Structural features of the ligand-binding domain of the serotonin 5HT3 receptor.
The nicotinic acetylcholine receptor (AChR) and the serotonin type 3 receptor (5HT3R) are members of the ligand-gated ion channel gene family. Both receptors are inhibited by nanomolar concentrations of d-tubocurarine (curare) in a competitive fashion. Chemical labeling studies on the AChR have identified tryptophan residues on the gamma (gammaTrp-55) and delta (deltaTrp-57) subunits that interact with curare. Comparison of the sequences of these two subunits with the 5HT3R shows that a tryptophan residue is found in the homologous position in the 5HT3R (Trp-89), suggesting that this residue may be involved in curare-5HT3R interactions. Site-directed mutagenesis at position Trp-89 markedly reduces the affinity of the 5HT3R for the antagonists curare and granisetron but has little effect on the affinity for the agonist serotonin. To further examine the role of this region of the receptor in ligand-receptor interactions, alanine-scanning mutagenesis analysis of the region centered on Trp-89 (Thr-85 to Trp-94) was carried out, and the ligand binding properties of the mutant receptors were determined. Within this region of the receptor, curare affinity is reduced by substitution only at Trp-89, whereas serotonin affinity is reduced only by substitution at Arg-91. On the other hand, granisetron affinity is reduced by substitutions at Trp-89, Arg-91, and Tyr-93. This differential effect of substitutions on ligand affinity suggests that different ligands may have different points of interaction within the ligand-binding pocket. In addition, the every-other-residue periodicity of the effects on granisetron affinity strongly suggests that this region of the ligand-binding site of the 5HT3R (and by inference, other members of the ligand-gated ion channel family) is in a beta-strand conformation. (+info)
S-16924 [(R)-2-[1-[2-(2,3-dihydro-benzo[1,4]dioxin-5-yloxy)-ethyl]- pyrrolidin-3yl]-1-(4-fluorophenyl)-ethanone], a novel, potential antipsychotic with marked serotonin1A agonist properties: III. Anxiolytic actions in comparison with clozapine and haloperidol.
S-16924 is a potential antipsychotic that displays agonist and antagonist properties at serotonin (5-HT)1A and 5-HT2A/2C receptors, respectively. In a pigeon conflict procedure, the benzodiazepine clorazepate (CLZ) increased punished responses, an action mimicked by S-16924, whereas the atypical antipsychotic clozapine and the neuroleptic haloperidol were inactive. Similarly, in a Vogel conflict paradigm in rats, CLZ increased punished responses, an action shared by S-16924 but not by clozapine or haloperidol. This action of S-16924 was abolished by the 5-HT1A antagonist WAY-100,635. Ultrasonic vocalizations in rats were inhibited by CLZ, S-16924, clozapine, and haloperidol. However, although WAY-100,635 abolished the action of S-16924, it did not affect clozapine and haloperidol. In a rat elevated plus-maze, CLZ, but not S-16924, clozapine, and haloperidol, increased open-arm entries. Like CLZ, S-16924 increased social interaction in rats, whereas clozapine and haloperidol were inactive. WAY-100,635 abolished this action of S-16924. CLZ, S-16924, clozapine, and haloperidol decreased aggressive interactions in isolated mice, but this effect of S-16924 was not blocked by WAY-100, 635. All drugs inhibited motor behavior, but the separation to anxiolytic doses was more pronounced for S-16924 than for CLZ. Finally, in freely moving rats, CLZ and S-16924, but not clozapine and haloperidol, decreased dialysis levels of 5-HT in the nucleus accumbens: this action of S-16924 was blocked by WAY-100,165. In conclusion, in contrast to haloperidol and clozapine, S-16924 possessed a broad-based profile of anxiolytic activity at doses lower than those provoking motor disruption. Its principal mechanism of action was activation of 5-HT1A (auto)receptors. (+info)
Intestinal prokinesia by two esters of 4-amino-5-chloro-2- methoxybenzoic acid: involvement of 5-hydroxytryptamine-4 receptors and dissociation from cardiac effects in vivo.
