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The overflow of tritium from stimulated rat hypothalamic slices preincubated with [3H]-serotonin (5-HT) was significantly enhanced by reducing the frequency of stimulation from 3 Hz to 1 Hz while keeping the number of impulses constant. The 5-HT receptor agonist 5-methoxytryptamine inhibited in a concentration-dependent manner the electrically-evoked release of [3H]-5-HT with IC50 values of 560 nmol/l and of 34 nmol/l when the stimulations were delivered at 3 Hz and 1 Hz, respectively. The terminal 5-HT autoreceptor antagonist methiothepin enhanced in a concentration-dependent manner the electrically-evoked release of [3H]-5-HT and this effect was greater at a frequency of stimulation of 3 Hz than at 1 Hz. In the same paradigm, the 5-HT reuptake inhibitors citalopram and paroxetine did not alter the overflow of radioactivity elicited by stimulation at 3 Hz but significantly decreased it at 1 Hz. In the presence of 5-HT autoreceptor blockade achieved with methiothepin, citalopram increased the overflow of [3H]-5-HT to the same extent at 1 Hz and at 3 Hz. The IC50 values for inhibition of [3H]-5-HT release by the selective alpha 2-adrenoceptor agonist UK 14.304 were 35 nmol/l at 3 Hz and 30 nmol/l at 1 Hz. It is concluded that modulation of 5-HT release by 5-HT autoreceptors, but not by alpha 2-adrenoceptors is dependent on the synaptic concentration of 5-HT as a function of the frequency of depolarization.

It has been suggested that the external carotid vasodilatation produced by serotonin (5-hydroxytryptamine; 5-HT) in anaesthetised dogs with intact vagosympathetic trunks is mediated by sympatho-inhibitory '5-HT1D' receptors and musculotropic '5-HT1-like' receptors. The present study has re-analysed this suggestion with regard to the classification schemes recently proposed by the NC-IUPHAR subcommittee on 5-HT receptors. In pentobarbital-anaesthetised dogs with intact vagosympathetic trunks, 1-min intracarotid (i.c.) infusions of 5-carboxamidotryptamine (5-CT; 0.01-0.3 microg/min), 5-HT (0.3-30 microg/ min), 5-methoxytryptamine (5-MeO-T; 1-100 microg/min) or sumatriptan (1-100 microg/min) dose-dependently increased the external carotid blood flow without affecting blood pressure or heart rate. The selective 5-HT1D receptor agonist, PNU-142633 (1-1000 microg/min), was essentially inactive. After mesulergine (300 microg/kg, i.v.), an antagonist at cardiovascular 5-HT7 receptors, the above responses to 5-HT, 5-CT and 5-MeO-T were blocked, whilst those to sumatriptan remained unaffected. In contrast, after the 5-HT1B/1D receptor antagonist, GR127935 (10 microg/kg, i.v.), the responses to 5-HT, 5-CT and 5-MeO-T were not affected, but those to sumatriptan were abolished. Furthermore, after the selective 5-HT1B receptor antagonist, SB224289 (300 microg/kg, i.v.), the responses to 5-HT, 5-CT and 5-MeO-T were significantly enhanced, whereas those to sumatriptan were abolished. Interestingly, the responses to all these agonists remained unmodified after the selective 5-HT1D receptor antagonist, BRL15572 (300 microg/kg, i.v.). The above results suggest that the '5-HT1-like' receptors, which mediate canine external carotid vasodilatation, display the pharmacological profile of sympatho-inhibitory 5-HT1B receptors and musculotropic 5-HT7 receptors, and confirm the existence of vasoconstrictor 5-HT1B receptors.

