Pergolide, terguride and N,N'-spacer-linked oligomers of both interact with 5-HT2A receptors of rat tail artery

Pergolide, terguride and N,N'-spacer-linked oligomers of both have been tested for their ability to interact with 5 hydroxytryptamine(HT)2A receptors of rat tail artery. Pergolide was a potent partial agonist (pEC50 7.5, Emax 55 %) and antagonized 5-HT-induced contractions (pKp 7.2). Pergolide dimer 3 with a p-xylene spacer between the indole nitrogens (N-1) displayed somewhat lower agonist potency than pergolide (pEC50 7.0, Emax 55 %, pKp 6.6). The contractile responses to pergolide and dimer 3 were antagonized by the 5-HT2A receptor antagonist ketanserin (pA2 9.4, 9.1). In contrast to pergolide dimer 3, pergolide dimers 5 and 9 with an alkyl and an aralkyl spacer between the piperidine nitrogens (N-6) lacked agonism and displayed low affinity at 5-HT2A receptors (pA2 < 5.5). Terguride behaved as an insurmountable antagonist of 5-HT (pA2 8.4). Oligomers of terguride showed 5 to 50-fold lower affinity. It is concluded that pergolide and terguride show a high affinity for 5-HT2A receptors, but dimerization (oligomerization) of both drugs fails to increase affinity.     

Effects of oral pergolide mesylate on nociceptive spinal cord reflexes in rats

The effects of orally administered pergolide mesylate on the flexor reflex were evaluated in chronic spinal rats. The mixed D1/D2 agonist pergolide (0.1 to 3.0 mg/kg) produced a dose-related decrease in the magnitude of the flexor reflex elicited by a tetanic stimulus. The effects of pergolide were blocked by haloperidol, demonstrating that the effects of pergolide were mediated through dopamine receptors. In contrast, the selective D2 agonist bromocriptine (3.0 to 30 mg/kg) had no effect on the flexor reflex. The present results are consistent with the interpretation that pergolide produces an antinociceptive action at the spinal cord level by stimulating both D1 and D2 dopamine receptors.     

Pharmacological characterization of mammalian D1 and D2 dopamine receptors expressed in Drosophila Schneider-2 cells

Mammalian D1 and D2 dopamine receptors were stably expressed in Drosophila Schneider-2 (S2) cells and screened for their pharmacological properties. Saturable, dose-dependent, high affinity binding of the D1-selective antagonist [3H]SCH-23390 was detected only in membranes from S2 cells induced to express rat dopamine D1 receptors, while saturable, dose-dependent, high affinity binding of the D2-selective antagonist [3H]methylspiperone was detected only in membranes from S2 cells induced to express rat dopamine D2 receptors. No specific binding of either radioligand could be detected in membranes isolated from uninduced or untransfected S2 cells. Both dopamine D1 and D2 receptor subtypes displayed the appropriate stereoselective binding of enantiomers of the nonselective antagonist butaclamol. Each receptor subtype also displayed the appropriate agonist stereoselectivities. The dopamine D1 receptor bound the (+)-enantiomer of the D1-selective agonist SKF38393 with higher affinity than the (-)-enantiomer, while the dopamine D2 receptor bound the (-)-enantiomer of the D2-selective agonist norpropylapomorphine with higher affinity than the (+)-enantiomer. At both receptor subtypes, dopamine binding was best characterized as occurring to a single low affinity site. In addition, the low affinity dopamine binding was also found to be insensitive to GTPgammaS and magnesium ions. Overall, the pharmacological profiles of mammalian dopamine D1 and D2 receptors expressed in Drosophila S2 cells is comparable to those observed for these same receptors when they are expressed in mammalian cell lines. A notable distinction is that there is no evidence for the coupling of insect G proteins to mammalian dopamine receptors. These results suggest that the S2 cell insect G system may provide a convenient source of pharmacologically active mammalian D1 and D2 dopamine receptors free of promiscuous G protein contaminants.     

