Pharmacological Research Communications, Vo/. 15, No. Z 1983 697

DIFFERENTIAL AFFINITY OF DOPAMINERGIC AGONISTS AND. ANTAGONISTS

FOR D 1 AND D 2 DOPAMINE RECEPTORS IN RAT STRIATUM.

E. Carboni, M. Memo and P.F. Spano

Department of Pharmacology and Pharmacognosy, University of Cagliari, Cagliari, Italy.

Rece~edmf~na/~rm 11 May;983

Dopamine (DA) receptors have been classified into two types, D 1 and D 2, on the basis of their phar- macological specifities and localization. We now report that lesions of striatal dopaminergic neu rons by kainic acid (KA) injection or cerebral ne ocortex (CN) ablation, differentially affect the potency of DA agonists and antagonists in displac ing H-spiroperidol specific binding. In particu: far, ago~ists show a preferential affinity in dis placing JH-spiroperidol from those receptors sit: uated on intrastriatal neurons, following CN le- sion. On the other hand, antagonists show a high- er displacing activity for those receptors mainly located on cortical terminals following KA lesion. These data support the hypothesis of the exis- tence of different types of DA receptors with a differential anatomical location.

During the last twenty years, dopamine (DA) receptors

have been intensively studied in both central and periphe-

ral nervous systems of man and other animals. Recently, in

attempts to clarify how dopamine is involved in physiolog!

cal and pharmacological mechanisms, the existence of diff£

rent classes of DA receptors has been suggested.

Although the concept of multiple receptors for dopamine

had been origJna!ly discussed by Cools and Van Rossum in

1976, a more suitable classification has recently proposed

by Spano et al. (19.79) and Kebabian and Calne (1979). This

can be summarized as follows: the D 1 receptor is linked to

adenylate cyclase stimulation and the D 2 receptor is not

linked to the s£imulation of this enzyme. This classifica-

tion has been modified by Seeman (1980) who takes into ac-

count the differing receptor affinities for DA-agonists

and antagonists. On the basis of experiments involving che

mical and surgical lesions, Creese (1982) has extended the

0031-6989183/070697-101503,00/0 C 1983 The Italian Pharmacological Society

898 R ~ n m ~ O / o g ~ 8~si~a~.l~ Communicatior~ V~I. I5.. No. Z 1983

t l leor~ ;0~ DA recell~tur subolass~lf icatl lon to - Incorpora te the it: different se~nsltivlty .to .guanine :.nucleotides and their anato~ica.l., lo~allzation as a further means of dlstlngui-

The use: o£ :such '.specific chemical and surgical lesions

has grea t l y .a lded the characterization o f central DA rece

ptors. Thus, after intrastrlatal kalnic acid (KA) injec-

tion DA-.st~ulated adenylate cyclase activity is virtually

abolished, while 3H-haloperidol or 3H-spiroperidol specif-

ic binding is reduced to about 60% (Govoni et al., 1978;

Schwarcz and Coyle, 1977}. On the other hand, lesions of

the cerebral cortex in the rat resulted in decreased stri-

atal 3H-splroperidol binding without any change of the DA-

-stlmulate4 adeny.late cyclase activity (Garau et al., 1978;

Schwercz et al., 1978). These results suggest the presence

Of DA receptors not linked to adenylate cyclase on the cot

tico-st.riatal nerve endings.

On these premises we have investigated 3H-spiroperidol

binding in several experimental conditlons~ in particular,

we have studied the displace~nent capacity of various dopa

mlnerglc compounds endowed with agonist or antagonist ac-

tivity.

Our exper.i:e.ents were performed using the two models

quoted above: the in~rastrlatal KA injection, to destroy

intrinsic striatal neurons hut spare axon terminals, and

the sur¢l.lcal lesion of the cerebral neocortex to destroy

cortJca]-innervatlon of ..he strlatum These lesions

should therefore provide, in the former case, striatal

%is.~ue is which the hA-receptors associated with adenyla

te cyclase Dave been substantia.lly eliminated (HA lesion)

a~~d, in the latter case, stflatal membranes iD which DA

receptors not associated with adenylate cyclase are selec

t~ve]y reduced (CN lesion).

M a t e r i a l s a n d ~ethods

Male Sprague-D~ley rets (Charles River, Calco, Italy)

w~Ighinq 275-300 g were used throughout all experiments.

Animals w~re fed ad llbltum with standard (Purina Chow

Pharmacological Research Communications; Vo/. 15. No. Z1983 699

Rat) diet and kept under a light regimen of 12 hours a

day.

Rats were operated under Equitesin anaesthesia in a

Kopf stereotaxic apparatus and received KA (3 pg/2 ul) as

two unilateral injections, (coordinates from Pellegrino

and Cushman stereotaxic atlas (1971): L 2.5, A 3.0, V 5.5

and L 2.5, A 2.0, V 5.5) at a rate of 1 ~l/min for each i

njection.

