3- and 4-O-sulfoconjugated and methylated dopamine: highly reduced binding affinity to dopamine D2 receptors in rat striatal membranes

Summary The binding properties of 3- and 4-O-sulfoconjugated dopamine (DA-3-0-S, DA-4-0-S) as well as 3-O-methylated dopamine (MT) to rat striatal dopamine D₂ receptors were investigated. ³H-spiperone was used as a radioligand in the binding studies. In saturation binding experiments (+)butaclamol, which has been reported to bind to dopaminergic D₂ and serotoninergic 5HT₂ receptors, was used in conjunction with ketanserin and sulpiride, which preferentially label 5HT₂ and D₂ receptors, respectively, in order to discriminate between ³H-spiperone binding to D₂ and to 5HT₂ receptors. Under our particular membrane preparation and assay conditions, ³H-spiperone binds to D₂ and 5HT₂ receptors with a maximal binding capacity (B _max) of 340 fmol/mg protein in proportions of about 75%:25% with similar dissociation constants K _D (35 pmol/l; 43 pmol/l). This result was verified by the biphasic competition curve of ketanserin, which revealed about 20% high (K _D = 24 nmol/l) and 80% low (K _D = 420 nmol/l) affinity binding sites corresponding to 5HT₂ and D₂ receptors, respectively. Therefore, all further competition experiments at a tracer concentration of 50 pmol/l were performed in the presence of 0.1 mol/l ketanserin to mask the 5HT₂ receptors. DA competition curves were best fitted assuming two binding sites, with high (K _H = 0.12 mol/l) and low (K_L = 18 mol/l) affinity, present in a ratio of 3:1. The high affinity binding sites were interconvertible by 100 mol/l guanyl-5-yl imidodiphosphate [Gpp(NH)p], resulting in a homogenous affinity state of DA receptors (K _D = 2.8 mol/l). Competition experiments with various compounds confirmed the binding of ³H-spiperone to D₂ receptors. DA-3-O-S, DA-4-O-S, and MT were more than 5,000-, more than 10,000-, and 530-fold less potent in competing for ³H-spiperone binding when compared with DA at the high affinity binding site which mediates biological effects. Therefore, it is concluded that these DA metabolites are biologically ineffective at central D₂ receptors. Send offprint requests to E. Werle at the above address     

Roles of conserved intracellular sequences regions for the proper expression of dopamine D2 and D3 receptors

The dopamine D(2) receptor and D(3) receptor (D(2)R, D(3)R) have high homology in both their amino acid composition and signaling pathways. Virtually all signaling pathways reported thus far overlap between the two receptors with the exception that the D(3)R signals are 2 approximately 5 times less efficient than D(2)R. Previous studies have suggested that conformational constraints of D(3)R might be responsible for the poor coupling with the G protein. To this hypothesis, point mutations were introduced into some of the conserved regions between D(2)R and D(3)R, and their effects on receptor expression were investigated. Among the four conserved intracellular receptor regions examined (TTT motif in the 1(st) intracellular loop, SS motif in the 2(nd) intracellular loop, YxxL and TxxS/xS motifs in the 3(rd) intracellular loop), a mutation of the Thr-Thr-Thr (TTT) motif in the first intracellular loop or the LxxY motif in the 3(rd) intracellular loop markedly decreased the level of D(3)R expression compared with D(2)R. The TTT motif was further mutated individually or in combination to test which residue plays a critical role on the expression of the receptor proteins. Different amino acids between D(2)R and D(3)R in the 1(st) intracellular loop were exchanged to determine if the adjacent amino acid residues are responsible for the differences between D(2)R and D(3)R. The first two threonine residues become more important when the individual threonine residue is mutated. However, all three intact threonine residues are essential for proper expression of the receptor proteins. The neighboring sequences around the triplet threonine residues in the 1(st) loop of D(3)R are not important for proper positioning of the receptor proteins on the plasma membrane. It was concluded that D(2)R has a more flexible overall conformation that can accept mutated residues in the intracellular region than D(3)R, which might be partly responsible for the quantitative differences in the signaling efficiency between D(2)R and D(3)R.     

