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. Competition of [(3)H]5-HT binding by various compounds confirmed that coexpression of the human 5-HT(1D) receptor with Galpha(i/o)beta(1)gamma(2) reconstitutes the receptor in a high affinity agonist binding state, having the same pharmacological profile as the receptor expressed in mammalian cells. Binding of the antagonist ocaperidone to the human 5-HT(1D) receptor in coupled or noncoupled state was analyzed. This compound competed with [(3)H]5-HT binding more potently on the human 5-HT(1D) receptor in the noncoupled state, showing its inverse agonistic character. Ocaperidone acted as a competitive inhibitor of [(3)H]5-HT binding when tested with the coupled receptor form but not so when tested with the noncoupled receptor preparation. Finally, [(35)S]GTPgammaS binding experiments using the inverse agonist ocaperidone revealed a high level of constitutive activity of the human 5-HT(1D) receptor. Taken together, the reconstitution of the human 5-HT(1D) receptor-G-protein coupling using baculovirus-infected Sf9 cells made possible the assessment of coupling specificity and the detection of different binding states of the receptor induced by G-protein coupling or ligand binding.

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. For receptor autoradiography, two new and highly selective radioligands were compared: [(3)H]prucalopride, which preferentially labels the G-protein coupled fraction of receptors, and [(3)H]R116712, which labels the entire receptor population at subnanomolar concentrations. [(3)H]Prucalopride and [(3)H]R116712 binding was performed on human brain hemisphere sections. The highest densities for both radioligands were found in the basal ganglia (caudate nucleus, putamen, nucleus accumbens, globus pallidus, substantia nigra). Moderate to low densities were detected in the hippocampal formation and in the cortical mantle. Mismatches between 5-HT(4) receptor mRNA and binding sites in the globus pallidus and the substantia nigra suggested that the binding sites may be localized on axonal projections originating from the striatum. To compare densities of binding sites, concentration binding curves with [(3)H]prucalopride, [(3)H]R116712 and [(3)H]GR113808 were performed on membranes from homogenates of several human brain regions. Comparison of B(max)-values obtained with [(3)H]prucalopride and [(3)H]R116712 indicated that the G-protein coupled fraction of 5-HT(4) receptors in the substantia nigra was exceptionally high (54%) in comparison with percentages (16-27%) found in the frontal cortex, the striatum and the hippocampus. Such a high percentage (40%) of [(3)H]prucalopride vs. [(3)H]R116712 binding was also observed in the substantia nigra in the receptor autoradiography experiments. The [(3)H]prucalopride binding was GppNHp-sensitive, whereas [(3)H]R116712 and [(3)H]GR113808 was not. These data indicate that in the substantia nigra 5-HT(4) receptors are more strongly coupled to their signal transduction pathway than in other brain regions.

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. We concentrated in this study on the functional characterization of the novel splice variant h, which leads to the insertion of 14 amino acids into the second extracellular loop of the receptor. The h variant was cloned as a splice combination with the C-terminal b variant; therefore, we call this receptor 5-HT4(hb). This novel receptor variant was expressed transiently in COS-7 cells, and its pharmacological profile was compared with those of the previously cloned 5-HT4(a) and 5-HT4(b) isoforms, with the latter being the primary reference for the h variant. In competition binding experiments using reference 5-HT4 ligands, no significant differences were detected. However, the broadly used 5-HT4 antagonist GR113808 discriminated functionally among the receptor variants investigated. As expected, it was an antagonist on the 5-HT4(a) and 5-HT4(b) variant but showed partial agonistic activity on the 5-HT4(hb) variant. These data emphasize the importance of variations introduced by splicing for receptor pharmacology and may help in the understanding of conflicting results seen with 5-HT4 ligands in different model systems.

