2L Receptors Research Articles

Dopamine D2 Antagonist-Induced Striatal Nur77 Expression Requires Activation of mGlu5 Receptors by Cortical Afferents

Dopamine D2 receptor antagonists modulate gene transcription in the striatum. However, the molecular mechanism underlying this effect remains elusive. Here we used the expression of Nur77, a transcription factor of the orphan nuclear receptor family, as readout to explore the role of dopamine, glutamate, and adenosine receptors in the effect of a dopamine D2 antagonist in the striatum. First, we investigated D2 antagonist-induced Nur77 mRNA in D2L receptor knockout mice. Surprisingly, deletion of the D2L receptor isoform did not reduce eticlopride-induced upregulation of Nur77 mRNA levels in the striatum. Next, we tested if an ibotenic acid-induced cortical lesion could block the effect of eticlopride on Nur77 expression. Cortical lesions strongly reduced eticlopride-induced striatal upregulation of Nur77 mRNA. Then, we investigated if glutamatergic neurotransmission could modulate eticlopride-induced Nur77 expression. A combination of a metabotropic glutamate type 5 (mGlu5) and adenosine A2A receptor antagonists abolished eticlopride-induced upregulation of Nur77 mRNA levels in the striatum. Direct modulation of Nur77 expression by striatal glutamate and adenosine receptors was confirmed using corticostriatal organotypic cultures. Taken together, these results indicate that blockade of postsynaptic D2 receptors is not sufficient to trigger striatal transcriptional activity and that interaction with corticostriatal presynaptic D2 receptors and subsequent activation of postsynaptic glutamate and adenosine receptors in the striatum is required. Thus, these results uncover an unappreciated role of presynaptic D2 heteroreceptors and support a prominent role of glutamate in the effect of D2 antagonists.

In vitro pharmacology of aripiprazole, its metabolite and experimental dopamine partial agonists at human dopamine D 2 and D 3 receptors

Aripiprazole is the first dopamine D 2/D 3 receptor partial agonist successfully developed and ultimately approved for treatment of a broad spectrum of psychiatric and neurological disorders. Aripiprazole's dopamine D 2 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, D 3 Ser-9 and D 3 Gly-9 for human dopamine receptors. All test compounds behaved as dopamine D 2/D 3 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 D 3 Ser-9 and D 3 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 D 2/D 3 receptors for clinical efficacy and safety.

Specific binding of photoaffinity-labeling peptidomimetics of Pro-Leu-Gly-NH 2 to the dopamine D 2L receptor: Evidence for the allosteric modulation of the dopamine receptor

The present study was undertaken to investigate the mechanistic role of l-prolyl- l-leucyl-glycinamide (PLG) in modulating agonist binding to the dopamine D 2L receptor. Competition and displacement assays indicate that the photoaffinity-labeling peptidomimetics of PLG, 3(R)-[(4(S)-(4-azido-2-hydroxy-benzoyl) amino-2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide hydrochloride ( 1a) and 3(R)-[(4(S)-(4-azido-2-hydroxy-5-iodo-benzoyl)amino-2( S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide hydrochloride ( 1b) bind at the same site as PLG. Autoradiography was used to establish the covalent binding of [ 125I]- 1b to a ∼ 51 kDa protein in bovine striatal membranes. Western blot analysis with a dopamine D 2L-specific antibody, in combination with autoradiography, following a two-dimensional gel separation, suggested this ∼ 51 kDa protein to be the dopamine D 2L receptor. Further evidence for binding of 1b to dopamine D 2L was provided by samples immunoprecipitated with the D 2L antibody. These samples were analyzed by western blotting in parallel with autoradiography of [ 125I]- 1b labeled protein. Both methods revealed bands at ∼ 51 kDa. Furthermore, PLG is shown to compete with 1b for binding to the dopamine D 2L receptor as determined by autoradiography, as well as competition experiments with PLG and 1a. Collectively, these findings suggest the successful development of a photoaffinity-labeling agent, compound 1b, that has been used to elucidate the interaction of PLG specifically with the dopamine D 2L receptor.

