Novel 5-HT(1A/1B/1D) receptors antagonists with potent 5-HT reuptake inhibitory activity

Novel 2-methyl-5-quinolinyl-1-piperazinylalkyl-3,4-dihydro-2H-1,4-benzoxazin-3-ones showing high affinities for the 5-HT(1A/1B/1D) receptors coupled with potent 5-HT reuptake inhibitory activity have been discovered. This is the first report describing docking of the lead compound 6-{2-[4-(2-methyl-5-quinolinyl)-1-piperazinyl]ethyl}-2H-1,4-benzoxazin-3(4H)-one 1, into a model of the 5-HT transporter and the 5-HT(1A) receptor model.     

New 5-HT1A receptor agonists possessing 1,4-benzoxazepine scaffold exhibit highly potent anti-ischemic effects

A series of new 3-substituted-4-(4-aminobutyl)-1,4-benzoxazepin-5(4H)-one derivatives (1-5) which showed a very high affinity for 5-HT1A receptor with good selectivity over dopamine D2 receptor was synthesized. Among these compounds, 3-chloro-4-[4-[4-(2-pyridinyl)-1,2,3,6-tetrahydropyridin-1-yl]butyl]-1,4-benzoxazepin-5(4H)-one (5: SUN N4057) exhibited remarkable neuroprotective activity in a transient middle cerebral artery occlusion (t-MCAO) model.     

Hypothalamic 5-HT functional regulation through 5-HT1A and 5-HT2C receptors during pancreatic regeneration

5-HT receptors are predominantly located in the brain and are involved in pancreatic function and cell proliferation through sympathetic nervous system. The objective of this study was to investigate the role of hypothalamic 5-HT, 5-HT1A and 5-HT2C receptor binding and gene expression in rat model of pancreatic regeneration using 60% pancreatectomy. The pancreatic regeneration was evaluated by 5-HT content, 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus of sham operated, 72 h and 7 days pancreatectomised rats. 5-HT content was quantified by HPLC. 5-HT1A receptor assay was done by using specific agonist [3H]8-OH DPAT. 5-HT2C receptor assay was done by using specific antagonist [3H]mesulergine. The expression of 5-HT1A and 5-HT2C receptor gene was analyzed by RT-PCR. 5-HT content was higher in the hypothalamus of 72 h pancreatectomised rats. 5-HT1A and 5-HT2C receptors were down-regulated in the hypothalamus. RT-PCR analysis revealed decreased 5-HT1A and 5-HT2C receptor mRNA expression. The 5-HT1A and 5-HT2C receptors gene expression in the 7 days pancreatectomised rats reversed to near sham level. This study is the first to identify 5-HT1A and 5-HT2C receptor gene expression in the hypothalamus during pancreatic regeneration in rats. Our results suggest the hypothalamic serotonergic receptor functional regulation during pancreatic regeneration.     

1-Aryl-4-(4-succinimidobutyl)piperazines and their conformationally constrained analogues: synthesis, binding to serotonin (5-HT1A, 5-HT2A, 5-HT7), alpha1-adrenergic, and dopaminergic D2 receptors, and in vivo 5-HT1A functional characteristics

