Receptors In Rat Striatum Research Articles

The kinetics of [ 3H]SCH 23390 dissociation from rat striatal dopamine D 1 receptors: effect of dopamine

The present study investigated possible allosteric interactions between dopamine and [ 3H]SCH 23390 (( R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1 H-3-benzazepin-7-ol)-labelled dopamine D 1 receptors in rat striatum. As previously described, dopamine prevented [ 3H]SCH 23390 binding in a mixed competitive/non-competitive manner, causing both a loss of ligand affinity and a decrease in B max. The effect of dopamine was largely reversed following pretreatment of the membranes with 100 μM Gpp(NH)p (5′-guanylylimidodiphosphate) and was significantly enhanced by omission of Na + from the incubation buffer. In dissociation kinetic studies, two methods of initiating ligand dissociation were used: dilution into 100-fold volume excess of buffer or addition of a molar excess of drug. Both methods yielded similar rates of [ 3H]SCH 23390 dissociation. Inclusion of dopamine in the volume excess of buffer did not alter the k −1 for [ 3H]SCH 23390 dissociation. However, when 100 μM dopamine was used instead of 1 μM piflutixol to initiate dissociation, a significant slowing of the rate of dissociation of [ 3H]SCH 23390 occurred. This effect of dopamine on k −1 was Na +-dependent since in the absence of Na + the dopamine-induced rate of dissociation was only slightly slower than control values. Under neither condition did dopamine accelerate the rate of ligand dissociation, indicating that dopamine does not interact allosterically with [ 3H]SCH 23390 binding sites. These data, therefore, preclude an allosteric mechanism to explain the dopamine-induced decrease in dopamine D 1 receptor density and provide direct evidence that dopamine masks ligand binding by binding to a high affinity site which can be modulated by Gpp(NH)p and Na +.

Binding and functional properties of hexocyclium and sila‐hexocyclium derivatives to muscarinic receptor subtypes

1. We have compared the binding properties of several hexocyclium and sila-hexocyclium derivatives to muscarinic M1 receptors (in rat brain, human neuroblastoma (NB-OK 1) cells and calf superior cervical ganglia), rat heart M2 receptors, rat pancreas M3 receptors and M4 receptors in rat striatum, with their functional antimuscarinic properties in rabbit vas deferens (M1/M4-like), guinea-pig atria (M2), and guinea-pig ileum (M3) muscarinic receptors. 2. Sila-substitution (C/Si exchange) of hexocyclium (-->sila-hexocyclium) and demethyl-hexocyclium (-->demethyl-sila-hexocyclium) did not significantly affect their affinities for muscarinic receptors. By contrast, sila-substitution of o-methoxy-hexocyclium increased its affinity 2 to 3 fold for all the muscarinic receptor subtypes studied. 3. The p-fluoro- and p-chloro-derivatives of sila-hexocyclium had lower affinities than the parent compound at the four receptor subtypes, in binding and pharmacological studies. 4. In binding studies, o-methoxy-sila-hexocyclium (M1 = M4 > or = M3 > or = M2) had a much lower affinity than sila-hexocyclium for the four receptor subtypes, and discriminated the receptor subtypes more poorly than sila-hexocyclium (M1 = M3 > M4 > M2). This is in marked contrast with the very clear selectivity of o-methoxy-sila-hexocyclium for the prejunctional M1/M4-like heteroreceptors in rabbit vas deferens. 5. The tertiary amines demethyl-hexocyclium, demethyl-sila-hexocyclium and demethyl-o-methoxy-sila-hexocyclium had 10 to 30 fold lower affinities than the corresponding quaternary ammonium derivatives.

The subcellular distribution of [3H]-CGS 21680 binding sites in the rat striatum: Copurification with cholinergic nerve terminals

The subcellular distribution of the adenosine A2a receptor in rat striatum has been investigated using specific binding of the A2a-selective ligand [3H]-CGS 21680. After subcellular fractionation, the distribution of [3H]-CGS 21680 binding was similar to that of the cholinergic nerve terminal marker acetylcholinesterase rather than the more general membrane marker 5'-nucleotidase, with 42% of binding associated with the synaptosomal sub-fraction and 19% with a light membrane fraction. Binding of [3H]-CGS 21680 was also found to co-purify with the cholinergic nerve terminal marker choline acetyltransferase during immunoaffinity purification of striatal cholinergic nerve terminals. These results demonstrate that some adenosine A2a receptors are present on cholinergic nerve terminals in rat striatum.