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. the interferon-inducible mouse Mxl promoter. Using the gene coding for chloramphenicol acetyltransferase (CAT) as a reporter gene, we tested the inducibility of this promoter in the murine cell line L929. We found that background expression was low and that a distinct interferon-induced expression could be obtained. CAT expression reached its maximum at approximately 15 ng CAT/mg protein after induction for 24 hr with 1000 U/ml murine interferon-beta; the induction ratio was 150-fold. Next, L929 cells were transfected with four different human serotonin (5HT) receptor cDNAs (5HT1A, 5HT2A, 5HT1D beta and 5HT1E) under the control of the same Mxl promoter fragment. Also in this case well-regulated serotonin receptor-expressing clones were isolated. Bmax values varied from 3100 fmol/mg protein for the 5HT2A receptor, 3300 fmol/mg protein for the 5HT1D beta receptor, 9800 fmol/mg protein for the 5HT1E receptor, and even up to 10,400 fmol/mg protein for the 5HT1A receptor. Furthermore, the expression levels were shown to remain stable during serial propagation for at least one year, demonstrating the usefulness of this expression system. In fact, the 5HT1D beta receptor-expressing cells were used in the characterization of a new antimigraine agent, viz. alniditan.

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. Moreover, we also found the same additional cytosine in the rat 5-HT4 sequence. We confirmed the occurrence of the sequence by examining this part of the sequence in genomic DNA of 10 human volunteers and in rat genomic DNA. Based on a part of the genomic 5-HT4 receptor sequence that was identified in the cloning process, there seem to be at least two possible splice sites in the coding region of the gene. The human 5-HT4 receptor, transiently expressed in COS-7 cells, showed radioligand binding properties similar to 5-HT4 receptors in guinea pig striatal tissue. [3H]GR 113808 revealed K(D) values of 0.15 +/- 0.01 nM for the human receptor and 0.3 +/- 0.1 nM in the guinea pig tissue. Binding constants were determined for four investigated 5-HT4 antagonists and three agonists, and appropriate binding inhibition constants were found in each case. Stimulation of transfected COS-7 cells with 5-HT4-specific agonists caused an increase in cyclic AMP levels.

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. We investigated the receptor-binding profile in vitro of alniditan compared with sumatriptan and dihydroergotamine for 28 neurotransmitter receptor subtypes, several receptors for peptides and lipid-derived factors, ion channel-binding sites, and monoamine transporters. Alniditan revealed nanomolar affinity for calf substantia nigra 5-HT1D and for cloned h5-HT1D alpha, h5-HT1D beta and h5-HT1A receptors (Ki = 0.8, 0.4, 1.1, and 3.8 nM, respectively). Alniditan was more potent than sumatriptan at 5-HT1D-type and 5-HT1A receptors. Alniditan showed moderate-to-low or no affinity for other investigated receptors; sumatriptan showed additional binding to 5-HT1F receptors. Dihydroergotamine had a much broader profile with high affinity for several 5-HT, adrenergic and dopaminergic receptors. In signal transduction assays using cells expressing recombinant h5-HT1D alpha, h5-HT1D beta, or h5-HT1A receptors, alniditan (like 5-HT) was a full agonist for inhibition of stimulated adenylyl cyclase (IC50 = 1.1, 1.3, and 74 nM, respectively, for alniditan). Therefore, in functional assays, the potency of alniditan was much higher at 5-HT1D receptors than at 5-HT1A receptors. We further compared the properties of [3H]alniditan, as a new radioligand for 5-HT1D-type receptors, with those of [3H]5-HT in membrane preparations of calf substantia nigra, C6 glioma cells expressing h5-HT1D alpha, and L929 cells expressing h5-HT1D beta receptors. [3H]Alniditan revealed very rapid association and dissociation binding kinetics and showed slightly higher affinity (Kd = 1-2 nM) than [3H]5-HT. We investigated 25 compounds for inhibition of [3H]alniditan and [3H]5-HT binding in the three membrane preparations; Ki values of the radioligands were largely similar, although some subtle differences appeared. Most compounds did not differentiate between 5-HT1D alpha and 5-HT1D beta receptors, except methysergide, ritanserin, ocaperidone, risperidone, and ketanserin, which showed 10-60-fold higher affinity for the 5-HT1D alpha receptor. The Ki values of the compounds obtained with 5-HT1D receptors in calf substantia nigra indicated that these receptors are of the 5-HT1D beta-type. We demonstrated that alniditan is a potent agonist at h5-HT1D alpha and h5-HT1D beta receptors; its properties probably underlie its cranial vasoconstrictive and antimigraine properties.

