Effect of the selective 5-HT3 receptor antagonists ICS 205-930 and MDL 72222 on 5-HTP-induced head shaking and behavioral symptoms induced by 5-methoxy-N,N,dimethyltryptamine in rats: comparison with some other 5-HT receptor antagonists

The effect of the selective 5-HT₃ receptor antagonists ICS 205-930 and MDL 72222 on head shaking behavior induced by l-5-HTP and behavioral symptoms induced with 5-methoxy-N,N,-dimethyltryptamine (5-MeODMT) in rats was evaluated. Both drugs dose-dependently reduced l-5-HTP-induced head shaking but were at least 600 times less potent than pirenperone and ketanserin and at least 50 times less potent than methysergide. ICS 205-930 and MDL 72222 were more than 1000 times less potent than pirenperone or methysergide and 100 times less potent than ketanserin in blocking 5-MeODMT-induced forepaw treading and tremor. Since it appears that head shakes induced by l-5-HTP are mediated by 5-HT₂ receptors, these data suggest that ICS 205-930 and MDL 72222 do not significantly interact with 5-HT₂ receptors in the brain. Furthermore, the data suggest that ICS 205-930 and MDL 72222 lack appreciable antagonistic activity at the 5-HT receptor(s) mediating those behavioral effects induced by 5-MeODMT.     

MDL 72222: a potent and highly selective antagonist at neuronal 5-hydroxytryptamine receptors

Summary The properties of MDL 72222 (1H,3,5H-tropan-3-yl-3,5-dichlorobenzoate), a novel compound with potent and selective blocking actions at certain excitatory 5-hydroxytryptamine (5-HT) receptors on mammalian peripheral neurones, are described.On the rabbit isolated heart, MDL 72222 was a potent antagonist of responses mediated through the receptors for 5-HT present on the terminal sympathetic fibres. The threshold for antagonism was approximately 0.1 nM and the negative logarithm of the molar concentration of MDL 72222 which reduced the chronotropic response of the isolated rabbit heart to twice an ED₅₀ of 5-HT to that of the ED₅₀ was 9.27. MDL 72222 was also highly selective since responses to the nicotine receptor agonist, dimethylphenylpiperazinum iodine (DMPP), were inhibited only at concentrations more than 1000 times those necessary to inhibit 5-HT.In the anaesthetised rat, MDL 72222 produced marked blockade of the Bezold-Jarisch effect of 5-HT. Again, inhibition was selective since much higher doses of MDL 72222 failed to alter the response to electrical stimulation of the efferent vagus nerves. In contrast, MDL 72222 proved only a weak and essentially non-selective antagonist of responses mediated by the 5-HT M-receptor present on the cholinergic nerves of the guinea-pig ileum.MDL 72222 does not block smooth muscle contractile responses elicited by oxytocin or mediated through 5-HT D-receptors, muscarinic or nicotinic cholinoceptors or histamine H₁-receptors except at relatively high concentrations. Similarly, in a number of radioligand binding assays carried out using brain tissue membranes, the displacing effects of MDL 72222 were absent or weak at sites identifying compounds with activity at ₁, ₂ or -adrenoceptors, 5-HT₁ or 5-HT₂ receptors, benzodiazepine receptors or histamine H₁-receptors.MDL 72222 is the first reported selective and potent antagonist of responses mediated through the 5-HT receptors present on the terminal sympathetic neurones of the rabbit heart and on the neurones subserving the afferent limb of the Bezold-Jarisch reflex. The compound should provide a useful means by which responses mediated through such sites can be distinguished.     

THE CLASSIFICATION OF 5-HYDROXYTRYPTAMINE RECEPTORS

1. The current classification of receptors for 5-hydroxytryptamine (5-HT) is based on functional studies, and encompasses three main receptor types. 2. 5-HT1-like receptors mediate inhibition of release of various neurotransmitters from central and peripheral sites, smooth muscle contraction and relaxation (and release of endothelium-derived relaxing factor), tachycardia, a variety of behavioural actions (for example, forepaw treading, hypothermia, hyperphagia, drug discriminative stimulus properties, nociceptive pathway modulation, and anxiolytic, anti-aggressive and prosexual effects), and central neuronal excitatory and inhibitory activity. Selective antagonists for this receptor are not yet available, but the 5-HT2 receptor antagonists methysergide and methiothepin have appreciable affinity for 5-HT1-like receptors, and 5-carboxamidotryptamine is a selective agonist. 3. 5-HT2 receptors mediate smooth muscle contraction, platelet aggregation, increased capillary permeability, some behavioural syndromes (for example, head twitch and wet-dog shakes) and drug discriminative stimulus properties, central neuroexcitatory effects, and some neuroendocrine functions. Ketanserin and cyproheptadine are selective antagonists. 4. 5-HT3 receptors mediate peripheral afferent and efferent neuroexcitatory actions, anxiogenic effects, and modulation of cytotoxic drug-induced emesis, gastric emptying, and dopamine-related mesolimbic hyperactivity. Selective antagonists include cocaine, MDL 72222 and ICS 205-930; 2-methyl-5-HT is a selective agonist.     

