Comparison of rat dopamine D2 receptor occupancy for a series of antipsychotic drugs measured using radiolabeled or nonlabeled raclopride tracer

Preclinical brain receptor occupancy measures have heretofore been conducted by quantifying the brain distribution of a radiolabeled tracer ligand using either scintillation spectroscopy or tomographic imaging. For smaller animals like rodents, the majority of studies employ tissue dissection and scintillation spectroscopy. These measurements can also be accomplished using liquid chromatography coupled to mass spectral detection to measure the brain distribution of tracer molecules, obviating the need for radioligands. In order to validate mass spectroscopy-based receptor occupancy methods, we examined dopamine D2 receptor dose-occupancy curves for a number of antipsychotic drugs in parallel experiments using either mass spectroscopy or radioligand-based approaches. Oral dose-occupancy curves were generated for 8 antipsychotic compounds in parallel experiments using either radiolabeled or unlabeled raclopride tracer. When curves generated by these two methods were compared and ED(50) values determined, remarkably similar data were obtained. Occupancy ED(50) values were (mg/kg): chlorpromazine, 5.1 and 2.7; clozapine, 41 and 40; haloperidol, 0.2 and 0.3; olanzapine, 2.1 and 2.2; risperidone, 0.1 and 0.4; spiperone, 0.5 and 0.4; thioridazine 9.2 and 9.5; and ziprasidone 1.4 and 2.1 (unlabeled and radiolabeled raclopride tracer, respectively). The observation that in vivo application of both techniques led to comparable data adds to the validation state of the mass spectroscopy-based approach to receptor occupancy assays.     

Brain neurotransmitter receptor-binding characteristics in rats after oral administration of haloperidol, risperidone and olanzapine

The present study was conducted to characterize the binding of neurotransmitter receptors (dopamine D(2), serotonin 5-HT(2), histamine H(1), adrenaline alpha(1) and muscarine M(l) receptors) in the rat's brain after the oral administration of haloperidol, risperidone, and olanzapine. Haloperidol at 1 and 3 mg/kg displayed significant activity to bind the D(2) receptor (increase in the Kd value for [(3)H]raclopride binding) in the corpus striatum with little change in the activity toward the 5-HT(2) receptor (binding parameters for [(3)H]ketanserin). In contrast, risperidone (0.1-3 mg/kg) showed roughly 30 times more affinity for the 5-HT(2) receptor than D(2) receptor. Also, olanzapine (1-10 mg/kg) was most active toward the H(1) receptor in the cerebral cortex, corpus striatum, and hippocampus, was less active in binding 5-HT(2) and D(2) receptors, and showed the least affinity for alpha(1) and M(1) receptors. In conclusion, haloperidol and risperidone administered orally selectively bind D(2) and 5-HT(2) receptors, respectively, in the rat brain, while olanzapine binds H(1), 5-HT(2), and D(2) receptors more than alpha(1) and M(1) receptors.     

Binding of antipsychotic drugs to human brain receptors focus on newer generation compounds

Using radioligand binding assays and post-mortem normal human brain tissue, we obtained equilibrium dissociation constants (K(d)s) for nine new antipsychotic drugs (iloperidone, melperone, olanzapine, ORG 5222, quetiapine, risperidone, sertindole, ziprasidone, and zotepine), one metabolite of a new drug (9-OH-risperidone), and three older antipsychotics (clozapine, haloperidol, and pimozide) at nine different receptors (alpha1-adrenergic, alpha2-adrenergic, dopamine D2, histamine H1, muscarinic, and serotonin 5-HT1A, 5-HT1D, 5-HT2A, and 5-HT2C receptors). Iloperidone was the most potent drug at the two adrenergic receptors. ORG 5222 was the most potent drug at dopamine D2 and 5-HT2c receptors, while ziprasidone was the most potent compound at three serotonergic receptors (5-HT1A, 5-HT1D, and 5-HT2A). At the remaining two receptors, olanzapine was the most potent drug at the histamine H1 receptor (Kd=0.087 nM); clozapine at the muscarinic receptor (Kd=9 nM). Certain therapeutic and adverse effects, as well as certain drug interactions can be predicted from a drug's potency for blocking a specific receptor. These data can provide guidelines for the clinician in the choice of antipsychotic drug.     

