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P Seeman

Publications and source records attributed to P Seeman.

At least 55 records · Page 3Linked to original sources
PubMed1999

Clozapine pre-treatment enhances raclopride catalepsy.

The clinical replacement of clozapine by another antipsychotic sometimes causes extrapyramidal signs, including dystonia, to appear suddenly. The present study was done, therefore, to test whether clozapine pre-treatment of rats could affect raclopride-induced catalepsy. Clozapine, at 5 mg/kg, given 2 h before a catalepsy-threshold dose of 0.1 mg/kg raclopride, markedly enhanced raclopride-induced catalepsy in the rats. The results are compatible with earlier in vitro data where pre-exposure of human cloned dopamine D2 receptors to clozapine resulted in an increased potency of raclopride in inhibiting the binding of [3H]clozapine to the receptors. The mechanism of clozapine potentiation of raclopride action may contribute to the clinically observed post-clozapine dystonia.

Animals↗
PubMed1999

Rapid release of antipsychotic drugs from dopamine D2 receptors: an explanation for low receptor occupancy and early clinical relapse upon withdrawal of clozapine or quetiapine.

OBJECTIVE: In an attempt to understand the basis of early relapse after antipsychotic withdrawal, the objective of this study was to determine whether the low occupancy of dopamine D2 receptors by clozapine and by quetiapine, as seen by brain imaging, could arise from a rapid release of some of the D2-bound clozapine or quetiapine by the brain imaging compounds and by the action of a physiological concentration of dopamine. METHOD: Human cloned D2 receptors were first pre-equilibrated with the [3H]antipsychotic drug, after which raclopride, iodobenzamide, or dopamine (at the physiological concentration in the synapse) was added, and the time course of release of the [3H]antipsychotic from the D2 receptor was measured. RESULTS: Within 5 minutes, low concentrations of raclopride and iodobenzamide displaced appreciable amounts of [3H]clozapine and [3H]quetiapine from the D2 receptors but, during the course of 1 hour, did not displace any of the other antipsychotic [[3H]ligands. [3H]Clozapine and [3H]quetiapine, moreover, were displaced by dopamine (100 nM) at least 100 times faster than the other antipsychotic [3H]ligands. CONCLUSIONS: Clozapine and quetiapine are loosely bound to the D2 receptor, and the injected radioactive ligand at its peak concentration may displace some of the D2-bound antipsychotic drug, resulting in apparently low D2 occupancies. Therefore, under clinical brain imaging conditions with [11C]raclopride, D2 occupancies by clozapine and by quetiapine may be higher than currently estimated. These considerations may result in high levels of the D2 receptors being occupied by therapeutic doses of clozapine or quetiapine. The rapid release of clozapine and quetiapine from D2 receptors by endogenous dopamine may contribute to low D2 receptor occupancy and to early clinical relapse upon withdrawal of these medications.

Animals↗
PubMed1998

Dopamine D2 receptor dimers in human and rat brain.

In order to determine whether dimers of dopamine D2 receptors can occur in mammalian brain, rat and human brain striatal membranes were photolabelled with two radioactive photoaffinity compounds selective for dopamine D2 receptors, [125I]azidophenethylspiperone and [125I]-4-azido-5-iodonemonapride. It was found that [125I]azidophenethylspiperone only labelled the D2 monomer, while [125I]-4-azido-5-iodonemonapride labelled both D2 monomers and dimers, despite the fact that very high concentrations (6 nM) of both radiocompounds were used. In addition, human cloned D2 receptors were probed with a D2-specific antibody, revealing multiple bands indicating the existence of trimers, tetramers and pentamers of D2 receptors. The different D2-binding patterns of the spiperone and benzamide congeners may explain the different densities of dopamine D2 receptors found with these two radioligands in human brain positron tomography in health and disease.

Animals↗
PubMed1998

A transmembrane domain-derived peptide inhibits D1 dopamine receptor function without affecting receptor oligomerization.

