Reconstitution of the human 5-HT(1D) receptor-G-protein coupling: evidence for constitutive activity and multiple receptor conformations.

The 5-hydroxytryptamine (5-HT) 1D/1B receptors have gained particular interest as potential targets for treatment of migraine and depression. G-protein coupling and other intrinsic properties of the human 5-HT(1D) receptor were studied using a baculovirus-based expression system in Sf9 cells. Coexpression of the human 5-HT(1D) receptor with Galpha(i1), alpha(i2), alpha(i3), or Galpha(o)-proteins and Gbeta(1)gamma(2)-subunits reconstituted a Gpp(NH)p-sensitive, high affinity binding of [(3)H]5-HT to this receptor, whereas the Galpha(q)beta(1)gamma(2) heterotrimer was ineffective in this respect.

Mapping of serotonin 5-HT(4) receptor mRNA and ligand binding sites in the post-mortem human brain.

The anatomical localization of 5-HT(4) receptor mRNA and 5-HT(4) receptor protein was examined in sections of post-mortem human brain by in situ hybridization histochemistry and radioligand receptor autoradiography. In the in situ hybridization study, the highest levels of 5-HT(4) receptor mRNA were found in caudate nucleus, putamen, nucleus accumbens, and in the hippocampal formation. No 5-HT(4) receptor mRNA was detected in globus pallidus and substantia nigra.

Structure of the human serotonin 5-HT4 receptor gene and cloning of a novel 5-HT4 splice variant.

Several variants of the serotonin 5-HT4 receptor are known to be produced by alternative splicing. To survey the existence and usage of exons in humans, we cloned the human 5-HT4 gene. Based on sequence analysis seven C-terminal variants (a-g) and one internal splice variant (h) were found.

Cloning and expression of a human serotonin 5-HT4 receptor cDNA.

Using a combination of library screening and nested PCR based on a partial human serotonin 5-HT4 receptor sequence, we have cloned the complete coding region for a human 5-HT4 receptor. The sequence shows extensive similarity to the published porcine 5-HT4A and rat 5-HT4L receptor cDNA; however, in comparison with the latter, we find an open reading frame corresponding to only 388 amino acids instead of 406 amino acids. This difference is due to a frame shift caused by an additional cytosine found in the human sequence after position 1,154.

Stable, high-level expression of human serotonin receptors in L929 cells using an inducible expression system.

Heterologous expression of cloned receptor subtypes for screening programs has become a real necessity for a modern pharmaceutical company. As the expression levels obtained so far are often low or unstable, we addressed this problem by using an inducible promoter system, i.e.

Alniditan, a new 5-hydroxytryptamine1D agonist and migraine-abortive agent: ligand-binding properties of human 5-hydroxytryptamine1D alpha, human 5-hydroxytryptamine1D beta, and calf 5-hydroxytryptamine1D receptors investigated with [3H]5-hydroxytryptamine and [3H]alniditan.

Alniditan is a new migraine-abortive agent. It is a benzopyran derivative and therefore structurally unrelated to sumatriptan and other indole-derivatives and to ergoline derivatives. The action of sumatriptan is thought to be mediated by 5-hydroxytryptamine (5-HT)1D-type receptors.

Seasonal variation in platelet [3H]paroxetine binding in healthy volunteers. Relationship to climatic variables.

Recently, our laboratory has reported significant seasonal differences in [3H]paroxetine binding to platelets in depressed subjects. This study aimed to examine the seasonal variation in [3H]paroxetine binding to platelets and the relationships between [3H]paroxetine binding and climatic variables in healthy volunteers. We took monthly blood samples during one calendar year from 26 healthy volunteers for assay of [3H]paroxetine binding and analyzed the data by means of univariate and multivariate spectral and cosinor analyses.

Risperidone compared with new and reference antipsychotic drugs: in vitro and in vivo receptor binding.

Risperidone and its active metabolite 9-OH-risperidone were compared to reference antipsychotic drugs (haloperidol, pipamperone, fluspirilene, clozapine, zotepine) and compounds under development (olanzapine, seroquel, sertindole, ORG-5222, ziprasidone) for in vitro binding to neurotransmitter receptors in brain tissue and on membranes of recombinant cells expressing cloned human receptors and for in vivo occupancy of neurotransmitter receptors in rat and guinea-pig brain following acute treatment (2 h., s.c.