It has been suggested that the late hypotensive response to serotonin (5-hydroxytryptamine; 5-HT) in vagosympathectomized rats is mediated by '5-HT1-like' receptors since this effect is mimicked by 5-carboxamidotryptamine (5-CT), is not modified by cyproheptadine, ketanserin or MDL 72222, but it is blocked by methysergide. The present study was set out to reanalyze this suggestion in terms of the classification schemes proposed in 1994 and 1996 by the NC-IUPHAR subcommittee on the classification and nomenclature of 5-HT receptors. I.v. bolus injections of 5-CT (0.01-0.3 microg x kg(-1)), 5-HT (1-30 microg x kg(-1)) and 5-methoxytryptamine (5-MeO-T; 1-30 microg x kg(-1)) produced dose-dependent hypotensive responses with a rank order of agonist potency: 5-CT5-HT5-methoxytryptamine with sumatriptan (30-1000 microg x kg(-1)) inactive. The depressor responses to 5-HT and 5-CT were not attenuated by i.v. GR127935 (300-3000 microg x kg(-1)) or equivalent volumes of saline. In contrast, lisuride, methiothepin, mesulergine, metergoline and clozapine dose-dependently antagonized the responses to 5-HT and 5-CT; the rank order of apparent pA2 values against 5-HT and 5-CT, respectively, was: lisuride (7.7; 7.8)methiothepin (6.8; 7.0)or = mesulergine (6.4; 6.6)clozapine (5.7; 5.8); metergoline displayed variable potencies (5.6; 6.4). Except for lisuride, which also affected isoprenaline-induced hypotension, the antagonism by the other drugs was selective. Based upon the above rank order of agonist potency, the blockade by a series of drugs showing high affinity for the cloned 5-ht7 receptor and the lack of blockade by GR127935, our results indicate that the 5-HT receptor mediating hypotension in vagosympathectomized rats is operationally similar to other putative 5-ht7 receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine coronary and external carotid arteries and guinea-pig ileum as well as feline tachycardia).

In segments of human right atrial appendages preincubated with [3H]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-MeOT), 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-HTID receptors in human brain and for cloned human 5-HT1D alpha and 5-HT1D beta receptors, but not with their affinity for 5-HT1B, 5-HT1E, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3, 5-HT5A, 5-HT5B and 5-HT7 receptors. The potency order 5-CT5-HT5-MeOT is opposite to the order of affinities reported for 5-HT6 binding sites. The preferential 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetraline (up to 0.3 microM) and the selective 5-HT4 receptor agonist cisapride (up to 1 microM) failed to inhibit tritium overflow. L-694,247, a potent 5-HT1D beta receptor agonist, did not inhibit tritium overflow, but counteracted the inhibitory effect of 5-HT. Ketanserin at a concentration which should block 5-HT1D alpha but not 5-HT1D beta receptors and methiothepin at a concentration which may be assumed to block both 5-HT1D alpha and 5-HT1D beta 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-HT1D subfamily. The ability of ketanserin to antagonize the inhibitory effect induced by activation of these receptors suggests that they can be subclassified as 5-HT1D alpha.

The response of a cell line of Xenopus laevis melanophores to serotonin was examined. Serotonin increased intracellular levels of cAMP and induced pigment dispersion in the cells. The responses depended on both the concentration of serotonin applied and on the time for which the cells were exposed to serotonin. Using a recently described, microtiter-plate-based bioassay, a series of serotonin receptor ligands were evaluated as agonists or antagonists at the melanophore serotonin receptor. The pharmacological profile suggests the presence of a receptor which shares some properties with but appears different from other previously described serotonin receptors.

