Studies on Muscarinic Binding Sites in Human Brain Identified with [3H]Pirenzepine

Pirenzepine, a potent antimuscarinic agent with apparent selectivity for a subtype (M1) of muscarinic receptors, was used in tritiated form to characterize its binding to human brain tissue. Specific [3H]pirenzepine binding showed rapid association and dissociation. From kinetic and competitive binding experiments, its KD was 5.5 nM and 9 nM, respectively. Regional distribution of [3H]pirenzepine binding determined in parallel with [3H]quinuclidinyl benzilate binding, a nonselective muscarinic antagonist, indicated a significant correlation for the maximum number of binding sites for the two radioligands in 13 brain regions, with the highest amount of binding for each in the putamen and the least in the cerebellum. Binding for [3H]pirenzepine averaged 57% of that for [3H]quinuclidinyl benzilate, with a range of 20% (cerebellum) to 77% (frontal cortex). Most antidepressants and neuroleptics tested had affinities for [3H]pirenzepine binding sites that were not significantly different from their previously reported values obtained with the use of [3H]quinuclidinyl benzilate.     

Visualization of Antipsychotic Drug Binding to Living Mesolimbic Neurons Reveals D2 Receptor, Acidotropic, and Lipophilic Components

Abstract: To examine the binding of antipsychotic drugs to living neurons, we applied fluoroprobe derivatives of the D2 antagonist spiperone to mesolimbic system neurons in postnatal culture. We found that rhodamine- N -( p -aminophenethyl)spiperone (rhodamine-NAPS) stereospecifically labeled the plasma membranes of 38 ± 6% of ventral tegmental area neurons, 22 ± 7% of which were dopaminergic, and 50 ± 6% of medium-sized putatively GABAergic nucleus accumbens neurons, with a time constant of ∼8 min. In contrast, the BODIPY derivative of NAPS rapidly labeled intracellular sites in all neurons in a punctate pattern, consistent with acidotropic uptake. Native antipsychotics also show acidotropic uptake, which we visualized by their displacement of the fluorescent weak base vital dye acridine orange from acidic intracellular compartments. We found that acidotropic uptake correlated best with the partition coefficients of the drugs. With a time constant of 23 min, rhodamine-NAPS labeled all neurons in a pattern suggestive of lipophilic solvation. Thus, initially rhodamine-NAPS makes possible visualization of D2 receptors on living neurons; however, acidotropic uptake and lipophilic solvation obscure receptor labeling and may account for time-dependent factors in the action of antipsychotic drugs, as well as affect their use as radioreceptor ligands.     

Dopamine D4-Like Receptor Elevation in Schizophrenia: Cloned D2 and D4 Receptors Cannot Be Discriminated by Raclopride Competition Against [3H]Nemonapride

Abstract: Three independent studies have found that the density of dopamine D4-like receptors is elevated in postmortem brain striata in schizophrenia. This elevation has been questioned by a fourth study that used a different method and failed to detect a biphasic component when raclopride was used to compete against the binding of 1 n M [³H]nemonapride to schizophrenia tissue. To test whether this competition method could distinguish between dopamine D2 and D4 receptors, the present study used mixtures of only these two cloned receptors, free of all other receptors. Using combinations of cloned dopamine D2 and D4 receptors, this competition method could not resolve these components up to a level of 48% D4 receptors. Thus, the objections raised by the findings of the fourth study, mentioned above, do not appear valid. Furthermore, the present results indicate that the data using such a competition method actually mask a manyfold marked elevation in the density of dopamine D4-like receptors in schizophrenia.     

Induction of c-fos mRNA Expression in Rat Striatum by Neuroleptic Drugs

The effect of selective dopamine D2 receptor-acting drugs on striatal c-fos mRNA expression in the rat has been investigated by Northern hybridization and autoradiography to determine a possible role for c-fos in the initiation of adaptive changes in D2 receptor number by neuroleptic drugs. The neuroleptic drug haloperidol, a D2 receptor antagonist, was found to produce a rapid and transient induction of c-fos mRNA expression as compared with the expression in animals treated with saline. This induction by haloperidol was found to be dose dependent and D2 receptor mediated, inasmuch as a D2 agonist completely reversed the induction and the inactive isomer of the neuroleptic butaclamol, which does not produce an increase in D2 receptors, had no effect on c-fos mRNA expression. From these data, it can be concluded that c-fos expression in striatum is under dopamine D2 receptor-mediated inhibitory control. It is suggested that c-fos may play a role in the initiation of the increase in D2 receptor number produced by chronic neuroleptic drug treatment.     

