Brian J. Jones

Binding of the 5-HT3 ligand, [3H]GR65630, to rat area postrema, vagus nerve and the brains of several species

The radiolabelled derivative of the potent and selective 5-HT3 receptor antagonist, GR65630, has been shown to label 5-HT3 receptors in homogenates of rat entorhinal cortex. We now report on the specific binding of this radioligand in homogenates of 16 discrete brain areas from several species and nine areas of human brain. We have further characterised specific binding to homogenates of rat area postrema and vagus nerve. In all species examined (rat, mouse, rabbit and ferret) the highest level of specific [3H]GR65630 (0.2 nM) binding was found in homogenates of the area postrema (26-83 fmol/mg protein). Binding in forebrain areas varied with species, Rat and mouse brain had relatively high levels of binding in cortical areas. The ferret and rat had relatively high levels in forebrain dopamine-containing areas. Of the areas of human brain examined the amygdala showed the highest level of specific [3H]GR65630 (0.2 nM) binding (3.0 fmol/mg protein). Specific [3H]GR65630 (0.05-2 nM) binding to homogenates of rat vagus nerve and area postrema was saturable (Bmax: vagus nerve 89.1 fmol/mg protein, area postrema 44.4 fmol/mg protein) and of high affinity (Kd: vagus nerve 0.50 nM, area postrema 0.24 nM). Selective 5-HT3 receptor agonists and antagonists potently inhibited [3H]GR65630 (0.2 nM) binding to homogenates of rat area postrema and vagus nerve. There was a close correlation between drug affinities in each area.

5-HT3 receptor antagonists injected into the area postrema inhibit cisplatin-induced emesis in the ferret.

1 The purpose of the present study was to identify and investigate the role of 5‐hydroxytryptamine3 (5‐HT3) receptors in the area postrema in the control of cisplatin‐induced emesis in the ferret. 2 Homogenate binding and autoradiography experiments using the high affinity 5‐HT3 receptor ligand, [3H]‐GR65630, identified the presence of a high concentration of 5‐HT3 receptors in the area postrema of the ferret. 3 Intraperitoneal injection of the 5‐HT3 receptor antagonists, GR38032F, GR65630A and MDL72222, at doses of 1, 0.1 and 1 mg kg−1 respectively, inhibited emesis induced by cisplatin, 9 mg kg−1 i.p. 4 Discrete injection of low doses of the 5‐HT3 receptor antagonists directly into the area postrema region also inhibited cisplatin‐induced (9 mg kg−1 i.p.) emesis. The dose ranges used were: GR38032F, 0.01–1 μg; GR65630A, 0.001–0.1 μg; MDL72222, 0.1–10 μg. 5 Cisplatin‐induced emesis was not inhibited by discrete injection of ketanserin (30 μg) or methiothepin (30 μg) into the area postrema. Injection of the 5‐HT3 receptor agonist, 2‐methyl‐5‐HT, directly into the area postrema produced an incomplete emetic response. 6 These results confirm a role of 5‐HT, and in particular 5‐HT3 receptors, in the control of cisplatin‐induced emesis, and show that at least one functional site for these receptors in modulating the emetic response is the area postrema, the locus of the chemoreceptor trigger zone.

SB-224289–a novel selective (human) 5-HT1B receptor antagonist with negative intrinsic activity

