Proc. Natl. Acad. Sci. USAVol. 79, pp. 4456-4460, July 1982Neurobiology

Agonist-induced subsensitivity of adenylate cyclase coupled with adopamine receptor in slices from rat corpus striatum

(D-1 dopamine receptor/down-regulation/modulator proteins)

MAURIZIO MEMO, WALTER LOVENBERG, AND INGEBORG HANBAUERSection on Biochemical Pharmacology, National Heart, Lung and Blood Institute, National Institutes of Health, Building 10, Room 7N-262, Bethesda, Maryland 20205

Communicated by Paul Greengard, April 12, 1982

ABSTRACT Incubation, for 30 min, of striatal slices with 10,sM dopamine, 10 IpM apomorphine, or 10 IpM SKF 38393 de-creases dopamine-stimulated adenylate cyclase activity by 50-60%.This loss in dopamine-stimulated enzyme activity appears to bemediated by a persistent occupancy of recognition sites of the D-1 receptor because: (i) at 10 IpM, SKF 38393, a selective D-1 re-ceptor agonist, facilitates desensitization and LY 141865, a selec-tive D-2 receptor agonist, fails to elicit desensitization of dopa-mine-dependent adenylate cyclase; and (ii) preincubation withdopamine in the presence of 1 paM haloperidol but not 1 pmM sul-piride curtails the desensitization of dopamine-dependent ade-nylate cyclase. In dopamine-desensitized striatal slices the K3 forN-propylnorapomorphine binding is increased but the content ofmembrane-bound calmodulin and the activation of adenylate cy-clase by NaF and cholera toxin are decreased significantly. Instriatal slices incubated with dopamine for prolonged time periodsthe coupling of the GTP-binding protein with adenylate cyclaseand dopamine recognition sites may be impaired and the contentof membrane-bound calmodulin is decreased.

In brain tissue the recognition sites for dopamine appear to becoupled to various kinds of amplifier systems by different mo-lecular mechanisms. This variety is the molecular basis for thediversity in pharmacological and biochemical profiles of differ-ent dopamine receptors located in various brain structures andin other tissues (for review see ref. 1). For instance, in D-1 re-ceptors, the coupling of the dopamine recognition site withadenylate cyclase (2) involves a GTP binding protein (3) andcalmodulin (4). The function of these modulator proteins in theamplification ofthe dopamine signal is not clear. In rat striatum,calmodulin has been shown to participate in the regulation ofadenylate cyclase sensitive to dopamine stimulation (5). In ad-dition, calmodulin and Ca2O were shown to down-regulate thecytosolic cyclicAMP content in rat striatum (4). This mechanismmay be operative in the subsensitivity of dopamine receptorselicited by their persistent stimulation.

In the case of -adrenergic receptors, persistent occupancyofthe recognition site by an agonist causes desensitization (6-8)and decreases the number of recognition sites for agonists andthe catecholamine-induced activation of adenylate cyclase(9-11). The present study examined whether persistent occu-pation ofdopamine recognition sites desensitizes the dopamine-induced activation of adenylate cyclase in striatal slices. Theexperiments were planned to determine whether calmodulinand the adenylate cyclase-coupling system change during do-pamine receptor desensitization. The data show that prolongedoccupancy of the dopamine recognition site decreases the do-pamine-induced activation of adenylate cyclase. The decreasein enzyme activation is associated with a decrease in membrane-

bound calmodulin content, with a loss in the efficiency of theadenylate cyclase-coupling system, and with a decrease in theaffinity of the dopamine recognition site for the agonist.

EXPERIMENTAL PROCEDURESMale Sprague-Dawley rats (100-200 g) were kept under stan-dard conditions with 12-hr light/dark cycle and had free accessto food and water. They were decapitated between 1100 and1200, the brain was rapidly removed, and the corpus striatumwas dissected out. Striatal slices (230 ,um thick; 35 mg oftissue,fresh weight) were prepared with a TC-2 Sorvall tissue sec-tioner. The slices were preincubated for 30 min (unless oth-erwise indicated) at 37°C in 1 ml of Krebs bicarbonate buffer(pH 7.4) supplemented with 10 mM dextrose and 1.14 mM as-corbic acid under constant oxygenation (95% 02/5% CO2). Afteraddition ofvarious drugs, as reported in Results, the incubationwas continued for an additional 30 min. Thereafter, the sliceswere removed from the incubation medium and washed inKrebs bicarbonate buffer that was free of drugs. The excessmedium was drained offon filter paper and homogenates oftheslices were prepared. When 0.1 mM [3H]dopamine was usedas a tracer, it was found that 14 nmol ofdopamine was taken upby striatal slices during 30 min of incubation. This amount ofdopamine present in the adenylate cyclase assay mixture failedto increase adenylate cyclase activity.

