Receptor Antagonist WAY-100635 Research Articles

Attenuation of CCK-induced aversion in rats on the elevated x-Maze by the selective 5-HT 1A receptor antagonists (+)WAY100135 and WAY100635

The present study determined the effect of pretreatment with “silent” selective 5-HT 1A receptor antagonists on cholecystokinin(CCK)-mediated effects on rat behaviour in the elevated x-maze model of anxiety. In the absence of 5-HT 1A receptor antagonists, non-sulphated cholecystokinin-octapeptide (CCK-8ns; 10 and 50 μg/kg, i.p.; 30 min prior to testing) produced an anxiogenic profile of behaviour on the x-maze, reducing the number of open arm entries and the number of exploratory head dips, while increasing the level of risk-assessment as measured by the number of stretched-attend postures. CCK-8ns did not, however, alter ambulatory activity. Two 5-HT 1A receptor antagonists were employed in these experiments: ( + )WAY100135 (the active enantiomer of N-tert-butyl-3-(4-(2-rnethoxyphenyl)piperzin-1-yl)-2-phenylpropronamide) and WAY 10063 5 ( N-[2-[4(2-methoxyphenyl)-1 -piperazinyIJethyl]-7 V-2-pyridinyl)cyclohexanecarbonate trihydrochloride). When administered 10 min prior to CCK-8ns, ( + )WAY100135 (1.0 mg/kg s.c.) or WAY100635 (0.03 and 0.3 mg/kg s.c.) significantly attenuated the anxiogenic profile of CCK-8ns. ( + )WAY100135 was also demonstrated to significantly inhibit postsynaptic 5-HT 1A receptor-mediated 8-OH-DPAT (8-hydroxy-2-(di-,/-Vpropylamino) tetralin)-induced 5-HT syndrome at the same dose used in the x-maze experiment. Neither ( + )WAY100135 nor WAY100635 had any effects on ambulatory activity. These results support a CCK/5-HT 1A receptor interaction in the modulation of aversion in rats exposed to the elevated x-maze.

Serotonin Upregulates Mitogen-Stimulated B Lymphocyte Proliferation through 5-HT1AReceptors

Serotonin is a well-known neurotransmitter and neuroimmunomodulator which has been reported to modulate T cell and NK cell proliferation. In this study we investigated whether serotonin could regulate mitogen-stimulated proliferation of the mature B lymphocyte. Mouse and rat spleen cells were cultured with serotonin in the presence or absence of a combination of Escherichia coli lipopolysaccharide and dextran sulfate, and proliferation was assessed by [3H]thymidine uptake or propidium iodide staining of DNA. Serotonin alone had no effect on spleen cell proliferation, while it increased mitogen-stimulated B cell proliferation in a dose- and time-dependent manner. These effects were reproduced by the selective 5-HT1A receptor agonist 8-OH-DPAT. Serotonin- or 8-OH-DPAT-induced increase in proliferation could be blocked by the 5-HT1A receptor antagonists (+)WAY 100135 and propranolol. Moreover, lipopolysaccharide-activated mouse spleen cells expressed specific binding sites for [3H]8-OH-DPAT. These results show that serotonin upregulates mitogen-stimulated B lymphocyte proliferation through 5-HT1A receptors, thus providing an important link between this neurotransmitter and the immune system.

Evidence for the involvement of the 5-HT1A receptor in CCK induced satiety in rats

The present study was designed to examine possible interactions between exogenous CCK and the 5-HT1A receptor subtype mediated serotonergic effects on feeding in rats. The somatodendritic 5-HT1A receptor agonist 8-OH-DPAT (0.32 mg/kg sc) evoked feeding in freely feeding rats. This effect was attenuated by treatment with CCK-8 (1, 5 and 25 micrograms/kg ip). In food deprived rats, CCK-8 (40 micrograms/kg ip) significantly reduced the size of a test meal. Treatment with the 5-HT1A receptor antagonist WAY-100135 (10 mg/kg ip) antagonized this anorectic effect of CCK-8. WAY-100135 on its own did not affect food intake. These results suggest the involvement of the 5-HT1A receptor subtype in mediating 5-HT-CCK interactions in the control of food intake in rats.