Starting with the structure of potent 5-HT(1A) ligands, that is, MM77 [1-(2-methoxyphenyl)-4-(4-succinimidobutyl)piperazine, 4] and its constrained version 5 (MP349), previously obtained in our laboratory, a series of their direct analogues with differently substituted aromatic ring (R=H, m-Cl, m-CF(3), m-OCH(3), p-OCH(3)) were synthesized. The flexible and the corresponding 1e,4e-disubstituted cyclohexane derivatives were designed in order to investigate the influence of rigidification on 5-HT(1A) affinity, selectivity for 5-HT(2A), 5-HT(7), D(1), and D(2) binding sites and functional profile at pre- and postsynaptic 5-HT(1A) receptors. The new compounds 19-25 were found to be highly active 5-HT(1A) receptor ligands (K(i)=4-44 nM) whereas their affinity for other receptors was: either significantly decreased after rigidification (5-HT(7)), or controlled by substituents in the aromatic ring (alpha(1)), or influenced by both those structural modifications (5-HT(2A)), or very low (D(2), K(i)=5.3-31 microM). Since a distinct disfavor towards rigid compounds was observed for 5-HT(7) receptors only, it seems that the bioactive conformation of chain derivatives at those sites should differ from the extended one. Several in vivo models were used to asses functional activity of 19-25 at pre- (hypothermia in mice) and postsynaptic 5-HT(1A) receptors (lower lip retraction in rats and serotonin syndrome in reserpinized rats). Unlike the parent antagonists 4 and 5, all the new derivatives tested were classified as partial agonists with different potency, however, similar effects were observed within pairs (flexible and rigid) of the analogues. The obtained results indicated that substitution in the aromatic ring, but not spacer rigidification, controls the 5-HT(1A) functional activity of the investigated compounds. Moreover, an o-methoxy substituent in the structure of 5 seems to be necessary for its full antagonistic properties. Of all the new compounds studied, trans-4-(4-succinimidocyclohexyl)-1-(3-trifluoromethylphenyl)piperazine 24 was the most potent 5-HT(1A) receptor ligand in vitro (K(i)=4 nM) and in vivo, with at least 100-fold selectivity for the other receptors tested.     

Dual 5-HT1A agonists and 5-HT re-uptake inhibitors by combination of indole-butyl-amine and chromenonyl-piperazine structural elements in a single molecular entity

The dual serotonin (5-HT) re-uptake inhibitor and 5-HT(1A) receptor agonist vilazodone was found to increase central serotonin levels in rat brain. In the course of structural modifications of vilazodone 3-[4-[4-(2-oxo-2H-1-benzopyran-6-yl)-1-piperazinyl]-butyl]-1H-indole-5-carbonitrile 8i and its fluorine analogue 6-[4-[4-(5-fluor-3-indolyl)-butyl]-1-piperazinyl]-2H-1-benzopyran-2-one have been identified. These unsubstituted chromenones are equally potent at the 5-HT(1A) receptor and 5-HT transporter. The implementation of nitrogen functionalities in position 3 of the chromenones resulted in compounds acting as agonists at the 5-HT(1A) receptor and as 5-HT re-uptake inhibitors like vilazodone. Ex vivo 5-HT re-uptake inhibition and in vitro 5-HT agonism were determined in the PCA- and GTPgammaS-assay, respectively. The potential of these chromenones to increase central 5-HT levels was measured in microdialysis studies and especially the derivatives 3-[4-[4-(3-amino-2-oxo-2H-chromen-6-yl)-piperazin-1-yl]-butyl]-1H-indole-5-carbonitrile 8f, ethyl (6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-carbamate 8h and N-(6-[4-[4-(5-cyano-1H-indol-3-yl)-butyl]-piperazin-1-yl]-2-oxo-2H-chromen-3-yl)-acetamide 8k give rise to rapid development of increased serotonin levels in rat brain cortex, lasting longer than 3h.     

The Influence of Gender and Age on Neonatal Rat Hypothalamic 5-HT1A and 5-HT2A Receptors

1.Rat hypothalamic 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) concentrations are transiently sexually differentiated in the second week postpartum (pp), with higher levels in the female. In this report we investigate the possibility that 5-HT receptors may also exhibit sexual dimorphism in the neonatal period.2.5-HT1A and 5-HT2A receptors were quantitated by radioligand binding of [³H]ketanserin and [³H]8-OH DPAT, respectively, in hypothalamus and amygdala from male and female rats at days 8–16 pp.3.There was no sexual dimorphism or change in the density of 5-HT2A binding in hypothalamus or amygdala over days 8–16 pp. There was also no sexual dimorphism of 5-HT1A receptors.4.There was an increase in 5-HT1A receptor density in both the hypothalamus and the amygdala. In the hypothalamus, but not the amygdala, this increase was interrupted on day 14 by a decrease in 5-HT1A receptors, which we suggest may be of physiological significance in modifying the eventual pattern of adult agonistic activity.5.The results suggest that the sexual dimorphism in 5-HT turnover is predominantly presynaptic, relating to altered synthesis and/or release, and is not of sufficient magnitude or duration to produce adaptive responses in postsynaptic 5-HT1A or 5-HT2A receptors.     