L-Tryptophan and prolactin release: Evidence for interaction between 5-HT1 and 5-HT2 receptors

The effects of the selective 5-HT₂ receptor antagonist, ritanserin, on the growth hormone (GH) and prolactin (PRL) responses to intravenous L-tryptophan (LTP) were assessed in 8 normal volunteers. Administration of ritanserin (40 mg orally) prior to infusion of LTP (5 g) significantly enhanced the PRL responses but not those of GH. The results are consistent with previous proposals that the endocrine responses to LTP are mediated by 5-HT₁ rather than 5-HT₂ receptors and also suggest that 5-HT₂ receptor antagonists may increase certain 5-HT₁ mediated responses in the human brain.     

Inhibition of noradrenaline release via presynaptic 5-HT1B receptors of the rat vena cava

Summary In the rat inferior vena cava preincubated with ³H-noradrenaline, the effects of nine serotonin (5-HT) receptor agonists and of eight antagonists (including two -adrenoceptor blocking agents) on the electrically evoked ³H overflow were determined. 1. 5-HT, 5-carboxamidotryptamine, 5-methoxy-3(1,2,3,6-tetrahydropyridine-4-yl)-1H-indole (RU 24969), 5-methoxytryptamine, N,Ndimethyl-5-HT, tryptamine and 5-aminotryptamine inhibited the evoked ³H overflow. The potencies of these agonists in inhibiting overflow were significantly correlated with their affinities for 5-HT_1B binding sites, but not with their affinities for 5-HT_1A, 5-HT_1C or 5-HT₂ binding sites. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5HT_1A receptor agonist, and ipsapirone, a partial agonist at these receptors, did not inhibit overflow. 2. Cyanopindolol facilitated the evoked ³H overflow, an effect which was abolished by propranolol. The maximum inhibition of overflow obtainable with 5-HT was diminished by cyanopindolol. 3. The concentration-response curve for 5-HT was shifted to the right by metitepine, metergoline, quipazine, 6-chloro-2-(1-piperazinyl)pyrazine (MK 212) and propranolol which, given alone, did not affect ³H overflow. The apparent pA₂ values of these antagonists tended to be correlated with their affinities for 5-HT_1B (but not 5-HT_1A, 5-HT_1c or 5-HT₂) binding sites. Ketanserin, a 5-HT₂ receptor antagonist, and spiperone, which blocks 5-HT₂ and 5-HT_1A but not 5-HT_1B or 5-HT_1C receptors, failed to antagonize the effect of 5-HT. These results suggest that the inhibitory presynaptic 5-HT receptors on the sympathetic nerve terminals of the rat vena cava appear to belong to the 5-HT_1B subtype. Cyanopindolol may act as a partial agonist at these receptors, as it does at the facilitatory prosynaptic -adrenoceptors.This study was supported by a grant of the Deutsche Forschungsgemeinschaft Send offprint requests to M. Göthert     

SDZ 205-557, a selective,surmountable antagonist for 5-HT4 receptors in the isolated guinea pig ileum

Summary A selective antagonist for the recently characterized 5-HT₄-receptor is lacking. The only surmountable antagonist available, ICS 205-930, is a weak antagonist and is far more potent at 5-HT₃-than at 5-HT₄ receptors. In this paper, SDZ 205-557 (2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino) ethyl ester) is characterized as the first potent, selective and surmountable antagonist at 5-HT₄ receptors in the isolated guinea pig ileum.SDZ 205-557 was investigated in the non-stimulated and in the field-stimulated guinea pig ileum longitudinal muscle preparation for its affinity for 5-HT₄-, 5-HT₃-, muscarine-, nicotine- and histamine H₁ receptors. The affinity for 5-HT₁-, 5-HT₂-, ₁-, ₂- and opiate () receptors was determined by binding assays. SDZ 205-557 was devoid of substantial affinity (pK_D values below 5.6) for all receptors investigated except for 5-HT₃- and 5-HT₄ receptors. At these two receptors, SDZ 205-557 acted as an antagonist without measurable intrinsic activity.At the 5-HT₄ receptors of the non-stimulated guinea pig ileum, responses to 5-HT and 5-methoxytryptamine were antagonized by SDZ 205-557 with identical pA₂ values of 7.4. The effect of renzapride was also blocked with no significant change in the maximum response; Schild analysis, however, revealed that the interaction was not competitive with an apparent pA₂ value of 7.6. A pA₂ of 6.8 was obtained using zacopride as a contractile agent; this value differed significantly from 7.4, the value obtained for 5-HT and 5-methoxytryptamine. The effects of metoclopramide were inhibited non-competitively with a pD₂' value of 5.4. 5-HT₄ receptor mediated effects in the field-stimulated guinea pig ileum were antagonized in a similar way by SDZ 205-557. The resulting pA₂ values were not significantly different from those obtained in the non-stimulated preparation.In the non-stimulated preparation, contractions induced by 5-HT and 2-methyl-5-HT via activation of 5-HT₃ receptors were antagonized by SDZ 205-557 only at concentrations of 10^–6 mol/l and above yielding pA₂ values of 5.6 and 5.9, respectively, which were considerably lower than those obtained for the blockade of 5-HT₄ receptors. At concentrations greater than 10^–6 mol/l, SDZ 205-557 caused a significant reduction of the maximum responses, indicating non-competitive antagonism.It is concluded that SDZ 205-557 is a potent, selective and surmountable antagonist at 5-HT₄ receptors in the isolated guinea pig ileum, and is a more practical tool for the characterization of 5-HT₄ receptors than ICS 205-930.A part of these results was presented at the 32nd Spring Meeting of the German Society for Pharmacology and Toxicology, Mainz, March 12–15, 1991, and published as Rapid communication (Buchheit et al. 1991) Send offprint requests to K. H. Buchheit at the above address     