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. There was a statistically highly significant seasonal pattern in [3H]paroxetine binding to platelets with significant annual, 4-monthly, and bimonthly rhythms, which were expressed as a group phenomenon. [3H]Paroxetine binding to platelets was significantly lower in fall and summer than in winter and spring; lows occurred in summer and peaks in spring. The peak-trough difference in this yearly variation, expressed as a percentage of the mean, was as large as 83.7%. A large part of the variance, that is, 32.5%, in [3H]paroxetine binding could be explained by weather variables, such as ambient temperature, relative humidity, and air pressure. Highly significant common annual rhythms were expressed in [3H]paroxetine binding and ambient temperature or humidity (both inversely related) and changes in temperature the 2 weeks preceding blood samplings (positively related).

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.). An ex vivo autoradiography technique was applied to determine the receptor occupancy by the drugs administered in vivo. Of particular interest are the central 5HT2A receptors and D2-type receptors. Predominant 5HT2A receptor antagonism is supposed to add to an atypical profile of the antipsychotics (treatment of the negative symptoms, low incidence of extrapyramidal side effects). D2 antagonism is required the treatment of positive symptoms. A contribution of the new dopamine receptor subtypes D3 and in particular D4 receptors has been proposed. In vitro, all compounds, except the 'typical' antipsychotics haloperidol and fluspirilene, showed higher affinity for 5HT2A than for D2 receptors. Subnanomolar affinity for human 5HT2A receptors was observed for ORG-5222, sertindole, risperidone, 9-OH-risperidone and ziprasidone. Fluspirilene, ORG-5222, haloperidol, ziprasidone, risperidone, 9-OH-risperidone and zotepine displayed nanomolar affinity for human D2 receptors. Sertindole and olanzapine were slightly less potent. Pipamperone, clozapine and seroquel showed 2 orders of magnitude lower D2 affinity in vitro. Clozapine, but even more so pipamperone, displayed higher affinity for D4 than for D2 receptors. For most other compounds, D4 affinity was only slightly lower than their D2 affinity. Seroquel was totally devoid of D4 affinity. None of the compounds had nanomolar affinity for D1 receptors; their affinity for D3 receptors was usually slightly lower than for D2 receptors. In vivo, ORG-5222, risperidone, pipamperone, 9-OH-risperidone, sertindole, olanzapine, zotepine and clozapine maintained a higher potency for occupying 5HT2A than D2 receptors. Risperidone and ORG-5222 had 5HT2A versus D2 potency ratio of about 20. Highest potency for 5HT2A receptor occupancy was observed for ORG-5222 followed by risperidone and olanzapine. Ziprasidone exclusively occupied 5HT2A receptors. ORG-5222, haloperidol, fluspirilene and olanzapine showed the highest potency for occupying D2 receptors. No regional selectivity for D2 receptor occupancy in mesolimbic versus nigrostriatal areas was detected for any of the test compounds. Risperidone was conspicuous because of its more gradual occupancy of D2 receptors; none of the other compounds showed this property. The various compounds also displayed high to moderate occupancy of adrenergic alpha 1 receptors, except fluspirilene and ziprasidone. Clozapine, zotepine, ORG-5222 and sertindole occupied even more alpha 1 than D2 receptors. Clozapine showed predominant occupancy of H1 receptors and occupied cholinergic receptors with equivalent potency to D2 receptors. A stronger predominance of 5HT2A versus D2 receptor occupancy combined with a more gradual occupancy of D2 receptors differentiates risperidone and its 9-OH-metabolite from the other antipsychotic compounds in this study. The predominant 5HT2A receptor occupancy probably plays a role in the beneficial action of risperidone on the negative symptoms of schizophrenia, whereas maintenance of a moderate occupancy of D2 receptors seems adequate for treating the positive symptoms of schizophrenia. A combined 5HT2A and D2 occupancy and the avoidance of D2 receptor overblockade are believed to reduce the risk for extrapyra

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. There was no correlation between [3H]paroxetine binding to platelet membranes and severity of depression. [3H]Paroxetine binding to platelets was significantly higher in spring than in summer, fall and winter.