Novel, flexible, and conformationally defined analogs of gepirone: synthesis and 5-HT1A, 5-HT2A, and D2 receptor activity

Novel, flexible arylpiperazine gepirone analogs (1a-3a) with a mixed 5-HT1A/5-HT2A receptor profile, low D2 receptor affinity, and agonistic (2a) or partial agonistic (1a, 3a) activity toward 5-HT1A receptor sites were synthesized. Their conformationally restricted counterparts (1b-3b) were selective 5-HT1A ligands (over 5-HT2A and D2 receptors), which turned out to be agonists (2b, 3b), or partial agonist (1b) of 5-HT1A receptors.     

Synthesis, SAR studies, and evaluation of 1,4-benzoxazepine derivatives as selective 5-HT1A receptor agonists with neuroprotective effect: Discovery of Piclozotan

A new series of 1,4-benzoxazepine derivatives was designed, synthesized, and evaluated for binding to 5-HT1A receptor and cerebral anti-ischemic effect. A lot of compounds exhibited nanomolar affinity for 5-HT1A receptor with good selectivity over both dopamine D2 and alpha1-adrenergic receptors. Among these compounds, 3-chloro-4-[4-[4-(2-pyridinyl)-1,2,3,6-tetrahydropyridin-1-yl]butyl]-1, 4-benzoxazepin-5(4H)-one (50: SUN N4057 (Piclozotan) as 2HCl salt) showed remarkable neuroprotective activity in a transient middle cerebral artery occlusion (t-MCAO) model.     

Synthesis and SAR of 3- and 4-substituted quinolin-2-ones: discovery of mixed 5-HT(1B)/5-HT(2A) receptor antagonists

Quinolin-2-ones bearing a heteroaryl-piperazine linked by a two carbon chain at the 3- or 4-position were synthesised and evaluated as mixed 5-HT(1B)/5-HT(2A) receptor antagonists. Potent mixed antagonists were obtained with thieno[3,2-c]pyridine derivatives. In this series, compound 2.1 (SL 65.0472) proved to be functional antagonist at both the 5-HT(2A) receptor (rat in vivo 5-HT-induced hypertension model) and the 5-HT(1B) receptor (dog in vitro saphenous vein assay).     

In vivo efflux of serotonin in the dorsal raphe nucleus of 5-HT1A receptor knockout mice

In the dorsal raphe nucleus (DR), extracellular serotonin (5-HT) regulates serotonergic transmission through 5-HT1A autoreceptors. In this work we used in vivo microdialysis to examine the effects of stressful and pharmacological challenges on DR 5-HT efflux in 5-HT1A receptor knockout (5-HT1A-/-) mice and their wild-type counterparts (5-HT1A+/+). Baseline 5-HT concentrations did not differ between both lines of mice, which is consistent with a lack of tonic control of 5-HT1A autoreceptors on DR 5-HT release. (R)-(+)-8-Hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT, 0.5 mg/kg) reduced 5-HT levels to 30% of basal values in 5-HT1A+/+ mice, but not in 5-HT1A-/- mice. The selective 5-HT1B receptor agonist 1,4-dihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo[3,2-b]pyridin-5-one dihydrochloride (CP 93129, 300 micro m) reduced dialysate 5-HT to the same extent (30-40% of baseline) in the two genotypes, which suggests a lack of compensatory changes in 5-HT1B receptors in the DR of such mutant mice. Both a saline injection and handling for 3 min increased DR dialysate 5-HT in mutants, but not in 5-HT1A+/+ mice. Fluoxetine (5 and 20 mg/kg) elevated 5-HT in a dose-dependent manner in both genotypes. However, this effect was markedly more pronounced in the 5-HT1A-/- mice. The increased responsiveness of the extracellular 5-HT in the DR of 5-HT1A receptor knockout mice reflects a lack of the autoinhibitory control exerted by 5-HT1A autoreceptors.     