In vivo rat brain opioid receptor binding of LY255582 assessed with a novel method using LC/MS/MS and the administration of three tracers simultaneously

LY255582 is a pan opioid selective receptor antagonist that has been shown to have high affinity for mu, delta, and kappa receptors in vitro. In order to better understand the in vivo opioid receptor selectivity of LY255582, we developed in vivo receptor occupancy assays in the rat for the opioid mu, kappa and delta receptors using the occupancy tracers naltrexone, GR103545 and naltriben respectively. Individual assays for each target were established and then a "triple tracer" assay was created where all three tracers were injected simultaneously, taking advantage of LC/MS/MS technology to selectively monitor brain tracer levels. This is the first report of a technique to concurrently measure receptor specific occupancy at three opioid receptors in the same animal. The opioid subtype selective antagonists cyprodime, JDTic and naltrindole were used to validate selectivity of the assay. Examination of LY255582 in dose-occupancy experiments demonstrated a relative order of potency of mu>kappa>delta, reproducing the previously reported order determined with in vitro binding.     

Extended treatment with typical and atypical antipsychotic drugs differential effects on the densities of dopamine D2-like and GABAA receptors in rat striatum

In situ radioligand binding and quantitative autoradiography have been used to measure the density of striatal D1-like, D2-like, and GABAA receptors in rats treated with haloperidol at 0.01 or 0.1 mg/kg/ day or chlorpromazine, olanzapine or clozapine at 0.1 or 1.0 mg/kg/day for 1, 3 or 7 months. [3H]SCH23390 binding to D1-like receptors was not changed by any drug treatments. There were significant increases in [3H]nemonapride binding to D2-like receptors at different time points due to treatment with haloperidol, chlorpromazine and olanzapine. By contrast, treatment with clozapine and olanzapine caused a time-dependent decrease in [3H]muscimol binding to the GABAA receptor. These data suggest that treatment with atypical antipsychotic drugs, but not typical antipsychotic drugs, affect striatal GABAergic neurons. In addition, it would appear that clozapine might be unique in that it does not increase dopamine-D2 like receptor density at doses which would be predicted to have antipsychotic effects in humans. The extent to which such changes are involved in the therapeutic effects of drugs such as olanzapine and clozapine remains to be determined.     

Pharmacological evaluation of (+)-2- [123I]A-69024: a radioligand for in vivo studies of dopamine D1 receptors

(+)-2-[123I] A-69024, [(+)-1-(2-[123I] iodo-4,5-dimethoxy-benzyl)-7-hydroxy-6-methoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline], is a specific and enantioselective dopamine D1 receptor radioligand. The in vivo biodistribution of this radioligand in rats showed high brain uptake and a distribution consistent with the density of dopamine D1 receptors. Highest uptake was observed in the striatum (0.65 %ID/g) at 5 min followed by clearance. As a measure of specificity the striatum/cerebellar ratio reached a maximum of 3.9 at 30 min post-injection. Radioactivity in the striatum was reduced by 68% by pre-administration of the D1 antagonist SCH 23390. Pre-administration of other dopamine binding drugs, spiperone (D2), 7-OH-DPAT (D3), and clozapine (D4) displayed no inhibitory effect on (+)-2-[123I]A-69024 accumulation in any brain region. Ketanserin (5-HT2/5-HT2C) and haloperidol (D2 receptor antagonist/sigma receptor ligand) also displayed no inhibitory effect in any brain region studied. With the pharmacologically inactive enantiomer, (-)-2-[123I]A-69024, the brain uptake was determined to be non-specific since a striatum/cerebellar ratio of near 1 was observed throughout the time course of the experiment. (+)-2-[123I]A-69024 displays enantioselectivity for dopamine D1 receptors and may deserve further investigation as a possible SPECT radioligand.     

