Molecular modeling of serotonin, ketanserin, ritanserin and their 5-HT2C receptor interactions

Molecular modeling techniques were used to build a three-dimensional model of the rat 5-HT2C receptor, which was used to examine receptor interactions for protonated forms of serotonin, ketanserin and ritanserin. Molecular dynamics simulations which were started with the fluoro benzene moiety of ketanserin and ritanserin oriented towards the cytoplasmic side of the receptor model, produced the strongest antagonist-receptor interactions. The fluoro bezene ring(s) of the antagonists interacted strongly with aromatic residues in the receptor model, which predicts slightly different orientations and ligand-receptor interactions of ketanserin and ritanserin at a putative binding site. The model suggests that Asn333 (transmembrane helix 6) is involved in a hydrogen-bonding interaction with ketanserin, but not with ritanserin. The model also also suggests that the position corresponding to Cys362 (transmembrane helix 7) may be an important determinant for specifying 5-HT2A receptor selectivity in ketanserin binding.     

The Arabidopsis thaliana ACT4/ACT12 actin gene subclass is strongly expressed throughout pollen development

We analyzed the displacement activity of sarpogrelate and its active metabolite (M-1) in the radiolabeled ligand binding to various 5-hydroxytryptamine (5-HT) receptor subtypes using rat brain cortical membranes. Sarpogrelate was shown to have the same affinity as ritanserin for 5-HT2A receptors, with a Ki value of 8.39 nM. The active metabolite of sarpogrelate, M-1, was more active than sarpogrelate itself and of ritanserin, with a Ki value of 1.70 nM. Both sarpogrelate and M-1 had no affinity for 5-HT1A receptors, but these substances, at a concentration of 10 microM, displaced the specific binding to the 5-HT1B receptors of [125I]iodocyanopindolol, resulting in Ki values of 0.881 and 0.859 microM, respectively. The Ki values of sarpogrelate and M-1 are almost the same as that of ritanserin, a specific 5-HT2 receptor antagonist. Sarpogrelate and M-1, as well as ritanserin, are shown to have very low affinity for 5-HT1B receptors. Both sarpogrelate and M-1 had no affinity for 5-HT3 receptor subtypes. In the 5-HT4 receptor binding experiments, sarpogrelate exhibited almost no affinity, while M-1, at the concentration of 10 microM, displaced the binding activity, resulting in a Ki value of 0.838 microM. Both drugs had a weak antagonistic effect on a 5-HT4 receptor-mediated function, i.e., the 5-HT-induced relaxation of rat isolated esophageal tunica muscularis mucosae. In conclusion, sarpogrelate and M-1 have high affinity for 5-HT2A receptors with a relatively high selectivity.     

2-aminotetralin-derived substituted benzamides with mixed dopamine D2, D3, and serotonin 5-HT1A receptor binding properties: a novel class of potential atypical antipsychotic agents

A new chemical class of potential atypical antipsychotic agents, based on the pharmacological concept of mixed dopamine D2 receptor antagonism and serotonin 5-HT1A receptor agonism, was designed by combining the structural features of the 2-(N,N-di-n-propylamino)tetralins (DPATs) and the 2-pyrrolidinylmethyl-derived substituted benzamides in a structural hybrid. Thus, a series of 35 differently substituted 2-aminotetralin-derived substituted benzamides was synthesized and the compounds were evaluated for their ability to compete for [3H]-raclopride binding to cloned human dopamine D2A and D3 receptors, and for [3H]-8-OH-DPAT binding to rat serotonin 5-HT1A receptors in vitro. The lead compound of the series, 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (12a), displayed high affinities for the dopamine D2A receptor (Ki = 3.2 nM), the dopamine D3 receptor (Ki = 0.58 nM) as well as the serotonin 5-HT1A receptor (Ki = 0.82 nM). The structure-affinity relationships of the series suggest that the 2-aminotetralin moieties of the compounds occupy the same binding sites as the DPATs in all three receptor subtypes. The benzamidoethyl side chain enhances the affinities of the compounds for all three receptor subtypes, presumably by occupying an accessory binding site. For the dopamine D2 and D3 receptors, this accessory binding site may be identical to the binding site of the 2-pyrrolidinylmethyl-derived substituted benzamides.     

