Agonist high- and low-affinity states of dopamine D2 receptors: methods of detection and clinical implications

Dopamine D₂ receptors, similar to other G-protein-coupled receptors, exist in a high- and low-affinity state for agonists. Based upon a review of the methods for detecting D₂ receptor agonist high-affinity states, we discuss alterations of such states in animal models of disease and the implications of such alterations for their labelling with positron emission tomography (PET) and single-photon emission computed tomography (SPECT) tracers. The classic approach of detecting agonist high-affinity states compares agonist competition for antagonist radioligands, in most cases using [³H]-spiperone as the radioligand; alternative approaches and radioligands have been proposed, but their claimed advantages have not been substantiated by other investigators. In view of the advantages and disadvantages of various techniques, we critically have reviewed reported findings on the detection of D₂ receptor agonist high-affinity states in a variety of animal models. These data are compared to the less numerous findings from human in vivo studies based on PET and SPECT tracers; they are interpreted in light of the finding that D₂ receptor agonist high-affinity states under control conditions may differ between rodent and human brain. The potential advantages of agonist ligands in studies of pathophysiology and as diagnostics are being discussed.     

Mapping of the beta 2-adrenoceptor on Chang liver cells. Differences between high- and low-affinity receptor states

Receptor mapping procedures based on the methodology of Crippen are used to study the beta 2-adrenergic receptor system in intact Chang liver cells. In cases of agonists, the presence of both a low- and high-affinity receptor state is assumed, whereas antagonists bind to the low-affinity state only. The high-affinity state is considered to contain the "functional" binding site responsible for formation of the second messenger (cAMP), whereas the low-affinity state is assumed to be the "true" (physiological) low-affinity state. Both receptor states are taken into account in the receptor mapping process. Characterization of the high- and low-affinity states made it possible to identify features that make a state an antagonist or agonist. The receptor model found for the low-affinity state of the beta-adrenergic receptor present in an intact cell system is compared to the low-affinity state previously obtained for this receptor present on a membrane preparation of the bovine skeletal muscle in the presence of high amounts of Gpp(NH)p guanosine 5'-(beta, gamma-imidotriphosphate). Remarkable differences are found between the two receptor models. The tentative conclusion is drawn that these differences in low-affinity states most probably are artificial and are caused by the different pharmacological properties (e.g., intrinsic activity) of the labeled ligands used in displacement experiments for determining the affinities of the drugs.     

3H]N-propylapomorphine and [3H]spiperone binding in brain indicate two states of the D2-dopamine receptor

[3H]N-propylapomorphine ([3H]NPA) a dopaminergic catecholamine derivative, labels a sub-set of D2-dopamine receptors in bovine caudate particulate preparation. [3H]Spiperone, a dopamine receptor antagonist, labels twice as many sites as [3H]NPA. Dopaminergic ergots and potent neuroleptics compete for both radioactive ligands with similar high affinities. Catecholamines and catecholamine derivatives compete more potently for [3H]NPA binding than for [3H]spiperone binding. Guanyl nucleotides reduce both [3H]NPA binding and the high affinity phase of catecholamine and catecholamine derivative competition for [3H]spiperone binding. These results are similar to binding results reported in studies of two-state receptors linked to adenylate cyclase such as the beta-adrenergic receptors. These observations indicate that the D2-dopamine receptor in the brain may exist in two states and may be inversely coupled to brain adenylate cyclase activity.     

Dependence of dopamine receptor conversion from agonist high- to low-affinity state on temperature and sodium ions

Previous workers found that the anterior pituitary dopamine receptors were inconsistent in converting from their high-affinity state (D2 high) into their low-affinity state (D2 low) for dopamine. We tested, therefore, whether temperature or sodium ion concentration could be factors accounting for such inconsistencies. We found that the proportion of sites which converted depended on the temperature of incubation. No conversion occurred at 4 degrees, despite the presence of guanine nucleotide and sodium ions. At room temperature (20 degrees) guanine nucleotide consistently induced complete conversion in the presence of sodium ions, but gave inconsistent conversion in the absence of sodium ions. At body temperature (37 degrees) guanine nucleotide consistently resulted in complete conversion without requiring sodium ions.     