In this study, we show that a peptide based on the sequence of transmembrane domain 6 of the D1 dopamine receptor (D1DR) specifically inhibited D1DR binding and function, without affecting receptor oligomerization. It has been shown that an analogous peptide from the beta2-adrenergic receptor disrupted dimerization and adenylyl cyclase activation in the beta2-adrenergic receptor (Hebert, T. E., Moffett, S., Morello, J. P., Loisel, T. P., Bichet, D. G., Barret, C., and Bouvier, M. (1996) J. Biol. Chem. 271, 16384-16392). Treatment of D1DR with the D1DR transmembrane 6 peptide resulted in a dose-dependent, irreversible inhibition of D1DR antagonist binding, an effect not seen in D1DR with peptides based on transmembrane domains of other G protein-coupled receptors. Incubation with the D1DR transmembrane 6 peptide also resulted in a dose-dependent attenuation of both dopamine-induced [35S]guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding and receptor-mediated dopamine stimulation of adenylyl cyclase activity. Notably, GTPgammaS binding and cAMP production were reduced to levels below baseline, indicating blockade of ligand-independent, intrinsic receptor activity. Immunoblot analyses of the D1DR revealed the receptor existed as monomers, dimers, and higher order oligomers and that these oligomeric states were unaffected after incubation with the D1DR transmembrane 6 peptide. These findings represent the first demonstration that a peptide based on the transmembrane 6 of the D1DR may represent a novel category of noncompetitive D1DR antagonists.

Animals↗
PubMed1998

A serotonin-4 receptor-like pseudogene in humans.

During a search for new G-protein-linked receptors for dopamine and serotonin, we found a serotonin-4 receptor-like pseudogene. This receptor-like pseudogene is intronless, contains an in-frame stop codon following transmembrane-3, and has two one-nucleotide insertions between transmembrane-5 and -6 regions which alter the reading frame. The predicted amino acid sequence of the human pseudogene is about 35% identical with that of the rat serotonin-4 receptor.

Amino Acid Sequence↗
PubMed1998

A human serotonin-7 receptor pseudogene.

Although the serotonin-7 receptor was cloned several years ago, its localization in brain tissues remains confusing because of the existence of a related expressed pseudogene, the sequence of which has not hitherto been reported. During the course of searching for related receptor genes, we also searched for this pseudogene to determine its sequence. Human genomic DNA was screened for dopamine and serotonin receptor-like genes, using the polymerase chain reaction method and degenerate oligonucleotide primers based on the similar sequences in the transmembrane-6 and -7 regions of the serotonin-5A, the serotonin-7, and the dopamine D2, D3 and D4 receptors. This resulted in one of the clones having a 115 bp fragment, of which 89% of the bases were identical to the transmembrane-6 and -7 regions of the serotonin-7 receptor sequence. The fragment was radiolabelled and used to screen a human fetal brain cDNA library. A novel cDNA clone of 1326 bp was isolated. Based on the nucleotide sequence, 88% of the bases in this sequence of the pseudogene are identical to the human serotonin-7 receptor coding sequence. However, compared to the serotonin-7 receptor DNA sequence, the pseudogene sequence has nucleotide deletions and insertions, resulting in frame-shifts and stop codons. It was concluded that this sequence represented a pseudogene related to the serotonin-7 receptor gene.

Amino Acid Sequence↗
PubMed1998

Antipsychotic drugs which elicit little or no parkinsonism bind more loosely than dopamine to brain D2 receptors, yet occupy high levels of these receptors.

