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.     

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 long-term administration of the 5-hydroxytryptamine1B receptor antagonist AR-A000002 to guinea pigs

Rationale Recently a new 5-hydroxytryptamine_1B (5-HT_1B)-receptor antagonist, AR-A000002, was described. It was shown that this compound dose dependently increased 5-HT metabolism and release in guinea pig brain following a single injection.Objectives The present study investigated effects of 3 weeks twice-daily treatment of guinea pigs with the 5-HT_1B-receptor antagonist AR-A000002 on the serotonergic neurons and receptor densities.Methods Guinea pigs were injected subcutaneously with AR-A000002, citalopram or saline twice daily for 3 weeks. Groups of animals were treated with challenge doses of AR-A000002 or saline 24 h after the last chronic treatment (citalopram group 48 h) and sacrificed 2 h thereafter. The effect on 5-HT metabolism and 5-HT release was assessed. Plasma and brain concentrations of AR-A000002 were analysed. The effects on binding of [³H]8-OH-DPAT to 5-HT_1A receptors, [³H]GR125743 to 5-HT_1B/1D receptors, [³H]ketanserin to 5-HT_2A receptors, and [³H]prazosin to ₁-adrenoceptors were determined.Results Repeated treatment of guinea pigs with AR-A000002 did not change the 5-HT_1B/1D, 5-HT_1A, 5-HT_2A or ₁-adrenergic receptor densities. Following repeated treatment of guinea pigs for 3 weeks with AR-A000002, the 5-HT_1B receptors were still receptive to a challenge with the same compound. Thus, an increase in the 5-HIAA/5-HT ratio and 5-HT release was seen following challenge doses of AR-A000002. No difference in the plasma and brain concentrations of AR-A000002 was found between the sub-chronic treated AR-A000002 and saline-treated guinea pigs.Conclusions It is concluded that AR-A000002 is a 5-HT_1B receptor antagonist, which enhances persistently the serotonergic neurotransmission in guinea pig brain.     

The effects of ageing on prejunctional 5-hydroxytryptamine receptors in the rat vas deferens

Summary The prejunctional inhibitory effects of a series of 5-HT₁ receptor agonists were examined against the isometric contraction of epididymal portions of rat vas deferens evoked by single stimulus pulses in the presence of nifedipine (10 mol/l). The 5-HT_1A ligand flesinoxan produced inhibition of contractions which was not inhibited by cyanopindolol or yohimbine. However, the prejunctional inhibitory concentration response curve for the 5-HT₁ agonist 5-carboxamidotryptamine (5-CT) was biphasic in tissues from 1.5 month old animals but monophasic in tissues from 24 months animals. Cyanopindolol (1 mol/l) antagonised the inhibitory effects of 5-CT in tissues from 1.5 and 3 month animals but not in tissues from 8 or 24 months animals. Inhibitory actions of 5-CT were not prevented by pretreating animals with pertussis toxin (6 g/kg i. v.), a dose which abolished the negative inotropic response to acetylcholine in rat left atria. It is concluded that the nerve terminals of vas deferens from 1.5 month old animals contain both 5-HT_1B and other as yet unclassified 5-HT₁ receptors, but that this 5-HT_1B-mediated response is lost in maturation and ageing.Send offprint requests to J. R. Docherty 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     

The pharmacological properties of the presynaptic serotonin autoreceptor in the pig brain cortex conform to the 5-HT1D receptor subtype