Differential voltage-sensitivity of D 2-like dopamine receptors

Agonist potency at some neurotransmitter receptors has been shown to be regulated by transmembrane voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by autoreceptors and in synaptic plasticity. We have recently described the voltage-sensitivity of the dopamine D 2L receptor and we now extend our studies to include the other members of the D 2-like receptor subfamily; the D 2S, D 3, and D 4 dopamine receptors. Electrophysiological recordings were performed on Xenopus oocytes coexpressing human dopamine D 2S, D 3, or D 4 receptors with G protein-coupled potassium (GIRK) channels. Comparison of concentration–response relationships at −80 mV and at 0 mV for dopamine-mediated GIRK activation revealed significant rightward shifts for both D 2S and D 4 upon depolarization. In contrast, the concentration–response relationships for D 3-mediated GIRK activation were not appreciably different at the two voltages. Our findings provide new insight into the functional differences of these closely related receptors.

Partial agonist actions at dopamine D 2L receptors are modified by co-transfection of D 3 receptors: Potential role of heterodimer formation

The effects of aripiprazole, S33592, bifeprunox, N-desmethylclozapine and preclamol acting as partial agonists on recombinant D 2L and D 3 receptors expressed both separately and concomitantly in COS-7 cells are evaluated here. Aripiprazole, S33592, bifeprunox, N-desmethylclozapine and preclamol behave as partial agonists on D 2L receptors coupled with adenylyl cyclase, but they behave as antagonists on co-expression of D 3 with D 2L receptors. These data raise the intriguing hypothesis that antipsychotic actions of “partial agonists” such as aripiprazole may not reflect inefficient stimulation of D 2 and/or D 3 receptors but, by analogy with other antipsychotics, may instead represent a blockade of D 2/D 3 heterodimers (and/or D 3 receptors) that are “weakly” coupled to transduction mechanisms postsynaptically of the dopaminergic pathway.

Aripiprazole: What Is the Role of Dopamine D2Receptor Partial Agonism?

Aripiprazole: What Is the Role of Dopamine D₂Receptor Partial Agonism?

Synthesis and in vitro binding studies of substituted piperidine naphthamides. Part I: Influence of the substitution on the basic nitrogen and the position of the amide on the affinity for D 2L, D 4.2, and 5-HT 2A receptors

A series of 1- and 2-naphthamides has been prepared and tested for in vitro binding to D 2L, D 4.2, and 5-HT 2A receptors. Different compounds display selectivity for D 4.2 and 5-HT 2A receptors versus D 2L receptors. N-(1-Arylalkyl-piperidin-4-yl) carboxamides have higher affinities than the corresponding N-(4-arylalkylamino-piperidin-1-yl) carboxamide analogues. A benzyl moiety in position 1 of the piperidine in the 2-naphthamide series ( 2) appears to be the best choice for a favorable interaction with D 4.2 and 5-HT 2A receptors. Increasing the linker length between the phenyl ring and the basic nitrogen led to a decreased affinity for these receptors. In the 1-naphthamide series, the most potent D 4.2 ligand ( 7) possesses a phenylpropyl moiety while its affinity for 5-HT 2A receptors is strongly reduced. All compounds with significant affinity for D 4.2 and 5-HT 2A receptors were antagonists.

