggest that ADTN and sulpiride are labelling the same receptor. Thus. the results with DTT are consistent with the proposal that ADTN and sulpiride attach to slightly different sites on the receptor. This is consistent with previou\ work. V-ETHYLMALEIMIDE

(Nbbl)

It seems likely that there is also an essential sulphydryl (SH) group on or near. the hinding zite

Functional dopamine receptors

245

!,. Zetidoltne labelled by dopamine antagonist ligands. Thus, al5 100 z kylating agents such as N-ethylmaleamide (NEM) f IL completely inhibit the binding of [“Hlsulpiride. Preincubation with various concentrations of the sulphydryl reagent inhibits specific [3H]sulpiride binding to rat striatal membranes in a dose-dependent manSKF 38393 ner (IC,,, 0.84 mM). Similarly, NEM pretreatment inhibits specific [3H]sulpiride binding to pig striatal membranes (IC, 0.4mM). These results are in contrast to those obtained with agonist binding where ligands such as [‘HI-ADTN are much more susLog cone (M) Fig. 1. The effect of zetidoline and SKF 38393 in protecting ceptible to NEM (ICYi I PM). Thus, there might be pig striatal membranes from alkylation by NEM. Dill’crent two essential SH groups. A “high affinity SH group”, concentrations of drugs were added to striatal metnbranc< susceptible to DTT and to small concentrations of and incubated for 15 min at 37 C. They were then subNEM and involved in agonist binding, and a second sequently incubated for 30 min with 3 mM NEM: DTT was “low affinity group”, involved in sulpiride binding. then added and the membranes washed 3 times. Binding of [‘Hlsulpiride was assessed with 15 nM [‘Hlsulpiride with the If the sulphydryl group alkylated by NEM is on, addition of 1 PM S-( - )-sulpiride to half of the tubes to or near to, the sulpiride binding site then addition of define specific binding. The results are expressed as perunlabelled sulpiride during the preincubation period centage control binding being an average of 3-6 obserwould be expected to protect against NEM alvations. kylation. This is indeed the case. Sulpiride, added during the preincubation period, prior to NEM, site which is in agreement with the lack of protects the binding site from NEM alkylation (EC,, effectiveness of this compound in displacing 26.3 nM) as was previously demonstrated (Freed[3H]sulpiride binding (Table 3). man, Poat and Woodruff, 1982). The results, shown In preliminary experiments this ability of sulpiride in Table 4 show that a variety of compounds which and zetidoline to protect the essential SH group on or are active in displacing [‘Hlsulpiride binding will also near the [3H]sulpiride binding site has been used in an protect from NEM (3 mM) alkylation, if added durattempt to attach a labelled group irreversibly to this ing the preincubation period. Thus, the agonist site. Thus, the presence of sulpiride or zetidoline prior ADTN, the ergot pergolide, and zetidoline all protect to the addition of NEM should protect the essential the SH group associated with [‘Hlsulpiride binding SH group leaving other SH groups on the membranes sites. Zetidoline has recently been shown to antagoto be alkylated. Removal of the protecting agent nise the effect of iontophoretically applied dopamine should theoretically free the essential SH group for on neurones in the substantia nigra. zona compacta subsequent alkylation by [3H]-NEM, effectively lahel(Harris and Woodruff, 1983). Zetidoline is also ling the binding site in an irreversible fashion. The effective in displacing specific [‘Hlsulpiride from its experimental method adopted is demonstrated in binding sites in rat and pig striatal membranes Fig. 2. In preliminary experiments the level of [‘HI(Holden-Dye, Poat, Senior and Woodruff, 1983). It has a similar potency to sulpiride in protection studies. The dose-related protection is shown in Fig. 1 (EC,, 22.8 nM). The Figure also shows the inability of SKF 38393 (2,3,4.5-tetrahydro-7,8-dihydroxy-l+ NEM phenyl- 1H-3-benzazepine) to afford protection to the

I

1

Table 4 Protection

agamst

NEM (3 mM) alkylation [‘H]Sulpiride binding fmol per mg protein

Control NEM NEM + NEM + NEM + NEM +

sulpwide retidolinc pergolidr ADTN

170* IO 42 * I 1x4* 15 18Ok9 168 * I4 I45 f I?