Unilateral cortical ablation was performed after remov

ing the meninges by suction of the neocortex. Animals w£

re sacrificed by decapitation five days after kainate i£

sion, and ten days after cortical ablation.

The validity of the lesions was verified by measuring

the enzyme activities of choline-o-acetyltransferase

(CHAT), adenylate cyclase (AC) and g!utamic acid uptake,

according to the methods of Fonnum (1975), Kebabian et

al. (1975) and Storm-Mathison (1977), respectively.

As previously demonstrated the kainate injection redu

ted ChAT activity to 12%, DA stimulated AC activity to

14% in the lesioned striatum while cortical ablation re-

duced glutamic acid uptake only to 69%.

3H-spiroperidol (3H-SPIR) binding was performed acco~

ding to Burtet al. (1976). Rat striata were homogenized

with a Polytron (setting 5, for 5 sec) in 50 volumes of

cold Tris-HCl 50 mM, pH 7.6 and centrifuged at 48.000xg

for 20 min. The pellet was resuspended and centrifuged

as before; final resuspension, was in incubation buffer

(TRIS-HCI 50 mM, pH 7.4, 0.1% ascorbic acid, 120 mM NaCI

and pargyline 1 ~M, 200 weight/volume).

After preincubation at 37"C for I0 min, binding assa-

yes were conducted using one ml aliquots of resuspended

striatal membranes. 3H-spiroperidol and test substances

were added in 50 ~i of 0,1% ascorbic acid, to make a f~

hal volume of i,[ ml. The incubation was allowed to pro

ceed for 10 mid at 37"C and stopped by filtration

through Whatman GF/B filters, followed by two washes

with 5 ml of cold incubation buffer. Non specific bind-

ing was obtained in the presence of 1 ~M (+)butaclamol.

7 0 0 Pt~rmm.:o/ogical Re$e~rch Communicatiom;. VoL 15, No Z I983

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I1~1,

&

-% ..g . ?

A

l e O -

D

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ed as a c o n t r o l ,

Results

Fiq. i shows the potency of various concentrations of

DA-agonlsts and -antagonists In displacing 3H-splroperi-

dol from striatal membranes of normal and KA lesloned

rats. As previously reported we found that intrastrlatal

KA Injections reduced 3M-spiroperidol blndlng to 6u% (da

TABLE 1

Inhibition of striatal 3H-spiroperidol specific binding in KA- and CN-lesioned rats

by various DA agonists and antagonists.

COMPOUNDS

CN-lesioned side

K i (nM)

Intact s~de

KA-lesioned side

KA/CS

DOPAMINE

592

+ 45

1,526

+ 120

9,582

+ 2B6

16.2

APOMORPHINE

56

+

8.3

152

+

13

445

+

39

7.9

FLUPHENAZINE

7.0 +

0.42

2.0 +

0.15

0.8

+

0.07

0.12

(+)BUTACLAMOL

9.3 +

0.71

2.3 +

0.21

i.i

+

0.15

0.ii

HALOPERIDOL

8.2 +

0.53

4.2 +

0.34

2.4

+

0.30

0.33

[-)SULPIRIDE

1,025

+ 97

270

+

25

261

+

18

0.25

SULTOPRIDE

832

+ 81

224

+

21

250

+

20

0.33

Five concentrations in triplicate were used for each drug to determine the concentration

required to inhibit specific binding by 50% (ICs0). K i values were calculated according

to the equation K i = IC50/(I+3H/KD) where 3H is the concentration of 3H-SPIR (0.5 nM)

and K D is the apparent dissociation constant at equilibrium (0.3 nM). Values are the

mean + S.D.

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702 Phawnacrdogk:al Research Communications. Vol. I 6, No, 7. I983

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}'~g. 2. CompetiZ~on by various DA agonlsts and antagonls~s [or 3H-SPIR binding to me~brane~ from control (o----~) and CN-lesi0ned (z~----a) striata. The ordinate represents 3H-SPIR specific b~nding. Each poirl~ i~ the ~ean for at lea~t three exper iments per[ormed in t r L i p l [ ca te (S .~ . ' .H . 1 0 ~ ) . C o ~ t r o l a t e r a l s t r i . a t t a ~ , f r o m u n i l a t e r a l l e s i o n e d r a t s , was used as a control.

ta not shown). KA lesion alters the potency of both DA-a

gonlsts and antagonists in inhibiting 3H-splroperidol

binding (fig. i and table i). In particular, DA and apo-

morphine showed a decreased affinity for the receptors

iabelledlby 3}I-spiroperi4ol in striatal membranes from

KA-treated rats. In contrast, in the same membranes,

classical neuroleptic8, such as fluphenazine, (÷)butacla

Pharmacological Resuarch Communicerions. VoL 15, No. 7, 1983 703

mol and haloperidol, showed an increased potency in inh~

biting 3H-spiroperidol binding. The ICs0'S of substitu-

ted benzamides such as (-)sulpiride and sultopride were

not affected by KA lesion.

