clozapine and Neuroblastoma

Recently, the expression changes of microRNAs (miRNAs) in the serum exosomes (EXO) of schizophrenia (SCZ) have been reported. The aim of this study was to investigate the global expression changes of miRNA derived from the plasma EXO of patients with treatment-resistant schizophrenia (TRS) and the effects of clozapine on miRNA expression.. Global miRNA expression changes in plasma EXO between TRS and controls were studied using microarray analysis. Then, miRNA expressions among TRS, non-TRS, and controls were confirmed with quantitative qPCR experiments. We also studied changes in EXO miRNA expression with in-vitro SH-SY5Y cells.. A microarray for miRNA expression analysis (nine controls vs. nine patients with TRS) revealed 13 up- and 18 downregulated miRNAs that were relevant to neuronal and brain development based on gene ontology analysis. Of those, upregulated miR-675-3p expression was successfully validated in the same cohort by qPCR experiments. Conversely, miR-675-3p expression levels were significantly decreased in the non-TRS cohort (50 controls vs. 50 patients without TRS without clozapine treatment).. We identified global miRNA changes in plasma EXO derived from patients with SCZ that were relevant to neuronal functions, among which, hsa-miR-675-3p expression was upregulated by clozapine treatment.

Topics: Clozapine; Exosomes; Humans; MicroRNAs; Neuroblastoma; Schizophrenia

Current antipsychotics (APs) effectively control positive psychotic symptoms, mainly by blocking dopamine (DA) D2 receptors, but have little effect on negative and cognitive symptoms. Increased glutamate (GLU) release would trigger neurotoxicity, leading to apoptosis and synaptic pruning, which is involved in the pathophysiology of schizophrenia. New pharmacological strategies are being developed such as positive allosteric modulators (PAMs) of the metabotropic GLU receptor 2 (mGluR2) that inhibit the presynaptic release of GLU. We previously reported that treatment of adult mice with JNJ-46356479 (JNJ), a recently developed mGluR2 PAM, partially improved neuropathological deficits and schizophrenia-like behavior in a postnatal ketamine mouse model. In the present study, we evaluated, for the first time, the putative neuroprotective and antiapoptotic activity of JNJ in a human neuroblastoma cell line and compared it with the effect of clozapine (CLZ) as a clinical AP with the highest efficacy and with apparent utility in managing negative symptoms. Specifically, we measured changes in cell viability, caspase 3 activity and apoptosis, as well as in the expression of key genes involved in survival and cell death, produced by CLZ and JNJ alone and in combination with a high DA or GLU concentration as apoptosis inducers. Our results suggest that JNJ is not neurotoxic and attenuates apoptosis, particularly by decreasing the caspase 3 activation induced by DA and GLU, as well as increasing and decreasing the number of viable and apoptotic cells, respectively, only when cultures were exposed to GLU. Its effects seem to be less neurotoxic and more neuroprotective than those observed with CLZ. Moreover, JNJ partially normalized altered expression levels of glycolytic genes, which could act as a protective factor and be related to its putative neuroprotective effect. More studies are needed to define the mechanisms of action of this GLU modulator and its potential to become a novel therapeutic agent for schizophrenia.

Topics: Adult; Allosteric Regulation; Animals; Caspase 3; Cell Culture Techniques; Clozapine; Glutamic Acid; Humans; Mice; Neuroblastoma; Neuroprotective Agents

Autophagy is a process preserving the balance between synthesis, degradation and recycling of cellular components and is therefore essential for neuronal survival and function. Several key proteins govern the autophagy pathway including beclin1 and microtubule associated protein 1 light chain 3 (LC3). Here, we show a brain-specific reduction in beclin1 expression in postmortem hippocampus of schizophrenia patients, not detected in peripheral lymphocytes. This is in contrast with activity-dependent neuroprotective protein (ADNP) and ADNP2, which we have previously found to be deregulated in postmortem hippocampal samples from schizophrenia patients, but that now showed a significantly increased expression in lymphocytes from related patients, similar to increases in the anti-apoptotic, beclin1-interacting, Bcl2. The increase in ADNP was associated with the initial stages of the disease, possibly reflecting a compensatory effect. The increase in ADNP2 might be a consequence of neuroleptic treatment, as seen in rats subjected to clozapine treatment. ADNP haploinsufficiency in mice, which results in age-related neuronal death, cognitive and social dysfunction, exhibited reduced hippocampal beclin1 and increased Bcl2 expression (mimicking schizophrenia and normal human aging). At the protein level, ADNP co-immunoprecipitated with LC3B suggesting a direct association with the autophagy process and paving the path to novel targets for drug design.