Membranes were incubated for 15 min at 37 C m the presence of I PM drugs and were subsequently incubated for 30 mln with 3 mM NEM DTT (3 mM) was added at the end of the second preincuhation periods and membranes were washed three times (washing was by dilution with 301111 Tris-Krebs’ buffer) and membranes collected by centrlfugation at 50,OOOg for I5 min. Bindmg was then assessed with I5 nM [‘Hlsulpiride with the addition of I I’M S-( -)-sulpride to half of the tubes to define specific bmding The results *SEM are from at least 6 observatlons from Z to 3 experiments

C3HI-NEM NEM S’

NEM

Fig. 2. Diagramatic representation of the method of [‘HINEM binding to striatal membranes. Sulpiride (1 1~M) was added for 15 min at 37’ C followed by NEM (2 mM) for a further 30 min. DTT (2 mM) was added if required at the end of the incubation. The protecting agent was then removed by dilution (30 ml Tris-Krebs’ buffer pH 7.4) and the membranes collected by centrifugation. [‘HI-NEM binding was assessed with 2mM [‘HI-NEM.

c;. N.

246 Table 5. [‘HI-NEM

bmdmg

;.

Control NEM Zetldoline

to pig btriatal membranes [‘HI-NEM bindmg (pmol, mg prowl”,

GRXlp I. ,

WOODRUFF

+ NEM

x2.9 I5 Y 31.Y + 3.6* 42.2 15.0

Group 3: membranes were mcubated with retidolme (I ,uM) for 15 m!n at 37 C, then wth I.5 mM NEM for a further 30 m,n At the end of this time DTT (2 mM) ~+as added and the membrane\ washed 3 mnes Ahquotb w’rre tested for [‘HI-NEM binding. The results are the mean of 5 observatmn~ from 3 experxnents. Group 1: membranes were incubated wth 2 mM NEM for 30 n11”. then washed and aliquots taken for [‘HI-NEM (?mM) bindmg. Group I. control membrane\ were used for [‘HI-NEM bindmg without pretreatment. *Slgnificantlp dXerent from group 3 using a paired r-test

NEM binding to striatal membranes was high and erratic. In order to overcome this problem DTT (2mM) was added at the end of the incubation to remove excess [‘HI-NEM. The addition of DTT was effective in reducing the blank rate (binding of [‘HINEM to membranes which had not been treated with sulpiride or zetidoline in the preincubation period). Table 5 demonstrates that zetidoline. added during the preincubation period, protected SH groups, which. upon removal of the protection agent. were available for alkylation by [‘HI-NEM. Similar results were obtained using sulpiride as the protecting agent. Although these results are of a preliminary nature they do suggest that this approach might be useful for irreversibly labelling the [‘Hlsulpiride site in striatal membranes. Such an approach might prove valuable in the isolation and characterisation of dopamine receptors.

REFERENCES Costall 9. and Naylor R. J. (1981) The hypotheses of different dopamine receptor mechanisms. Lifi Sci. 28: 215-229. Edwards J. G.. Alexander J. R.. Alexander M. S.. Gordon A. and Zutcix T. (1980) Controlled trial of sulpiridc in chronic schizophrenia patients. J. Psyhicrf. 137: 522-529. Freedman S. 9. and WoodruB G. N. (1982) Effects of cations on [‘Hlsulpiride binding. Br. J. Pharmuc. 74: 228P. Freedman S. 9.. Poat J. A. and Woodruff G. 1\1. ( 1981) Effect of guanosine nucleotides on dopaminergic agomsts and antagomsts affinity of [‘Hlsulpiride binding sites in rat striatal mcmbranc preparations. d. h’eurochwn 3: 608-612. Freedman S. 9.. Poat J. A. and Woodruff G. N. (1982) Influence of sodium and sulphydryl groups on

et

(I/

[‘Hlsulpiride bindmg sites in rat stnatal membrane\. J Neurochem. 38: 1459-1464. Freedman S. 9.. Templeton W. W. and Woodrull’ G. N. (1982) Importance of wlphydryl groups In the bIndIng 01 dopaminergic agonists and antagomhts. Bv. .I. Plwmtrcc 75: 39P. Harris N. C. and Woodrult‘
Rcwpror

Agonist.\.

AC/U Plftrn,icrt,c,lcr/~,~~ Swc/c i/ .Yupp/

(Carlsson A. and Lars J.. Eds). pp. I IX I?‘). G. Nils\on. Swedish Pharmaceutical Prc\\. Stockholm.

I