Specific Ligand Binding to Dopamine Receptor. N-[3H]Propylnorapomorphine (58.5 Ci/mmol; 1 Ci = 3.7 X 1010becquerels; New England Nuclear) and [3H]spiroperidol (39.5Ci/mmol; New England Nuclear) binding was determined asdescribed by Creese et al. (12) and Burt et al. (13), respectively.Striatal slices were homogenized at 4°C for 30 sec in 40 vol (wt/vol) of 50 mM Tris'HCl pH 7.6 buffer in a Polytron homoge-nizer (setting 6) and centrifuged at 50,000 X g for 10 min. Thepellet was washed in the same volume of buffer and centrifugedagain. The resultant pellet was resuspended in the same volumeof 50 mM Tris-HCl pH 7.1 buffer containing 120 mM NaCl, 5mM KC1, 2 mM CaC12, and 1 mM MgCl2. The incubation mix-ture (1 ml) contained [3H]spiroperidol (0.2-1.2 nM) or N-[3H]propylnorapomorphine (0.2-1.5 nM) and an aliquot ofmembrane suspension corresponding to 10 or 2 mg of tissue(fresh weight), respectively. Under these incubation conditions,high- and low-affinity binding sites for [3H]spiroperidol wereassessed. The mixtures were incubated for 10 min at 37°C andthen rapidly filtered over a Whatman GF/B filter and rinsedtwo times with 5 ml ofcold buffer. Filters were placed in count-ing vials containing 10 ml of Aquasol (New England Nuclear)and the radioactivity was determined by liquid scintillationspectroscopy. Specific [3H]spiroperidol or N-[3H]pro-pylnorapomorphine binding was derived from the difference

Abbreviation: ELISA, enzyme-linked immunosorbent assay.

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Proc. NatL Acad. Sci. USA 79 (1982) 4457

between total binding and the binding observed in presence of0.1 mM dopamine (nonspecific binding). Scatchard analysis (14)of the specific radioligand binding was used to determine thetotal~number of binding sites (BmX) and the apparent dissocia-tion constant (KId).

Adenylate Cyclase Activity. Adenylate cyclase activity wasmeasured as described by Clement-Cormier et al. (15) withsmall modifications. In brief, the washed slices were homoge-nized in 15 vol (wt/vol) of 10 mM Tris maleate, pH 7.5/1.2 mMEGTA in a glass/Teflon homogenizer. The incubation mediumcontained 82.5 mM Tris maleate buffer (pH 7.5), 20 mMMgSO4, 2 mM cyclic AMP, 5 mM theophylline, 0.3 mM EGTA(carried over from tissue preparation), 0.01 mM GTP, 20 kgof pyruvate kinase, 4 mM phosphoenolpyruvate, 5 mM[2,8- H]ATP (28.3 Ci/mmol, New England Nuclear), and analiquot of homogenate corresponding to 1 mg of tissue, freshweight. The mixtures were incubated with and without 0.1 mMdopamine- (unless otherwise indicated) in a final volume of300/.l for 3 min at 30'C. The reaction was stopped by adding 500p1 ofstopping solution containing 2 mM ATP and 0.7 mM cyclicAMP followed by heating the samples at 900C for 3 min. Whenmeasurement ofthe kinetic properties ofadenylate cyclase wascarried out the Mg/ATP concentration ratio was held constantat 4:1. The amount of cyclic AMP formed was measured as de-scribed by Salomon et al. (16) by using subsequent chromatog-raphy on Dowex 5OW X4 (100-200 mesh, hydrogen form, Bio-Rad) and alumina oxide (alumina, Woelm neutral, activity 1,ICN). The recovery of cyclic AMP was 75-80%. [3H]ATP waspurified on a Dowex 1 X8 column (200-400 mesh, chlorideform, 1 x 4 cm) equilibrated with water. The eluate containing[3H]ATP was desalted on a Sephadex G-10 column (1.9 x 40cm) with water as solvent (17).