5-HT(2A) and D(2) receptor blockade increases cortical DA release via 5-HT(1A) receptor activation: a possible mechanism of atypical antipsychotic-induced cortical dopamine release

Atypical antipsychotic drugs (APDs), all of which are relatively more potent as serotonin (5-HT)(2A) than dopamine D(2) antagonists, may improve negative symptoms and cognitive dysfunction in schizophrenia, in part, via increasing cortical dopamine release. 5-HT(1A) agonism has been also suggested to contribute to the ability to increase cortical dopamine release. The present study tested the hypothesis that clozapine, olanzapine, risperidone, and perhaps other atypical APDs, increase dopamine release in rat medial prefrontal cortex (mPFC) via 5-HT(1A) receptor activation, as a result of the blockade of 5-HT(2A) and D(2) receptors. M100907 (0.1 mg/kg), a 5-HT(2A) antagonist, significantly increased the ability of both S:(-)-sulpiride (10 mg/kg), a D(2) antagonist devoid of 5-HT(1A) affinity, and R:(+)-8-OH-DPAT (0.05 mg/kg), a 5-HT(1A) agonist, to increase mPFC dopamine release. These effects of M100907 were abolished by WAY100635 (0.05 mg/kg), a 5-HT(1A) antagonist, which by itself has no effect on mPFC dopamine release. WAY100635 (0.2 mg/kg) also reversed the ability of clozapine (20 mg/kg), olanzapine (1 mg/kg), risperidone (1 mg/kg), and the R:(+)-8-OH-DPAT (0.2 mg/kg) to increase mPFC dopamine release. Clozapine is a direct acting 5-HT(1A) partial agonist, whereas olanzapine and risperidone are not. These results suggest that the atypical APDs via 5-HT(2A) and D(2) receptor blockade, regardless of intrinsic 5-HT(1A) affinity, may promote the ability of 5-HT(1A) receptor stimulation to increase mPFC DA release, and provide additional evidence that coadministration of 5-HT(2A) antagonists and typical APDs, which are D(2) antagonists, may facilitate 5-HT(1A) agonist activity.     

A definite measure of occupancy exposures,seeking with non-radiolabeled in vivo 5-HT2A receptor occupancy and in vitro free fractions

Abstract

Unbound drug concentration in the brain would be the true exposure responsible for specific target occupancy. Drug exposures from preclinical are total concentrations of those over/underestimate the clinical dose projection. With the application of mass spectrometry, the current work proposes a definite measure of test drug exposures at serotonin-2A occupancy. The 5-HT_2A occupancy of antagonist in the rat brain has determined with non-radiolabeled tracer MDL-100,907 at an optimized dose (3?µg/kg) and treatment time (30?min). Equilibrium dialysis method determines the in vitro free fraction of the test antagonist in untreated rat brain homogenates and plasma. Drug-free fractions derived the unbound concentration (EC₅₀) in plasma and brain at test doses. The corresponding binding affinities (Ki) correlated with the unbound concentrations. Except for quetiapine, the ED₅₀ values in the dose-occupancy curves of antagonists are close and ranged from 1 to 3?mg/kg. The test drug quetiapine, eplivanserin, and clozapine showed high free fractions in plasma, but for ketanserin and olanzapine, the brain free fraction was higher. The correlation between the unbound EC₅₀ of the antagonists and corresponding Ki values was good (r²=0.828). The improved EC₅₀ accuracy with unbound concentrations was 10–250 folds in plasma and 10–170 folds in the brain. Further, the free fractions (f_{u, plasma}/f_{u, brain}) of test drugs had shown a correlation of ～83% with brain permeability (C_{total brain}/C_{total plasma}), a limiting factor. Thus, correlating the occupancy with unbound exposure and pharmacology would result in an accurate measurement of drug potency and optimizes in selecting the clinical dose.     

Assessment of sigma-1 receptor occupancy in mice with non-radiolabelled FTC-146 as a tracer