1 Human 5‐HT1B (h5‐HT1B) and human 5‐HT1D (h5‐HT1D) receptors show remarkably similar pharmacology with few compounds discriminating the receptors. We report here on a novel compound, SB‐224289 (1′‐Methyl‐5‐[[2′‐methyl‐4′‐(5‐methyl‐1,2,4‐oxadiazol‐3‐yl)biphenyl‐4‐yl]carbonyl]‐2,3,6,7‐tetrahydrospiro [furo [2,3‐f]indole‐3,4′‐piperidine] oxalate), which has high affinity for h5‐HT1B receptors (pK1=8.16±0.06) and displays over 75 fold selectivity for the h5‐HT1B receptor over all other 5‐HT receptors including the h5‐HT1D receptor and all other receptors tested thus far. 2 Functional activity of SB‐224289 was measured in a [35S]GTPγS binding assay on recombinant h5‐HT1B and h5‐HT1D receptors expressed in Chinese Hamster Ovary (CHO) cells. SB‐224289 displayed negative intrinsic activity at both receptors with higher potency at h5‐HT1B receptors. SB‐224289 caused a rightward shift of agonist concentration response curves consistent with competitive antagonism and generated affinities comparable with those obtained from competition radioligand receptor binding studies. 3 SB‐224289 potentiated [3H]5‐HT release from electrically stimulated guinea‐pig cerebral cortical slices to the same extent as as the non‐selective 5‐HT1 antagonist methiothepin. SB‐224289 also fully reversed the inhibitory effect of exogenously superfused 5‐HT on electrically stimulated release. 4 Using SB‐224289 as a tool compound, we confirm that in guinea‐pig cerebral cortex the terminal 5‐HT autoreceptor is of the 5‐HT1B subtype.

The distribution of specific binding of the 5-HT3 receptor ligand [3H]GR65630 in rat brain using quantitative autoradiography

We have examined the distribution of specific binding of the 5-HT3 receptor ligand [3H]GR65630 to rat brain using quantitative autoradiography. Brain tissue was frozen and thin sections (20 microns) cut. Slide-mounted tissue sections were incubated with [3H]GR65630 (0.2 nM) in the presence or absence of metoclopramide (30 microM, to define non-specific binding). After drying, sections were apposed to photographic film for 3 months. Films were quantified for radioactivity using image analysis. The highest level of specific binding was found in the area postrema (34.0 fmol/mg tissue). Specific binding was high in intermediate layers of the cortex, particularly the entorhinal, temporal and pyriform cortex (5.2-7.0 fmol/mg tissue). Amongst sub-cortical areas, binding was evident in the olfactory lobes, olfactory tubercle, hippocampus and basolateral amygdala (1.3-3.3 fmol/mg tissue). Specific binding was below measurable levels in other sub-cortical areas.

Evidence that the amygdala is involved in the disinhibitory effects of 5-HT3 receptor antagonists

The effects of various 5-HT3 receptor antagonists were examined in the social interaction (SI) test following discrete microinjection into either the dorsal raphe nucleus (DRN) or amygdala of the rat. Following DRN injection, ondansetron, ICS205-930, and MDL72222 (5–500 ng) all failed to modify SI under high light/unfamiliar (HLU) test conditions relative to vehicle pretreated controls. The 5-HT3 receptor agonist, 2-Me 5-HT (100–2500 ng), was similarly ineffective under both HLU and low light/familiar (LLF) conditions, although 5-HT (20–100 ng) increased SI under the HLU paradigm. After amygdaloid injection, ondansetron (10–100 ng), granisetron (1–10 ng), ICS205-930 (10–100 ng), GR 65630 (1–10 ng), and MDL72222 (100–1000 ng) all significantly increased SI under the HLU but not LLF condition. Furthermore, a detailed behavioural analysis revealed that the behaviours underlying this increase were similar to those seen in vehicle pretreated animals tested in the LLF compared to HLU condition. The benzodiazepine, flurazepam (200 ng), increased both SI (HLU condition) and punished responding in a modified water-lick conflict model, after amygdaloid injection. Both ondansetron (10–1000 ng) and ICS205-930 (1–100 ng) were ineffective in the conflict test. Finally, 2-Me 5-HT and 5-HT (100–10 000 ng) reduced SI under the LLF test condition with no concomitant change in locomotor activity. It is concluded that the amygdala, but not the DRN, may represent an important neuroanatomical locus for the disinhibitory, perhaps anxiolytic, properties of 5-HT3 receptor antagonists.