In vitro pharmacology of aripiprazole, its metabolite and experimental dopamine partial agonists at human dopamine D2 and D3 receptors

Aripiprazole is the first dopamine D₂/D₃ receptor partial agonist successfully developed and ultimately approved for treatment of a broad spectrum of psychiatric and neurological disorders. Aripiprazole's dopamine D₂ and serotonin 5-HT_1A receptor partial agonist activities have been postulated to confer clinical efficacy without marked sedation, and a relatively favorable overall side-effect profile. Using aripiprazole's unique profile as a benchmark for new dopamine partial agonist development may facilitate discovery of new antipsychotics. We conducted an in vitro comparative analysis between aripiprazole, and its human metabolite OPC-14857 (7-(4-[4-(2,3-dichlorophenyl)-1-piperazinyl)butoxy)-2(1H)-quinolinone)); RGH-188 (trans-1-[4-[2-[4-(2,3-dichlorophenyl)piperazine-1-yl]ethyl]cyclohexyl]-3,3-dimethylurea), and its metabolite didesmethyl-RGH-188 (DDM-RGH-188); as well as bifeprunox, sarizotan, N-desmethylclozapine (NDMC; clozapine metabolite), and SDZ 208-912 (N-[(8α)-2-chloro-6-methylergolin-8-yl]-2,2-dimethylpropanamide). In vitro pharmacological assessment included inhibition of forskolin-stimulated cAMP accumulation and the reversal of dopamine-induced inhibition in clonal Chinese hamster ovary cell lines expressing D_2S, D_2L, D₃ Ser-9 and D₃ Gly-9 for human dopamine receptors. All test compounds behaved as dopamine D₂/D₃ receptor partial agonists. Aripiprazole's intrinsic activity at dopamine D_2S and D_2L receptors was similar to that of OPC-14857 and RGH-188; lower than that of dopamine and bifeprunox; and higher than that of DDM-RGH-188, SDZ 208-912, sarizotan, and NDMC. Aripiprazole's intrinsic activity at dopamine D₃ Ser-9 and D₃ Gly-9 receptors was similar to that of OPC-14857 and sarizotan; lower than that of dopamine, bifeprunox, RGH-188 and DDM-RGH-188; and higher than that of SDZ 208-912 and NDMC. A consolidated assessment of these findings may help defining the most appropriate magnitude of intrinsic activity at dopamine D₂/D₃ receptors for clinical efficacy and safety.     

Marked changes in signal transduction upon heteromerization of dopamine D1 and histamine H3 receptors

Background and purpose:

Functional interactions between the G protein-coupled dopamine D₁ and histamine H₃ receptors have been described in the brain. In the present study we investigated the existence of D₁–H₃ receptor heteromers and their biochemical characteristics.

Experimental approach:

D₁–H₃ receptor heteromerization was studied in mammalian transfected cells with Bioluminescence Resonance Energy Transfer and binding assays. Furthermore, signalling through mitogen-activated protein kinase (MAPK) and adenylyl cyclase pathways was studied in co-transfected cells and compared with cells transfected with either D₁ or H₃ receptors.

Key results:

Bioluminescence Resonance Energy Transfer and binding assays confirmed that D₁ and H₃ receptors can heteromerize. Activation of histamine H₃ receptors did not lead to signalling towards the MAPK pathway unless dopamine D₁ receptors were co-expressed. Also, dopamine D₁ receptors, usually coupled to G_s proteins and leading to increases in cAMP, did not couple to G_s but to G_i in co-transfected cells. Furthermore, signalling via each receptor was blocked not only by a selective antagonist but also by an antagonist of the partner receptor.

Conclusions and implications:

D₁–H₃ receptor heteromers constitute unique devices that can direct dopaminergic and histaminergic signalling towards the MAPK pathway in a G_s-independent and G_i-dependent manner. An antagonist of one of the receptor units in the D₁–H₃ receptor heteromer can induce conformational changes in the other receptor unit and block specific signals originating in the heteromer. This gives rise to unsuspected therapeutic potentials for G protein-coupled receptor antagonists.     

Relative roles of ventral striatal D1 and D2 dopamine receptors in responding with conditioned reinforcement

Several experiments investigated the involvement of D₁ and D₂ dopamine receptors in the ventral striatum in the control over behaviour by a conditioned reinforcer using an acquisition of new response procedure. Intra-accumbens infusion of either the D₁ receptor antagonist, SCH 23390, or the D₂ receptor antagonist, raclopride, completely blocked the potentiative effects of intra-accumbensd-amphetamine on responding with conditioned reinforcement and reduced responding to control levels. SCH 23390 was more potent than raclopride. At higher doses in the absence ofd-amphetamine, both antagonists also blocked the preference for responding on the lever producing the conditioned reinforcer. Intra-accumbens infusions of either the D₁ receptor agonist, SKF 38393, or the D_2/3 receptor agonist, LY 171555 (quinpirole), selectively potentiated responding on the lever producing the conditioned reinforcer. Various combined infusions of the D₁ and D₂ agonists in specific low doses had additive, but not synergistic, effects on responding with conditioned reinforcement. None of the drugs affected the drinking of water in deprived subjects when infused intra-accumbens. These results suggest that both D₁ and D₂ receptors in the nucleus accumbens are involved in mediating the effects of dopamine in potentiating the control over behaviour by conditioned reinforcers.     