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.     

Sequence and functional analysis of cloned guinea pig and rat serotonin 5-HT1D receptors: common pharmacological features within the 5-HT1D receptor subfamily

This study was undertaken to investigate the pharmacology of cloned guinea pig and rat 5-hydroxytryptamine (serotonin; 5-HT)1D receptor sites. Guinea pig, rat, and mouse 5-HT1D receptor genes were cloned, and their amino acid sequences were compared with those of the human, dog, and rabbit. The overall amino acid sequence identity between these 5-HT1D receptors is high and varies between 86 and 99%. The sequence homology is slightly more divergent (13-27%) in the N-terminal extracellular region of these 5-HT1D receptors. Guinea pig and rat 5-HT1D receptors, stably and separately expressed in rat C6 glial cells, are negatively coupled to cyclic AMP formation upon stimulation with agonists, as previously found for cloned human 5-HT1D receptor sites. The cyclic AMP data show some common pharmacological features for the 5-HT1D receptors of guinea pig, rat, and human: an almost similar rank order of potency for the investigated 5-HT1D receptor agonists, stereoselectivity for the binding affinity and agonist potency of R(+)-8-hydroxy-2-(di-n-propylamino)tetralin, and equal 5-HT1D receptor-mediated antagonist potency for methiothepin and the 5-HT2 receptor antagonists ritanserin and ketanserin. In conclusion, the pharmacology of the cloned 5-HT1D receptor subtype seems, unlike the 5-HT1B receptor subtype, conserved among various mammal species such as the human, guinea pig, and rat.     

2-(1-Naphthyloxy)ethylamines with enhanced affinity for human 5-HT1D beta (h5-HT1B) serotonin receptors

Although the beta-adrenergic antagonist propranolol (1) binds at rodent 5-HT1B serotonin receptors, it displays low affinity (Ki > 10,000 nM) for its species homologue 5-HT1D beta (i.e., h5-HT1B) receptors. The structure of propranolol was systematically modified in an attempt to enhance its affinity for the latter population of receptors. Removal of the alkyl hydroxyl group, shortening of the O-alkyl chain from three to two methylene groups, and variation of the terminal amine substituent resulted in compounds, such as N-monomethyl-2-(1-naphthyloxy)-ethylamine (11; Ki = 26 nM), that display significantly higher h5-HT1B affinity than propranolol. Compound 11 was shown to bind equally well at human 5-HT1D alpha (h5-HT1D) receptors (Ki = 34 nM) and was further demonstrated to possess h5-HT1B agonist character in an adenylate cyclase assay. It would appear that such (aryloxy)alkylamines may represent a novel class of 5-HT1D receptor agonists.     

The binding of propranolol at 5-hydroxytryptamine1D beta T355N mutant receptors may involve formation of two hydrogen bonds to asparagine

