Biomedical subjects

S J Peroutka

Publications and source records attributed to S J Peroutka.

At least 19 recordsLinked to original sources

PubMed1992

A single amino-acid difference confers major pharmacological variation between human and rodent 5-HT1B receptors.

Neuropsychiatric disorders such as anxiety, depression, migraine, vasospasm and epilepsy may involve different subtypes of the 5-hydroxytryptamine (5-HT) receptor. The 1B subtype, which has a unique pharmacology, was first identified in rodent brain. But a similar receptor could not be detected in human brain, suggesting the absence in man of a receptor with equivalent function. Recently a human receptor gene was isolated (designated 5-HT1B receptor, 5-HT1D beta receptor, or S12 receptor) which shares 93% identity of the deduced protein sequence with rodent 5-HT1B receptors. Although this receptor is identical to rodent 5-HT1B receptors in binding to 5-HT, it differs profoundly in binding to many drugs. Here we show that replacement of a single amino acid in the human receptor (threonine at residue 355) with a corresponding asparagine found in rodent 5-HT1B receptors renders the pharmacology of the receptors essentially identical. This demonstrates that the human gene does indeed encode a 1B receptor, which is likely to have the same biological functions as the rodent 5-HT1B receptor. In addition, these findings show that minute sequence differences between homologues of the same receptor from different species can cause large pharmacological variation. Thus, drug-receptor interactions should not be extrapolated from animal to human species without verification.

Alprenolol↗

PubMed1992

Differential radioligand binding properties of [3H]5-hydroxytryptamine and [3H]mesulergine in a clonal 5-hydroxytryptamine1C cell line.

[3H]5-Hydroxytryptamine ([3H]5-HT) and [3H]mesulergine were used to label 5-HT1C receptors expressed in NIH 3T3 mouse fibroblast cells. Using a rapid filtration assay, saturation analysis of the [3H]5-HT radioligand data indicate that the binding is biphasic. Based on computerized analysis of the data, a 2-site model of radioligand binding is significantly more consistent with the data than a one-site model (P less than 0.01). The KD values of [3H]5-HT for the 2 populations are 0.5 +/- 0.1 nM and 31 +/- 15 nM, while the Bmax values are 400 +/- 90 pmol/g protein and 3,000 +/- 600 pmol/g protein, respectively. A biphasic binding pattern is also observed with [3H]5-HT using a centrifugation assay (KD1 = 0.6 +/- 0.06 nM, KD2 = 60 +/- 10 nM; Bmax1 = 740 +/- 90 pmol/g, Bmax2 = 4,000 +/- 700 pmol/g). By contrast, saturation analysis of [3H]mesulergine binding is monophasic (KD = 4.7 +/- 0.7 nM) with a Bmax value (6,800 +/- 1,000 pmol/g protein) that is significantly greater than that obtained using [3H]5-HT (P less than 0.01). Drug competition studies confirm that both [3H]5-HT and [3H]mesulergine label at least 2 subpopulations of expressed 5-HT1C receptors in NIH 3T3 cells. 10(-4) M GTP eliminates the high affinity [3H]5-HT-labeled binding sites with minimal effect on the low affinity [3H]5-HT-labeled sites and no effect on [3H]mesulergine-labeled sites. These data demonstrate that at least 2 distinct subpopulations of 5-HT1C receptors in NIH 3T3 cells can be differentiated using radioligand binding techniques.

Animals↗

PubMed1992

Characterization of the human 5-hydroxytryptamine1B receptor.

We report the cloning of a human gene encoding the 5-hydroxytryptamine1B receptor. The receptor has the characteristics of a G-protein-linked receptor and is most homologous to the human 5-HT1D receptor. This human 5-HT receptor gene, most abundantly expressed in striatum, is localized on chromosome 6, at 6q13, and the gene encoding the 5-HT1D receptor is localized on chromosome 1. Radioligand studies indicate that the affinity of [3H]5-HT is 16 +/- 2 nM. Drug competition studies indicate that the receptor displays high affinity (i.e. less than 40 nM) for 5-HT, 5-carboxyamidotryptamine, methiothepin, and metergoline.