This review addresses two questions. First, why does clozapine apparently occupy low levels of dopamine D2 receptors in patients, in contrast to all other antipsychotic drugs which occupy 70-80% of brain dopamine D2 receptors? Second, what is the receptor basis of action of antipsychotic drugs which elicit low levels of Parkinsonism? Antipsychotic doses of clozapine occupy between 0% and 50% of D2 receptors, as measured in patients by a variety of radioligands. It has recently been found, however, that the percent occupancy of a receptor by a drug depends on the radioligand used to measure that receptor. Based on this new finding, this review concludes that clozapine clinically occupies high levels of D2 receptors in the absence of any radioligand. This occupancy is estimated to be of the order of 70-80% in the dopamine-rich region of the human striatum, and even higher in the limbic D2-containing regions which are low in endogenous synaptic dopamine. This conclusion arises from two different approaches. One approach is to relate the reported clozapine occupancies in the human striatum with the dissociation constants of the various radioligands at the D2 receptor. This relation extrapolates to approximately 70-80% occupancy by clozapine when clozapine competes with endogenous dopamine at the D2 receptor. The second approach is to calculate the D2 occupancy of each antipsychotic drug, using the average spinal fluid concentration and the correct dissociation constant of the antipsychotic, thereby revealing that all antipsychotic drugs, including clozapine, occupy approximately 70-80% of dopamine D2 receptors in the human striatum, and possibly higher in the limbic regions. As determined by the new dissociation constants, antipsychotic drugs which elicit Parkinsonism (trifluperazine, chlorpromazine, raclopride, haloperidol, fluphenazine, risperidone) bind more tightly than dopamine to D2, while those antipsychotic drugs which elicit little or no Parkinsonism (melperone, seroquel, perlapine, clozapine, remoxipride, molindone, sulpiride, olanzapine, sertindole) bind more loosely than dopamine to D2 receptors. Compared to the tightly bound antipsychotic drugs, the more loosely bound antipsychotics generally require higher clinical doses, require fewer days for clinical adjustment, but may dissociate from the D2 receptor more rapidly and could lead to clinical relapse somewhat earlier than that found with the traditional tightly bound antipsychotic drugs.

Antipsychotic Agents↗
PubMed1998

Anti-hyperactivity medication: methylphenidate and amphetamine.

How do 'stimulants' reduce hyperactivity in children and adults? How can drugs which raise extracellular dopamine result in psychomotor slowing of hyperactive children when dopamine is known to enhance motor activity, such as in Parkinson's disease? These apparent paradoxes are the focus of this brief review on the mechanism of action of stimulant medications used in the treatment of children, and of an increasing number of adults who meet diagnostic criteria for attention deficit hyperactivity disorder.

Adult↗
PubMed1998

Simple PCR amplification of the entire glucocerebrosidase gene (GBA) coding region for diagnostic sequence analysis.

Mutations in the human glucocerebrosidase gene (GBA) may lead to Gaucher disease-an autosomal recessive, lysosomal storage disease. In about 15-25% of Caucasian patients with Gaucher disease yet the disease-causing mutations remain to be identified. There exists 16kb downstream from the functional GBA gene (chromosome 1q21) a highly homologous transcribed pseudogene (GBAP) with some sequence differences to GBA. These sequence differences might erroneously imitate a true mutation in the functional gene if an unintentional co-investigation of the pseudogene occurred. We describe a protocol which allows the selective analysis of a PCR-amplified 7.1 kb genomic GBA-fragment encompassing the entire GBA coding region. Direct, nonradioactive double stranded cycle-sequencing procedure of nested PCR fragments from this long range GBA-specific product allowed the sequencing of the coding exons including the flanking splice sites. Several, so far unknown coding mutation were identified in non-Jewish families with Gaucher disease. This protocol allows the rapid detection of new GBA mutations.

Chromosomes, Human, Pair 1↗
PubMed1998

The human serotonin-7 receptor pseudogene: variation and chromosome location.

We report a variation of the pseudogene for the serotonin-7 receptor in human DNA. Human genomic DNA was amplified, using the polymerase chain reaction method and degenerate oligonucleotide primers for serotonin receptor-like genes. A novel gene DNA sequence of 1325 bp was found. Based on nucleotides, this gene is 88% identical to the serotonin-7 receptor coding sequence. Compared with the previously known serotonin-7 receptor pseudogene, this pseudogene has 1 nucleotide deletion and 4 nucleotide mutations. The gene is located on human chromosome 12 at 12p12.3-p13.2.

