Modification of dopamine D(1) receptor knockout phenotype in mice lacking both dopamine D(1) and D(3) receptors

Experimental evidence suggests that dopamine D(1) and D(3) receptors may interact in an opposing or synergistic fashion. To investigate interactions between both receptors in behaviour, we have used dopamine D(1) and D(3) receptor knockout mice to generate mice lacking both receptors. D(1)(-/-)D(3)(-/-) mice were viable, fertile and showed no gross morphological abnormalities. In an open field, they exhibited lower activity than wild-type, D(1)(-/-) and D(3)(-/-) mice. D(1)(-/-)D(3)(-/-) mice performed equally poorly in the rotarod and Morris water maze tasks as their D(1)(-/-) littermates. Basal locomotor activity and anxiety-like behaviour were normal in D(1)(-/-)D(3)(-/-) mice. Combined deletion of both receptors abolished the exploratory hyperactivity and anxiolytic-like behaviour of dopamine D(3) receptor mutant phenotype and further attenuated the low exploratory phenotype of D(1)(-/-) mice. These results imply an interaction of both receptors in the expression of exploratory behaviour in a novel environment, and the need for the presence of intact dopamine D(1) receptor for the expression of certain behaviours manifested in dopamine D(3) receptor mutant phenotype. In addition, dopamine D(1) receptor, but not dopamine D(3) receptor, is involved in the ability to perform on the rotarod and spatial learning.     

Structural determinants of pharmacological specificity between D(1) and D(2) dopamine receptors

To test the hypothesis that pharmacological differentiation between D(1) and D(2) dopamine receptors results from interactions of selective ligands with nonconserved residues lining the binding pocket, we mutated amino acid residues in the D(2) receptor to the corresponding aligned residues in the D(1) receptor and vice versa and expressed the receptors in human embryonic kidney 293 cells. Determinations of the affinity of the 14 mutant D(2) receptors and 11 mutant D(1) receptors for D(1)- and D(2)-selective antagonists, and rhodopsin-based homology models of the two receptors, identified two residues whose direct interactions with certain ligands probably contribute to ligand selectivity. The D(1) receptor mutant W99(3.28)F showed dramatically increased affinity for several D(2)-selective antagonists, particularly spiperone (225-fold), whereas the D(2) receptor mutant Y417(7.43)W had greatly decreased affinity for benzamide ligands such as raclopride (200-fold) and sulpiride (125-fold). The binding of the D(1)-selective ligand R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) was unaffected, indicating that SCH23390 makes little contact with these ancillary pocket residues. Mutation of A/V(5.39) caused modest but consistent and reciprocal changes in affinity of the receptors for D(1) and D(2)-selective ligands, perhaps reflecting altered packing of the interface of helices 5 and 6. We also obtained some evidence that residues in the second extracellular loop contribute to ligand binding. We conclude that additional determinants of D(1)/D(2) receptor-selective binding are located either in that loop or in the transmembrane helices but, like residue 5.39, indirectly influence the interactions of selective ligands with conserved residues by altering the shape of the primary and ancillary binding pockets.     

Saturation of striatal D(2) dopamine receptors by clozapine

Positron emission tomography (PET) studies have demonstrated low striatal D2 dopamine receptor occupancy in clozapine-treated schizophrenic patients. The aim of this pilot study was to explore if this low receptor occupancy indeed represents partial saturability of striatal D2 dopamine receptors by clozapine. Three anaesthetized Cynomolgus monkeys were examined during one day with PET and [11C]raclopride at baseline and after intravenous injections of clozapine 1.5 mg/kg followed by 18.5 mg/kg. The estimated corresponding human oral doses were approx. 210 mg/d and 2800 mg/d. D2 dopamine receptor occupancy was calculated using an equilibrium-ratio analysis and ranged from 54 to 58% after 1.5 mg/kg and 87 to 89% after the total dose 20 mg/kg. The calculated maximal occupancy was 93%. We conclude that PET-measured D2 dopamine receptor occupancy by clozapine can be described using a model based on the law of mass action, previously validated for conventional antipsychotics. Therefore, sufficiently high doses of clozapine are expected to produce complete striatal D2 dopamine receptor occupancy. The findings further support our previous findings of low D2 dopamine receptor occupancy in patients treated with standard doses of clozapine.     

