Agonist-Induced Subsensitivity of Adenylate Cyclase Coupled with A

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

Agonist-induced subsensitivity of adenylate cyclase coupled with a dopamine receptor in slices from rat corpus striatum (D-1 dopamine receptor/down-regulation/modulator proteins) MAURIZIO MEMO, WALTER LOVENBERG, AND INGEBORG HANBAUER Section 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 bound calmodulin content, with a loss in the efficiency of the ,sM dopamine, 10 IpM apomorphine, or 10 IpM SKF 38393 de- adenylate cyclase-coupling system, and with a decrease in the creases dopamine-stimulated adenylate cyclase activity by 50-60%. affinity ofthe dopamine recognition site for the agonist. This loss in dopamine-stimulated enzyme activity appears to be mediated by a persistent occupancy of recognition sites of the D- 1 receptor because: (i) at 10 IpM, SKF 38393, a selective D-1 re- EXPERIMENTAL PROCEDURES ceptor agonist, facilitates desensitization and LY 141865, a selec- Male Sprague-Dawley rats (100-200 g) were kept under stan- tive D-2 receptor agonist, fails to elicit desensitization of dopa- dard conditions with 12-hr light/dark cycle and had free access mine-dependent adenylate cyclase; and (ii) preincubation with to food and water. They were decapitated between 1100 and dopamine in the presence of 1 paM haloperidol but not 1 pmM sul- 1200, the brain was rapidly removed, and the corpus striatum piride curtails the desensitization of dopamine-dependent ade- was dissected out. Striatal slices (230 ,um thick; 35 mg oftissue, nylate cyclase. In dopamine-desensitized striatal slices the K3 for fresh weight) were prepared with a TC-2 Sorvall tissue sec- N-propylnorapomorphine binding is increased but the content of tioner. The slices were preincubated for 30 min (unless oth- membrane-bound calmodulin and the activation of adenylate cy- erwise indicated) at 37°C in 1 ml of Krebs bicarbonate buffer clase by NaF and cholera toxin are decreased significantly. In (pH 7.4) supplemented with 10 mM dextrose and 1.14 mM as- striatal slices incubated with dopamine for prolonged time periods corbicacidunderconstantoxygenation (95% 02/5% CO2). After the coupling of the GTP-binding protein with adenylate cyclase addition ofvarious drugs, as reported in Results, the incubation and dopamine recognition sites may be impaired and the content was continued for an additional 30 min. Thereafter, the slices of membrane-bound calmodulin is decreased. were removed from the incubation medium and washed in In brain tissue the recognition sites for dopamine appear to be Krebs bicarbonate buffer that was free of drugs. The excess coupled to various kinds ofamplifier systems by different mo- medium was drained offon filter paper and homogenates ofthe lecular mechanisms. This variety is the molecular basis for the slices were prepared. When 0.1 mM [3H]dopamine was used diversity in pharmacological and biochemical profiles ofdiffer- as a tracer, it was found that 14 nmol ofdopamine was taken up ent dopamine receptors located in various brain structures and by striatal slices during 30 min of incubation. This amount of in other tissues (for review see ref. 1). For instance, in D-1 re- dopamine present in the adenylate cyclase assay mixture failed ceptors, the coupling of the dopamine recognition site with to increase adenylate cyclase activity. adenylate cyclase (2) involves a GTP binding protein (3) and Specific Ligand Binding to Dopamine Receptor. N- calmodulin (4). The function ofthese modulator proteins in the [3H]Propylnorapomorphine (58.5 Ci/mmol; 1 Ci = 3.7 X 1010 amplification ofthe dopamine signal is not clear. In rat striatum, becquerels; New England Nuclear) and [3H]spiroperidol (39.5 calmodulin has been shown to participate in the regulation of Ci/mmol; New England Nuclear) binding was determined as adenylate cyclase sensitive to dopamine stimulation (5). In ad- described by Creese et al. (12) and Burt