Derek N. Middlemiss

8-hydroxy-2-(di-n-propylamino) tetralin is devoid of activity at the 5-hydroxytryptamine autoreceptor in rat brain. Implications for the proposed link between the autoreceptor and the [3H] 5-HT recognition site

SummaryExperiments have been carried out to provide direct evidence for the proposed presynaptic 5-HT autoreceptor agonist activity of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) a compound with selectivity for the 5-HT1A subtype of the 5-HT1 binding site. Rat brain frontal cortex slices were preincubated with [3H] 5-hydroxytryptamine and continuously stimulated with Krebs solution containing paroxetine and elevated K+ ions (25 mmol/l). The elevated efflux of tritium caused by exposure to K+ Krebs was inhibited in a dose related manner by 5-hydroxytryptamine and this inhibition was attenuated in the presence of quipazine and methiothepin.In slices of the rat frontal cortex, 8-OH-DPAT was without agonist or antagonist activity at the 5-HT autoreceptor at concentrations up to 1 μmol/l. Higher concentrations caused an increase in basal efflux of tritium. 8-OH-DPAT (1 μmol/l) was also without inhibitory activity in the piriform cortex, striatum and the hippocampus.These experiments have therefore failed to provide direct evidence for agonist activity of 8-OH-DPAT at the 5-HT autoreceptor and alternative explanations must be sought for its biochemical and behavioural effects in vivo. Moreover, the fact that 8-OH-DPAT is inactive at the autoreceptor at concentrations selective for the 5-HT1A recognition site suggests that this subtype of the 5-HT1A binding site may not correspond to the 5-HT autoreceptor.

Centrally active 5-HT receptor agonists and antagonists.

Eleven subtypes of central 5-HT receptor have so far been postulated, four of which have been cloned (5-HT1A, 5-HT1C, 5-HT1D and 5-HT2) and a fifth (the 5-HT3 receptor) purified. The present review discusses the agonists and antagonists which act at these subtypes with respect to their degree of selectivity and in vivo potency. Selective agonists exist for the 5-HT1A, 5-HT1B and 5-HT3 receptors and selective antagonists for the 5-HT2 and 5-HT3 receptors.

The 5-HT1B Receptors

The 5-HT1B receptors have been identified by radioligand binding techniques predominantly in the basal ganglia of the rat and mouse brain. A number of 5-HT receptor agonists have been shown to display high affinity but limited selectivity for the 5-HT1B recognition site. These include 5-CT, 5-HT, RU 24969, TFMPP, MCPP, and CGS 12066B. Antagonists at the 5-HT1B site include the drugs metitepin, metergoline, cyanopindolol, isamoltane, and propranolol but none of these drugs are selective for this receptor. Functional correlates of 5-HT1B receptor activation have been most closely defined in vitro. These include inhibition of transmitter release, inhibition of forskolin-stimulated adenylate cyclase and actions on the mouse urinary bladder strip and the rat vena cava. Many functional correlates of 5-HT1B receptor activation in vivo have been proposed, but convincing evidence from antagonist studies is generally lacking. The development of selective 5-HT1B receptor agonists and antagonists will be a key step in defining the physiological role of this receptor site in the brain and periphery of the mouse and rat although it must be realized that these compounds, if they are developed, are unlikely to have functional effects in man since the 5-HT1B recognition site is absent in the human CNS. Nevertheless many of these studies on the 5-HT1B receptor may aid the development of drugs acting at the 5-HT1D site since this receptor has been identified as being the equivalent of the 5-HT1B site in species other than the rat and mouse.

A pharmacological analysis of the 5-HT receptor mediating inhibition of 5-HT release in the guinea-pig frontal cortex

The release of [3H]5-HT from guinea-pig frontal cortex slices was elicited by continuous exposure to Krebs solution containing elevated K+ ions (30 mM) and 10 microM fluvoxamine. K+-stimulated release was inhibited by 5-carboxamidotryptamine (pIC25 8.1), 5-HT (7.4), RU 24969 (6.5) and GR 43175 (6.4). 8-OH-DPAT was without effect on K+-evoked release of [3H]5-HT at concentrations up to 1 microM. The inhibitory effects of 5-HT were antagonised by metitepine (pA2 8.2), metergoline (7.0), methysergide (6.5), cyanopindolol (6.5), yohimbine (6.5) and mesulergine (6.2) but not by the 5-HT3 antagonist, ICS 205-930 (1 microM). The results are discussed in the context of the known pharmacology of 5-HT receptor subtypes. It is concluded that the 5-HT receptor modulating 5-HT release in the guinea-pig frontal cortex does not correspond to any of the 5-HT1-subtype recognition sites or to 5-HT2 or 5-HT3 receptors.