Investigation of the alternatively spliced insert region of the D 2L dopamine receptor by epitope substitution

Alternatively spliced variants of the D 2 dopamine receptor have distinct neuronal function and localization. The long isoform (D 2L) of this heptahelical transmembrane receptor differs from the short form only by the presence of a 29-amino acid insert in the third intracellular loop—a region known to be important for G protein coupling. Short and long isoforms have been shown to have distinct Gα i/o protein coupling specificities. However, the exact role of the alternatively spliced insert region in D 2 dopamine receptor function needs a more comprehensive examination. One way to address this is to substitute the entire insert region with an equivalent length, yet nonhomologous protein sequence. This report demonstrates the feasibility of replacing the 29-amino acid insert with a hemagglutinin double epitope tag with no recognizable functional consequences. The D 2L mutant is indistinguishable from the wild type D 2L receptor in terms of its ligand binding characteristics, as well as two effector responses: the agonist-mediated inhibition of forskolin-stimulated cAMP production, and agonist-stimulated MAPK phosphorylation. These data demonstrate that the epitope substitution generates a functional receptor, and that the alternatively spliced insert region, itself, does not appear to play a direct role in signal transduction. The epitope substitution permits dissection of sequence-mediated effects from structural effects due to the presence of the alternatively spliced insert region. Thus, this new construct could be a valuable tool for the study of D 2 receptor function.

KKHA-761, a potent D 3 receptor antagonist with high 5-HT 1A receptor affinity, exhibits antipsychotic properties in animal models of schizophrenia

KKHA-761, 1-{4-[3-(3,4-dimethoxy-phenyl)-isoxazol-5-yl]-butyl}-4-(2-methoxy-phenyl)-piperazine, has a high affinity ( K i = 3.85 nM) for human dopamine D 3 receptor with about 70-fold selectivity over the human dopamine D 2L receptor ( K i = 270 nM). KKHA-761 also showed high affinity for cloned human 5-HT 1A receptor ( K i = 6.4 nM). KKHA-761 exhibited D 3 and 5-HT 1A receptor antagonist activities in vitro, reversing dopamine- or 5-HT-mediated stimulation of [ 35S]GTPrS binding. The in vivo pharmacological profile of KKHA-761 was compared with both typical and atypical antipsychotics including clozapine and haloperidol. Apomorphine-induced dopaminergic behavior, cage climbing, in mice was potently blocked by a single administration (i.p.) of KKHA-761 (ID 50 = 4.06 mg/kg) or clozapine (ID 50 = 4.0 mg/kg). Cocaine- or MK-801-induced hyperactivity in animals was markedly inhibited by KKHA-761 or clozapine. In addition, KKHA-761 significantly reversed the disruption of prepulse inhibition (PPI) produced by apomorphine in mice, indicating the antidopaminergic or antipsychotic activity of KKHA-761 in mice. However, KKHA-761 was inactive in the forced swimming behavioral despair model in mice, suggesting lack of antidepressant properties. KKHA-761 attenuated the hypothermia induced by a selective dopamine D 3 agonist, 7-OH-DPAT, in mice, whereas clozapine enhanced it. Moderate doses of both KKHA-761 and clozapine did not increase serum prolactin levels in rats. Lower doses of, however, haloperidol significantly increased prolactin secretion. KKHA-761 did not induce cataleptic response up to 20 mg/kg, but significant catalepsy was shown at lower doses of clozapine and haloperidol. Furthermore, KKHA-761 showed a low incidence of rotarod ataxia (TD 50 = 34.4 mg/kg, i.p.) in mice. The present results, therefore, suggest that KKHA-761 is a potent antipsychotic agent with combined dopamine D 3 and serotonin 5-HT 1A receptors modulation activity, which may further enhance its therapeutic potential for anxiety, psychotic depression, and other related disorders.