Unique roles of G protein-coupled histamine H2 and gastrin receptors in growth and differentiation of gastric mucosa

Disruption of histamine H2 receptor and gastrin receptor had different effects growth of gastric mucosa: hypertrophy and atrophy, respectively. To clarify the roles of gastrin and histamine H2 receptors in gastric mucosa, mice deficient in both (double-null mice) were generated and analyzed. Double-null mice exhibited atrophy of gastric mucosae, marked hypergastrinemia and higher gastric pH than gastrin receptor-null mice, which were unresponsive even to carbachol. Comparison of gastric mucosae from 10-week-old wild-type, histamine H2 receptor-null, gastrin receptor-null and double-null mice revealed unique roles of these receptors in gastric mucosal homeostasis. While small parietal cells and increases in the number and mucin contents of mucous neck cells were secondary to impaired acid production, the histamine H2 receptor was responsible for chief cell maturation in terms of pepsinogen expression and type III mucin. In double-null and gastrin receptor-null mice, despite gastric mucosal atrophy, surface mucous cells were significantly increased, in contrast to gastrin-null mice. Thus, it is conceivable that gastrin-gene product(s) other than gastrin-17, in the stimulated state, may exert proliferative actions on surface mucous cells independently of the histamine H2 receptor. These findings provide evidence that different G-protein coupled-receptors affect differentiation into different cell lineages derived from common stem cells in gastric mucosa.     

Characterization of functional roles of DRY motif in the 2nd intracellular loop of dopamine D2 and D3 receptors

Dopamine D₂R and D₃R (D₂R, D₃R) show very high sequence homology and employ virtually identical signaling pathways even though D₂R is 2 ∼ 5 times more active. Among the structural motifs identified, a triplet sequence, Asp-Arg-Tyr (DRY motif), plays critical roles in the determination of receptor conformations for signaling and intracellular trafficking of G protein-coupled receptors by forming intramolecular interactions. Thus, it is possible that different signaling efficiencies of D₂R and D₃R might be caused by the receptor activation levels stabilized by their own DRY motifs. In this study, the Arg and Asp residues of D₂R and D₃R were mutated, and resulting changes in their signaling and intracellular trafficking properties were comparatively studied. Mutation of the Arg residues of D₂R and D₃R abolished their signaling but differently affected their intracellular localizations. The wildtype and R132H-D₂R were expressed mainly on the plasma membrane. On the other hand, compared with the wildtype D₃R, a substantial amount of R128H-D₃R was localized intracellularly. The expression of receptor proteins on the plasma membrane and their signaling efficiencies were more drastically affected by the mutation of the Asp residue of D₃R than D₂R. Therefore, it was concluded that the different levels of conformational strain exerted by the DRY motif might partly determine the quantitative differences in the signaling efficiencies between D₂R and D₃R. These authors contributed equally to this work.     

Pre-synaptic histamine H3 receptors regulate glutamate, but not GABA release in rat thalamus

We have investigated the presence of histamine H(3) receptors (H(3)Rs) on rat thalamic isolated nerve terminals (synaptosomes) and the effect of their activation on glutamate and GABA release. N-alpha-[methyl-(3)H]histamine ([(3)H]-NMHA) bound specifically to synaptosomal membranes with dissociation constant (K(d)) 0.78+/-0.20 nM and maximum binding (B(max)) 141+/-12fmol/mg protein. Inhibition of [(3)H]-NMHA binding by histamine and the H(3)R agonist immepip fit better to a two-site model, whereas for the H(3)R antagonist clobenpropit the best fit was to the one-site model. GTPgammaS (30 microM) decreased [(3)H]-NMHA binding by 55+/-4% and made the histamine inhibition fit better to the one-site model. Immepip (30 nM) induced a modest, but significant increase (113+/-2% of basal) in [(35)S]-GTPgammaS binding to synaptosomal membranes, an effect prevented by clobenpropit (1 microM) and by pre-treatment with pertussis toxin. In thalamus synaptosomes depolarisation-induced, Ca(2+)-dependent glutamate release was inhibited by histamine (1 microM, 25+/-4% inhibition) and immepip (30 nM, 38+/-5% reduction). These effects were reversed by clobenpropit (1microM). Conversely, immepip (up to 1 microM) had no effect on depolarisation-evoked [(3)H]-GABA release. Extracellular synaptic responses were recorded in the thalamus ventrobasal complex by stimulating corticothalamic afferents. H(3)R activation reduced by 38+/-7% the glutamate receptor-mediated field potentials (FPs), but increased the FP2/FP1 ratio (from 0.86+/-0.03 to 1.38+/-0.05) in a paired-pulse paradigm. Taken together, our results confirm the presence of H(3)Rs on thalamic nerve terminals and show that their activation modulates pre-synaptically glutamatergic, but not GABAergic neurotransmission.     

