The cloned dopamine D2 receptor reveals different densities for dopamine receptor antagonist ligands. Implications for human brain positron emission tomography

Eutz~pean Journal of Pharmacology - Moh'cuht r Pharmacology Section. 227 (1992) 139-146 /~ 1992 Elsevier Science Publishers B.V. All righls reserved t/922-4106/92/$05,00

139

E J P M O L 90346

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for dopamine receptor antagonist ligands. Implications for human brain positron emission tomography Philip S e e m a n

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H o n g - c h a n g G u a n ", Olivier Civelli ~, H u b e r t H,M. V a n T o l ,,.h,d R o g e r K. S u n a h a r a ~' a n d H y m a n B. Niznik b,d

Departments r~l" " Pharmacoh~gy and I, Psychiuto', Faculty ~[" Medicine. Medical Science Buihling. U~;irersity ¢~1"Toronto, Torot~fo MIlS/,4& C~1~1~t¢h1, ' Vollum Institute .¢or Adtanced Biomedical Research, DtTmrtment ~['(_'ell Biology and .4natomy. Oregon tb'atth Scie;ues Unilerwity, Portland. OR 97201, USA and ,t Laboratot3' of Molecuiar Neurol)ioh)g,v, Ch~rke htstitute ¢~ft{,;ychiato', Toronto MS T 1R& ('anoda Received I 1 March 1992, revised MS received 19 May 1992, accepted 26 May 1992

Since [3H]cmonapridc ([3H]yMo09151-2), a benzamidc ncuroleptic, consistently detects more dopaminc 11, receptors than [3H]spiperonc in the same tissue, wc tested whether this property was inherent in the cloned dopaminc D e receptor. Wc found that thc density of dopamine D e rcccptors labellcd by [SH]cmonapridc was 1.5-fold to 2-lklld (mean of 1.S-fold) highcr than the density of dc,paminc D, receptors labelled by [~H]spipcrone in cells expressing cloned dopaminc D z rcccplors (either the short form (from rat} or tile Ring form (from human)), matching similar findings in anterior pituitaly tissue (rat or pig) or in post-mortcm human caudatc nucleus tissuc. The situation was similar for another bcnzamide, [3H]raclopridc, which revealed 1.3-fold to 1.8-fold (mean of 1.5-fold) more binding sites than that for [3H]spipcronc in cell membranes containing cloned dopamine D: receptors. The apparently different dopaminc D: receptor densities revealed by these two typcs of 31ot-ligands(i.e. [3H]spipcronc and the [3H]bcnzamides), thereh)rc, arise from an inherent property of tile dopaminc D, receptor protein. These findings for the cloned dopaminc D~ receptor, therefore, partly explain the higher dopaminc D, receptor density measured in human brain (by positron emission tomography) when using radioactive raclopridc compared to rcsu!!s using radioactive mcthylspiperone. Hence, the apparent density of dopamine D e receptors measured by radioactive raclopridc nil) '.:c lower or higher than thai revealed by radioactive spipcrone or methylspipcrone, depending on the magnitude of cndogcn(ms dopamine (which inhibits tile binding of radioactive raclopridc) and the inherent properly of dopaminc 11, receptor protcir~s to exhibit more binding sites (D 2 monomers, possibly) for radioactive benzamidcs than for radioactive spipcronc or nacthylspipcrone (D 2 directs, possibly). Dopaminc D: receptors; Dopamine receptor antagonist ligands; Po:dlron emission tomography (Imman brain)

i. Introduction

Emonapride * (YM-09151-2; lwanami et al., 19811 is a benzamide ncuroteptic with a typical antipsychoticlike action in schiz,,,,phrenic patients (Kudo, 1990) and in animals (e.g. catalepsy, and related actions; Usuda et al., 19~';1).Of the many receptors tested (Terai et al., 1983; TerM, 1989), emonapride has its highest affinity

Correspondcrme to: Philip Seeman, Department of Pharmacology, Medical Sciences Building, R o o m 4344.8 Taddlecreek Road, Toronto M53 lAX, Canada. * E m o n a p r i d e (or YM-ilgl 5 I-2; eis-N-[(2RS,3RS)- I -benzyl-2-melhy!pyrrolidin-3-y!]-5-chhm)-2-metht~xy-4-[(iraethyhmfino]-be nzamidc) ccmtains two asymnlelrie carbon atom~ alld is, |]lcre['tlre, a lilct.-mate (lwanan{i el al.. 1981; H a t a n o cl el., 1990) of tkmr cnantiomers, two of which, (R,R)YM-09151-2 and (S,S)YM-09151.2, are aclive with identical potency (Terai el el., 1989).

fur the dopamine D 2 receptor, its dissociation constant for inhibiting tile binding of ligands to dopamine D, receptors has been reported as 21 pM (Feral, 1989; Terai el al., 19891, 57 pM (Niznik ct al., !985), 82 pM (tissue slices; Cox and Waszczak, 1991). 100 pM (Terai et el., 1983) and 626 pM (),issue sliccs; Unis et al., 199(I). Emonapridc had a iaigher dissociation constant of 10.5 nM to inhibit dopamine-inhibited adenylate cyclase (Grewe et al., 19821. The high affinity of emonapride for tile dopamine D.? receptor has led io its use in positron emission tomography for in viw) studies (Hatano et al,, 1989, 19911; l-latazawa et al., 1991). Niznik et al. (1985) noliced that [qq]emorm0ride consistently labelled 4(1%, more dopamine D 2 receptor sitcs than [3H]spiperone in all tissucs tested. This puzzling anomaly was confirmed by Terai ctal, (1989). These "additional' receptor sites wcrc dopaminergic in nature and nol serotonc~'gic (Niznik ct el,, 1985). No