WAY-100635 has high selectivity for serotonin 5-HT(1A) versus dopamine D(4) receptors

The serotonin 5-HT(1A) receptor antagonist WAY-100635 was recently reported to have potent agonist properties at dopamine D(4) receptors (Chemel et al., 2006, Psychopharmacology 188, 244-251.). Herein WAY-100635 (pK(i) at human (h) serotonin 5-HT(1A) receptors=9.51; pK(i) at dopamine hD(4.4) receptors=7.42) stimulated [(35)S]GTPgammaS incorporation in membranes of Chinese Hamster Ovary cells expressing dopamine hD(4.4) receptors with only moderate potency and modest efficacy (pEC(50)=6.63; E(max)=19% of dopamine). Moreover, in antagonism experiments, WAY-100635 had a much lower potency at dopamine hD(4.4) receptors (pK(B)=7.09), than at serotonin h5-HT(1A) receptors (pK(B)=9.47). These data demonstrate that WAY-100635 has high selectivity for serotonin h5-HT(1A)versus dopamine hD(4.4) receptors.     

Psychomotor-activating effects mediated by dopamine D(2) and D(3) receptors in the nucleus accumbens

The contribution made by specific dopamine receptor subtypes to the induction of motor behaviors has not been firmly established. Here, we first characterized the behavioral effects induced by a D(2)-class receptor agonist, bromocriptine, following injections into the nucleus accumbens (Acb). Bromocriptine showed an atypical D(2)-class receptor agonist profile, having no observable effect on a range of motor behaviors. However, when coadministered with the D(1)-class receptor agonist SKF 38393, bromocriptine showed a typical D(2)-class receptor agonist profile, enhancing locomotor activity and suppressing spontaneous yawning. We then administered the dopamine receptor antagonists L-741626 and nafadotride, which possess relative selectivity for D(2) and D(3) receptors, respectively, prior to injections of dopamine agonists into the Acb. Nafadotride significantly reduced the locomotor-enhancing effects elicited by the coadministration of SKF 38393 and the D(2)-class receptor agonist (+)-PD 128907 into the Acb, and also attenuated the effects induced by the combination of SKF 38393 and bromocriptine, although not significantly so. L-741626 mildly attenuated the locomotor effects elicited by both drug combinations. Taken together, these results suggest that both D(2) and D(3) receptors in the Acb contribute to the expression of heightened psychomotor activation.     

The dopamine D(4) receptor: one decade of research

Dopamine is an important neurotransmitter involved in motor control, endocrine function, reward, cognition and emotion. Dopamine receptors belong to the superfamily of G protein-coupled receptors and play a crucial role in mediating the diverse effects of dopamine in the central nervous system (CNS). The dopaminergic system is implicated in disorders such as Parkinson's disease and addiction, and is the major target for antipsychotic medication in the treatment of schizophrenia. Molecular cloning studies a decade ago revealed the existence of five different dopamine receptor subtypes in mammalian species. While the presence of the abundantly expressed dopamine D(1) and D(2) receptors was predicted from biochemical and pharmacological work, the cloning of the less abundant dopamine D(3), D(4) and D(5) receptors was not anticipated. The identification of these novel dopamine receptor family members posed a challenge with respect to determining their precise physiological roles and identifying their potential as therapeutic targets for dopamine-related disorders. This review is focused on the accomplishments of one decade of research on the dopamine D(4) receptor. New insights into the biochemistry of the dopamine D(4) receptor include the discovery that this G protein-coupled receptor can directly interact with SH3 domains. At the physiological level, converging evidence from transgenic mouse work and human genetic studies suggests that this receptor has a role in exploratory behavior and as a genetic susceptibility factor for attention deficit hyperactivity disorder.     