Although the beta-adrenergic receptor antagonist (-)-propranolol binds with relatively low affinity at human 5-hydroxytryptamine1D beta receptors (Ki = 10,200 nM), it displays significantly higher affinity (Ki = 17 nM) at its species homolog, 5-HT1B receptors, and at a mutant 5-HT1D beta receptor (Ki = 16 nM), where the threonine residue at position 355 (T355) is replaced with an asparagine residue (i.e., a T355N mutant). Propranolol contains two oxygen atoms, an ether oxygen atom and a hydroxyl oxygen atom, and it has been speculated that the enhanced affinity of propranolol for the T355N mutant receptor is related to the ability of the asparagine residue to hydrogen bond with the ether oxygen atom. However, the specific involvement of the propranolol oxygen atoms in binding to the wild-type and T355N mutant 5-HT1D beta receptors has never been addressed experimentally. A modification of a previously described 5-HT1D beta receptor graphic model was mutated by replacement of T355 with asparagine. Propranolol was docked with the wild-type and T355N mutant 5-HT1D beta receptor models in an attempt to understand the difference in affinity of the ligand for the receptors. The binding models suggest that the asparagine residue of the mutant receptor can form hydrogen bonds with both oxygen atoms of propranolol, whereas the threonine moiety of the wild-type receptor can hydrogen-bond only to one oxygen atom. To test this hypothesis, we prepared and examined several analogues of propranolol that lacked either one or both oxygen atoms. The results of radioligand binding experiments are consistent with the hypothesis that both oxygen atoms of propranolol could participate in binding to the mutant receptor, whereas only the ether oxygen atom participates in binding to the wild-type receptor. As such, this is the first investigation of serotonin receptors that combines the use of molecular modeling, mutant receptors generated by site-directed mutagenesis, and synthesis to investigate structure/affinity relationships.     

Detailed mapping of serotonin 5-HT1B and 5-HT1D receptor messenger RNA and ligand binding sites in guinea-pig brain and trigeminal ganglion: clues for function

The similar pharmacology of the 5-HT1B and 5-HT1D receptors, and the lack of selective compounds sufficiently distinguishing between the two receptor subtypes, have hampered functional studies on these receptors. In order to provide clues for differential functional roles of the two subtypes, we performed a parallel localization study throughout the guinea-pig brain and the trigeminal ganglia by means of quantitative in situ hybridization histochemistry (using [35S]-labelled riboprobes probes for receptor messenger RNA) and receptor autoradiography (using a new radioligand [3H]alniditan). The anatomical patterns of 5-HT1B and 5-HT1D receptor messenger RNA were quite different. While 5-HT1B receptor messenger RNA was abundant throughout the brain (with highest levels in the striatum, nucleus accumbens, olfactory tubercle, cortex, hypothalamus, hippocampal formation, amygdala, thalamus, dorsal raphe and cerebellum), 5-HT1D receptor messenger RNA exhibited a more restricted pattern; it was found mainly in the olfactory tubercle, entorhinal cortex, dorsal raphe, cerebellum, mesencephalic trigeminal nucleus and in the trigeminal ganglion. The density of 5-HT(1B/1D) binding sites (combined) obtained with [3H]alniditan autoradiography was high in the substantia nigra, superior colliculus and globus pallidus, whereas lower levels were detected in the caudate-putamen, hypothalamus, hippocampal formation, amygdala, thalamus and central gray. This distribution pattern was indistinguishable from specific 5-HT1B receptor labelling in the presence of ketanserin under conditions to occlude 5-HT1D receptor labelling; hence the latter were below detection level. Relationships between the regional distributions of the receptor messenger RNAs and binding sites and particular neuroanatomical pathways are discussed with respect to possible functional roles of the 5-HT1B and 5-HT1D receptors.     

Differential distribution of [3H]sumatriptan binding sites (5-HT1B, 5-HT1D and 5-HT1F receptors) in human brain: focus on brainstem and spinal cord

We report on the autoradiographic distribution of 5-HT1B, 5-HT1D and 5-HT1F receptor subtypes in human brain, focusing on the brainstem and cervical spinal cord. We have used [3H]sumatriptan as a radioligand in the presence of suitable concentrations of 5-CT (5-carboxamidotryptamine) to define 5-HT1F receptors, and ketanserin, to discriminate between 5-HT1B and 5-HT1D receptors. In the brainstem the highest concentrations of [3H]sumatriptan binding sites were seen in substantia nigra. The spinal trigeminal nucleus, substantia gelatinosa of the spinal cord, nucleus of the tractus solitarius and periaqueductal grey, also showed significant levels of [3H]sumatriptan binding sites. In the brainstem and spinal cord the total population of 5-CT-insensitive receptors, corresponding to 5-HT1F receptors, ranged from 9.8% in the periaqueductal grey to 53.4% in the substantia gelatinosa. This population represented 67.0% of binding in layer V of the frontal cortex. The decrease in [3H]sumatriptan binding in the presence of 200 nM ketanserin, indicative of the presence of 5-HT1D receptors, was very limited throughout the human brain, only reaching 20% of total specific binding over the periaqueductal grey. The proportion of [3H]sumatriptan binding sites displaced by 5-CT and insensitive to ketanserin, corresponding to 5-HT1B receptors, was, in general, the most abundant, ranging from 43.8% in substantia gelatinosa to 69.9% in the periaqueductal grey. Significant levels of 5-HT1B and 5-HT1D receptors found in migraine control pain areas suggest their involvement in antinociceptive mechanisms.     