Activation of histamine H3 receptors in human nasal mucosa inhibits sympathetic vasoconstriction

The peripheral histamine H3 receptor is a presynaptic heterologous receptor located on postganglionic sympathetic nerve fibers innervating sympathetic effector systems such as blood vessels and the heart. An extensive body of evidence shows that activation of the histamine H3 receptor attenuates sympathetic tone by presynaptic inhibition of noradrenaline release. It is proposed that this sympathoinhibitory action, in vivo, leads to reduced vasoconstriction, thereby eliciting a vasodilatory effect. In humans, the peripheral histamine H3 receptor has also been shown to exert a sympathoinhibitory function on specific peripheral autonomic effector systems. For example, human saphenous vein and heart possess functional presynaptic histamine H3 receptors on the sympathetic nerve terminals that upon activation decrease the sympathetic tone to these respective organs. The present studies were conducted to define the role of histamine H3 receptors on neurogenic sympathetic vasoconstrictor responses in human nasal turbinate mucosa. Contractility studies were conducted to evaluate the effect of histamine H3 receptor activation on sympathetic vasoconstriction in surgically isolated human nasal turbinate mucosa. We found that the histamine H3 receptor agonist, (R)-alpha-methylhistamine (30 and 300 nM), inhibited electrical field stimulation-induced (neurogenic) sympathetic vasoconstriction in a concentration-dependent fashion. Pretreatment with the selective histamine H3 receptor antagonist, clobenpropit (100 nM), blocked the sympathoinhibitory effect of (R)-alpha-methylhistamine on the neurogenic sympathetic vasoconstriction. In addition, analysis of Taqman mRNA expression studies showed a specific, high level of distribution of the histamine H3 receptor localized in the human nasal mucosa.Taken together, these studies indicate that histamine H3 receptors modulate vascular contractile responses in human nasal mucosa most likely by inhibiting noradrenaline release from sympathetic nerve terminals in nasal mucosa. It is further suggested that histamine H3 receptors may play a role in the regulation of vascular tone and nasal patency in histamine-dependent allergic nasal congestive disease.     

Occupancy of dopamine D2 receptors by the atypical antipsychotic drugs risperidone and olanzapine: theoretical implications

Rationale To examine the D₂ occupancy of two commonly used antipsychotic medications and relate this to the D₂ occupancy by endogenous dopamine in schizophrenia.Objectives The aim of this study is to compare the occupancy of striatal D₂ receptors by the atypical antipsychotic medications risperidone and olanzapine at fixed dosages and to estimate the effect on D₂ occupancy by dopamine as a result of these treatments.Methods Seven patients with schizophrenia taking risperidone 6 mg/day and nine patients with schizophrenia taking olanzapine 10 mg/day underwent an [¹²³I]IBZM SPECT scan after 3 weeks of treatment. The specific to non-specific equilibrium partition coefficient (V₃) after bolus plus constant infusion of the tracer was calculated as [(striatal activity)/(cerebellar activity)]–1. D₂ receptor occupancy was calculated by comparing V₃ measured in treated patients to an age-corrected V₃ value derived from a group of untreated patients with schizophrenia, previously published, according to the following formula: OCC=1–(V₃ treated/V₃ drug free).Results V₃ was significantly lower in risperidone treated patients compared with olanzapine treated patients (0.23±0.06 versus 0.34±0.08, P=0.01), which translated to a significantly larger occupancy in schizophrenic patients treated with risperidone compared to olanzapine (69±8% versus 55±11%, P=0.01). Data from our previous study were used to calculate the occupancy of striatal D₂ receptors by antipsychotic medications required to reduce the occupancy of these receptors by endogenous dopamine to control values. In medication-free patients with schizophrenia, the occupancy of striatal D₂ receptors by endogenous dopamine is estimated at 15.8%. In healthy controls, the occupancy of striatal D₂ receptors by dopamine is estimated at 8.8%. In order to reduce the dopamine occupancy of striatal D₂ receptors in patients with schizophrenia to control values, 48% receptor occupancy by antipsychotic medications is required.Conclusions These data indicate that the dosage of these medications, found to be effective in the treatment of schizophrenia, reduces DA stimulation of D₂ receptors to levels slightly lower than those found in unmedicated healthy subjects.     