Topics: Adult; Aged; Aged, 80 and over; Animals; Antipsychotic Agents; Apoptosis Regulatory Proteins; Autophagy; bcl-Associated Death Protein; Beclin-1; Case-Control Studies; Cell Line, Transformed; Clozapine; Female; Hippocampus; Homeodomain Proteins; Humans; Lymphocytes; Male; Mice; Mice, Knockout; Microtubule-Associated Proteins; Middle Aged; Nerve Tissue Proteins; Neuroblastoma; Rats; Rats, Sprague-Dawley; Schizophrenia; Young Adult

Second-generation antipsychotic drugs, such as clozapine, were reported to enhance neurite outgrowth by nerve growth factor in PC12 cells. The authors previously showed that chloride channel 4 (CLC-4) is responsible for nerve growth factor-induced neurite outgrowth in neuronal cells. In this study, we examined whether clozapine induces CLC-4 in neuroblastoma and glioma cells.. The effect of clozapine on CLC-4 expression was examined in neuroblastoma (SH-SY5Y) and glioma (U87) cells. To investigate the signaling pathway responsible for clozapine-induced CLC-4 expression, the phosphorylation of cAMP response element-binding protein (CREB), which binds CRE in the promoter of the human CLC-4 gene, was examined. To identify the target of clozapine induced CLC-4, CLC-4 siRNA was introduced to neuroblastoma and glioma cells for functional knockdown.. We observed that clozapine increased CLC-4 expression in both SH-SY5Y and U87 cells. Clozapine induced CREB phosphorylation, but in the presence of inhibitor of protein kinase A (an upstream kinase of CREB) clozapine-induced CLC-4 expression was suppressed. Finally, we found that CLC-4 knockdown suppressed clozapine-induced cyclin-dependent kinase 5 (CDK5) expression in SH-SY5Y and U-87 cells suggesting CDK5 as potential molecular target of clozapine induced CLC-4 expression.. The results of the present study suggest that clozapine's therapeutic effect may include the induction of CLC-4 which is dependent on CREB activation via PKA. We also found that functional knockdown of CLC-4 resulted in reduction of CDK5 expression, which may also be implicated in clozapine's therapeutic effect.

Topics: Analysis of Variance; Antipsychotic Agents; Cell Line, Tumor; Clozapine; CREB-Binding Protein; Cyclic AMP-Dependent Protein Kinases; Cyclin-Dependent Kinase 5; Dose-Response Relationship, Drug; Enzyme Activation; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Nerve Growth Factor; Neuroblastoma; RNA, Small Interfering

Metabolic syndrome (MetS) is a constellation of factors including abdominal obesity, hyperglycemia, dyslipidemias, and hypertension that increase morbidity and mortality from diabetes and cardiovascular diseases and affects more than a third of the population in the US. Clozapine, an atypical antipsychotic used for the treatment of schizophrenia, has been found to cause drug-induced metabolic syndrome (DIMS) and may be a useful tool for studying cellular and molecular changes associated with MetS and DIMS. Mitochondria dysfunction, oxidative stress and inflammation are mechanisms proposed for the development of clozapine-related DIMS. In this study, the effects of clozapine on mitochondrial function and inflammation in insulin responsive and obesity-associated cultured cell lines were examined.. Cultured mouse myoblasts (C2C12), adipocytes (3T3-L1), hepatocytes (FL-83B), and monocytes (RAW 264.7) were treated with 0, 25, 50 and 75 µM clozapine for 24 hours. The mitochondrial selective probe TMRM was used to assess membrane potential and morphology. ATP levels from cell lysates were determined by bioluminescence assay. Cytokine levels in cell supernatants were assessed using a multiplex array. Clozapine was found to alter mitochondria morphology, membrane potential, and volume, and reduce ATP levels in all cell lines. Clozapine also significantly induced the production of proinflammatory cytokines IL-6, GM-CSF and IL12-p70, and this response was particularly robust in the monocyte cell line.. Clozapine damages mitochondria and promotes inflammation in insulin responsive cells and obesity-associated cell types. These phenomena are closely associated with changes observed in human and animal studies of MetS, obesity, insulin resistance, and diabetes. Therefore, the use of clozapine in DIMS may be an important and relevant tool for investigating cellular and molecular changes associated with the development of these diseases in the general population.