Survey on the pharmacodynamics of the new antipsychotic risperidone

This review reports on the pharmacodynamics of the new antipsychotic risperidone. The primary action of risperidone is serotonin 5-HT2 receptor blockade as shown by displacement of radioligand binding (Ki: 0.16 nM), activity on isolated tissues (EC50: 0.5 nM), and antagonism of peripherally (ED50: 0.0011 mg/kg) and centrally (ED50: 0.014 mg/kg) acting 5-HT2 receptor agonists in rats. Risperidone is at least as potent as the specific 5-HT2 receptor antagonist ritanserin in these tests. Risperidone is also a potent dopamine D2 receptor antagonist as indicated by displacement of radioligand binding (Ki: 1.4 nM), activity in isolated striatal slices (IC50: 0.89 nM), and antagonism of peripherally (ED50: 0.0057 mg/kg in dogs) and centrally acting D2 receptor agonists (ED50: 0.056-0.15 mg/kg in rats). Risperidone shows all effects common to D2 antagonists, including enhancement of prolactin release. However, some central effects such as catalepsy and blockade of motor activity occur at high doses only. Risperidone is 4-10 times less potent than haloperidol as a central D2 antagonist in rats and it differs from haloperidol by the following characteristics: predominant 5-HT2 antagonism; LSD antagonism; effects on sleep; smooth dose-response curves for D2 antagonism; synergism of combined 5-HT2/D2 antagonism; pronounced effects on amphetamine-induced oxygen consumption; increased social interaction; and pronounced effects on dopamine (DA) turnover. Risperidone displays similar activity at pre- and postsynaptic D2 receptors and at D2 receptors from various rat brain regions. The binding affinity for D4 and D3 receptors is 5 and 9 times weaker, respectively, than for D2 receptors; interaction with D1 receptors occurs only at very high concentrations. The pharmacological profile of risperidone includes interaction with histamine H1 and alpha-adrenergic receptors but the compound is devoid of significant interaction with cholinergic and a variety of other types of receptors. Risperidone has excellent oral activity, a rapid onset, and a 24-h duration of action. Its major metabolite, 9-hydroxyrisperidone, closely mimics risperidone in pharmacodynamics. Risperidone can be characterized as a potent D2 antagonist with predominant 5HT2 antagonistic activity and optimal pharmacokinetic properties.     