Positron-labeled dopamine agonists for probing the high affinity states of dopamine subtype 2 receptors

It is well documented that guanidine nucleotide-coupled dopamine subtype 2 receptors (D2) are configured in high and low affinity states for the dopamine agonist in vitro. However, it is still unclear whether these functional states exist in vivo. We hypothesized that positron-labeled D2 agonist and Positron Emission Tomography can be used to probe these functional states noninvasively. Recently, we demonstrated in nonhuman primates that N-[11C]propyl-norapomorphine (NPA), a full D2 agonist, is a suitable tracer for imaging the high affinity states of D2 receptors in vivo. We also developed kinetic modeling method to derive receptor parameters, such as binding potential (BP) and specific uptake ratios (V3'). When coupled with a dopamine releasing drug, amphetamine, NPA was found to be more sensitive than antagonist tracers, such as [11C]raclopride (RAC), to endogenous dopamine concentration changes (by about 42%). This finding suggests that NPA is a superior tracer for reporting endogenous DA concentration. In addition, the difference of the BP or V3' of NPA and RAC under control and amphetamine challenge conditions could be used to estimate the functional states of D2 receptors in vivo. On the basis of our findings and the assumptions that NPA binds only to the high affinity states and RAC binds equally to both affinity states, we proposed that about 70% of the D2 receptors are configured in the high affinity states in vivo.     

Identification of Dopamine "D3'' and "D4'' Binding Sites, Labelled with [3H]2-Amino-6,7-Dihydroxy- 1,2,3,4- Tetrahydronaphthalene, as High Agonist Affinity States of the D1 and D2 Dopamine Receptors, Respectively

On the basis of affinity differences for spiperone, two binding sites for [3H](+/-)-2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene ([3H]ADTN) in the rat brain could be distinguished: "D3" with a low and "D4" with a high affinity for spiperone. Evidence is provided that D3 and D4 sites are related to high agonist affinity states of the D1 and D2 dopamine receptors, respectively. Various well-known selective D1 and D2 agonists and antagonists showed potencies at these sites in agreement with this hypothesis. A comparison of the Bmax values for [3H]ADTN binding to D3 and D4 sites with the numbers of D1 receptors (labelled by [3H]SCH 23390) and of D2 receptors (labelled by [3H]spiperone), both in the striatum and in the mesolimbic system, indicated that under the conditions used for 3H-agonist binding experiments, both populations of D1 and D2 receptors were converted to their high agonist affinity states to a considerable, although different extent. In fact, when competition experiments with [3H]spiperone were performed under the conditions otherwise used for [3H]ADTN binding experiments (instead of the conditions usually used for antagonist binding), substantial shifts of the displacement curves of 3,4-dihydroxyphenylethylamine (dopamine) and ADTN toward higher affinities were observed. A comparison of the effects of various agonists and antagonists in the [3H]ADTN binding experiments and in functional tests revealed a significant correlation between their potencies at D4 binding sites and at D2 receptors modulating the release of [3H]acetylcholine from striatal slices. However, in the situation of the D1/D3 pair, when the measurement of adenylate cyclase activity was taken as a functional test for D1 receptors, agonists were more active in the binding than in the functional test, whereas for many antagonists the opposite was found. The results are discussed with regard to the classification and functional aspects of brain dopamine receptors.     

Positive interaction between alpha-1 adrenergic and dopamine-2 receptors in locomotor activity of normo and supersensitive mice

In normosensitive mice either the D1 antagonist SCH 23390 or the D2 antagonist sulpiride inhibited the reversion of reserpine-induced akinesia elicited by the mixed D1/D2 agonist pergolide. In mice rendered supersensitive by a five days' reserpine treatment, sulpiride did not prevent the pergolide-induced reversal of akinesia while SCH 23390 disclosed two subpopulations of mice. One population responded to pergolide with marked locomotor activity whereas in the other subpopulation this response was absent. However, all mice challenged with pergolide failed to reverse reserpine-akinesia after alpha-methyl-p-tyrosine (AMPT) pretreatment. The alpha 1/alpha 2 agonist clonidine restored the ability of pergolide to overcome reserpine akinesia in supersensitive mice pretreated with SCH 23390. Clonidine reversed the akinesia in supersensitive mice but in normal animals it did not. However, in these last conditions, the combined use of clonidine plus the D2 agonist LY 171555 was effective to induce locomotion. Neither AMPT nor SCH 23390 inhibited this response whereas the alpha-adrenergic antagonists prazosin and yohimbine did prevent it. The alpha 2 agonist B-HT 920 failed to induce locomotor responses when given together with LY 171555. The same occurred with the D1 agonist SKF 38393 when given together with clonidine. The combined use of SCH 23390 plus prazosin in chronic reserpinized mice prevented pergolide-induced locomotion. Adrenergic stimulation, acting on alpha 1 receptors, could be an alternative to D1 stimulation as a necessary factor to obtain D2-induced motor responses under normo and supersensitive conditions.     