Topics: 3T3-L1 Cells; Adenosine Triphosphate; Adipocytes; Animals; Antipsychotic Agents; Brain; Cell Line; Clozapine; Cytokines; Hepatocytes; Inflammation; Inflammation Mediators; Insulin; Membrane Potential, Mitochondrial; Metabolic Syndrome; Mice; Mitochondria; Myoblasts; Neuroblastoma

G-protein coupled designer receptors that are specifically activated by designer drugs have been developed. Here, we have analyzed the regulation of gene transcription following activation of Gα(q)-coupled designer receptor (Rα(q)). Stimulation of human embryonic kidney (HEK) 293 cells expressing Rα(q) with clozapine-N-oxide (CNO), a pharmacologically inert compound, induced the expression of biologically active Egr-1, a zinc finger transcription factor. Expression of a dominant-negative mutant of the ternary complex factor (TCF) Elk-1, a key transcriptional regulator of serum response element (SRE)-driven gene transcription, prevented Egr-1 expression. Stimulation of Rα(q) with CNO increased the transcriptional activation potential of Elk-1 and enhanced transcription of an SRE regulated reporter gene. In addition, AP-1 transcriptional activity was significantly elevated. AP-1 activity was controlled by TCFs and c-Jun in cells expressing an activated Gα(q)-coupled designer receptor. CNO stimulation did not increase Egr-1 and AP-1 activity in neuroblastoma cells expressing endogenous M3 muscarinic acetylcholine receptors, indicating that CNO did not function as a ligand for these receptors. Rα(q) stimulation also increased the transcriptional activation potential of CREB and cAMP response controlled gene transcription. Pharmacological and genetic experiments revealed that the protein kinases Raf and ERK were essential to connect Rα(q) stimulation with enhanced Egr-1 and AP-1 controlled transcription. In contrast, MAP kinase phosphatase-1 functioned as a nuclear shut-off device of stimulus-transcription coupling. The fact that Rα(q) stimulation activates the transcription factors Egr-1, Elk-1, AP-1, and CREB indicates that regulation of gene transcription is an integral part of Gα(q)-coupled receptor signaling.

Topics: Cell Line, Tumor; Clozapine; CREB-Binding Protein; Cyclic AMP Response Element-Binding Protein; Dual Specificity Phosphatase 1; Early Growth Response Protein 1; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Genes, Immediate-Early; GTP-Binding Protein alpha Subunits, Gq-G11; HEK293 Cells; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mutation; Neuroblastoma; raf Kinases; Receptor, Muscarinic M3; Receptors, G-Protein-Coupled; Serum Response Element; Transcription Factor AP-1; Transcription, Genetic; Transcriptional Activation

N-Acetylaspartate (NAA) and N-acetylaspartylglutamate (NAAG) are related neuronal metabolites associated with the diagnosis and treatment of schizophrenia. NAA is a valuable marker of neuronal viability in magnetic resonance spectroscopy, a technique which has consistently shown NAA levels to be modestly decreased in the brains of schizophrenia patients. However, there are conflicting reports on the changes in brain NAA levels after treatment with antipsychotic drugs, which exert their therapeutic effects in part by blocking dopamine D(2) receptors. NAAG is reported to be an agonist of the metabotropic glutamate 2/3 receptor, which is linked to neurotransmitter release modulation, including glutamate release. Alterations in NAAG metabolism have been implicated in the development of schizophrenia possibly via dysregulation of glutamate neurotransmission. In the present study we have used high performance liquid chromatography to determine the effects of the antipsychotic drugs haloperidol and clozapine on NAA and NAAG levels in SH-SY5Y human neuroblastoma cells, a model system used to test the responses of dopaminergic neurons in vitro. The results indicate that the antipsychotic drugs haloperidol and clozapine increase both NAA and NAAG levels in SH-SY5Y cells in a dose and time dependant manner, providing evidence that NAA and NAAG metabolism in neurons is responsive to antipsychotic drug treatment.