Cyclic AMP Content. At the end of the incubation, the me-dium containing the slices was heated for 3 min at 900C. Thestriatal slices were removed from the media, homogenized witha glass/Teflon homogenizer in 1 ml of 4 M formic acid, andcentrifuged at 3,000 x g for 15 min; the supernatant was col-lected and Iyophilized. The residue was dissolved in 1 ml of50mM sodium acetate buffer (pH 6.2) and the cyclicAMP contentwas measured radioimmunochemically (New England Nuclear)in 100-ul aliquots.

Enzyme-Linked Immunosorbent Assay (ELISA) for Cal-modulin. Striatal slices were homogenized (glass/Teflon ho-mogenizer) in 10 vol (wt/vol) of0.32 M sucrose and centrifugedat 1,000 x g for 30 min. The supernatant fraction was removedand centrifuged at 100,000 x g for 30 min. The resultant pelletwas homogenized in half the original volume of 50 mM Trisbuffer, pH 7.4/1.0% Lubrol WX (Sigma), incubated on ice for

30 min, and centrifuged at 100,000 x g for 30 min. The cal-modulin content was measured by ELISA in the supernatantfraction obtained by the first high-speed centrifugation (re-ferred to as "soluble") and in the supernatant fraction of thesecond high-speed centrifugation (referred to as "membrane-bound"). As reported (18), the ELISA was carried out with aspecific antibody directed toward calmodulin and produced inrabbits. As second antibody, horseradish peroxidase-labeledanti-rabbit Ig conjugate was used (Miles) and the degradationof o-phenylenediamine in the presence of H202 was assessedspectrophotometrically at 488 nm with an ELISA microreader(Biomedical Engineering and Instrumentation Branch, Na-tional Institutes ofHealth, Bethesda, MD). The protein contentwas determined by the method of Lowry et al. (19) with spec-trophotometrically standardized bovine serum albumin as areference.

RESULTSAdenylate Cyclase Responsiveness to Dopamine Stimula-

tion After Persistent Occupancy of Dopamine RecognitionSites. When striatal slices were incubated with dopamine forlonger than 15 min, the responsiveness of adenylate cyclaseactivity to dopamine stimulation in homogenates prepared fromthese slices was decreased, although the basal activity of thisenzyme was unchanged (Fig. 1A). This inhibition was dose de-pendent and was maximal with 0.1mM dopamine. With thisconcentration a significant desensitization of striatal adenylatecyclase to dopamine stimulation appeared after 15 min of in-cubation and the maximal inhibition, 66% of control response,was reached after 30 min of incubation (Fig. 1B).A similar loss in the stimulation of adenylate cyclase by do-

pamine was also obtained when 10 AuM apomorphine or SKF38393 was added to the incubation medium (Table 1). In con-trast, when slices were incubated with 10 AM LY 141865, ahighly specific D-2 receptor agonist (20), the responsiveness ofadenylate cyclase to dopamine stimulation was unaffected.When slices were preincubated with 1,uM haloperidol beforethe addition of 10 AuM dopamine, the desensitization processwas partially prevented. In contrast, preincubation with 1 AuMsulpiride, which has high affinity for the D-2 receptor, failedto block the desensitization. When slices were incubated with10 ,uM isoproterenol, the subsequent activation of adenylatecyclase by 0.1 mM dopamine was not impaired. Preincubationof striatal slices with 0.5 mM propranolol failed to prevent thedesensitization ofadenylate cyclase elicited by 10AM dopamine(10-5M), indicating that possible occupancy of P-adrenergicrecognition sites was not involved in the desensitization of stria-tal dopamine receptors. The possibility that prolonged incu-

500 r

400

300 F Dopamine

200- Basal

100F

10 20 30 40 50 60 70Time, min

FG. 1. Desensitization of dopa-mine-sensitive adenylate cyclase. (A)Dose-response relationship of dopamineinduced desensitization of adenylate cy-clase. Striatal slices were preincubatedfor 30 min; then various concentrations(1-500 pM) of dopamine were added tothe media and incubation was continuedfor 30 min. Dopamine-stimulated (0.1mM) adenylate cyclase was measured inhomogenates. (B) Time course of.dopa-mine-induced desensitization of adenyl-ate cyclase. After preincubation, striatalslices were incubated with 0.1. mM do-pamine for different periods of time. Do-pamine-stimulated adenylate cylase wasmeasured in homogenates. Each pointrepresents the mean ± SEM of four sim-ilar experiments.

