The agonist activities of the putative antipsychotic agents, L‐745,870 and U‐101958 in HEK293 cells expressing the human dopamine D4.4 receptor
Lucien GaziIonel BobirnacM DanzeisenE. SchüpbachAnne T. BruinvelsSabine GeisseBernd SommerDaniël HoyerMark D. TricklebankPhilippe Schoeffter
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1. Dopamine D4 receptor antagonists are being developed by several pharmaceutical companies as putative novel antipsychotics, possibly with low propensity to side-effects. Two such compounds, L-745,870 and U-101958 have been recently introduced. 2. The radioligand binding and functional activities of L-745,870 and U-101958 were investigated in human embryonic kidney (HEK)293 cells expressing the human recombinant dopamine D4.4 receptor (HEK293/D4 cells). [3H]-spiperone binding experiments were performed and inhibition of forskolin-stimulated cyclic AMP accumulation was used as the functional response. 3. [3H]-spiperone was found to label a homogeneous and saturable population of specific binding sites in HEK293/D4 cell homogenates (Bmax 505+/-90 fmol mg(-1) protein, pK(D) 9.5+/-0.1, n=3). Inhibition of specific [3H]-spiperone binding was observed with spiperone (pKi 9.6+/-0.1, n=3), clozapine (pKi 7.4+/-0.1, n=4), L-745,870 (pKi 8.5+/-0.1, n=3) and U-101958 (pKi 8.9+/-0.1, n=3). By contrast, raclopride was very weak (pKi < 5, n=3). 4. Dopamine inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D4 cells in a concentration-dependent fashion (Emax 71+/-2% inhibition of forskolin-stimulated levels, pEC50 8.7+/-0.1, n=10). This effect was mimicked by the dopamine D2-like receptor agonists, quinpirole and 7-hydroxy-2-dipropylaminotetralin (7-OH-DPAT). 5. L-745,870 and U-101958 also inhibited forskolin-stimulated cyclic AMP accumulation in HEK293/D4 cells in a concentration-dependent way. L-745,870 was less efficacious than dopamine (71% the efficacy of dopamine), whereas U-101958 behaved as a full agonist compared to dopamine. Potencies (pEC50) values of L-745,870 and U-101958 were 9.0+/-0.2 (n=4) and 8.7+/-0.3 (n=3), consistent with pKi values determined in radioligand binding studies. 6. Dopamine, L-745,870 and U-101958 (up to 1 microM) were devoid of effect on forskolin-stimulated cyclic AMP accumulation in control, non-transfected HEK293 cells. 7. The agonist effects of dopamine, L-745,870 and U-101958 in HEK293/D4 cells could be antagonized by spiperone (pK(B) 8.2-8.8) and clozapine (pK(B) 7.1), but not by raclopride (pK(B) < 5). None of these antagonists had any significant agonist activity at concentrations up to 10 microM. 8. These results show that the putative dopamine D4 receptor antagonists, L-745,870 and U-101958 are not devoid of intrinsic activity at human recombinant dopamine D4.4 receptors. Therefore, they may not represent the most appropriate drugs for testing the benefit of D4 receptor antagonism in schizophrenic patients, if agonism should translate in vivo.Keywords:
Spiperone
HEK 293 cells
Raclopride
Quinpirole
Pharmacological manipulations that alter dopamine (DA) at DA receptor subtypes produce reductions in feeding behaviour. What remains uncertain is the exact way in which these reductions in feeding are achieved as a consequence of differing drug actions at separate receptor subtypes. In this study our aim was to compare the anorectic effects of the preferential D3/D2 agonists 7-hydroxy-2-(di-n-propylamino)tetralin (7-OH-DPAT) and quinpirole and the non-selective D2/D3 antagonist raclopride on the microstructure of licking responses in non-deprived rats. In a 20-min test, trained adult, male hooded rats had access to one of three solutions: 1%, 3% or 10% sucrose. 7-OH-DPAT (0.1-1.0 mg/kg, i.p.), quinpirole (0.03–0.3 mg/kg, s.c.), raclopride (0.03–0.3 mg/kg, i.p.) or vehicle were injected 20 min prior to the start of the licking test. A lickometer recorded the timing of each lick, from which the microstructural parameters of bout frequency and bout duration were also computed. All compounds reduced the mean bout duration, while 7-OH-DPAT and raclopride also brought about a compensatory increase in bout number. Analysis of the licking rates over the test session showed that 7-OH-DPAT, quinpirole and raclopride decreased the initial rate, without affecting the rate of decline of licking. Changes in licking microstructure (i.e. initial rate of licking and mean bout duration) after the administration of 7-OH-DPAT, quinpirole and raclopride, are consistent with an action of these dopaminergic compounds to reduce palatability.
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The D2/D3 dopamine receptor agonist quinpirole (QNP) produces biphasic effects on locomotion and can induce oral behaviour. To determine whether or not these behaviours result from actions of QNP at D2 receptors located in the dorsal raphe, nucleus (DRN), rats were pretreated intra-DRN with the selective dopamine D2 receptor antagonist, raclopride (0, 1 or 2 μg) prior to an acute peripheral injection of QNP (0 or 0.5 mg/kg, s.c.). Intra-DRN raclopride did not affect QNP-induced oral activity or QNP-induced locomotor inhibition. However, the high dose of raclopride potentiated QNP-induced locomotor excitation. These data suggest that D2 receptor activation in the DRN suppresses QNP-induced locomotor excitation, an effect that may be related to D2 receptor-mediated alterations in serotonin transmission.