A-412997 is a selective dopamine D 4 receptor agonist in rats

A-412997 (2-(3′,4′,5′,6′-tetrahydro-2′ H-[2,4′] bipyridinyl-1′-yl)- N- m-tolyl-acetamide) is a highly selective dopamine D 4 receptor agonist that binds with high affinity to rat dopamine D 4 and human dopamine D 4.4 receptors ( K i = 12.1 and 7.9 nM, respectively). In contrast to the dopamine D 4 receptor agonists PD168077 and CP226269, A-412997 showed a better selectivity profile and no affinity < 1000 nM for other dopamine receptors or any other proteins in a panel of seventy different receptors and channels. In functional assays using calcium flux, A-412997 was a potent full agonist at rat dopamine D 4 receptors (28.4 nM, intrinsic activity = 0.83) and did not activate rat dopamine D 2L receptors, unlike CP226269. Dopamine D 4 receptor selective agonists have been shown to induce penile erection in rats by central mechanisms. A-412997 induces penile erection in a conscious rat model (effective dose = 0.1 μmol/kg, s.c.) with comparable efficacy as the nonselective D 2-like agonist, apomorphine. When dosed systemically, A-412997 crossed the blood brain barrier rapidly and achieved significantly higher levels than PD168077. A-412997 is a highly selective dopamine D 4 receptor agonist and a useful tool to understand the role of dopamine D 4 receptors in rat models of central nervous system processes and disease.

α 2C-Adrenoceptor blockade by clozapine and other antipsychotic drugs

The noradrenergic system may play a role in antipsychotic modulation of schizophrenia symptoms. Therefore, the antagonistic potencies of the antipsychotics clozapine, chlorpromazine, risperidone, olanzapine, haloperidol, quetiapine, ziprasidone, iloperidone and aripiprazole were quantified using cell lines expressing the recombinant human α 2C-adrenoceptor, α 2A-adrenoceptor, or dopamine D 2L receptor. The α 2-adrenoceptor antagonists, yohimbine and idazoxan, were also tested. Alterations in cAMP were measured as changes in luminescence. In the α 2A-adrenoceptor cell line, the agonist 5-bromo-6-(2-imidazolin-2-ylamino)quinoxaline (UK14,304) induced a concentration-dependent increase in luminescence. In cell lines expressing α 2C and D 2L receptors, agonists induced a concentration-dependent reduction in luminescence. Yohimbine and idazoxan were the most potent α 2A-adrenoceptor antagonists, yohimbine and iloperidone were the most potent α 2C-adrenoceptor antagonists, and haloperidol and olanzapine were the most potent dopamine D 2 receptor antagonists. Clozapine had the highest α 2C/D 2 selectivity, and iloperidone the highest α 2C/α 2A ratio. It is hypothesised that α 2C-adrenoceptor blockade contributes to improvement of cognitive function.

Estrogen regulation of gene expression in the brain: a possible mechanism altering the response to psychostimulants in female rats

Acute behavioral responses to cocaine are more pronounced in female than in male rats. We have shown that 3 weeks of treatment with 17β-estradiol (E 2) implants significantly enhanced the hyperactivity induced by cocaine in ovariectomized (OVX) rats. The ligand-bound estrogen receptor (ER) functions as a transcription factor to regulate the expression of E-responsive genes. Thus, we hypothesized that estrogen (E) modulates the behavioral response to cocaine via regulation of expression of components of dopamine (DA) and serotonin (5-HT) systems in mesolimbic nuclei important in the response to cocaine as well as the hypothalamus, a brain area known to be E-responsive. Adult female Sprague–Dawley rats were OVX; half of them then received E 2 implant (OVX+E) ( n=8/group, two groups). Twenty-seven days later, brain tissue was collected to study E 2 effects on mRNA expression for DA D 1-like (D 1) and D 2-like (D 2S, D 2L, D 3) receptors, DA transporter (DAT), 5-HT 1A, 5-HT 1B, 5-HT 2A, 5-HT 2C receptors, and 5-HT transporter (SERT) as well as ERα and ERβ in amygdala, hypothalamus, nucleus accumbens, midbrain, and ventral tegmental area (VTA). We found that E 2 implants in OVX rats increased mRNA levels for D 1 receptor in hypothalamus, D 2L receptor in midbrain, and D 3 receptor in VTA, and decreased D 3 receptor mRNA levels in midbrain relative to OVX controls. E 2 also increased 5-HT 2C receptor mRNA levels in midbrain and hypothalamus. In addition, E 2 decreased mRNA levels for ERα in amygdala and hypothalamus and ERβ in amygdala. The present study demonstrates that E can regulate mRNA expression for specific DA and 5-HT receptors in a region-specific manner and suggests that such modifications may contribute to the behavioral response to cocaine.