In five fasting, conscious dogs, we compared the prokinetic action of two selective 5-hydroxytryptamine-4 (5-HT4) receptor agonists with low affinity for 5-HT3 receptors ML10302 (2-piperidinoethyl 4-amino-5-chloro-2-methoxybenzoate) and SR59768 (2-[(3S)-3-hydroxypiperidino]ethyl 4-amino-5-chloro-2-methoxybenzoate) in the duodenum and jejunum, using cisapride as a reference compound. Heart rate and rate-corrected QT (QTc) also were monitored to assess whether or not the cardiac effects of cisapride are shared by other 5-HT4 receptor agonists. Both ML10302 and SR59768 dose-dependently stimulated spike activity in the duodenum with similar potencies (dose range, 3-300 nmol/kg i.v.; ED50 values: 24 and 23 nmol/kg i.v., respectively), mimicking the effect of cisapride (30-3000 nmol/kg i.v.). The maximal effect was achieved with the dose of 100 nmol/kg i.v. for both compounds. Similar findings were obtained in the jejunum. Atropine and GR125487 (1-[2-[(methylsulfonyl)amino]ethyl]-4-piperidinyl-methyl 5-fluoro-2-methoxy-1H-indole-3-carboxylate, selective 5-HT4 receptor antagonist), at doses having no effect per se, antagonized intestinal prokinesia by maximal doses of ML10302 and SR59768. Neither ML10302 nor SR59768 had any effect on heart rate or QTc at any of the doses tested, whereas cisapride, at the highest dose (3000 nmol/kg), induced tachycardia and lengthened the QTC (p <.01). In conclusion, ML10302 and SR59768 share with cisapride a similar prokinetic action in the canine duodenum and jejunum in vivo. This effect is mediated by pathways involving activation of 5-HT4 and muscarinic receptors. Unlike cisapride, which induces tachycardia and prolongs the QTc by a mechanism probably unrelated to 5-HT4 receptor activation, ML10302 and SR59768 are devoid of cardiac effects in this model. (+info)
Selective antiaggressive effects of alnespirone in resident-intruder test are mediated via 5-hydroxytryptamine1A receptors: A comparative pharmacological study with 8-hydroxy-2-dipropylaminotetralin, ipsapirone, buspirone, eltoprazine, and WAY-100635.
The present study characterized the effects of the novel, selective, and potent 5-hydroxytryptamine1A (serotonin) (5-HT1A) receptor agonist, alnespirone [S-20499, (S)-N-4-[5-methoxychroman-3-yl)propylamino)butyl- 8-azaspiro-(4,5)-diacetamide, hydrochloride] on offensive and defensive resident-intruder aggression in wild-type rats and compared its actions with those of the prototypical full 5-HT1A agonist 8-hydroxy-2- dipropylaminotetralin (8-OH-DPAT), the partial 5-HT1A agonists ipsapirone and buspirone, and the mixed 5-HT1A/1B agonist eltoprazine. All five agonists exerted effective dose-dependent decreases of offensive aggressive behavior in resident rats; 8-OH-DPAT was the most potent (ID50 = 0.074 mg/kg), followed by eltoprazine (0.24), buspirone (0.72), ipsapirone (1.08), and alnespirone (1.24). However, in terms of selectivity of the antiaggressive effects as determined by the absence of decrements in social interest and general motor activity, alnespirone appeared to be superior. In the defensive aggression test, neither alnespirone nor any of the other four agonists changed defensive behaviors in the intruder rats. The involvement of 5-HT1A receptors in the antiaggressive actions of these drugs was confirmed by showing that the selective 5-HT1A receptor antagonist WAY-100635 (N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-N-(2- pyridinyl)cyclohexanecarboxamide trihydrochloride), which was inactive alone, fully prevented the antiaggressive effects of alnespirone, 8-OH-DPAT, and buspirone and partly reversed those of ipsapirone and eltoprazine. The data clearly indicate that alnespirone effectively suppresses offensive aggression with an advantageous profile of action compared with other full or partial 5-HT1A agonists. These selective antiaggressive actions of alnespirone are mediated by stimulating 5-HT1A receptors, presumably the somatodendritic autoreceptors at the raphe nuclei. Furthermore, the data provide evidence for a major involvement of these 5-HT1A receptors in the modulation of aggressive behavior by 8-OH-DPAT, ipsapirone, buspirone, and eltoprazine. (+info)
Differential addressing of 5-HT1A and 5-HT1B receptors in epithelial cells and neurons.