Binding characteristics of remoxipride and its metabolites to dopamine D 2 and D 3 receptors

The substituted benzamide, remoxipride, is a new atypical antipsychotic agent with good clinical efficacy and low extrapyramidal side-effect potential. In the present study, the in vitro receptor binding properties of remoxipride and several of its metabolites to rat striatal dopamine D 2 and cloned human dopamine D 2A and D 3 receptors were investigated. Remoxipride bound to [ 3H]raclopride-labelled dopamine D 2 receptors in rat striatum with an affinity (K i) of 113 nM. The significantly lower affinities of remoxipride reported when [ 3H]spiperone was used as a radioligand are suggested to be due to methodological problems associated with the use of very high-affinity radioligands. Some of the phenolic metabolites of remoxipride found mainly in rat exhibited considerably higher affinities to dopamine D 2 and D 3 receptors than remoxipride itself. The pyrrolidone metabolites found mainly in the human had very low dopamine D 2 and D 3 affinities. The present in vitro results suggest that the behavioural effects of remoxipride in rats may reflect the effect of remoxipride and some of its high-affinity metabolites.

O-321 - Alteration of dopamine D, receptor in rat striatum following exposure to hypoxia.

O-321 - Alteration of dopamine D, receptor in rat striatum following exposure to hypoxia.

Effects of mono- and divalent ions on the binding of the adenosine analogue CGS 21680 to adenosine A 2 receptors in rat striatum

The effect of monovalent and divalent cations on equilibrium binding of the adenosine A 2-selective agonist ligand CGS 21680 (2-[ p-(2-carbonylethyl)phenylethylamino]-5′- N-ethylcarboxamidoadenosine) to membranes prepared from rat striatum was examined. Competition experiments with cyclohexyladenosine, 2-chloroadenosine, N-ethylcarboxamidoadenosine and CGS 21680 suggest that at 2 nM [ 3H]CGS 21680 binds to a single site with the pharmacology of an A 2a receptor. Magnesium and calcium ions caused a concentration-dependent increase in binding that reached about 10-fold at 100 mM. Manganese ions had a biphasic effect on binding with a maximal increase at 5 mM. Lithium, sodium and potassium ions all caused a concentration-dependent decrease of binding. Sodium was most potent, potassium least. At 200 mM ion concentration, the inhibition of binding was 88% by sodium, 47% by lithium and 29% by potassium ions. The effect of sodium chloride was the same as that of sodium acetate. The effect of sodium ions was essentially similar to that of Gpp(NH)p. However, sodium ions produced a larger effect than even maximally effective concentrations of Gpp(NH)p. The maximal inhibition by Gpp(NH)p was about 55% at 2 nM radioligand concentration irrespective of the magnesium concentration. The maximal effect of sodium ions was reduced by increasing concentrations of magnesium ions. Increasing magnesium ion concentration from 1 to 100 mM increased the half-maximally effective concentration of Gpp(NH)p almost 10-fold and that of sodium ions less than 2-fold. Furthermore, sodium ions and Gpp(NH)p had additive effects. The binding of an agonist to striatal A 2a receptors shows an unusually large dependence on both divalent and monovalent cations that can only partly be explained by a change in the coupling to G s proteins.

Molecular cloning of the rat A 2 adenosine receptor: selective co-expression with D 2 dopamine receptors in rat striatum

A cDNA fragment homologous to other G protein-coupled receptors was isolated from rat brain using the PCR method and demonstrated to be abundantly expressed in striatum. Using this fragment as a probe, a 2.1 kb full-length cDNA was isolated from a rat striatal cDNA library. This cDNA encodes a protein of 410 amino acids and is highly homologous to previously isolated adenosine receptor cDNAs. Expression of this cDNA in COS cells revealed high affinity ( K d = 38.6 nM) and saturable binding of the A 2 adenosine receptor-selective ligand [ 3H]CGS 21680. Agonist displacement profile of [ 3H]CGS 21680 binding was consistent with an adenosine receptor of the A 2 subtype (NECA > ( R)- PIA > CPA > ( S)- PIA). In situ hybridization demonstrated that rat A 2 adenosine receptor mRNA was co-expressed in the same striatal neurons as D 2 dopamine receptor mRNA, and never co-expressed with striatal D 1 dopamine receptor mRNA. Several lines of evidence have previously suggested that dopamine-induced changes in motor behavior can be modulated by adenosine analogs acting at the A 2 subtype of adenosine receptor in the forebrain. The co-expression of D 2 dopamine and A 2 adenosine receptors in a subset of striatal cells provides an anatomical basis for dopaminergic-adenosinergic interactions on motor behavior.