A selective dopamine D 4 receptor antagonist, NRA0160: A preclinical neuropharmacological profile

NRA0160, 5 - [2- (4- (3 - fluorobenzylidene) piperidin-1-yl) ethyl] - 4 -(4-fluorophenyl) thiazole-2-carboxamide, has a high affinity for human cloned dopamine D 4.2, D 4.4 and D 4.7 receptors, with Ki values of 0.5, 0.9 and 2.7 nM, respectively. NRA0160 is over 20,000-fold more potent at the dopamine D 4.2 receptor compared with the human cloned dopamine D 2L receptor. NRA0160 has negligible affinity for the human cloned dopamine D 3 receptor (Ki = 39 nM), rat serotonin (5-HT) 2A receptors (Ki = 180 nM) and rat α 1 adrenoceptor (Ki = 237 nM). NRA0160 and clozapine antagonized locomotor hyperactivity induced by methamphetamine (MAP) in mice. NRA0160 and clozapine antagonized MAP-induced stereotyped behavior in mice, although their effects did not exceed 50% inhibition, even at the highest dose given. NRA0160 and clozapine significantly induced catalepsy in rats, although their effects did not exceed 50% induction even at the highest dose given. NRA0160 and clozapine significantly reversed the disruption of prepulse inhibition (PPI) in rats produced by apomorphine. NRA0160 and clozapine significantly shortened the phencyclidine (PCP)-induced prolonged swimming latency in rats in a water maze task. These findings suggest that NRA0160 may have unique antipsychotic activities without the liability of motor side effects typical of classical antipsychotics.

Heterocyclic congeners of PD 128,907 with a partially hydrogenated benzomorpholine moiety as potential dopamine D 3-receptor ligands

With a straightforward seven-step synthesis, racemic perhydro[1,4]benzoxazin-6-on was synthesized in overall good yields via regioselective epoxid ring-opening to the corresponding β-aminoalcohol. The oxazine derivative was the key intermediate for the preparation of heteroaromatic analogues of the dopamine D 3-receptor preferring agonist PD 128,907. The morpholine moiety of PD 128,907 was incorporated in diazole and diazine compounds obtained by different ring closure reactions. The target compounds obtained were structurally related to non-ergot heteroaromatic dopamine agonists which display preferential activity at the D 3 receptor, e.g., quinpirole, quinerolane, or pramipexole. The five membered aminothiazole, aminoselenazole, and pyrazole derivatives showed at least one order of magnitude higher binding at the human D 3 receptor than that at the D 2L receptor. Although the novel compounds displayed K i values only in the micromolar concentration range, the most active ones showed full agonist activity in a functional assay on mitogenesis.

Comparison of the ability of dopamine receptor agonists to inhibit forskolin-stimulated adenosine 3′5′-cyclic monophosphate (cAMP) accumulation via D 2L (long isoform) and D 3 receptors expressed in Chinese hamster ovary (CHO) cells

The pharmacological properties of the human D 2L (long isoform) and rat D 3 dopamine receptors in functional assays were examined. A range of dopamine agonists were assessed for their ability to inhibit adenosine 3′5′-cyclic monophosphate (cAMP) accumulation via the two receptors expressed stably in Chinese hamster ovary cells. Dopamine caused a significantly greater maximal inhibition ( P < 0.05) of cAMP accumulation via the D 2L receptor (∼70%) as compared to the D 3 receptor (∼50%). The pattern of agonist effects was different at the two receptors. The absolute and relative potencies for inhibition of cAMP accumulation were different for a range of agonists acting at the two receptors. Similarly, the maximal inhibitions achieved by a range of agonists were different for the two receptors.