Amino Acid Sequence↗

PubMed1992

Molecular structural basis of ligand selectivity for 5-HT2 versus 5-HT1C cortical receptors.

A molecular structural criterion of ligand selectivity for the 5-HT2 versus 5-HT1C receptor was hypothesized on the basis of radioligand binding data. Despite the large number of compounds which have been tested at both receptors, analysis of published data led to the identification of only five agents which are greater than 10-fold selective for the 5-HT2 versus the 5-HT1C receptor. Comparison of the two-dimensional structures revealed that, although these five compounds represent three distinct structural classes, they share a common structural feature located in the region hypothesized to be involved in receptor binding: a carbonyl or carboxyl oxygen interposed spatially between an aromatic ring and nitrogen atom. This structural feature was used to predict the relative selectivity of compounds that had not previously been analyzed at both the 5-HT2 and 5-HT1C receptors. All six drugs tested which contain the identified reactive carbonyl or carboxyl group were found to be selective for the 5-HT2 versus the 5-HT1C receptor with selectivity ratios ranging from 26 to 380. By contrast, three agents which are structurally similar but do not contain the reactive carbonyl or carboxyl group displayed equally high affinity for both receptor binding sites. Since the physiological roles of the 5-HT2 and 5-HT1C receptor are markedly different, it would be of potential clinical and scientific value to utilize this molecular structural feature to further identify chemical compounds which would selectively interact with only one of the two receptors.

Animals↗

PubMed1992

Phylogenetic tree analysis of G protein-coupled 5-HT receptors: implications for receptor nomenclature.

The nomenclature system used to characterize 5-hydroxytryptamine receptor subtypes remains controversial. To date, the majority of nomenclature systems have been on the differential pharmacological properties of the receptors. However, the availability of molecular biological data allows for a nomenclature system based on the structural properties of the receptors. The evolutionary relationships between the known G protein-coupled 5-HT receptor subtypes were determined by a phylogenetic tree analysis. The data indicate that 2 major classes of G protein-coupled 5-HT receptors have evolved. Each of the 2 branches differentiate into additional 5-HT receptor subtypes. The most recent branching of 5-HT receptor subtypes occurs at the level of individual species. These data also indicate that the degree of structural similarity (e.g. 93% identify between human and rat 5-HT1B receptors) does not necessarily correlate with pharmacological similarity. Phylogenetic tree analysis allows for a nomenclature framework that can be easily expanded to incorporate additional 5-HT receptor subtypes that have yet to be identified.

Animals↗

PubMed1992

Binding potency of paroxetine analogues for the 5-hydroxytryptamine uptake complex.

The in-vitro inhibition constants (Ki) of 14 structural analogues of the potent 5-hydroxytryptamine (5-HT)-uptake inhibitor paroxetine were determined to assess the structure-affinity relationship of these derivatives. A goal of these studies was to determine those positions on paroxetine which could be derivatized without significantly decreasing the affinity of the drug for the binding site, so that radiolabels such as [18F]fluoroalkyl groups might be appended for future in-vivo imaging studies of the 5-HT uptake system. Using the methyl moiety as a steric probe for these studies, it was found that the rank order of potency of various methyl-substituted paroxetine analogues for inhibiting the binding of [3H]paroxetine to the 5-HT re-uptake site was: 4'-approximately equal to 3'-approximately equal to 2''- > 2'-approximately equal to 1- > 5''- > 6''-methyl. The in-vitro equipotent molar ratios (EPMR, Ki(analogue)/Ki(paroxetine)) of the analogues were determined, and the EPMRs of the 4'-, 3'-, and 2''-methyl derivatives were 1.9, 2.2 and 2.2, respectively. The 4'- and 2''-fluoromethyl and -fluoroethyl analogues were synthesized, and the EPMRs of the 4'- and 2''-fluoromethyl derivatives were determined to be 2.0 and 3.5, and those of the 4'- and 2''-fluoroethyl analogues were 5.2 and 6.2, respectively. The 2''-fluoromethyl analogue was unstable in aqueous solutions, and it is not a promising ligand for in-vivo studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Identification of a single amino acid residue responsible for the binding of a class of beta-adrenergic receptor antagonists to 5-hydroxytryptamine1A receptors.