et al. (13), respectively. dition, calmodulin and Ca2O were shown to down-regulate the Striatal slices were homogenized at 4°C for 30 sec in 40 vol (wt/ cytosolic cyclic AMP content in rat striatum (4). This mechanism vol) of 50 mM Tris'HCl pH 7.6 buffer in a Polytron homoge- may be operative in the subsensitivity of dopamine receptors nizer (setting 6) and centrifuged at 50,000 X g for 10 min. The elicited by their persistent stimulation. pellet was washed in the same volume of buffer and centrifuged In the case of -adrenergic receptors, persistent occupancy again. The resultant pelletwas resuspended in the same volume ofthe recognition site by an agonist causes desensitization (6-8) of 50 mM Tris-HCl pH 7.1 buffer containing 120 mM NaCl, 5 and decreases the number ofrecognition sites for agonists and mM KC1, 2 mM CaC12, and 1 mM MgCl2. The incubation mix- the catecholamine-induced activation of adenylate cyclase ture (1 ml) contained [3H]spiroperidol (0.2-1.2 nM) or N- (9-11). The present study examined whether persistent occu- [3H]propylnorapomorphine (0.2-1.5 nM) and an aliquot of pation ofdopamine recognition sites desensitizes the dopamine- membrane suspension corresponding to 10 or 2 mg of tissue induced activation of adenylate cyclase in striatal slices. The (freshweight), respectively. Underthese incubation conditions, experiments were planned to determine whether calmodulin high- and low-affinity binding sites for [3H]spiroperidol were and the adenylate cyclase-coupling system change during do- assessed. The mixtures were incubated for 10 min at 37°C and pamine receptor desensitization. The data show that prolonged then rapidly filtered over a Whatman GF/B filter and rinsed occupancy of the dopamine recognition site decreases the do- two times with 5 ml ofcold buffer. Filters were placed in count- pamine-induced activation of adenylate cyclase. The decrease ing vials containing 10 ml of Aquasol (New England Nuclear) in activation is associated with adecrease in membrane- and the radioactivity was determined by liquid scintillation enzyme spectroscopy. Specific [3H]spiroperidol or N-[3H]pro- was The publication costs ofthis article were defrayed in partby page charge pylnorapomorphine binding derived from the difference payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: ELISA, enzyme-linked immunosorbent assay. 4456 Downloaded by guest on September 27, 2021 Neurobiology: Memo et al. Proc. NatL Acad. Sci. USA 79 (1982) 4457 between total binding and the binding observed in presence of 30 min, and centrifuged at 100,000 x g for 30 min. The cal- 0.1 mM dopamine (nonspecific binding). Scatchard analysis (14) modulin content was measured by ELISA in the supernatant of the specific radioligand binding was used to determine the fraction obtained by the first high-speed centrifugation (re- total~number ofbinding sites (BmX) and the apparent dissocia- ferred to as "soluble") and in the supernatant fraction of the tion constant (KId). second high-speed centrifugation (referred to as "membrane- Adenylate Cyclase Activity. Adenylate cyclase activity was bound"). As reported (18), the ELISA was carried out with a measured as described by Clement-Cormier et al. (15) with specific antibody directed toward calmodulin and produced in small modifications. In brief, the washed slices were homoge- rabbits. As second antibody, horseradish peroxidase-labeled nized in 15 vol (wt/vol) of10 mM Tris maleate, pH 7.5/1.2 mM anti-rabbit Ig conjugate was used (Miles) and the degradation EGTA in aglass/Teflon homogenizer. The incubation medium of o-phenylenediamine in the presence of H202 was assessed contained 82.5 mM Tris maleate buffer (pH 7.5), 20 mM spectrophotometrically at 488 nm with an ELISA microreader MgSO4, 2 mM cyclic AMP, 5 mM theophylline, 0.3 mM EGTA (Biomedical Engineering and Instrumentation Branch, Na- (carried over from tissue preparation), 0.01 mM GTP, 20 kg tional Institutes ofHealth, Bethesda, MD). The