The role of glutamate receptors in antipsychotic drug action

Summary. It has recently been postulated that disturbances in glutamatergic neurotransmission may contribute to the pathophysiology of schizophrenia. Therefore the aim of the present study was to evaluate the role of glutamate NMDA and group II metabotropic receptors in the antipsychotic drug action. To this aim the influence of some well-known neuroleptics on cortical NMDA receptors was examined. Furthermore, their behavioral effects were compared with those of the novel agonist of group II glutamate metabotropic receptors, LY 354740, in some animal models of schizophrenic deficits. We found that long-term administration of the typical neuroleptic haloperidol and the atypical one clozapine increased the number of NMDA receptors labelled with [³H]CGP 39653 in different cortical areas. Long-, but not short-term, treatment with haloperidol and raclopride diminished the deficit of prepulse inhibition produced by phencyclidine, which is a model of sensorimotor gating deficit in schizophrenia. In contrast, neither short- nor long-term treatment with clozapine influenced the phencyclidine effect in that model. Acute treatment with LY 354740 reversed neither (1) the deficit of prepulse inhibition produced by phencyclidine or apomorphine, nor (2) the impairment in a delayed alternation task induced by MK-801, which is commonly used to model the frontal lobe deficits associated with schizophrenia. The present study suggests that an increase in the density of cortical NMDA receptors may be important to a longterm neuroleptic therapy. Conversely, the results do not support the role of group II metabotropic glutamate receptors in the antipsychotic drug action. Received August 31, 1999 Accepted September 20, 1999     

Differential Effect of Subchronic Treatment with Various Neuroleptic Agents on Serotonin2 Receptors in Rat Cerebral Cortex

In addition to antidepressant drugs, some neuroleptic (NL) drugs reduce serotonin2 (5-HT2) receptor binding sites after chronic administration. The present study was undertaken to characterize further this property of NL drugs. Scatchard analysis of [3H]spiperone binding in rat cerebral cortex revealed that 21-day treatment with chlorpromazine (CPZ), cis-flupenthixol, and thioridazine reduced 5-HT2 radioligand binding density by 60, 27, and 18%, respectively. The more selective dopamine-D2 antagonists haloperidol and sulpiride were totally ineffective in this regard. No reduction in 5-HT2 ligand binding sites occurred after 1 day of treatment with CPZ but 3-days of treatment was effective and this reduction persisted, although diminished, for at least 72 h after the last injection. cis-Flupenthixol and d-butaclamol were also effective after 3 days of treatment but trans-flupenthixol and l-butaclamol were not, indicating stereo-specificity of the response mechanism. Female rats showed the same response to CPZ as did male rats. Central 5,7-dihydroxytryptamine-induced lesions of 5-HT neurons demonstrated that intact 5-HT neurons were not required for the reduction of 5-HT2 receptor ligand binding by CPZ. Since CPZ has high affinity for many receptors, including alpha 1, histamine1, and muscarinic receptors, the role of these effects in producing 5-HT2 receptor down-regulation was considered by studying the effects of prazosin, atropine, and pyrilamine administration on 5-HT2 radioligand binding. Results indicate that no one of these actions appears to account for the down-regulation of 5-HT2 receptors by CPZ. Several of these effects, in combination, or some unique mechanism, may be involved.     

Evaluation of electrospray ionisation liquid chromatography-tandem mass spectrometry for rational determination of a number of neuroleptics and their major metabolites in human body fluids and tissues

A study of liquid chromatography-triple quadrupole mass spectrometry (LC-MS-MS) with positive electrospray ionisation (ESI) for the determination of selected drugs in human tissues and body fluids such as blood, urine and hair is described. The possibility to screen for and quantify the 19 most commonly prescribed neuroleptics on the Swedish market and determine the presence of their major metabolites within a single LC-MS-MS analysis was evaluated on a PE Sciex API2000 instrument. Chromatographic conditions were optimised and the best separation, with individual retention times for most of the analytes, was obtained on a Zorbax SB-CN column within a 9-min gradient run. The MS-MS fragmentation conditions were optimised for each compound in order to obtain both specific fragments and high signal intensity. Since neuroleptics are a heterogeneous group of compounds, a markedly difference in collision energy needed to achieve fragments of the selected parent ions was seen and the number of fragments achieved varied as well. For sensitive quantification the transition of the most intense fragment of the protonated molecular ion (M+1)(+) was selected for multiple reaction monitoring analysis. More than 70 transitions were finally included in the assay. Detection levels down to the lower ng/ml level were achieved for all analytes, but between analytes more than a 10-fold difference in signal response was seen. By evaluation of extracted ion chromatograms from the analysis of authentic human blood, urine and hair sample the proposed concept for rational drug analysis was found to be both selective and sensitive for the neuroleptics included. A great number of metabolites could be determined in blood, urine and hair as well. A full method validation was not performed since the objective was to evaluate the method design rather than to validate a final method set-up.     