Comparative receptor mapping of serotoninergic 5-HT3 and 5-HT4 binding sites

The clinical use of currently available drugs acting at the 5-HT4 receptor has been hampered by their lack of selectivity over 5-HT3 binding sites. For this reason, there is considerable interest in the medicinal chemistry of these serotonin receptor subtypes, and significant effort has been made towards the discovery of potent and selective ligands. Computer-aided conformational analysis was used to characterize serotoninergic 5-HT3 and 5-HT4 receptor recognition. On the basis of the generally accepted model of the 5-HT3 antagonist pharmacophore, we have performed a receptor mapping of this receptor binding site, following the active analog approach (AAA) defined by Marshall. The receptor excluded volume was calculated as the union of the van der Waals density maps of nine active ligands (pKi > or = 8.9), superimposed in pharmacophoric conformations. Six inactive analogs (pKi < 7.0) were subsequently used to define the essential volume, which in its turn can be used to define the regions of steric intolerance of the 5-HT3 receptor. Five active ligands (pKi > or = 9.3) at 5-HT4 receptors were used to construct an antagonist pharmacophore for this receptor, and to determine its excluded volume by superimposition of pharmacophoric conformations. The volume defined by the superimposition of five inactive 5-HT4 receptor analogs that possess the pharmacophoric elements (pKi < or = 6.6) did not exceed the excluded volume calculated for this receptor. In this case, the inactivity may be due to the lack of positive interaction of the amino moiety with a hypothetical hydrophobic pocket, which would interact with the voluminous substituents of the basic nitrogen of active ligands. The difference between the excluded volumes of both receptors has confirmed that the main difference is indeed in the basic moiety. Thus, the 5-HT3 receptor can only accommodate small substituents in the position of the nitrogen atom, whereas the 5-HT4 receptor requires more voluminous groups. Also, the basic nitrogen is located at ca. 8.0 A from the aromatic moiety in the 5-HT4 antagonist pharmacophore, whereas this distance is ca. 7.5 A in the 5-HT3 antagonist model. The comparative mapping of both serotoninergic receptors has allowed us to confirm the three-component pharmacophore accepted for the 5-HT3 receptor, as well as to propose a steric model for the 5-HT4 receptor binding site. This study offers structural insights to aid the design of new selective ligands, and the resulting models have received some support from the synthesis of two new active and selective ligands: 24 (Ki(5-HT3) = 3.7 nM; Ki(5-HT4) > 1000 nM) and 25 (Ki(5-HT4) = 13.7 nM; Ki(5-HT3) > 10,000 nM).     

The pharmacological characterization of a novel selective 5-hydroxytryptamine1A receptor antagonist, NAD-299

The pharmacological properties of a novel selective 5-hydroxytryptamine1A (5-HT1A) receptor antagonist, NAD-299 [(R)-3-N,N-dicyclobutylamino-8-fluoro-3,4-dihydro-2H-1-benzopyran-5-carboxamide hydrogen (2R,3R)-tartrate monohydrate] were examined in vitro and in vivo and compared with the reference 5-HT1A receptor antagonist, WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazin-yl))ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride]. The new compound had high affinity for 5-HT1A receptors in vitro with a Ki value of 0.6 nM. The only other receptors for which NAD-299 had affinity less than 1 microM were alpha-1 and beta adrenoceptors with Ki values of 260 and 340 nM, respectively. Thus, the selectivity of NAD-299 for 5-HT1A receptors was more than 400 times. WAY-100635 had considerably higher affinity than NAD-299 for alpha-1 adrenoceptors (Ki = 45 nM) and dopamine D2 and D3 receptors (Ki = 79 and 67 nM, respectively). Like WAY-100635, NAD-299 competitively blocked 5-HT-induced inhibition of vasoactive intestinal peptide-stimulated cAMP production in GH4ZD10 cells and had no intrinsic activity. Both compounds were therefore 5-HT1A receptor antagonists in vitro and also behaved as such in in vivo experiments. Thus, they competitively antagonized the 8-hydroxy-2-(di-n-propylamino)tetralin-induced 5-HT behavioral effects, hypothermia, corticosterone secretion and inhibition of passive avoidance behavior without causing any actions of their own. The effective dose of NAD-299 varied between 0.03 and 0.35 micromol/kg s.c., depending on the test and the dose of 8-hydroxy-2-(di-n-propylamino)tetralin.     

Alpha 1-adrenergic receptor subtype determinants for 4-piperidyl oxazole antagonists

Mutational studies in conjunction with ligand binding assays were used to examine the basis of alpha1-adrenergic receptor subtype selectivity for a series of 4-piperidyloxazole antagonists. A set of chimeric alpha 1A receptors were created by systematically substituting individual transmembrane domains from alpha 1D adrenergic receptors. The oxazole antagonists exhibited significant reductions in affinity against the receptor construct alpha 1A/D(TM2), and moderate reductions in affinity versus constructs alpha 1A/D(TM5), alpha 1A/B(TM5), and alpha 1A/D(TM6). Antagonist affinities for these chimeras exceeded those found for wild type alpha 1D and alpha 1B. Site-directed mutagenesis methods were then used to explore the role that individual residues in TM2 and TM5 play in ligand binding affinity and selectivity. These studies revealed that mutations at position 86 in the second transmembrane domain and position 185 in the fifth transmembrane domain of the alpha 1A receptor have a major impact on receptor subtype selectivity.     