Delineation of the structural basis for the activation properties of the dopamine D1 receptor subtypes.

To delineate the structural determinants involved in the constitutive activation of the D1 receptor subtypes, we have constructed chimeras between the D1A and D1B receptors. These chimeras harbored a cognate domain corresponding to transmembrane regions 6 and 7 as well as the third extracellular loop (EL3) and cytoplasmic tail, a domain referred herein to as the terminal receptor locus (TRL). A chimeric D1A receptor harboring the D1B-TRL (chimera 1) displays an increased affinity for dopamine that is indistinguishable from the wild-type D1B receptor. Likewise, a chimeric D1B receptor containing the D1A-TRL cassette (chimera 2) binds dopamine with a reduced affinity that is highly reminiscent of the dopamine affinity for the wild-type D1A receptor. Furthermore, we show that the agonist independent activity of chimera 1 is identical to the wild-type D1B receptor whereas the chimera 2 displays a low agonist independent activity that is indistinguishable from the wild-type D1A receptor. Dopamine potencies for the wild-type D1A and D1B receptor were recapitulated in cells expressing the chimera 2 or chimera 1, respectively. However, the differences observed in agonist-mediated maximal activation of adenylyl cyclase elicited by the D1A and D1B receptors remain unchanged in cells expressing the chimeric receptors. To gain further mechanistic insights into the structural determinants of the TRL involved in the activation properties of the D1 receptor subtypes, we have engineered two additional chimeric D1 receptors that contain the EL3 region of their respective cognate wild-type counterparts (hD1A-EL3B and hD1B-EL3A). In marked contrast to chimera 1 and 2, dopamine affinity and constitutive activation were partially modulated by the exchange of the EL3. Meanwhile, hD1A-EL3B and hD1B-EL3A mutant receptors display a full switch in the agonist-mediated maximal activation, which is reminiscent of their cognate wild-type counterparts. Overall, our studies suggest a fundamental role for the TRL in shaping the intramolecular interactions implicated in the constitutive activation and coupling properties of the dopamine D1 receptor subtypes.     

Dopamine D1 receptors characterized with [3H]SCH 23390. Solubilization of a guanine nucleotide-sensitive form of the receptor

Dopamine D1 receptors were solubilized from canine and bovine striatal membranes with the detergent digitonin. The receptors retained the pharmacological characteristics of membrane-bound D1 receptors, as assessed by the binding of the selective antagonist [3H]SCH 23390. The binding of [3H]SCH 23390 to solubilized receptor preparations was specific, saturable, and reversible, with a dissociation constant of 5 nM. Dopaminergic antagonists and agonists inhibited [3H]SCH 23390 binding in a stereoselective and concentration-dependent manner with an appropriate rank order of potency for D1 receptors. Moreover, agonist high affinity binding to D1 receptors and its sensitivity to guanine nucleotides was preserved following solubilization, with agonist dissociation constants virtually identical to those observed with membrane-bound receptors. To ascertain the molecular basis for the existence of an agonist-high affinity receptor complex, D1 receptors labeled with [3H] dopamine (agonist) or [3H]SCH 23390 (antagonist) prior to, or following, solubilization were subjected to high pressure liquid steric-exclusion chromatography. All agonist- and antagonist-labeled receptor species elute as the same apparent molecular size. Treatment of brain membranes with the guanine nucleotide guanyl-5'-yl imidodiphosphate prior to solubilization prevented the retention of [3H]dopamine but not [3H]SCH 23390-labeled soluble receptors. This suggests that the same guanine nucleotide-dopamine D1 receptor complex formed in membranes is stable to solubilization and confers agonist high affinity binding in soluble preparations. These results contrast with those reported on the digitonin-solubilized dopamine D2 receptor, and the molecular mechanism responsible for this difference remains to be elucidated.     