Partial brain dopamine D2 receptor agonists in the treatment of schizophrenia

Partial dopamine agonists showing high affinity but low efficacy at D2 receptors can act as dopaminergic "buffers," reducing dopaminergic activity when it is excessive, and promoting it when reduced. This makes them of interest as potential therapeutic agents for the treatment of both positive and negative symptoms in schizophrenia, where they should also result in fewer or less severe motor disturbances than classical neuroleptics. SDZ 208-911 and SDZ 208-912 are amino-ergolines exhibiting partial agonistic properties in the rat, where they inhibit apomorphine-induced stereotypies, are only weakly cataleptogenic, induce varying degrees of circling behavior after unilateral lesioning of the nigrostriatal pathway, and strongly suppress prolactin secretion. The least agonistically acting agent, SDZ 208-912, should be effective against positive symptoms, whereas SDZ 208-911 could be suitable for the treatment of negative symptoms. In addition to possible therapeutic effects, the clinical testing of this class of agent should help to elucidate the status of central dopaminergic function in schizophrenic psychosis.     

Autoradiographic localisation of D3-dopamine receptors in the human brain using the selective D3-dopamine receptor agonist (+)-[3H]PD 128907

The selective D₃-dopamine receptor agonist 4aR,10bR-(+)-trans-3,4,4a,10b-tetrahydro-4-[N-propyl-2,3-³H]-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol ([³H]PD 128907) was used to visualise D₃-dopamine receptors in whole hemisphere cryosections from post-mortem human brain. [³H]PD 128907 has an 18- to 40-fold selectivity for D₃- over D₂-dopamine receptors as compared to a 7- to 24-fold selectivity of the more commonly used ligand [³H]7-OH-DPAT. [³H]PD 128907 accumulated markedly in the nucleus accumbens and in the ventral parts of caudate nucleus and putamen, with a slightly heterogeneous (patch-matrix like) distribution. The binding in the lateral parts of caudate nucleus and putamen was much less dense. No binding was obtained in any other regions. A very high proportion of [³H]PD 128907 was specifically bound, as judged from the low binding remaining in the presence of the D₂/D₃-dopamine receptor antagonist raclopride. This gives the ligand a potential for the detection of low density D₃-dopamine receptors in the human brain. The binding obtained with [³H]PD 128907 was qualitatively similar to that using [³H]7-OH-DPAT in the presence of GTP. However, [³H]7-OH-DPAT labelled, in contrast to [³H]PD 128907, also D₃-dopamine receptors in neocortex. The new compound [³H]PD 128907 appears to be a suitable radioligand for autoradiographic examination of the D₃-dopamine receptor localisation in the human brain, and should also be useful for pharmacological studies of this receptor subtype. Received: 20 November 1995/Final version: 2 May 1996     

An open label trial of raclopride in acute schizophrenia. Confirmation of D2-dopamine receptor occupancy by PET

Raclopride, a highly selective D2-dopamine receptor antagonist, was administered in doses up to 4 mg b.i.d. to ten schizophrenic patients in an open label non-comparative study lasting 4 weeks. Safety, tolerability, potential antipsychotic effect, prolactin response and drug effect on plasma homovanillic acid were evaluated. Central D2-dopamine receptor occupancy was determined by positron emission tomography (PET). No major deviations were found in biochemical and physiological safety parameters. Raclopride was well tolerated. The mean BPRS score was reduced by 55% at endpoint. In the global evaluation seven patients were very much or much improved. Extrapyramidal side effects were recorded in four patients and disappeared after dose reduction or single doses of biperiden. An increase in plasma prolactin of short duration was observed in both sexes. A significant decrease of plasma HVA was obtained after 4 weeks of treatment. In two of the patients the central D2-dopamine receptors occupancy was measured using PET. The receptor occupancy was 68 and 72% which is the same as that found in patients treated with conventional neuroleptics.     