The 5-hydroxytryptamine1A (5-HT1A) receptor can bind certain beta-adrenergic receptor antagonists, such as pindolol, with high affinity. Such pharmacological cross-reactivity suggests a structural similarity in the ligand binding site between the two receptors. To identify this structural entity, we mutated Asn385 in the seventh transmembrane domain of the human 5-HT1A receptor, based on the observation that this residue is conserved in all 5-HT1A and beta-adrenergic receptors of different species but is absent in all other cloned guanine nucleotide-binding protein-coupled receptors. This single point mutation (Asn385 to valine) causes a highly selective decrease in the affinity of pindolol and other aryloxyalkylamines for the mutant receptor (about 100-fold), while producing only minor changes in the binding of other 5-HT agonists and antagonists. The results provide direct evidence that Asn385 is responsible for the high affinity interaction between 5-HT1A receptors and aryloxyalkylamine beta-adrenergic antagonists but is not required for the binding of other chemical classes of ligands.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1991

Cortical and striatal variations in drug competition studies with putative 5-hydroxytryptamine1D binding sites.

The ability of 5-hydroxytryptamine (5-HT), 5-carboxytryptamine (5-CT) and sumatriptan to compete for [3H]5-HT binding sites in various brain tissues was analyzed in bovine, guinea pig, pig and human cortex and caudate. Radioligand binding conditions were designed to allow for the selective labeling of putative 5-HT1D binding sites. 5-HT competed monophasically with putative 5-HT1D binding sites in each of the 8 tissues studied. By contrast, both 5-CT and sumatriptan competed with markedly shallow displacement curves in each of the 8 tissues. In the case of sumatriptan, complete displacement of [3H]5-HT could not be achieved even at concentrations as high as 2000 times its IC50 value. These data indicate that 10(-5) M 5-HT should not be used to define specific binding to 5-HT1D receptors in radioligand binding assays. Instead, 5-HT1D receptor binding sites should be redefined as [3H]5-HT-labeled binding sites displaced by 10(-5) M sumatriptan.

Animals↗

PubMed1991

Differential interactions of dimethyltryptamine (DMT) with 5-HT1A and 5-HT2 receptors.

The interactions of the indolealkylamine N,N-dimethyltryptamine (DMT) with 5-hydroxytryptamine1A (5-HT1A) and 5-HT2 receptors in rat brain were analyzed using radioligand binding techniques and biochemical functional assays. The affinity of DMT for 5-HT1A sites labeled by [3H]-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]-8-OH-DPAT) was decreased in the presence of 10(-4) M GTP, suggesting agonist activity of DMT at this receptor. Adenylate cyclase studies in rat hippocampi showed that DMT inhibited forskolin-stimulated cyclase activity, a 5-HT1A agonist effect. DMT displayed full agonist activity with an EC50 of 4 x 10(-6) M in the cyclase assay. In contrast to the agonist actions of DMT at 5-HT1A receptors, DMT appeared to have antagonistic properties at 5-HT2 receptors. The ability of DMT to compete for [3H]-ketanserin-labeled 5-HT2 receptors was not affected by the presence of 10(-4) M GTP, suggesting antagonist activity of DMT at 5-HT2 receptors. In addition, DMT antagonized 5-HT2-receptor-mediated phosphatidylinositol (PI) turnover in rat cortex at concentrations above 10(-7) M, with 70% of the 5-HT-induced PI response inhibited at 10(-4) M DMT. Micromolar concentrations of DMT produced a slight PI stimulation that was not blocked by the 5-HT2 antagonist ketanserin. These studies suggest that DMT has opposing actions on 5-HT receptor subtypes, displaying agonist activity at 5-HT1A receptors and antagonist activity at 5-HT2 receptors.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1991

Structural determinants of 5-HT1A versus 5-HT1D receptor binding site selectivity.