Density and function of central serotonin (5-HT) transporters, 5-HT1A and 5-HT2A receptors, and effects of their targeting on BTBR T+tf/J mouse social behavior

BTBR mice are potentially useful tools for autism research because their behavior parallels core social interaction impairments and restricted-repetitive behaviors. Altered regulation of central serotonin (5-HT) neurotransmission may underlie such behavioral deficits. To test this, we compared 5-HT transporter (SERT), 5-HT(1A) and 5-HT(2A) receptor densities among BTBR and C57 strains. Autoradiographic [(3) H] cyanoimipramine (1 nM) binding to SERT was 20-30% lower throughout the adult BTBR brain as compared to C57BL/10J mice. In hippocampal membrane homogenates, [(3) H] citalopram maximal binding (B(max) ) to SERT was 95 ± 13 fmol/mg protein in BTBR and 171 ± 20 fmol/mg protein in C57BL/6J mice, and the BTBR dissociation constant (K(D) ) was 2.0 ± 0.3 nM versus 1.1 ± 0.2 in C57BL/6J mice. Hippocampal 5-HT(1A) and 5-HT(2A) receptor binding was similar among strains. However, 8-OH-DPAT-stimulated [(35) S] GTPγS binding in the BTBR hippocampal CA(1) region was 28% higher, indicating elevated 5-HT(1A) capacity to activate G-proteins. In BTBR mice, the SERT blocker, fluoxetine (10 mg/kg) and the 5-HT(1A) receptor partial-agonist, buspirone (2 mg/kg) enhanced social interactions. The D(2) /5-HT(2) receptor antagonist, risperidone (0.1 mg/kg) reduced marble burying, but failed to improve sociability. Overall, altered SERT and/or 5-HT(1A) functionality in hippocampus could contribute to the relatively low sociability of BTBR mice.     

5-HT2A and 5-HT2C receptors and their atypical regulation properties

The 5-HT(2A) and 5-HT(2C) receptors belong to the G-protein-coupled receptor (GPCR) superfamily. GPCRs transduce extracellular signals to the interior of cells through their interaction with G-proteins. The 5-HT(2A) and 5-HT(2C) receptors mediate effects of a large variety of compounds affecting depression, schizophrenia, anxiety, hallucinations, dysthymia, sleep patterns, feeding behaviour and neuro-endocrine functions. Binding of such compounds to either 5-HT(2) receptor subtype induces processes that regulate receptor sensitivity. In contrast to most other receptors, chronic blockade of 5-HT(2A) and 5-HT(2C) receptors leads not to an up- but to a (paradoxical) down-regulation. This review deals with published data involving such non-classical regulation of 5-HT(2A) and 5-HT(2C) receptors obtained from in vivo and in vitro studies. The underlying regulatory processes of the agonist-induced regulation of 5-HT(2A) and 5-HT(2C) receptors, commonly thought to be desensitisation and resensitisation, are discussed. The atypical down-regulation of both 5-HT(2) receptor subtypes by antidepressants, antipsychotics and 5-HT(2) antagonists is reviewed. The possible mechanisms of this paradoxical down-regulation are discussed, and a new hypothesis on possible heterologous regulation of 5-HT(2A) receptors is proposed.     

3,4-Dihydro-2H-benzoxazinones are 5-HT(1A) receptor antagonists with potent 5-HT reuptake inhibitory activity

Starting from a high throughput screening hit, a series of 3,4-dihydro-2H-benzoxazinones has been identified with both high affinity for the 5-HT(1A) receptor and potent 5-HT reuptake inhibitory activity. The 5-(2-methyl)quinolinyloxy derivative combined high 5-HT(1A/1B/1D) receptor affinities with low intrinsic activity and potent inhibition of the 5-HT reuptake site (pK(i)8.2). This compound also had good oral bioavailability and brain penetration in the rat.     

The influence of substitution at aromatic part of 1,2,3,4-tetrahydroisoquinoline on in vitro and in vivo 5-HT(1A)/5-HT(2A) receptor activities of its 1-adamantoyloaminoalkyl derivatives.