Chromosomes↗
PubMed1997

Dopamine D2-like sites in schizophrenia, but not in Alzheimer's, Huntington's, or control brains, for [3H]benzquinoline.

Although the basis of schizophrenia is not known, evidence indicates a possible overactivity of dopamine pathways. In order to detect any new dopamine receptor-like sites which may be altered in schizophrenia, the present study used a new radioligand, a [3H]benzo[g]quinoline. The receptors were labelled by this ligand in the presence of other drugs to block the known dopamine D1, D2, D3, or D5 receptors (no D4-selective ligands are available to block D4). Using this method, we found that schizophrenia brain striata had elevated levels of a D2-like site not detected in control human postmortem brains or in Alzheimer's, Huntington's, or Parkinson's disease brains. The ligand acted as an agonist at this D2-like site, because binding was abolished by guanine nucleotide. The binding of the ligand to the D4 receptor, however, was not sensitive to guanine nucleotide. The site differed from D2 itself, because S- and R-sulpiride were equally potent at the D2-like site. The D2-like sites were present in rat and mouse brain but were absent in brain slices from transgenic mice where D2 had been knocked out. The abundance of the receptor was not related to premortem use of antipsychotic drugs. Future research should examine the biochemical differences between the D2 dopamine receptor and these D2-like sites in schizophrenia.

Adult↗
PubMed1997

Clozapine occupies high levels of dopamine D2 receptors.

An important discrepancy has been noted concerning the number of dopamine D2 receptors which must be occupied in patients by clozapine, in contrast to other antipsychotic drugs, in order to achieve an antipsychotic effect. For example, when D2 receptors are labelled by radioactive raclopride or spiperone congeners in patients, psychosis-controlling doses of all antipsychotic drugs occupy about 70% or more of the D2 receptors in patients. However, equi-effective psychosis-controlling doses of clozapine (approximately 400 mg/day; approximately 70-130 nM in spinal fluid) only occupy between 20% and 50% of the D2 receptors in patients. This discrepancy of a consistently lower occupancy of D2 by psychosis-controlling doses of clozapine may be resolved when one considers that the neuroleptic concentration for half-occupancy of D2 receptors in vitro (i.e. the inhibition constant) depends on the radioligand used to label the receptor. Radioligands with higher tissue/buffer partition coefficients are less displaced by clozapine. This principle applies both in vitro and in vivo. Thus, allowing for this principle, psychosis-controlling doses of clozapine can be shown to occupy over 70% of the brain dopamine D2 receptors in patients, as found with other neuroleptics.

Antipsychotic Agents↗
PubMed1997

Atypical neuroleptics have low affinity for dopamine D2 receptors or are selective for D4 receptors.

This review examines the possible receptor basis of the atypical action of those atypical antipsychotic drugs that elicit low levels of Parkinsonism. Such an examination requires consistent and accurate dissociation constants for the antipsychotic drugs at the relevant dopamine and serotonin receptors. It has long been known, however, that the dissociation constant of a given antipsychotic drug at the dopamine D2 receptor varies between laboratories. Although such variation depends on several factors, it has recently been recognized that the radioligand used to measure the competition between the antipsychotic drug and the radioligand is an important variable. The present review summarizes information on this radioligand dependence. In general, a radioligand of low solubility in the membrane (i.e., low tissue:buffer partition) results in a low value for the antipsychotic dissociation constant when the drug competes with the radioligand. Hence, by first obtaining the antipsychotic dissociation constants using different radioligands of different solubility in the membrane, one can then extrapolate the data to low or "zero" ligand solubility. The extrapolated value represents the radioligand-independent dissociation constant of the antipsychotic. These values are here given for dopamine D2 and D4 receptors, as well as for serotonin 5-HT2A receptors. These values, moreover, agree with the dissociation constant directly obtained with the radioactive antipsychotic itself. For example, clozapine revealed a radioligand-independent value of 1.6 nM at the dopamine D4 receptor, agreeing with the value directly measured with [3H]-clozapine at D4. However, because clozapine competes with endogenous dopamine, the in vivo concentration of clozapine (to occupy dopamine D4 receptors) can be derived to be about 13 nM, agreeing with the value of 12 to 20 nM in the plasma water or spinal fluid observed in treated patients. The atypical neuroleptics remoxipride, clozapine, perlapine, seroquel, and melperone had low affinity for the dopamine D2 receptor (radioligand-independent dissociation constants of 30 to 90 nM). Such low affinity makes these latter five drugs readily displaceable by high levels of endogenous dopamine in the caudate or putamen. Most typical neuroleptics have radioligand-independent values of 0.3 to 5 nM at dopamine D2 receptors, making them more resistant to displacement by endogenous dopamine. Finally, a relation was found between the neuroleptic doses for rat catalepsy and the D2:D4 ratio of the radioligand-independent K values for these two receptors. Thus, the atypical neuroleptics appear to fall into two groups, those that have a low affinity for dopamine D2 receptors and those that are selective for dopamine D4 receptors.