Abstract

The use of liquid chromatography coupled with mass spectrometry (LC-MS/MS) is advantageous in in-vivo receptor occupancy assays at pre-clinical drug developmental stages. Relatively, its application is effective in terms of high throughput, data reproducibility, sensitivity, and sample processing. In this perspective, we have evaluated the use of FTC-146 as a non-radiolabelled tracer to determine the sigma-1 receptor occupancy of test drugs in mice brain. Further, the brain and plasma exposures of test drug were determined at their corresponding occupancies. In this occupancy method, the optimized tracer treatment (sacrification) time after intravenous administration was 30?min. The tracer dose was 3?µg/kg and specific brain regions of interest were frontal cortex, pons and midbrain. Mice were pretreated orally with SA4503, fluspidine, haloperidol, and donepezil followed by tracer treatment. Among the test drugs, SA4503 was used as positive control group at its highest test dose (7?mg/kg, intraperitoneal). There was a dose-dependent decrease in brain regional FTC-146 binding in pretreated mice. From the occupancy curves of SA4503, fluspidine, haloperidol, and donepezil the effective dose (ED₅₀) value ranges are 0.74–1.45, 0.09–0.11, 0.11–0.12, and 0.07–0.09?mg/kg, respectively. Their corresponding brain effective concentration (EC₅₀) values are 74.3–132.5, 3.4–3.7, 122.5–139.5, and 8.8–11.0?ng/g and plasma EC₅₀ values are 34.3–53.7, 0.08–0.10, 7.8–9.5, and 0.6–0.7?ng/mL. Brain regional distribution and binding inhibition upon pretreatment were comparable with data reported with labeled [¹⁸F]FTC-146. Drug exposures were simultaneously determined and correlated with sigma-1 occupancy from the same experiment. Wide category drugs can be assayed for sigma-1 receptor engagement and their correlation with exposures aid in clinical development.     

The influence of repeated administration of clozapine and haloperidol on the effects of the activation of 5-HT(1A), 5-HT(2) and 5-HT(4) receptors in rat frontal cortex.

The effects of a repeated treatment with antipsychotic drugs, clozapine and haloperidol, on the modulation of network activity ex vivo by 5-HT receptors were examined in rat frontal cortical slices using extracellular recording. Rats were treated for 21 days with clozapine (30 mg/kg p.o.), or haloperidol (1 mg/kg p.o.). Spontaneous bursting activity was induced in slices prepared 3 days after the last drug administration by perfusion with a medium devoid of Mg(2+) ions and with added picrotoxin (30 mM). The application of 2-3 microM 8-OH-DPAT, acting through 5-HT(1A) receptors, resulted in a reversible decrease of bursting frequency. In the presence of 1 microM DOI, the 5-HT(2) agonist, or 5 microM zacopride, the 5-HT(4) agonist, bursting frequency increased. Chronic clozapine treatment resulted in an attenuation of the effect of the activation of 5-HT(2) receptors, while the effects related to 5-HT(1A) and 5-HT(4) receptor activation were unchanged. Treatment with haloperiol did not influence the reactivity to the activation of any of the three 5-HT receptor subtypes. These data are consistent with earlier findings demonstrating a selective downregulation of 5-HT(2A) receptors by clozapine and indicate that chronic clozapine selectively attenuates the 5-HT-mediated excitation in neuronal circuitry of the frontal cortex while leaving the 5-HT-mediated inhibition intact.     

In vitro and in vivo pharmacological profile of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562), a novel and putative atypical antipsychotic

In vitro and in vivo pharmacological properties of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562), a novel atypical antipsychotic, were investigated. NRA0562 showed high affinities for human cloned dopamine D(1), D(2), D(3) and D(4) receptors with Ki values of 7.09, 2.49, 3.48 and 1.79 nM. In addition, NRA0562 had high affinities for the 5-HT(2A) receptor and the alpha(1) adrenoceptor with Ki values of 1.5 and 0.56 nM, and moderate affinity for the histamine H(1) receptor. Using in vivo and ex vivo receptor binding studies in rats, we showed NRA0562 occupied frontal cortical 5-HT(2A) receptors and alpha(1) adrenoceptor potently, while occupancy of striatal dopamine D(2) receptor was moderate as were other atypical antipsychotics. NRA0562 dose-dependently inhibited methamphetamine (MAP)-induced locomotor hyperactivity in rats. At higher dosage, NRA0562 dose-dependently antagonized MAP-induced stereotyped behavior and induced catalepsy dose-dependently and significantly in rats. But, the ED(50) value in inhibiting MAP-induced locomotion hyperactivity was 10 times lower than that in inhibiting MAP-induced stereotyped behavior, and 30 times lower than that in inducing catalepsy. In addition, the potency of NRA0562 in antagonizing MAP-induced hyperactivity in rats was higher than that of other antipsychotics, clozapine, risperidone and olanzapine. NRA0562 had favorable properties in view of prediction of extrapyramidal side effects. As this antipsychotic has a unique profile with affinity and occupancy for receptors, we propose that NRA0652 may have unique atypical antipsychotic activities, and a moderate liability of extrapyramidal motor side effects seen in the treatment with classical antipsychotics.     