Topics: Antipsychotic Agents; Aspartic Acid; Cell Line, Tumor; Clozapine; Dipeptides; Dose-Response Relationship, Drug; Haloperidol; Humans; Neuroblastoma; Neurons

The present study examined the effects of N-desmethylclozapine (NDMC), a biologically active metabolite of the atypical antipsychotic clozapine, at cloned human opioid receptors stably expressed in Chinese hamster ovary (CHO) cells and at native opioid receptors present in NG108-15 cells and rat brain. In CHO cells expressing the delta-opioid receptor (CHO/DOR), NDMC behaved as a full agonist both in stimulating [(35)S]GTPgammaS binding (pEC(50)=7.24) and in inhibiting cyclic AMP formation (pEC(50)=6.40). NDMC inhibited [(3)H]naltrindole binding to CHO/DOR membranes with competition curves that were modulated by guanine nucleotides in an agonist-like manner. Determination of intrinsic efficacies by taking into consideration both the maximal [(35)S]GTPgammaS binding stimulation and the extent of receptor occupancy at which half-maximal effect occurred indicated that NDMC had an efficacy value equal to 82% of that of the full delta-opioid receptor agonist DPDPE, whereas clozapine and the other clozapine metabolite clozapine N-oxide displayed much lower levels of agonist efficacy. NDMC exhibited poor agonist activity and lower affinity at the kappa-opioid receptor and was inactive at mu-opioid and NOP receptors. In NG108-15 cells, NDMC inhibited cyclic AMP formation and stimulated the phosphorylation of extracellular signal-regulated kinase 1/2 by activating the endogenously expressed delta-opioid receptor. Moreover, in membranes of different brain regions, NDMC stimulated [(35)S]GTPgammaS binding and regulated adenylyl cyclase activity and the effects were potently antagonized by naltrindole. These data demonstrate for the first time that NDMC acts as a selective and efficacious delta-opioid receptor agonist and suggest that this unique property may contribute, at least in part, to the clinical actions of the atypical antipsychotic clozapine.

Topics: Adenylyl Cyclases; Analgesics, Opioid; Animals; Antipsychotic Agents; CHO Cells; Clozapine; Cricetinae; Cricetulus; Cyclic AMP; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Male; Mice; Neuroblastoma; Protein Binding; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Recombinant Proteins

We previously identified Neuregulin1 (NRG1) as a gene contributing to the risk of developing schizophrenia. Furthermore, we showed that NRG1+/- mutant mice display behavioral abnormalities that are reversed by clozapine, an atypical antipsychotic drug used for the treatment of schizophrenia. We now present evidence that ErbB4 (v-erb-a erythroblastic leukemia viral oncogene homolog 4), the tyrosine kinase receptor for NRG1 in hippocampal neurons, interacts with two nonreceptor tyrosine kinases, Fyn and Pyk2 (proline-rich tyrosine kinase 2). NRG1 stimulation of cells expressing ErbB4 and Fyn leads to the association of Fyn with ErbB4 and consequent activation. Furthermore, we show that NRG1 signaling, through activation of Fyn and Pyk2 kinases, stimulates phosphorylation of Y1472 on the NR2B subunit of the NMDA receptor (NMDAR), a key regulatory site that modulates channel properties. NR2B Y1472 is hypophosphorylated in NRG1+/- mutant mice, and this defect can be reversed by clozapine at a dose that reverses their behavioral abnormalities. We also demonstrate that short-term synaptic plasticity is altered and theta-burst long-term potentiation is impaired in NRG1+/- mutant mice, and incubation of hippocampal slices from these mice with NRG1 reversed those effects. Attenuated NRG1 signaling through ErbB4 may contribute to the pathophysiology of schizophrenia through dysfunction of NMDAR modulation. Thus, our data support the glutamate hypothesis of schizophrenia.