The selective 5-HT 1A antagonist radioligand [ 3H]WAY 100635 labels both G-protein-coupled and free 5-HT 1A receptors in rat brain membranes

The tritiated derivative of the novel silent 5-HT 1A receptor antagonist WAY 100635 [ N-(2-(4-(2-methoxyphenyl)-1-pineperazinyl)ethyl)- N-(2-pridinyl) cyclohexane carboxamide] was tested as a potential radioligand of 5-HT 1A receptors in the rat br brain. Binding assays with membranes from various brain regions showed that [ 3H]WAY 100635 specifically bound to a homogenous population of sites, with a K d of 0.10nM. The regional distribution of [ 3H]WAY 100635 specific binding sites, as assessed in membrane binding assays and by autoradiography of labelled brain sections, superimposed exactly over that of 5-HT 1A receptors specifically labelled by [ 3 H]8- hydroxy-2-( di-n- propylamino) tetralin ([ 3H]-8-OH-DPAT). Futhermore, the positive correlation ( r = 0.96 A) between the respective p K l values of a large series of ligands as inhibitors of the specific binding of [ 3H]WAY 100635 and [ 3H]8-OH-DPAT in hippocampal membranes indicated that their pharmacological properties were similar. Nevertheless, marked differences also existed between [ 3H]8-OH-DPAT and [ 3H]WAY 100635 specific binding, as the former was inhibited by 1–100 μM GTP and GppNHp, whereas the latter was enhanced by these guanic nucleotides. In contrast, Mn 2+ (1–10 mM) increased the specific binding of [ 3H]8-OH-DPAT, but inhibited that of [ 3H]WAY 100635. Treatment of membranes with N-ethylmalcimide (1–5 mM) markedly reduced their capacity to specifically bind [ 3H]8-OH-DPAT, but slightly increased (at 1 mM) or did not affect (at 5 mM) their [ 3H]WAY 100635 specific binding capacity. Finally, the B max of [ 3H]WAY 100635 specific binding sites was regularly 50–60% higher than that of [ 3H]8-OH-DPAT in the same membrane preparations from various brain regions (hippocampus, septum, cerebral cortex). These data are compatible with the idea that whereas [ 3H]8-OH-DPAT only binds to G-protein-coupled 5-HT 1A receptors, [ 3H]WAY 100635 is a high affinity ligand of both G-protein-coupled and free 5-HT 1A receptor binding subunits in brain membranes.

Inhibition of 8-OH-DPAT-induced elevation of plasma corticotrophin by the 5-HT1A receptor antagonist WAY100635.

Numerous studies have demonstrated the stimulatory effect of 5-HT1A receptor agonists, such as 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), on plasma corticotrophin (ACTH) levels in the rat. However, until recently the lack of a selective 5-HT1A receptor antagonist has hampered mechanistic studies in this area. In this study we examined the effects of the selective 5-HT1A receptor antagonist N-[2-[4-(2-methoxyphenyl)-1- piperazinyl]ethyl]-N-(2-pyridinyl) cyclohexanecarboxamide trihydrochloride (WAY100635) on plasma ACTH levels and on the elevation of ACTH induced by the 5-HT1A receptor agonist 8-OH-DPAT in the conscious rat. The basal plasma ACTH level was 41.0 +/- 1.8 pg/ml. 8-OH-DPAT increased ACTH levels at doses of 100 and 300 micrograms/kg with maximum increases of 551 and 546% respectively occurring 10 min post-injection. WAY100635 had no effects per se on plasma ACTH at doses up to 100 micrograms/kg, indicating it has no 5-HT1A receptor agonist properties. WAY100635 dose-dependently blocked the elevation of ACTH induced by 8-OH-DPAT, the minimum effective dose being 10 micrograms/kg. The present results indicate that 8-OH-DPAT elevates plasma ACTH levels by stimulating 5-HT1A receptors, a conclusion that is consistent with the findings of previous studies using non-selective 5-HT1A receptor antagonists such as pindolol.