Effects of serotonin-3 receptor antagonists on the intracranial self-administration of ethanol within the ventral tegmental area of Wistar rats

Abstract Rationale. Previous work from our laboratory indicated that Wistar rats will self-administer ethanol (EtOH) directly into the posterior ventral tegmental area (VTA) and that 5-HT₃ antagonists will inhibit EtOH-stimulated somatodendritic release of dopamine within the VTA. Objectives. The objective of this study was to use the intracranial self-administration procedure to determine the involvement of 5-HT₃ receptors in mediating the reinforcing effects of EtOH within the VTA, and to increase our understanding of central nervous system mechanisms involved in the rewarding effects of EtOH. Methods. Adult female Wistar rats were stereotaxically implanted with guide cannulae aimed at the posterior VTA. After 1 week, rats were placed into standard two-lever experimental chambers for a total of seven sessions (4-h sessions separated by 48 h) and allowed to self-administer vehicle alone, a 5-HT₃ antagonist alone, 200 mg% EtOH alone, or combinations of 200 mg% EtOH with different concentrations of a 5-HT₃ antagonist (n=6–9 per group). Results. Throughout all seven sessions, Wistar rats self-infused more 200 mg% ETOH (25±5 infusions) than vehicle (5±4 infusions) or 5-HT₃ antagonist (6±4 infusions) (P<0.05), and responded significantly more (P<0.05) on the active than inactive lever (e.g., 50±12 vs 12±8 responses in session 1). Co-administration of 50 μM or 100 μM ICS 205,930 with 200 mg% EtOH completely prevented the acquisition and maintenance of EtOH self-infusion into the posterior VTA. Similarly, co-administration of either 25–100 μM LY-278–584 or 10–100 μM zacopride with 200 mg% EtOH completely blocked EtOH-maintained intracranial self-administration behavior. Conclusions. The results of this study suggest that the reinforcing effects of EtOH within the posterior VTA of Wistar rats require activation of local 5-HT₃ receptors. Electronic Publication     

Inhibition of noradrenaline release from the sympathetic nerves of the human saphenous vein via presynaptic 5-HT receptors similar to the 5-HT1D subtype

Summary The human saphenous vein preincubated with [³H]noradrenaline was used to determine the pharmacological properties of the release-inhibiting presynaptic serotonin (5-HT) receptor on the sympathetic nerves. The overflow of tritium evoked by transmural electrical stimulation (2 Hz) was concentration-dependently inhibited by drugs known to stimulate 5-HT receptors in the following rank order: oxymetazoline 5-HT 5-carboxamidotryptamine = 5-methoxytryptamine = sumatriptan > tryptamine > N,N(CH₃)₂-5-HT = yohimbine = 8-hydroxy-2-(di-n-propylamino)-tetraline. The potencies of these agonists in inhibiting overflow were significantly correlated with their affinities for 5-HT_1B and 5-HT_1D binding sites, but not with those for 5-HT_1A or 5-HT_1C binding sites. 5-Aminotryptamine, methysergide, ipsapirone, cyanopindolol, SDZ 21009 and metergoline dit not produce a significant inhibition. Metitepine and methysergide antagonized the inhibitory effect of 5-HT, whereas spiroxatrine, propranolol, ketanserin and ICS 205-930 did not.These data exclude the idea that the inhibitory presynaptic 5-HT receptor on the sympathetic nerves belongs to the 5-HT₂ and 5-HT₃ receptor class; the pattern of agonist potencies suggests that the receptor is very similar to the 5-HT_1D receptor subtype. Send offprint requests to M. Gothert at the above address     

Effects of selective h5-HT1B (SB-216641) and h5-HT1D (BRL-15572) receptor ligands on guinea-pig and human 5-HT auto- and heteroreceptors