Involvement of neurotransmitters in the action of apelin-13 on passive avoidance learning in mice

The widespread distribution of apelin-13 and apelin receptors in the brain suggests an important function of this neuropeptide in the brain that has not been explored extensively so far. In the present work, apelin-13 was found to facilitate the consolidation of passive avoidance learning in mice. In order to assess the possible involvement of transmitters in this action, the animals were pretreated with the following receptor blockers in doses which themselves did not influence the behavioral paradigm: phenoxybenzamine (a nonselective α-adrenergic receptor antagonist), propranolol (a β-adrenergic receptor antagonist), cyproheptadine (a nonselective 5-HT2 serotonergic receptor antagonist), atropine (a nonselective muscarinic acetylcholine receptor antagonist), haloperidol (a D2, D3 and D4 dopamine receptor antagonist), bicuculline (a γ-aminobutyric acid subunit A (GABA-A) receptor antagonist), naloxone (a nonselective opioid receptor antagonist), and nitro-l-arginine (a nitric oxide synthase inhibitor). Phenoxybenzamine, cyproheptadine, atropine, haloperidol, bicuculline and nitro-l-arginine prevented the action of apelin-13. Propranolol and naloxone were ineffective. The data suggest that apelin-13 elicits its action on the consolidation of passive avoidance learning via α-adrenergic, 5-HT2 serotonergic, cholinergic, dopaminergic, GABA-A-ergic and nitric oxide mediations.     

Tachykinin receptors and the airways.

The tachykinins, substance P, neurokinin A and neurokinin B, belong to a structural family of peptides. In mammalian airways, substance P and neurokinin A are colocalized to afferent C-fibres. Substance P-containing fibres are close to bronchial epithelium, smooth muscle, mucus glands and blood vessels. Sensory neuropeptides may be released locally, possibly as a result of a local reflex, and produce bronchial obstruction through activation of specific receptors on these various tissues. Three types of tachykinin receptors, namely NK-1, NK-2 and NK-3 receptors, have been characterized by preferential activation by substance P, neurokinin A and neurokinin B respectively. NK-1 and NK-2 receptors were recently cloned. The determination of receptor types involved in the effects of tachykinins in the airways has been done with synthetic agonists and antagonists binding specifically to NK-1, NK-2 and NK-3 receptors. Although the existence of species differences, the conclusion that bronchial smooth muscle contraction is mainly related to activation of NK-2 receptors on bronchial smooth muscle cell has been drawn. The hypothesis of a NK-2 receptor subclassification has been proposed with NK-2A receptor subtype in the guinea-pig airways. Other effects in the airways are related to stimulation of NK-1 receptors on mucus cells, vessels, epithelium and inflammatory cells. A non-receptor-mediated mechanism is also involved in the effect of substance P on inflammatory cells and mast cells.     

The actions of neurokinins and substance P in canine pylorus, antrum and duodenum

Analogues highly selective for receptors for substance P [beta-Ala4,Sar9,Met(02)11]-SP(4-11), for neurokinin A, [Nle10]-NKA(4-10), and for neurokinin B, [beta-Asp4,MePhe7]-NKB(4-10), were administered intraarterially before and after atropine or tetrodotoxin, to characterize the locations on nerve and muscle of the different receptor subtypes in the canine antrum, pylorus and duodenum. Circular muscle strips from each region were also studied in vitro. The NK-2 receptors in the antrum and the pylorus were located postsynaptically on smooth muscle. The NK-3 receptors, on the other hand, were located on neuronal sites in the antrum and duodenum. NK-1 receptors were located on neuronal and nonneuronal sites in the antrum, pylorus and duodenum. Only nonneural receptors could be activated in vitro.     