[3H]Dopamine Labeling of D3 Dopaminergic Sites in Human, Rat, and Calf Brain

Abstract: The binding of [³H]dopamine to brain regions of calf, rat, and human was investigated. The calf caudate contained the highest density of [³H]dopamine binding sites, with a B_max value of 185 fmol/mg protein, whereas rat and human striatum contained one-third this number of sites. The K_D values for [³H]dopamine in all tissues were 2–3 nM. Dopaminergic catecholamines (dopamine, apomorphine, 6,7-dihydroxy-2-aminotetralin, and N-propylnorapomorphine) inhibited the binding of [³H]dopamine in all three species, at low concentrations, with IC₅₀ values of 1.5 to 6 nM. Neuroleptics, in contrast, inhibited the binding at high concentrations (with IC₅₀ values of 200 to 40,000 nM). The [³H]dopamine binding sites were saturable, heat-labile, and detectable only in dopamine-rich brain regions; these sites differed from D₂ dopamine sites (labeled by [³H]butyrophenone neuroleptics), and from D_l dopamine sites (labeled by [³H]thioxanthene neuroleptics) associated with the dopamine-stimulated adenylate cyclase. We have, therefore, called these high-affinity [³H]dopamine binding sites D₃ sites. [³H]Apomorphine and [³H]ADTN also appeared to label D₃ sites. These ligands however, were less selective than [³H]dopamine, and labeled sites other than D₃ as well. Assay conditions were important in determining the parameters of [³H]dopamine binding. The optimum conditions for selective labeling of the D₃ dopaminergic sites, using [³H]dopamine, required the presence of EDTA and ascorbate.     

The effects by neuroleptics, antimycotics and antibiotics on disulfide reducing enzymes from the human pathogens Acanthamoeba polyphaga and Naegleria fowleri

This paper discusses the effects of two neuroleptic agents, chlorpromazine and trifluoperazine; three antimycotics, amphotericin B, ketoconazole and miconazole and four antibiotics, pentamidine, rifampicin, mepacrine and metronidazole on the NADPH-dependent disulfide reducing enzymes cystine reductase (CysR), glutathione reductase (GR) trypanothione reductase (TR) and a putative disulfide reductase for compound X in Acanthamoeba polyphaga from the human pathogens A. polyphaga and Naegleria fowleri. Against A. polyphaga, all nine drugs studied had the capacity to inhibit the putative disulfide reductase from the trophozoites at a concentration of 32microg/ml during a 24h incubation and they were: the neuroleptics trifluoperazine (100%) and chlorpromazine (96%), the antimycotics miconazole (89%) ketoconazole (81%) and amphotericin B, (53%) and the antibiotics pentamidine (89%), rifampicin (64%), mepacrine (57%) and metronidazole (14%). Only six of the nine drugs simultaneously inhibited CysR, GR and the putative disulfide reductase. In N. fowleri, the most potent inhibitors of trypanothione reductase were amphotericin B and miconazole which inhibited 100% at a concentration of 32microg/ml during the 24h incubation followed by the neuroleptics trifluoperazine (92%) and chlorpromazine (80%) and the antibiotic mepacrine (70%). All these also inhibited CysR and GR from the trophozoites other than mepacrine which inhibited only CysR and TR. Ketoconazole, rifampicin (which did not affect CysR), pentamidine and metronidazole had opposite effects since they did not inhibit but increased the amount of the three thiols.     

Monoclonal Antibodies with High Affinity for Spiroperidol

A diverse panel of monoclonal antibodies was obtained from BALB/c mice immunized with two haptens structurally related to spiroperidol (SPD). Bromoacetyl derivatives of aminospiroperidol (NH2SPD) and N-amino-phenethylspiroperidol (NAPS) were synthesized to couple the haptens covalently to a protein carrier for immunization, thereby maintaining the butyrophenone portion of the immunogen. Hybridomas were selected based on their ability to secrete antibody that binds [3H]SPD with high affinity. Equilibrium dissociation constants for these antibodies ranged from 0.2 to greater than 100 nM. The antigen binding sites of the anti-NH2SPD and anti-NAPS antibodies were characterized in studies of the inhibition of the binding of [3H]-SPD by a series of ligands that are either (a) structurally related to SPD or (b) structurally unrelated to the butyrophenones but known to be selective antagonists of the D2 subtype of dopamine receptor. Based on the patterns of inhibition of the binding of [3H]SPD by these compounds, 12 classes of antibody combining sites were identified. Most of these antibodies bound butyrophenones with high affinity. One anti-NH2SPD and four anti-NAPS antibodies also bound domperidone, a nonbutyrophenone that has a high affinity for D2 receptors. None of the antibodies bound clebopride or sulpiride, D2-selective antagonists of the benzamide class, or the agonist dopamine.