The possibility that the terminal serotonin (5-HT) autoreceptor in the rat hippocampus is coupled to Gi, Go or Gs regulatory proteins was investigated using the electrically evoked overflow of [3H]5-HT from preloaded slices. Pertussis toxin, which inactivates Gi/o or cholera toxin, which stimulates GS, was injected directly in the hippocampus 3 to 11 days prior to the experiments. Hippocampus slices were prepared, loaded with [3H]5-HT, superfused continuously, and stimulated electrically 72 min (S1) and 116 min (S2) after the beginning of superfusion. In the absence of any drug, the evoked overflow of [3H]5-HT in S1 was not altered by either toxin. The enhancing effect of the 5-HT reuptake blocker paroxetine (1 μmol/I) on the evoked [3H]5-HT overflow was also unaltered by these toxins. 5-Carboxyamidotryptamine, a 5-HT autoreceptor agonist, inhibited in a concentration-dependent manner the stimulation-evoked release of [3H]5-HT The concentration-effect curve (0.001–0.1 μmol/I) for this drug was not altered by pretreatment with either pertussis or cholera toxin. Similarly, the effect of another 5-HT autoreceptor agonist, 5-methoxytryptamine (0.1 and I μmol/I), was not altered in the pretreated rats. In addition, the reduction of [3H]5-HT overflow obtained by increasing the stimulation frequency from 1 Hz to 5 Hz, which is due to an increase in terminal 5-HT autoreceptor activation at the higher frequency, was not altered by either toxin. The enhancing effect of the 5-HT autoreceptor antagonist methiothepin (1 μmol/I) on stimulation-evoked [3H]5-HT overflow was not changed by either pretreatment. N-Ethylmaleimide inactivates Gi/o proteins by alkylation. Preincubation with 30 μmol/I N-ethylmaleimide for 30 min did not alter the efficacy of the 5-HT reuptake blocker paroxetine to enhance [3H]5-HT overflow nor did it alter the attenuating effect of 5-methoxytryptamine and the differential effectiveness of 1 and 5 Hz stimulations on the overflow of [3H]5-HT In conclusion, the results suggest that the terminal 5-HT autoreceptor in the rat hippocampus may not be coupled to Gi, Go or Gs proteins.

This study describes a component of 5-HT-evoked depolarization of the rat isolated vagus nerve which was unaffected by the 5-HT3 receptor antagonist ondansetron. A grease-gap extracellular recording technique was used. Ondansetron (10-100 nmol/l) displaced the 5-HT concentration-response curve to the right yielding a pA2 value of 8.6 (8.5-8.8), consistent with 5-HT3 receptor antagonism, and revealing a component of the 5-HT response which was resistant to ondansetron blockade. In the presence of ondansetron (100 nmol/l) the maximum depolarization in the resistant phase was 15.5 (12.6-19.2)% of the initial maximum response to 5-HT and the pEC50 value was 7.0 (6.7-7.3). The mechanism of the ondansetron-resistant component of the 5-HT response resembled a 5-HT4-receptor-effect in being absent in preparations equilibrated with 5-methoxytryptamine (10 mumol/l) and antagonised by ICS 205930 (tropisetron, pA2 6.4). 5-Methoxytryptamine alone was an agonist in the vagus nerve with a maximum response similar to that of the ondansetron resistant phase of the 5-HT response. Similarly renzapride alone evoked small depolarizations of this preparation but antagonized the ondansetron resistant phase of the 5-HT response (pA2 7.3-7.4). These effects of 5-methoxytryptamine and renzapride are also consistent with a 5-HT4 receptor mechanism. Ketanserin (1 mumol/l) and methysergide (1 mumol/l) had little effect on responses to 5-HT. The depolarization evoked by this putative 5-HT4 receptor mechanism was small but prolonged and appears to mask and after-hyperpolarizing phase of the 5-HT response in this tissue.