Dopaminergic regulation of the serotonergic raphe-striatal pathway: microdialysis studies in freely moving rats

Morphological evidence demonstrates the existence of dopaminergic afferent pathways and dopamine (DA)-containing neurons in the dorsal raphe nucleus (DRN). In a recent report, a DA D2-like receptor-mediated regulation of serotonin (5-HT) extracellular concentration in DRN has been found. Given the existence of somatodendritic 5-HT1A autoreceptors in the DRN, changes of the extracellular concentration of 5-HT in the vicinity of cell bodies and dendrites may be relevant for the control of the activity of ascending serotonergic pathways. In the present brain microdialysis study we have used a chromatographic method (HPLC) enabling the simultaneous measurement of DA, 5-HT, and their main metabolites dihydroxyphenylacetic acid (DOPAC) and 5-hydroxyindoleacetic acid (5-HIAA). The presence of a neuronal pool of DA within the DRN was revealed by the local infusion of amphetamine (10 microM), which significantly increased the extracellular concentration of both amines. The local striatal infusion (10 microM) of the selective DA D1-like agonist SKF-38393, the selective DA D2-like agonist quinpirole (LY 171,555), or the nonselective DA agonist apomorphine markedly decreased DA and DOPAC extracellular concentrations and failed to modify 5-HT or 5-HIAA in the striatum, indicating the lack of terminal (striatal) control of 5-HT release by dopaminergic transmission. In contrast, the systemic administration of apomorphine (2.8 mumol/kg, s.c.) significantly increased the extracellular concentration of 5-HT in the DRN and decreased it in the striatum. The reduction of striatal 5-HT extracellular concentration was prevented by the previous administration of the selective 5-HT1A receptor antagonist WAY 100135 (17.6 mumol/kg, s.c.), which by itself did not change extracellular 5-HT in striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of the selective 5-HT 1A receptor antagonist WAY100135 and its enantiomers on 8-OH-DPAT-induced hyperglycaemia in conscious rats

8-Hydroxy-2-(di- n-propylamino)tetralin (8-OH-DPAT) increases plasma glucose levels in conscious rats probably by stimulation of central 5-HT 1 receptors. We have examined the effects of WAY100135 ( N-tert-butyl-3-[4-(2-methoxyphenyl)piperazin-1-yl]-2- phenylpropanamide), a selective 5-HT 1A receptor antagonist and its enantiomers on plasma glucose levels and on the hyperglycaemia induced by 8-OH-DPAT. ( R, S)-WAY100135 (minimum effective dose (MED) 3 mg/kg i.v.) and ( S)-WAY100135 (MED 1 mg/kg i.v.) dose-dependently attenuated 8-OH-DPAT-induced hyperglycaemia. In contrast, ( R)-WAY100135 at doses up to 3 mg/kg i.v. was unable to block hyperglycaemia induced by 8-OH-DPAT. When the antagonist were examined for intrinsic effects on plasma glucose levels only ( S)-WAY100135 (3 mg/kg i.v.) caused a significant but transient hyperglycaemia (20% increase). These results are consistent with previous suggestions that ( R, S)-WAY100135 and ( S)-WAY100135 are selective 5-HT 1A receptor antagonist and that 8-OH-DPAT-induced hyperglycaemia is mediated by 5-HT 1A receptors. The antagonist action of WAY100135 is stereoselective, the more potent activity being observed with the ( S) enantiomer.