5-HT1B receptor antagonist properties of novel arylpiperazide derivatives of 1-naphthylpiperazine

A new series of arylpiperazide derivatives of 1-naphthylpiperazine of general formula 4 has been prepared and evaluated as 5-HT1B antagonists. Binding experiments at cloned human 5-HT1A, 5-HT1B, and 5-HT1D receptors show that these derivatives are potent and selective ligands for 5-HT1B/1D subtypes with increased binding selectivity versus the 5-HT1A receptor when compared to 1-naphthylpiperazine (1-NP). Studies of inhibition of the forskolin-stimulated cAMP formation mediated by the human 5-HT1B receptor demonstrate that the nature of the arylpiperazide substituent modulates the intrinsic activity of these 1-NP derivatives. Among them, 2-[[8-(4-methylpiperazin-1-yl)naphthalen-2-yl]oxy] -1-(4-o-tolylpiperazin-1-yl)ethanone (4a) was identified as a potent neutral 5-HT1B antagonist able to antagonize the inhibition of 5-HT release induced by 5-CT (5-carbamoyltryptamine) in guinea pig hypothalamus slices. Moreover, 4a was found to potently antagonize the hypothermia induced by a selective 5-HT1B/1D agonist in vivo in the guinea pig following oral administration (ED50 = 0.13 mg/kg).     

5-HT autoreceptors in the regulation of 5-HT release from guinea pig raphe nucleus and hypothalamus

5-HT autoreceptors involved in the regulation of 5-HT release in the guinea pig dorsal raphe nucleus have been studied in comparison with those in the hypothalamus. In vitro release was measured in slices of raphe and hypothalamus prelabelled with [3H]5-HT, superfused with Krebs solution and depolarized electrically. The non-selective 5-HT receptor agonist, 5-carboxamidotryptamine (5-CT) (0.1-10 nM for raphe: 1-100 nM for hypothalamus) and antagonist, methiothepin (10-1000nM), decreased and increased, respectively, the release of [3H]5-HT evoked by electrical stimulation in either of these regions when given alone. The selective 5-HT1B/D receptor antagonist, GR127935 (100-1000 nM), and the 5-HT1D receptor antagonist, ketanserin (300-1000 nM), had no significant effect on this release in either of these regions. Methiothepin and GR127935 (100-1000 nM) shifted to the right the concentration-effect curve of 5-CT in both the raphe and the hypothalamus. At 300 nM, ketanserin shifted to the right the concentration-effect curve of 5-CT in the raphe but did not modify the 5-CT curve in the hypothalamus. In microdialysis experiments ketanserin, applied locally at 10 microM, increased the extracellular levels of 5-HT in the dorsal raphe nucleus of the freely moving guinea pig, whereas 5-HT levels were unchanged in the hypothalamus. Ketanserin at 1 microM did not affect the decrease in 5-HT output induced by the selective 5-HT1B/D receptor agonist, naratriptan (used at 10 microM in raphe and 0.1 microM in hypothalamus), in the raphe or the hypothalamus. In the raphe, WAY100635, a 5-HT1A receptor antagonist, at 1 microM, did not prevent naratriptan (10 microM) from reducing the extracellular levels of 5-HT. These results suggest that, in the conditions used in this study, the release of 5-HT in the dorsal raphe nucleus is possibly modulated in part by 5-HT1B receptors but essentially the control is through 5-HT receptors whose subtype is still to be determined. In the hypothalamus, however, it is clear that only 5-HT1B receptors are involved in the modulation of 5-HT neurotransmission.     