protein content of pyruvate kinase, 4 mM phosphoenolpyruvate, 5 mM was determined by the method of Lowry et al. (19) with spec- [2,8- H]ATP (28.3 Ci/mmol, New England Nuclear), and an trophotometrically standardized bovine serum albumin as a aliquot of homogenate corresponding to 1 mg of tissue, fresh reference. weight. The mixtures were incubated with and without 0.1 mM dopamine- (unless otherwise indicated) in a final volume of300 RESULTS /.l for 3 min at 30'C. The reaction was stopped by adding 500 Adenylate Cyclase Responsiveness to Dopamine Stimula- p1 ofstopping solution containing 2 mM ATP and 0.7 mM cyclic tion After Persistent Occupancy of Dopamine Recognition AMP followed by heating the samples at 900C for 3 min. When Sites. When striatal slices were incubated with dopamine for measurement ofthe kinetic properties ofadenylate cyclase was longer than 15 min, the responsiveness of adenylate cyclase carried out the Mg/ATP concentration ratio was held constant activity to dopamine stimulation in homogenates prepared from at 4:1. The amount ofcyclic AMP formed was measured as de- these slices was decreased, although the basal activity of this scribed by Salomon et al. (16) by using subsequent chromatog- enzyme was unchanged (Fig. 1A). This inhibition was dose de- raphy on Dowex 5OW X4 (100-200 mesh, hydrogen form, Bio- pendent and was maximal with 0.1mM dopamine. With this Rad) and alumina oxide (alumina, Woelm neutral, activity 1, concentration a significant desensitization of striatal adenylate ICN). The recovery ofcyclic AMP was 75-80%. [3H]ATP was cyclase to dopamine stimulation appeared after 15 min of in- purified on a Dowex 1 X8 column (200-400 mesh, chloride cubation and the maximal inhibition, 66% ofcontrol response, form, 1 x 4 cm) equilibrated with water. The eluate containing was reached after 30 min ofincubation (Fig. 1B). [3H]ATP was desalted on a Sephadex G-10 column (1.9 x 40 A similar loss in the stimulation of adenylate cyclase by do- cm) with water as solvent (17). pamine was also obtained when 10 AuM apomorphine or SKF Cyclic AMP Content. At the end of the incubation, the me- 38393 was added to the incubation medium (Table 1). In con- dium containing the slices was heated for 3 min at 900C. The trast, when slices were incubated with 10 AM LY 141865, a striatal slices were removed from the media, homogenized with highly specific D-2 receptor agonist (20), the responsiveness of a glass/Teflon homogenizer in 1 ml of 4 M formic acid, and adenylate cyclase to dopamine stimulation was unaffected. centrifuged at 3,000 x g for 15 min; the supernatant was col- When slices were preincubated with 1,uM haloperidol before lected and Iyophilized. The residue was dissolved in 1 ml of50 the addition of 10 AuM dopamine, the desensitization process mM sodium acetate buffer (pH 6.2) and the cyclic AMP content was partially prevented. In contrast, preincubation with 1 AuM was measured radioimmunochemically (New England Nuclear) sulpiride, which has high affinity for the D-2 receptor, failed in 100-ul aliquots. to block the desensitization. When slices were incubated with Enzyme-Linked Immunosorbent Assay (ELISA) for Cal- 10 ,uM isoproterenol, the subsequent activation of adenylate modulin. Striatal slices were homogenized (glass/Teflon ho- cyclase by 0.1 mM dopamine was not impaired. Preincubation mogenizer) in 10 vol (wt/vol) of0.32 M sucrose and centrifuged ofstriatal slices with 0.5 mM propranolol failed to prevent the at 1,000 x g for 30 min. The supernatant fraction was removed desensitization ofadenylate cyclase elicitedby 10 AM dopamine and centrifuged at 100,000 x g for 30 min. The resultant pellet (10-5M), indicating that possible occupancy of P-adrenergic was homogenized in half the original volume of 50 mM Tris recognition sites was not involved in the desensitization ofstria- buffer, pH 7.4/1.0% Lubrol WX (Sigma), incubated on ice for tal dopamine receptors. The possibility that prolonged incu-