Topics: Animals; Antineoplastic Agents; Antipsychotic Agents; Cell Differentiation; Cell Line, Tumor; Chlorocebus aethiops; CHO Cells; Clozapine; COS Cells; Cricetinae; Cricetulus; ErbB Receptors; Hippocampus; Humans; Kidney; Mice; Mice, Knockout; Nerve Tissue Proteins; Neuregulin-1; Neuroblastoma; Neuronal Plasticity; Phosphorylation; Proto-Oncogene Proteins c-fyn; Receptor, ErbB-4; Receptors, N-Methyl-D-Aspartate; Schizophrenia; Signal Transduction; Synapses; Tretinoin

Loss of mitochondrial membrane integrity and consequent release of apoptogenic factors may be involved in mediating striatal neurodegeneration after prolonged treatment with the typical antipsychotic drug haloperidol. Apoptosis-inducing factor (AIF), an intramitochondrial protein, may have a large influence on mediating haloperidol-induced striatal neuron destruction. Translocation of this protein from mitochondria to the nucleus promotes cell death independently of the caspase cascade. To examine how AIF may contribute to haloperidol-induced apoptosis, AIF translocation was observed in three haloperidol treatment paradigms. SH-SY5Y cells were treated with both haloperidol and clozapine and examined for AIF immunofluorescence. Immunohistochemistry was also performed on human striatal sections obtained from the Stanley Foundation Neuropathology Consortium and on rat brain sections following 28 days of antipsychotic drug treatment. In the cellular model haloperidol, but not clozapine treatment increased the nuclear AIF immunofluorescent signal and decreased cell viability. Corollary to these findings, striatal sections from patients who had taken haloperidol and rats who were administered haloperidol both had an elevated nuclear AIF signal. The results provide novel evidence implicating the involvement of AIF in haloperidol-associated apoptosis and its relevance to the development of typical antipsychotic drug-related adverse effects such as tardive dyskinesia.

Topics: Animals; Antipsychotic Agents; Apoptosis; Apoptosis Inducing Factor; Case-Control Studies; Cell Line, Tumor; Cell Nucleus; Cell Survival; Clozapine; Corpus Striatum; Haloperidol; Humans; Immunohistochemistry; Male; Mitochondria; Neuroblastoma; Rats; Rats, Sprague-Dawley

The goal of the present study was to determine the effects of clozapine (Cloz) and its metabolites norclozapine (Norcloz) and clozapine-N-oxide (Cloz-N-oxide) on the 5-HT(2) receptor system on the levels of protein and gene expression in in vitro systems of primary cortical cells of the rat and human hippocampal SHS5Y5 neuroblastoma cells.. Clinically relevant concentrations of Cloz (200/400 ng/ml) and its metabolites (200 ng/ml) were used for the examination of the effects of Cloz and its metabolites on serotoninergic 5-HT(2) receptor parameters (density, affinity and mRNA levels) as well as on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels in primary cortical cells of the rat after treatment for 24 h under in vitro conditions. To compare the results to human cells, we also measured treatment-induced changes in 5-HT(2) and GAPDH mRNA levels in human hippocampal SHS5Y5 cells.. A significant decrease was found in primary cortical cells for 5-HT(2) receptor density (Cloz 200/Cloz 400/Norcloz 200 and Cloz-N-oxide 200 vs. control) and 5-HT(2A) receptor mRNA levels (Cloz 200 vs. control). 5-HT(2A) receptor mRNA levels were also significantly reduced (Norcloz 200 vs. control) in SHS5Y5 cells. GAPDH mRNA levels were not affected.. The results of the present study show that Cloz and Norcloz induce significant alterations on the 5-HT(2) receptor system in primary cortical cells of the rat and in human hippocampal cells.