Human cerebral cortical slices and synaptosomes, guinea-pig cerebral cortical slices and human right atrial appendages were used to study the effects of SB-216641, a preferential h5-HT_1B receptor ligand, and of BRL-15572, a preferential h5-HT_1D receptor ligand, on the presynaptic h5-HT_1B and h5-HT_1B-like autoreceptors in the human and guinea-pig brain preparations, respectively, and on the presynaptic h5-HT_1D heteroreceptors in the human atrium. The brain preparations, preincubated with [³H]serotonin ([³H]5-HT), and the segments of atrial appendages, preincubated with [³H]noradrenaline, were superfused with modified Krebs’ solution and tritium overflow was evoked electrically (human and guinea-pig cerebral cortex slices and human atrial appendages) or by high K⁺ (human cerebral cortex synaptosomes). The electrically evoked tritium overflow from guinea-pig cerebral cortex slices was reduced by the 5-HT receptor agonist 5-carboxamidotryptamine (5-CT). This effect was not modified by BRL-15572 (2μM; concentration 154 times higher than its K_i at h5-HT_1D receptors) but was antagonized by SB-216641 (0.1μM; concentration 100 times higher than its K_i at h5-HT_1B receptors; apparent pA₂ 8.45). SB-216641 (0.1μM) by itself facilitated, whereas BRL-15572 (2μM) did not affect, the evoked overflow. In human cerebral cortex slices SB-216641 (0.1μM) also facilitated, and BRL-15572 (2μM) again failed to affect, the electrically evoked tritium overflow. In human cerebral cortical synaptosomes, 5-CT reduced the K⁺-evoked tritium overflow. This response was unaffected by BRL-15572 (300nM) but antagonized by SB-216641 (15nM; drug concentrations 23 and 15 times higher than their K_i at h5-HT_1D and h5-HT_1B receptors, respectively). Both drugs, given alone, did not modify the K⁺-evoked tritium overflow. In human atrial appendages, the electrically evoked tritium overflow was inhibited by 5-HT in a manner susceptible to antagonism by BRL-15572 (300nM; 23 times K_i at h5-HT_1D receptors) but not by SB-216641 (30nM; 30 times K_i at h5-HT_1B receptors). Both drugs by themselves did not change the electrically evoked tritium overflow. In conclusion, SB-216641 behaves as a preferential antagonist at native human 5-HT_1B receptors and BRL-15572 as a preferential antagonist at native human 5-HT_1D receptors. These compounds are clearly useful tools for the differentiation between human 5-HT_1B and 5-HT_1D receptors in functional studies. Received: 14 March 1997 / Accepted: 18 May 1997     

3,4-Methylenedioxymethamphetamine (MDMA): Stereoselective interactions at brain 5-HT1 and 5-HT2 receptors

3,4-Methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxy-amphetamine (MDA), and their optical isomers, were assayed for their affinities at radiolabeled brain serotonin (5-HT₁, 5-HT₂) and dopamine (D₂) binding sites. (R(–)-MDA and R(–)-MDMA) displayed moderate affinities for ³H-ketanserin-labeled 5-HT₂ sites (Ki=3425 and 3310 nM, respectively) whereas the affinities for their S(+)-enantiomers were lower (Ki=13,000 and 15,800 nM, respectively). Similar absolute and relative affinities were obtained at ³H-serotonin-labeled 5-HT₁ sites; binding at D₂ sites was very low (Ki>25,000 nM in each case). The (–)>(+) order of potency at 5-HT₂ sites is consistent with the observation that R(–)-MDA is a more potent psychoactive agent than its S(+)-enantiomer, but contrasts with the reported finding that S(+)-MDMA is more potent than R(–)-MDMA in humans. These results suggest that MDMA, unlike MDA and other hallucinogenic phenylisopropylamines, does not work primarily through a direct interaction at 5-HT sites.     

Analysis of the heart rate effects of 5-hydroxytryptamine in the cat; mediation of tachycardia by 5-HT1-like receptors