Neurochemical profile of the selective and silent 5-HT 1A receptor antagonist WAY100135: an in vivo microdialysis study

The neurochemical profile of the selective 5- HT 1A receptor antagonist WAY100135 [ N- tert- butyl 3-4-(2- methoxyphenyl) piperazin-1- yl-2- phenylpropanamide dihydrochloride] and its enantiomers at the somatodenditic 5- HT 1 A eceptor was determined by studying the effects of these compounds on 5-HT (5-hydroxytryptamine, serotonin) release in the rat hippocampus using in vivo microdialysis. (±)-WAY100135, (+)-WAY100135 and (−)-WAY100135 (all at 10 mg/kg s.c.) had no significant effect on extracellular levels of 5-HT in the hippocampus demonstrating that these compounds are devoid of 5-HT 1A receptor agonist properties. In contrast, the 5-HT 1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) (0.1 mg/kg s.c.) and the partial agonists BMY 7378 (1.0 mg/kg s.c.) and buspirone (5 mg/kg s.c.) significantly decreased hippocampal 5-HT. Pretreatment with (±)-WAY100135 (at 10 mg/kg s.c.) and (+)-WAY100135 (at 1.0–10 mg/kg s.c.) completely blocked the 8-OH-DPAT-induced decrease in 5-HT release demonstrating that these compounds are antagonists at the somatodendritic 5- HT 1 A autoreceptor. (−)-WAY100135 at a dose of 10 mg/ kg s. c. had no significant effect on the 8- OH- DPAT- induced inhibition of 5- HT release.(±)-WAY100135 had no significant effect on extracellular levels of dopamine in the rat hippocampus but significantly increased extracellular levels of noradrenaline. The mechanism underlying the increase in noradrenaline is unknown at present. These data suggest that WAY100135 is a silent and selective receptor antagonist at the somatodendritic 5-HT 1A receptor with activity residing in the (+)-enantiomer. In addition, these findings confirm that WAY100135 is likely to be a useful tool for further investigation of 5-HT 1A receptor function.

Effect of repeated neuroleptic treatment on spontaneously active dopamine neurons in the VTA and SNC area in rats. Correlation to antipsychotic activity in the clinic?

The mechanism by which the psychostimulant methamphetamine (METH) increases locomotor activity may be attributable to indirect activation of serotonin (5-HT) and dopamine (DA) receptors. In the present study, the ability of the serotonin reuptake inhibitor fluvoxamine, 5-HT1A, 5-HT1B, 5-HT2A and 5-HT2C receptor antagonists WAY100635, GR127935, M100907 and SB242084, and the 5-HT2C receptor agonists WAY163909 and Ro 60-0175 or the 5-HT synthesis inhibitor para-chlorophenylalanine (pCPA) to alter METH-induced hyperactivity was analysed. Further, for comparative purposes, the involvement of the DA D1 and D2 receptor antagonists SCH23390 and haloperidol, D2 partial agonists terguride, (−)3PPP and aripiprazole and finally clozapine were assessed. Doses of pCPA that attenuated 5-HT levels reduced METH activity. The 5-HT1B antagonist GR127935 had no effect on METH-induced locomotor activity but blocked that induced by MDMA. The 5-HT1A antagonist WAY100635 reduced activity but this did not reach significance. In contrast, M100907 (minimal effective dose; MED = 0.125 mg/kg), WAY163909 (MED = 3 mg/kg), Ro 60-0175 (MED = 3 mg/kg), haloperidol (MED = 0.1 mg/kg), clozapine (MED = 5 mg/kg), aripiprazole (MED = 1 mg/kg), (−)3PPP (MED = 3 mg/kg), terguride (MED = 0.2 mg/kg) and SCH23390 (MED = 0.001325 mg/kg) attenuated METH-induced locomotor activity. Administration of 20 mg/kg fluvoxamine attenuated, while SB242084 (MED = 0.25 mg/kg) potentiated METH-induced activity.These results contribute significantly to the understanding of the mechanism of action of this psychostimulant and suggest for the first time, that METH-induced locomotor stimulation is modulated by 5-HT2A and 5-HT2C receptors, but demonstrate that 5-HT1B receptors are not directly involved. The involvement of the dopaminergic system was also demonstrated.