Further structure-activity relationship (SAR) studies with the 1,2,3,4-tetrahydroisoquinoline (THIQ) class of 5-HT(1A) ligands led to the synthesis of new 1-adamantoyloaminoalkyl derivatives. The impact of substituent variations in the aromatic part of THIQ moiety on 5-HT(1A) and 5-HT(2A) receptor affinities, as well as in vivo functional properties of the investigated compounds were discussed. It was found that those modifications reduced the binding affinity for 5-HT(1A) receptors (in comparison with unsubstituted THIQ derivatives); however, the majority of new compounds still remained potent 5-HT(1A) ligands (K(i)=4.9-46 nM) and most of them showed features of partial agonists of postsynaptic 5-HT(1A) receptors. At the same time, their 5-HT(2A) receptor affinity was slightly increased (K(i)=40-1475 nM), which resulted in a loss of 5-HT(2A)/5-HT(1A) selectivity. 5-Br,8-OCH3 derivative-the most potent, mixed 5-HT(1A)/5-HT(2A) ligand-produced activation of presynaptic 5-HT(1A) receptors and showed properties of a 5-HT(2A) receptor antagonist.     

The influence of repeated administration of clozapine and haloperidol on the effects of the activation of 5-HT(1A), 5-HT(2) and 5-HT(4) receptors in rat frontal cortex.

The effects of a repeated treatment with antipsychotic drugs, clozapine and haloperidol, on the modulation of network activity ex vivo by 5-HT receptors were examined in rat frontal cortical slices using extracellular recording. Rats were treated for 21 days with clozapine (30 mg/kg p.o.), or haloperidol (1 mg/kg p.o.). Spontaneous bursting activity was induced in slices prepared 3 days after the last drug administration by perfusion with a medium devoid of Mg(2+) ions and with added picrotoxin (30 mM). The application of 2-3 microM 8-OH-DPAT, acting through 5-HT(1A) receptors, resulted in a reversible decrease of bursting frequency. In the presence of 1 microM DOI, the 5-HT(2) agonist, or 5 microM zacopride, the 5-HT(4) agonist, bursting frequency increased. Chronic clozapine treatment resulted in an attenuation of the effect of the activation of 5-HT(2) receptors, while the effects related to 5-HT(1A) and 5-HT(4) receptor activation were unchanged. Treatment with haloperiol did not influence the reactivity to the activation of any of the three 5-HT receptor subtypes. These data are consistent with earlier findings demonstrating a selective downregulation of 5-HT(2A) receptors by clozapine and indicate that chronic clozapine selectively attenuates the 5-HT-mediated excitation in neuronal circuitry of the frontal cortex while leaving the 5-HT-mediated inhibition intact.     

Identification and role of serotonin 5-HT1A and 5-HT1B receptors in primary cultures of rat embryonic rostral raphe nucleus neurons

Autoregulatory mechanisms affecting serotonin [5-hydroxytryptamine (5-HT)] release and synthesis during the early period of development were investigated in dissociated cell cultures raised from embryonic rostral rat rhombencephalon. The presence of 5-HT1A and 5-HT1B receptors in serotoninergic neurons was assessed using binding assays. The involvement of 5-HT1A and 5-HT1B receptors in the control of the synthesis and release of [3H]5-HT was studied using biochemical approaches with several serotoninergic receptor ligands. A mean decrease of 30% in [3H]5-HT synthesis and release was observed in the presence of 5-HT (10(-8) M), the 5-HT1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), the 5HT1B/1A agonist 5-methoxy-3-(1,2,5,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969), the 5-HT1B agonist 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP-93,129), and the 5-HT(1D/1B) agonist sumatriptan. Inhibition of 5-HT synthesis and release induced by 8-OH-DPAT was blocked by chiral N-tert-butyl-3-[1-[1-(2-methoxy)phenyl]piperazinyl]-1-phenylpropionam ide dihydrochloride quaternary-hydrate (WAY 100135) (10(7) M) or methyl 4-[4-[4-(1,1,3-trioxo-2H-1,2-benzoisothiazol-2-yl)butyl]-1-p iperazinyl]-1Hindole-2-carboxylate (SDZ 216-525) (10(-7)M), and that of CP-93,129 was blocked by methiothepin (10(-7) M). Paradoxically, extracellular levels of [3H]5-HT increased in the presence of 8-OH-DPAT and RU 24969 at 10(-6) M. 5-HT uptake experiments showed that these two agonists interacted with the 5-HT transporter. 5-HT1 binding sites (620 fmol/mg of protein) and 5-HT1A (482 fmol/mg of protein) and 5-HT1B (127 fmol/mg of protein) receptors were detected in 12-day in vitro cell cultures. Experiments carried out with tetrodotoxin suggested that 5-HT1A receptors are located on nerve cell bodies, whereas 5-HT1B receptors are located on the nerve terminals. We concluded that autoregulatory mechanisms involving 5-HT1A and 5-HT1B autoreceptors are functionally mature in cells from rostral raphe nuclei during the early period of development.     

