BW 723C86 Research Articles

Differential expression of serotonin2B receptors in GABAergic and serotoninergic neurons of the rat and mouse dorsal raphe nucleus

The central serotonin2B receptor (5-HT2BR) modulates 5-HT and dopamine (DA) neuronal function in the mammalian brain and has been suggested as a potential target for the treatment of neuropsychiatric disorders involving derangements of these monoamine systems, such as schizophrenia, cocaine abuse and dependence and major depressive disorder. Studies in rats and mice yielded contrasting results on the control of 5-HT/DA networks by 5-HT2BRs, thereby leading to opposite views on the therapeutic potential of 5-HT2BR agents for treating the above disorders. These discrepancies may result from anatomo-functional differences related to a different cellular location of 5-HT2BRs in rat and mouse brain. Using immunohistochemistry, we assessed this hypothesis by examining the expression of 5-HT2BRs in 5-HT and GABAergic neurons of rats and mice within different subregions of the dorsal raphe nucleus (DRN), currently considered as the main site of action of 5-HT2B agents. Likewise, using in vivo microdialysis, we examined their functional relevance in the control of DRN 5-HT outflow, a surrogate index of 5-HT neuronal activity.In the DRN of both species, 5-HT2BRs are expressed in 5-HT cells expressing tryptophan hydroxylase 2 (TPH2), in GABAergic cells expressing glutamic acid decarboxylase 67 (GAD67), and in cells expressing both markers (GAD67 & TPH2; i.e., GABA-expressing 5-HT neurons). The proportion of 5-HT2BR-positive cells expressing only TPH2 was significantly larger in mouse than in rat DRN, whereas the opposite holds true for the expression in cells expressing GAD67 & TPH2. No major species differences were found in the dorsal and ventral subregions. In contrast, the lateral subregion exhibited large differences, with a predominant expression of 5-HT2BRs in TPH2-positive cells in mice (67.2 vs 19.9 % in rats), associated with a lower expression in GAD67 & TPH2 cells (7.9 % in mice vs 41.5 % in rats). Intra-DRN (0.1 μM) administration of the preferential 5-HT2BR agonist BW 723C86 decreased and increased DRN 5-HT outflow in rats and mice respectively, both effects being prevented by the intra-DRN perfusion of the selective 5-HT2BR antagonist RS 127445 (0.1 μM).Altogether, these results show the existence of anatomical differences in the cellular expression of 5-HT2BRs in the rat and mouse DRN, which translate into an opposite control of 5-HT outflow. Also, they highlight the relevance of the subset of GAD67-positive 5-HT neurons as a key factor responsible for the functional differences between rats and mice in terms of 5-HT neuronal activity modulation.

Evaluation of a 5-HT2B receptor agonist in a murine model of amyotrophic lateral sclerosis

Degeneration of brainstem serotonin neurons has been demonstrated in ALS patients and mouse models and was found responsible for the development of spasticity. Consistent with involvement of central serotonin pathways, 5-HT2B receptor (5-HT2BR) was upregulated in microglia of ALS mice. Its deletion worsened disease outcome in the Sod1G86R mouse model and led to microglial degeneration. In ALS patients, a polymorphism in HTR2B gene leading to higher receptor expression in CNS, was associated with increased survival in patients as well as prevention of microglial degeneration. Thus, the aim of our study was to determine the effect of a 5-HT2BR agonist : BW723C86 (BW), in the Sod1G86R mouse model. Despite good pharmacokinetic and pharmacological profiles, BW did not ameliorate disease outcome or motor neuron degeneration in a fast progressing mouse model of ALS despite evidence of modulation of microglial gene expression.

The influence of fluoxetine on interleukin-6 and interleukin-1β production by dendritic cells in multiple sclerosis in vitro

Was to evaluate the effect of selective serotonin reuptake inhibitor fluoxetine on the production of cytokines interleukin-6 (IL-6) and interleukin-1β (IL-1β) by dendritic cells in multiple sclerosis (MS). 5 patients with relapsing-remitting MS and five healthy subjects were examined. Levels of IL-6 and IL-1β were measured by ELISA in culture supernatants obtained from lipopolysaccharide stimulated dendritic cells. To assess the effect of fluoxetine on cytokine production, dendritic cells were stimulated in the presence of fluoxetine and antagonists of 5-HT1A-, 5-HT2A-, 5-HT2B-receptors or agonist of 5-HT2B-receptors. Cytokine production by dendritic cells was comparable in both groups. Fluoxetine suppressed IL-6 and IL-1β production in both groups. Blockade of 5-HT2B-receptors with specific antagonist RS 127445 reduced the inhibitory effect of fluoxetine on IL-1β production in both groups and IL-6 production in healthy subjects. In contrast, activation of 5-HT2B-receptors by specific agonist BW 723C86 increased the inhibitory effect of fluoxetine on IL-6 production by dendritic cells in both groups, but did not affect on IL-1β production. These data suggest an anti-inflammatory effect of fluoxetine in MS by modulating pro-inflammatory cytokines production by dendritic cells. This effect could be mediate by activation of 5-HT2B-receptors.