140

such discrepancy in dopaminc D, receptor density occurred when comparing [~l]]spipcrone and [-~H]sulpiridc, both ligands apparently yielding the same density (Zahniser and Dubocovich, 1983: Urwylcr and Coward, 19871. Although metabotitcs of emonapride exist (Higuchi et al., [986), they have lower potencies "~t dopamine D: rocep!or,: tTerai oI al._ 1989) and would not be expected to interfere with ligand binding at these receptors (Hatano ctal.. 19891. particularly in vitro. In measuring the density of dopaminc D, receptors in the living human brain by means of positron emission tomography, a similar discrepancy exists belwcen [liC]methylspiperone ° which reveals a receptor density in the caudate nucleus of 16.6_+2.5 pmol/ml ** (Wong el al., 1986), and [tlC]raclopride. which reveals a dopaminc l), receptor density of 22.3 ± 6 pmoi/nll 134c;- higher than that measured by [llC]mcthylspiperone) (Fardc et al., 19901. The two bcnzamides ([ 3H]emonapride and [ t tC]racIopride), therefore, cons|sternly reveal more dopaminc I), receptor binding sites than the two butyrophenones ([~'H]spipcronc and [J'(']N-methy!spipcrone). Thc different dopamine D~ receptor dens|tics detected by the two pairs of ligant]s, therefore, raised the possibility that there may be a "bcnzamidc neuroleptic subtype" of dopamine D, receptor, a concept often mentioned (Ogrc|l and l-i~igbcrg, t9881. Hence, in order to test for tile possible existence of a benzamide-type dopamine I), receptor, or to determine whether thc property of two apparently different dens|tics was inherent to the pure dopaminc 1)2 receptor', we recast.red tile density of the cloncd dopanline D~ receptor (Bunzow ct at.. tt,~8), using three 31.tligands: [ ~H]spipcronc, [~1 t]emonapridc, and [3 H]racIopridc. The findings show thal the chined dopaminc D~ receptor consistently revealed higher apparent dopaminc D., receptor densities for the two [~HJbenzamides, compared to [ ~H]spiperone,

2. Materials and methods 2.1. Material.~ 2. I. 1. ~ll-l.igand.s [N-methyl-~H]lLnlon:,tpridc (71-75 Ci/mnu~l; 2t~27-2775 GBq/mmol) and [~t-t]raclol)ride (fill--70 (:'i/retool)

* * T h e ut~il tff r ¢ c c p l o r dcn:,ity m D~sl-morlcm tissue n l e a s u r c m ¢ l l l is Drool p e r g of oril~illal lissuc, or n m o l / k g . T h e latter tlnit is virtually identica! to nM. smcc lhc Sl~ccific gravity ~f the |lttlll;In *,lrialunl is 1.(}3 g / m l (Scemlm c t a l , . 1987J. "l'h¢ unit ~)f i ¢ c e p t o r densily hi positron emission l o m o g r a p h y is pmol p e r illl [~1'living brain region; this is identical to nM.

were purchased from New England Nuclear, E.I. du Pont de Nemours & Co., Boston. MA. USA. Two types of radioactive spiperone were tested (with identical results): [bcnzene ring-3H]spiperone 121t-411 Ci/mmol; 740 G B q / m m o l - 1.48 TBq/mmol) from New England Nuclear, and [ 3H]spiperone (60-100 Ci/mmol: .~._-3.7 TBa/mmol) from Amersham Life Scienccs° Oakville, Ontario, Canada. 2. L Z Tissues Human post-mortcm brain tissue (at -70°C) was obtained from the Canadian Brain Tissue Bank (Basting lnstitu~c, University of Toronto, Toronto, Canada: Dr. T.P. Morley, Medical Director: M. Pataki, Coordinator). Pig anterior pituitary ~issucs were from Bocknek Organi: Materials, Rexdale, Ontario, Canada). Rat anterior pituitary tissues were from Sprague-Daw° Icy rats (200 g). Both GH4 (first generation, CI, rat somatomammotrophs) and mouse fibroblast Ltk tissue cultuce cells were used. Both long and short forms of the chined dopamine D~ receptor (Grandy et al,, 1989; O'Dowd ct al., 199()) wcre examined. Thc following combinations wcrc uscd: Des (rat) in GH4 cells (Bunzow ct :.iI., l¢.}g?,): D2,~ (human) in L t k cells {Grandy ct al., 19891; D2t ' (human) in GH4 cells and in Ltk cclls (Grandy et at., 1989). 2.2. Melhods 2.2.1. Mea~'ureme'nt o f the ~hJpamhw D 2 recq)tor densio' The d¢~pamine Dr receptor densities were determined by minor nlodificalM,ls ~ff a centrifugation method, previously described (Seeman et al., lt1871. Human brain frozen tissues, from which major myelin tr~lcts were trimmed off, wcrc lighlly blotted and weighed froze,1. Buffer was added to yield 4 mg tissue per ml suspensi,.m. The buffer contained 5(1 mM TrisI-iCI (pH 7.4 at 20"C), 1 mM EDTA. 5 mM KCI, 1.5 mM 17aCI2, 4 mM MgCI 2 and 12(1 mM NaCI. The suspension was homogenized with an A.H. Thomas glass homogenizer with ]'eflon piston {rotating at 650 rpm: ten up-down strokes). The homogenate was not washed and was not prcincuhated, ~ince such steps consistently result in a 10--3(1q loss of receptors (Seeman el al., 198,1: Secman. 1~)87). The density of dopamine 1), receptors was measured by saturating the tissue witil it:creasing amounts of [:~|ul]emonapride, [~H]raclopridc or [~H]spiperone, as follows. Polypropylone microcentrifuge tubes (1.8 ml with screw cap; Sarstedt Co., Montreal. Canada) received aliquots in tile fi)ilowing order: 0.25 ml buffer, 0.25 ml of 3H-ligand (12 final concentrations, ranging from 10 pM to 4000 pM for [3H]cmonapride and [~H]spiperone, and from 0.2 to 2(1 nM fi~r [3tl]raclopride), and (1.5 ml of tile tissue homogenate. In order to determine nonspecific