Partial agonism at serotonin 5-HT 1B and dopamine D 2L receptors using a luciferase reporter gene assay

We have used a luciferase reporter gene assay to study the functional responses of two G-protein-coupled receptors in Chinese hamster ovary (CHO) cells. The rank order of potency of drugs for the endogenous 5-HT 1B receptor was 5-Hydroxytryptamine (5-HT)>zolmitriptan>dihydroergocristine>(−)lisuride (with no response to bromocriptine). However, only 5-HT and (−)lisuride produced a full functional response, with zolmitriptan and dihydroergocristine achieving 69±2% and 50±1% of the maximal response. In the same cells stably transfected with the rat dopamine D 2L receptor, dopamine and bromocriptine produced a full agonist functional response, whilst (−)lisuride produced a biphasic response curve, indicating activity at both the endogenous 5-HT 1B and exogenous dopamine D 2L receptors. Using the receptor specific antagonists, pindolol and (+)butaclamol, (−)lisuride was shown to produce 52% of the maximal response at the dopamine D 2 receptor relative to dopamine. In comparison to a cAMP accumulation assay, the rank orders of potency and intrinsic activity were the same for all compounds used. These results demonstrate that this reporter gene assay is capable of discriminating both potency and efficacy of drugs and can be used to characterise partial agonists at endogenously and heterologously expressed receptors in CHO cells.

Pharmacological Characterization of PD 152255, a Novel Dimeric Benzimidazole Dopamine D 3 Antagonist

152255 ( E-1, 1′-(2-butene-1,4-diyl)bis [2-[4-[3-(1-piperidinyl)propoxy]-phenyl]-1H-benzimidazole]) exhibited high affinity ( K i = 12.7 nM) for human dopamine (DA) D 3 receptors expressed in CHO K1 cells but not for DA D 2L receptors ( K i = 565 nM), DA D 4.2 or DA D 1 receptors ( K i > 3 μM) and a number of other neurotransmitter receptors. Affinity for human muscarinic receptors was seen in vitro but no functional muscarinic agonist and/or antagonist action was observed in vivo. Antagonist activity at DA D 3 receptors was demonstrated by blockade of quinpirole-stimulated [ 3H]-thymidine uptake in D 3 transfected cells, an effect that was 28-fold more potent than in D 2-transfected cells. Unlike classical DA D 2 antagonists, PD 152255 did not increase rat brain DA synthesis and it increased locomotion in habituated rats. However, like antipsychotics, PD 152255 reduced locomotor activity in mice and reduced spontaneous and amphetamine-stimulated locomotion in nonhabituated rats. These results demonstrate that PD 152255 is a DA D 3 antagonist that may have antipsychotic activity.

Dopamine receptor agonist potencies for inhibition of cell firing correlate with dopamine D 3 receptor binding affinities

The potencies for in vivo inhibition of substantia nigra pars compacta dopamine single cell firing were determined for apomorphine, BHT 920, N-0923, (±)-7-hydroxy-dipropylaminotetralin (7-OH-DPAT), (+)-3-(3-hydroxyphenyl)- N-propylpiperidine (3-PPP), pramipexole, quinelorane, quinpirole, RU 24926, U-86170, and U-91356. Significant correlation was obtained between the potencies of these 11 highly efficacious dopamine receptor agonists and the in vitro binding affinities at dopamine D 3 receptors, but not at dopamine D 2L receptors. These results support a functional role for the dopamine D 3 receptor subtype in the autoreceptor-mediated regulation of dopamine cell activity, while a role for dopamine D 2 receptors awaits further analysis. In addition, the results demonstrate the limitations of using currently available dopamine receptor agonists to delineate relative in vivo roles for the dopamine D 2 and D 3 receptor subtypes.