L‐694,247: a potent 5‐HT1D receptor agonist

1 The 5‐hydroxytryptamine (5‐HT) receptor binding selectivity profile of a novel, potent 5‐HT1D receptor agonist, L‐694,247 (2‐[5‐[3‐(4‐methylsulphonylamino)benzyl‐1,2,4‐oxadiazol‐5‐yl]‐1H‐indole‐3‐yl]ethylamine) was assessed and compared with that of the 5‐HT1‐like receptor agonist, sumatriptan. 2 L‐694,247 had an affinity (pIC50) of 10.03 at the 5‐HT1D binding site and 9.08 at the 5‐HT1B binding site (sumatriptan: pIC50 values 8.22 and 5.94 respectively). L‐694,247 retained good selectivity with respect to the 5‐HT1A binding site (pIC50 = 8.64), the 5‐HT1C binding site (6.42), the 5‐HT2 binding site (6.50) and the 5‐HT1E binding site (5.66). The pIC50 values for sumatriptan at these radioligand binding sites were 6.14, 5.0, < 5.0 and 5.64 respectively. Both L‐694,247 and sumatriptan were essentially inactive at the 5‐HT3 recognition site. 3 L‐694,247, like sumatriptan, displayed a similar efficacy to 5‐HT in inhibiting forskolin‐stimulated adenylyl cyclase in guinea‐pig substantia nigra although L‐694,247 (pEC50 = 9.1) was more potent than sumatriptan (6.2) in this 5‐HT1D receptor mediated functional response. L‐694,247 (pEC50 = 9.4) was also more potent than sumatriptan (6.5) in a second 5‐HT1D receptor mediated functional response, the inhibition of K+‐evoked [3H]‐5‐HT release from guinea‐pig frontal cortex slices. 4 The excellent agreement observed for L‐694,247 between the 5‐HT1D radioligand binding affinity and the functional potency confirm that the two functional models (the inhibition of forskolin‐stimulated adenylyl cyclase in guinea‐pig substantia nigra and the inhibition of K+‐evoked [3H]‐5‐HT release from guinea‐pig frontal cortex) do indeed reflect 5‐HT1D‐mediated events. 5 L‐694,247 is a novel, highly potent 5‐HT1D/5‐HT1B receptor ligand which should prove useful for the exploration of the physiological role of these receptors in animals.

An investigation of the 5-HT1D receptor binding affinity of 5-hydroxytryptamine, 5-carboxyamidotryptamine and sumatriptan in the central nervous system of seven species

The ability of three 5-HT1 receptor agonists, 5-HT (5-hydroxytryptamine), 5-CT (5-carboxyamidotryptamine) and sumatriptan to inhibit the binding of [3H]5-HT, in the presence of cyanopindolol and mesulergine, from cerebral cortical and/or caudate membranes in seven species (dog, guinea-pig, rabbit, pig, human, hamster and calf) has been investigated. Under the experimental conditions used, 5-CT and sumatriptan consistently yielded displacement curves best fit to a two-site model whereas 5-HT always gave a monophasic displacement curve. The pIC50 values obtained with 5-HT displacement gave a mean of 8.1 +/- 0.1 (mean +/- S.E.M.). In contrast the biphasic displacement curves for 5-CT and sumatriptan yielded high and low affinity pIC50 values of 8.3 +/- 0.1, 5.5 +/- 0.1 and 7.6 +/- 0.1, 5.0 +/- 0.1, respectively. These data indicate that under these experimental conditions the high affinity component labelled by [3H]5-HT is the same receptor subtype, previously denoted the 5-HT1D receptor, in all seven species.

In vivo labelling of the NMDA receptor channel complex by [3H]MK-801

An in vivo radioligand binding assay for the N-methyl-D-aspartate (NMDA) receptor channel complex in the mouse brain has been developed using the non-competitive NMDA receptor antagonist [3H]MK-801. In vivo binding of [3H]MK-801 was displaced by MK-801 (ED50 = 0.17 mg/kg i.p.), (-)-MK-801 (1.0 mg/kg), thienylcyclohexylpiperidine (1.8 mg/kg), etoxadrol (5.1 mg/kg) and (+)-SKF 10,047 (34.5 mg/kg). The potency of these drugs in this in vivo binding assay was highly correlated (r = 0.97) with their functional effects as antagonists of N-methyl-DL-aspartate-induced tonic convulsions.