Regulation of excitatory amino acid release by [formula omitted] receptors in rat striatum: in vivo microdialysis studies

The microdialysis technique was utilized to study the effects of N- methyl- d-aspartate (NMDA) receptor ligands on the in vivo release of endogenous glutamate (Glu) and aspartate (Asp) from the rat striatum. Addition of NMDA (250 and 500 μM) to the dialysis perfusion solution resulted in a striking dose-dependent increase in extracellular concentrations of Glu and Asp in the striatum. The NMDA-induced effects were reduced in a dose-related way by prior perfusion with 75 μM dizocilpine (MK-801), a non-competitive NMDA receptor antagonist. MK-801, at 75 μM, produced no changes on basal levels of Glu and Asp. However, 100 μM MK-801 did increase Glu and Asp extracellular concentrations. Local infusion with 500 μM d-serine, an agonist at the glycine site associated to the NMDA receptor, significantly increased basal level of Glu, but not Asp. Such d-serine-induced effects were reduced by 7-Cl-kynurenic acid (200 μM), a selective blocker of the glycine site present in the NMDA receptor. It is proposed that activation of NMDA receptors by endogenous Glu and Asp enhances the subsequent release of these excitatory amino acids in the striatum. Part of these NMDA receptors might be located presynaptically on cortico-striatal nerve endings. In addition, postsynaptic NMDA receptors present in the striatum may also indirectly modulate the release of Glu and Asp, through trans-synaptic mechanism.

Magnesium‐Dependent Enhancement of Endogenous Agonist Binding to A1 Adenosine Receptors: A Complicating Factor in Quantitative Autoradiography

Quantitative autoradiography was used to investigate the effects of Mg2+ on agonist and antagonist binding to A1 receptors in rat striatum. A1 receptors were labelled with the selective agonist N6-[3H]cyclohexyladenosine ([3H]CHA) or the selective antagonist 1,3-[3H]dipropyl-8-cyclopentylxanthine ([3H]DPCPX). Mg2+ had no significant effect on equilibrium binding constants for [3H]CHA [control: KD (95% confidence interval) of 0.34 (0.15-0.80) nM and Bmax of 267 +/- 8 fmol/mg of gray matter; with 10 mM Mg2+: KD of 0.8 (0.13-4.9) nM and Bmax of 313 +/- 8.9 fmol/mg of gray matter] or [3H]DPCPX [control: KD of 0.54 (0.30-0.99) nM and Bmax of 256 +/- 2.3 fmol/mg of gray matter; with 10 mM Mg2+: KD of 1.54 (0.2-11.0) nM and Bmax of 269 +/- 35.7 fmol/mg of gray matter]. In contrast, Mg2+ slowed the apparent association rate for both ligands; this was observed as a shift from a one-component to a two-component model for [3H]DPCPX. Mg2+ also affected the dissociation rates of both ligands; for [3H]CHA, dissociation in the presence of Mg2+ was not detected. Mg2+ produced a concentration-dependent inhibition of [3H]CHA binding only prior to equilibrium. HPLC was performed on untreated sections, sections preincubated with adenosine deaminase (ADA), and sections preincubated with ADA and incubated with ADA in the absence or presence of Mg2+. Adenosine was found in measurable quantities under all conditions, and the concentration was not influenced by Mg2+ or by the inclusion of GTP in the preincubation medium. From these data, we conclude the following: (a) adenosine is present and may be produced continuously in brain sections; (b) ADA is not capable of completely eliminating the produced adenosine; (c) Mg2+ apparently does not influence adenosine production or elimination; (d) A1 receptor-guanine nucleotide binding protein coupling is maximal in this preparation; and (e) Mg2+ decreases the dissociation rate of bound endogenous adenosine from A1 receptors, thus limiting the access of [3H]CHA and [3H]DPCPX to the receptors. Thus, enhancement of endogenous adenosine binding to A1 receptors by Mg2+ is a complicating factor in receptor autoradiography and may be so in other preparations as well.