-4 4000.bo

0 3000

m 200

*u 100c.l0

Dopamine

Basal

0 10-6 10-5 10-4 10-3Dopamirne, M

Neurobiology: Memo et al.

4458 Neurobiology: Memo et al.

Table 1. Decrease of dopamine-stimulated adenylate cyclaseactivity after incubation of rat striatal slices withvarious drugs

Dopamine-elicitedincrease in cyclic AMP

Addition to medium pmol/rg ofPreincubation Incubation* protein/mint %

None None 208 ± 24 105None Dopamine 108 ± 6* 54None SKF 38393 105 ± 8* 53None LY 141865 188 ± 12 95None Apomorphine 118 ± 8* 59'None Isoproterenol 168 ± 11 85.Haloperidol (1 M) Dopamine 158 ± 12§ 80Sulpiride (1 pM) Dopamine 104 ± 8* 52Propranolol'(0.5 mM) Dopamine 112 ± 9* 56* All drugs listed were at 10 pM.t Numbers are the mean + SEM of three separate experiments intriplicate. Basal enzyme activity is 198 20 pmol of cyclic AMP per

mg of protein per min.*P < 0.01 for difference from control.*P < 0.01.for difference from dopamine-incubated group.

bation.of striatal slices with 0.1 mM dopamine could modify thecatalytic activity of adenylate cyclase was ruled out by experi-ments showing that 20 mM Mg2e elicited half-maximal acti-vation ofthe enzyme from normal and desensitized striatal slices(results not shown).

The'responsiveness ofadenylate cyclase in homogenates fromcontrol and dopamine-treated slices to various concentrationsofdopamine is shown in Fig. 2. Incubation ofslices with 0.1mMdopamine for 30 min slightly decreased the V. of adenylatecyclase (- 15%; statistically insignificant) and caused a shift tothe right in the dose-response curve for adenylate cyclase ac-tivation by dopamine. The ED50for the stimulation ofadenylatecyclase by dopamine increased by about 5-fold from 17 ,M incontrol slices to 88 AM in slices desensitized by dopamine in-cubation, based on comparable Vma. values. This loss of re-

sponsiveness was due to a change in the Ka for the enzyme fordopamine (control, 1.2 AM; desensitized, 4.1 AM)..

Changes in the Calmodulin Content of Membranes Pre-pared from Striatal Slices Incubated with Dopamine ReceptorAgonists. Incubation of striatal slices with 10 pM dopamine or

10 pM apomorphine for 30 min decreased the amount of cal-

4.2

P 400

Normal

, 300 Desensitized

Ax2001I

10-6 1o-5 1m-41n-3Dopamine, M

FIG. 2. Concentration-effect relationship. for dopamine-stimu-lated adenylate cyclase activity in homogenates from normal (e) anddesensitized (o) striatal slices. Slices were preincubated for 30 min; theincubation was continued for 30 min in presence or absence of 0.1 mM.dopamine. Then, adenylate cyclase -activity in homogenates was de-termined in presence of the indicated concentrations of dopamine.

modulin present in the membrane fraction and increased it inthe cytosol fiaction of homogenates prepared from these slices(Table 2). hi addition, there was a time-dependent increase ofthe soluble calmodulin and cyclic AMP contents in homoge-nates prepared from striatal slices incubated in presence or ab-sence of dopamine (Fig. 3). After 30 min of preincubation theamount of cyclic AMP formed in the slices (Fig. 3 Upper) or

released into the incubation medium (data not shown) was atsteady state. Five minutes after the addition of 10 ,uM dopaminethe cyclic AMP content in the slices increased by 3.5-fold, butit returned almost to base line 30 min thereafter. Another ad-dition of dopamine to. the incubation medium after 30 min ofincubation with dopamine failed to enhance the formation ofcyclic AMP significantly, indicating that the decline in theamount ofcyclic AMP released during dopamine exposure was