500 r FG. 1. Desensitization of dopamine-sensitive adenylate cyclase. (A) Dose-response relationship ofdopamine

-4 400 induced desensitization of adenylate cy- 400 clase. Striatal slices were preincubated bo0. for 30 min; then various concentrations 300 F Dopamine (1-500 pM) of dopamine were added to 0 300 Dopamine the media and incubation was continued 0 for 30 min. Dopamine-stimulated (0.1 m 200- Basal mM) adenylate cyclase was measured in 200 Basal homogenates. (B) Time course of.dopamine-induced desensitization of adenylate *u 100 100F cyclase. Afterpreincubation, striatal c.l slices were incubated with 0.1. mM do- 0 pamine for different periods of time. Do- pamine-stimulated adenylate cylase was measured in homogenates. Each point 0 10-6 10-5 10-4 10-3 10 20 30 40 50 60 70 represents the mean ± SEM of four sim- Dopamirne, M Time, min ilar experiments. Downloaded by guest on September 27, 2021 4458 Neurobiology: Memo et al. Proc. Nad Acad. Sci. USA 79 (1982) Table 1. Decrease of dopamine-stimulated adenylate cyclase modulin present in the membrane fraction and increased it in activity after incubation of rat striatal slices with the cytosol fiaction ofhomogenates prepared from these slices various drugs (Table 2). hi addition, there was a time-dependent increase of Dopamine-elicited the soluble calmodulin and cyclic AMP contents in homoge- increase in cyclic AMP nates prepared from striatal slices incubated in presence or ab- Addition to medium pmol/rg of sence of dopamine (Fig. 3). After 30 min of preincubation the Preincubation Incubation* protein/mint % amount of cyclic AMP formed in the slices (Fig. 3 Upper) or released into the incubation medium (data not shown) was at None None 208 ± 24 105 steady state. Five minutes after the addition of 10 ,uM dopamine None Dopamine 108 ± 6* 54 the cyclic AMP content in the slices increased by 3.5-fold, but None SKF 38393 105 ± 8* 53 it returned almost to base line 30 min thereafter. Another ad- None LY 141865 188 ± 12 95 dition to. the incubation medium after 30 min of None Apomorphine 118 ± 8* 59' of dopamine None Isoproterenol 168 ± 11 85. incubation with dopamine failed to enhance the formation of Haloperidol (1 M) Dopamine 158 ± 12§ 80 cyclic AMP significantly, indicating that the decline in the Sulpiride (1 pM) Dopamine 104 ± 8* 52 amount ofcyclic AMP released during dopamine exposure was Propranolol'(0.5 mM) Dopamine 112 ± 9* 56 not due to the decreased availability of the agonist at the receptor sites but was due to the reduced functional capacity of * All drugs listed were at 10 pM. adenylate cyclase. Fig. 3 also shows that the decrease was not t Numbers are the mean + SEM of three separate experiments in due to a nonspecific decay of the preparation because striatal triplicate. Basal enzyme activity is 198 20 pmol of cyclic AMP per slices incubated in absence ofdopamine for a period of60 min mg of protein per min. *P < 0.01 for difference from control. still responded maximally to 10 AM dopamine. The calmodulin *P < 0.01.for difference from dopamine-incubated group. content in the supernatant fraction of homogenates prepared from striatal slices was significantly increased after incubation with 10 ,uM dopamine for 30 or 60 min (Fig. 3 Lower). The bation.ofstriatal slices with 0.1 mM dopamine could modify the cytosolic calmodulin content of slices incubated without dopa- catalytic activity of adenylate cyclase was ruled out by experi- mine did not change significantly from 30 min (0;27 ± 0.02 pug/ ments showing that 20 mM elicited half-maximal acti- Mg2e mg of protein) to 90 min (0.22 ± 0.03 pg/mg of protein) of vation ofthe enzyme from normal and desensitized striatal slices incubation. (results not shown). Stimulation ofAdenylate Cyclase by Cholera Toxin and NaF The'responsiveness ofadenylate cyclase in homogenates from in Normal and. Dopamine-Desensitized Striatal Slices. It has control and dopamine-treated slices to various concentrations