BIMG 80, a Novel Potential Antipsychotic Drug: Evidence for Multireceptor Actions and Preferential Release of Dopamine in Prefrontal Cortex

Abstract: In radioligand binding studies, BIMG 80, a new putative antipsychotic, displayed good affinity at certain serotonin (5-HT_1A, 5-HT_2A, 5-HT₆), dopamine (D₁, D_2L, D₄), and noradrenergic (α₁) receptors. The effect of acute subcutaneous BIMG 80, clozapine, haloperidol, risperidone, amperozide, olanzapine, and Seroquel was then investigated on dopamine release in medial prefrontal cortex, nucleus accumbens, and striatum in freely moving rats using the microdialysis technique. Four different neurochemical profiles resulted from the studies: (a) Systemic administration of BIMG 80, clozapine, and amperozide produced greater percent increases in dopamine efflux in medial prefrontal cortex than in the striatum or the nucleus accumbens. (b) Haloperidol induced a similar increase in dopamine concentrations in the striatum and nucleus accumbens with no effect in the medial prefrontal cortex. (c) Risperidone and olanzapine stimulated dopamine release to a similar extent in all brain regions investigated. (d) Seroquel failed to change significantly dopamine output both in the medial prefrontal cortex and in the striatum. Because an increase in dopamine release in the medial prefrontal cortex may be predictive of effectiveness in treating negative symptoms and in the striatum may be predictive of induction of extrapyramidal side effects, BIMG 80 appears to be a potential antipsychotic compound active on negative symptoms of schizophrenia with a low incidence of extrapyramidal side effects.     

Effect of typical and atypical antipsychotic drugs on 5-HT2 receptor density in rat cerebral cortex

The effect of acute treatment with seven atypical antipsychotic drugs and four typical antipsychotic drugs on serotonin2 (5-HT2) receptor binding sites in rat cerebral cortex was studied. Among the atypical antipsychotic drugs examined, clozapine, fluperlapine, RMI-81582 and setoperone decreased the density of 5-HT2 receptors, but ticspirone, amperozide and melperone did not. None of the drugs affected the Kd value. Among the typical antipsychotic drugs, loxapine decreased Bmax and increased the Kd of 5-HT2 receptor binding sites, whereas chlorpromazine and cis-flupenthixol had no effect. Clothiapine, a typical antipsychotic drug of the same chemical class as clozapine, decreased Bmax without increasing Kd. The downregulation of 5-HT2 receptor binding sites following a single injection of clozapine, 20 mg/kg, remained almost unchanged during the first 72 hrs and was still significantly decreased for up to 120 hrs. There was no relationship between the affinity for the downregulation of rat cortical 5-HT2 receptor binding site and 5-HT2 receptor density. Coadministration of the D1 dopamine agonist, SKF-38393, did not affect the clozapine-induced downregulation. It is suggested that rapid and prolonged downregulation of 5-HT2 receptor sites is characteristic of some but not all atypical antipsychotic drugs and is not specific to atypical antipsychotic drugs. Dibenzo-epines (clozapine, loxapine, amoxapine, chlothiapine) consistently downregulate 5-HT2 receptors in frontal cortex after acute treatment.     

Diversity in mammalian tachykinin peptidergic neurons: multiple peptides, receptors, and regulatory mechanisms

The tachykinins comprise a family of closely related peptides that participate in the regulation of diverse biological processes. The tachykinin peptides substance P, neurokinin A, neurokinin A(3-10), neuropeptide K, and neuropeptide gamma are produced from a single preprotachykinin gene as a result of differential RNA splicing and differential posttranslational processing. Another tachykinin, neurokinin B, is produced from a separate preprotachykinin gene. These preprotachykinin mRNAs and peptide products are differentially distributed throughout the nervous system. Three distinct G protein-coupled tachykinin receptors exist for these tachykinin peptides. The three receptors interact differentially with the tachykinin peptides and are uniquely distributed throughout the nervous system. The NK-1 receptor preferentially interacts with substance P, the NK-2 receptor prefers neurokinin A, neuropeptide K, and neuropeptide gamma, and the NK-3 receptor interacts best with neurokinin B. Examples of the roles of tachykinin peptidergic neuronal systems are taken from the spinal cord sensory system and the nigrostriatal extrapyramidal motor system. Analysis of the functional significance of multiple tachykinin peptide systems, receptor-second messenger coupling mechanisms, and developmental and regulatory mechanisms underlying peptide mRNA and receptor expression represent areas of current and future investigation.     