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. Binding studies with [3H]pyrilamine on membranes from transfected cells revealed saturable specific binding with a KD of 1.2 nM and a Bmax of 3400 fmol/mg protein. Binding affinities of histamine and known histamine antagonists were similar to those for histamine H1 receptors in guinea-pig cerebellum. In transfected COS-7 cells, histamine induced inositol phosphate formation, that was inhibitable by pyrilamine.

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:0.056-0.122 nM) for cloned human D2S and D2L receptors and for the D2 receptors in the three rat brain areas. Binding affinities of 25 dopamine antagonists and of 10 dopamine agonists belonging to different chemical classes were measured. The IC50 values of the antagonists were virtually identical in the five preparations: spiperone was the most potent compound (pIC50 approximately 9.9), remoxipride the least potent one (pIC50 approximately 5.7). The agonists showed similar IC50 values for the cloned human D2S and D2L receptors but their affinity for rat brain D2 receptors was 2- to 5-fold higher. Dopamine showed shallow inhibition curves, the high affinity binding was 10-fold lower for the cloned human D2 receptors than for the rat brain D2 receptors. Addition of stable guanosine-5'-triphosphate (GTP) analogues shifted the D2 receptors in the rat brain tissues to the "low" affinity state, the low affinity binding of dopamine was equal to the affinity for the cloned human receptor. None of the dopamine antagonists or agonists could differentiate between the two splice forms of the cloned human D2 receptors or between the D2 receptors in rat striatal and mesolimbic tissues. The lower apparent affinity of some agonists and of dopamine in the absence of stable GTP analogues suggests a less appropriate receptor G-protein coupling for the cloned human D2 receptors expressed in the 293 human kidney cells. Unexpectedly, guanosine-5'-O-(3-thiotriphosphate) (GTP-gamma-S) reduced the [125I]2'-iodospiperone binding to the D2 receptors by 20-35% in the rat brain tissues and the cloned human D2L receptor, and by 75% to the cloned human D2S receptor. The inhibition in the last case could be prevented partly by submicromolar concentrations of dopamine. The GTP-gamma-S effect is suggested to be due to reduction of disulphide bonds in the receptor. Recent molecular modelling studies indicated an important role of the disulphide bridge between Cys107 at the start of transmembrane domain three and Cys182 in the third extracellular loop, for the binding of dopamine to the D2 receptor.

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. A high density of D3 receptors was noticed in the islands of Calleja. When preincubation of the sections was omitted, no D3 receptor labelling could be achieved, indicating tight binding to the receptor of an endogenous inhibitor. Such a tight receptor occupancy was not observed for the D2 receptor and various other neurotransmitter receptors. The occlusion of the D3 receptor could be prevented by tetrabenazine-induced monoamine depletion of the rats. It can be concluded, therefore, that D3 receptors are massively occupied by a monoamine, likely to be dopamine. This observation prompts the question to what extent dopamine D3 receptors can become occupied in vivo by systematically applied exogenous compounds.