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

Peripheral serotonin receptor 2B and transient receptor potential channel 4 mediate pruritus to serotonergic antidepressants in mice

Specific binding and characteristics of geissoschizine methyl ether, an indole alkaloid of Uncaria Hook, in the rat brain

Ethnopharmacological relevanceGeissoschizine methyl ether (GM) is an indole alkaloid that is a component of Uncaria Hook, and has been identified as the active component responsible for the anti-aggressive effects of the Uncaria Hook-containing traditional Japanese medicine, yokukansan. Recently, GM was shown to reach the brain by crossing the blood–brain barrier in rats following the oral administration of yokukansan. This finding suggested that there may be specific binding sites for GM in the brain. Here we show evidence that tritium-labeled GM ([3H]GM) binds specifically to several brain areas of rats. Materials and methodsMale rats were used. [3H]GM was synthesized from a demethylated derivative of GM. Specific binding sites of [3H]GM on brain sections were determined by quantitative autoradiography, and maximum binding densities (Bmax) and dissociation constants (Kd) were calculated. Several chemical compounds were used to clarify the molecules that recognize [3H]GM in the completion-binding assay. Emulsion microautoradiography was also performed to identify the cells that bind [3H]GM. ResultsSpecific binding of [3H]GM was observed in the frontal cortex, including the prefrontal cortical region (e.g., prelimbic cortex (PrL)), hippocampus, caudate putamen, amygdala, central medial thalamic nucleus, dorsal raphe nucleus (DR), and cerebellum. Bmax ranged between 0.65 and 8.79pmol/mg tissue, and Kd was between 35.0 and 232.6nM. Specific binding with relatively high affinity (Kd less than 62nM) was dense in the frontal cortical region, moderate in the DR, and sparse in the cerebellum. The specific binding of [3H]GM in the PrL was significantly replaced by the serotonin 1A (5-HT1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (DPAT), 5-HT2A receptor antagonist ketanserin, 5-HT2B receptor agonist BW 723C86, 5-HT2C receptor agonist RO60-0175, adrenergic α2A receptor antagonist yohimbine, L-type Ca2+ channel blocker verapamil, and μ-opioid receptor antagonist naloxone. Similar results were obtained in the frontal cortex and DR, but not in the cerebellum. Microautoradiography revealed that [3H]GM signals were distributed throughout the frontal cortex, which included neuron-like large cells. ConclusionThese results demonstrate that specific binding sites for GM exist in rat brain tissue, and suggest that the pharmacological actions of GM are mainly associated with 5-HT receptors in the frontal cortex and DR. These results provide an insight into the neuropharmacology of GM and GM-containing herbal medicines.

Transient, 5-HT2B receptor–mediated facilitation in neuropathic pain: Up-regulation of PKCγ and engagement of the NMDA receptor in dorsal horn neurons

Spinal nociception can be facilitated by 5-HT2 receptors in neuropathic pain. We investigated the involvement of glutamate receptors in dorsal neuron hyperexcitation that is promoted by 5-HT2B receptor (5-HT2BR) after spinal nerve ligation (SNL) in the rat. Augmentation of C-fiber–evoked potentials by spinal superfusion with 5-HT2BR agonist BW 723C86 in nerve-ligated rats was impeded by co-administration of NMDA receptor (NMDAR) antagonist D-AP5, but not by mGluR1/5 antagonist AIDA or mGluR2/3 antagonist LY 341495. Evoked potentials were increased by cis-ACPD in nerve-injured rats, irrespective of simultaneous 5-HT2BR blockade by SB204741. In uninjured rats, NMDAR agonist cis-ACPD enhanced evoked potentials in the presence of BW 723C86 but not if administered alone or during exposure to protein kinase C γ (PKCγ) inhibitor peptide. Triple immunofluorescence labelings revealed co-localization of NMDAR and 5-HT2BR in PKCγ-expressing perikarya in lamina II neurons. As a result of SNL, PKCγ was transiently and bilaterally up-regulated in synaptic fraction from dorsal horn homogenates, peaking at day 2 and returning to basal levels by day 9. Chronic blockade of 5-HT2BR with selective antagonist SB 204741 after SNL bilaterally decreased the following: (i) PKCγ up-regulation in synaptic fraction, (ii) phosphorylation of NMDAR subunit NR1 (serine 889) in synaptic fraction, and (iii) co-localization of both PKCγ and phosphorylated NR1 with postsynaptic marker PSD-95. Chronic delivery of SB 204741 bilaterally attenuated thermal and mechanical allodynia occurring after SNL, particularly at day 2 post injury. These findings suggest that transient activation of the PKCγ/NMDAR pathway is critically involved in 5-HT2BR-mediated facilitation in the SNL model of neuropathic pain.