Effect of 5-HT1A receptor agonists in two models of anxiety after dorsal raphe injection

The purpose of the present study was two-fold. Firstly, to present a more comprehensive analysis of the disinhibitory effects of 5-HT1A receptor agonists after discrete dorsal raphe (DRN) injections (Higgins et al. 1988). Secondly, the effects of the 5-HT1B receptor agonist CGS12066B and the 5-HT1B/1C agonist mCPP were examined following injection into this nucleus. The increases in social interaction (SI) induced by intra-raphe injections of 8-OH DPAT (0.02–1 μg), buspirone (0.04–0.2 μg), ipsapirone (0.2 μg) and gepirone (0.2–1 μg) under a high light unfamiliar paradigm (HLU) were typically due to increased bout frequency, duration and a higher incidence of sniff, follow, allogroom behaviour. These increases were qualitatively similar to those seen in control animals tested under low light/familiar (LLF) conditions, thus supporting the belief that the drug-induced increases in SI reflected decreases in anxiety. Furthermore, at doses effective under the HLU condition, 8-OH DPAT, buspirone and gepirone failed to modify SI under conditions of minimal suppression (LLF paradigm). At doses which significantly increased punished responding in a water-lick conflict test 8-OH DPAT, ipsapirone and gepirone tended to also increase unpunished rates of drinking. However, in drug untreated rats, prior habituation to the test apparatus also increased unpunished drinking, suggesting some neophobia-induced suppression. At a comparatively high dose, the 5-HT1B agonist CGS12066B (2.5 μg), but not the putative 5-HT1B/1c agonist mCPP (0.5–12.5 μg), increased SI under the HLU condition. Considered along-side the other compounds described in this report, the relative potency of CGS12066B may be reflective of a 5-HT1A receptor interaction. Together, these data support the proposal that the DRN is an important site through wich 5-HT1A receptor agonists express their anxiolytic actions.

GR127935 acts as a partial agonist at recombinant human 5-HT1Dα and 5-HT1Dβ receptors

In this study we have investigated the functional activity of GR127935 (2-methyl-4-(5-methyl-1,2,4 oxadiazol-3-yl)-biphenyl-[4-carboxylic acid 4-methoxy-3-(4-methyl-piperazine-1-yl)-phenyl]-amide) at human 5-HT1Dα and 5-HT1Dβ receptors which have been expressed in a Chinese Hamster Ovary (CHO) cell line. Using [35S]GTPγS binding to cell membranes as a measure of receptor-G protein coupling, GR127935 showed partial agonist activity in both 5-HT1Dα and 5-HT1Dβ receptor expressing cells (Emax: 29 and 31% above basal control; pEC50: 8.6 and 9.7, respectively). GR127935 also acted as a potent antagonist at the 5-HT1Dα (app. pA2 8.5) and 5-HT1Dβ (app. pA2 9.1) receptors. From studies measuring cAMP accumulation in cultured CHO cells GR127935 also displayed partial agonism, as well as acting as a potent antagonist at the 5-HT1Dα receptors which stimulate cAMP levels and 5-HT1Dβ receptors which inhibit cAMP levels (app. pA2 8.6 and 9.7, respectively). The 5HT1-like receptor antagonist methiothepin showed negative intrinsic activity at both receptors in the [35S]GTPγS binding assay only. From studies using the receptor alkylating agent EEDQ (N-ethoxycarbonyl-2-methoxy-1,2-dihydroquinoline) the 5-HT1Dα cell line displayed a lack of receptor reserve but it was evident in the 5-HT1Dβ cell line. In previous studies we have also shown that agonist stimulation of 5-HT1Dα receptors increases cAMP levels which may be due to high receptor expression. Further investigation using up to 1 μM EEDQ to reduce 5-HT1Dα receptor number did not reveal an underlying inhibitory adenylyl cyclase response. In conclusion, GR127935 acts as a partial agonist, aswell as a potent antagonist, at the human 5-HT1Dα and 5-HT1Dβ receptors when expressed in CHO cells.

Functional characterization of the 5-HT terminal autoreceptor in the guinea-pig brain cortex.