Head and whole-body jerking in guinea pigs are differentially modulated by 5-HT1A, 5-HT1B/1D and 5-HT2A receptor antagonists

The effects of a serotonin (5-HT) releasing drug, p-chloroamphetamine, on plasma glucose levels were investigated in rats. p-Chloroamphetamine elicited a significant hyperglycemia. The hyperglycemic effects of p-chloroamphetamine were completely prevented by the 5-HT synthesis inhibitor, p-chlorophenylalanine. Prior adrenodemedullation abolished the hyperglycemia elicited by p-chloroamphetamine. p-Chloroamphetamine-induced hyperglycemia was prevented by methysergide, which blocks the 5-HT1 and 5-HT2 receptor, the 5-HT1A/1B/2C receptor antagonist, (-)-propranolol, the selective 5-HT1A receptor antagonist, 4-(2'-methoxyphenyl-1-[2'-n-2"pyridinyl)-p-iodobenzamido]-ethyl-pi perazine (p-MPPI), the 5-HT2A/2B/2C receptor antagonists, ritanserin and 4-isopropyl-7-methyl-9-(2-hydroxy-1-methyl-propoxycarbonyl)-4,6A,7 ,8,9,10,10A-octahydro-indolo[4,3-FG]quinolone maleate(LY 53857). However, the 5-HT3 and 5-HT4 receptor antagonist, tropisetron, the 5-HT4 receptor antagonist, 2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino) ethyl ester (SDZ 205-557), and the 5-HT2A receptor antagonist, ketanserin, did not affect the p-chloroamphetamine-induced hyperglycemia. These results suggest that p-chloroamphetamine-induced hyperglycemia is elicited by an enhanced 5-HT release and facilitated adrenaline release. Moreover, our results indicate that p-chloroamphetamine-induced hyperglycemia is mediated by 5-HT1A and 5-HT2B/2C receptors.     

Synthesis and structure-activity relationships of 3,7-dimethyl-1-propargylxanthine derivatives, A2A-selective adenosine receptor antagonists

A series of 8-substituted derivatives of 3,7-dimethyl-1-propargylxanthine (DMPX) was synthesized and investigated as A2A adenosine receptor antagonists. Different synthetic strategies for the preparation of DMPX derivatives and analogues were explored. A recently developed synthetic procedure starting from 3-propargyl-5,6-diaminouracil proved to be the method of choice for the preparation of this type of xanthine derivatives. The novel compounds were investigated in radioligand binding studies at the high-affinity adenosine receptor subtypes A1 and A2A and compared with standard A2A adenosine receptor antagonists. Structure-activity relationships were analyzed in detail. 8-Styryl-substituted DMPX derivatives were identified that exhibit high affinity and selectivity for A2A adenosine receptors, including 8-(m-chlorostyryl)-DMPX (CS-DMPX, Ki A2A = 13 nM, 100-fold selective), 8-(m-bromostyryl)-DMPX (BS-DMPX, Ki A2A = 8 nM, 146-fold selective), and 8-(3,4-dimethoxystyryl)-DMPX (Ki A2A = 15 nM, 167-fold selective). These and other novel compounds are superior to the standard A2A adenosine receptor antagonists KF17837 (4) and CSC (5) with respect to A2A affinity and/or selectivity.     

Functional characterisation of the human cloned 5-HT7 receptor (long form); antagonist profile of SB-258719