Alterations in serotonin receptor-induced contractility of bovine lateral saphenous vein in cattle grazing endophyte-infected tall fescue12

As part of a 2-yr study documenting the physiologic impact of grazing endophyte-infected tall fescue on growing cattle, 2 experiments were conducted to characterize and evaluate effects of grazing 2 levels of toxic endophyte-infected tall fescue pastures on vascular contractility and serotonin receptors. Experiment 1 examined vasoconstrictive activities of 5-hydroxytryptamine (5HT), α-methylserotonin (ME5HT; a 5HT(2) receptor agonist), d-lysergic acid (LSA), and ergovaline (ERV) on lateral saphenous veins collected from steers immediately removed from a high-endophyte-infected tall fescue pasture (HE) or a low-endophyte-infected mixed-grass (LE) pasture. Using the same pastures, Exp. 2 evaluated effects of grazing 2 levels of toxic endophyte-infected tall fescue on vasoconstrictive activities of (±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI), BW 723C86 (BW7), CGS-12066A (CGS), and 5-carboxamidotryptamine hemiethanolate maleate (5CT), agonists for 5HT(2A),( 2B), 5HT(1B), and 5HT(7) receptors, respectively. One-half of the steers in Exp. 2 were slaughtered immediately after removal from pasture, and the other one-half were fed finishing diets for >91 d before slaughter. For Exp. 1, maximal contractile intensities were greater (P < 0.05) for steers grazing LE pastures than HE pastures for 5HT (73.3 vs. 48.9 ± 2.1%), ME5HT (52.7 vs. 24.9 ± 1.5%), and ERV (65.7 vs. 49.1 ± 2.6%). Onset of contractile response did not differ for 5HT (P = 0.26) and ERV (P = 0.93), but onset of ME5HT contraction was not initiated (P < 0.05) in HE steers until 10(-4) compared with 10(-5) M in LE-grazing steers. For Exp. 2, maximal contractile intensities achieved with DOI were 35% less (P < 0.05), whereas those achieved with 5CT were 37% greater (P < 0.05), in steers grazing HE pastures. Contractile response to CGS did not differ between pasture groups, and there was an absence of contractile response to BW7 in both groups. There were no differences between endophyte content in contractile responses after animals were finished for >91 d. Experiment 1 demonstrated that grazing of HE pastures for 89 to 105 d induces functional alterations in blood vessels, as evidenced by reduced contractile capacity and altered serotonergic receptor activity. Experiment 2 demonstrated that grazing HE pastures alters vascular responses, which may be mediated through altered serotonin receptor activities, and these alterations may be ameliorated by the removal of ergot alkaloid exposure as demonstrated by the absence of differences in finished steers.

Hyperthyroidism enhances 5-HT-induced contraction of the rat pulmonary artery: Role of calcium-activated chloride channel activation

Experimentally-induced hyperthyroidism in rodents is associated with signs and symptoms of pulmonary hypertension. The main objective of the present study was to investigate the effect of thyroxine-induced pulmonary hypertension on the contractile response of the pulmonary artery to 5-HT and the possible underlying signaling pathway. 5-HT concentration-dependently contracted artery segments from control and thyroxine-treated rats with pD(2) values of 5.04 ± 0.19 and 5.34 ± 0.14, respectively. The maximum response was significantly greater in artery segments from thyroxine-treated rats. Neither BW 723C86 (5-HT(2B)-receptor agonist) nor CP 93129 (5-HT(1B)-receptor agonist) contracted ring segments of the pulmonary artery from control and thyroxine-treated rats at concentrations up to 10(-4)M. There was no significant difference in the level of expression of 5-HT(2A)-receptor protein between the two groups. Ketanserin (3 × 10(-8)M) produced a rightward shift of the concentration-response curve to 5-HT in both groups with equal potency (-logK(B) values were 8.1 ± 0.2 and 7.9 ± 0.1 in control and thyroxine-treated rats, respectively). Nifedipine (10(-6)M) inhibited 5-HT-induced contractions in artery segments from control and thyroxine-treated rats and was more effective against 5-HT-induced contraction in artery segments for thyroxine-treated rats. The calcium-activated chloride channel blocker, niflumic acid (10(-4)M) also inhibited 5-HT-induced contractions in artery segments from control and thyroxine-treated rats and was more effective against 5-HT-induced contraction in artery segments for thyroxine-treated rats. It was concluded that hyperthyroidism enhanced 5-HT-induced contractions of the rat pulmonary artery by a mechanism involving increased activity of calcium-activated chloride channels.