not due to the decreased availability of the agonist at the re-

ceptor sites but was due to the reduced functional capacity ofadenylate cyclase. Fig. 3 also shows that the decrease was notdue to a nonspecific decay of the preparation because striatalslices incubated in absence of dopamine for a period of 60 minstill responded maximally to 10 AM dopamine. The calmodulincontent in the supernatant fraction of homogenates preparedfrom striatal slices was significantly increased after incubationwith 10 ,uM dopamine for 30 or 60 min (Fig. 3 Lower). Thecytosolic calmodulin content of slices incubated without dopa-mine did not change significantly from 30 min (0;27 ± 0.02 pug/mg of protein) to 90 min (0.22 ± 0.03 pg/mg of protein) ofincubation.

Stimulation ofAdenylate Cyclase by Cholera Toxin and NaFin Normal and. Dopamine-Desensitized Striatal Slices. It hasbeen suggested that the GTP-binding protein mediates the reg-ulation ofadenylate cyclase by neurohormones, NaF, and chol-era' toxin (21-23). These experiments were designed to deter-mine whether a persistent occupancy of striatal dopaminerecognition sites by the agonist alters the coupling function ofGTP-binding protein for adenylate cyclase. Incubation of sliceswith cholera toxin, (100 pug/ml) for 40 min increased the cyclicAMP accumulation 2-fold- (Table 3). In contrast, in slices in-cubated with 0.1 mM dopamine for 30 min, the addition ofchol-era toxin elicited-only a33% increase ofthe cyclicAMP content,which was not statistically significant. A similar reduction in thedegree of adenylate cyclase stimulation (42%) was observedwhen homogenates of. dopamine-desensitized striatal sliceswere incubated with 10 mM NaF (Table 4).

Dopamine Recognition Sites in Normal and Dopamine-De-sensitized Striatal Slices. Incubation of striatal slices with 0.1mM dopamine for 30 min down-regulated the affinity of do-pamine recognition sites. This effect resembles that seen on the

Table- 2. Changes of soluble and membrane-bound calmodulincontent after incubation of striatal slices with variousdopamine receptor agonists

Calmodulin, Wdg/mg proteinAgonist (10 /AM) Soluble, Membrane-boundNone 0.20 ± 0.0 1.4 ±+0.1Dopamine 0.30 ± 0.02* 1.0 ± 0.1*Apomorphine. 0.34 ± 0.02* 1.1 ± 0.1*

Striatal slices were. preincubated for 30 min. The incubation wascontinuedfor 30 min-in the absence or presence ofan agonist. The sliceswere homogenized in 0.32 M' sucrose and centrifuged at 1,000 x g for10 min. The supernatant was removed and centrifuged at 100,000 xg for 30 min. The calmodulin content of the supernatant and mem-brane fraction was. assayed by ELISA. (18). The values are the mean(± SEM) of four experiments.*P < 0.01 for difference from value in absence of agonist.

Proc. Nad Acad. Sci. USA 79 (1982)

Proc. Natl Acad. Sci. USA 79 (1982) 4459

8

0.6

0.5

10 20 30 40 50 60 70 80 90

Time, min

FIG. 3. Time-dependent changes in cyclic AMP and calmedulin in

striatal slices elicited by incubation with 10 ,uM dopamine. Striatal

slices were preincubated for 30 min (e) or 60 min (a). Dopamine was

added to the incubation medium, as indicated by arrows. The cyclic

AMP content was measured in the supersnatant fraction of homoge-

nates prepared from striatal slices (each point represents four similar

experiments). Calmodulin content was assayed in the supernatant

fraction of homogenates prepared from slices after 30, 60, and -90 minof incubation in Krebs bicarbonate buffer (hatched bars) and 30 or 60

min after addition of dopamine to the incubation medium (open bars).

Each bar represents the mean SEM of six experiments. *, P < 0.05

for diffierence between open and hatched bars.

binding of N-[3H]propylnorapomorphmne to crude synaptic

membrane preparations after the addition of GTP. Incubation

of striatal slices with 0.1 mM dopamine for 30 min decreased

the binding affinity forN-[3H]propylnorapomorphine but failed

to change the number of specific binding sites present in mem-

branes prepared from homogenates of striatal slices (Table 5).