Functional study of rat 5-HT2A receptors using antisense oligonucleotides

We studied the effects in rats of a 6-day intracerebroventricular (i.c.v) infusion of four different end-capped phosphorothioate-modified antisense oligonucleotides (AOs), specifically targeting different regions of the 5-hydroxytryptamine2A (5-HT2A) receptor mRNA, on central 5-HT2A receptor expression and 5-HT2A receptor-mediated behaviours. Only one of the AOs (sequence 4), directed against the 5'-untranslated region (from + 557 to + 577), specifically affected central 5-HT2A receptor expression and receptor-mediated behaviour. This AO (sequence 4) reduced binding of the 5-HT2A agonist 1-(2,5-dimethoxy-4-[125I]iodophenyl)-2-aminopropane ([125I]DOI) up to 25% in cortical areas, as measured by quantitative autoradiography. Cortical binding of the antagonist [3H]ketanserin was not affected. As the specific AO treatment presumably affects the synthesis of new receptor, we hypothesize that this newly synthesized receptor represents the major part of the functionally active, G protein coupled receptor. A 5-day infusion of AO (sequence 4) resulted in profound inhibition of the head-twitch response (HTR) to 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). In contrast, treatment with vehicle, sense oligonucleotides (SOs) and other AOs (sequences 1, 2 and 3) caused an increased DOM-induced HTR as well as a spontaneous HTR. The latter was abolished by treatment with the 5-HT2 receptor antagonist, ritanserin. Systematic investigation of the surgical and infusion procedures revealed that the enhanced HTR already appeared following drilling of the skull. This wounding can probably damage the blood-brain barrier and cause a stress-induced increase in serotonergic transmission. AO (sequence 4) treatment also abolished the spontaneous HTR. AO (sequence 4) treatment allowed the identification of specific central 5-HT2A receptor-mediated behaviours in the complex serotonergic syndrome induced by tryptamine in rats. Only bilateral convulsions and body tremors were significantly inhibited. The backward locomotion, hunched back and Straub tail were not affected, nor was cyanosis, an index of vasoconstriction induced by peripheral 5-HT2A receptor activation. Labelling of central 5-HT2C receptors by [3H]mesulergine, and 5-HT2C receptor-mediated anxiety were not attenuated by AO or SO treatment. Rats treated with AO (sequence 4) showed increased locomotor activity and a strong reactivity towards touching. We hypothesize that the down-regulation of functional 5-HT2A receptors may shift the balance between various 5-HT receptor subtypes. Our analysis of the behavioural consequences of AO treatment and the use of different AOs and SOs has shown that specific receptor-mediated behaviour can be identified.     