A structure-activity analysis was used to identify selective 5-HT1A versus 5-HT1D receptor agents. An analysis of published data identified 13 drugs which display nanomolar affinity for the 5-HT1A receptor and that have been analyzed at 5-HT1D receptor binding sites. Four agents display greater than or equal to 100-fold selectivity for the 5-HT1A receptor. Two structural features were identified which hypothetically result in selectivity for 5-HT1A versus 5-HT1D binding sites. The linkage of an indole ring to a basic nitrogen atom via the 4 position on the indole ring or the absence of an indole ring are two features which lower the affinity for the 5-HT1D receptor, but do not necessarily lower the affinity for the 5-HT1A receptor. A series of 7 agents (5 indoles, 2 quinolines) was identified which met these hypothetical selectivity criteria. These compounds were then analyzed in radioligand binding studies. These 7 agents display affinities of 1.3-170 nM for the 5-HT1A receptor binding site, and 1,800-13,000 nM for the 5-HT1D receptor binding site. All 7 agents display greater than or equal to 47-fold selectivity for the 5-HT1A versus 5-HT1D site and 4 of the agents are greater than 100-fold selective. Compound No. 1 (N,N'-bis[3-(4-indolyloxy)-2-hydroxypropyl]-(Z)-1,8-diamino-p-meth ane) and compound No. 2 (N8-[3-(4-indolyloxy)-2-hydroxypropyl]-N1-(propioloyl)-(Z)-1 ,8-diamino-p-methane) are the most selective agents yet described for 5-HT1A versus 5-HT1D receptor binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

PubMed1991

Identification of 5-hydroxytryptamine1A receptor agents using a composite pharmacophore analysis and chemical database screening.

A composite pharmacophore analysis and computer-assisted chemical database screening were used to identify a previously unrecognized class of 5-hydroxy-tryptamine1A (5-HT1A) receptor active agents. An analysis of published data led to the identification of 20 different chemical structures which share nanomolar affinity for the 5-HT1A receptor. From a composite pharmacophore analysis of all 20 potent agents, we hypothesized that compounds containing a novel (in terms of 5-HT1A receptor analysis) 3 ring structure might be active at the 5-HT1A receptor. To test this hypothesis, the Chemical Abstracts database, which contains over 10 million compounds, was screened electronically for compounds that contain this core structure. A series of 319 agents was identified which contain this core structure. A total of 6 compounds was then obtained commercially and evaluated in radioligand binding studies. A single agent (Compound 69/183) conformed most closely to the composite 5-HT1A pharmacophore and displayed an affinity of 20 nmol/l for the 5-HT1A receptor binding site. Two other agents displayed affinities of 170 and 500 nmol/l, respectively, for the 5-HT1A receptor site. The 3 agents which differed most significantly from the composite 5-HT1A pharmacophore displayed affinities of 1,200- greater than 10,000 nmol/l for the 5-HT1A receptor binding site. These data suggest that a composite pharmacophore analysis and computer-assisted chemical database screening can be an effective technique for the identification of previously unrecognized receptor active agents.

Computer Simulation↗

PubMed1991

Postsynaptic localization of 5-HT1D receptor binding sites in human caudate.

5-hydroxytryptamine1D (5-HT1D) receptor binding sites were quantified by saturation studies in the postmortem caudate nucleus and frontal cortex of individuals with Huntington's disease and control individuals with no known neurological disease. 3H-5-HT was used as the radioligand in the presence of 100 nM 8-OH-DPAT and 100 nM mesulergine in order to restrict radioligand binding to 5-HT1D receptors. No alteration in KD value was detected in Huntington's disease as compared to control brain tissue. However, the density (Bmax) of the 5-HT1D site was significantly decreased (P less than 0.01) in the caudate, but not the frontal cortex, of Huntington's disease versus control individuals. By contrast, no significant difference was found in Bmax or KD of [3H]paroxetine binding between control and Huntington's caudates. These data suggest that a significant number of caudate 5-HT1D receptors are located on the intrinsic neurons of the striatum as opposed to 5-HT nerve terminals.

Aged↗

PubMed1991

5-benzyloxytryptamine: a relatively selective 5-hydroxytryptamine 1D/1B agent.