Modulation by dopamine receptors of the histamine release in the rat hypothalamus

Summary The involvement of dopaminergic neurons of the hypothalamus in the modulation of histamine release was studied by the push-pull technique. The posterior hypothalamus of the conscious, freely moving rat was superfused with artificial cerebrospinal fluid (CSF) and the release of histamine was determined radioenzymatically in the superfusate. Agonists and antagonists of dopamine D₁-, D₂- and D₃-receptors were dissolved in CSF and applied to the hypothalamus through the push-pull cannula.Hypothalamic superfusion with the D₁-, D₂- and D₃-receptor agonists dopamine or R(–)-apomorphine enhanced the release rate of histamine. (±) Apomorphine also enhanced the release of histamine, but to a lesser extent than did equimolar concentration of R(–)apomorphine. The D₃-agonist quinpirole inhibited the release of histamine, while the D₁-receptor agonist SKF 82958 [(±)-6-chloro-7,8-dihydroxy-3-allyl-l-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine] did not virtually influence the release of the neurotransmitter. On the other hand, [–]-sulpiride which predominantly blocks D₂-receptors, decreased histamine release. Hypothalamic superfusion with SKF 83566 [(±)-7-bromo-8-hydroxy-3-methyl-l-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine], which seems to be a selective antagonist of D₁-receptors, enhanced the release rate of histamine.These findings suggest that dopaminergic neurons of the hypothalamus influence the release of histamine from its neurons in a dual way. D₂-heteroreceptors stimulate the release of histamine, while D₃-heteroreceptors seem to inhibit the release of this neurotransmitter. Both types of dopamine receptors might be located presynaptically on histaminergic neurons. Alternatively, D₃- and D₂-receptors might be located on histaminergic and non-histaminergic neurons, respectively.This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung Correspondence to H. Prast at the above address     

The expression and function of histamine H3 receptors in pancreatic beta cells

BACKGROUND AND PURPOSE

Histamine and its receptors in the CNS play important roles in energy homeostasis. Here, we have investigated the expression and role of histamine receptors in pancreatic beta cells, which secrete insulin.

EXPERIMENTAL APPROACH

The expression of histamine receptors in pancreatic beta cells was examined by RT-PCR, Western blotting and immunostaining. Insulin secretion assay, ATP measurement and calcium imaging studies were performed to determine the function and signalling pathway of histamine H₃ receptors in glucose-induced insulin secretion (GIIS) from MIN6 cells, a mouse pancreatic beta cell line. The function and signalling pathway of H₃ receptors in MIN6 cell proliferation were examined using pharmacological assay and Western blotting.

KEY RESULTS

Histamine H₃ receptors were expressed in pancreatic beta cells. A selective H₃ receptor agonist, imetit, and a selective inverse H₃ receptor agonist, JNJ-5207852, had inhibitory and facilitatory effects, respectively, on GIIS in MIN6 cells. Neither imetit nor JNJ-5207852 altered intracellular ATP concentration, or intracellular calcium concentration stimulated by glucose and KCl, indicating that GIIS signalling was affected by H₃ receptor signalling downstream of the increase in intracellular calcium concentration. Moreover, imetit attenuated bromodeoxyuridine incorporation in MIN6 cells. The phosphorylation of cAMP response element-binding protein (CREB), which facilitated beta cell proliferation, was inhibited, though not significantly, by imetit, indicating that activated H₃ receptors inhibited MIN6 cell proliferation, possibly by decreasing CREB phosphorylation.

CONCLUSIONS AND IMPLICATIONS

Histamine H₃ receptors were expressed in mouse beta cells and could play a role in insulin secretion and, possibly, beta cell proliferation.     