been suggested that the GTP-binding protein mediates the reg- ofdopamine is shown in Fig. 2. Incubation ofslices with 0.1 mM and chol- dopamine for 30 min slightly decreased the of adenylate ulation ofadenylate cyclase by neurohormones, NaF, V. era' toxin (21-23). These experiments were designed to deter- cyclase (- 15%; statistically insignificant) and caused a shift to mine whether a persistent occupancy of striatal dopamine the right in the dose-response curve for adenylate cyclase ac- recognition sites by the agonist alters the coupling function of tivation by dopamine. The ED50for the stimulation ofadenylate GTP-binding protein for adenylate cyclase. Incubation ofslices cyclase by dopamine increased by about 5-fold from 17 ,M in with cholera toxin, (100 pug/ml) for 40 min increased the cyclic control slices to 88 AM in slices desensitized by dopamine in- AMP accumulation 2-fold- (Table 3). In contrast, in slices incubation, based on comparable values. This loss of re- Vma. cubated with 0.1 mM dopamine for 30 min, the addition ofchol- was to a in the Ka for the enzyme for sponsiveness due change era toxin a 33% increase ofthe AMP content, 1.2 4.1 elicited-only cyclic dopamine (control, AM; desensitized, AM).. was A similar reduction in the in the Calmodulin Content of Membranes Pre- which not statistically significant. Changes degree of adenylate cyclase stimulation (42%) was observed pared from Striatal Slices Incubated with Dopamine Receptor when homogenates of. dopamine-desensitized striatal slices Agonists. Incubation of striatal slices with 10 pM dopamine or were incubated with 10 mM NaF (Table 4). 10 pM apomorphine for 30 min decreased the amount of cal- Dopamine Recognition Sites in Normal and Dopamine-De- sensitized Striatal Slices. Incubation of striatal slices with 0.1 mM dopamine for 30 min down-regulated the affinity of dopamine recognition sites. This effect resembles that seen on the 4.2 P 400 Table- 2. Changes of soluble and membrane-bound calmodulin Normal content after incubation of striatal slices with various dopamine receptor agonists , 300 Desensitized Calmodulin, Wdg/mg protein Agonist (10 /AM) Soluble, Membrane-bound None 0.20 ± 0.0 1.4 ±+0.1 Ax 2001I Dopamine 0.30 ± 0.02* 1.0 ± 0.1* 10-6 1o-5 1m-41n-3 Apomorphine. 0.34 ± 0.02* 1.1 ± 0.1* Dopamine, M Striatal slices were. preincubated for 30 min. The incubation was continuedfor 30 min-inthe absence orpresence ofan agonist. The slices FIG. 2. Concentration-effect relationship. for dopamine-stimu- were homogenized in 0.32 M' sucrose and centrifuged at 1,000 x g for lated adenylate cyclase activity in homogenates from normal (e) and 10 min. The supernatant was removed and centrifuged at 100,000 x desensitized (o) striatal slices. Slices were preincubated for 30 min; the g for 30 min. The calmodulin content of the supernatant and mem- incubation was continued for 30 min in presence or absence of 0.1 mM. brane fraction was. assayed by ELISA.(18). The values are the mean dopamine. Then, adenylate cyclase-activity in homogenates was de- (± SEM) of four experiments. termined in presence of the indicated concentrations of dopamine. * P < 0.01 for difference from value in absence of agonist. Downloaded by guest on September 27, 2021 Neurobiology: Memo et al. Proc. Natl Acad. Sci. USA 79 (1982) 4459 Table 4. Activation of adenylate cyclase by NAF after incubation of rat striatal slices with dopamine 8 Cyclic AMP, pmol/mg protein/min Addition Normal Desensitized None 216 ± 9 198 ± 18 NaF 650 ± 44* 430 ± 32 Slices were incubated for 30 min in Krebs bicarbonate buffer at pH 7.4(95% 02/5% C02). The incubation was continued for 30 min in ab- 0.6 sence (normal) or in presence (desensitized) of 0.1 mMdopamine. Stim- ulation of adenylate cyclase by 10 mM NaF was measured in the ho- 0.5 mogenate prepared from striatal slices. Values are the mean ± SEM of three separate experiments in triplicate. *P < 0.01 for difference between normal and desensitized.