Conformationally-flexible benzamide analogues as dopamine D3 and sigma 2 receptor ligands

A series of conformationally-flexible analogues was prepared and their affinities for D2-like dopamine (D2, D3 and D4) were determined using in vitro radioligand binding assays. The results of this structure-activity relationship study identified one compound, 15, that bound with high affinity (K(i) value=2nM) and moderate selectivity (30-fold) for D3 compared to D2 receptors. In addition, this series of compounds were also tested for affinity at sigma1 and sigma2 receptors. We evaluated the affinity of these dopaminergic compounds at sigma receptors because (a) several antipsychotic drugs, which are high affinity antagonists at dopamine D2-like receptors, also bind to sigma receptors and (b) sigma receptors are expressed ubiquitously and at high levels (picomoles per mg proteins). It was observed that a number of analogues displayed high affinity and excellent selectivity for sigma2 versus sigma1 receptors. Consequently, these novel compounds may be useful for characterizing the functional role of sigma2 receptors and for imaging the sigma2 receptor status of tumors in vivo with PET.     

The A1 adenosine receptor. Solubilization and characterization of a guanine nucleotide-sensitive form of the receptor

Adenosine acting through membrane-bound A1 receptors is capable of inhibiting the enzyme adenylate cyclase. A1 adenosine receptors from rat cerebral cortex have been solubilized in high yield and in an active form with the detergent digitonin. The solubilized receptors bind the agonist radioligand (-)-N6-3-[125I] iodo-4-hydroxyphenylisopropyl)adenosine (HPIA) with the same high affinity, demonstrate the same agonist and antagonist potency series and stereo-specificity as the membrane-bound A1 receptor. In addition to maintaining high affinity agonist binding, soluble A1 receptors' affinity for agonists is still modulated by guanine nucleotides. This result contrasts with other adenylate cyclase coupled receptors (beta 2, alpha 2, D2) wherein high affinity agonist binding is lost subsequent to solubilization. To investigate the molecular basis for this difference, solubilized A1 receptors which were labeled with [125I]HPIA either prior to or subsequent to solubilization, were compared by sucrose density gradient centrifugation. Both labeled species demonstrated exactly the same sedimentation properties and display guanine nucleotide sensitivity. This suggests that the same guanine nucleotide-sensitive receptor complex formed in membranes in stable to solubilization and can form a high affinity agonist complex in soluble preparation. The molecular mechanism responsible for the stable receptor complex in this system compared to the beta 2, alpha 2, and D2 systems remains to be determined.     

Synthesis of 2-(5-bromo-2,3-dimethoxyphenyl)-5-(aminomethyl)-1H-pyrrole analogues and their binding affinities for dopamine D2, D3, and D4 receptors

A series of 2-(5-bromo-2,3-dimethoxyphenyl)-5-(aminomethyl)-1H-pyrrole analogues was prepared and their affinity for dopamine D(2), D(3), and D(4) receptors was measured using in vitro binding assays. The results of receptor binding studies indicated that the incorporation of a pyrrole moiety between the phenyl ring and the basic nitrogen resulted in a significant increase in the selectivity for dopamine D(3) receptors. The most selective compound in this series is 2-(5-bromo-2,3-dimethoxyphenyl)-5-(2-(3-pyridal)piperidinyl)methyl-1H-pyrrole (6p), which has a D(3) receptor affinity of 4.3 nM, a 20-fold selectivity for D(3) versus D(2) receptors, and a 300-fold selectivity for D(3) versus D(4) receptors. This compound is predicted to be a useful ligand for studying the functional role of dopamine D(3) receptors in vivo.     

Imidazoline-modified benzylimidazolines as h5-HT(1D/1B) serotonergic ligands

Sumatriptan, a h5-HT1D and h5-HT1B receptor agonist used clinically as a migraine-abortive, produces certain side effects thought to result from its affinity for h5-HT1B receptors. The present investigation extends our work with benzylimidazolines as novel non-tryptamine h5-HT(1D/1B) ligands. The effect of N-methylation, N-benzylation, ring-aromatization, and variation of the imidazoline ring on affinity both at h5-HT1D and h5-HT1B receptors was examined. Several compounds were identified with good affinity and enhanced (i.e., > 100-fold) h5-HT1D versus hS-HT1B selectivity.     