Summary The effects of serotonin receptor agonists and antagonists on the electrically (3 Hz) evoked ³H overflow were determined on pig brain cortex slices preincubated with ³H-serotonin and superfused with physiological salt solution containing indalpine (an inhibitor of serotonin uptake) plus phentolamine. The potencies of the serotonin receptor agonists and antagonists were compared with their affinities for 5-HT_1A, 5-HT_1B, 5-HT_1c, and 5-HT_1D binding sites in pig or rat tissue membranes; in addition, the potencies of the agonists were compared to their potencies in inhibiting adenylate cyclase activity in membranes of calf substantia nigra. In the superfusion experiments on pig brain cortex slices the following rank orders of potencies were obtained: agonists, serotonin > 5-methoxytryptamine = 5-carboxamidotryptamine >R U 24969 (5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole) > SDZ 21009 (4(3-terbutylamino- 2-hydroxypropoxy)indol- 2-carbonic-acid-isopropylester) yohimbine cyanopindolol > 8-OHDPAT (8-hydroxy-2-(di-n-propylamino)tetralin) CGS 12066 B (7-trifluoromethyl-4(4-methyl-l-piperazinyl)-pyrrolo[1,2-a]quinoxaline); ipsapirone and urapidil were ineffective; antagonists (antagonism determined against 5methoxytryptamine as an agonist), metitepine > metergoline > mianserin. Propranolol, spiperone or mesulergine did not produce a shift of the concentration-response curve for 5-methoxytryptamine. The potencies of the serotonin receptor agonists in pig brain cortex slices were significantly correlated with their affinities for 5-HT_1c and 5-HT_1D binding sites in membranes of the pig choroid plexus and caudate nucleus, respectively, but not with their affinities for 5-HT_1A and 5-HT_1B sites in membranes of the cerebral cortex of pig and rat, respectively. The agonist potencies in decreasing ³H overflow were also significantly correlated with their potencies in inhibiting adenylate cylase activity in calf substantia nigra (i.e., a 5-HT_1D receptor-mediated effect). In conclusion, the pig brain cortical 5-HT autoreceptor probably belongs to the 5-HT_1D subtype. The involvement of 5-HT_1c recognition sites was excluded by the low potency of mianserin as an antagonist and, in particular, by the ineffectiveness of the 5-HT_1c receptor antagonist mesulergine.E. S. and M. G. were supported by grants of the Deutsche ForschungsgemeinschaftSend offprint requests to M. Göthert at the above address     

Pharmacological characterisation of the agonist radioligand binding site of 5-HT<Subscript>2A</Subscript>, 5-HT<Subscript>2B</Subscript> and 5-HT<Subscript>2C</Subscript> receptors

In the present study we compared the affinity of various drugs for the high affinity agonist-preferring binding site of human recombinant 5-HT_2A, 5-HT_2B and 5-HT_2C receptors stably expressed in monoclonal mammalian cell lines. To ensure that the agonist-preferring conformation of the receptor was preferentially labelled in competition binding experiments, saturation analysis was conducted using antagonist and agonist radiolabels at each receptor. Antagonist radiolabels ([³H]-ketanserin for 5-HT_2A receptor and [³H]-mesulergine for 5-HT_2B and 5-HT_2C receptor) bound to a larger population of receptors in each preparation than the corresponding agonist radiolabel ([¹²⁵I]-DOI for 5-HT_2A receptor binding and [³H]-5-HT for 5-HT_2B and 5-HT_2C receptor binding). Competition experiments were subsequently conducted against appropriate concentrations of the agonist radiolabels bound to the agonist-preferring subset of receptors in each preparation. These studies confirmed that there are a number of highly selective antagonists available to investigate 5-HT₂ receptor subtype function (for example, MDL 100907, RS-127445 and RS-102221 for 5-HT_2A, 5-HT_2B and 5-HT_2C receptors respectively). There remains, however, a lack of highly selective agonists. (–)DOI is potent and moderately selective for 5-HT_2A receptors, BW723C86 has poor selectivity for human 5-HT_2B receptors, while Org 37684 and VER-3323 display some selectivity for the 5-HT_2C receptor. We report for the first time in a single study, the selectivity of numerous serotonergic drugs for 5-HT₂ receptors from the same species, in mammalian cell lines and using, exclusively, agonist radiolabels. The results indicate the importance of defining the selectivity of pharmacological tools, which may have been over-estimated in the past, and highlights the need to find more selective agonists to investigate 5-HT₂ receptor pharmacology.     

Modulation of cortical acetylcholine release by serotonin: the role of substance P interneurons

The cholinergic system exerts an important modulatory effect on hippocampal functions. Presynaptic inhibition of hippocampal and neocortical acetylcholine (ACh) release by serotonin (5-HT) has been reported in both rat and human brain. There is some controversy, however, concerning the 5-HT receptor which mediates the inhibitory effects of 5-HT. Using slices of the hippocampal formation of rat prelabelled with [³H]-choline, superfused and depolarized electrically (2 min, 3 Hz, 2 ms, 24 mA) or by K⁺ (20 mM) we observed that 5-HT inhibits hippocampal and entorhinal [³H]-overflow ([³H]-ACh release) by 5-HT_1B receptors located on cholinergic terminals. However, this inhibition requires the functional elimination of substance P/-aminobutyric acid (SP/GABA) interneurons which express 5-HT_2A receptors as shown by in situ hybridisation histochemistry. Activation of these somadendritically located 5-HT_2A receptors facilitates SP release. SP, in turn, stimulates hippocampal [³H]-ACh release through NK₁ receptors present on cholinergic terminals. These findings suggest close links between cholinergic afferents, SP interneurons and 5-HT₂ receptors. A loss of cholinergic afferents and 5-HT₂ receptors, along with a reduction in substance P-immunoreactive neurons, have been observed in the brains of patients suffering from Alzheimer's disease, suggesting the concept that these three alterations reflect a disruption of a functional unit. The present findings might help to explain early pathological changes in Alzheimer's disease.     