Microdialysis of rat preoptic area: evidence that noradrenaline (NA) stimulates release of GABA : A.E. Herbison, R.P. Heavens and R.G. Dyer. Department of Neuroendocrinology, AFRG Institute of Animal Physiology and Genetics Research, Babraham, Cambridge CB2 4AT, UK

In this study, we use in vivo microdialysis to investigate the influence of endogenous serotonin (5-HT) on striatal dopamine (DA) and 5-hydroxyidoleacetic acid (5-HIAA) efflux in both basal and activated conditions. The selective serotonin reuptake inhibitors citalopram and fluoxetine were used to mobilize endogenous 5-HT.In halothane-anaesthetized rats, citalopram (5 mg/kg, i.p.), administered either alone or in combination with the 5-HT1A receptor antagonist WAY 100635 (0.1 mg/kg, s.c.), while reducing striatal 5-HIAA outflow (−25 and −15%, respectively), had no effect on basal DA output. When locally applied into the striatum, citalopram had no effect at 1 μM concentration, but enhanced DA release after its perfusion at 25 and 100 μM concentrations (+27% and +67%, respectively). However, the injection of the neurotoxin 5,7-dihydroxytryptamine into the dorsal raphe nucleus, which markedly depleted 5-HT in the striatum, failed to modify the effect of 25 μM citalopram.In freely-moving rats, the intrastriatal infusion of citalopram or fluoxetine (1 μM each), had no effect on its own, but significantly enhanced the increase in DA outflow induced by the subcutaneous administration of 0.01 mg/kg haloperidol (+31% and +30% for citalopram and fluoxetine, respectively).These findings indicate that, in the striatum, endogenous 5-HT has no influence on DA release under basal conditions, but positively modulates DA outflow when nigro-striatal DA transmission is activated.

THE ARTIFICALLY PRODUCED RETINAL PULSE

Both orexin and serotonin (5-HT) have important roles in the regulation of sleep-wakefulness, as well as in feeding behavior. We examined the effects of 5-HT on orexin/hypocretin neurons, using hypothalamic slices prepared from orexin/enhanced green fluorescent protein (EGFP) transgenic mice in which EGFP is expressed exclusively in orexin neurons. Patch-clamp recording from EGFP-expressing cells showed that 5-HT hyperpolarized all orexin neurons in a concentration-dependent manner. The response was inhibited by the 5-HT_1A receptor antagonist WAY100635. A 5-HT_1A receptor agonist, 8-hydroxy-2-(dl-<i>N</i>-propyl-amino)tetralin, also evoked hyperpolarization on orexin neurons with potency comparable with 5-HT. A low concentration of Ba²⁺ (30 μm) inhibited 5-HT-induced hyperpolarization. Single-channel recording revealed that the conductance of 5-HT-induced channel activity was 33.8 pS, which is in good agreement with that of the G-protein-coupled inward rectifier potassium channel (GIRK). Moreover, 5-HT_1A receptor-like immunoreactivity was observed on orexin neurons, and 5-HT transporter immunoreactive nerve endings are in close apposition to orexin neurons. Intracerebroventricular injection of the 5-HT_1A receptor-selective antagonist WAY100635 (100 ng) increased locomotor activity during the latter half of dark phase in wild-type mice but not in <i>orexin/ataxin-3</i> mice in which orexin neurons are specifically ablated, suggesting that activation of orexin neurons is necessary for the WAY100635-induced increase in locomotor activity. These results indicate that 5-HT hyperpolarizes orexin neurons through the 5-HT_1A receptor and subsequent activation of the GIRK and that this inhibitory serotonergic input to the orexin neurons is likely to be important for the physiological regulation of this neuropeptide system.