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 0.14 and 0.12 nM, respectively). Further, the drugs bound at nanomolar concentrations to the following receptors (Ki values, in nM, for ocaperidone and risperidone, respectively): alpha 1-adrenergic (0.46 and 0.81), dopamine D2 (0.75 and 3.0), histamine H1 (1.6 and 2.1), and alpha 2-adrenergic (5.4 and 7.3). In contrast, haloperidol showed nanomolar affinity for D2 receptors (1.55) and haloperidol-sensitive sigma sites (0.84) only. The in vitro binding affinity of ocaperidone, risperidone, and haloperidol for D2 receptors was exactly the same when measured in membranes from rat striatum, nucleus accumbens, tuberculum olfactorium, and human kidney cells expressing the cloned human D2 receptor (long form). In vivo binding in rats, using intravenous administration of [3H]spiperone, revealed very potent occupation by ocaperidone and risperidone of 5HT2 receptors in the frontal cortex (ED50 of 0.04-0.03 mg/kg); in this respect, they were 6, 30, and 100 times more potent than ritanserin, haloperidol, and clozapine, respectively. Ocaperidone occupied D2 receptors in the striatum and the nucleus accumbens with similar potency as did haloperidol (ED50 of 0.14-0.16 mg/kg). Risperidone revealed biphasic inhibition curves in the latter brain areas, indicating that [3H] spiperone labeled both 5HT2 receptors (occupied by risperidone at less than 0.04 mg/kg) and D2 receptors (risperidone ED50 of approximately 1 mg/kg). In the tuberculum olfactorium, 5HT2 and D2 receptors were also distinguished with risperidone. The ED50 values for occupation of the latter were for ocaperidone and risperidone 2 times lower and for haloperidol 2 times higher than in the striatum. Ocaperidone, risperidone, and haloperidol readily increased the levels of the dopamine metabolites 3,4-dihydroxybenzene acetic acid and homovanillic acid in the striatum, the nucleus accumbens, the tuberculum olfactorium, and, to some extent, the frontal cortex. Dose-response curve shapes were markedly different; with ocaperidone maximal levels were reached at 0.16 mg/kg and maintained to 10 mg/kg; with risperidone the levels tended to increase continuously up to 10 mg/kg. Haloperidol produced dome-shaped curves (maximum at 0.16-0.63 mg/kg).(ABSTRACT TRUNCATED AT 400 WORDS)

[Non-sedative antihistaminics and binding to central and peripheral H1 histamine receptors]. / Antihistaminiques non-sédatifs et liaison aux récepteurs histaminergiques-H1 centraux et périphériques.

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 0.2 microM. Terfenadine, cetirizine and loratadine bound with 30-, 80- and 100-times lower affinity to H1 receptors. The occupancy of lung and cerebellar H1 receptors was investigated after oral administration of various dosages of the drugs and at several times after drug administration, using ex vivo binding techniques. Astemizole was a very potent compound showing complete differentiation between lung and cerebellar receptor occupation (with 0.63 mg/kg: 70% of lung H1 receptors were occupied, with less than 10% of cerebellar H1 receptor occupancy). A 7-times higher dose of terfenadine was required to induce the same effect. Astemizole produced a rapid and complete occupancy of lung receptors, which was maintained up to 72 h after administration. In contrast, terfenadine produced a peak effect at 1 h and was completely eliminated from lung receptors in 24 h. Loratadine and cetirizine only poorly differentiated between lung and cerebellar receptor occupancy (at 2.5 mg/kg: 70 and 60% of lung receptor occupancy, 50 and 70% of cerebellar receptor occupancy).(ABSTRACT TRUNCATED AT 250 WORDS)

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. [3H]Ketanserin binding in the presence of BW501 was inhibited by the tetrabenazine derivative RO-4-1284 (IC50 = 1.4 nM), tetrabenazine (IC50 = 8.6 nM) and the ketanserin derivatives R 71,278 (IC50 = 6.3 nM), R 47,288 (IC50 = 17 nM) and R 71,428 (IC50 = 100 nM). Ketanserin revealed an IC50 = 32 nM. The drugs were found to trigger the release of 3H from [3H]5-HT-loaded human platelets in superfusion experiments in vitro. The amount of 3H released by the drugs correlated with their binding affinities for the non-serotonergic sites. The non-serotonergic [3H]ketanserin binding sites on human platelets and their possible role in triggering monoamine release corresponded to the properties of non-serotonergic ketanserin binding sites previously characterized in rat striatum. The possible role of the action of ketanserin on the non-serotonergic sites in the reported partial reduction by ketanserin of the monoamine content in cardiovascular tissues is discussed.