Behavioural and biochemical evidence of the interaction of the putative antipsychotic agent, BMY 14802 with the 5-HT1A receptor

The behavioural and biochemical profile of the sigma ligand and putative antipsychotic agent, BMY 14802 (alpha-(4-fluorophenyl)-4-(5-fluoro-2- pyrimidinyl)-1-piperazine butanol) has been determined in the mouse and rat. In mice, pretreatment with BMY 14802 attenuated both amphetamine-induced hyperactivity and conditioned avoidance responding, consistent with its previously reported antipsychotic potential. In common with 5-HT1A receptor agonists or partial agonists, BMY 14802 induced (a) a dose-dependent hypothermia in mice; (b) aspects of the 5-HT behavioural syndrome in rats, (c) antagonised mescaline-induced head twitches in mice and (d) generalised to the 8-hydroxy-2-(di-n-propylamino)tetralin discriminative stimulus over the dose range of 3-15 mg/kg. BMY 14802 had appreciable affinity for the 5-HT1A receptor (pIC50 = 6.7 compared to 7.3 for sigma binding) and antagonised forskolin-stimulated adenylate cyclase activity with a pEC50 of 6.2, consistent with an agonist action at this receptor. The results support the involvement of 5-HT1A receptors, but not the sigma binding site, in the behavioural profile of BMY 14802.

Destruction of a sub-population of cortical neurones by suicide transport of volkensin, a lectin from Adenia volkensii

A method for the destruction of a sub-population of neocortical pyramidal neurones is described. The technique uses unilateral striatal injections of volkensin, a toxic lectin from Adenia volkensii, which undergoes retrograde axonal transport from the site of injection to destroy subcortically projecting pyramidal cells within the neocortex. Striatal volkensin injections produce a significant reduction in the number of large pyramidal neurones of the infragranular layer. The selectivity of the lesion was demonstrated by the preservation of glutamic acid decarboxylase mRNA positive cells, considered to be cortical interneurones within ipsilateral cortex. The binding of the serotonin 1A receptor agonist [3H]-8-hydroxy-2-(n-dipropylamino)tetralin, visualised by autoradiography, was reduced in areas showing loss of large cells, indicating that these receptors may be present on subcortically projecting pyramidal cells. Ricin, another toxin lectin, but effective as a suicide transport agent in only the peripheral nervous system, produced local striatal damage but no cortical cell loss. The selective destruction by volkensin of neocortical pyramidal neurones with subcortical projection targets should aid the neurobiological investigation of such cells. Additionally, data obtained using this technique may provide insights into the interpretation of biochemical findings in neurodegenerative disease in which pyramidal cell loss is a significant feature.

Metitepine distinguishes two receptors mediating inhibition of [3H]-5-hydroxytryptamine release in guinea pig hippocampus

SummaryInhibition of [3H]-5-hydroxytryptamine ([3H]-5-HT) release from guinea pig brain slices via activation of the terminal 5-HT autoreceptor has previously been characterised as a model of 5-HT1D receptor activation, based on the rank potencies of a range of agonists, and the potent antagonism of the inhibitory effects of 5-HT by metitepine. The present study uses this model, in slices of the guinea pig hippocampus, to examine the antagonist potency of metitepine against the 5-HT receptor agonists sumatriptan, 5-carboxamidotryptamine (5-CT) and 5-HT Addition of metitepine to the perfusion buffer (30, 300 and 1000 nmol/l) significantly shifted the concentration-response curve to 5-HT, producing a Schild slope of 1.1, and a pA2 value of 7.6. However, the ability of metitepine to antagonise the effects of sumatriptan or 5-CT in this model was less marked. A clear-cut shift in the concentration-response curve to sumatriptan was only achieved at1000 nmol/l metitepine (apparent pA2 = 6.7),and this was similar to the ability of metitepine to attenuate the effects of 5-CT (apparent pA2 7.0 at 300 nmol/l and 6.7 at 1000 nmol/l). These findings suggest heterogeneity in the receptor mediating inhibition of [3H]-5-HT release in guinea pig hippocampus.