Subtype-specific changes in 5-HT receptor-mediated modulation of C fibre-evoked spinal field potentials are triggered by peripheral nerve injury

Neurotransmitter serotonin (5-HT) released from descending pain modulation pathways to the dorsal horn is crucial to spinal nociception processing. This study sought to gain insight into the modulatory roles of specific serotonin receptor subtypes in experimentally induced neuropathic pain. In rats subjected to spinal nerve ligation (SNL) surgery, we recorded field potentials evoked in the spinal dorsal horn by C fibre-input, during spinal superfusion with subtype-selective drugs. In neuropathic rats, subtype 5-HT1A agonist 8-OH-DPAT (100 nM) was found to potently depress evoked field potentials, as opposed to 5-HT2A or 5-HT2B subtype agonists TCB-2 (100 nM) or BW 723C86 (1 μM), respectively, which consistently enhanced evoked potentials. All three failed to alter spinal field potentials in sham operated rats. CP 94253 (1 μM), WAY 161503 (1 mM) or SR 57227 (at 1 μM in SNL rats, and 100 μM in sham rats), selective agonists for 5-HT1B, 5-HT2C and 5-HT3 receptors, respectively, significantly depressed evoked field potentials in both animal groups. The 5-HT4 agonist RS 67333 (1 μM) was depressant only in sham operated animals. Only after SNL, spinal superfusion with 5-HT1A- or 5-HT1B receptor-antagonists (S)-WAY 100135 (100 μM) or SB 224289 (100 μM), respectively, disinhibited C fibre-evoked potentials, whereas 5-HT2A or 5-HT2B receptor-antagonists 4F 4PP (100 μM) or SB 204741 (100 μM) depressed evoked potentials, suggesting tonic activity of all four subtypes as a consequence of experimental nerve injury. The present findings reveal profound subtype-specific changes in the functional modulatory activities of spinal serotonin receptors following peripheral nerve injury. In particular, spinal hyperexcitation promoted by receptors 5-HT2A and 5-HT2B is suggested as a novel pathogenic pathway contributing to neuropathic pain.

5-HT 2 receptor-mediated reversal of the inhibition of hippocampal long-term potentiation by acute inescapable stress

The serotonergic system is known to modulate and mediate many of the central nervous system effects of stress. Here we investigated the ability of serotonergic agents to reverse the inhibition of the induction of hippocampal long-term potentiation (LTP) caused by prior exposure to inescapable stress. Elevated platform stress prevented the induction of LTP in the CA1 area of anaesthetized rats. An agent that increases extracellular 5-HT concentration, fenfluramine (5 mg/kg, i.p.) enabled the induction of LTP in previously stressed animals. Consistent with a role for enhanced activation of 5-HT 2 receptors, the facilitatory effect of fenfluramine was prevented by the 5-HT 2 receptor antagonist cinanserin (30 mg/kg). Agents that directly activate 5-HT 2 receptors, including the 5-HT 2B receptor agonist BW 723C86 (30 mg/kg) and the 5-HT 2C receptor agonist MK-212 (3 mg/kg), mimicked the restorative effect of fenfluramine. Fenfluramine also opposed inhibition of LTP caused by the NMDA-receptor antagonist D-AP5 (100 nmol, i.c.v.) which suggests that the facilitatory action of serotonergic agents is not restricted to stress-mediated inhibition of LTP. These findings support an important role for activation of 5-HT 2 receptors by systemically applied agents to enable recovery from the inhibition of LTP by stress.

Stimulation of Growth Hormone Release by 5-Hydroxytryptamine (5-HT) in Cultured Rat Anterior Pituitary Cell Aggregates: Evidence for Mediation by 5-HT2B, 5-HT7, 5-HT1B, and Ketanserin-Sensitive Receptors