The 5-HT1A and 5-HT1B serotonin receptors are expressed in a variety of neurons in the central nervous system. While the 5-HT1A receptor is found on somas and dendrites, the 5-HT1B receptor has been suggested to be localized predominantly on axon terminals. To study the intracellular addressing of these receptors, we have used in vitro systems including Madin-Darby canine kidney (MDCK II) epithelial cells and primary neuronal cultures. Furthermore, we have extended these studies to examine addressing in vivo in transgenic mice. In epithelial cells, 5-HT1A receptors are found on both apical and basolateral membranes while 5-HT1B receptors are found exclusively in intracellular vesicles. In hippocampal neuronal cultures, 5-HT1A receptors are expressed on somatodendritic membranes but are absent from axons. In contrast, 5-HT1B receptors are found on both dendritic and axonal membranes, including growth cones where they accumulate. Using 5-HT1A and 5-HT1B knockout mice and the binary tTA/tetO system, we generated mice expressing these receptors in striatal neurons. These in vivo experiments demonstrate that, in striatal medium spiny neurons, the 5-HT1A receptor is restricted to the somatodendritic level, while 5-HT1B receptors are shipped exclusively toward axon terminals. Therefore, in all systems we have examined, there is a differential sorting of the 5-HT1A and 5-HT1B receptors. Furthermore, we conclude that our in vivo transgenic system is the only model that reconstitutes proper sorting of these receptors. (+info)
[3H]-Mesulergine labels 5-HT7 sites in rat brain and guinea-pig ileum but not rat jejunum.
1. The primary aim of this investigation was to determine whether binding sites corresponding to the 5-HT7 receptor could be detected in smooth muscle of the rat jejunum. Binding studies in rat brain (whole brain minus cerebellum) and guinea-pig ileal longitudinal muscle were also undertaken in order to compare the binding characteristics of these tissues. Studies were performed using [3H]-mesulergine, as it has a high affinity for 5-HT7 receptors. 2. In the rat brain and guinea-pig ileum, pKD values for [3H]-mesulergine of 8.0 +/- 0.04 and 7.9 +/- 0.11 (n = 3) and Bmax values of 9.9 +/- 0.3 and 21.5 +/- 4.9 fmol mg(-1) protein were obtained respectively, but no binding was detected in the rat jejunum. [3H]-mesulergine binding in the rat brain and guinea-pig ileum was displaced with the agonists 5-carboxamidotryptamine (5-CT) > 5-hydroxytryptamine (5-HT) > or = 5-methoxytryptamine (5-MeOT) > sumatriptan and the antagonists risperidone > or = LSD > or = metergoline > ritanserin > > pindolol. 3. Despite the lack of [3H]-mesulergine binding in the rat jejunum, functional studies undertaken revealed a biphasic contractile response to 5-HT which was partly blocked by ondansetron (1 microM). The residual response was present in over 50% of tissues studied and was found to be inhibited by risperidone > LSD > metergoline > mesulergine = ritanserin > pindolol, but was unaffected by RS 102221 (3 microM), cinanserin (30 nM), yohimbine (0.1 microM) and GR 113808 (1 microM). In addition, the agonist order of potency was 5-CT > 5-HT > 5-MeOT > sumatriptan. 4. In conclusion, binding studies performed with [3H]-mesulergine were able to detect 5-HT7 sites in rat brain and guinea-pig ileum, but not in rat jejunum, where a functional 5-HT7-like receptor was present. (+info)
Effect of 5-HT4 receptor stimulation on the pacemaker current I(f) in human isolated atrial myocytes.