The ability of nineteen tryptamine derivatives to interact with putative 5-hydroxytryptamine1D (5-HT1D) receptor binding sites in bovine caudate was analyzed. Sixteen of the nineteen agents competed, with variable potency, for these binding sites with Hill slopes of approximately unity. By contrast, 5-carboxyamidotryptamine (5-CT), sumatriptan and 5-benzyloxytryptamine (5-BT) competed with Hill slope values significantly less than unity. These three drugs share, in comparison to the sixteen other tryptamines, relatively large substitutions at the 5-position of the indole moiety. Additional radioligand binding studies with 5-BT indicate that the drug shows relative selectivity for 5-HT1D/1B binding sites. Functionally, 5-BT and sumatriptan inhibit 3H-5-HT release from guinea pig cortical synaptosomes with equal potency but 5-BT is significantly less efficacious than sumatriptan. These data indicate that 5-BT is a relatively selective partial agonist at 5-HT1D receptors.

Animals↗

PubMed1991

Characterization of a novel and potent 5-hydroxytryptamine1A receptor antagonist.

A series of pindolol derivatives (n = 7) was analyzed in radioligand binding, biochemical and behavioral studies. Three of these drugs (Compounds A, B, and C) are extremely potent (i.e., Ki values less than 1.0 nM) at 5-hydroxytryptamine1A (5-HT1A) sites labeled by [3H] 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT). Moreover, these drugs are selective in that they are approximately an order of magnitude less potent at beta-adrenergic receptors labeled by 3H-dihydroalprenolol (DHA). Compound A (N1-(bromoacetyl)-N8-[3-(4-indolyloxy)-2-hydroxypropyl]-(Z)-1,8-di amino-p- methane) is also significantly less potent at 10 other neurotransmitter receptor sites analyzed. In addition, Compound A (10(-10) M to 10(-3) M) has no effect on baseline forskolin-stimulated adenylate cycalse activity in rat hippocampus. By contrast, nanomolar concentrations of the drug significantly (p less than 0.01) reverse 8-OH-DPAT-induced inhibition of forskolin-stimulated activity. In behavioral studies. Compound A (0.5 mg/kg) alone has no effect on baseline measures of reciprocal forepaw treading in the rat. Pretreatment with Compound A, however, significantly (p less than 0.05) inhibits the reciprocal forepaw treading induced by 8-OH-DPAT. These data suggest that Compound A is a potent and selective antagonist of 5-HT1A receptors in the CNS.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1991

Molecular graphics applied to 5-HT3 ligands.

The recent surge of scientific interest in 5-hydroxytryptamine3 (5-HT3) receptors can be attributed largely to the rapid increase in the number of potent and selective 5-HT3 receptor antagonists. The availability of a large variety of chemically diverse 5-HT3 compounds allows for a unique opportunity to apply computer-based modelling techniques to the analysis of the 5-HT3 receptor pharmacophore. The present review summarises recent studies performed in this laboratory. The primary goal of these studies was to elucidate the chemical requirements needed for molecular recognition at the 5-HT3 receptor binding site. A secondary goal was to determine if this type of molecular information could be assessed using computerised chemical databases.

Computer Simulation↗

PubMed1991

New vistas on 5-HT receptors and migraine.

1. A number of serotonergic agents have been shown to be effective in the treatment of migraine. At the present time, migraine drug interactions have been analyzed most extensively at 5-hydroxytryptamine1D (5-HT1D), 5-HT2 and 5-HT3 receptor subtypes. 2. This review will summarize the current status of 5-HT receptor subtypes as they relate to anti-migraine agents. 3. The available data suggest that drug interactions with specific 5-HT receptor subtypes may be the basis for their efficacy in both the acute and prophylactic treatment of migraine.

Animals↗

PubMed1991

Further characterization of the putative 5-HT receptor which mediates blockade of neurogenic plasma extravasation in rat dura mater.