141 binding in this dopamine D, receptor assay, the 025 ml buffer aliquot (for half the tubes) contained 40 # M S-(-)-sulpiride (Ravizza, Milan, Italy), such that tile final concentration was 10 y.M, Dcfining nonspedfic binding as that occurring in the presence of 30 p,M dopamine yielded identical results, The tubes were screw-capped, vortexed, and left to incubate at room [ei]lpt2iiliLiiG 1,5i# L J it}i ,.. i l . d t ~a, i q ~ - i i [[~&C . . . . : l : ~ - - : . . _ _ had been achieved. ]'tie tubes were centrifuged in tile horizor, lal position at 11,000 × g for 6 min in a Beckman 12 microfugc at room temperature. (These conditions were sufficicnt to rccover all the doDamine D , rcceptors, since centrifugaiion at 28,0(1(1 × g for 15 rain (Sorvall RC2B) or at 1t,(1(}(1× g for 15 rain did not increase the density of sites in the pcllc:ts of tissues that had becn hom,.igenized by the glass-Teflon homogenizer.) The stlpcl'natanls were ,genii), renloved by aspiration, using a Pasteur pipette and a water-fancet vacuum. The pellets wcrc not rinsed, since this lowered the dopamine D, receptor density by 5c~. Using an electrically, heated wire, the nellet-cowui-lng, ............ ,,,,~ ,,,"r each tube wag cut off and put into a scintillation minivial (Packard Instruments, Chicago, IL, USA). The minMals received 4 ml each of scintiflant (Ready Solve, Beckman ('o,, CA, USA), ant! were Inonitored 6 h later tor tritium in a Packard 4660 scintillation spectrometer at 55% efficiency. Thc values for thc density of ~Hiigand bindh'lg sitcs (B,,,,~) arid the dissociatioll constants, K i), were obtained by Scatchard analysis. Pig pituitaries, freshly removed and individually frozen on dry ice, were obtained from a local abattoir (Bocknck Organic Mater(also Rcxdalc, Ontario, Canada) and slorcd at -711~(7' until used. Mcmbrancs from the anterior pitt, itary gland were prepared as foik)ws. The pituitary was thawed, and the anterior pituitaiy was dissected frcc of the ncumintcrmediate lobe and the altachcd hypophyscal stalk. The anterior pituitary lobes wcle finely minced with scissors, and homogenized with a Polytron (Brh~kmann Instruments, Westbui3,, NY, USA; setting 6 onl of a maximum of 1(I) for 20 s in 20 vols. buffer (as above). Thc homogenate was passed through two layers of cheesecloth and centrifuged tit 480 × g for 5 min lit (I"C. The supcrmitant was collccted, and the pellet was resuspended in 10 wlls. buffer arid centrifuged again at 480 x g for 5 rain. The sccond supernatanl was combined with the first and the pellet discarded. The combined supernatants were ccntrift,ged at 49,5{10 × g for 30 rain al 0°C, and the pellet was rcsuspcndcd ill buffcr at a concentration of 200 mg original wci wcight/ml of final suspension. This suspension was brictly rehofnogcnizcd by thc Polytmn for 10 s, and thcn chilled on ice until used in a binding :
Dopaminc D, receptor densities were also determined on the GH4CI and IAk tissue culture cells containing the cloned forms of D2s or Dzi from thc rat (Bunzow et al., t988) or the human (Grandy et al,, 1089). The cells were scraped from the botlom of the Pclri dishes, poolcd and homogcnized (by hand. using .'l Teflon piston-glass homogenizer) in bnffcr (as above, ~t lrifugcd for 15 rain at ,~),000 × £, and the resulting pellets rcsuspcnded in buffer at a concentration of 150-25(I /~g protein/ml. The D, densities in these Imm~gcn,ate.'; were dctcrmiricd by tl,c ccntrifugation method, given above, or, in some cases by the standard filtration method, using a Skatron cell harvester (see ncxl seclioil ).