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 = 0.9 nM and beta 2/beta 1 ratio = 50). The drug dissociated slowly from these receptor sites. The activity resided in the (S,R,R,R)-enantiomer (R 67 138); the (R,S,S,S)-enantiomer (R 67 145) revealed 175 times lower beta 1-adrenergic binding affinity. Within the series of stereoisomers, nebivolol and R 67 138 showed the best combination of high affinity and selectivity. Among the reference compounds, only CGP 20712-A shared these properties. Nebivolol bound to S1A binding sites with a Ki value of 20 nM. The stereospecific requirements for interaction with these sites were different from those for the beta 1-adrenergic receptor site. S1A binding site affinity was also observed with the potent but nonselective beta-adrenergic blockers carvedilol, pindolol, and propranolol. In the various other investigated radioligand binding and neurotransmitter uptake assays, nebivolol and its stereoisomers showed activity only at micromolar concentrations or were inactive. Clinical studies have shown an interesting hemodynamic profile of nebivolol, offsetting the negative effects on left ventricular performance generally observed with classical beta-adrenergic blockers. Several hypotheses regarding the mechanism of action of nebivolol are summarized.

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 = 0.16 and 0.30 nM, respectively) and a slow dissociation (half-time, 31 and 160 min). In accordance, risperidone (IC50 = 0.5 nM) and ritanserin (IC50 = 1.8 nM) potently blocked serotonin-induced 32P-phosphatidic acid formation in human blood platelets. Risperidone showed, like haloperidol, high binding affinity for dopamine-D2 receptors (Ki = 3.13 and 1.55 nM, respectively) and rapid dissociation (half-time, 2.7 and 5.8 min). Risperidone displayed higher binding affinity than ritanserin and haloperidol for alpha-1 adrenergic (Ki = 0.8 nM), histamine-H1 (Ki = 2.23 nM) and alpha-2 adrenergic receptors (Ki = 7.54 nM). In in vitro superfusion experiments, risperidone and haloperidol reversed at nanomolar concentrations the inhibition by LY 171555 (a dopamine-D2 agonist) and by amphetamine of potassium and electrically evoked release of [3H]acetylcholine from striatal slices (postsynaptic dopamine-D2 effects). Both drugs reversed with similar potency the inhibition by LY 171555 of electrically evoked release of [3H]dopamine (a presynaptic dopamine-D2 effect). Risperidone did not affect the activation by amphetamine of [3H]dopamine efflux from rat striatal slices. Risperidone enhanced at nanomolar concentrations the stimulated [3H]norepinephrine efflux from cortical slices and it similarly reversed the inhibition by clonidine, at concentrations corresponding to its binding affinity for alpha-2 adrenergic receptors. The in vitro biochemical properties of risperidone are in agreement with the reported in vivo pharmacological profile, the relation to clinical findings is discussed.

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. However, these substances also interacted in differing degrees with alpha-1, alpha-2, beta-adrenergic, 5-HT-1, 5-HT-2, and opiate sites. Sulpiride, alizapride, SL 74205, TER 1546 and tiapride were specific for D-2 receptors, but these drugs were active only in the 10(-7)-10(-6) M range. Raclopride, amisulpiride and sultopride showed a 100-1000 differentiation between action on dopamine sites compared with other neurotransmitter receptors. No such selectivity was observed for clebopride or YM 09151-2. Specific substituted benzamides such as alizapride, may be appropriate in high concentrations for defining the interaction of PET ligands with brain dopamine receptors. More potent, but selective, drugs such as raclopride and amisulpiride, may be effective in low concentrations as ligands for labelling dopamine receptor sites. However, the ability of these various substituted benzamide drugs to penetrate into brain and in-vivo to identify dopamine receptors in all brain areas must be assessed.