1. We describe the effects of pretreatment with 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on neurogenically-mediated plasma protein extravasation ([125I]-albumin) in rat dura mater and in extracranial tissues (temporalis muscle fascia, conjunctiva, eyelid and lip) induced by electrical stimulation of the right trigeminal ganglion. 2. Leakage of [125I]-bovine serum albumin from blood vessels in dura mater following high intensity stimulation (1.2 mA, 5 ms, 5 Hz for 5 min) was significantly reduced by the intravenous administration of drugs active at 5-HT receptors with some selectivity for the 5-HT1 receptor subtypes: 5-carboxamidotryptamine (5-CT) (threshold dose, 1 ng kg-1); 5-benzyloxytryptamine (5-BT) (10, 30 or 100 micrograms kg-1); 8-hydroxydipropylaminotetralin (8-OH-DPAT) (300 micrograms kg-1); and as previously reported, sumatriptan (100 micrograms kg-1), dihydroergotamine (DHE) (50 micrograms kg-1); ergotamine tartrate (100 micrograms kg-1) and chronically administered methysergide (1 mg kg-1). 3. The putative 5-HT receptor antagonist, metergoline 100 micrograms kg-1, inhibited partially the effect of sumatriptan in dura mater providing additional evidence for a 5-HT1 receptor subtype-mediated mechanism, although it was not effective against 5-CT (1 ng kg-1). Methiothepin (300 micrograms kg-1) did not affect the response to sumatriptan. When administered at high concentrations (1 mg kg-1) methiothepin and metergoline decreased plasma protein extravasation in rat dura mater. 4. Pretreatment with the 5-HT2 receptor antagonists pizotifen, 300pugkg 1, or ketanserin, 300,ugkg ', or the 5-HT3 receptor antagonists MDL 72222, 300,ugkg-1, or ICS 205-930, 300pgkg-1, did not affect plasma protein leakage following electrical trigeminal stimulation. Blockade by sumatriptan of plasma protein extravasation was not inhibited by pizotifen (300,ug kg-1) or MDL 72222 (300pg kg- '). 5. The 5-HT receptor(s) mediating this response were present only on intracranial tissues innervated by the trigeminal nerve; plasma protein extravasation in extracranial tissues was not blocked by pretreatment with the equivalent or higher concentrations of the above drugs following low intensity trigeminal stimulation (0.1 mA, 5 ms, 5 Hz). 6. The putative 5-HT receptor(s) mediating this response were not present on sympathetic fibres innervating dura mater since unilateral removal of the superior cervical ganglion did not prevent the development of plasma protein extravasation nor did it affect the blockade by sumatriptan IOOpug kg- '. 7. The above pharmacological data suggest that intracranial vessels possess 5-HT receptor(s) which are coupled to inhibition of neurogenically-mediated plasma protein extravasation. These receptors cannot be detected on extracranial cephalic blood vessels innervated by the trigeminal nerve, although available evidence strongly suggests that the 5-HT receptors reside on perivascular trigeminal nerve fibres. The rank order of effective doses (threshold concentrations; 5-CT < 5-BT < DHE < sumatriptan < 8-OHDPAT) is most consistent with a 5-HTlB- or 5-HTlD-mediated response, among the known 5-HT1 family of receptors. However, the lack of effect of methiothepin against the actions of sumatriptan, or metergoline against the effects of 5-CT suggest important differences and the possibility that a previously unrecognized 5-HT receptor(s) is involved in this response.

8-Hydroxy-2-(di-n-propylamino)tetralin↗

PubMed1991

5-Hydroxtryptamine1D receptor agonism predicts antimigraine efficacy.

The interactions of four abortive anti-migraine agents and four prophylactic anti-migraine agents with 5-HT1D receptors in bovine brain were analyzed using radioligand binding techniques and adenylate cyclase assays. In bovine caudate, the affinities of abortive anti-migraine agents (i.e. 5-hydroxytryptamine, ergotamine, dihydroergotamine, sumatriptan) for 5-HT1D receptors range from 4.0-34 nM while the affinities of prophylactic anti-migraine agents (i.e. methysergide, amitriptyline, (-)propranolol, verapamil) range from 46-11,000 nM. In adenylate cyclase studies in bovine substantia nigra, all four abortive anti-migraine agents dose-dependently inhibit forskolin-stimulated adenylate cyclase activity, a biochemical effect mediated by 5-HT1D receptors. No agonist effect on cyclase activity is observed with the four prophylactic anti-migraine agents. These data support the hypothesis that abortive anti-migraine agents are 5-HT1D receptor agonists and that this effect may underlie their anti-migraine efficacy.

Adenylyl Cyclase Inhibitors↗