2.2. Z (bmpetition hetuven 111-I(qand and drugs Thc competition between a drug and a tH-ligand for binding at the dopaminc D, reccp!or was done as follows. Each incubation tube (12 :< 75 mm glass) received, in the following order, (I.5 mi buffer (with or wilhout S-sulpirid,-, the final concentration of which was It) /xM), 05 ml ~H-ligand (final concentration of about 200 pM), and 0,5 till nlcmbrane suspension, The tubes were incubated for 2 h at room len]perature (20°C). after which the incubates wore filtered, using a 12-well celt harvester (Titertek, Skatron, Lier, Norway) and buffer-presoaked glass fiber filter mats (No, 7034, Skatron, Sterling, VA, USA). After fillcring the incubate, the filter mat was rinsed with buffer for t5 s (7.5 ml bufl'cr). The filters wcrc pushed out and placed in scintillalion rain(vials (Packard) and monitored for lritiunl, as above. Nonspecific bhading was defined as that Ifinding which occurfcd ill ihc presence t)f 10 /xM S-sulpiride (lAst and Sccnlan, I/}~ I ). These eonlf~ciition data were analyzed by the weighted, nonlinear leastsquares culw¢-fitling program LIGAND (Munson mid Rodbard, 1980) on a Digital microPDP-ll computer, The data were filled to one* or two-site models. A two-site model was retained as apprt+l+riato only if a statistically significant (by t: lest) inlprovetllcnl of the fit was obtail~ed over a one-silt model.

3. Resulls

J." 1. [ ]]][:mo, laI #'tde u:id Inl,v,,,,,',<>,,,'

t'ompari,~on

[~H]Spipcrone :aid [~H]enmnapridc revealed two different densities for dopanlinc 1-71, receptors in rat anlerior piltlilary tissue (fig. 1, top) i,s well as in hunian brain tissue (fig. 1. boiionl). The reeeplor densities difl'erod by 1.5-fokt to two-fold (fig. I). An almost identical situation occurred in the cloned fornls of lhc dopamil,c [), ioccpior. "trhe [ll-rl]enlorlapride dcnsily wcls 1.5- to I-fold higher th:m the

142 8 0 ~. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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10 15 20 25 Bound pmot/g l:i~L 1, Th,: den'.it3, of lit,sue dopamine l), receptor r,ile~, detected by [~ltlcim~r~apritte was about 2-fokJ higher lh;in !hal delected hy ['~11jspil'~enmein ral ~*nteriorpiluila~" tissue (top) or human caudalc nucleus (bottom). I ne[ ll]emonapnde (hssoClal~ofleonstanl was 5~ pM (rat anterior piluilao') and 51) pM (hunmn caudate nucleus), v,hite thai for [;ll]spipcrone was 70 pM (rat unlerinr pitnilaq.) and fill pM (human eaud;lle nucleus). The dilti{ o n each tissue were obtadned lhe same day. u;i=lg '~,.', ,,. satn¢ tissue ......... pr. ~.,.,.,';,..,,.

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Fig. 2. The density of cloned dopmnine I), receptor sites measured by [~lt]emonapride wz;s higher lhan thai revealed hyl~lljspiperone for the cloned Dzs (ra!) in (il{ ~,('1 lissue culture cells (lop)or for the cloned t):l (human) in GIt4('l cells (l~oltor'n). The lwo ~tt-ligaml dllll~;l~'t

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[~H]spiperone density in I) m (rat) a n d D : r ( h u m a n ) (fig. 2), the average heing 1.8-fold ( + (1.09; n = 5) higher. T h e dissociation c o n s t a n t s of each 3H-ligand were thc same for the native a n d cloned h)rms of the d o p a m i n c D , receptor. All the [3H]spiperone sites were inhibited by e m o n apride over two d c c a d e s of c m o n a p r i d e c o n c e n t r a t i o n ( 1 0 - i ( i 0 pM), as shown in fig. 3 (top) fi)r pig a n t e r i o r pituitary tissuc, and in fig. 3 ( b o t t o m ) for c h m c d l)2s (human). T h c sitcs labclicd by 200 p M [ 3 H ] c m o n a p r i d e in native tissues, however, were inhibited by s p i p c r o n c in two phases over four d e c a d e s of s p i p e r o n e c o n c e n t r a tion (20 p M - 1 0 0 0 nM), This p a t t e r n was o b s c r v c d for pig a n t e r i o r pituita~" tissues ;rod for eight h u m a n stxiata (fig. 4, top, middle). In native tissues, thcreli)re, s p i p e r o n e revealed two dissociation c o n s t a n t s (vcrsus {~H]cmonapridc) with a h i g h - a f f i n i t y site of 0 0 - 9 8 p M and a low-r, ffinity site of 18,-,29 riM. No such l w o phases, howev.:,r, were found in the D2-con!aining tissue culture cells, w h e n using 21tl) pM [ ~ H ] e m o n a p r M c (fig, 4, bottom), t ) and/H]slnperone ¢ ' 3.2-. [~,t]Raclopride comparison