A similar decrease in binding affinity for the labeled agonist was

obtained in membranes prepared from normal slices when 0.1

mM GTP was present in the assay mixture. Interestingly, in-

cubation of dopamine-desensitized slices with 0.1 mM GTP

failed to increase the 1Kd 'for N-[3H]propylnorapomorphine fuar-ther. The number ofbinding sites and the 1Kd for [3H]spiroperidol

Table 3. Increase in cyclic AMP formation elicited by cholera

toxin after incubation of rat striatal slices with dopamine

Cyclic AMP, pmol/mg proteinAddition Normal Desensitized

None 1.8 t 0.2 2.1 t 0.2Cholera toxin 3.8 0.2* 2.8 t 0.2

Slices were preincubated for 30 min in Krebs bicarbonate buffer atpH 7.4 (95% 02/5% C02). The incubation was continued for 30 min inabsence (normal) or in presence (desensitized) of 0.1mM dopamine. Toone set of culture dishes, cholera toxin (100 Ag/ml) was added and theincubation was continued for 40 min. Thereafter, slices were heatedat 90°C for 3 min and homogenized in 4MHCOOH and the cyclicAMPcontent of the supernatant fraction was measured by radioimmu-noassay. Values are the mean SEM of three experiments intriplicate.* P < 0.001 for difference from control.

Table 4. Activation of adenylate cyclase by NAF after incubationof rat striatal slices with dopamine

Cyclic AMP,pmol/mg protein/min

Addition Normal DesensitizedNone 216 ± 9 198 ± 18NaF 650 ± 44* 430 ± 32

Slices were incubated for 30 min in Krebs bicarbonate buffer at pH7.4(95% 02/5% C02). The incubation was continued for 30 min in ab-sence (normal) or in presence (desensitized) of 0.1 mMdopamine. Stim-ulation of adenylate cyclase by 10 mM NaF was measured in the ho-mogenate prepared from striatal slices. Values are the mean ± SEMof three separate experiments in triplicate.*P < 0.01 for difference between normal and desensitized.

binding to crude synaptic membranes was similar in control anddopamine-desensitized slices. However, measurements of theIC50 of [3H]spiroperidol binding with dopamine as a displacerindicated that in dopamine-desensitized slices the affinity fordopamine (Kd = 3.8 ± 0.21 uM) was only about 1/7th that incontrol slices (0.56 ± 0.12 ,uM).

DISCUSSIONIn the present report we extend previous reports on the reg-ulation ofthe function ofstriatal dopamine receptor (24, 25) andprovide evidence that prolonged incubation of slices with 0.1mM dopamine desensitizes dopamine receptors, causing achange in those functions that couple dopamine recognitionsites with the catalytic function of adenylate cyclase. In slicesofcorpus striatum incubated for at least 15 min with dopaminevarying from 1 to 100 ,uM the D-1 receptors are desensitizedby a direct action ofdopamine on the D-1 receptor. 'This infer-ence is supported by the finding that incubation with LY 141865and isoproterenol for 30 min failed to decrease the stimulationof enzyme activity by dopamine and that pretreatment withhaloperidol but not with sulpiride curtailed the desensitizationelicited by incubation with dopamine. Sulpiride was shown tobe potent in inhibiting D-2 receptors but extremely weak inacting on D-1 receptors (26, 27), whereas haloperidol blocksboth types ofdopamine receptors (1). Dopamine receptor sitesthat are located mainly on neurons postsynaptic to dopaminer-gic axons (28) remain functional in preparations ofstriatal slices.A similar receptor-specific desensitization by prolonged expo-

Table 5. Specific binding of [3H]spiroperidol and N-[3H]-propylnorapomorphine to membranes prepared fromrat striatal slices incubated with dopamine

N-[3H]Propyl-[3H]Spiroperidol norapomorphine

GTP Kd Kd BNormal

No 1.1 ± 0.1 350 ± 38 0.91 ± 0.1 410 ± 53Yes 1.1 ± 0.1 371 ± 36 1.4 ± 0.1* 405 ± 42

DesensitizedNo 1.1 ± 0.1 368 ± 37 1.3 ± 0.1t 420 ± 42Yes 11 ± 0.1 355 ± 35 1.4 ± 0.1 405 ± 30

Kd is expressed as nM and B,,, as fmol/mg protein. After prein-cubation of striatal slices in Krebs bicarbonate buffer at pH 7.4 for 30min the incubation was continued for 30 min in presence or in absenceof 0.1 mM dopamine. The binding was measured in the presence orabsence of 0.1 mM GTP. Results are mean ± SEM of six experimentsin each group.*P < 0.05 for difference from value in absence of GTP.tP < 0.05 for difference from corresponding normal value.