P-018 - The effect of in vivo hypoxia on dopamine receptors in rat striatum

P-018 - The effect of in vivo hypoxia on dopamine receptors in rat striatum

3H]SCH 39166, A New D1‐Selective Radioligand: In Vitro and In Vivo Binding Analyses

SCH 39166 [(-)-trans-6,7,7a,8,9, 13b-hexahydro-3-chloro-2-hydroxy-N-methyl-5H-benzo-[d]naphtho[2, 1b]azepine] has recently been described as a selective D1 antagonist and has entered clinical trials for the treatment of schizophrenia. The tritiated analogue of this compound, [3H]SCH 39166, has now been synthesized and characterized for its in vitro and in vivo binding profiles. [3H]SCH 39166 binds to D1 receptors in a saturable, high-affinity fashion, with a KD of 0.79 nM. In competition studies, D1-selective antagonists like SCH 23390 displaced the binding of [3H]SCH 39166 with nanomolar affinities, whereas antagonists of other receptors exhibited poor affinity. In vivo, [3H]SCH 39166 bound to receptors in rat striatum in a fashion suggestive of D1 selectivity. Further, when the time course for the binding of [3H]SCH 39166 was compared with the behavioral time course of the unlabeled compound, the two durations of action were virtually indistinguishable. Similar studies were performed for SCH 23390 and its tritiated analogue, but the in vivo binding of this radioligand exhibited a duration of action far greater than the behavioral activity of the unlabeled drug. In concert, these data demonstrate that [3H]SCH 39166 selectively labels D1 receptors in vitro and in vivo, and that this drug is superior for in vivo imaging of the D1 receptor.

Decrease in mRNA levels but not in the density of D 2 dopamine receptors in rat striatum after transient forebrain ischemia

D 2 dopamine receptor mRNA was analyzed by in situ hybridization histochemistry in rat striatum 7 days after transient forebrain ischemia. A patchy disappearance of the D 2 receptor mRNA was observed in the dorsolateral striatum. In the same area, a disappearance of D 1 binding sites occurred in the absence of significant changes in D 2 receptor density. These results suggest that, although D 2 receptors seem to be apparently unaffected after forebrain ischemia, a long-lasting impairment of their neosynthesis may be present in striatal D 2 dopaminoceptive neurons.

Characterization of anti-peptide antibodies for the localization of D2 dopamine receptors in rat striatum.

Seven different peptides of 14-23 residues in length based on the predicted amino acid sequence of the cloned rat D2 receptor cDNA were used as immunogens to develop antibodies in rabbits. Two of these peptides were derived from the amino terminus and four were from the third cytoplasmic loop, including one to the splice variant insertion sequence and one to the carboxyl terminus of the receptor protein. These peptides were conjugated to bovine thyroglobulin prior to rabbit immunization. Antibody production was monitored by a solid-phase ELISA. With the exception of the carboxyl-terminal peptide, all of the peptide immunogens produced antiserum of high titer ranging from 1:10(4) to 1:10(6) on ELISA. Specificity of the reaction was demonstrated by the absence of a response in the preimmune serum and by the absence of cross-reactivity between the various antisera and the nonimmunization peptides. Moreover, preincubation of the antiserum with the immunization peptide, but not other peptides, blocked the subsequent ELISA reactions. Some of the antisera were additionally characterized by immunodot assays using solubilized rat striatal membranes blotted onto nitrocellulose. Positive reactions with antiserum dilutions of 1:500 were observed that were dependent on the presence and concentration of membrane protein and were not observed using preimmune serum. Additionally, immunofluorescent staining by the D2 receptor antiserum was observed on cells that had been transfected with the D2 receptor cDNA but not on untransfected cells. Immunoprecipitation of the photoaffinity-labelled and solubilized D2 receptor also suggested that the antisera were able to directly recognize the native receptor protein. Immunohistochemical localization of the D2 receptor in slices of fresh frozen and perfusion-fixed rat brain was performed using these antisera. Within the striatum, about 50% of the medium-sized neurons were labeled as well as large, putatively cholinergic interneurons.