Two affinity states of Ri adenosine receptors in brain membranes. Analysis of guanine nucleotide and temperature effects on radioligand binding

The binding of agonists and antagonists to Ri adenosine receptors of synaptosomal membranes from rat and bovine brain was studied. The effects of guanine nucleotides and temperature were analyzed with the aid of computerized curve fitting. Evidence is presented for two different states of the receptor: one of high and one of low affinity for agonists. Antagonists bind to both states with the same affinity. The two states are characterized by saturation, competition, and kinetic experiments with very similar results. Guanine nucleotides cause transition of the high- to the low-affinity state. The ratio of the KD values for the two affinity states is 90-150 in rat brain but only 10 in bovine brain. The proportions of the two affinity states are the same for all agonists tested; in the absence of exogenous guanine nucleotides, 75% of the total receptor population is in the high-affinity state, whereas in the presence of guanine nucleotides only 5% remain in the high-affinity state. Binding of antagonists to the receptor is enthalpy-driven whereas binding of the agonist (-)-N6-phenylisopropyladenosine to the high-affinity state of the receptor is entropy-driven. Binding of the agonist to the low-affinity state is enthalpy-driven and thus similar to the binding of antagonists. Our data indicate that guanine nucleotides convert the Ri adenosine receptor from a high- to a low-agonist affinity state and that agonist binding shows thermodynamic differences from antagonist binding only when it is to the high-affinity state of the receptor.     

Cooperation between D1- and D2-dopamine receptors in the nucleus accumbens.

Apomorphine, used in small doses (20-50 micrograms/kg), induced an increase in the activity of an endogenous inhibitor of cAMP dependent protein kinases (Walsh inhibitor, type I inhibitor) in nucleus accumbens of the rat. The action of apomorphine was blocked by sulpiride and aminophylline and enhanced by SCH-23390. Pretreatment with 6-OH-dopamine resulted in a shift of the dose-response curve for apomorphine to the left, suggesting supersensitivity of D2 receptors. Moreover, stimulation of D2 receptors induced a decrease in phosphorylation of DARPP-32, a specific protein, located in neurones containing D1 receptors. Large doses of apomorphine (over 0.5 mg/kg) provoked a decrease in type I inhibitor activity, blocked by SCH-23390 and enhanced by sulpiride and aminophylline. Moreover, SCH-23390 blocked a decrease in type I inhibitor activity induced by large doses of sulpiride and sulpiride blocked an increase in type I inhibitor activity produced by large doses of SCH-23390. The results suggest that D1 and D2 receptors in the nucleus accumbens could cooperate with the same adenylate cyclase and could be located on the same neurones.     

Identification of the binding subunit of the D1-dopamine receptor by photoaffinity crosslinking

The D1-dopamine receptor from rat striatum has been successfully identified by photoaffinity crosslinking using a newly synthesized radioiodinated derivative of the selective D1-antagonist SCH-23390. This compound, (R,S)-5-(3'-aminophenyl)-8-chloro-2,3,4,5-tetrahydro-3-methyl-[1H]-3- benzazepin-7-ol(SCH-38548), has been radioiodinated by a chloramine T procedure yielding three radioiodinated products. One of these separated congeners (with Rf = 0.35 on thin layer chromatography; CH2Cl2/MeOH/triethylamine; 82.5:17.5:0.01) binds reversibly to rat striatal membranes with high affinity (KD approximately equal to 200 pM), appropriate stereoselectivity, and D1-dopaminergic specificity. [125I]SCH-38548 can be covalently incorporated into a peptide of Mr approximately equal to 72,000 using the heterobifunctional crosslinking reagent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Covalent incorporation of [125I]SCH-38548 into the Mr approximately equal to 72,000 peptide can be blocked by dopaminergic agents with D1-dopaminergic specificity (for agonists: SKF-38393 greater than apomorphine greater than dopamine; for antagonists: SCH-23390 much much greater than, SCH-23388 and cis-flupentixol much much greater than trans-flupentixol). The D1-dopaminergic selectivity and specificity of the labeling were further demonstrated by the fact that other antagonists such as domperidone, ketanserin, phentolamine, and alprenolol did not compete for the covalent labeling of the Mr approximately equal to 72,000 peptide. These results indicate that the ligand-binding subunit of the D1-dopamine receptor resides on peptide distinct from that of the D2-dopamine receptor (Mr = 94,000). This new radioligand should be useful in the molecular characterization of the D1-dopaminergic receptor from various sources.     