Summary The effects of 5-hydroxytryptamine (5-HT) on heart rate in anaesthetized cats were analysed both in intact animals and after spinal section plus vagotomy.The intact cat responded to 5-HT (3, 10 and 30 g·kg^–1, i.v.) with a brief, but intense, bradycardia and a longerlasting hypotension. Administration of MDL 72222, a selective antagonist of M-type 5-HT receptors, blocked bradycardia elicited by 5-HT without affecting that caused by stimulation of the vagus nerve.In spinal cats the same doses of 5-HT increased heart rate and blood pressure. These effects remained essentially unchanged after bilateral adrenalectomy, guanethidine, propranolol and burimamide, suggesting that 5-HT acted directly on the myocardium and blood vessels. The tachycardic responses to 5-HT in spinal cats were little affected by 0.5 mg·kg^–1 doses of MDL 72222 or of the 5-HT₂ receptor antagonists, ketanserin, ritanserin or cyproheptadine. In contrast, the non-selective 5-HT receptor antagonist, methysergide, which binds to both 5-HT₁ and 5-HT₂ recognition sites in rat brain membranes, potently antagonized the 5-HT-induced tachycardia in doses of 0.05 to 0.5 mg·kg^–1. However pizotifen and mianserin, two other 5-HT₂ antagonists which show poor affinity for 5-HT₁ recognition sites, were also effective against the tachycardic response to 5-HT in doses of 0.5–4.5 mg·kg^–1. The pressor responses to 5-HT in the spinal cat were markedly inhibited by all six 5-HT₂ antagonists at a dose of 0.5 mg·kg^–1.5-Carboxamido-tryptamine, which has a high and selective affinity for 5-HT₁ recognition sites, elicited marked tachycardia in doses of 0.1–10 g/kg^–1 in spinal cats treated with saline. These responses were not affected in animals treated with 4.5 mg·kg^–1 of ketanserin, which was able to shift the dose-response curve for 5-HT to the right. On the other hand, methysergide (0.5 mg·kg^–1) displaced the dose-response curves for both 5-carboxamidotryptamine and 5-HT to a similar extent.Unlike on the dog saphenous vein, methysergide showed no agonist effects on heart rate in the spinal cat.On the basis of the above results, we conclude that: (i) the reflexogenic bradycardic response elicited by 5-HT overshadows its direct tachycardic response on heart rate in the intact cat; (ii) M-type 5-HT receptors mediate the bradycardic response; (iii) the pressor response to 5-HT in the spinal cat involves 5-HT₂ receptors; (iv) tachycardia by 5-HT in the spinal cat is mediated mainly by 5-HT₁-like receptors, but an additional, though less important, non-5-HT₁ mechanism may also be involved; (v) the cardiac 5-HT₁ receptors are similar, but perhaps not identical, to those delineated in the dog saphenous vein or rat brain membrane preparations; and (vi) the tachycardic responses to 5-HT and, in particular the more selective, 5-carboxamidotryptamine may be conveniently utilized to characterize new chemical compounds designed for potential 5-HT₁ receptor antagonist activity.     

Long-term 5-HT reuptake blockade,but not monoamine oxidase inhibition,decreases the function of terminal 5-HT autoreceptors: an electrophysiological study in the rat brain

Summary 5-HT-containing terminals possess autoreceptors which modulate the release of 5-HT into the synaptic cleft. Tritiated imipramine ([³H]IMI), and more specifically [³H]citalopram and [³H]paroxetine, bind to a site associated with the 5-HT reuptake carrier on the 5-HT terminals. The function of terminal 5-HT autoreceptors is decreased following long-term treatment with the 5-HT reuptake blocker citalopram. The present study was undertaken to determine whether an increased synaptic availability of 5-HT or, the occupation of the [³H]IMI site, were responsible for this modification. Unitary extracellular recordings were obtained from CA₃ dorsal hippocampus pyramidal neurons under chloral hydrate anesthesia in rats treated daily with fluoxetine (10 mg/kg/day × 14 days), a selective 5-HT reuptake blocker, or clorgyline (1 mg/kg/day × 21 days), an inhibitor of type A monoamine oxidase. The function of the terminal 5-HT autoreceptors was assessed by comparing the effectiveness of the electrical stimulation of the ascending 5-HT pathway on the firing activity of hippocampus pyramidal neurons prior to, and following, the administration of methiothepin, an antagonist of the terminal 5-HT autoreceptor, and, by determining the ratio of effectiveness of 0.8 Hz (S1) and 5 Hz (S2) stimulations. Long-term administration of fluoxetine or clorgyline both increased the efficacy of the stimulation of the 5-HT pathway. However, the enhancing effect of methiothepin on the efficacy of the stimulation was attenuated by the fluoxetine, but not by the clorgyline, treatment. The reduction of the function of the terminal 5-HT autoreceptor by the long-term fluoxetine treatment was further indicated by the decreased ratio of effectiveness of the 0.8 and 5 Hz stimulations as compared to that of control rats. To verify that the reuptake blockade per se could not account for the increased synaptic efficacy of 5-HT projections following long-term fluoxetine, the drug was administered acutely to naive rats. It did not increase the efficacy of the stimulation of the 5-HT pathway. Two conclusions are drawn from these results: 1) the increased efficacy of 5-HT synaptic transmission by long-term treatment with antidepressant 5-HT reuptake blockers is not directly due to 5-HT reuptake blockade, but rather, to a reduced function of the terminal 5-HT autoreceptor; 2) the latter phenomenon cannot be ascribed to an increased availability of 5-HT in the synaptic cleft as it was not produced by long-term clorgyline treatment. Hence, these results suggest that long-term occupation of the [³H]IMI site might result in a decreased function of terminal 5-HT autoreceptors.Send offprint requests to Claude de Montigny     

Neuropeptide S selectively inhibits the release of 5-HT and noradrenaline from mouse frontal cortex nerve endings

Background and purpose:

Neuropeptide S (NPS) is a recently identified neurotransmitter/neuromodulator able to increase arousal and wakefulness while decreasing anxiety-like behaviour. As several classical transmitters play a role in arousal and anxiety, we here investigated the possible presynaptic regulation of transmitter release by NPS.

