Erik H.F. Wong

Central 5-HT4 receptors

Activation of the 5-HT4 receptor mediates widespread effects in central and peripheral nervous systems. Recent developments, such as the identification of novel, selective agonists and antagonists, as well the cloning of the receptor, have provided insights into the physiological role of the receptor. In this article, Richard Eglen and colleagues assess the emerging evidence relating to the function of the 5-HT4 receptor in the brain. The cerebral distribution of the receptor, along with neurochemical and electrophysiological data, suggests a role in cognition. The role of the receptor in modulation of dopamine transmission and anxiolysis is also addressed.

The Effects of Novel, Selective 5-Hydroxytryptamine (5-HT)4 Receptor Ligands in Rat Spatial Navigation

Activation of central 5-hydroxytryptamine (5-HT4) receptors may enhance cognitive performance. In the present study, the effects of two novel, potent and selective 5-HT4 receptor agonists, RS 67333 (1-(4-amino-5-chloro-2-methoxyphenyl)-3-(1-n-burtl-4-piperidinyl)- 1-propanone) and RS 67506 (1-(4-amino- 5-chloro-2-methoxyphenyl)-3-[1-[2-[(methylsulfonyl)amino]ethyl]-4- piperidinyl]-1-propanone), were studied in a rat model of spatial learning and memory; the Morris water maze. RS 67333 (0.1, 10 and 1000 micrograms/kg, intraperitoneally (i.p.)), a highly potent, selective and hydrophobic 5-HT4 receptor agonist, reversed the decrements in cognitive performance induced by atropine (30 mg/kg, i.p.). By contrast, no effect was seen to RS 67506 (0.1, 10 and 1000 micrograms/kg, i.p.), a hydrophilic 5-HT4 receptor agonist, of equivalent potency and selectivity to RS 67333. This differential effect may reflect the enhanced ability of RS 67333 to enter the CNS, with respect to RS 67506. The ameliorative actions of RS 67333 on cognitive dysfunction were abolished by prior treatment with a selective 5-HT4 receptor antagonist, RS 67532 [1-(4-amino-5-chloro-2-(3, 5-dimethoxy benzyloxyphenyl)-5-(1-piperidinyl)-1-pentanone; 1 mg/kg, i.p.]. When given alone, or in naive rats, RS 67532 (0.1, 10 and 1000 micrograms/kg, i.p.), was without effect. None of the compounds tested affected the swim speed at any of the doses used. In separate locomotor studies, RS 67532 reduced activity at 1 and 10 mg/kg, i.p., although no effect was seen with RS 67333 or RS 67506 (0.01-10 mg/kg, i.p.). These data suggest that RS 67333 reversed the cognitive deficit induced by atropine and support a role of 5-HT4 receptors in rat spatial learning and memory.

Quantitative autoradiography of 5-HT4 receptors in brains of three species using two structurally distinct radioligands, [3H]GR113808 and [3H]BIMU-1

Recent radioligand binding studies have demonstrated the presence of 5-HT4 receptors throughout the nigrostriatal and mesolimbic systems of mammalian brain. In many regions, the binding has not yet been correlated with functional responses. The present study was carried out to fully characterize the regional distribution of 5-HT4 receptors in brain sections from three species using two structurally distinct radioligands, [3H]GR113808, and [3H]BIMU-1. The highest density of 5-HT4 receptors labeled with [3H]GR113808 was found in the olfactory tubercle, substantia nigra, ventral pallium and striatum of rat and guinea pig, and similar regions of pig-tail macaque monkey. A similar distribution of 5-HT4 receptors was observed in guinea pig brain using [3H]BIMU-1. With either ligand, the binding was saturable and of high affinity (Kd = 0.08-0.53 nM for [3H]GR113808; 1.4-3.0 nM for [3H]BIMU-1). These results extend previous distribution studies, confirm the heterogenous distribution of 5-HT4 receptors throughout the nigrostriatal and mesolimbic systems of three species, and demonstrate a similar distribution using two structurally distinct 5-HT4 radioligands.

Enhanced delayed matching performance in younger and older macaques administered the 5-HT4 receptor agonist, RS 17017.