Kinetic analysis of agonist-induced down-regulation of the beta(2)-adrenergic receptor in BEAS-2B cells reveals high- and low-affinity components

We examined the interrelationships of internalization and down-regulation of the beta(2)-adrenergic receptor in response to treatment of the BEAS-2B human epithelial cell line with both a series of agonists at high occupancy and with various concentrations of fenoterol that gave occupancies from 0.93 to 0.001. We found that the extent of internalization measured after a 30-min treatment increased as a function of coupling efficiency, with ephedrine, dobutamine, albuterol, fenoterol, and epinephrine giving 0, 7, 17, 48, and 55% internalization, respectively. With the exception of dobutamine, the rates of down-regulation (k(deg)) also showed a dependence on agonist coupling efficiency, giving (in terms of fraction of receptors lost/h) 0.082 with ephedrine, 0.250 with dobutamine, 0.148 with albuterol, 0.194 with fenoterol, and 0.212 with epinephrine. Comparison of down-regulation to internalization showed that weak agonists caused down-regulation in the absence of significant internalization. The extent of internalization caused by fenoterol over a 1000-fold range of occupancy was proportional to agonist occupancy. However, although no internalization was observed with the low concentrations (0.2 and 2 nM fenoterol), these concentrations did cause significant down-regulation. Thus, as with partial agonists, it was clear that down-regulation occurred in the absence of measurable internalization. The kinetics of agonist-induced down-regulation are consistent with a scheme in which down-regulation proceeds by two pathways; a high-affinity, low-capacity component (EC(50) = 0.5 nM) clearly dissociated from internalization and a low-affinity, high-capacity component (EC(50) = 160 nM) closely correlated with internalization.     

Thermodynamically distinct high and low affinity states of the A(1) adenosine receptor induced by G protein coupling and guanine nucleotide ligation states of G proteins

The influence of the receptor-G protein coupling state and the guanine nucleotide ligation state of the G protein on the binding mechanism of A(1) adenosine receptor ligands has been investigated in [(3)H]-1,3-dipropyl-8-cyclopentylxanthine ([(3)H]-DPCPX) binding studies in rat brain membranes. Thermodynamic parameters of binding of A(1) adenosine receptor ligands of different intrinsic activities were determined in the absence or presence of GDP and compared to the binding mechanism after receptor-G protein uncoupling. In agreement with previous studies, it was found that xanthine and non-xanthine antagonists showed an enthalpy- or enthalpy- and entropy-driven binding mechanism under all conditions. In contrast to antagonists, the binding mechanism of agonists was strongly affected by the G protein coupling state or the absence or presence of guanine nucleotides. Binding of full and partial agonists to the high-affinity state of the A(1) receptor was entropy-driven in the absence of GDP, and a good correlation between intrinsic activities and the contribution of entropy was observed. In the absence of GDP, binding of full and partial agonists and antagonists to the high affinity state of the receptor was thermodynamically discriminated. In contrast, no such discrimination was found in the presence of GDP. The binding mechanism of agonists to the low-affinity state of the receptor was identical to that of antagonists only after uncoupling of the receptor from G proteins by pretreatment with N-ethylmaleimide or guanosine-5'-(gamma-thio)-triphosphate (GTPgammaS). These results indicate the existence of two thermodynamically distinct high- and low-affinity states of the A(1) adenosine receptor.     

Characterization of D2-dopamine receptor modification in rat striatum after calcium channel blocker treatment

A wide variety of chemically heterogenous substances selectively inhibit the flow of Ca2+ ions through the calcium-conducting channels. The possible involvement of brain D2-dopamine receptor modulation in chronic treatment with verapamil, nicardipine and cinnarizine was investigated in rats. None of the drugs modified the affinity constant, but nicardipine and cinnarizine increased significantly the number of D2 binding sites. These results confirm the possible association of calcium antagonist receptors with selected synaptic areas and their different modulation related to their therapeutic relevance.     