Topics: Analysis of Variance; Animals; Cells, Cultured; Cerebral Cortex; Clozapine; Dose-Response Relationship, Drug; Gene Expression Regulation; Hippocampus; Humans; In Vitro Techniques; Neuroblastoma; Neurons; Protein Binding; Radioligand Assay; Rats; Receptors, Serotonin, 5-HT2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serotonin Antagonists

We investigated the effect of 10 microM clozapine on the activity of glycogen synthase kinase-3beta (GSK-3beta) and its upstream and downstream molecules in SH-SY5Y human neuroblastoma cells. Clozapine activates both Akt- and Dvl-mediated phosphorylation of GSK-3beta through phosphorylation at Ser9, and increased total cellular and intranuclear levels of beta-catenin. Pretreatment with the specific inhibitor of the phosphatidylinositol 3-kinase (PI3K)-Akt pathway, LY294002 (20 microM), prevented the phosphorylation of Akt but did not affect the phosphorylation of GSK-3beta. These results suggest that clozapine regulates the phosphorylation of GSK-3beta through Wnt signal pathways involving Dvl upstream but not through the PI3K-Akt pathway in SH-SY5Y cells.

Topics: Apoptosis; beta Catenin; Cell Division; Cell Line, Tumor; Cell Nucleus; Chromones; Clozapine; Culture Media, Serum-Free; Cytoskeletal Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; Morpholines; Neuroblastoma; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Serine; Signal Transduction; Time Factors; Trans-Activators; Wnt Proteins; Zebrafish Proteins

Previous studies have shown D2-like dopamine receptor involvement in the regulation of phospholipid methylation (PLM), while others have documented impaired methionine and folate metabolism in schizophrenia. Utilizing [14C]formate labeling in cultured neuroblastoma cell lines, we now show that D4 dopamine receptors (D4R) mediate the stimulatory effect of dopamine (DA) on PLM. The effect of DA was potently blocked by highly D4R-selective antagonists and stimulated by the D4R-selective agonist CP-226269. DA-stimulated PLM was dependent upon the activity of methionine cycle enzymes, but DA failed to increase PLM in [3H]methionine labeling studies, indicating that a methionine residue in the D4R might be involved in mediating PLM. A direct role for MET313, located on transmembrane helix No. 6 immediately adjacent to phospholipid headgroups, was further suggested from adenosylation, site-directed mutagenesis and GTP-binding results. A comparison of PLM in lymphocytes from schizophrenia patients vs control samples showed a four-fold lower activity in the schizophrenia group. These findings reveal a novel mechanism by which the D4R can regulate membrane composition. Abnormalities in D4R-mediated PLM may be important in psychiatric illnesses such as schizophrenia.

Topics: Amino Acid Sequence; Aminopyridines; Animals; Benzazepines; Binding Sites; Carbon Radioisotopes; CHO Cells; Clozapine; Cricetinae; Dopamine Agonists; Dopamine Antagonists; Dopamine D2 Receptor Antagonists; Formates; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Methionine; Mutagenesis, Site-Directed; Neuroblastoma; Phospholipids; Phosphorylation; Piperidines; Psychotic Disorders; Pyridines; Pyrroles; Raclopride; Receptors, Dopamine D2; Receptors, Dopamine D4; Recombinant Proteins; S-Adenosylmethionine; Salicylamides; Schizophrenia; Transfection; Tumor Cells, Cultured

In radioligand binding studies, BIMG 80, a new putative antipsychotic, displayed good affinity at certain serotonin (5-HT1A, 5-HT2A, 5-HT6), dopamine (D1, D2L, D4), and noradrenergic (alpha1) 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.

Topics: Animals; Antipsychotic Agents; Benzodiazepines; CHO Cells; Clozapine; Corpus Striatum; COS Cells; Cricetinae; Dibenzothiazepines; Dopamine; Female; Guinea Pigs; Haloperidol; Indoles; Kidney; Male; Microdialysis; Neuroblastoma; Nucleus Accumbens; Olanzapine; Piperazines; Pirenzepine; Prefrontal Cortex; Pyridines; Quetiapine Fumarate; Radioligand Assay; Rats; Rats, Sprague-Dawley; Risperidone; Tritium; Tumor Cells, Cultured

In NG 108-15 cells expressing the recombinant human D3 receptor, dopamine agonists enhance [3H]thymidine incorporation and decrease cAMP accumulation. In these cells, but not in wild type cells, haloperidol, fluphenazine, and various other antipsychotics inhibited basal [3H]thymidine incorporation in a concentration-dependent manner. In contrast, other dopamine antagonists such as nafadotride or (+)AJ 76, two D3-preferring antagonists, were without effect. The concentration-response curve of haloperidol was shifted to the right in presence of nafadotride, with a potency compatible with its nanomolar apparent affinity as neutral antagonist. Pertussis toxin treatment abolished or markedly reduced the responses to haloperidol or fluphenazine. In contrast, no significant enhancement of cAMP accumulation could be observed, under the influence of haloperidol or eticlopride. These data indicate that some dopamine antagonists behave as inverse agonists, and thus appear to inhibit an agonist-independent activity of the D3 receptor on [3H]thymidine incorporation pathway, but not on the cAMP pathway.