Molecular dynamics of buspirone analogues interacting with the 5-HT1A and 5-HT2A serotonin receptors

Three-dimensional (3-D) models of the human serotonin 5-HT1A and 5-HT2A receptors were constructed, energy refined, and used to study the interactions with a series of buspirone analogues. For both receptors, the calculations showed that the main interactions of the ligand imide moieties were with amino acids in transmembrane helix (TMH) 2 and 7, while the main interactions of the ligand aromatic moieties were with amino acids in TMH5, 6 and 7. Differences in binding site architecture in the region of highly conserved serine and tyrosine residues in TMH7 gave slightly different binding modes of the buspirone analogues at the 5-HT1A and 5-HT2A receptors. Molecular dynamics simulations of receptor-ligand interactions indicated that the buspirone analogues did not alter the interhelical hydrogen bonding patterns upon binding to the 5-HT2A receptor, while interhelical hydrogen bonds were broken and others were formed upon ligand binding to the 5-HT1A receptor. The ligand-induced changes in interhelical hydrogen bonding patterns of the 5-HT1A receptor were followed by rigid body movements of TMH2, 4 and 6 relative to each other and to the other TMHs, which may reflect the structural conversion into an active receptor structure.     

Imipramine and citalopram reverse corticosterone-induced alterations in the effects of the activation of 5-HT(1A) and 5-HT(2) receptors in rat frontal cortex.

Using extracellular recording we studied changes in the reactivity of rat frontal cortical slices to the 5-HT(1A), 5-HT(2) and 5-HT(4) receptor agonists, (+/-)-2-dipropyloamino-8-hydroxy-1,2,3,4-tetrahydronaphtalene hydrobromide (8-OH-DPAT), (+/-)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) and zacopride, respectively, induced by an earlier treatment of animals with corticosterone lasting 1 or 3 weeks. Spontaneous bursting activity was recorded in ex vivo slices incubated in a medium devoid of Mg(2+) ions and containing picrotoxin (30 microM). Repetitive, but not single, corticosterone administration resulted in an attenuation of the effect of the activation of 5-HT(1A) receptors and in an enhancement of the effect related to 5-HT(2) receptors. The effect of 5-HT(4) receptor activation remained unchanged. In separate two sets of experiments rats were treated with corticosterone for 3 weeks and additionally with imipramine or citalopram, beginning on the eighth day of corticosterone administration. In the corticosterone plus imipramine as well as corticosterone plus citalopram groups the effects of 8-OH-DPAT and DOI were not different from control indicating that corticosterone-induced functional modifications in the reactivity of 5-HT(1A) and 5-HT(2) receptors were reversed by antidepressant treatments.     

The serotonin 5-HT2A and 5-HT2C receptors interact with specific sets of PDZ proteins

The 5-hydroxytryptamine type 2A (5-HT(2A)) receptor and the 5-HT(2C) receptor are closely related members of the G-protein-coupled receptors activated by serotonin that share very similar pharmacological profiles and cellular signaling pathways. These receptors express a canonical class I PDZ ligand (SXV) at their C-terminal extremity. Here, we have identified proteins that interact with the PDZ ligand of the 5-HT(2A) and 5-HT(2C) receptors by a proteomic approach associating affinity chromatography using immobilized synthetic peptides encompassing the PDZ ligand and mass spectrometry. We report that both receptor C termini interact with specific sets of PDZ proteins in vitro. The 5-HT(2C) receptor but not the 5-HT(2A) receptor binds to the Veli-3.CASK.Mint1 ternary complex and to SAP102. In addition, the 5-HT(2C) receptor binds more strongly to PSD-95 and MPP-3 than the 5-HT(2A) receptor. In contrast, a robust interaction between the 5-HT(2A) receptor and the channel-interacting PDZ protein CIPP was found, whereas CIPP did not significantly associate with the 5-HT(2C) receptor. We also show that residues located at the -1 position and upstream the PDZ ligand in the C terminus of the 5-HT(2A) and 5-HT(2C) receptors are major determinants in their interaction with specific PDZ proteins. Immunofluorescence and electron microscopy studies strongly suggested that these specific interactions also take place in living cells and that the 5-HT(2) receptor-PDZ protein complexes occur in intracellular compartments. The interaction of the 5-HT(2A) and the 5-HT(2C) receptor with specific sets of PDZ proteins may contribute to their different signal transduction properties.