Regulation of human D(1), d(2(long)), d(2(short)), D(3) and D(4) dopamine receptors by amiloride and amiloride analogues

1. The modulatory effects of the allosteric effectors methylisobutylamiloride (MIA), benzamil and amiloride have been examined at human D(1), D(2), D(3) and D(4) dopamine receptors. The subtype selectivity and the mechanism of action of this allosteric regulation was examined. 2. In radioligand dissociation experiments each modulator accelerated dissociation from all four receptor subtypes indicating allosteric regulation. MIA displayed selectivity for the D(3) subtype for acceleration of radioligand dissociation. 3. In equilibrium binding (pseudo-competition) experiments the three compounds inhibited radioligand binding at the four receptor subtypes. Inhibition curves for D(1), D(2(short)), D(2(long)) and D(3) receptors were described by Hill coefficients exceeding unity and data were fitted best by a model that assumes binding of modulator to both the primary and allosteric binding sites of the receptor (the allosteric/competitive model). 4. At the D(4) subtype, Hill coefficients of unity described the binding data for amiloride and benzamil, consistent with competitive inhibition. The Hill coefficient for MIA at the D(4) subtype was less than unity and data could be fitted well by the allosteric/competitive model, but it was not possible to define unambiguously the modulatory mechanism. For this effect a better definition of the mechanism could be obtained by simultaneous analysis of data obtained in the presence of a range of concentrations of a purely competitive ligand. 5. MIA reduced the potency with which dopamine stimulated [(35)S]-GTPgammaS binding at the D(2) receptor. The effects of MIA could be described by the allosteric/competitive model with effects of MIA to inhibit the binding of dopamine but not its ability to induce a response.     

Association of dopamine D(3) receptors with actin-binding protein 280 (ABP-280)

Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280.     

Effect of repeated treatment with tianeptine and fluoxetine on central dopamine D(2) /D(3) receptors

Tianeptine (TIA) is an antidepressant drug that has been shown to decrease extracellular serotonin level and reveals no affinity for neurotransmitter receptors. The present study was aimed at determining whether repeated TIA treatment induced any adaptive changes in the central dopamine D(2)/D(3) system (behavioural and biochemical) similar to those reported earlier for tricyclic antidepressants. Experiments were carried out on male Wistar rats. TIA was administered at a dose of 5 and 10 mg/kg once or repeatedly (twice daily for 14 days). Fluoxetine (FLU), used as a reference compound, was also administered at a dose of 10 mg/kg. The results obtained showed that TIA or FLU administered repeatedly increased the hyperlocomotion induced by D-amphetamine and 7-hydroxy-dipropylaminotetralin (7-OH-DPAT). Biochemical study revealed a decrease in the [(3)H]7-OH-DPAT binding sites after acute and repeated treatment with TIA or FLU in the islands of Calleja minor, as well as in the shell part of nucleus accumbens septi. On the other hand, both TIA and FLU administered repeatedly increased the binding of [(3)H]quinpirole (a D(2)/D(3) receptor agonist) in the nucleus caudatus as well as in the core part of the nucleus accumbens septi. Similar effects have been observed when dopamine D(2)/D(3) receptors were visualized with the use of [3H]raclopride, a dopamine D(2)/D(3) receptor antagonist. However, TIA and FLU induced a decrease in the level of mRNA encoding for dopamine D(2) receptors, not only after repeated but also after acute treatment. These results indicate that repeated TIA and FLU administration induces adaptive changes in the dopaminergic D(2)/D(3) system and especially enhances the functional responsiveness of dopamine D(2) and D(3) receptors. However, the question of whether this increased responsiveness is important for clinical antidepressant efficacy remains open.     