Experimental approach:

Synaptosomes purified from mouse frontal cortex were prelabelled with [³H]5-hydroxytryptamine (5-HT), noradrenaline, dopamine, choline, D-aspartate or GABA and depolarized in superfusion with 12–15 mmol·L⁻¹ KCl to evoke [³H]neurotransmitter exocytosis. NPS was added at different concentrations (0.001 to 100 nmol·L⁻¹).

Key results:

NPS behaved as an extremely potent inhibitor of the evoked overflow of [³H]5-HT and [³H]noradrenaline exhibiting EC₅₀ values in the low picomolar range. The inhibitory action of NPS on [³H]5-HT release was mimicked by [Ala²]NPS that was, however, about 100-fold less potent than the natural peptide. NPS (up to 100 nmol·L⁻¹) was unable to affect the depolarization-evoked overflow of [³H]D-aspartate and [³H]GABA. The neuropeptide only weakly reduced the overflow of [³H]dopamine and [³H]ACh when added at relatively high concentrations.

Conclusions and implications:

NPS, at low picomolar concentrations, can selectively inhibit the evoked release of 5-HT and noradrenaline in the frontal cortex by acting directly on 5-hydroxytryptaminergic and noradrenergic nerve terminals. These direct effects may explain only in part the unique behavioural activities of NPS, while an indirect involvement of other transmitters, especially of glutamate, must be considered.     

Inhibition of noradrenaline release via presynaptic 5-HT1Da receptors in human atrium

In segments of human right atrial appendages preincubated with [3^H]noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone, we determined the effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by transmural electrical stimulation (2 Hz).Tritium overflow was inhibited by 5-HT, 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-McOT), 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)-1H indole succinate (RU 24969) and sumatriptan. Yohimbine and oxymetazoline (in the presence of idazoxan) also inhibited tritium overflow. The inhibitory potency of the drugs was significantly correlated with their affinity for 5-HT_ID receptors in human brain and for cloned human 5-HT_1D and 5-HT_1D receptors, but not with their affinity for 5HT_1B, 5-HT_1E, 5-HT_1F, 5-HT_2A, 5-HT_2B, 5-HT_2C, 5-HT₃, 5-HT_5A, 5-HT_5B and 5-HT₇ receptors. The potency order 5-CT >5-HT >5-MeOT is opposite to the order of affinities reported for 5-HT₆ binding sites. The preferential 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetraline (up to 0.3 M) and the selective 5-HT₄ receptor agonist cisapride (up to 1 M) failed to inhibit tritium overflow. L-694,247, a potent 5-HT_ID receptor agonist, did not inhibit tritium overflow, but counteracted the inhibitory effect of 5-HT. Ketanserin at a concentration which should block 5-HT_1Da but not 5-HT_1D receptors and methiothepin at a concentration which may be assumed to block both 5-HT_1D and 5-HT_1D receptors antagonized the inhibitory effect of 5-HT. Propranolol and ondansetron did not modify the 5-HT-induced inhibition of release. In conclusion, noradrenaline release in human right atrial appendages is inhibited via 5-HT receptors which are located on the noradrenergic axon terminals. These inhibitory presynaptic 5-HT receptors belong to the 5-HT_ID subfamily. The ability of ketanserin to antagonize the inhibitory effect induced by activation of these receptors suggests that they can be subclassified as 5-HT_1D.     

Activation of 5-HT(1A) autoreceptors enhances the inhibitory effect of galanin on hippocampal 5-HT release in vivo

The microdialysis technique was used to examine interactions between 5-HT(1A) and galanin receptors in the dorsal raphe nucleus (DRN), by measuring the extracellular levels of 5-HT in the ventral hippocampus of awake rats. The rats were pretreated with the 5-HT(1A) receptor agonist (R,S)-8-OH-DPAT (0.3 mg/kg, s.c.) or saline. 8-OH-DPAT caused a time-dependent reduction of basal 5-HT levels down to 43-48% at 40 min while at 140 min, the hippocampal 5-HT had returned to control values. At that time point, the rats received a second injection of 8-OH-DPAT or galanin (0.15, 0.5 and 1.5 nmol/0.5 microl) infused into the lateral ventricle. The second injection of 8-OH-DPAT caused a significantly smaller reduction of hippocampal 5-HT levels. In contrast, galanin at all three doses in the 8-OH-DPAT-pretreated groups, was significantly more potent in reducing 5-HT levels (maximal reduction to 74%, 52% and 49%, respectively) than it was in saline-pretreated rats (maximal reduction to 96%, 85% and 69%, respectively). The inhibitory effect of galanin (1.5 nmol) on extracellular 5-HT levels in the rat hippocampus was significantly attenuated by co-administration of the 5-HT(1A) receptor antagonists WAY-100635 (0.3 and 0.6 mg/kg s.c.) and, to a lesser extent, with pindolol (20 mg/kg s.c.). These data provide direct in vivo evidence of agonistic 5-HT(1A)-galanin receptor interaction at the presynaptic level. Furthermore, the findings indicate that a down-regulation of the somato-dendritic 5-HT(1A) autoreceptors, following their stimulation with 8-OH-DPAT and possibly also indirectly with 5-HT reuptake inhibitors, may be compensated by a subsequent 'sensitization' of the inhibitory galanin receptors in the DRN. Thus, the enhanced galanin receptor-mediated inhibition of 5-HT neurotransmission may contribute to the pathophysiology of depression or to the reduced and delayed efficacy of antidepressant therapies.     