Abstract Recent evidence indicates that the 5-HT4 subtype of serotonin receptor may modulate central cholinergic activity in regions of the mammalian CNS important to memory such as the frontal cortex, hippocampus and amygdala. These receptors could represent targets for drugs designed for the symptomatic therapy of Alzheimer’s disease (AD) and other disorders of memory. In the present study, the binding activity of RS 17017 (previously described as a selective 5-HT4 agonist) was assessed across a number of neurotransmitter receptors and binding sites, pharmacokinetic data were obtained, and the compound was evaluated in macaques for mnemonic effects via a computer-assisted delayed matching-to-sample task (DMTS). Binding data confirmed the 5-HT4 selectivity of the compound, while pharmacokinetic results revealed low oral bioavailability, but a large volume of distribution of the compound. Significant and reproducible improvements in DMTS accuracy were observed after oral administration of the compound across a dose-effect series in both younger and older monkeys. The results suggest that RS 17017 offers a potential for memory enhancement in disorders involving cognitive decline, and are consistent with a role for central 5-HT4 receptors in memory. Improvements in DMTS performance in aged monkeys may have particular implications for neurodegenerative conditions such as AD, whereas positive results in the younger monkeys indicate that RS 17017 (or similar compounds) may have additional potential in the therapeutics of memory disorders not necessarily associated with advanced age.

The effect of the 5-HT3 receptor antagonist, RS-42358-197, in animal models of anxiety

The S-isomer of the novel 5-HT3 receptor antagonist RS-42358 ((S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-2,4,5,6-tetrahydro-1-H- benzo[de]isoquinolin-1-one, RS-42358-197) disinhibited behaviour in the mouse suppressed by the aversive situation of the light/dark test box. RS-42358-197 was effective at sub-ng/kg dose levels and the efficacy was maintained over a 100 million-fold dose range. In contrast, the R-isomer was ineffective at all doses studied. The S-isomer also disinhibited a suppressed behaviour in social interaction and elevated X-maze tests in the rat and reduced anxiety-related behaviours in a marmoset human threat test. RS-42358-197 prevented the exacerbation of the suppression of behaviour in the mouse light/dark test following withdrawal from treatment with alcohol, nicotine, cocaine and diazepam. Thus, the S-isomer of RS-42358 has a consistent non-sedating anxiolytic profile in rodent and primate models. It is exceptionally potent and a maintained efficacy at high doses distinguishes its actions from many other 5-HT3 receptor antagonists.

Ondansetron improves cognitive performance in the Morris water maze spatial navigation task.

In the present studies we investigated the actions of ondansetron, a prototypic 5-hydroxytryptamine3 (5-HT3) receptor antagonist, on performance in a complex spatial navigation/memory task in rats. Specifically, we compared the activity of ondansetron to that of the cholinesterase inhibitor physostigmine in attenuating two distinct cognitive deficits in the Morris water maze. In the first model, rats treated with the muscarinic receptor antagonist atropine (30 mg/kg) had significantly longer latencies to find the submerged platform across two days of testing. Physostigmine (0.03, 0.1 and 0.3 mg/kg) and ondansetron (0.03–1 mg/kg) significantly reduced the latencies to find the submerged platform in atropine-treated animals, suggesting an increase in cognitive performance. There was little evidence of a dose-response relationship for either compound, and a loss of efficacy for ondansetron was seen at 3 mg/kg. In the second model, pre-screened, aged (23 months), cognition-impaired and nonimpaired rats were tested. Ondansetron (0.1 mg/kg), but not physostigmine (0.1 mg/kg), decreased the latencies to find the submerged platform in the aged-impaired rats, while neither compound improved performance of aged-nonimpaired rats. These data suggest that ondansetron may have cognition enhancing properties in animal models of aging and cholinergic hypofunction.

Pharmacological Characterization of 5‐Hydroxytryptamine3Receptors in Murine Brain and Ileum Using the Novel Radioligand [3H]RS‐42358–197: Evidence for Receptor Heterogeneity

Abstract: Previous studies have demonstrated species‐specific differences in 5‐hydroxytryptamine3 (5‐HT3) receptors, but unequivocal evidence of 5‐HT3 receptor subtypes, within a species, has not yet been obtained. The purpose of the current study was to test for heterogeneity in 5‐HT3 receptors in murine tissues. 5‐HT3 receptors in membranes derived from brain cerebral cortex of CD‐1, C57BI/6, and Swiss Webster mice and ileum of CD‐1 mice were labeled with the 5‐HT3 receptor antagonist [3H]RS‐42358–197. Structurally diverse competing ligands were then used to characterize the binding site. [3H]RS‐42358‐197 bound with similar affinity in each of the cortical tissues (mean KD= 0.14 nM; range, 0.06–0.32 nM) but bound with lower affinity in ileal tissue (2.5 nM). The density of sites labeled with [3H]RS‐42358–197 ranged from 10.4 fmol/mg of protein in Swiss Webster mouse cortex to 44.2 fmol/mg of protein in Sprague‐Dawley rat cortex. Displacing ligands produced a pharmacologic profile of the [3H]RS‐42358–197 binding site consistent with it being a 5‐HT3 receptor: (R)‐YM060 > (S)‐zacopride > (R)‐zaco‐pride > MDL 72222 > 2‐methyl‐5‐HT. However, 10‐fold differences in the affinity of certain ligands were found when comparing 5‐HT3 binding sites in membranes from cerebral cortex of the different strains of mice and when comparing 5‐HT3 binding sites in brain and ileal membranes prepared from the CD‐1 mouse strain. Ligands demonstrating selectivity included RS‐42358–197, (R)‐za‐copride, 1‐(m‐chlorophenyl) biguanide, (R)‐YM060, and MDL 72222. These studies demonstrate tissue‐and strain‐dependent differences in murine 5‐HT3 binding sites. This suggests that 5‐HT3 receptors exist as multiple subtypes within species and that subtype‐selective 5‐HT3 ligands may be identified.