5-Hydroxytryptamine (5-HT) promotes the release of GH by a hypothalamic site of action. The present study explores a putative pituitary action in a perifused rat anterior pituitary aggregate cell culture system. In aggregates cultured with 1 nM estradiol for expression of the 5-HT4, -5, and -6 receptor (R), 5-HT promptly stimulated GH secretion with a dose dependency between 1 and 10 nM. The effect of 5-HT was partially blocked by methiothepin and methysergide; by SB-206553, a 5-HTR2B/C antagonist; SB-269970, a 5-HTR7/5A antagonist; and SB-224289, a 5-HTR1B antagonist. The GH response was fully blocked by combined administration of SB-206553+SB-269970 and SB-206553+ketanserin but not by SB-206553+spiperone. Culturing the aggregates without estradiol diminished the magnitude of the GH response to 5-HT as well as the impact of 5-HTR7/5 blockade on the response. Basal GH release was stimulated by the 5-HTR2 agonists 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, m-chlorophenyl piperazine, and alpha-methyl 5-HT; 5-carboxytryptamine (agonist at 5-HTR1, -5, and -7); tryptamine (preferential 5-HTR7 agonist); and the selective 5-HTR1B agonist CP93129 but not the 5-HTR1A agonists 7-(dipropylamino)tetralin-1-ol-8-hydroxy-2-(di-n-propylamino)tetralin and the 5-HTR1B/D agonist sumatriptan. The selective 5-HTR2B agonist BW 723C86 stimulated GH release, and the selective 5-HTR2B antagonist SB-204741 attenuated the GH response to 5-HT. The present data suggest that 5-HT may release GH through a pituitary site of action, and that the 5-HTR2B, 5-HTR7 and 5-HTR1B mediate this response, with possibly an inhibitory component of the 5-HTR1D. The relative contribution of these receptors may be modulated by estrogen.

Exogenous Serotonin Regulates Proliferation of Interstitial Cells of Cajal in Mouse Jejunum Through 5-HT2B Receptors

Interstitial cells of Cajal (ICC) are required for normal gastrointestinal motility. Loss of ICC is associated with several motility disorders. The mechanisms modulating ICC survival and proliferation are poorly understood. This study aimed to establish whether 5-hydroxytryptamine (5-HT) plays a role in regulating ICC proliferation. Expression of 5-HT receptor mRNA was investigated in muscle strips, in purified populations of ICC, and in identified single cells. The effect of 5-HT(2B) receptor ligands on ICC numbers was studied in primary cell cultures. Proliferation of ICC was determined by counting Ki67-positive cells in culture. Of the 5-HT receptors known to be involved in proliferation, 5-HT(2B) receptor mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in jejunal muscle, whereas 5-HT(1A), 5-HT(1D), and 5-HT(2C) receptor mRNAs were not. 5-HT(2B) receptor mRNA was found in single ICC and cells purified by flow cytometry. Exogenous 5-HT (1 micromol/L) increased (66% +/- 9%, P < .005) ICC numbers in culture. The 5-HT(2) receptor antagonist, ritanserin, and the 5-HT(2B) receptor antagonist, SB204741, inhibited the effect of 5-HT. The 5-HT(2B) receptor agonist BW 723C86 induced a concentration-dependent increase in ICC number (50% +/- 6% at 50 nM, P < .04) and increased ICC proliferation (25% +/- 3% vs 19 +/- 1% in controls, P < .03). These studies establish that 5-HT(2B) receptors are expressed on ICC. Exogenous 5-HT regulates ICC numbers through 5-HT(2B) receptors in part by increasing ICC proliferation. The 5-HT(2B) receptor may serve as a novel pathway to regulate ICC numbers.

The contractile effect of the ghrelin receptor antagonist, D-Lys 3-GHRP-6, in rat fundic strips is mediated through 5-HT receptors

Ghrelin is an orexigenic peptide present in the stomach with gastroprokinetic properties. Previous in vivo studies have shown that the ghrelin receptor antagonist, D-Lys 3-GHRP-6, reduced food intake and delayed gastric emptying in rodents but these effects are at variance with the normal phenotype of the ghrelin knockout mice. To verify the specificity of the effects observed with D-Lys 3-GHRP-6 this study aimed to investigate the pharmacology of D-Lys 3-GHRP-6 in vitro. Rat fundic strips were suspended in a tissue bath and the contraction of strips to 10 − 5 M of ghrelin, GHRP-6 or D-Lys 3-GHRP-6 was measured isometrically in the absence and presence of blockers. Neither ghrelin, nor GHRP-6, induced significant contractions in the absence of electrical field stimulation thereby excluding the presence of ghrelin receptors on smooth muscle cells. In contrast D-Lys 3-GHRP-6, induced a pronounced biphasic contraction of 13.9 ± 1.8% and 40.5 ± 3.2% relative to the response to 60 mM KCl. The contraction was blocked by the 5-HT 1,2 receptor antagonist methysergide and was markedly reduced by the 5-HT 2B receptor antagonist, yohimbine, which also profoundly affected 5-HT induced contractions in fundic strips. The existence of 5-HT 2B receptors in the fundus was confirmed by use of the 5-HT 2B receptor agonist, BW 723C86. In contrast to ghrelin and GHRP-6, the ghrelin receptor antagonist, D-Lys 3-GHRP-6, induced pronounced smooth muscle contractions in the rat fundus by interacting with 5-HT 2B receptors. This may question the role of endogenous ghrelin in the effects observed with D-Lys 3-GHRP-6 on food intake and gastric emptying in vivo.