Inhibition of jaw opening reflex and single neurons in the trigeminal subnucleus caudalis by activation of striatal D2 dopamine receptors

The influence of striatal dopaminergic receptors on the inhibitory action of the striatum on the jaw opening reflex (JOR) was studied in anesthetized rats. Single unit activity was recorded at the subnucleus caudalis of the trigeminal nerve. Dopamine agonists and antagonists were microinjectd into the striatum. The striatal administration of apomorphine inhibits the JOR evoked by dental pulp stimulation. Similar results were observed by microinjections of quinpirole, an agonist of D2 receptors, but not by microinjection of SKF 38393, a D1 agonist. The effect of quinpirole was only inhibited by intrastriatal microinjection of haloperidol, a blocker of D2 receptors and reversed by systemic administration of 1 mg/kg of naloxone. The evoked neuronal responses in subnucleus caudalis, by tooth pulp stimulation, were also suppressed by microinjection of quinpirole into the striatum and reversed by naloxone (1 mg/kg, i.v.). Based on the above results, we conclude that the activation of striatal D2 dopamine receptors is responsible for the inhibition of the JOR possibly by action on the subnucleus caudalis of the trigeminal nerve.     

Chronic haloperidol treatment leads to an increase in the intramembrane interaction between adenosine A2 and dopamine D2 receptors in the neostriatum

Stimulation of adenosine A₂ receptors (with the selective adenosine A₂ agonist CGS 21680) in rat striatal membrane preparations, produces a decrease in both the affinity of D₂ receptors and the transduction of the signal from the D₂ receptor to the G protein. This intramembrane A₂-D₂ interaction might be responsible for the behavioural depressant effects of adenosine agonists and for the behavioural stimulant effects of adenosine antagonists such as caffeine and theophylline. Dopamine denervation induces an increase in the intramembrane A₂-D₂ interaction, which may underlie the observed higher sensitivity to the behavioural effects induced by adenosine antagonists found in these animals. The present study was designed to examine if chronic treatment with haloperidol, which also produces dopamine receptor supersensitivity, is also associated with an increase in the intramembrane A₂-D₂ interaction in the neostriatum and with a higher sensitivity to the behavioural effects induced by adenosine antagonists. The data showed that: (i) haloperidol pretreatment causes a higher binding of the D₂ antagonist [³H] raclopride in striatal membrane preparations due to an increase in the number of D₂ receptors without changes in their affinity for the antagonist (increase in B_max without changes in k_d); (ii) GCS 21680 decreases the affinity of dopamine for the D₂ receptor, by increasing the equilibrium dissociation constants of high (K_h) and low affinity (K₁) dopamine D₂ binding sites and increases the proportion of high affinity binding sites (R_h); (iii) a low dose of CGS 21680 (3 nM), which is ineffective in membrane preparations from neostriatum of nontreated animals, is effective in membranes from the striatum of haloperidol-pretreated animals; (iv) the nonselective adenosine antagonist theophylline (20 mg/kg SC) causes a higher motor activation in rats pretreated with haloperidol. The possible relevance of these results for the pathophysiology and treatment of tardive dyskinesias is discussed.     

Action of novel antipsychotics at human dopamine D3 receptors coupled to G protein and ERK1/2 activation

The effects of new generation antipsychotic drugs (APDs) targeting dopamine D(2) and serotonin 5-HT(1A) receptors were compared with typical and atypical APDs on phosphorylation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and measures of G protein activation in CHO cell lines stably expressing the human dopamine D(3) receptor. The preferential dopamine D(3) agonists (+)-7-OH-DPAT and PD128907, like dopamine and quinelorane, efficaciously stimulated ERK 1/2 phosphorylation at dopamine D(3) receptors. In contrast, in [(35)S]GTPgammaS binding experiments, (+)-7-OH-DPAT exhibited partial agonist properties, while PD128907 and quinelorane maintained full agonist properties. The preferential dopamine D(3) ligand BP 897 and the antidyskinetic sarizotan partially activated ERK 1/2 phosphorylation while exerting no agonist activity on GTPgammaS binding, suggesting signal amplification at the MAP kinase level. Antipsychotics differed in their ability to inhibit both agonist-stimulated GTPgammaS binding and ERK 1/2 phosphorylation, but all typical and atypical compounds tested acted as dopamine D(3) receptor antagonists with the exception of n-desmethylclozapine, the active metabolite of clozapine, which partially activated dopamine D(3) receptor-mediated ERK 1/2 phosphorylation. Among the new generation dopamine D(2)/serotonin 5-HT(1A) antipsychotics, only F 15063 and SLV313 acted as pure dopamine D(3) receptor antagonists, bifeprunox was highly efficacious whereas SSR181507 and aripiprazole showed marked partial agonist properties for ERK 1/2 phosphorylation. In contrast, in the GTPgammaS binding study, aripiprazole was devoid of agonist properties and bifeprunox, and to an even lesser extent SSR181507, only weakly stimulated GTPgammaS binding. In summary, these findings underline the differences of dopamine D(3) properties of new generation antipsychotics which may need to be considered in understanding their diverse therapeutic actions.     