The A2 adenosine receptor: guanine nucleotide modulation of agonist binding is enhanced by proteolysis

Agonist binding to the A2 adenosine receptor (A2AR) and its regulation by guanine nucleotides was studied using the newly developed radioligand 125I-2-[4-(2-[2-[(4-aminophenyl)methylcarbonylamino] ethylaminnocarbonyl]ethyl)phenyl]ethylamino-5'-N- ethylcarboxamidoadenosine (125I-PAPA-APEC) and its photoaffinity analog 125I-azido-PAPA-APEC. A single protein of Mr 45,000, displaying the appropriate A2AR pharmacology, is labeled in membranes from bovine striatum, PC12 cells, and frog erythrocytes. In DDT1 MF2 cells the labeled protein has a slightly lower molecular weight. Incorporation of 125I-azido-PAPA-APEC into membranes from rabbit striatum, however, reveals two specifically labeled peptides (Mr approximately 47,000 and 38,000), both of which display A2AR pharmacology. Inhibition of protease activity leads to a decrease in the amount of the Mr 38,000 protein, with only the Mr 47,000 protein remaining. This suggests that the Mr 38,000 peptide is a proteolytic product of the Mr 47,000 A2AR protein. In membranes containing the intact undigested A2AR protein, guanine nucleotides induce a small to insignificant decrease in agonist binding, which is atypical of stimulatory GS-coupled receptors. This minimal effect is observed in rabbit striatal membranes prepared in the presence of protease inhibitors, as well as in the other tissues studied. Binding to rabbit striatal membranes that possess the partially digested receptor protein, however, reveals a 50% reduction in maximal specific agonist binding upon addition of guanine nucleotides. Inhibition of proteolysis in rabbit striatum, on the other hand, results in a diminished ability of guanine nucleotides to regulate agonist binding. Thus, the enhanced effectiveness of guanine nucleotides in rabbit striatal membranes is associated with the generation of the Mr 38,000 peptide fragment. Guanosine 5'-(beta,gamma-imido)triphosphate reduces photoaffinity labeling by 55% in the Mr 38,000 protein, whereas the labeling is decreased by only 28% in the Mr 47,000 receptor protein. Our data, therefore, suggest that, unless proteolysis occurs, the A2AR in all tissues studied is tightly associated with the GS protein and displays minimal guanine nucleotide modulation of agonist binding, which makes the A2AR an atypical stimulatory receptor.     

The new selective D2-dopamine receptor antagonist raclopride--pharmacokinetics, safety and tolerability in healthy males

Raclopride, a new potential antipsychotic drug, with high selectivity and affinity for central D2-dopamine receptors, was in this first human study administered to 8 healthy male volunteers in single oral doses from 0.1 to 16 mg. Two subjects, known to be slow metabolizers of debrisoquine, were also included. Pharmacokinetics, safety, tolerability, and effect on plasma prolactin levels were evaluated. The maximum plasma concentrations of raclopride (Cmax) and the area under the raclopride curve vs time (AUC) increased proportionally with dose. No deviant kinetic parameters were seen in the slow debrisoquine metabolizers. Only minor deviations in biochemical and physiological safety parameters were found. Raclopride was well tolerated by all subjects at doses up to 8 mg but not at 16 mg because of akathisia. No other extrapyramidal side-effects were recorded. The drug induced a rapid and transient increase of plasma prolactin concentrations.     

Regulation of high- and low-affinity [3H]imipramine recognition sites in rat brain by chronic treatment with antidepressants

Specific binding of [3H]imipramine to its recognition sites in frontal cortex and levels of serotonin (5-HT), 5-hydroxyindoleacetic acid (5-HIAA) and norepinephrine (NE) as well as uptake of serotonin by crude synaptosomal (P2) fraction were determined in a group of rats chronically (for 21 days) treated with different types of antidepressant drugs: nortriptyline, fluoxetine, iprindole, phenelzine (10 mg/kg per day), maprotiline (20 mg/kg per day) and vehicle only (controls). Quantitative analysis of imipramine competition curves confirmed the existence of two classes of [3H]imipramine sites: high-affinity with IC50 and 11.2 nM and low-affinity with IC50 of 630 nM for the competing ligand. The proportion of high- and low-affinity sites was 73 +/- 4 and 26 +/- 4%, respectively. Chronic treatment with all antidepressant drugs except iprindole significantly decreased the affinity but not the proportion of high-affinity sites for imipramine. IC50 of imipramine at low-affinity sites was even more markedly increased at low-affinity sites by all treatments except for phenelzine. Fluoxetine was by far the most effective in altering the affinity of both high- and low-affinity [3H]imipramine recognition sites. Both NE and 5-HT levels were significantly enhanced only by phenelzine treatment, whereas 5-HT and 5-HIAA levels were found to be lower after fluoxetine. Kinetics of 5-HT uptake were altered significantly only in rats treated with fluoxetine: rate of 5-HT uptake (Vmax) was decreased by 43% and Km value increased from 104 to 184 nM. Changes in affinity of imipramine for its binding sites were not found to be associated with the effect of tested drugs on 5-HT levels or uptake. They may be due to adaptive alterations in physico-chemical properties of binding proteins although the presence of residual drug interfering with the binding assay cannot be excluded. The observed changes are likely to represent the condition during chronic administration of these drugs in clinical therapy of depression.     