Dopamine D3 receptor binding by D3 agonist 7-OH-DPAT (7-hydroxy-dipropylaminotetralin) and antipsychotic drugs measured ex vivo by quantitative autoradiography

Because the dopamine D3 receptor is primarily expressed in regions of the limbic system of brain, it was proposed that it may represent a target for antipsychotic drugs that is free of extrapyramidal side effects. An ex vivo receptor binding technique employing [3H]7-OH-DPAT was used to evaluate in vivo occupancy of dopamine D3 receptors in the rat nucleus accumbens by selective D3 agonist 7-OH-DPAT (7-hydroxy-dipropylaminotetralin) and various antipsychotic drugs. With an ID50 value of 0.07 mg/kg, the selective D3 agonist (+)-7-OH-DPAT had the most potent inhibitory effect on ex vivo binding of [3H]7-OH-DPAT among all drugs tested. Clinical doses of phenothiazine drugs, such as chlorpromazine and levomepromazine, induce binding to D3 receptors in vivo, while atypical antipsychotic drugs, such as clozapine, pimozide, and sulpiride, are very weak in inhibiting ex vivo binding of [3H]7-OH-DPAT, indicating that the role of D3 receptors as targets of antipsychotic drugs free of extrapyramidal side effects may not be important.     

Comparative biochemical pharmacology of central nervous system dopamine D1 and D2 receptors

The biochemical properties of central nervous system (CNS) dopamine (DA) D1 and D2 receptors were examined using the specific antagonists [3H]SCH23390 and [3H]raclopride, respectively. There is a different participation of sulfhydryl (-SH) and disulfide (-SS-) groups in the binding site and/or coupling to second messenger systems of D1 and D2 receptors. The ionic studies with [3H]SCH23390 showed slight agonist and antagonist affinity shifts for the D1 receptor. On the other hand, the D2 receptor is very sensitive to cations; even if lithium and sodium influence specific [3H]raclopride binding in a similar manner, there appear to be quantitative differences between these two ions that cannot be explained by surface charge mechanisms. The distribution of D1 and D2 receptors was heterogenous in both species, with the greatest densities in the neostriatum, where the highest concentrations of DA and metabolites were measured. Regions with low endogenous DA content (cerebral cortex and hippocampus) had lower densities of DA receptors. Furthermore, these binding sites were differentially localized within the various regions, and there were substantially more D1 than D2 receptors. The functional significance and heterogeneities in the distribution of D1 and D2 receptors can be related to dopaminergic innervation and turnover.     

Modulation of dopamine D1 receptor binding by neurotensin in the rat striatum

The effect of neurotensin on binding characteristics of dopamine D1 receptors was examined in the rat striatal membranes through radioreceptor assay. Neurotensin or its analogs were added to incubation medium of[³H]SCH 23390 saturation or dopamine/[³H]SCH 23390 inhibition experimental systems. Neurotensin did not modulate D1 antagonist binding but converted a part of D1 agonist high affinity binding sites to a low affinity state. Neurotensin_8–13 had the same potency as neurotensin itself, whereas neurotensin_1–8 had only weak activity in modulating D1 agonist binding. GTP and neurotensin had the same effect on D1 agonist binding. However, when both neurotensin and GTP were added, the result was the same as with either alone.

These data suggest that neurotensin modulates the functional state of D1 receptors probably via a GTP binding protein in the rat striatum.     

8,9-dihydroxy-1,2,3,11b-tetrahydrochromeno[4,3,2,-de]isoquinoline (dinoxyline), a high affinity and potent agonist at all dopamine receptor isoforms

The synthesis and preliminary pharmacological evaluation of 8,9-dihydroxy-1,2,3,11b-tetrahydrochromeno[4,3,2,-de]isoquinoline (5, now named dinoxyline) is described. This molecule was designed as a potential bioisostere that would conserve the essential elements of our beta-phenyldopamine D(1) pharmacophore (i.e., position and orientation of the nitrogen, hydroxyls, and phenyl rings). Previously, we have rigidified these elements using alkyl bridges, as exemplified in the dopamine D(1) full agonist molecules dihydrexidine (1) and dinapsoline (2). This approach has been modified and we now show that it is possible to tether these elements using an ether linkage. Preliminary pharmacology has revealed that 5 is a potent full D(1) agonist (K(0.5) <10 nM; EC(50)=30 nM), but also has high affinity for brain D(2)-like and cloned D(2) and D(3) receptors. Interestingly, whereas 1 and 2 and their analogues have only moderate affinity for the human D(4) receptor, 5 also has high affinity for this isoform. Moreover, although N-alkylation of 1 and 2 increases D(2) affinity, the N-allyl (15) and N-n-propyl (17) derivatives of 5 had decreased D(2) affinity. Therefore, 5 may be engaging different amino acid residues than do 1 and 2 when they bind to the D(2) receptor. This is the first example of a ligand with high affinity at all dopamine receptors, yet with functional characteristics similar to dopamine. These rigid ligands also will be useful tools to determine specific residues of the receptor transmembrane domains that are critical for agonist ligand selectivity for the D(4) receptor.     