1. The functional profile of the long form of the human cloned 5-HT7 receptor (designated h5-HT7(a)) was investigated using a number of 5-HT receptor agonists and antagonists and compared with its binding profile. Receptor function was measured using adenylyl cyclase activity in washed membranes from HEK293 cells stably expressing the recombinant h5-HT7(a) receptor. 2. The receptor binding profile, determined by competition with [3H]-5-CT, was consistent with that previously reported for the h5-HT7(a) receptor. The selective 5-HT7 receptor antagonist SB-258719 ((R)-3,N-Dimethyl-N-[1-methyl-3-(4-methylpiperidin-1-yl)propyl]ben zene sulfonamide) displayed high affinity (pKi 7.5) for the receptor. 3. In the adenylyl cyclase functional assay, 5-CT and 8-OH-DPAT were both full agonists compared to 5-HT and the rank order of potency for agonists (5-CT > 5-HT > 8-OH-DPAT) was the same in functional and binding studies. 4. Risperidone, methiothepin, mesulergine, clozapine, olanzapine, ketanserin and SB-258719 antagonised surmountably 5-CT-stimulated adenylyl cyclase activity. Schild analysis of the antagonism by SB-258719 gave a pA2 of 7.2+/-0.2 and slope not significantly different from 1, consistent with competitive antagonism. 5. The same antagonists also inhibited basal adenylyl cyclase activity with a rank order of potency in agreement with those for antagonist potency and binding affinity. Both SB-258719 and mesulergine displayed apparent partial inverse agonist profiles compared to the other antagonists tested. These inhibitory effects of antagonists appear to be 5-HT7 receptor-mediated and to reflect inverse agonism. 6. It is concluded that in this expression system, the h5-HT7(a) receptor shows the expected binding and functional profile and displays constitutive activity, revealing inverse agonist activity for a range of antagonists.     

Structure functional expression and spatial distribution of a cloned cDNA encoding a rat 5-HT1D-like receptor

Using polymerase chain reaction (PCR) a complementary DNA (cDNA) encoding a 5-hydroxytryptamine (5-HT) receptor was isolated from rat forebrain. The amplified cDNA specifies an open reading frame of 374 amino acids comprising seven putative transmembrane regions. Expression of the cloned cDNA in human embryonic kidney cells (HEK 293) was used to establish the pharmacological profile of the encoded receptor polypeptide. Membranes containing the cloned receptor showed high affinity binding of [3H]-5-HT. Competition binding experiments with a variety of serotonin receptor ligands displayed a rank order of affinities corresponding to a 5-HT1D subtype: 5-CT > 5-HT = metergoline > CGS 12066 > methysergide > sumatriptan > mianserin = (-)alpha-Me-5-HT = yohimbine > 8-OH-DPAT > or = rauwolscine > spiperone > DOI > propranolol > or = 2-Me-5-HT > or = ICS 205930. Ketanserin and ritanserin displaced [3H]-5-HT-binding in a biphasic manner. In situ hybridization revealed highest expression of the corresponding mRNA in the pyramidal layer of the olfactory tubercle and the nucleus caudatus and accumbens.     

Trazodone is a potent agonist at 5-HT2C receptors mediating inhibition of the N-methyl-D-aspartate/nitric oxide/cyclic GMP pathway in rat cerebellum

The effects of trazodone on the cyclic GMP elevation elicited by N-methyl-D-aspartate in rat cerebellar slices were analyzed. Trazodone inhibited in a concentration-dependent manner (EC50 = 0.82 nM) the cyclic GMP response evoked by 0.1 microM N-methyl-D-aspartate. The inhibition was near complete at 10 nM trazodone. The effect of 10 nM trazodone was unaffected by 0.3 microM spiperone or rauwolscine, antagonists with selectivity for the 5-HT(serotonin)2A or the 5-HT2B subtype, respectively, but it was totally prevented by 0.01 microM mesulergine, a 5-HT2A/5-HT2B/5-HT2C receptor antagonist. Trazodone was potently counteracted (IC50 = 2.7 nM) by the selective 5-HT2B/5-HT2C receptor antagonist N-(1-methyl-5-indolyl)-N-(3-pyridil) urea HCl and, less potently (IC50 = 95 nM), by ketanserin, a 5-HT2A/5-HT2C receptor blocker. It is concluded that trazodone behaves as a potent full agonist at the 5-HT2C receptor mediating inhibition of the cerebellar N-methyl-D-aspartate/nitric oxide/cyclic GMP system.