5‐HT2B receptors do not modulate sensitivity to colonic distension in rats with acute colorectal hypersensitivity

The 5-HT4 receptor agonist tegaserod is an effective prokinetic agent that increases gastrointestinal secretion and reduces visceral sensitivity. Tegaserod has both 5-HT4 receptor agonist and 5-HT2B receptor antagonist activity, the latter being a less potent effect of the drug. In a rat model of colonic hypersensitivity, selective 5-HT4 receptor antagonists only partially reversed the antihyperalgesic effect of tegaserod suggesting that non-5-HT4 receptor-mediated mechanisms may also be involved in its overall antihyperalgesic action. The objective of this study was to determine whether 5-HT2B receptors play a role in colonic hypersensitivity. A visceromotor response (VMR) in acutely sensitized animals (intracolonic acetic acid, 0.6%, 1.5 mL) quantified colonic hypersensitivity. Acetic acid produced an increase in the VMR at all distension pressures. However, neither the 5-HT2B receptor agonist BW 723C86, the 5-HT2B antagonist SB204741 or the 5-HT2B/2C antagonist SB 206553 caused any significant inhibition of the VMR. In summary, in the same rodent model in which tegaserod has previously been shown to produce a potent antihyperalgesic effect, 5-HT2B receptors do not appear to mediate colonic hypersensitivity. We conclude that 5-HT2B receptor-mediated mechanisms are unlikely to play a role in the antihyperalgesic action of tegaserod in man.

Implication of 5-HT 2A subtype receptors in DOI activity in the four-plates test–retest paradigm in mice

The four-plates test (FPT) is an animal model of anxiety which allows the detection of anxiolytic effect not only of benzodiazepines (BZDs) but also of other non-BZDs anxiolytic compounds such as antidepressants (ADs). Furthermore, DOI, a 5-HT 2A/2C agonist, has been shown to exert an anxiolytic-like effect in this model. Retesting mice in animal models of anxiety (test–retest paradigm) induces an anxiogenic-like and a loss of anxiolytic-like effects in response to BZDs and ADs. On the contrary, DOI has been reported to oppose the fear potentiation induced by trial 1 in the FPT. Despite DOI is considered as one of the most selective 5-HT 2A available, it acts as agonist at all three 5-HT 2 receptor subtypes (5-HT 2A, 5-HT 2B and 5-HT 2C). The aim of this study was thus to investigate in the FPT test–retest paradigm, which 5-HT 2 receptor subtype(s) was involved in the DOI-induced effect in experienced mice. The effect of DOI (0.25–4 mg/kg) and the agonists, 5-HT 2B, BW 723C86 (1–16 mg/kg) and 5-HT 2C, RO 60-0175 (0.25–4 mg/kg) have also been studied. Then, antagonism studies were conducted combinating the 5-HT 2A receptor antagonist SR 46349B, the 5-HT 2B/2C receptor antagonist SB 206553 or the selective 5-HT 2C receptor antagonist RS 10-2221 (at the doses of 0.1 and 1 mg/kg) with the DOI (1 mg/kg). Our study shows that the BW 723C86 had no effect on retesting mice, whereas it exerted an anxiolytic-like effect in naive mice. By contrast to DOI, the RO 60-0175 had no effect neither in naive nor experienced mice. Furthermore, only the SR 46349B antagonized the DOI-induced anti-punishment effect. Diazepam included as a positive control also increased in each case the number of punished passages in naive mice. Our findings altogether also suggest that DOI exerts its anxiolytic-like effect in the FPT test–retest paradigm through 5-HT 2A receptors.

Evidence for 5-HT2B and 5-HT7 receptor-mediated relaxation in pulmonary arteries of weaned pigs

This study characterizes the relaxant response to 5-hydroxytryptamine (5-HT) in prostaglandin F(2alpha) (PGF(2alpha))-precontracted pulmonary arteries of weaned pigs. In arterial rings with intact endothelium, the relaxation to 5-HT was biphasic. The high affinity component of relaxation to 5-HT (0.1-10 nM) was abolished by mechanical removal of the endothelium or after the addition of L: -NAME (200 microM), and was inhibited by the 5-HT(2B/2C) receptor antagonist SB 206553 (1 microM), but not the 5-HT(2C) receptor antagonist SB 242084 (0.1 microM). Endothelium-intact arteries were also relaxed by the selective 5-HT(2B) receptor agonist BW 723C86 (pD(2) 7.7). The relaxant response to BW 723C86 was inhibited by 1 microM SB 206553 (pK(B) 6.8). The low affinity component of relaxation to 5-HT (>/=30 nM) remained unaffected after mechanical removal of the endothelium or the addition of L: -NAME. In endothelium-denuded arterial rings, 5-HT, 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT), and frovatriptan produced monophasic relaxations with pD(2) values of 6.5, 7.5, 5.9, and 4.7 respectively. Relaxant responses to the agonists were antagonized by the selective 5-HT(7) receptor antagonist SB 269970 (pK(B) 8.2-8.9). The relaxant response to the potent 5-HT(7) receptor agonist 5-CT was also antagonized by methiothepin (pK(B) 9.6), pimozide (pK(B) 8.2), mesulergine (pK(B) 7.7), methysergide (pK(B) 7.4), clozapine (pK(B) 7.6), and spiperone (pK(B) 7.4). The estimated pK(B) values argue in favor of an involvement of 5-HT(7) receptors in the direct vasorelaxant action of 5-HT in the pulmonary arteries of weaned pigs. The relaxant response to 5-CT was associated with an increase in cAMP that was surmountably antagonized by SB 269970 (pK(B) 8.6). The present in vitro bioassay can be used to characterize new drugs with potential agonist or antagonist properties at functional 5-HT(7) receptors.