5-HT3 receptor ligands lack modulatory influence on acetycholine release in rat entorhinal cortex

SummaryThe objective of this study was to explore the role of 5-HT3 receptors in modulating potassium (K+)-evoked release of [3H]-acetylcholine ([3H]-ACh) from superfused slices of rat entorhinal cortex previously loaded with [3H]-choline. Rat entorhinal cortices were cross-chopped into 300 μm slices, superfused with oxygenated Krebs buffer containing 2.5 mmol/1 Ca2+ and stimulated with two consecutive exposures of 20 mmol/l K+ for 4 min (S1 and S2, respectively). Compounds were added 20 min before S2 stimulation and remained in the superfusion buffer for the duration of the experiment. The S2/S1 ratio was then calculated.Stimulated release of [3H]-ACh was dependent on extracellular Ca2+ and K+ concentration. In Sprague Dawley rats, 2-methyl-5-HT (10-9−10-6 mol/l), in the presence of 1 μmol/l ritanserin or 1 gmmol/l ondansetron, had no influence on K+-evoked release of [3H]-ACh. In slices prepared from Hooded Lister rats, 2 μmol/l 5-HT but not 2-Me-5-HT significantly (P<0.05) inhibited K+-evoked [3H]-ACh release only 17% in the presence of 1 μmol/l ritanserin. However, 2 μmol/l 2-Me-5-HT plus 1 nmol/l ondansetron had no effect. High performance liquid chromatography coupled to electrochemical detection (HPLC-ECD) was used to monitor endogenous release of ACh in the above conditions to confirm data from the radiolabelled experiments. No significant inhibition or increase in K+-evoked ACh release was observed with either 5-HT3 receptor agonists or antagonists. 2-Me-5-HT (10−9 – 10−5 mol/l) or 1-(m-chlorophenyl)-biguanide (10−9 – 10−5 mol/l), when added simultaneously at the S2 stimulation, in the presence of 1 όl/l methysergide, also showed no effect on [3H]ACh release.In entorhinal cortex slices from aged Wistar rats, neither 1-(m-chlorophenyl)-biguanide (2 or 10 μol/l) nor 2-Me-5-HT (2 μmol/l) in combination with ritanserin (1 μmol/l) or ondansetron (1 nmol/l) elicited any effect on K+-evoked [3H]-ACh release. However, release of [3H]-ACh was inhibited by carbachol (10 μmol/l) and adenosine (10 μmol/l). DuP 996 (3,3-bis(4- pyridinyl-methyl)-1-phenylindolin-2-one) (10−7 – 10−5 mol/l), a known releaser of ACh, markedly augmented K+-evoked [3H]-ACh release.These studies have failed to confirm the postulated role of 5-HT3 receptors in modulating cortical ACh release in rat entorhinal cortex slices and suggest that a critical reexamination of the interaction of 5-HT3 receptor and cortical cholinergic function needs to be addressed.

Labelling of 5‐Hydroxytryptamine3 Receptors with a Novel 5‐HT3 Receptor Ligand, [3H]RS‐42358–197