Histamine H3 receptor activation inhibits glutamate release from rat striatal synaptosomes

The release of glutamate from striatal synaptosomes induced by depolarisation with 4-aminopyridine (4-AP) was studied by a method based on the fluorescent properties of the NAPDH formed by the metabolism of the neurotransmitter by glutamate dehydrogenase.Ca²⁺-dependent, depolarisation-induced glutamate release was inhibited in a concentration-dependent manner by the selective histamine H₃ agonist immepip. Best-fit estimates were: maximum inhibition 60±10% and IC₅₀ 68±10 nM. The effect of 300 nM immepip on depolarisation-evoked glutamate release was reversed by the selective H₃ antagonist thioperamide in a concentration-dependent manner (EC₅₀ 23 nM, K_i 4 nM).In fura-2-loaded synaptosomes, the increase in the intracellular concentration of Ca²⁺ ([Ca²⁺]_i) evoked by 4-AP-induced depolarisation (resting level 167±14 nM; Δ[Ca²⁺]_i 88±15 nM) was modestly, but significantly reduced (29±5% inhibition) by 300 nM immepip. The action of the H₃ agonist on depolarisation-induced changes in [Ca²⁺]_i was reversed by 100 nM thioperamide.Taken together, our results indicate that histamine modulates the release of glutamate from corticostriatal nerve terminals. Inhibition of depolarisation-induced Ca²⁺ entry through voltage-dependent Ca²⁺ channels appears to account for the effect of H₃ receptor activation on neurotransmitter release. Modulation of glutamatergic transmission in rat striatum may have important consequences for the function of basal ganglia and therefore for the control of motor behaviour.     

Characteristics of recombinantly expressed rat and human histamine H3 receptors

Human and rat histamine H(3) receptors were recombinantly expressed and characterized using receptor binding and a functional cAMP assay. Seven of nine agonists had similar affinities and potencies at the rat and human histamine H(3) receptor. S-alpha-methylhistamine had a significantly higher affinity and potency at the human than rat receptor, and for 4-[(1R*,2R*)-2-(5,5-dimethyl-1-hexynyl)cyclopropyl]-1H-imidazole (Perceptin) the preference was the reverse. Only two of six antagonists had similar affinities and potencies at the human and the rat histamine H(3) receptor. Ciproxifan, thioperamide and (1R*,2R*)-trans-2-imidazol-4 ylcyclopropyl) (cyclohexylmethoxy) carboxamide (GT2394) had significantly higher affinities and potencies at the rat than at the human histamine H(3) receptor, while for N-(4-chlorobenzyl)-N-(7-pyrrolodin-1-ylheptyl)guanidine (JB98064) the preference was the reverse. All antagonists also showed potent inverse agonism properties. Iodoproxyfan, Perceptin, proxyfan and GR175737, compounds previously described as histamine H(3) receptor antagonists, acted as full or partial agonists at both the rat and the human histamine H(3) receptor.     

Functional crosstalk and heteromerization of serotonin 5-HT2A and dopamine D2 receptors

The serotonin 5-HT_2A receptor (5-HT_2AR) and dopamine D₂ receptor (D₂R) are high-affinity G protein-coupled receptor targets for two different classes of antipsychotic drugs used to treat schizophrenia. Interestingly, the antipsychotic effects are not based on the regulation of same signaling mediators since activation of the 5-HT_2AR and of the D₂R regulate G_q/11 protein and G_i/o protein, respectively. Here we use radioligand binding and second messenger production assays to provide evidence for a functional crosstalk between 5-HT_2AR and D₂R in brain and in HEK293 cells. D₂R activation increases the hallucinogenic agonist affinity for 5-HT_2AR and decreases the 5-HT_2AR induced inositol phosphate production. In vivo, 5-HT_2AR expression is necessary for the full effects of D₂R antagonist on MK-801-induced locomotor activity. Co-immunoprecipitation studies show that the two receptors can physically interact in HEK293 cells and raise the possibility that a receptor heterocomplex mediates the crosstalk observed. The existence of this 5-HT_2AR-D₂R heteromer and crosstalk may have implications for diseases involving alterations of serotonin and dopamine systems and for the development of new classes of therapeutic drugs.     