Dopamine D2 receptors retain agonist high-affinity form and guanine nucleotide sensitivity after removal of sialic acid

The ligand binding subunit of the D2 dopamine receptor (Mr approximately equal to 94,000) can be visualized by autoradiography following photoaffinity labeling with [125I]N-azidophenethylspiperone and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following removal of sialic acids with the exoglycosidase, neuraminidase, [125I]N-azidophenethylspiperone photoincorporated into a protein of Mr = 54,000 with the appropriate pharmacological profile for D2 receptors. The desialylated D2 receptor bound dopaminergic agonists with high affinity and was capable of coupling to a functional G-protein as indexed by: 1) pertussis-toxin mediated [32P]ADP ribosylation of proteins of Mr = 42,000 and 39,000, and 2) the conversion of the agonist high affinity form of D2 receptors to one displaying low affinity for agonists in the presence of guanine nucleotides. These data suggest that sialic acid residues do not contribute significantly to the ligand binding characteristics of D2 receptors despite the large change produced in the estimated molecular mass of the binding subunit.     

Dopamine D5 receptor agonist high affinity and constitutive activity profile conferred by carboxyl-terminal tail sequence

The mammalian dopamine D1-like receptor gene family is comprised of two members, termed D1/D1A and D5/D1B. In an attempt to define the role of the carboxyl terminal (CT) tail in the expression of D5 subtype-specific pharmacological and constitutive activity profiles, we examined a series of D5 receptor chimeras in which only the CT tail was swapped with corresponding sequences encoding human/vertebrate D1-like receptors. D5/D1(CT) or D5/D1D(CT) tail substitution mutants displayed a rank order of potency and agonist affinities virtually mimicking wild-type (wt) D1 receptors, as indexed by both ligand binding and dopamine-stimulated cAMP accumulation assays, and, similar to wt D1 receptors, did not exhibit receptor constitutive activity or responsiveness to inverse agonists. D1/D5(CT) or D1/D1D(CT) tail receptor mutants displayed agonist pharmacological and functional characteristics not significantly different from parental D1 or mutant D5/D1(CT) and D5/D1D(CT) receptors. The affinities for numerous antagonists remained essentially unchanged for all receptor chimeras relative to parental wt receptors. A series of stepwise D5-CT-tail truncation/deletion mutants identified the region encoded by amino acids 438-448 and particularly Gln(439), as necessary and sufficient for the full expression of high affinity agonist and functional D5 receptor characteristics. Site-directed mutagenesis of the highly conserved D5/D1B receptor residue Gln(439)-(Ala/Ile), converts the full-length D5 receptor to one displaying "super" D5 characteristics with expressed affinities for discriminating agonists approximately 4- to 5-fold higher than wt D5 but without any concomitant increases of agonist-independent basal cAMP accumulation or intrinsic activity. Taken together, these data suggest that, in addition to other well characterized receptor domains, the agonist pharmacological and functional signature of the D5/D1B receptor is modulated by sequence-specific motifs within the CT tail and that one conserved amino acid in this region can further regulate D5 agonist high affinity binding interactions independent of receptor constitutive activity.     

Dopamine D1 receptors with enhanced agonist affinity and reduced antagonist affinity revealed by chemical modification

In order to investigate the possibility that there may be two conformationally distinct dopamine D1 binding sites, the effect of lysine-modifying agents on striatal dopamine D1 receptors was investigated. Treatment with the distilbene derivative, 4,4'-diisothiocyanostilbene-2,2'-disulfonate, (DIDS), resulted in an irreversible D1 receptor inactivation that was associated with a 70% loss of binding sites. The remaining DIDS-insensitive sites displayed both a decreased affinity (approximately 5 fold) for the D1 antagonist SCH-23390 and an enhanced affinity of dopaminergic agonists (approximately 10 fold) for the agonist high-affinity form of the receptor. Pretreatment with Gpp(NH)p, a non-hydrolysable guanine nucleotide, prevented the formation of the agonist high-affinity form, indicating that these sites are G-protein-linked. Prior occupancy of D1 receptors with dopaminergic agonists and antagonists afforded no protection against DIDS inactivation, suggesting that a site outside the ligand binding subunit of the D1 receptor was modified. Taken together, these data suggest that [3H]SCH-23390 labels two conformationally distinct populations of dopamine D1 receptors.