三氯乙醇对5－HT和ICS_（205－930）与［~3H］BRL_（46470

用放射配体受体结合分析法，观察了三氯乙醇（ＴＣＥｔ）对５－ＨＴ３受体激动剂５－ＨＴ和５－ＨＴ３受体拮抗剂ＩＣＳ２０５－９３０与［３Ｈ］ＢＲＬ１６１７０竞争大鼠脑皮质５－ＨＴ３受体能力的影响。结果证实，ＴＣＥｔ（４ｍｍｏｌ·Ｌ－１）能使５－ＨＴ竞争受体的浓度－反应曲线左移，半数抑制结合浓度降低（Ｐ＜０．０１），斜率无显著改变；而对ＩＣＳ２０５－９３Ｏ未见明显影响。表明ＴＣＥｔ能增强５－ＨＴ与５－ＨＴ３受体结合的能力，提示它可能通过这一机制促进５－ＨＴ３配体闸门离子通道开放而发挥作用。     

[3H]Rauwolscine: an antagonist radioligand for the cloned human 5-hydroxytryptamine2B (5-HT2B) receptor

In previous reports, [³H]5-HT has been used to characterize the pharmacology of the rat and human 5-HT_2B receptors. 5-HT, the native agonist for the 5-HT_2B receptor, has a limitation in its usefulness as a radioligand since it is difficult to study the agonist low-affinity state of a G protein-coupled receptor using an agonist radioligand. When using [³H]5-HT as a radioligand, rauwolscine was determined to have relatively high affinity for the human receptor (K_i human = 14.3 ± 1.2 nM, compared to K_i rat = 35.8 ± 3.8 nM). Since no known high affinity antagonist was available as a radioligand, these studies were performed to characterize [³H]rauwolscine as a radioligand for the cloned human 5-HT_2B receptor expressed in AV12 cells. When [³H]rauwolscine was initially tested for its usefulness as a radioligand, complex competition curves were obtained. After testing several α₂-adrenergic ligands, it was determined that there was a component of [³H]rauwolscine binding in the AV12 cell that was due to the presence of an endogenous α₂-adrenergic receptor. The α₂-adrenergic ligand efaroxan was found to block [³H]rauwolscine binding to the α₂-adrenergic receptor without significantly affecting binding to the 5-HT_2B receptor and was therefore included in all subsequent studies. In saturation studies at 37° C, [³H]rauwolscine labeled a single population of binding sites, K_d = 3.75 ± 0.23 nM. In simultaneous experiments using identical tissue samples, [³H]rauwolscine labeled 783 ± 10 fmol of 5-HT_2B receptors/mg of protein, as compared to 733 ± 14 fmol of 5-HT_2B receptors/mg of protein for [³H]5-HT binding. At 0° C, where the conditions for [³H]5-HT binding should label mostly the agonist high affinity state of the human 5-HT_2B receptor, [³H]rauwolscine (B_max = 951 ± 136 fmol/ mg), again labeled significantly more receptors than [³H]5-HT (B_max = 615 ± 34 fmol/mg). The affinity of [³H]rauwolscine for the human 5-HT_2B receptor at 0° C did not change, K_d = 4.93 ± 1.27 nM, while that for [³H]5-HT increased greatly (K_d at 37° C = 7.76 ± 1.06 nM; K_d at 0° C = 0.0735 ± 0.0081 nM). When using [³H]rauwolscine as the radioligand, competition curves for antagonist structures modeled to a single binding site, while agonist competition typically resulted in curves that best fit a two site binding model. In addition, many of the compounds with antagonist structures displayed higher affinity for the 5-HT_2B receptor when [³H]rauwolscine was the radioligand. Typically, ∼ 85% of [³H]rauwolscine binding was specific binding. These studies display the usefulness of [³H]rauwolscine as an antagonist radioligand for the cloned human 5-HT_2B receptor. This should provide a good tool for the study of both the agonist high- and low-affinity states of the human cloned 5-HT_2B receptor. Received: 26 June 1997 / Accepted: 30 August 1997     

Clomipramine enhances the cortisol response to 5-HTP: implications for the therapeutic role of 5-HT2 receptors