A sin]ilar situation was found for [ 3 H ] r a c l o p r i d e and ['~H]spiperonc w h e n m e a s u r i n g the d o p a m i n e I ) , rec e p t o r density in d o n c d d o p a m i n c 132 receptor-con-

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10-u' 10" 10 u" tO.q 10~ 10 ; t0 4~ 10 s Emenapride, rno{es4~ter Fig. ?,. Emonapridc mhihilcd all the tlol~anfine D. receptor silts occupied hy 200 pM [~lllspipcrone in both pig anterior pituita~T (top) and cloned I)2s (human).

t43

taining tissue culturc cells. An example is she,we in fig. 5, where it may bc seen that [3H]raclopride revealed a dopamine D, receptor density which wa,'; 46% higher than that measured by [3H]spiperone. The average density for [3H]raclopride was 1.5-fifld (+_0.1: n = 5) higher than that fl~r [3H]spiperone. Two experiments were done on the ek;ncd dopamine D, receptor using all three ~H-ligap.ds on the same prcparation. Thc ratio (Bin,,.' [3H]cmonapridc)/(Bm,, [3H]spiperonc) was idcntical to that for the ratio (B,,,,,, [XH]raclopride)/(B ...... [3H]spiperone); this ralia was

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10 <,

Fig, 4. The dopanlinc De rcccptor sites labelled by J ~1liculonal~i'ide were inhibiled by spiperonc in I,,Vo phases over four decades of spipcronc ¢Ollcentralion (20 pM--100{) nM) ill pig anterior pituilai3' tissue (top) and in cighl human ~lri;ita (mktdlc). }lowcver, in lissuc cullurt2 cells conlaieint' clorit'd thll)imliue [)~ rt2cel)lOrs, Spil'lt'rolle inhibited file binding of I~tlienlollaprid¢ i/1 only a si|iglc phaise, Thus, ill COlllpeling versus [~ll]cn~onapridc in ilalive lissllt.'s oely, spiperone revealed Iwo tlissocialion ¢onshlills. ;i high+affinity site of 60-98 pM and il low-affinity site of 18-29 nM. Data from eight human lissues (bottom) were obtained using the cilutlalc nuclei: the perceelage of high-affildly sites with dissociation conslanl of 98 pM was 870:, while lhc percentage ot h~w-illTinity sites with dismcialion co[Islanl c,f 28 nM was 13G+

The results indicate that the cloned dopaminc D, receptor (long or short) revealed a density which was 3(1% to i0(iC~ higher when using a [~H]bcnzamidc ligand, coml~ared to the [~H]huiyrophenone [igand, [-*|t]spiperone. In tile case of [3H]emonapridc, tile dopamine D, recep|o,: apparem density wlts its high as 2-1old more than that nleasured b y [3H]spiperone, matching tile difference found in nalivc tissues (como pare figs. 1 a,ld 2), These rcsults Oll the cloned ctopamine l ) : receptor-containing cells indicate, therefore, that this property of two app'trcntly different densities, as measured by these two ciasscs of radioactive ligands, is inherent to lhe dopamine D e receptor protein. One possible interpretation o1" these data is that the tertiary nitrogen of boil1 tile bcnzanlide and spiperonc lleuroleptics hinds Io the same a m i l l o acid in tile D, rcccptor (asparhilc', N e w ei al., U)gl), bul thai ihc rcmahldcr of lhcsc ncurolcp!ics llllt), associate with different amino aeids of ihe dopan!ine D, receptor. Howcver, there is ill p r e s e n t nt) C O l l s c n s u s a s it) which a n l i l l O a c i d s o f tile d o p a n l i n e [ ) , l C C C p t o r .are a s s o c i a t e d with cilhcr tile antagonist or tile agonist (e.g, dopamine may be associaled with either lraulsnlenlbrane helices 2 anti 7 (I)ahl ei al., 1991) or helices 3 and 5 (loliberi ctal., It)91 )). A second possible interpretation is lh:ll [:It]spillerone hhlds to dimers of the dopamine D~ receptor, while the [3H]benzamidc binds to reccplor nlonomers. This hl!erprclalion is s t l p p o r l c d by the facl that tile l~!l]emunapridc density in some experiments coukl be as higl/ as twice Ilult delcclcd by [3tl]spiperone. The