Binding of [3H]paroxetine to platelets of depressed patients: seasonal differences and effects of diagnostic classification.

[3H]Paroxetine is a more reliable ligand for studying the serotonin (5-HT) transporter complex than [3H]imipramine. The present study investigates [3H]paroxetine binding to platelets in 54 depressed in-patients (18 minor, 16 simple major and 20 melancholic depressed patients) and 16 healthy controls. There were no significant differences in maximal number of binding sites between depressed subjects and normal controls.

Genomic cloning, heterologous expression and pharmacological characterization of a human histamine H1 receptor.

A human histamine H1 receptor gene lacking introns was isolated by screening a human genomic library with a bovine histamine H1 receptor probe. The deduced protein of 487 amino acids showed characteristic properties of G-protein-coupled receptors. The coding region was subcloned into the expression vector pSVL (Pharmacia), and the resulting construct transfected into COS-7 cells.

Comparison of in vitro binding properties of a series of dopamine antagonists and agonists for cloned human dopamine D2S and D2L receptors and for D2 receptors in rat striatal and mesolimbic tissues, using [125I] 2'-iodospiperone.

We investigated the ligand binding properties in vitro of two splice variants of the cloned human dopamine D2 receptor (the 443 and 414 amino acids long forms called D2L and D2S, respectively), expressed in 293 human kidney cells, in comparison with those of the dopamine D2 receptors in rat striatum, nucleus accumbens and tuberculum olfactorium. The new radioligand, [125I]2'-iodospiperone, showed a similar high binding affinity (KD:0.056-0.

Autoradiographic evidence for the occlusion of rat brain dopamine D3 receptors in vivo.

[125I]Iodosulpride binding was studied in frontal rat brain sections by quantitative autoradiography. Using preincubated (= washed) sections, selective labelling and identification of dopamine D3 receptors was obtained using 0.2 nM [125I]iodosulpride in the presence of 100 nM domperidone for the occlusion of the D2 receptors.

In vitro and in vivo receptor binding and effects on monoamine turnover in rat brain regions of the novel antipsychotics risperidone and ocaperidone.

Risperidone and ocaperidone are new benzisoxazol antipsychotics with particularly beneficial effects in schizophrenia. We report a comprehensive study on the in vitro and in vivo receptor binding profile of the new compounds, compared with haloperidol, and on the drug effects on monoamine and metabolite levels in various brain areas. The in vitro receptor binding and monoamine uptake inhibition profiles, comprising 29 receptors and four monoamine uptake systems, revealed that ocaperidone and risperidone bound primarily, and with the highest affinity thus far reported, to serotonin 5HT2 receptors (Ki values of 0.

[Non-sedative antihistaminics and binding to central and peripheral H1 histamine receptors].

Four non-sedating antihistamines (astemizole, cetirizine, loratadine and terfenadine) were investigated for in vitro and ex vivo binding to histamine-H1 receptors in guinea-pig cerebellum and lung. In vitro, all the drugs dissociated slowly from H1 receptors (half-times greater than or equal to 100 min), Ki,app-values decreased with longer incubation times for potent lipophylic agents (astemizole and terfenadine) Ki,app-values were lower with more dilute tissue suspensions. In optimized assay conditions astemizole showed a Ki,app-value of 0.

Identification of non-serotonergic [3H]ketanserin binding sites on human platelets and their role in serotonin release.

[3H]Ketanserin was found to label (besides a low amount of 5-HT2 receptors) non-serotonergic binding sites on human platelet membranes. The latter binding was detected in the presence of excess of the 5-HT2 antagonist BW501, and was potently inhibited by tetrabenazine. [3H]Ketanserin revealed a KD value = 19 +/- 4 nM and a Bmax = 425 +/- 82 fmol/10(9) platelets for these binding sites.

The receptor binding profile of the new antihypertensive agent nebivolol and its stereoisomers compared with various beta-adrenergic blockers.