Effect of acute and chronic stress restraint on amphetamine-associated place preference: involvement of dopamine D(1) and D(2) receptors

Fenfluramine + phentermine was a widely used combination for weight loss. Fenfluramine and phentermine are believed to act via serotonin and catecholamines, respectively. To what extent these drugs interact has not been well-established. We compared the anorectic efficacy of a range of doses of the combination (using dexfenfluramine instead of fenfluramine) relative to a range of doses of the individual drugs in 90 min sweetened milk intake tests in deprived and nondeprived rats. Results were plotted on isobolograms to determine whether the anorectic effects of the combination were either additive or synergistic. Collectively, the isobolographic analysis revealed that: (1) under acute conditions, dexfenfluramine and phentermine interact for the most part in a synergistic manner, and (2) with the exception of phentermine alone, deprivation state at time of testing did not alter the efficacy of the anorectics. These findings suggest that combined drug treatment for obesity is a theoretical approach that merits further investigation.     

Short- and long-term heterologous sensitization of adenylate cyclase by D4 dopamine receptors

The D₄ dopamine receptor, a member of the D₂-like dopamine receptor family, may be important in the etiology and treatment of schizophrenia. The present study was designed to examine the effects of dopamine agonist exposure on adenylate cyclase activity in HEK293 cells stably expressing recombinant-D₄ receptors. Two hour pretreatment with dopamine receptor agonists resulted in heterologous sensitization of forskolin-stimulated cyclic AMP accumulation in intact cells expressing the D_4.2, D_4.4, or D_4.7 dopamine receptor variant. The potency and efficacy of dopamine for sensitization of cyclic AMP accumulation was comparable at all D₄ receptor variants. D₄ dopamine receptor-mediated sensitization was blocked by the D₄ antagonist, clozapine, and prevented by overnight pretreatment with pertussis toxin, implying a role for G_i/G_o proteins in heterologous sensitization. Further, long-term (18 h) agonist exposure resulted in a greater degree of sensitization of forskolin-stimulated cyclic AMP accumulation in both intact cells and membrane preparations of cells expressing the D₄ receptor, compared to 2 h agonist exposure, without altering the density of the receptors. In addition, long-term agonist exposure decreased the abundance of G_iα without altering the abundance of G_sα, whereas short-term agonist treatment had no effect on the immunoreactivity of either G protein. In summary, long-term agonist-induced sensitization of adenylate cyclase by the D₄ receptor may involve mechanisms that do not contribute to short-term sensitization. Received: 26 March 1998/Final version: 6 May 1998     

Pharmacological and functional characterisation of dopamine D4 receptors in the rat retina

In the retina, activation of dopamine receptors, particularly the D2-like family (D2, D3, D4 receptor subtypes), with quinpirole suppresses the light sensitive cAMP pool and inhibits melatonin synthesis in photoreceptor cells. We have characterised rat retinal D4 receptors using the D4 selective radioligand [¹²⁵I] L-750667 which bound specifically and saturably to rat retinal membranes with high affinity (K_d 0.06±0.02 nM) and exhibited a D4 receptor pharmacology. Comparison of the binding kinetics of [¹²⁵I] L-750667 and [³H] spiperone revealed B_max values of 134±27 fmol/mg and 219±47 fmol/mg respectively, indicating that the dopamine D4 receptor is a major component of D2-like dopamine receptors in the rat retina. Modulation of retinal cAMP levels by quinpirole was used to evaluate the functional relevance of rat retinal dopamine D4 receptors. Quinpirole (0.03–3 μM) produced a dose-related decrease of the light sensitive cAMP pool which was reversed by haloperidol, clozapine and the D4 selective antagonist, L-745870 with a rank order of potency suggesting that the quinpirole effect is due to activation of the dopamine D4 receptors. The D2 selective ligand L-741626 had no effect on the quinpirole response confirming that the D4 receptor is the major receptor subtype mediating dopamine induced suppression of adenylate cyclase in the retina.     

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.