Neuroleptics and Dopamine Transporters

The effects of neuroleptic treatments on dopamine transporters and on dopamine receptors was investigated in the forebrain of adult rats treated for 21 days with either haloperidol, clozapine or saline. The dopamine D₁receptors, labeled with [³H]SCH23390, increased in nucleus accumbens, latero-dorsal rostral neostriatum and substantia nigra, after clozapine but not haloperidol. The dopamine D₂receptors, studied with [³H]raclopride, increased in nucleus accumbens and in dorsolateral, ventro-medial and dorso-medial quadrants of the rostral neostriatum after either haloperidol or clozapine treatments, and also in latero-ventral rostral neostriatum but only after haloperidol. Haloperidol also up-regulated D₂receptors in rostral and caudal neostriatum, but clozapine produced a more uneven increase, especially in caudal neostriatum. In contrast, the densities of dopamine uptake sites, or transporters, labeled with [^I25I]RTI-121, remained unchanged after both neuroleptic treatments. The observation that dopamine transporters are resistant to treatments that modify D₁and D₂receptors indicates that these uptake sites can probably be ruled out as the target of neuroleptic drugs, and that dopamine receptor up-regulations can indeed occur independently of the densities of nerve endings at the terminal fields of innervation.     

Pharmacology of [3H]R(+)-7-OH-DPAT binding in the rat caudate-putamen

Dopamine D3 receptors may be involved in drug addiction and in disorders such as schizophrenia and Parkinson's disease. To determine the pharmacological properties of dopamine D3 receptors in the rat caudate-putamen, we have investigated R(+)-[3H]7-hydroxy-N,N-di-n-propyl-2-aminotetralin ([3H]R(+)-7-OH-DPAT) binding to membrane preparations from the rat caudate-putamen. Kinetic analyses showed that [3H]R(+)-7-OH-DPAT binding reached equilibrium in approximately 1 h and that both association and dissociation curves were composed of at least two components. Likewise, saturation curves showed at least two binding components with a combined Bmax value of about 600 fmol/mg protein, which is three times higher than what is present in the subcortical limbic area. Competition curves were performed with agonists such as R(-)-propylnorapomorphine, dopamine, PD 128907, quinpirole, and bromocriptine, and antagonists such as haloperidol, raclopride, clozapine, GR 218231x, remoxipride, and U99194A. These experiments revealed that [3H]R(+)-7-OH-DPAT binding could be resolved into three specific binding sites (R1-R3) and one nonspecific binding site, with R1-R2 probably representing D3 receptor binding and the minor R3 representing D2 receptor binding. The low affinities of (+/-)-8-OH-DPAT and 1,3-di(2-tolyl)guanidine to inhibit [3H]R(+)-7-OH-DPAT binding indicate negligible involvement of 5-HT1A or sigma binding sites, respectively. The pharmacological profile of [3H]R(+)-7-OH-DPAT (2 nM) binding in the caudate-putamen was similar to that of dopamine on [125I]iodosulpride binding in the cerebellar lobule X, which contain D3 but not D2 receptors. Mg2+ increased and GTP and Na+ decreased the binding of [3H]R(+)-7-OH-DPAT, suggesting a coupling of endogenous D3 receptors to G proteins. Taken together, these results suggest that dopamine D3 receptors display multiple agonist binding states, and that D3 receptors are present in high concentrations in the rat caudate-putamen. These results may have implications for the physiological and pathological roles of dopamine D3 receptors in the brain.     

The antisecretory effects of clozapine,a dopamine D4 receptor antagonist,are blocked by the dopamine D1 receptor antagonist,SCH23390

Dopaminergic compounds affect gastric secretion and response to experimental gastric mucosal injury. We showed previously that the novel dopamine D₄ receptor antagonist, clozapine, significantly reduces gastric acid secretion and restraint stress-induced gastric lesions. Because the selectivity of clozapine for D₄ receptors has recently been questioned, we tested the ability of a known d₁ receptor blocker, SCH23390, to affect clozapine-induced reduction in gastric acid secretion. SCH23390 given i.p. or i.c.v., at doses that did not affect gastric acid secretion, significantly blocked the anti-secretory effect of clozapine, administered either peripherally or centrally. These data suggest that neither clozapine nor SCH23390 exhibit as high a degree of selectivity for the dopamine D₄ and d₁ receptor, respectively, as previously believed.