Lack of a dopamine autoreceptor selective profile of B-HT 920 in functional in vitro model systems of D 2 receptors in rat striatum

Based on the results of in vivo studies, the thiazoloazepine derivative B-HT 920 has been proposed to be a selective agonist of dopamine autoreceptors. In the present study, we investigated the effects of B-HT 920 in tow functional in vitro model systems of D 2 receptors and compared these effects with the effects of the classical D 2 agonist LY 171555. B-HT 920 and LY 171555 concentration dependently inhibited the electrically evoked release of radiolabeled dopamine and acetylcholine and the forskolin-induced stimulation of adenylate cyclase activity in rat striatal tissue slices with comparable efficacies. In striatal tissue slices prepared after 6-hydroxydopamine-induced destruction of dopaminergic terminals, both drugs were still able to inhibit forskolin-stimulated adenylate cyclase activity with a efficacy similar to that in tissue obtained from unlesioned rats. It is concluded that, in vitro, B-HT 920 is an agonist at both presynaptic and ‘normosensitive’ postsynaptic D 2 receptors showing relatively high intrinsic activity.

Autoradiographic evidence for G-protein coupled A2-receptors in rat neostriatum using [3H]-CGS 21680 as a ligand

Recently [3H]-CGS 21680 (2-[p-(2-carbonyl-ethyl)-phenylethylamino]- 5'-N-ethylcarboxamidoadenosine) has been identified as a selective adenosine A2-receptor agonist. In this study the binding of [3H]-CGS 21680 to 10 microns sections of rat neostriatum was investigated with quantitative autoradiography. Specific, saturable binding was detectable, and Scatchard analysis of saturation experiments gave estimates for KD and Bmax of 1.7 nM and 322 fmol/mg protein, respectively. The rank order of potency for inhibition of [3H]-CGS 21680 binding was 5'-N-ethylcarboxamidoadenosine (1.9 nM) greater than 2-chloroadenosine (18 nM) greater than R-N6-phenylisopropyladenosine (59 nM) greater than S-N6-phenylisopropyladenosine (460 nM) greater than 1,3-dipropyl-8-cyclopentylxanthine (700 nM). The binding of [3H]-CGS 21680 was sensitive to GTP, since 1 microM GTP reduced binding to 4.7% of control. These data support the identity of CGS 21680 as an agonist at high affinity adenosine A2-receptors and indicate these receptors in rat striatum are coupled to guanine nucleotide binding proteins.

Modulation of acetylcholine release by D1, D2 dopamine receptors in rat striatum under freely moving conditions

Using in vivo brain dialysis under freely moving conditions, we have studied the effects of dopamine (DA) agonists and antagonists on acetylcholine (ACh) and DA release in rat striatum. The striatal infusion of the D1 DA receptor specific agonist, SKF38393, increased striatal ACh release in a dose-dependent manner (10−6 to 10−4 M), and 3 × 10−5 M SKF38393 elicited a 60% augmentation in the level of ACh release. The level of ACh was increased with perfusion of 10−4 M SCH23390, a D1 specific antagonist, but decreased with 10−3 M SCH23390. The D2 specific agonist, LY171555, and the antagonist, sulpiride, slightly altered the level of ACh in the striatum. On the other hand the level of DA dramatically increased in a dose-dependent manner with SKF38393 or SCH23390 and decreased with LY171555. LY171555 inhibited the effect of 10−4 M SKF38393 on ACh release, and enhanced the effect of SKF38393 on DA release. These results suggest that the D1 DA receptor mainly mediates ACh release and the D2 DA receptor modifies the effects of the D1 receptor.

Chronic treatment with MK-801 increases the quinolinic acid-induced loss of D-1 dopamine receptors in rat striatum

Following withdrawal from short-term treatment with the non-competitive N-methyl-d-aspartate receptor antagonist MK-801 (1 mg/kg per day) there was no significant change in the B max or K D of [ 3H]SCH23390 binding to dopamine D-1 receptors in rat striatum. Intrastriatal injection of the excitotoxin quinolinic acid (100 nmol) produced a significant decrease in [ 3H]SCH23390 binding. In rats withdrawn from chronic MK-801 treatment quinolinic acid produced a significantly greater loss of [ 3H]SCH23390 binding sites than in rats not treated with MK-801. These data indicated that striatal neurons are hypersensitive to the neurotoxic actions of quinolinic acid following withdrawal from chronic MK-801 treatment.

Interactions of 7-[3-(4-[2,3-dimethylphenyl]piperazinyl)-propoxy]-2(1H)-quinolinone binding in rat striatum: effects of lesions.