Implication of 5-HT2 receptor subtypes in the mechanism of action of antidepressants in the four plates test

The selective serotonin reuptake inhibitors (SSRIs) and the serotonin and noradrenaline reuptake inhibitors (SNRIs) increase synaptic levels of serotonin, leading to an increased activation of a multitude of specific postsynaptic 5-HT receptors. However, it is not yet known which 5-HT receptor subtypes mediate the therapeutic effects of antidepressants. The effects of the SSRI, paroxetine and the SNRI, venlafaxine were evaluated in the mouse four plates test (FPT). Paroxetine administered intraperitoneally (IP) (0.5, 2-8 mg/kg) potently augmented the number of punished passages accepted by mice in this paradigm. The effects of paroxetine (8 mg/kg) were not reversed by the selective 5-HT2C receptor antagonist, RS 10-2221 (0.1 mg/kg and 1 mg/kg) or the selective 5-HT2B/2C receptor antagonist SB 206553 (0.1 mg/kg and 1 mg/kg), at doses which lack an effect when administered alone. In contrast, the selective 5-HT2A receptor antagonist, SR 46349B (0.1 mg/kg and 1 mg/kg) completely abolished the paroxetine-induced increase in punished passages. The acute administration of venlafaxine induced an anxiolytic-like effect in the FPT at the doses of 2-16 mg/kg. This effect was reversed by the 5-HT2B/2C receptor antagonist as did SR 46349B, for both doses administered. Our results strongly suggest that activation of 5-HT2A receptors is critically involved in the anxiolytic activity of paroxetine, whereas the 5-HT2A and 5-HT2B receptors are involved in the anti-punishment action of venlafaxine in the FPT. The co-administration of selective 5-HT2A, 2B, 2C receptor agonists (DOI, 0.06 mg/kg and 0.25 mg/kg; BW 723C86, 0.5 mg/kg and 2 mg/kg and RO 60-0175, 0.06 mg/kg and 0.25 mg/kg), respectively, was subsequently investigated. The effects of sub-active doses of paroxetine (0.25 mg/kg and 1 mg/kg) were augmented by BW 723C86 and RO 60-0175 receptor agonist challenge. The anti-punishment effects of venlafaxine (0.25 mg/kg and 1 mg/kg) were potentialised only by DOI co-administration. These results indicate that the co-administration of 5-HT2 receptor agonists with paroxetine and venlafaxine may provide a powerful tool for enhancing the clinical efficacy of these antidepressants.

The 5-HT4 receptor agonist, tegaserod, is a potent 5-HT2B receptor antagonist in vitro and in vivo.

1 Tegaserod (Zelnorm) is a potent 5-hydroxytryptamine4 (5-HT4) receptor agonist with clinical efficacy in disorders associated with reduced gastrointestinal motility and transit. The present study investigated the interaction of tegaserod with 5-HT2 receptors, and compared its potency in this respect to its 5-HT4 receptor agonist activity. 2 Tegaserod had significant binding affinity for human recombinant 5-HT2A, 5-HT2B and 5-HT2C receptors (pKi=7.5, 8.4 and 7.0, respectively). The 5-HT2B receptor-binding affinity of tegaserod was identical to that at human recombinant 5-HT4(c) receptors (mean pKi=8.4) in human embryonic kidney-293 (HEK-293) cells stably transfected with the human 5-HT4(c) receptor. 3 Tegaserod (0.1-3 microm) inhibited 5-HT-mediated contraction of the rat isolated stomach fundus potently (pA2=8.3), consistent with 5-HT(2B) receptor antagonist activity. Tegaserod produced, with similar potency, an elevation of adenosine 3',5' cyclic monophosphate in HEK-293 cells stably transfected with the human 5-HT4(c) receptor (mean pEC50=8.6), as well as 5-HT4) receptor-mediated relaxation of the rat isolated oesophagus (mean pEC50=8.2) and contraction of the guinea-pig isolated colon (mean pEC50=8.3). 4 Following subcutaneous administration, tegaserod (0.3 or 1 mg kg(-1)) inhibited contractions of the stomach fundus in anaesthetized rats in response to intravenous dosing of alpha-methyl 5-HT (0.03 mg kg(-1)) and BW 723C86 (0.3 mg kg(-1)), selective 5-HT2B receptor agonists. At similar doses, tegaserod (1 and 3 mg kg(-1) subcutaneously) evoked a 5-HT4 receptor-mediated increase in colonic transit in conscious guinea-pigs. 5 The data from this study indicate that tegaserod antagonizes 5-HT2B receptors at concentrations similar to those that activate 5-HT4 receptors. It remains to be determined whether this 5-HT2B receptor antagonist activity of tegaserod contributes to its clinical profile.