Independent mediation of unconditioned motor behavior by striatal D1 and D2 receptors in rats depleted of dopamine as neonates

The effects of systemic administration of DA receptor antagonists suggest that unconditioned motor behavior in rats depleted of DA as neonates continues to be dependent upon dopaminergic transmission, yet the specific contribution of D₁ and D₂ receptors to these behaviors has been altered. The purpose of the present study was to determine whether these depletion-induced receptor changes are occurring at the level of striatal DA terminals and their targets. The ability of bilateral intrastriatal injections (0.5 µl) of DA receptor antagonists to induce motoric deficits was determined in adult rats treated with vehicle or 6-OHDA (100 µg, intraventricular) on postnatal day 3. Administration of the D₁-like antagonist SCH 23390 (0.5–2.0 µg) or the D₂-like antagonist clebopride (1.0–4.0 µg) induced dose-dependent akinesia, catalepsy, and somatosensory neglect in vehicle-treated controls. In contrast, neither antagonist produced deficits in rats depleted of forebrain DA as neonates. However,combined administration of SCH 23390+clebopride induced similar akinesia, catalepsy, and somatosensory neglect in both controls and DA depleted animals. Animals depleted of DA were more sensitive than controls to the low doses of this combined D₁+D₂ antagonism. These results demonstrate that activation of striatal DA receptors remains necessary for unconditioned motor behavior in rats depleted of DA as neonates. However, the specific contributions of D₁- and D₂-like receptors to these behaviors differ between intact animals and those depleted of DA as neonates. The ability of endogenous DA acting at either D₁ or D₂ receptors to support spontaneous motor behavior in rats depleted of DA as neonates may contribute to their relative sparing from parkinsonian deficits.     

The role of dopamine D<Subscript>3</Subscript> compared with D<Subscript>2</Subscript> receptors in the control of locomotor activity: a combined behavioural and neurochemical analysis with novel,selective antagonists in rats

Background The role of dopamine D₃/D₂ receptors in the control of locomotion is poorly understood.Objectives To examine the influence of selective antagonists at D₃ or D₂ receptors on locomotion in rats, alone and in interaction with the preferential D₃ versus D₂ receptor agonist, PD128,907.Methods Affinities of ligands at rat D₂ and cloned, human hD₃, hD_2S, hD_2L and hD₄ sites were determined by standard procedures. Locomotion was monitored automatically in rats pre-habituated for 30 min to an open-field environment. Extracellular levels of dopamine (DA) were determined by dialysis in the nucleus accumbens and striatum. Drugs were given acutely via the systemic route.Results PD128,907, which preferentially recognised D₃ versus D₂ sites, biphasically reduced and enhanced locomotion at low (0.01–0.63 mg/kg) and high (2.5–10 mg/kg) doses, respectively. L741,626 and S23199, which behaved as preferential D₂ versus D₃ receptor antagonists, enhanced the reduction in locomotion evoked by the low dose of PD128,907, blocked the increase provoked by the high dose and suppressed spontaneous locomotion alone. Analogous findings were obtained with haloperidol and raclopride which showed equilibrated affinity at D₂ and D₃ receptors. UH232 and AJ76, which showed a mild preference for D₃ versus D₂ sites, did not modify the effect of a low dose of PD128,907, slightly enhanced the hyperlocomotion elicited by the high dose and exerted little influence on locomotion alone. S14297 and U99194, which acted as preferential D₃ versus D₂ receptor antagonists, abolished the reduction in locomotion elicited by a low dose of PD128,907, potentiated the induction of locomotion by a high dose, and failed to influence locomotion alone. The actions of S14297 were stereoselective inasmuch as they were mimicked by the racemic form, S11566, but not by the inactive enantiomer, S17777. In contrast to S14297, S11566 and U99194, however, S33084, SB269,652, GR218,231 and N-[-4-[-(1-naphtyl)piperazine-1-yl]butyl] anthracene-2-carboxamide (NGB-1), highly selective D₃ versus D₂ receptor antagonists, were inactive under all conditions. PD128,907 (0.01–10.0 mg/kg) suppressed dialysate levels of DA in the nucleus accumbens and striatum, actions blocked by L741,626 and haloperidol, yet unaffected by S14297 and S33084.Conclusions The facilitatory influence of a high dose of PD128,907 upon locomotion is mediated by postsynaptic D₂ receptors and, possibly, countered by their D₃ counterparts. Correspondingly, selective blockade of D₂ but not of D₃ receptors alone suppresses motor function. The reduction in locomotion provoked by a low dose of PD128,907 may be mediated by D₂ autoreceptors, but a role of postsynaptic D₃ receptors cannot be excluded. Finally, mechanisms underlying the contrasting influence of chemically diverse D₃ receptor antagonists upon locomotion remain to be elucidated.