144 attachment of a single ligand '~o a receptor dimcr is known to exist for other receptors (e.g. human growth hormone: Cunningham et al., 1991). Such a dimer hypothcsis would rcquire diffcrcnt amino acid binding sites tor the two types of 3H-ligands. It is known, for cxample, that replacemcnt of aspartatc-80 (in the tion constant of cpidcpride (a bcnzamidc) by 25-fold, while only altering tl.lat for spipcronc by 2-fold (Nevc et al.. 1091). These findi.,:gs sugge,:t two different attachment sites for these classes of neurolcptics on the D, receptor. Since both cmonapride al~d raclopridc arc bcnzamides, while spiperonc is a butyrophenonc, it is possible that the additional dopaminc D, receptor sites (i.e., over and above the density measured by [-aH]spiperon,:) is a site for "bcnzamide neuroleptics" in general. Howc~cr, no apparc~'~ discrepancy was found between the dopaminc I)~ receptor densities detected by [3H]spipcrone and [3t-/]sulpiride, also a bcnzamide (Zahniscr and Dubocovich, 1983; U~vylcr and Coward, 1987). These prcvious studies, howcvcr, did not usc any guanine nucleotide to remove endogenous dopaminc from thc brain tissues. It is essential to rcmovc cndogcnous dopaminc by means of guanine nucleotide (or reserpine in viw~) in order to prevent a 30¢~ underestimate of thc dopaminc D, receptor density, when using ligands with dissociation constants higher than 2 nM (Ross and Jackson, 1989; Sccman et al., 1989a, b, 1990: lnoue et al., 1991a, b; Young ct al.. 1991; l-iall c t a l . , 1992). Thus, it is likcly that the density of dopaminc D, receptor sites measurcd by [~H]sulpiridc in those studics (Zahniser and Dubocovich, 1983; Urwylcr and Coward, 1987) was undcrcstimatcd by at least 30q~, since thc dissociation constant for [3H]sulpiridc is about 10-20 nM. Whatever the precise amino acid nature of the I3H]cmonapridc binding sitcs in thc cloncd dopaminc D 2 reccptor, fig. 4 indicatcs that spipcrc~':c, when used as the competing drug, did not recognize two populations of [3H]emonapridc sitcs. This suggests that all the [3H]cmonapridc sites wcrc rccognizcd with cqual ~fffinity as a single population by spipcrone.

tor density of 16.6 + 2.5 pmol/ml, while Farde et al. (1990) measured a dopamine D, receptor density of 22.3 + 6 p m o l / m l by mcans of-[lIC]raclopride, the latter density being 34% higher than that measured by [lIC]methylspiperone. It is likely, therefore, that the two ~LC-ligands are both measuring dopamine D2 receptors, bu! that the 34r/c hi~her value with [J~C]raclopride reflects an inherent property of the dopamine D e receptor protein, as shown above for the cloned dopaminc D, receptor. In measuring the dopamine D2 receptor density in various brain diseases, thercfore, it will be necessary to consider this inherent l~rnperty of the dopamine D, receptor protein to exhibit diffcrent densities, depending on the ligand. For example, the data of Wong et al. (1986) revealed a dopaminc D, reccptor density of 41.6 p m o l / m l in thc caudate nucleus of ncver-medicatcd schizophrenic patients, an elevation of 150% abovc the control value of 16.6 pmol/ml. Farde ct al. (199{1), howcver, using [lIC]raclopride, found a dopamine D~ receptor density of 30.9 + 5 p m o l / g (left putamen), which, although elcvated by 12% above thc control putamcn value of 27.7 pmol/g, was not found to be statistically significantly elevated. In gcncral, therefore, compared to the dopaminc D 2 rcceptor densities measured by [3H]spiperone, it appears that radioactive raclopridc underestimates the dopaminc D_, receptor dcnsity by 30% or so as a rcsult of endogenous dopamine, but overestimates the dopaminc D~ rcccptor density by 30-100% as a result of the inhcrent property of the dopamine D 2 receptor proteins to bind more benzamide moleculcs than spipcrone molecules. Hence, the apparent density of dopaminc D, rcccptor sites mcasurcd by the bcnzamidc ligands may bc lower or higher than that revealed by radioactive spipcronc or mcthylspiperonc, depending on thc balance of these two factors (endogenous dopamine and the inherent property of the dopamine D_, ~-cccptor protein to cxhibit diffcrcnt densities with diffcrcnt ligands).

Acknowledgements 4.1. hnplk'ations f t : Imman brain positron emission tomography Diffcrcnt clinical findings have emerged betwecn different positron tomography centers using different dopamine antagonist ~C-ligands for measuring brain dopamine D~ receptors in control human subjects and patients. For examplc, in the human caudate nucleus of control subjects, Wong ct al. (1986) found that [~lC]mcthylspiperonc dctccted a dopamine D 2 rccep-

We thank Carla Ulpianfi~rexcellentassislanceand MichioTerai (Research ('enter, Yamanouchi Phanl~aceulical Co., Tokyo) for helpful discussion.This work was stJpported by tile Constance and Stephen Licher Prize for research in schizophrenia, the Medical Research ('ouncil of Canada, I[lc Ontario Mental ltealth Foundation. the Jocy and Toby Tanenhaumaward of the Canadian Psychian'it Research Foundation, |he National Institutes of Ilealth (l~ O.C.) and NARSAD (Ihc National Alliance for Research in Schizophreniaand Depression),('hicagoand New York, USA. R.K.S. was supported by a Studenlshipfi'om |he Medical Research Council ¢~f Canada. It.It.MV.T. and II.B.N. are Career Scicnlists of the Ontario Ministryof tlcalth.