The effects of 5-methoxytryptamine and 5-hydroxytryptamine (5-HT) on both basal and electrically evoked outflow of tritium were studied in guinea-pig myenteric plexus preparations preincubated with [3H]-choline. Basal outflow. 5-Methoxytryptamine caused a transient and calcium-dependent increase in basal outflow of [3H]acetylcholine that was abolished by tetrodotoxin. Ondansetron (1 μmol/1) did not affect the stimulatory response of 5-methoxytryptamine but ICS 205-930 (1 and 3 μmol/1) produced parallel rightward displacements of the concentration-response curve to 5-methoxytryptamine. The PKB value for ICS 205-930 was 6.6 suggesting an involvement of 5-HT4 receptors. 5-HT caused an increase in basal outflow of [3H]acetylcholine and a biphasic concentration-response curve was obtained. The maximal response of the first phase to 5-HT (release of 0.98% of tissue tritium) and the maximal response to 5-methoxytryptamine (0.94% of tissue tritium) were similar but 5-methoxytryptamine (-log EC50: 6.9) was less potent than 5-HT (-log EC50 of the high affinity component: 7.9). ICS 205-930 (0.01–1.0 μmol/1) acted as a competitive antagonist against the low affinity component of the 5-HT concentration-response curve with a pA2 value of 8.0. It is concluded that stimulation of both 5-HT4 receptors (by 5-methoxytryptamine and submicromolar concentrations of 5-HT) and 5-HT3 receptors (by micromolar concentrations of 5-HT) causes a release of acetylcholine which in turn leads to smooth muscle contraction. Electrically evoked outflow. This outflow of [3H]acetylcholine was concentration-dependently inhibited by both 5-methoxytryptamine and 5-HT. ICS 205-930 (1 μmol/1) reinforced the inhibitory effect of 5-methoxytryptamine but not that of 5-HT. In the presence of methiothepine (0.1 μmol/1) 5-methoxytryptamine enhanced the evoked outflow of [3H]acetylcholine, an effect which was attenuated by 3 μmol/1 ICS 205-930. These results suggest that 5-methoxytryptamine may both inhibit (via 5-HT1 receptors) and facilitate (via 5-HT4 receptors) the evoked release of acetylcholine from guinea-pig myenteric neurones. The facilitatory action is unmasked when the 5-HT1 receptor is blocked by methiothepine.

The emetic effects of 5-hydroxytryptamine (5-HT) and 5-HT3 receptor agonists were investigated in the house musk shrew, Suncus murinus. 5-Hydroxytryptamine (5-HT; i.p., i.v., s.c.) and 2-methyl-5-HT (2-Me-5-HT; i.p.) but not 5-hydroxyindoleacetic acid (i.p.) or 5-methoxytryptamine (i.p.) induced emesis with very short latency. Tropisetron (ICS 205-930, a 5-HT3 receptor antagonist, s.c.) blocked the emesis induced by 5-HT (10 mg/kg, i.p.) and 2-Me-5-HT (5 mg/kg, i.p.) with respective ID50 values of 7.8 and 70.9 micrograms/kg. Pindolol (5-HT1 receptor antagonist) and ketanserin (5-HT2 receptor antagonist) were about 100 times less potent than tropisetron. The emesis induced by 5-HT was prevented by surgical vagotomy but not by pretreatment with a combination of atropine (0.1 mg/kg, s.c.) and hexamethonium (10 mg/kg, s.c.). These results clearly indicate that 5-HT is emetogenic probably through a stimulation of peripheral 5-HT3 receptors.

The mechanism of the attenuation, by serotonin uptake blockers, of the release-inhibiting effect of exogenous serotonin autoreceptor agonists was studied in rabbit brain cortex and rat hypothalamus slices. The slices were preincubated with 3H-serotonin and then superfused and stimulated electrically. In rabbit brain slices stimulated by trains of 4 pulses at 100 Hz, 5-carboxamidotryptamine and 5-methoxytryptamine reduced the evoked overflow of tritium, and their concentration-response curves were not changed by any of three serotonin uptake inhibitors, namely citalopram, fluvoxamine and 6-nitroquipazine. In contrast, when the slices were stimulated by trains of 10 pulses at 0.033 Hz, fluvoxamine shifted the concentration-response curve of 5-methoxytryptamine to the right. Experiments with the autoreceptor antagonist metitepine indicated that little, if any, endogenous autoinhibitory tone developed in the course of trains of 4 pulses/100 Hz, irrespective of the absence or presence of uptake inhibitors, as well as during trains of 10 pulses/0.033 Hz in the absence of uptake inhibitors, whereas marked autoinhibition developed when 10 pulses/0.033 Hz were applied in the presence of fluvoxamine. In rat hypothalamic slices stimulated by trains of 4 pulses at 100 Hz, citalopram also failed to change the concentration-response curve of 5-methoxytryptamine. These results indicate that serotonin uptake blockers attenuate the effect of exogenous autoreceptor agonists by an increase in the biophase concentration of released serotonin and, hence, in endogenous autoinhibitory tone, and not by some direct “molecular link” unrelated to the biophase concentration of released serotonin.