We measured the cortisol response to the 5-HT precursor, 5–hydroxytryptophan, (5-HTP) in seven patients with major depression before and after 8 weeks treatment with the tricyclic antidepressant, clomipramine. The cortisol response to 5-HTP was significantly increased following clomipramine treatment, suggesting that clomipramine, like selective serotonin re-uptake inhibitors (SSRIs), enhances this 5-HT₂ receptor mediated response. Because other tricyclic antidepressants do not increase 5-HTP-mediated cortisol release, it seems unlikely that enhancement of 5-HT₂ receptor function is a critical mechanism for antidepressant action. However, facilitation of neurotransmission at 5-HT₂ receptors could account for the efficacy of clomipramine and SSRIs in the treatment of obsessive compulsive disorder and also for their liability to cause orgasmic dysfunction. Received: 20 May 1998/Final version: 5 July 1998     

Thermoregulatory responses to serotonin (5-HT) receptor stimulation in the rat. Evidence for opposing roles of 5-HT2 and 5-HT1A receptors

The effects of serotonergic agonists and antagonists on the body temperatures of rats were investigated. The administration of the serotonin (5-HT) agonist 6-chloro-2(1-piperazinyl)-pyrazine (MK-212) produced a dose-related increase in body temperature. A maximal increase in body temperature of approx. 1.1°C was observed 30min after the administration of 3 mg/kg of MK-212. In contrast, administration of the putative 5-HT_1A agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) resulted in marked, dose-related hypothermic responses. Body temperatures were decreased approx. 3°C 30 min after an injection of 0.3 mg/kg of 8-OH-DPAT. Body temperatures were affected differentially by 5-methoxy-N,N-dimethyltryptamine (5-MeODMT). Large doses (3–10 mg/kg) of 5-MeODMT elicited hyperthermic responses, whereas small doses (0.5–1.0 mg/kg) produced hypothermie responses. Treatment of rats with ketanserin (3 mg/kg) completely prevented the hyperthermic effects of 5-MeODMT, and, in fact, converted a hyperthermic response to 5-MeODMT into a marked hypothermie response. Ketanserin (0.1–1.0 mg/kg) selectively antagonized the hyperthermic response to MK-212 but did not alter the hypothermic effect of 8-OH-DPAT. Mianserin (10 mg/kg) and pirenperone (0.03 mg/kg) also selectively antagonized hyperthermia induced by MK-212. In contrast, pindolol (0.03–0.1 mg/kg) and methiothepin (10 mg/kg) selectively antagonized hypothermia induced by 8-OH-DPAT but did not alter hyperthermia induced by MK-212. Spiperone (0.1–3 mg/kg) and pizotifen (10 mg/kg) attenuated the effects of both 8-OH-DPAT and MK-212. Xylamidine, a peripheral 5-HT antagonist, had no significant effect on hyperthermia induced by MK-212 or hypothermia induced by 8-OH-DPAT. It also was found that treatment of rats with the 5-HT₂ antagonists ketanserin or pirenperone alone resulted in a decrease in body temperature, whereas the administration of pindolol produced an increase in body temperature. On the basis of the present findings, it is concluded that the hyperthermic responses following the administration of MK-212 are mediated by central 5-HT₂ receptors and that the hypothermic responses to 8-OH-DPAT involve the activation of central 5-HT_1A receptors. The differential effects of 5-MeODMT on body temperature suggest that this indoleamine can activate both subtypes of 5-HT receptor.     

Pharmacological characterization of presynaptic α-adrenoceptors modulating [3H]noradrenaline and [3H]5-hydroxytryptamine release from slices of the hippocampus of the rat

The experiments served to characterize the receptors mediating the inhibitory effect of α-adrenergic drugs on K⁺ (20 mM)-induced [³H]noradrenaline (NA) and [³H]5-hydroxytryptamine ([³H]5-HT) release from slices of the dorsal part of rat hippocampus. Dose-response curves were constructed using the cumulative dose-response technique (Frankhuyzen and Mulder, 1982). All of the adrenergic agonist drugs examined inhibited the K⁺-induced [³H]NA release. NA appeared to have the highest intrinsic activity followed by adrenaline. Clonidine and adrenaline had similar intrinsic activities, while that of oxymetazoline was lowest. The highest pD₂ values were observed for oxymetazoline and clonidine, being slightly higher than that of adrenaline followed by NA. By far the lowest pD₂ values was observed for phenylephrine. With the exception of phenylephrine, all of the agonists also inhibited the K⁺-induced [³H]5-HT release. NA, adrenaline and oxymetazoline appeared to have similar intrinsic activities, while that of clonidine was considerably lower. The pD₂ values of NA and adrenaline were not significantly different but were somewhat lower than those of oxymetazoline and clonidine. Similar antagonistic effects of phentolamine and yohimbine were observed with respect to the adrenergic inhibition of K⁺-induced [³H]NA and [³H]5-HT release. Prazosin, however, appeared to be ineffective in both instances. It is concluded from these results that the presynaptic adrenergic inhibition of [³H]NA as well as [³H]5-HT release is mediated by α₂-adrenoceptors located on noradrenergic and serotonergic varicosities, respectively. Furthermore, our data suggest that these α₂-adrenoceptors are not pharmacologically identical.