1 In guinea‐pig cerebral cortical slices in vitro we have shown that the rank order of potency of 5‐hydroxytryptamine (5‐HT), 5‐carboxamidotryptamine and sumatriptan for inhibition of electrically stimulated [3H]‐5‐HT release correlates well with published data on their 5‐HT1D receptor binding affinites. 2 Both the non‐selective 5‐HT1D receptor antagonist, methiothepin and the selective 5‐HT1D receptor antagonist, N‐[4‐methoxy‐3‐(4‐methyl‐1‐piperazinyl]phenyl]‐2′‐methyl‐4′‐(5‐methyl‐1,2,4‐oxadiazole‐3‐yl) [1,1‐biphenyl]4‐carboxamide (GR127935) increased stimulated [3H]‐5‐HT release per se and also attenuated agonist‐induced inhibition of [3H]‐5‐HT release. GR127935 (10nM‐100nM) produced a pA2 of 9.0 against 5‐HT, which is consistent with its 5‐HT1D receptor binding affinity. 3 From these findings we conclude that, in guinea‐pig cerebral cortex, the 5‐HT terminal autoreceptor is of the 5‐HT1D receptor subtype. However, three observations suggest the presence of multiple terminal autoreceptors: shallow inhibition curves to the agonists; a shallow Schild slope of GR127935 antagonism and differences in the maximal responses to 5‐HT between whole cortex and frontal cortex.

In vivo determination of efficacy of pyrazoloquinolinones at the benzodiazepine receptor

The pyrazoloquinolinones CGS 9896, CGS 9895 and CGS 8216 potently displace the benzodiazepines from their CNS binding site in vitro but have been reported to display agonist, partial agonist and antagonist activity respectively in a number of in vivo tests in rats. We found CGS 9896 to have only weak antipentylenetetrazole activity in mice at doses which produced near maximal displacement of [3H]flunitrazepam in vivo and were not able to detect any antipentylenetetrazole activity of CGS 9895. However, CGS 9895 blocked the anticonvulsant effect of diazepam at doses closely related to those which inhibited the in vivo binding of [3H]flunitrazepam and CGS 9896 also showed some reversal of the antipentylenetetrazole action of diazepam. This is consistent with the notion that CGS 9896 is a partial agonist and CGS 9895 an antagonist at the benzodiazepine receptor.

(+)‐WAY 100135, a partial agonist, at native and recombinant 5‐HT1B/1D receptors

We have studied the effects of the purportedly selective 5‐HT1A receptor antagonist (+)‐WAY100135 on electrically stimulated 5‐hydroxytryptamine (5‐HT) efflux in the ventrolateral geniculate nucleus (vLGN), and its affinity at human 5‐HT1B and 5‐HT1D receptors stably expressed in Chinese hamster ovary (CHO) cells. On short ‘pseudo single pulse’ stimulations (20 pulses at 100 Hz, 190 ms train duration), (+)‐WAY100135 (1.0 μM) decreased 5‐HT efflux in the vLGN to 68±8% of pre‐drug values (P<0.01). This decrease could be blocked by the 5‐HT1D/1B receptor antagonist GR 127935 (50 nM). Conversely, when long stimulations (20 pulses at 20 Hz, 950 ms train) were used, (+)‐WAY100135 had no effect on 5‐HT efflux (84±8% of pre‐drug values) although both methiothepin (200nM) and GR 127935 (50 nM) caused significant increases (to 175±18 and 130±10% of pre‐drug values, respectively). Paroxetine (100 nM), the selective 5‐HT reuptake inhibitor, increased stimulated 5‐HT efflux and re‐uptake half‐life (to 145±18% and 649±121%, respectively) on pseudo single pulse stimulations. When (+)‐WAY 100135 was added in combination with the uptake blocker, the effect of paroxetine on stimulated 5‐HT efflux was potentiated to 282±48% (P<0.01) without further effect on the 5‐HT re‐uptake half‐life. The affinity and intrinsic activity of (+)‐WAY 100135 were determined at recombinant human 5‐HT1B and 5‐HT1D receptors expressed in CHO cells, by use of radioligand binding and [35S]‐GTPγS binding. (+)‐WAY 100135 was a partial agonist at human 5‐HT1B and 5‐HT1D receptors with moderately high affinity for 5‐HT1D receptors (pEC50=7.61). In conclusion, (+)‐WAY 100135 was found to be not a selective 5‐HT1A autoreceptor antagonist but may act as a partial agonist at the 5‐HT1B/1D receptor, displaying agonist or antagonist properties depending on the stimulation protocol used and the resultant 5‐HT ‘tone’ at the receptor.