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Biomedical subjects

P Seeman

Publications and source records attributed to P Seeman.

At least 91 records · Page 5Linked to original sources
PubMed1993

A human somatostatin receptor (SSTR3), located on chromosome 22, displays preferential affinity for somatostatin-14 like peptides.

We report here on the cloning of a human intronless gene encoding a member of the G-protein linked somatostatin (SST) receptor subfamily, termed SSTR3. Based on the deduced amino acid sequence, this gene encodes a 418 amino acid protein displaying sequence similarity, particularly within putative transmembrane domains, with the recently cloned human SSTR1 (62%), SSTR2 (64%) and SSTR4 (58%) receptors. Membranes prepared from COS-7 cells transiently expressing the human SSTR3 gene bound [125I]Leu8,D-Trp22,Tyr25 SST-28 in a saturable manner with high affinity (approximately 200 pM) and with rank order of potency (D-Trp8 SST-14 > SST-14 > SMS-201-995 > SST-28) indicative of a somatostatin-14 selective receptor. The pharmacological profile of the expressed human SSTR3 receptor is similar but not identical to that reported for the rat homolog [(1992) J. Biol. Chem. 267, 20422] where the peptide selectivity is SST-28 > or = SST-14 >>> SMS-201-995. Northern blot analysis reveals the presence of an SSTR3 mRNA species of approximately 5 kb in various regions of the monkey brain, including the frontal cortex, cerebellum, medulla, amygdala, with little or no SSTR3 mRNA detectable in brain regions such as the striatum, hippocampus, and olfactory tubercle. The SSTR3 receptor gene maps to human chromosome 22. The existence of at least four distinct human genes encoding somatostatin-14 selective receptors with diverse pharmacological specificities may help to account for some of the multiple biological actions of somatostatin under normal and pathological conditions.

Amino Acid Sequence↗
PubMed1993

Dopamine D1 receptor mutagenesis: role of amino acids in agonist and antagonist binding.

We investigated the role of amino acids, by site-directed mutagenesis, in five of the seven transmembrane regions of the human dopamine D1 receptor. The results demonstrate a role for an aspartic acid (Asp70) in transmembrane 2 mediating the sodium ion effect on receptor conformation. Amino acids residues in transmembrane 3 and 5 are important for optimum dopamine binding to the receptor. Mutant receptors involving amino acids in transmembrane 7 suggest that binding sites for agonists and antagonists have distinct binding determinants within the dopamine D1 receptor.

Amino Acid Sequence↗
PubMed1993

Dopamine D4 receptors bind inactive (+)-aporphines, suggesting neuroleptic role. Sulpiride not stereoselective.

In order to identify new atypical antipsychotic drugs which are more selective for the human dopamine D4 receptor than for the human dopamine D2 (long) receptor, we tested enantiomer pairs of dopamine agonists and dopamine antagonists on the expressed proteins of these cloned receptors. The (+)-aporphines ((+)-N-propyl-norapomorphine, 11-OH-N-propyl-norapomorphine and (+)-apomorphine) bound to the dopamine D4 receptor with selectivities up to 20 times greater than to the dopamine D2 receptor, suggesting that these pharmacologically inactive enantiomers may succeed as atypical neuroleptics.

Antipsychotic Agents↗
PubMed1993

Serotonin receptor cDNA cloned from Lymnaea stagnalis.

Serotonin (5-HT) is a major neurotransmitter that influences various behaviors, neuronal plasticity, learning, and memory in molluscs. Although the physiology of 5-HT transmission in molluscs is well studied, little is known about the pharmacology and diversity of the 5-HT receptor system. Based on the high homology of genes coding for guanine nucleotide-binding protein (G protein)-coupled receptors, we have cloned a gene for the Lymnaea stagnalis 5-HT (5HTlym) receptor. The putative receptor protein, 509 amino acids long, has highest homology with the Drosophila 5-HT receptors and mammalian 5HT1 receptors. As revealed by RNA blot-hybridization analysis, two mRNA species of 2.3 and 3.2 kb are detected in the central nervous system of Lymnaea. Transient expression of 5HTlym in COS-7 cells showed saturable [3H]lysergic acid diethylamide binding with an estimated dissociation constant of 0.9 nM. The 5HTlym receptor exhibited a mixed 5HT-like pharmacology that cannot be precisely categorized with existing mammalian classification nomenclature. However, the 5HTlym receptor does display some characteristics that have been attributed to the putative mammalian vascular 5HT1-like receptor.