Contribution of serotonin (5-hydroxytryptamine; 5-HT) 5-HT2 receptor subtypes to the hyperlocomotor effects of cocaine: acute and chronic pharmacological analyses.

The role of serotonin (5-hydroxytryptamine; 5-HT) 5-HT2 receptor subtypes (5-HT2AR, 5-HT2BR, and 5-HT2CR) in acute cocaine-evoked hyperactivity was compared with their contribution to the development and expression of locomotor sensitization upon repeated, intermittent treatment with cocaine (10 mg/kg/day for 5 days) in male Wistar rats. Cocaine-evoked hyperactivity was significantly enhanced by pretreatment with the preferential 5-HT2AR agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and the 5-HT2CR antagonist SDZ SER-082 [(+)-cis-4,5,7a,8,9,10,11,11a-octahydro-7H-10-methylindolo(1,7-BC)(2,6) naphthyridine fumarate]. The 5-HT2AR antagonist SR 46349B [1(Z)-[2-(dimethylamino)ethoxyimino]-1(2-fluorophenyl)-3-(4-hydroxyphenyl)-2(E)-propene] and the preferential 5-HT2CR agonist MK 212 [6-chloro-2-(1-piperazinyl)pyrazine HCl] (2 mg/kg) significantly attenuated acute cocaine-evoked hyperactivity; however, a lower dose of MK 212 (0.3 mg/kg) enhanced cocaine-evoked hyperactivity. The 5-HT2BR agonist BW 723C86 (1-[5-(2-thienylmethoxy)-1H-3-indolyl]propan-2-amine HCl) and the 5-HT2BR antagonist SB 204741 [N-(1-methyl-5-indolyl)-N'-(3-methyl-5-isothiazolyl) urea] had no effect on cocaine-evoked hyperactivity. Repeated treatment with cocaine alone resulted in a 2-fold increase in hyperactivity upon challenge with cocaine 5 days after termination of the cocaine regimen (sensitization). The 5-HT2AR antagonist SR 46349B also blocked cocaine-evoked hyperactivity following repeated cocaine treatment, whereas the other 5-HT2R ligands were ineffective. When any of the 5-HT2R ligands was coadministered with cocaine during the treatment regimen (10 mg/kg/day for 5 days), the development of sensitization was unchanged as measured by the level of cocaine-evoked hyperactivity upon challenge 5 days after termination of the treatment. The present study implies that 5-HT2AR and 5-HT2CR exert oppositional influence upon hyperactivity evoked by acute administration of cocaine; this balance is altered following repeated cocaine administration.

Vasoconstrictor Responses to 5-Hydroxytryptamine in the Autoperfused Hindquarters of Spontaneously Hypertensive Rats

In this work we studied the responses and receptors involved in the effects of intra-arterial 5-hydroxytryptamine (5-HT) in the in situ autoperfused hindquarters of spontaneously hypertensive rats (SHR). Intra-arterial administration of the highest doses (50–1,000 ng/kg) produced a vasoconstrictor effect that was inhibited by ritanserin (a selective 5-HT₂ receptor antagonist), SB 206553 (a selective 5-HT_2B/2C receptor antagonist) and spiperone (a nonspecific 5-HT_1/2A receptor antagonist), and was mimicked by α-methyl-5-HT (a selective 5-HT₂ receptor agonist) and m-CPP (a selective 5-HT_2C receptor agonist), but not by the intra-arterial administration of BW 723C86, a selective 5HT_2B receptor agonist. SB 206553 and spiperone inhibited α-methyl-5HT-induced vasoconstriction in the hindquarters of SHR. Our data suggest that the vasoconstrictor response induced by 5-HT in the autoperfused hindquarters of SHR is mainly mediated by the activation of 5-HT_2A and 5-HT_2C receptors.