Neurobiology: Memo et al.

4460 Neurobiology: Memo etal.P

sure to an agonist has been shown for a number of -adrenergicreceptors (8, 11, 29, 30). A common feature is the rapid uncou-pling of the recognition site from the adenylate cyclase (7). Thedesensitization of (adrenergic receptors is followed by an in-ternalization of the recognition, sites occurring at different ratesin different (3-adrenergic receptors from various tissues (9, 11,30).The present experiments show that, in striatal slices, expo-

sure to dopamine for 15-30 min results in an uncoupling of thedopamine recognition site from dopamine-sensitive adenylatecyclase. This phenomenon is associated with a reduced affinityof N-propylnorapomorphine binding to striatal membranes. Itis of interest that the Kd for the N-propylnorapomorphine bind-ing in desensitized slices increased by an extent comparable tothe increase elicited by the addition of GTP to control slices.In contrast, the- Kd for spiroperidol binding to striatal mem-branes was similar in control and in desensitized dopamine re-ceptors tested in presence or absence- of 'GTP. These findingsare in agreement with reports showing that in striatum GTPdecreases the binding affinity of specific recognition sites fordopamine agonists but not for the dopamine antagonists (3, 31).Because GTP uncouples the GTP-binding protein from.the rec-ognition. site and couples it to the enzyme, it can be.surmisedthat incubation ofstriatal slices with dopamine may cause a sim-ilar uncoupling.

These results further support the view that desensitizationofdopamine receptors could involve a modification ofthe GTP-binding protein-dopamine recognition site complex. A func-tional deficit of GTP-binding protein could extend also to ade-nylate cyclase because the activation of adenylate cyclase re-quires the availability of GTP and GTP-binding protein (22, 23).The decreased enzyme activation by NaF and cholera toxin instriatal slices desensitized to dopamine suggests that some stepsin the binding or in the dissociation ofGTP from the GTP-bind-ing. protein, complex (32) may be impaired. Such an inferenceappears to be reasonable because in. -adrenergic receptors theuncoupling (7) and internalization (30) of the recognition sitehave been proven to be associated with decreased response ofthe regulatory and catalytic components.

In striatum, in addition to these regulatory components, achange in calmodulin appears to be associated with the down-regulation of the dopamine receptor. In fact, physiological con-ditions such as dopamine receptor stimulation which result inactivation of striatal adenylate cyclase (33) also. elicit the releaseof calmodulin from striatal membranes into the cytosol (24, 34).The present data indicate that exposure of striatal. slices to do-pamine results not only in adenylate cyclase desensitization anda change in the affinity of agonist recognition sites but also ina change in.the compartmentation of calmodulin.

Because exposure of striatal slices to dopamine elicits an in-crease in cytosolic calmodulin and a decrease in the membrane-bound calmodulin pool, it is logical to infer that the release ofcalmodulin may be a consequence of dopamine receptor stim-ulation. The mechanism that elicits this dopamine-mediatedrelease ofcalmodulin from the membrane is not yet understood.It has been shown that in vitro phosphorylation of subcellularfractions of rat brain homogenates can accelerate the release ofmembrane-bound calmodulin (35). Preliminary results from ourlaboratory indicate that in desensitized striatal slices the incor-poration of 32pO43 into specific proteins is significantly en-hanced. It will be important to characterize further the sub-strate for this phosphorylating mechanism activated by dopaminereceptor agonists because it could be part of a physiological re-

sponse to stimulation ofdopamine receptors linked to adenylatecyclase.

This work was in part supported by a grant from the ScottishRite Schizophrenia Research Program, Northern Masonic Jurisdiction,U.S.A.

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