Alpha 2-adrenoceptor- and D2-dopamine receptor-mediated analgesic response of B-HT 920.

The involvement of D2-dopamine receptors in the antinociceptive action of B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-(4H) thiazolo-(4,5d)-azepine) has been investigated in mice. B-HT 920 (0.1-2.0 mg kg-1) and apomorphine (0.1-2.0 mg kg-1) produced a dose-dependent increase in tail flick latency. Analgesia induced by apomorphine was blocked by the D2-antagonist, haloperidol (1 mg kg-1) but not by the opioid antagonist, naloxone (1 mg kg-1). The antinociceptive action of B-HT 920 was potentiated by the D1-agonist SKF 38393 (5 mg kg-1), an action antagonized by haloperidol. The selective alpha 2-adrenoceptor blocking drug yohimbine (1 mg kg-1) and naloxone (1 mg kg-1) blocked the antinociceptive action of B-HT 920 (1 mg kg-1). Haloperidol, however, failed to modify the B-HT 920-induced increase in tail flick latency. B-HT 920 and apomorphine reversed reserpine (2 mg kg-1) 4 h-induced hyperalgesia. The reversing action of apomorphine was blocked by haloperidol but not by yohimbine. Thus, a role of alpha 2-adrenoceptors and D2-dopamine receptors is postulated in the antinociceptive action of B-HT 920.     

Effects of tryptamine mediated through 2 states of the 5-HT2 receptor in calf coronary artery

Summary The mode of action of tryptamine was investigated on strips of left circumflex coronary artery of calf. 1) Exposure to (–)-deprenyl, an irreversible inhibitor of monoamine oxidase B, markedly potentiated the contractions caused by tryptamine but not those by 5-hydroxytryptamine (5-HT). Experiments were therefore carried out on arteries treated with (–)-deprenyl. 2) Tryptamine, administered non-cumulatively, elicited fast developing contractions, which partially faded. The intrinsic activity for the peak response to tryptamine was 0.8 compared to 5-HT. Ketanserin competitively antagonized the tryptamine-induced contractions with a K _B of (–log mol/1) 9.9. Methysergide antagonized the effects of tryptamine in a noncompetitive manner by depressing the maximum response with an IC₅₀ (–log mol/1) >9.0. 3) Tryptamine caused unsurmountable depression of 5-HT-induced contractions with an IC₅₀ (–log mol/1) of 6.4. Ketanserin also competitively antagonized the depressant effects of tryptamine on 5-HT-induced contractions with a K _B of (–log mol/1) 9.9. 4) At high concentrations of tryptamine (0.2–1 mmol/l), the fast developing contractions were followed by slowly developing contractions. Methysergide 1 nmol/l enhanced maximally the slow developing contractions. 5) These findings are consistent with an interaction of tryptamine at different sites of the allosteric 5-HT₂-receptor system: (I) Tryptamine competes with ketanserin for the 5-HT₂-receptor in the highly active R state. Binding of tryptamine to the R state would cause the fast contraction. (II) Tryptamine competes with ketanserin for the allosteric site. Tryptamine induces allosterically a shift from R to the low active R state as methysergide does, thereby depressing the response to 5-HT. Ketanserin restores the response to 5-HT depressed by tryptamine. (III) Tryptamine appears to bind also to the R state of the 5-HT₂ receptor thereby causing the slowly developing contractions.[/p] Send offprint requests to A. J. Kaumann at the above address