Nebivolol [the (S,R,R,R)- + (R,S,S,S)-racemic mixture], the 10 stereoisomers, and known beta-adrenergic blockers were investigated in vitro for binding to beta 1- and beta 2-adrenergic receptor sites and various neurotransmitter, peptide, and ion channel binding sites and for inhibition of neurotransmitter uptake. Selective labeling of beta 1- and beta 2-adrenergic receptor sites in rabbit and rat lung, respectively, was obtained with [3H]CGP-12177 and [3H] dihydroalprenololin the presence of an appropriate concentration of the selective beta 2-adrenergic blocker ICI 118-551 or the selective beta 1-adrenergic blocker CGP 20712-A. Nebivolol revealed high affinity and selectivity for beta 1-adrenergic receptor sites in the rabbit lung membrane preparation (Ki value = 0.

Biochemical profile of risperidone, a new antipsychotic.

Risperidone was compared to the 5-hydroxytryptamine2 antagonist ritanserin and to the dopamine-D2 antagonist haloperidol. The in vitro receptor binding (neurotransmitter-, peptide- and ion channel binding sites) and neurotransmitter uptake profile were investigated. Risperidone revealed, like ritanserin, a very high binding affinity for 5-hydroxytryptamine2 receptors (Ki = 0.

Comparison of the in-vitro receptor selectivity of substituted benzamide drugs for brain neurotransmitter receptors.

The in-vitro selectivity of a group of substituted benzamide drugs for brain neurotransmitter receptors was determined to assess the most appropriate drugs for use in human PET studies. All substituted benzamide drugs studied inhibited [3H]haloperidol and [3H]spiperone binding to rat striatal membranes. The most potent compounds were YM 09151-2, clebopride and raclopride.

The dissociation rate of unlabelled drugs from receptor sites: a poorly investigated, yet important aspect in receptor research. Studies on the serotonin-S2 receptor.

The labelling by 3H-spiperone of serotonin-S2 receptors in rat frontal cortex tissue adsorbed to glass fibre filters was investigated. For 12 unlabelled serotonin antagonists the dissociation time from serotonin-S2 receptors was measured using rat frontal cortex tissue preparations adsorbed to glass fibre filters. The dissociation half-time varied from 4.

Identification of nonserotonergic [3H]ketanserin binding sites associated with nerve terminals in rat brain and with platelets; relation with release of biogenic amine metabolites induced by ketanserin- and tetrabenazine-like drugs.

In mammalian striatal tissue and cat platelets, [3H]ketanserin labels besides serotonin-S2 receptors nonserotonergic saturable binding sites. The sites have been distinguished and characterized in [3H]ketanserin binding assays by selective inhibition with tetrabenazine (Ki = 4 nM), a monoamine depleting agent. In rats, the nonserotonergic ketanserin sites were enriched in the striatum (KD = 12.

Receptor interactions of dopamine and serotonin antagonists: binding in vitro and in vivo and receptor regulation.

The advent of receptor binding techniques has provided new ways of studying the mechanism of action of drugs. In vitro radioligand binding is now currently applied to investigate the specificity or multiple action of compounds. By using the same technique, the binding affinity of a drug can be measured for a variety of neurotransmitter, drug, peptide and ion channel receptor binding sites, providing the drug's receptor binding profile (LEYSEN et al.

Distinction between adrenergic and serotonergic receptor subtypes: specificity of drugs and absence of cooperative interactions between adrenergic and serotonergic receptor binding sites.

In light of observed amplificatory interactions between serotonergic and adrenergic stimuli in functional studies on vascular tissue and platelets, we investigated the distinction and possible interactions between alpha 1-, alpha 2-, beta 1-, and beta 2-adrenergic and 5-HT1A-, 5-HT1B-, and 5-HT2-serotonergic receptor binding sites. Therefore, the binding affinities of archetypes of adrenergic and serotonergic agonists and antagonists for the various receptors were measured. Only the alpha 1-blocker prazosin revealed great specificity for alpha 1-adrenergic receptors; the other investigated antagonists and agonists showed cross-reactivity with adrenergic and serotonergic receptors in various combinations.