Amino Acid Sequence↗
PubMed1993

Low density of dopamine D4 receptors in Parkinson's, schizophrenia, and control brain striata.

The purpose of this study was to determine whether dopamine D4 receptors could be detected in the human brain striatum by means of an indirect ligand-binding method, because no dopamine D4 receptor-selective ligand presently exists. The antipsychotic clozapine is more selective for the dopamine D4 receptor than for other dopamine receptors. Although most antipsychotic drugs act in the striatum to elicit Parkinson-like side-effects, clozapine is atypical in that it does not produce Parkinsonism. To understand this atypical action of clozapine, it would be helpful to know whether the presumed target for clozapine, the dopamine D4 receptor, is or is not present in the human striatum. We measured dopamine D4 receptors indirectly, using [3H]emonapride and [3H]raclopride. Emonapride has a high affinity (K = 90 pM) for the dopamine D4 receptor, while raclopride has a very low affinity for this receptor (K = 240 nM); thus, any difference in the densities of these two [3H]ligands (in the absence of dopamine) could be attributed to the presence of dopamine D4 receptors. Since the binding of [3H]raclopride is sensitive to endogenous dopamine, we used Parkinson-diseased tissue which has little dopamine. We found that the densities of the two ligands were identical in Parkinson striata, indicating a low density (< 1 pmol/g) for dopamine D4 receptors in the human striatum. This low or undetectable density of dopamine D4 receptors in the striatum is consistent with other data indicating that clozapine does not have its major action in the human striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Dopamine receptors labelled by PHNO.

Since the high-affinity state of dopamine D2 receptors may be abnormal in psychomotor diseases, it is desirable to develop a radioactive agonist to label this high-affinity site for possible clinical diagnostic use. (+)PHNO is a selective D2 agonist used to treat Parkinson's disease. We prepared [3H](+)PHNO from allyl-des-propyl(+)PHNO. In binding to dopamine receptors in homogenates of canine brain striata, [3H](+)PHNO had a dissociation constant of 0.35 nM in the absence of NaCl, and 0.56 nM in the presence of NaCl. Dopamine agonists and antagonists inhibited the binding of [3H](+)PHNO at drug concentrations similar to those inhibiting other [3H]ligands at D2 receptors, but not similar to those acting at D4 receptors. Approximately 90% of the total [3H](+)PHNO binding was specific. Guanilylimidodiphosphate markedly inhibited [3H](+)PHNO binding, suggesting that [3H](+)PHNO was binding primarily to the high-affinity state of dopamine D2 receptors rather than to D3 receptors. The density of the [3H](+)PHNO binding sites was equal to that of [3H]emonapride (or [3H]YM-09151-2), both densities of which were 1.5- to 2-fold higher than that of [3H]spiperone, compatible with the idea that [3H](+)PHNO binds to monomers of D2, while [3H]spiperone binds to dimers of D2. Although [3H](+)PHNO has good selectivity and affinity for the high-affinity state of D2, the [3H]ligand was sensitive to endogenous dopamine, since washing the tissue lowered the dissociation constant. For future in vivo labelling of D2 by an agonist, therefore, it will be essential to search for a related [3H]ligand with an even lower dissociation constant.

Animals↗
PubMed1993

Schizophrenia: dopamine D1 receptor sequence is normal, but has DNA polymorphisms.

Genes that regulate dopamine (DA) receptors may underlie the overactive DA system in schizophrenia. Since it is known that there is an abnormally reduced or absent regulation of the DA D2 receptor by the DA D1 receptor in the postmortem schizophrenia brain, the human DA D1 receptor gene was sequenced from genomic deoxyribonucleic acid (DNA) of seven schizophrenic individuals. The tissues from two schizophrenics had previously been found to have a reduced link between DA D1 and D2 receptors. The D1 receptor genes were amplified by the polymerase chain reaction, subcloned, and sequenced. Although three DNA polymorphisms were found, the deduced amino acid sequence of the DA D1 receptor was normal in these tissues.

Adult↗
PubMed1993

Schizophrenia: normal sequence in the dopamine D2 receptor region that couples to G-proteins. DNA polymorphisms in D2.

Because dopamine (DA) D2 receptors are a target in neuroleptic therapy and have been found to be elevated in schizophrenia, the human DA D2 receptor gene was examined for possible abnormalities in schizophrenia. Moreover, since D2 receptors in psychosis have a reduced coupling to D1 receptors, the cytoplasmic third loop of D2 was chosen for deoxyribonucleic acid (DNA) sequencing, since this region is essential for coupling to G-proteins. This region also contains exon 5, which is expressed in the long form of D2, but not in the short form of D2. In eight schizophrenia cases, this region had normal exon sequences (exons 4, 5 and 6), and normal sequences at its intron-exon junctions. However, exon 6 contained three DNA polymorphic base changes, and introns 4 and 5 revealed three missing bases and two polymorphic base changes, none of which would be expected to alter the D2 receptor protein in schizophrenia.