Agonist-induced desensitization has been utilized to discriminate and independently “isolate” the neuronal excitatory receptors to 5-hydroxytryptamine (5-HT) in the guinea pig ileum (5-HT3 and putative 5-HT4 receptors). Electrically stimulated longitudinal muscle myenteric plexus preparations, and non-stimulated segments of whole ileum were used. Exposure to 5-methoxytryptamine (10 μmol/l) inhibited completely responses to 5-HT at the putative 5-HT4 receptor without affecting 5-HT3-mediated responses. Conversely, exposure to 2-methyl-5-HT (10 μmol/l) inhibited completely responses to 5-HT at the 5-HT3 receptor without affecting putative 5-HT4-mediated responses. The inhibition with 5-methoxytryptamine and 2-methyl-5-HT, either alone or in combination, appeared selective as responses to KCI, DMPP, carbachol, histamine, and substance P were unaffected or only very slightly modified. Furthermore, the pA2 values for ICS 205–930 at the putative 5-HT4 (pA2 = 6.2 to 6.5) and 5-HT3 (pA2 = 7.6 to 8.1) receptors (estimated in the presence of 2-methyl-5HT and 5-methoxytryptamine, respectively) were consistent with those estimated in the absence of desensitization. 5-Methoxytryptamine, but not 2-methyl-5-HT, suppressed completely but reversibly the concentration-effect curve to renzapride, suggesting that responses to this agent are mediated exclusively via agonism at the putative 5-HT4 receptor. It is concluded that 5-methoxytryptamine and 2-methyl-5-HT can be utilized as selective probes to discriminate the putative 5-HT4 receptor from the 5-HT3 receptor in guinea pig ileum. This finding is of importance as no selective antagonist exists for the putative 5-HT4 receptor. Furthermore, the presently described method of agonist-induced desensitization and 5-HT receptor discrimination may be useful for the identification and characterization of the putative 5-HT4 receptor in other tissues and species.

Isolated neurointermediate lobes (NILs) or isolated neural lobes (NLs) of the rat pituitary gland were incubated in Krebs-HEPES solution which contained pargyline and the dopamine uptake inhibitor GBR 12921. The release of endogenous dopamine was determined by HPLC with electrochemical detection. Electrical stimulation of the pituitary stalk induced a frequency-dependent release of dopamine. The release of dopamine from the combined NIL evoked by stimulation at 15 Hz was increased by 130% in the presence of the dopamine D2 receptor antagonist, (-)-sulpiride; the (+)-enantiomer of sulpiride had virtually no effect. When the stimulation frequency was 3 Hz (-)-sulpiride caused an increase in dopamine release by 230%. A similar increase was observed in the presence of domperidone, another dopamine D2 receptor antagonist. The dopamine receptor agonists, apomorphine and quinpirole, had no significant effects on the evoked release of dopamine indicating that under the present incubation conditions endogenous dopamine may have been maximally activating the autoinhibition. However, in the presence of 1 mumol/l (-)-sulpiride, apomorphine as well as quinpirole reduced the evoked release of dopamine in a concentration-dependent manner. The dopamine D1 receptor selective antagonist, SCH 23390, had no effect on the evoked release of dopamine at a concentration of 1 mumol/l. Only at a concentration of 10 mumol/l did SCH 23390 cause a small increase in dopamine release; this effect was, however, abolished in the presence of 1 mumol/l (-)-sulpiride.(ABSTRACT TRUNCATED AT 250 WORDS)