Topics: Animals; Antipsychotic Agents; Calcium; CHO Cells; Clozapine; Colforsin; Cricetinae; Cyclic AMP; DNA Replication; Dopamine Agonists; Dopamine Antagonists; Dose-Response Relationship, Drug; Flupenthixol; Glioma; Haloperidol; Humans; Hybrid Cells; Naphthalenes; Neuroblastoma; Pertussis Toxin; Phenothiazines; Pimozide; Pyrrolidines; Quinpirole; Receptors, Dopamine D2; Receptors, Dopamine D3; Recombinant Proteins; Risperidone; Salicylamides; Second Messenger Systems; Sulpiride; Tetrahydronaphthalenes; Tumor Cells, Cultured; Virulence Factors, Bordetella

Dopamine receptors belong to the family of G protein-coupled receptors. On the basis of the homology between these receptors, three different dopamine receptors (D1, D2, D3) have been cloned. Dopamine receptors are primary targets for drugs used in the treatment of psychomotor disorders such as Parkinson's disease and schizophrenia. In the management of socially withdrawn and treatment-resistant schizophrenics, clozapine is one of the most favoured antipsychotics because it does not cause tardive dyskinesia. Clozapine, however, has dissociation constants for binding to D2 and D3 that are 4 to 30 times the therapeutic free concentration of clozapine in plasma water. This observation suggests the existence of other types of dopamine receptors which are more sensitive to clozapine. Here we report the cloning of a gene that encodes such a receptor (D4). The D4 receptor gene has high homology to the human dopamine D2 and D3 receptor genes. The pharmacological characteristics of this receptor resembles that of the D2 and D3 receptors, but its affinity for clozapine is one order of magnitude higher. Recognition and characterization of this clozapine neuroleptic site may prove useful in the design of new types of drugs.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Blotting, Northern; Brain; Cell Line; Cloning, Molecular; Clozapine; Gene Library; Humans; Kinetics; Molecular Sequence Data; Molecular Weight; Neuroblastoma; Organ Specificity; Protein Conformation; Rats; Receptors, Dopamine; Receptors, Dopamine D2; Receptors, Dopamine D4; Restriction Mapping; RNA, Messenger; Sequence Homology, Nucleic Acid; Transfection

Tricyclic antidepressants and some structurally related compounds were tested for their ability to antagonize histamine H1 and muscarinic acetylcholine receptors of cultured mouse neuroblastoma cells. As a group, tertiary amine tricyclic antidepressants tended to be more potent than secondary amine drugs at both receptors. The most potent antihistamine, doxepin hydrocholoride, was about 4 times more potent than amitriptyline hydrochloride, about 800 times more potent than diphenhydramine hydrochloride, and about 8,000 times more potent than desipramine hydrochloride, the least potent tricyclic antidepressant at both the histamine H1 and the muscarinic acetylcholine receptors. All tricyclic drugs except desipramine hydrochloride were more potent as antihistamines than as anticholinergics. Doxepin hydrochloride and amitriptyline hydrochloride may be the most potent antihistamines known, and the antihistaminic potencies of these and the other tricyclic antidepressant drugs may relate directly to their ability to cause sedation and drowsiness in patients.

Topics: Amitriptyline; Animals; Antidepressive Agents, Tricyclic; Atropine; Benztropine; Cells, Cultured; Clozapine; Cyproheptadine; Desipramine; Doxepin; Haloperidol; Histamine H1 Antagonists; Hydroxyzine; Imipramine; Mice; Neoplasms, Experimental; Neuroblastoma; Nortriptyline; Protriptyline; Receptors, Histamine; Receptors, Histamine H1; Receptors, Muscarinic