binding to crude synaptic membranes was similar in control and dopamine-desensitized slices. However, measurements of the IC50 of [3H]spiroperidol binding with dopamine as a displacer indicated that in dopamine-desensitized slices the affinity for dopamine (Kd = 3.8 ± 0.21 uM) was only about 1/7th that in control slices (0.56 ± 0.12 ,uM). 10 20 30 40 50 60 70 80 90

Time, min DISCUSSION In the present report we extend previous reports on the reg- FIG. 3. Time-dependent changes in cyclic AMP and calmedulin in ulation ofthe function ofstriatal dopamine receptor (24, 25) and striatal slices elicited by incubation with 10 ,uM dopamine. Striatal slices were preincubated for 30 min (e) or 60 min (a). Dopamine was provide evidence that prolonged incubation of slices with 0.1 added to the incubation medium, as indicated by arrows. The cyclic mM dopamine desensitizes dopamine receptors, causing a AMP content was measured in the supersnatant fraction of homoge- change in those functions that couple dopamine recognition nates prepared from striatal slices (each point represents four similar sites with the catalytic function of adenylate cyclase. In slices experiments). Calmodulin content was assayed in the supernatant ofcorpus striatum incubated for at least 15 min with dopamine fraction of homogenates prepared from slices after 30, 60, and -90 min varying from 1 to 100 ,uM the D-1 receptors are of incubation in Krebs bicarbonate buffer (hatched bars) and 30 or 60 desensitized min after addition of dopamine to the incubation medium (open bars). by a direct action ofdopamine on the D-1 receptor. 'This infer- Each bar represents the mean SEM of six experiments. *, P < 0.05 ence is supported by the finding thatincubation with LY 141865 for diffierence between open and hatched bars. and isoproterenol for 30 min failed to decrease the stimulation of enzyme activity by dopamine and that pretreatment with binding of N-[3H]propylnorapomorphmne to crude synaptic haloperidol but not with sulpiride curtailed the desensitization membrane preparations after the addition of GTP. Incubation elicited by incubation with dopamine. Sulpiride was shown to of striatal slices with 0.1 mM dopamine for 30 min decreased be potent in inhibiting D-2 receptors but extremely weak in the binding affinity forN-[3H]propylnorapomorphine but failed acting on D-1 receptors (26, 27), whereas haloperidol blocks to change the number of specific binding sites present in mem- both types ofdopamine receptors (1). Dopamine receptor sites branes prepared from homogenates of striatal slices (Table 5). that are located mainly on neurons postsynaptic to dopaminer- A similar decrease in binding affinity for the labeled agonist was gic axons (28) remain functional in preparations ofstriatal slices. obtained in membranes prepared from normal slices when 0.1 A similar receptor-specific desensitization by prolonged expo- mM GTP was present in the assay mixture. Interestingly, incubation of dopamine-desensitized slices with 0.1 mM GTP Table 5. Specific binding of [3H]spiroperidol and N-[3H]- failed to increase the 1Kd 'for N-[3H]propylnorapomorphine fuar- propylnorapomorphine to membranes prepared from ther. The number of binding sites and the 1Kd for [3H]spiroperidol rat striatal slices incubated with dopamine N-[3H]Propyl- Table 3. Increase in cyclic AMP formation elicited by cholera [3H]Spiroperidol norapomorphine toxin after incubation of rat striatal slices with dopamine GTP Kd Kd Cyclic AMP, pmol/mg protein B Normal Addition