OBJECTIVE: 5-HT4 receptors are present in human atrial cells and their stimulation has been implicated in the genesis of atrial arrhythmias including atrial fibrillation. An I(f)-like current has been recorded in human atrial myocytes, where it is modulated by beta-adrenergic stimulation. In the present study, we investigated the effect of serotonin (5-hydroxytryptamine, 5-HT) on I(f) electrophysiological properties, in order to get an insight into the possible contribution of I(f) to the arrhythmogenic action of 5-HT in human atria. METHODS: Human atrial myocytes were isolated by enzymatic digestion from samples of atrial appendage of patients undergoing coeffective cardiac surgery. Patch-clamped cells were superfused with a modified Tyrode's solution in order to amplify I(f) and reduce overlapping currents. RESULTS AND CONCLUSIONS: A time-dependent, cesium-sensitive increasing inward current, that we had previously described having the electrophysiological properties of the pacemaker current I(f), was elicited by negative steps (-60 to -130 mV) from a holding potential of -40 mV. Boltzmann fit of control activation curves gave a midpoint (V1/2) of -88.9 +/- 2.6 mV (n = 14). 5-HT (1 microM) consistently caused a positive shift of V1/2 of 11.0 +/- 2.0 mV (n = 8, p < 0.001) of the activation curve toward less negative potentials, thus increasing the amount of current activated by clamp steps near the physiological maximum diastolic potential of these cells. The effect was dose-dependent, the EC50 being 0.14 microM. Maximum current amplitude was not changed by 5-HT. 5-HT did not increase I(f) amplitude when the current was maximally activated by cAMP perfused into the cell. The selective 5-HT4 antagonists, DAU 6285 (10 microM) and GR 125487 (1 microM), completely prevented the effect of 5-HT on I(f). The shift of V1/2 caused by 1 microM 5-HT in the presence of DAU 6285 or GR 125487 was 0.3 +/- 1 mV (n = 6) and 1.0 +/- 0.6 mV (n = 5), respectively (p < 0.01 versus 5-HT alone). The effect of 5-HT4 receptor blockade was specific, since neither DAU 6285 nor GR 125487 prevented the effect of 1 microM isoprenaline on I(f). Thus, 5-HT4 stimulation increases I(f) in human atrial myocytes; this effect may contribute to the arrhythmogenic action of 5-HT in human atrium. (+info)
Vasoconstriction in human isolated middle meningeal arteries: determining the contribution of 5-HT1B- and 5-HT1F-receptor activation.
AIMS: Sumatriptan is a 5-HT1B/1D-receptor agonist which also has affinity for 5-HT1F-receptors. The vasoconstrictor effects of sumatriptan are thought to be 5-HT1B-receptor mediated and these receptors have been shown to be expressed in human cranial blood vessels. However, in the same tissue mRNA coding for 5-HT1F-receptors has also been identified and this study addresses the possibility of whether 5-HT1F-receptor activation contributes to vasoconstriction. METHODS: The ability of two selective 5-HT1B/1D-receptor antagonists (GR125,743 and GR127,935) with no affinity for 5-HT1F-receptors, to inhibit sumatriptan evoked contractions in human isolated middle meningeal artery was investigated. Using a series of 5-HT1B/1D-receptor agonists (sumatriptan, zolmitriptan, CP122,288, L-741,519 and L-741,604), some with high affinity for 5-HTIF-receptors and the non-selective 5-HT-receptor agonists 5-HT and 5-CT, we compared the vasoconstrictor potency of these drugs in human isolated middle meningeal artery with their affinities at cloned human 5-HT1B-, 5-HT1D-and 5-HT1F-receptors expressed in CHO cell lines. RESULTS: GR125,743 antagonized sumatriptan evoked contractions in a competitive manner (apparent pA2 9.1) and GR127,935 antagonized sumatriptan-induced responses in a non-competitive manner (reducing the maximum contraction to 27%). There was a significant correlation between vasoconstrictor potency and 5-HT1B-receptor affinity (r=0.93, P=0.002) but not with 5-HT1D- or 5-HT1F-receptor affinity (r=0.74, P=0.06; r= 0.31, P= 0.49, respectively). CONCLUSIONS: These experiments show that in human middle meningeal artery vasoconstriction to sumatriptan-like agents is 5-HT1B-receptor mediated with little if any contribution from 5-HT1F-receptor activation. (+info)