Amino Acid Sequence↗
PubMed1993

Beta adrenoceptor recovery after heart failure in the dog.

OBJECTIVE: The role of myocardial beta receptors in pacing induced congestive heart failure is uncertain. The aims of the study were therefore (1) to examine total beta adrenoceptor density in canine left ventricle during pacing induced heart failure; and (2) to evaluate whether there is a relationship between beta adrenoceptor numbers and circulating noradrenaline during progression of heart failure. METHODS: Heart failure was examined at different stages: early (after 1 week of pacing at 250 beats.min-1); peak (after 4 weeks of pacing); and after recovery (4 weeks after pacing was discontinued). Myocardial beta adrenoceptor density was assessed by radioligand binding with iodocyanopindolol. Plasma noradrenaline was determined by high performance liquid chromatography with electrochemical detection. RESULTS: Compared to control, left ventricular beta adrenoceptor density was: (1) not significantly different in early heart failure, at 2540(SEM 460) v 3160(260) fmol.g-1 wet weight in control; (2) decreased 63% at peak heart failure [1180(100) fmol.g-1, p < 0.01]; and (3) restored to control density after recovery from heart failure [3430(540) fmol.g-1]. The beta adrenoceptor dissociation constant was unaltered throughout. There was an inverse correlation between myocardial beta adrenoceptor density and plasma noradrenaline (r = -0.785, p < 0.01). CONCLUSIONS: The progressive decline in beta adrenoceptor density as heart failure develops and the capacity of beta adrenoceptors to return to control levels after recovery from the experimental heart failure state suggests that there is a reversible abnormality in beta adrenoceptors during heart failure.

Animals↗
PubMed1993

Dopamine receptors and antipsychotic drug response.

Dopamine receptors have been divided into two major types--D1 and D2--based primarily on pharmacological and biochemical criteria. Recent advances in the molecular biology of the dopamine receptor system have allowed the identification and characterisation of at least five distinct neuronal dopamine receptor genes (D1 to D5). These genes encode dopamine receptors belonging to the D1 receptor family, termed D1 and D5, and three D2-like receptors, termed D2, D3 and D4. These receptors are distinguished on the basis of their primary structure, chromosomal location, mRNA size and tissue distribution, and biochemical and pharmacological differences. Although individually these receptor subtypes may not be directly and exclusively involved in the maintenance or expression of schizophrenia, alterations of any of the receptors may contribute to the perturbation or instability of dopaminergic homeostasis in the brain. What was once thought to be a simple two-receptor system seems to have emerged as an intricate and interactive entity. This review summarises what is currently understood about dopamine receptors, their role in antipsychotic drug action, and their association with psychosis.

Animals↗
PubMed1993

Cloning and expression of a human somatostatin-14-selective receptor variant (somatostatin receptor 4) located on chromosome 20.

Based on pharmacological, biochemical, and molecular criteria, multiple somatostatin receptor (SSTR) subtypes selective for somatostatin (SST)-14 and -28 have been postulated to exist in both the brain and periphery. We report here on the cloning and characterization of a human gene encoding a new member of the guanine nucleotide-binding protein-linked SSTR family, termed human (h)SSTR4. The 388-amino acid protein, with a predicted molecular mass of approximately 42 kDa, displays sequence similarity, particularly within putative transmembrane domains, with the recently cloned hSSTR1 (69%), hSSTR2 (56%), and hSSTR3 (58%). Membranes prepared from COS-7 cells transiently expressing the hSSTR4 gene bound 125I-[Leu8,D-Trp22,Tyr25]SST-28 in a saturable manner with high affinity (approximately 60 pM) and with a pharmacological profile and rank order of potency ([D-Trp8]SST-14 > SST-14 > SMS 201-995 > SST-28 > MK-678) indicative of a SST-14-selective receptor. Ki values for the inhibition of 125I-[Leu8,D-Trp22,Tyr25]SST-28 binding to the expressed receptor by these somatostatinergic peptides were 0.3, 1.1, 1.4, 2.2, and 6.5 nM, respectively. High affinity agonist binding to hSSTR4 was significantly reduced by GTP and pertussis toxin, indicating association of the expressed receptor with pertussis toxin-sensitive guanine nucleotide-binding proteins. Northern blot analysis revealed the presence of an SSTR4 mRNA species of approximately 4 kilobases in select regions of the monkey brain, including the hippocampus, hypothalamus, cortex, and striatum, with little or no receptor mRNA detected in either the olfactory tubercle, medulla, cerebellum, or amygdala. The SSTR4 gene maps to human chromosome 20. These findings document the existence of a novel human SSTR gene. Although the hSSTR4 displays an overall deduced amino acid homology of 86% with the recently reported rat homolog [Proc. Natl. Acad. Sci. USA 89:11151-11155 (1992)], the two gene products possess distinctive pharmacological profiles and affinities for the SST agonists SMS 201-995 and MK-678.