145

References Bunzow, J.R., tl.ll.M. Van Tel, D,K. Grandy, P. Albert, J. Sahm, M. Christie, ('.A. Machida. K.A. Ncvc and O. ('ivctli, 1988. ('loning and expression of a rat D, dopanaine receptor eDNA, Nature 336, 783. Cox, R.F. and B.L. Waszczak, 1991. Aumradiography of dopaminc D z receptors using[3lf]YM-09151-2, Eur J. Pharmacol. 199, 103. Cunuingham. B.C,. M. Uhsch. A.M. De Vos, M.G. Mulkcrrim K.R, Clauser and J.A. Wells, 1991. Dimerizalion of the extracclhflar donlain of the buman growth hormone receptor by a single hormone molecule, Science 254. 82. Dahl, S.G.. (?). Edvardscn and 1. Sylte, 1991, Molecular dymnnics of dopamine at the D2 receptor, Prec. Na~l. Acad. Sci. USA 88, 8111. Farde, L , F.-A. Wiesel, S. Stonc-Elandcr. C. thdldin, A.-L. Nordstr6m, tt. ltall and G. Sedvall. 1990, I) 2 dopamine receptors in neuroleplic-nai,..e schizophrenic patients: a positron emission Iomography study with [UC]raclopridc, Arch. Gen. PsychiatD' 47, 213. Grandy, D.K., M.A. Marchionni, It. Makam, R.E. SIolko. M. AIland, L Frolhingham, J.B. Fischcr, K.J. Burke-|lowie, J.R. Bunzow, A.C. Server and O. Civelli. 1989. Cloning of the eDNA and gene for a human D2 dopaminc rcceplor, Prec. Natl. Aca& Sci. t ISA 86, 9762. Grcwe, C.W., E.A. Frey, T.E. Cole and J.W. Kcbabian, 1982, YM09151-2: a potent antagonist for a peripheral D2-dopamiue receptor, Eur. J. Pharrnacol. 81, 149. lhdl, II., C. ltalldin and O. Sedvall, 1992, Gpp(Nlllp stimulater, [31t]raclopride binding to homogenates from human pulamen and accumbens. Neurosci. Left. 136. 79. tlatano, K., K. Ishiwata, K. Kawashima, J. l latazawa, M. Itoh and T. ldo, 1989, D2-Dopamine rcceptor specific brain uptake of carbon-I I-labeled YM-09151-2. J. Nucl. Mcd..sl), 515. l latano, K., T. ldo. K. Ishiwata, J. ttatazawa, M. lloh. K. Kawashima and R. Iw-ata, 1990, Synthesis of omega-[ISF]fluoroalkyl analogs of YM-t19151-2 for the measurcnlent of Dz-dopamine rcceptol:~ with PET, Appl. Radial. Isol. 41,551. I lalazawa, J., K. Itatam), K. lshiwata. M. lloh. T. ldo. K. Kawashima, K. Meguro, S. Walanuki and S. Set}. 1991, Measurement of D2 dopamine receptor-specific carbon- I I-YM-I)915 I-2 binding in tllc canine brain by PET: importance of partial volume correction. J. Nucl. Mcd. 32, 713. tliberl. M.F., S. Trumpp-Kalhncycr, A. Brninvcls and J. ltoflack. 1991, Three-dimensional models of neurolransmillcr (i-binding protein-coupled receptors, Mol. Pharmacol. 411, N. ttiguchi, S., K. Yokel, Y. Socishi aml S. Kawamura, 1986, Cc)ruf)aralive pharmacokinetics of a new I~enzamide ueuroleplie drug in rats, dogs and monkeys using a stable isotope technique, Xcuobiotica 16, 79. Indue, O., K. Kobayashi, Ii. Tsukuda. T. ltoh and B. Langstrom, 1991a. Difference in in vivo rcccptor binding between [~lt]Nmethylspiperone and [~lt]raclopride in reserpine-treated mouse brain, J. Neural Transm. N5, I. Indue, O., It. Tsukada. 11. Yonczawa. T. Suhara and B. Langstrom, 1991b. Rcscrpinc-induecd reduction of in vk,o binding of S('|I 23391) and N-methylspiperonc and its reversal by d-mnphelanfip.e, Eur. J. Pharnmcol. 197, 143. Iwanami, S., M. Takashima. Y. Ilirata, O. ttasegawa and S. Usuda, 1981, Synthesis and neuroleptic activity of benzamidcs, cis-N4 ll',enzyl-2-methylpyrrolidin-3-yl)-5-chh)r,:-2-methoxy-4-(n~et hylamino)benzamide and related compounds, J. Mcd. Chem. 24, 1224. Kudo, Y., 199(I, Trcalment of a new bcnzamide deriw~tive emonapride (YM-09151) lk~r schizophrenia, Cliu. Neuropharmacol. 13 (Suppl. 2), H~,0. List, S.J, and P. Seeman, 1981, Resolutkm of the dopamine and