Animals↗
PubMed1997

Resistance of the dopamine D2L receptor to desensitization accompanies the up-regulation of receptors on to the surface of Sf9 cells.

Dopamine D2 receptor agonists are commonly used in the control of PRL-secreting adenomas, and the sensitivity of dopamine agonists during long term therapy is exquisite. However, the molecular mechanisms responsible for the maintenance of this cellular sensitivity to dopamine agonists remain poorly understood. In the present study, we examined the agonist-induced regulation of the human D2L receptor expressed to a specific activity of approximately 1 pmol receptor/mg protein in Sf9 insect cells. Treatment of D2L receptor-expressing cells with dopamine for up to 3 h resulted in no detectable change in the ligand-binding properties of the receptor and a approximately 120-fold reduction in the potency, but not the efficacy, of D2L receptors to mediate dopamine inhibition of forskolin-stimulated adenylyl cyclase activity. This resistance of the D2L receptor to agonist-induced desensitization was accompanied by a approximately 28% translocation of intracellular D2L receptors to the cell surface, as quantified by cellular fractionation and radioligand binding and visualized by whole cell immunocytochemical staining and confocal microscopy. Immunoblot analysis of the P2 membrane fraction revealed that surface D2L receptors comprised monomers and dimers. Treatment of D2L receptor-expressing cells with the protein synthesis inhibitor cycloheximide significantly reduced the basal expression level of receptors, but did not block the agonist-induced up-regulation of receptors. Longer periods of dopamine exposure for 24 h brought about a small increase in surface receptor density. However, when these studies were conducted in the presence of cycloheximide, receptor density was marginally reduced, suggesting that receptor synthesis accounts for the maintenance of cellular receptor density under these conditions. We conclude that the resistance of the D2L receptor-coupled adenylyl cyclase system to agonist-induced desensitization is attributed to the up-regulation of surface receptors after the translocation of existing intracellular receptors and de novo receptor synthesis.

Animals↗
PubMed1996

Dopamine D2 receptor dimers and receptor-blocking peptides.

Dopamine D2 receptors exist as dimers in whole cell lysate, crude membranes prepared from human caudate, and following solubilization and immunoprecipitation of the receptor from these tissues. Photoaffinity labelling experiments confirmed that D2 receptors exist either as monomers that are selective targets for spiperone or as dimers that are targets for nemonapride. Incubation of D2 dimers with peptides derived from the putative transmembrane (TM) domains of the D2 receptor, or incubation under high temperatures or low pH resulted in the dissociation of the dimer to monomer. D2-TM peptides were unable to dissociate dopamine D1 and serotonin 5-HT1B receptor dimers, suggesting that receptor dimers are formed by specific intermolecular noncovalent interactions involving TM regions. This opens a path to new selective therapeutic receptor-blocking compounds based on this principle of mimicking transmembrane portions of neurotransmitter receptors.

Animals↗