Abstract: RS‐42358–197{(S)‐N‐(1‐azabicyclo[2.2.2]oct‐3‐yl)‐2,4,5,6‐tetrahydro‐1H‐benzo[de]isoquinolin‐1‐one hydrochloride} displaced the prototypic 5‐hydroxytryptamine3 (5‐HT3) receptor ligand [3H]quipazine in rat cerebral cortical membranes with an affinity (pKi) of 9.8 ± 0.1, while having weak affinity (pKi < 6.0) in 23 other receptor binding assays. [3H]RS‐42358–197 was then utilized to label 5‐HT3 receptors in a variety of tissues. [3H]RS‐42358–197 labelled high‐affinity and saturable binding sites in membranes from rat cortex, NG108–15 cells, and rabbit ileal myenteric plexus with affinities (KD) of 0.12 ± 0.01, 0.20 ± 0.01, and 0.10 ± 0.01 nM and densities (Bmax) of 16.0 ± 2.0, 660 ± 74, and 88 ± 12 fmol/mg of protein, respectively. The density of sites labelled in each of these tissues with [3H]RS‐42358–197 was similar to that labelled with [3H]GR 65630, but was significantly less than that found with [3H]‐quipazine. The binding of [3H]RS‐42358–197 had a pharmacological profile similar to that of [3H]quipazine, as indicated by the rank order of displacement potencies: RS‐42358–197 > (S)‐zacopride > tropisetron > (R)‐zacopride > ondansetron > MDL72222 > 5‐HT. However, differences in 5‐HT3 receptors of different tissues and species were detected on the basis of statistically significant differences in the affinities of phenylbiguanide, and 1‐(m‐chlorophenyl)biguanide when displacing [3H]RS‐42358‐197 binding. [3H]RS‐42358–197 also labelled a population (Bmax= 91 ± 17 fmol/mg of protein) of binding sites in guinea pig myenteric plexus membranes, with lower affinity (KD= 1.6 ± 0.3 nM) than those in the other preparations. Moreover, the rank order of displacement potencies of 15 5‐HT3 receptor ligands in guinea pig ileum was found not to be identical to that in other tissues. Binding studies carried out with [3H]RS‐42358–197 have detected differences in 5‐HT3 receptor binding sites in tissues of different species and further underscore the unique nature of the guinea pig 5‐HT3 receptor.

Comparison of 5-HT4 receptors in guinea-pig colon and rat oesophagus: effects of novel agonists and antagonists

Abstract5-HT4 receptors in isolated distal colon myenteric plexus of guinea-pig, mediating contraction of longitudinal smooth muscle, have been further characterized by selective agonists and antagonists. The indole agonists, 5-HT and 5-methoxytryptamine (5-MeOT), were full agonists (relative to 5-HT) with potency values (pEC50) of 8.0 ± 0.1 (n = 50) and 7.8 ± 0.1 (n = 12), respectively. 5-HT4 receptor agonists of other structural classes, including benzimidazolones (BIMU 1 and BIMU 8), and benzamides ((S)-zacopride, (R)-zacopride, renzapride, SC 49518) were partial agonists with intrinsic activities less than that of 5-HT. In general, the potencies for these compounds at 5-HT4 receptors in guinea-pig colon were similar to the potencies seen in the rat isolated oesophagus, where 5-HT4 receptors mediate relaxation.GR 113808 {[1-[2-[(methylsulfonyl)amino]ethyl]-4-piperidinyl]methyl1-methyl-1H-indole-3-carboxylat}, RS 39604 {1-[4-amino-5-chloro-2-(3,5-dimethoxybenzyloxy)phenyl]-3-[1-[2-[(methylsulfonyl)amino] ethyl]-4-piperidinyl]-1-propanone hydrochloride and SB 204070 {(1-n-butyl-4-piperidinyl)methyl 8-amino-7-chloro-1,4-benzodioxane-5-carboxylate} antagonized 5-HT responses with pA2 values of 9.1 ± 0.1, 9.0 ± 0.2 and 11.0 ± 0.1, respectively. These affinity values were similar to those obtained at 5-HT4 receptors in isolated rat oesophagus (9.0 ± 0.4, 9.3 ± 0.1 and 10.6 ± 0.1, respectively).Despite these operational similarities between 5-HT4 receptors in guinea-pig colon and rat oesophagus, several novel compounds have revealed important differences between 5-HT4 receptors in the two tissues. For example, the substituted benzoate, RS 23597 {3-(piperidine-1-yl) propyl-4-amino-5-chloro-2-methoxybenzoate hydrochloride, acted as a partial agonist (intrinsic activity 0.5) in guinea-pig colon with a potency of 7.6 ± 0.1 (n = 16). In isolated rat oesophagus, however, this compound was a surmountable antagonist (pA2 = 7.8 ± 0.1) with no intrinsic activity. In contrast, the substituted naphthalimide (S)RS 56532 {(S)6-amino-5-chloro-2-(1-azabicyclo[2, 2, 2]octan-3-yl)2,3-dihydro-1H-benz isoquinoline-1,3-dione hydrochloride}, was a potent (pEC50 = 7.9 ± 0.1), efficacious partial agonist (intrinsic activity = 0.8) in the rat oesophagus. However, in guinea-pig colon, it was a surmountable antagonist with an affinity (pKB) of 9.4 ± 0.1. Furthermore, several novel, selective, 5-HT4 compounds also showed opposing patterns of intrinsic activities similar to those described for RS 23597 and (S)RS 56532.It is concluded that these differences are inconsistent with differences in 5-HT4 receptor reserves, and may suggest that 5-HT4 receptors in the guinea-pig colon and the rat oesophagus can be operationally distinguished.