Amino Acid Sequence↗
PubMed1993

Dopamine receptor pharmacology.

Although antipsychotic drugs originally helped to discover dopamine receptors, the five dopamine receptors presently identified and cloned are facilitating the search for and discovery of more selective antipsychotic and antiparkinson drugs. The D1-like dopamine receptors, D1 and D5, are sensitive to the same drugs as the D1 receptor in native tissues, but D5 is about 10 times more sensitive to dopamine than D1. The D2-like receptors, D2, D3, and D4, have approximately similar sensitivities to dopamine, but bromocriptine and raclopride are both about two orders of magnitude weaker at D4, whereas clozapine is one order more potent at D4, as compared with D2 and D3. The human dopamine D4 receptor has many variants. The sensitivities to clozapine of human variants D4.2, D4.4, and D4.7 are approximately similar, with dissociation constants between 5 and 24 nM, matching the spinal fluid concentration of clozapine under therapeutic conditions. Thus antipsychotic action may be effected through blockade of either dopamine D2 or D4 receptors.

Animals↗
PubMed1992

The cloned dopamine D2 receptor reveals different densities for dopamine receptor antagonist ligands. Implications for human brain positron emission tomography.

Since [3H]emonapride ([3H]YM-09151-2), a benzamide neuroleptic, consistently detects more dopamine D2 receptors than [3H]spiperone in the same tissue, we tested whether this property was inherent in the cloned dopamine D2 receptor. We found that the density of dopamine D2 receptors labelled by [3H]emonapride was 1.5-fold to 2-fold (mean of 1.8-fold) higher than the density of dopamine D2 receptors labelled by [3H]spiperone in cells expressing cloned dopamine D2 receptors (either the short form (from rat) or the long form (from human)), matching similar findings in anterior pituitary tissue (rat or pig) or in post-mortem human caudate nucleus tissue. The situation was similar for another benzamide, [3H]raclopride, which revealed 1.3-fold to 1.8-fold (mean of 1.5-fold) more binding sites than that for [3H]spiperone in cell membranes containing cloned dopamine D2 receptors. The apparently different dopamine D2 receptor densities revealed by these two types of 3H-ligands (i.e. [3H]spiperone and the [3H]benzamides), therefore, arise from an inherent property of the dopamine D2 receptor protein. These findings for the cloned dopamine D2 receptor, therefore, partly explain the higher dopamine D2 receptor density measured in human brain (by positron emission tomography) when using radioactive raclopride compared to results using radioactive methylspiperone.(ABSTRACT TRUNCATED AT 250 WORDS)

Antipsychotic Agents↗
PubMed1992

Multiple dopamine D4 receptor variants in the human population.

The dopamine D4 receptor structurally and pharmacologically resembles the dopamine D2 and D3 receptors. Clozapine, an atypical antipsychotic that is relatively free of the adverse effects of drug-induced parkinsonism and tardive dyskinesia, binds to the D4 receptor with an affinity 10 times higher than to the D2 and D3 receptors. This may explain clozapine's atypical properties. Here we report the existence of at least three polymorphic variations in the coding sequence of the human D4 receptor. A 48-base-pair sequence in the putative third cytoplasmic loop of this receptor exists either as a direct-repeat sequence (D4.2), as a fourfold repeat (D4.4) or as a sevenfold repeat (D4.7). Two more variant alleles were detected in humans. Expression of the complementary DNA for the three cloned receptor variants showed different properties for the long form (D4.7) and the shorter forms (D4.2, D4.4) with respect to clozapine and spiperone binding. To our knowledge, this is the first report of a receptor in the catecholamine receptor family that displays polymorphic variation in the human population. Such variation among humans may underlie individual differences in susceptibility to neuropsychiatric disease and in responsiveness to antipsychotic medication.