The 5-hydroxytryptamine (5HT) receptor agonist, 5-methoxytryptamine, inhibited in a concentration-dependent manner the electrically-evoked release of 3H-5HT from superfused rat hypothalamic slices, with an IC50 of 560 nmol/l, without affecting the spontaneous outflow of radioactivity. In the presence of the selective monoamine oxidase B (MAO B) inhibitor, (-)-deprenyl (1 mumol/l), the concentration-effect curve for 5-methoxytryptamine was shifted significantly to the left, and the IC50 was decreased to 25 nmol/l. Under the same experimental conditions, the potency of the 5HT receptor agonist lysergic acid diethylamide (LSD) at inhibiting the electrically-evoked release of 3H-5HT was the same in the presence as well as in the absence of (-)-deprenyl. The IC50 values for LSD were 34 nmol/l in the absence of deprenyl, and 31 nmol/l in the presence of the MAO B inhibitor. It is concluded that deprenyl potentiates the inhibition by 5-methoxytryptamine of 3H-5HT release, by preventing its inactivation through MAO B. Since 5-methoxytryptamine may be present in the pineal gland of some species, the potent effects of this 5-HT receptor agonist on serotoninergic neurotransmission may be of physiological relevance.

In segments of the rat inferior vena cava preincubated with 3H-noradrenaline, it was examined whether presynaptic serotonin (5-HT) receptors exist on the postganglionic sympathetic nerves of the circulatory system; for this purpose the effects of 5-HT receptor agonists and antagonists on the electrically evoked 3H overflow were studied. Furthermore, vagotomized pithed rats (treated with atropine and captopril) were used to investigate the effects of these drugs on heart rate and on the tachycardia induced by electrical stimulation of the preganglionic sympathetic nerves (C7−T1) via the pithing rod; these experiments were carried out to provide evidence that the presynaptic 5-HT receptors are operative in vivo. 1. 5-HT and 5-methoxytryptamine (5-OCH3-T) concentration-dependently inhibited the electrically evoked 3H overflow from the vena cava. The inhibitory effect was more pronounced at 0.66 Hz than at 2 Hz. 8-Hydroxy-2-(di-n-propylamino)tetralin did not alter the evoked overflow. The inhibitory effect of 5-HT or 5-OCH3-T was antagonized by metitepin but not affected by ketanserin or rauwolscine. 2. In pithed rats 5-HT and 5-OCH3-T by themselves dose-dependently increased heart rate. The positive chronotropic effect of 5-HT 10 μmol/kg, which was not affected by ketanserin, was considerably decreased by desipramine, indicating that 5-HT at least at this high dose acts predominantly by a tyramine-like indirect sympathomimetic effect. The tachycardic response to 5-OCH3-T was reversed to a very slight negative chronotropic effect by ketanserin or methysergide, but was not affected by desipramine or propranolol indicating that the increase in heart rate induced by 5-OCH3-T is mediated via 5-HT2-receptors on the pacemaker cells. 3. Ketanserin-treated pithed rats were used to study the effect of 5-OCH3-T on the tachycardia induced by stimulation of the preganglionic sympathetic nerves via the pithing rod. The nerve stimulation-induced tachycardia was inhibited by 5-OCH3-T to a significantly higher extent than the tachycadia induced by infusion of noradrenaline. The inhibitory effect of 5-OCH3-T on nerve stimulation-induced tachycardia could not be antagonized by metitepin or rauwolscine; it decreased with increasing frequency of stimulation.

The occurrence and concentration of the four 5-methoxyindoles: 5-methoxtryptamine, 5-methoxytryptophol, 5-methyoxyindole-3-acetic acid and melatonin in the pineal gland of pig, cow, sheep and rat was investigated. The analytical method involved the use of deuterated analogues as internal standards and capillary column gas chromatography - mass spectrometry. The analyses of pineal glands obtained during the morning hours revealed the presence of 5-methoxyindole-3-acetic acid and melatonin in nmoles/g and 5-methyoxytryptophol and 5-methoxytryptamine in pmoles/g amounts in pig, cow and sheep. In Wistar and Sprague-Dawley strain of rat, melatonin was present at a concentration of about 0.5 pmoles/pineal, a level which was elevated more than five times by treatment with a monoamine oxidase inhibitor. 5-Methoxytryptamine was found at a concentration of about 0.03 pmoles/pineal, and was elevated by monoamine oxidase inhibition.