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The in vitro dopamine-D2 receptor binding profiles of two substituted benzamides (3H-sulpride and 3H-raclopride) and two butyrophenones (3H-spiperone and 3H-domperidone) were compared. 3H-Raclopride, 3H-domperidone and 3H-sulpride labelled approximately the same number of binding sites in the rat striatum, while 3H-spiperone labelled a higher number of binding sites. This latter finding was suggested to be due to the labelling of 5-HT2 receptors in addition to the dopamine-D2 binding sites since the labelling of these 5-HT2 receptors with 3H-spiperone could be avoided by the addition of the 5-HT2 receptor antagonist ketanserin. The inhibition constants (Ki) of the unlabelled compounds were similar to the Kd values obtained in the saturation analyses with 3H-raclopride, 3H-domperidone and 3H-sulpiride but were significantly higher when 3H-spiperone was used as radioligand. Addition of ketanserin to 3H-spiperone made the Ki values more similar to those obtained with the other radioligands. The implications of these findings in testing antidopaminergic activity is discussed.
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Abstract We recently investigated the binding properties of the antagonists [ 3 H]‐raclopride and [ 3 H]‐spiperone to intact Chinese hamster ovary cells expressing recombinant human D 2long ‐dopamine receptors (CHO‐D 2L cells). Compared with saturation binding with [ 3 H]‐raclopride, raclopride reduced [ 3 H]‐spiperone binding with to low potency in competition binding experiments. The present findings illustrate the ability of spiperone to inhibit [ 3 H]‐raclopride binding non‐competitively. While raclopride only decreases the apparent K D of [ 3 H]‐raclopride in saturation binding experiments, spiperone only decreases the number of sites to which [ 3 H]‐raclopride binds with high affinity. Also, while the IC 50 of raclopride depends on the concentration of [ 3 H]‐raclopride in competition experiments, this is not the case for spiperone. Kinetic studies reveal that the binding of raclopride at its high affinity sites does not affect the association of subsequently added [ 3 H]‐spiperone nor the rebinding of freshly dissociated [ 3 H]‐spiperone to the same or surrounding receptors. Yet, spiperone does not affect the dissociation rate of [ 3 H]‐raclopride and raclopride does not affect the (genuine) dissociation rate of [ 3 H]‐spiperone. The easiest way to interpret the present findings in molecular terms is to assume that D 2L ‐receptors or their dimeric complexes possess two distinct binding sites: one with high affinity/accessibility for [ 3 H]‐raclopride and the other one with high affinity/accessibility for [ 3 H]‐spiperone. The ability of bound spiperone to inhibit high affinity raclopride binding while the reverse is not the case suggests for the occurrence of non‐reciprocal allosteric interactions. These new findings could point at the occurrence of allosteric interactions between different classes of D 2 ‐receptor antagonists.
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The effects of the mixed dopamine D2/D3 agonist, quinpirole and antagonist, raclopride on ethanol consumption were examined within both a limited and a continuous access paradigm. The goals of these studies were to determine if decreases in ethanol intake after administration of the dopaminergic agents were the result of the type of paradigm employed, reductions of DA-mediated reinforcement or rather to non-specific drug effects, such as locomotor suppression. Experiment 1 demonstrated that quinpirole (0.1 mg/kg) failed to alter ethanol intake in rats exposed to a 1hr-limited access period. Raclopride-treated (0.5 mg/kg) rats significantly decreased their ethanol intake but were ataxic for the entire 1-hr limited access period. These results suggested that manipulations of the D2/D3 receptors at the dose tested had non-specific effects on the consumption of ethanol. Experiment 2 assessed whether the effects obtained within a limited access paradigm were comparable to a 22-hr continuous access paradigm. Similar, to Experiment 1, quinpirole (0.1 mg/kg) failed to attenuate ethanol consumption within a continuous access paradigm. Contrary to Experiment 1, raclopride (0.5 mg/kg) significantly increased ethanol intake during the treatment and post-treatment periods. Taken together, the findings of Experiment 1 and 2 demonstrated that the D2/D3 agents employed did not produce reductions in ethanol intake specific to ethanol reinforcement. Further, the raclopride induced increase in ethanol intake in Experiment 2 is contradictory to previous reports that systemic injections of DA antagonists produce reductions in ethanol intake. The results suggested that the D2/D3 receptors are not likely to be the primary mediators of ethanol intake.
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Abstract The neurotransmitter dopamine plays an important role in the processing of emotional memories, and prior research suggests that dopaminergic manipulations immediately after fear learning can affect the retention and generalization of acquired fear. The current study focuses specifically on the role of dopamine D2 receptors (D2Rs) in adult, male Wistar rats. In a series of five experiments, D2R (ant)agonists were injected systemically immediately after differential cued fear conditioning (CS+ followed by shock, CS− without shock). All five experiments involved the administration of the D2R agonist quinpirole at different doses versus saline (n = 12, 16 or 44 rats/group). Additionally, one of the studies administered the D2R antagonist raclopride (n = 12). One day later, freezing during the CS+ and CS− was assessed. We found no indications for an effect of quinpirole or raclopride on fear generalization during this drug-free test. Importantly, and contradicting prior research in mice, the evidence for the absence of an effect of quinpirole (1 mg/kg) on fear generalization was substantial according to Bayesian analyses and was observed in a highly powered experiment (N = 87). We did find acute behavioral effects in line with the literature, for both quinpirole and raclopride in a locomotor activity test.
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