7-[3-(4-[2,3-Dimethylphenyl]piperazinyl)propoxy]-2(1H)-quinolinone (OPC-4392), a presynaptic dopamine autoreceptor agonist and postsynaptic D-2 receptor antagonist (Yasuda et al., Life Sci. 42: 1941-1954, 1988), was studied for its binding characteristics at 3H-SCH 23390-labeled dopamine D-1 receptors and 3H-spiperone-labeled dopamine D-2 receptors in rat striatum. The binding affinity of OPC-4392 for 3H-spiperone-labeled D-2 receptors was 500 times higher than for 3H-SCH 23390-labeled D-1 receptors. 6-Hydroxydopamine lesions of striatum and high-frequency current lesions of medial forebrain bundle did not affect the competition of OPC-4392 for 3H-spiperone binding. Kainic acid lesions of striatum significantly changed the one-site model fit to a two-site model fit of the competition curve of OPC-4392 for 3H-spiperone binding, suggesting that OPC-4392 competed with 3H-spiperone binding differently for postsynaptic dopamine D-2 receptors and for presynaptic dopamine autoreceptors.

N-linked oligosaccharides are responsible for rat striatal dopamine D2 receptor heterogeneity

The glycoprotein nature of the binding subunit of the dopamine D2 receptor in rat striatum has been examined by photoaffinity labeling receptor preparations with N-( p-azido- m-[ 125I]iodophenethyl)spiperone followed by treatment of crude membrane receptor or receptor fractions isolated from sodium dodecyl sulfate (SDS) polyacrylamide gels with endo- and exoglycosidases. The major photoaffinity labeled protein migrates as a heterogeneous species on 10% SDS polyacrylamide gels and ranges from 130,000 to 75,000 relative molecular mass ( M r). This heterogeneity can be explained by glycosylation of the receptor by complex-type N-linked oligosaccharides. Three fractions of labeled receptor were isolated from SDS polyacrylamide gels over a range of 130,000 to 75,000 M r; after digestion with peptide- N 4-[ N-acetyl-β-glucosaminyl]asparagine amidase, all fractions yielded a single peptide ~ 40,000 M r. Treatment of photoaffinity labeled membranes with α-mannosidase was without effect. The dopamine D2 receptor appears to contain substantial amounts of sialic acid as treatment of photoaffinity labeled membranes with neuraminidase increased the receptor mobility on SDS polyacrylamide gels to a species of 50,000–54,000 M r. Treatment of the receptor with neuraminidase followed by endo-α- N-acetylgalactosaminidase did not change the electrophoretic migration pattern from that seen after neuraminidase treatment alone, suggesting that the binding peptide contains no serine- or threonine-linked oligosaccharides. A smaller binding peptide of ~31,000 M r is also apparent in crude photoaffinity labeled membranes. This material also contains N-linked oligosaccharide. Complete removal of N-linked oligosaccharide from the dopamine D2 receptor did not change the rank order potency of agonist and antagonist compounds to compete for [ 3H]spiperone binding to crude membrane fractions. The dopamine D2 receptor represents a highly glycosylated neural receptor.

Investigation of rat striatal dopamine D-1 receptors solubilized by digitonin with a precipitation method

[ 3H]SCH 23390 binding sites solubilized from rat striatal membranes by the detergent digitonin were investigated by using a polythylene glycol precipitation method to separate the bound [ 3H]SCH 23390 from the free [ 3H]SCH 23390. The binding of [ 3H]SCH 23390 to the solubilized preparations was specific and saturable with a K D of 4.99 ± 0.03 nM and a B max of 619 ± 13 fmol/mg protein. The rank order of potency of dopamine agonists and antagonists for competing with [ 3H]SCH 23390 binding for the solubilized preparations was appropriate for dopamine D-1 receptors. The competition of SCH 23390 and S(-)-SCH 23388 with [ 3H]SCH 23390 binding for the solubilized preparations was stereoselective. However, the sensitivity of the dopamine agonist high-affinity binding to guanine nucleotide GTP was almost lost upon digitonin solubilization. Preincubating the membranes with dopamine preserved the guanine nucleotide sensitivity of agonist binding for membranes in solubilized preparations. These results proved that the polyethylene glycol recipitation method can be used for assay of digitonin-solubilized dopamine D-1 receptors in rat striatum.