:gerotonin receph'~r ¢onlponelllS o1" ~)|-spipert~l~ bifidhlg to rat brain regions. Proc. Natl. Acad. So). USA 7~. 2~20. Munsom P.J., and D. Rodbard. 1981L IJGANI): a versatile computerized approach for ctuuacterizathm of ligand-biuding systems. Anal. Biochem. 1t)7. 22tl. Neve. K.A., B.A. Cox. R.A. tlenningseu, A. Spanoyannis and R.L. Neve, 1991, PNotal role fl~r aspartalc-8(I in the regulation of dopamine D2 receptor affinity for drugs and inhibition of adenyl cyclasc, Mol, Pharmac(d. 39, 733. Niznik, tt.I3.. D.E. (higoriatJis, L ?ri-13ar, O. Buchman and P. Seeman, 1'485, Dopamine I) 2 receptors selectively labeled by a bcnzamide ncuroleptic: [~II]-YM-flgI51-2, Naunyn-Schmied. Arch, Pharmacol, 329. 333. O'Dov.'d, B.F., T. Nguycn, A. Tirpak, K.R. Jar¢ie, Y. Israel, P. Secman and H.B. Niznik, 1990, Cloning of two addiliomd calecholamine receptors from rat bre'n FEBS Left. 262. 8. (~)grcn. S.O. and T. tt6gberg. 11t88, Novel dopamine D-2 antagonists fi)r the treatment of schizophrenia. Ibl Alias Sei. Pharmacol. 2, 141 Ross, S.B. and D.M. Jackson. U,~89, Kinetic properties ~ff the accumulation of ~tl-raclopride in the mouse brain in viw) NaunynSchmied. Arch. Pharmaeol. 3411. 6 Seeman. P.. 1987, The absohlle density of neurotranxmitler receptors in brain: example for dopaminc receptors. J. Pharmacol. Methods 17. 347. Sccmam P., C. Ulpian, K.A. Wrcggctt and J. Welts. It)84. Dopamine receptor parameters detecled by ~ll-spiperone depend tm tissue concentration: amdysis and examDtes, J. Neur(~ct~em. 43, 22t. Sccmam P., N.II. Bzowej, tt.-C. Guan, ('. Bergemn, L.E. Bccker, G.P. Reynolds. E.D. Bird, P. Ricdcrer. K..lellingcr. S. Watanabe and W.W. T¢)urtellotte, 1987, l lnman brain dopamine receptors in children and aging adults, Synapse 1,399. Seeman, P., t1.-('. Guan and ll.B. Niznik, 19,q9~c Endogenous dopumine lowers the dopamine D2 receptor density as measured by [31t]raclopride: implications for positron cmissMn tomography of the human brain, Synapse 3. 9B, Seeman, P.. ll.B. Niznik, It.-('. Guan. G. Flooth and C. Ulpian, t989b. Link between DI and D2 dopamine receptors ix reduced in schizophrenia and lluntiugton diseased brain. Proc. Natl. Acad. Sci. USA ~6, 10156. Secman, P.. FI.B. Niznik and tt.-C. Guan. 19911, Elevation of D2 dopamine receptors in schizof~hrcnia is underestimated by radioactive raclopride. Arch. Gcn. Psychiatry 47. 1171L Tcrai. M., 1989, Selective biuding ~)f ['II}YM-(}9iSI-2 to the l)~ doparnine receptor in rat brain, l)uPont Biolech l.!pdale 4 (Nov.), 7. Tcrai. M.. S. Usuda. I. Kuroiv.a, O. Noshiro and It. Maeno, 1983, Selective binding (ff YM-119151-2, a nev." potcnl neuroleptie, to I)2-dopaminergie receptors, Jpu. ,I. Pharmacol. 23. 741L Tcrai. M., K. llidaka and Y. Nakamura, t981L ('omparisou of [~1t]YM-0915t-2 with [~lt]spip~ronc and [~l|]raclopridc for dopamine D-2 receptor binding to ral slriatum, l:ur. J. Pbarmacol. 173, t77. Unis. A.S.. J.G. Vincent and B. [)ill~n. 1990. Brain reccphn" autoradiography v, ith [ ~1t]-YM-ID 151..2: a hgand 1or lahelmg dopamine D-2 receptors, Life Sci. 47, PL 151. U~'ylcr, S. arm D. ('oward, 1987, Binding of ~|l-spipcrone and ~1t-( - )-sulpirMc to dopamine 17'2 receptors in r;~t slriata] membranes: mcthodtflogical cousideralions and defflonstration of the identical nature of the binding sites fi~r lhc two iigands, NaunynSchmied. Arch. Pharmacol. 335. 115. U~uda. S., K. Nisifikori, O. Noshiro :,aid tt. Macn(~, 1951, Neurotcptie properties of cis-N-( 1-benzyl-2-methylpyrr()lidin-3-yl)-5-chtoro2-mcthoxy-4-methylaminobenzamidc (YM-119151-2) wilh selective antidopamincrgic activity, Psychopharmacology 73, i03. Wong, D.F.. tt,N, Wagner. Jr,, L,E. Tune. R.F. [)annals. G.D. Pcarlson, J.M, Link,,. ('.A. Tanm3inga, E.P. lSmuz,soite. ILT.

146 Raverl. A.A. Wilson. J.K.T. Toung. J. Malal. J.A. Williams. L.A. O'Tuama. SJI. Snyder, M.I|. Kuhar and A. Gjcdde. 19S6. Positron emission tomography reveals elevated D2 dopaminc receptors in drug-naive schizophrenics. Science 234. 1558. Young, L.T., D.F. Wong, S. Goklman, E. Minkin, C. ('hen, K. Matsumura, U. Scheffcl and II.N, Wagner. ,Ir., 1991. Effects of

endogenous dopamine on kinetics of [31t]N-methylspiperone and [~tl]raciopride binding in the rat brain, Synapse 9, 188. Z~lhniser, N.R. and M.L. Dubocovich, 1983, Comparison of dopamine receptor sites labeled by [~H]-sulpiride and [~ll]-spiperone in striatum, J. Pharmacol. Exp. Ther. 227, 5¢)2.