Normal Desensitized No 1.1 ± 0.1 350 ± 38 0.91 ± 0.1 410 ± 53 None 1.8 t 0.2 2.1 t 0.2 Yes 1.1 ± 0.1 371 ± 36 1.4 ± 0.1* 405 ± 42 Cholera toxin 3.8 0.2* 2.8 t 0.2 Desensitized No 1.1 ± 0.1 368 ± 37 1.3 ± 0.1t 420 ± 42 Slices were preincubated for 30 min in Krebs bicarbonate buffer at Yes 11 ± 0.1 355 ± 35 1.4 ± 0.1 405 ± 30 pH 7.4 (95% 02/5% C02). The incubation was continued for 30 min in absence (normal) or in presence (desensitized) of 0.1 mM dopamine. To Kd is expressed as nM and B,,, as fmol/mg protein. After prein- one set of culture dishes, cholera toxin (100 Ag/ml) was added and the cubation of striatal slices in Krebs bicarbonate buffer at pH 7.4 for 30 incubation was continued for 40 min. Thereafter, slices were heated min the incubation was continued for 30 min in presence or in absence at 90°C for 3 min and homogenized in 4 M HCOOH and the cyclic AMP of 0.1 mM dopamine. The binding was measured in the presence or content of the supernatant fraction was measured by radioimmu- absence of 0.1 mM GTP. Results are mean ± SEM of six experiments noassay. Values are the mean SEM of three experiments in in each group. triplicate. *P < 0.05 for difference from value in absence of GTP. * P < 0.001 for difference from control. tP < 0.05 for difference from corresponding normal value. Downloaded by guest on September 27, 2021 4460 Neurobiology: Memo et al.PProc. Nad Acad. Sci. USA 79 (1982)' sure to an agonist has been shown for a number of -adrenergic sponse to stimulation ofdopamine receptors linked to adenylate receptors (8, 11, 29, 30). A common feature is the rapid uncou- cyclase. pling ofthe recognition site from the adenylate cyclase (7). The desensitization of (adrenergic receptors is followed by an in- This work was in part supported by a grant from the Scottish ternalization of the recognition, sites occurring at different rates Rite Schizophrenia Research Program, Northern Masonic Jurisdiction, in different (3-adrenergic receptors from various tissues (9, 11, U.S.A. 30). 1. Seeman; P. (1981) Pharmacol Rev. 32, 229-313. The present experiments show that, in striatal slices, expo- 2. Kebabian, J. W. & Calne, D. B. (1979) Nature (London) 277, sure to dopamine for 15-30 min results in an uncoupling ofthe 93-96. dopamine recognition site from dopamine-sensitive adenylate 3. Creese, I., Prosser, T. & Snyder, S. H. (1978) Life Sci. 23, cyclase. This phenomenon is associated with a reduced affinity 495-500. 4. Hanbauer, I. &8 Costa, E. (1980) in Calcium and Cell Function, of N-propylnorapomorphine binding to striatal membranes. It ed. Cheung, W. Y. (Academic, New York), Vol. 1,. pp. 253-271. is ofinterest that the Kd for the N-propylnorapomorphine bind- 5. Gnegy, M. E., Uzunov, P. & Costa,. E. (1977)J. Pharmacol. Exp. ing in desensitized slices increased by an extent comparable to Ther. 202, 558-564. the increase elicited by the addition of GTP to control slices. 6. Shear, M., Insel, P. A., Melmon, K. L. & Coffimo, P. (1976) J. In contrast, the- Kd for spiroperidol binding to striatal mem- Biol Chem. 251, 7572-7576. branes was similar in controland in desensitized re- 7. Su, Y. F., Harden, T. K. & Perkins, J. P. (1979) J. Biol Chem. dopamine 254, 38-41. ceptors tested in presence or absence- of'GTP. These findings 8. Homburger, V., Lucus, M., Cantau, B., Barabe, J., Penit, J. & are in agreement with reports showing that in striatum GTP Bockaert, J. (1980)J. Biol. Chem. 255, 10436-10444. decreases the binding affinity of specific recognition sites for 9. Perkins, J. P., Johnson, G. L. & Harden, T. K. (1978) Adv. Cyclic dopamine agonists but not for the dopamine antagonists (3, 31). Nucleotide Res. 9, 19-32. Because GTP uncouples the GTP-binding protein from.the rec- 10. Terasaki, W. L.; Brooker, G., deVellis, J., Hsu, C. Y. & Moylan, ognition. site and couples it to the enzyme, it can be.surmised R. D. (1978) Adv. Cyclic Nucleotide Res. 9, 33-52. 