Base Sequence↗
PubMed1992

A human serotonin 1D receptor variant (5HT1D beta) encoded by an intronless gene on chromosome 6.

An intronless gene encoding a serotonin receptor (5HT1D beta) has been cloned and functionally expressed in mammalian fibroblast cultures. Based on the deduced amino acid sequence, the gene encodes a 390-amino acid protein displaying considerable homology, within putative transmembrane domains (approximately 75% identity) to the canine and human 5HT1D receptors. Membranes prepared from CHO cells stably expressing the receptor bound [3H]serotonin with high affinity (Kd 4 nM) and displayed a pharmacological profile consistent, but not identical, with that of the characterized serotonin 5HT1D receptor. Most notably, metergoline and serotonergic piperazine derivatives, as a group, display 3- to 8-fold lower affinity for the 5HT1D beta receptor than for the 5HT1D receptor, whereas both receptors display similar affinities for tryptamine derivatives, including the antimigraine drug sumatriptan. Northern blot analysis revealed an mRNA of approximately 5.5 kilobases expressed in human and monkey frontal cortex, medulla, striatum, hippocampus and amygdala but not in cerebellum, olfactory tubercle, and pituitary. The 5HT1D beta gene maps to human chromosome 6. The existence of multiple neuronal 5HT1D-like receptors may help account for some of the complexities associated with [3H]serotonin binding patterns in native membranes.

Amino Acid Sequence↗
PubMed1992

Autoradiographic localization of [3H]quinpirole binding to dopamine D2 and D3 receptors in rat brain.

A radiolabelled form of the dopamine agonist, quinpirole (LY17155), has been evaluated as a ligand for dopamine receptors in the rat brain. Quinpirole has been reported to be a selective D2 dopamine agonist; however, a recent report has indicated that it may have high affinity for a novel dopamine binding site which has been termed D3. In rat brain sections, [3H]quinpirole binding exhibited a distribution similar to that described for dopamine D2 receptors using either agonist or antagonist labelling. High densities of binding could be found in caudate-putamen, nucleus accumbens, olfactory tubercle and islands of Calleja. When the labelling was done in the presence of 10 microM guanylyl-5'-imidodiphosphate to convert the dopamine D2 receptor to a 'low affinity agonist conformation', binding was inhibited in most brain regions with the notable exception of the islands of Calleja which retained most of the [3H]quinpirole binding. The guanine nucleotide insensitivity of this binding and distribution of this site indicates that [3H]quinpirole is binding to dopamine D3 receptors in this region of the brain. Therefore, these results indicate that [3H]quinpirole labels a high affinity agonist conformation of dopamine D2 receptors as well as dopamine D3 receptors in rat brain. In addition, this study provides the first detection the dopamine D3 receptor protein in the brain.

Animals↗
PubMed1992

Visualization of dopamine D1, D2 and D3 receptor mRNAs in human and rat brain.

Using 32P-labelled oligonucleotides derived from the coding regions of dopamine D1, D2 and D3 receptor mRNAs we localized cells containing transcripts for these receptors in the human (hD1, hD2) and rat brain (rD1, rD2, rD3). Dopamine D1 receptor mRNA was detected at high levels in neurons of the caudate and putamen as well as in the nucleus accumbens in both human and rat brain. In the rat brain D1 receptor mRNA was also abundant in the olfactory tubercles and several thalamic nuclei. In both species D1 mRNA was absent from the neurons of the substantia nigra and the ventral tegmental area as well as from the globus pallidus medialis in humans and entopeduncular nucleus in rats. In contrast, dopamine D2 receptor mRNA was found in dopaminergic neurons of the substantia nigra pars compacta and of the ventral tegmental area. In addition high levels of D2 mRNA were detected in neurons of the caudate, putamen and accumbens nuclei, the olfactory tubercle and the anterior lobe of pituitary gland. In the rat the highest level of hybridization was found in the intermediate lobe of the pituitary gland. In the rat brain dopamine D3 mRNA was mainly detected in the Islands of Calleja and at lower levels in the anterior nucleus accumbens, the medial mammillary nucleus as well as in the bed nucleus of the stria terminalis. In general, a good agreement was found between the distribution of transcripts and binding sites labelled with the D1 antagonist SCH 23390 or with the D2 ligand SDZ 205-502. For D1 receptors, the main exceptions were the absence of mRNA in the globus pallidus and the substantia nigra despite the high densities of binding sites in these regions. For D2 receptors, regions where binding sites but not mRNA were detected included the olfactory bulb, neocortex, hippocampus and superior colliculus.

Animals↗