Fig. 2 shows the inhibitory potency of various concert

trations of DA-agonists and antagonists in 3H-spiroperi-

dol binding to striatal membranes from normal and CN-Ie

sioned rats. As previously found cortical ablation redu

ted about 3H-spiroperidol binding to 62% (data not shown).

Using striatal membranes from CN-lesioned rats, 3H-sp~

roperldol binding was inhibited by concentrations of DA

and apomorphine virtually inactive in membranes from con

trol rats. Therefore the lesion causes a shift to the

left of the DA-agonist inhibitory curves. In contrast,

the IC50 of all the DA-antagonists tested was~significan~

ly higher in me/nbranes from CN-lesioned than in normal rats.

This phenomenon is demonstrated by a shift to the right of

%he displacement curves.

Table 1 summarizes the results obtained after different

lesions and expresses the inhibitory potency of both DA-a~

onists and antagonistson 3H-spiroperidol bindingas K iva !

UeS.

Discussion

Since current concepts concerning DA-receptor classifi~

ation are both controversial and confusing we maintain that

DA receptors should be classified in accordance with their

function and pharmacological specificity. Hence, DA is able

to stimulate adenylate cyclase activity by interaction with

D 1 receptor sites.

A key piece of evidence in the characterization of D 1

and D 2 receptors was the fact that some DA-agonists e.g.

the ergot derivatives and DA-antagonists e.g. the substitu~

ed benzamides are-completely unable to stimulate or inhibit

respectively, DA-sensitive adenylate cyclase activity (Sp£

no et al., 1979a; Spano et al., 1980).

Recently, different groups reported that DA may even i£

704 PIM,macologicat Reseamh Communications. VoL I5. No. Z 1983

hlblt adenylate cyclase activity through stimulation o f D 2

recognition sites (Onali et al., 1981; Cote et al., 1981).

On the other hand, more recent results have provided sub-

stantlal evidence that a subset of D 2 receptors are asso-

ciated with neither stimulation nor inhibition of aden~

late cyclase activity (Uzumaki et al., 1982; Spano et ai.,

1983).

Since striatal membranes from normal rats contain all

types of OA receptors, the present study was directed at

verifying whether i) Selective lesions could provide evi

dence for tt~e existence of a particular class of DA rece~

tots, ii) the different types of DA receptors possess a

characteristic state of affinity for agonists and antago

hi.sis.

Y~ lesion is known to virtually abolish DA-s.timulated

adenylate cyclase activity, indicating that almost all D 1

receptors present in the •intrinsic neurons of striatum

re destroyed. In the striatum, no data is yet available

on the precise localization of DA receptors associated

with adenylate cyclase inhibition although indirect stu-

dies suggest that their main location is on the intrinsic

neurons (Stool and Kebabian, 1981). Thus, DA receptor bin

ding sites remaining after F~ lesion may represent the

subclass of D 2 receptors not linked to any adenylate cy-

clase moiety. The pharmacological characterization of

this type of D 2 receptor suggests that their binding site

has high affinity for antagonists and low affinity for a-

gonists. It is interesting to note that the substituted

benzamides, (-)sulpiride and sultopride do not change

their potency .in inhibiting 3H-spiroperidol binding after

KA-leslon. In other words, the KA induced elimination o~

D 1 receptors does not alter the action of sulpirlde and

sultoprlde at the DA receptor level indicating, once

again, the selectivity of these drugs for D 2 receptors.

On the othe[ hand, cortical ablation apparently re-

sults in the removal of those D2 ;eceptors not linked

to adenylate cyclase. The remaining DA receptors are

D 1 and those D 2 linked to inhibitory adenylyl cyclase

Pharmacological Research Communications. VoL 15, No. Z I983 705

-D2(i)-. Our results suggest that DA-agonists have a

higher affinity for D 1 and D2(i) receptors than for D 2-

not linked to adenyl cyclase.

Interestingly, DA-antagonlsts have less affinity for

D 1 and D2(i)receptors than for the D 2 receptors remain-

ing after KA-lesion. The reduced ability of sulpiride

and sultopride to displace 3H-spiroperldol binding fo !

lowing CN lesion suggests that a certain number of D 2

receptors are still present after the lesion. These r£

sults, together with those obtained from the measure-

ment of adenylate cyclase activity, could be interpre~

ed as suggestive of different DA receptors being diff£

rentially localized within the striatum. Furthermore

their recognition sites could have different affinities

for agonists and antagonists. Thus DAergic agonists ha-

ve higher affinity for the receptors linked to adenyla

te cyc!ase present in the intrinsic neurons of the sir !

atum -both D 1 and D2(i)- than for D 2 receptors -not lin

ked to adenyl cyclase- on the nerve terminals of corti-

costriatal pathways. In contrast DA-antagonists have

the opposite effect, they have higher affinity for the

D 2 receptors present in the nerve terminals of cortico

striatal pathways than for other DA receptors.

The interaction of sulpiride and sultopride with DA

receptors as measured by 3H-spiroperidol binding appears

to be insensitive to the presence of D 1 receptors.

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