11. Su, Y. F., Harden, T K. & Perkins, J. P. (1980) J. Biol Chem. that incubation ofstriatal slices with dopamine may cause a sim- 255, 7410-7419. ilar uncoupling. 12. Creese, I., Padgett, L., Fazzini, E. & Lopez,;F. (1979) Eur. J. These results further support the view that desensitization Pharmacol 59, 411-412. ofdopamine receptors could involve a modification ofthe GTP- 13. Burt, D., Creese, I. & Snyder, S. H. (1977) Science 196, binding protein-dopamine recognition site complex. A func- 326-328. extend to ade- 14. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672. tional deficit of GTP-binding protein could also 15. Clement-Cormier, Y. C., Kebabian, J. W., Petzold, G. L. & nylate cyclase because the activation of adenylate cyclase re- Greengard, P. (1974) Proc. NatL Acad. Sci. USA 71, 1113-1117. quires the availability ofGTP and GTP-binding protein (22,23). 16. Salomon, Y., Londos, C. & Rodbell,. M. (1974) AnaL Biochem. The decreased enzyme activation by NaF and cholera toxin in 58, 541-548. striatal slices desensitized to dopamine suggests that some steps 17. Butcher, F. R. (1971) Horm.. Metab. Res. 3, 336-340. in the binding or in the dissociation of GTP from the GTP-bind- 18. Hanbauer, I., Prdhan, S. & Yang, H.-Y. T. (1980) Ann. N.Y. Such an inference Acad. Sci. 356, 292-303. ing. protein, complex (32) may be impaired. 19. Lowry, 0. H., Rosenbrough, M. J., Farr, A. C. & Randall, R. J. appears to be reasonable because in. -adrenergic receptors the (1951)J. Biol Chem. 193, 265-275. uncoupling (7) and internalization (30) of the recognition site 20. Tsuruta, K., Frey, E. A., Grewe, C. W., Cote, T. E., Eskay, R. have been proven to be associated with decreased response of L. & Kebabian, J. W. (1981) Nature (London) 292, 463-465. the regulatory and catalytic components. 21. Pfeuffer, T. (1977)J. Biol Chem. 252,7224-7234. In striatum, in addition to these regulatory components, a 22. Gill, D. M. & Meren, R. (1978) Proc. NatL Acad. Sci. USA 75, to be associated with the down- 3050-3054. change in calmodulin appears 23. Spiegel; A., Downs, R. & Aurbach, G. (1979)J. Cyclic Nucleotide regulation ofthe dopamine receptor. In fact, physiological con- Res. 5, 3-17. ditions such as dopamine receptor stimulation which result in 24. Hanbauer, I., Gimble, J. & Lovenberg, W. (1979) Neurophar- activation ofstriatal adenylate cyclase (33) also.elicit the release macology 18, 851-857. ofcalmodulin from striatal membranes into the cytosol (24, 34). 25. Memo, M., Pradhan, S. & Hanbauer, I. (1981) Neuropharma- The present data indicate that exposure of striatal. slices to do- cology 20, 1145-1150. desensitization and 26. Trabucchi, M., Longoni, R., Fresia, P. & Spano, P. F. (1975) Life pamine results not only in adenylate cyclase Sci. 17, 1551-1556. a change in the affinity of agonist recognition sites but also in 27. Scatton, B., Bischoff, S., Dedek, J. & Korf, J. (1977) Eur.J. Phar- a change in.the compartmentation of calmodulin. macol, 44, 287-292. Because exposure of striatal slices to dopamine elicits an in- 28. Minneman, K. P., Quick, M. & Emson, P. C. 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