Evidence for presynaptic location of inhibitory 5-HT1D\-like autoreceptors in the guinea-pig brain cortex

The effects of 5-hydroxytryptamine (5-HT) receptor agonists and antagonists on tritium overflow evoked by high K⁺ were determined in superfused synaptosomes and slices, preincubated with [³H]5-HT, from guinea-pig brain cortex. In addition, we estimated the potencies of 5-HT receptor ligands in inhibiting specific [³H]5-HT binding (in the presence of 8-hydroxy-2(di-n-propylamino)tetralin and mesulergine to prevent binding to 5-HT_1A and 5-HT_2C sites) to guinea-pig cortical synaptosomes and membranes.5-HT receptor agonists inhibited the K⁺-evoked tritium overflow from synaptosomes and slices. In synaptosomes the rank order of potencies was 2-[5-[3-(4-methylsulphonylamino)benzyl-1,2,4-oxadiazol-5-yl]-1H-indole-3-yl] ethylamine (L-694,247) >5-carboxamidotryptamine (5-CT) > oxymetazoline (in the presence of idazoxan) 5-HT > sumatriptan 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole (RU 24969). The potencies of the agonists in inhibiting tritium overflow from slices correlated with those in synaptosomes, suggesting that the same site of action is involved in both preparations. In synaptosomes the nonselective antagonist at cloned human 5-HT_1D, and 5-HT_1D receptors, methiothepin, shifted the concentration-response curve for 5-CT to the right (apparent pA₂: 7.87). In contrast, ketanserin at a concentration which should block the 5-HT_1D, but not the 5-HT_1D\, receptor did not alter the inhibitory effect of 5-CT on tritium overflow. In cortical synaptosomes and membranes, [³H]5-HT bound to a single site with high affinity. In competition experiments, 5-HT receptor agonists and antagonists inhibited specific [³H]5-HT binding. In synaptosomes the rank order was L-694,247 > methiothepin >5-CT >5-methoxytryptamine >5-HT sumatriptan oxymetazoline > RU 24969 > ketanserin > ritanserin. A very similar rank order was obtained in cerebral cortical membranes. The potencies of the 5-HT receptor agonists in inhibiting tritium overflow from synaptosomes and slices correlated with their potencies in inhibiting [³H]5-HT binding to synaptosomes and membranes.In conclusion, the 5-HT receptors mediating inhibition of 5-HT release in the guinea-pig cortex are located on the serotoninergic axon terminals and, hence, represent presynaptic inhibitory autoreceptors. The [³H]5-HT binding sites in cerebral cortical synaptosomes and membranes exhibit the pharmacological properties of 5-HT_1D receptors. The correlation between the functional responses and the binding data confirms the 5-HT_1D character of the presynaptic 5-HT autoreceptors. According to the results of the interaction experiment of ketanserin and methiothepin with 5-CT on 5-HT release, the presynaptic 5-HT autoreceptors can be subclassified as 5-HT_1D\-like.     

5-HT1B receptor-mediated contractions in human temporal artery: evidence from selective antagonists and 5-HT receptor mRNA expression

1. In the human temporal artery both 5-HT_1-like and 5-HT_2A receptors mediate the contractile effects of 5-hydroxytryptamine (5-HT) and we have suggested that the 5-HT_1-like receptors resemble more closely recombinant 5-HT_1B than 5-HT_1D receptors. To investigate further which subtype is involved, we investigated the blockade of 5-HT-induced contractions by the 5-HT_1B-selective antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2-methyl-4′[(5-methyl-1,2,4-oxadiazole-3-yl) biphenyl-4-yl] carbonyl} furo[2,3-f]indole-3-spiro-4′-piperidine oxalate) and the 5-HT_1D-selective antagonist BRL-15572 (1-phenyl-3[4-3-chlorophenyl piperazin-1-yl] phenylpropan-2-ol). We also used RT-PCR to search for the mRNA of 5-HT_1B, 5-HT_1D and other 5-HT receptors.
2. The contractile effects of 5-HT in temporal artery rings were partially antagonized by SB-224289 (20, 200 nM) (apparent K_B=1 nM) and ketanserin (1 μM) but not by BRL-15572 (500 nM).
3. Sumatriptan evoked contractions (EC₅₀, 170 nM) that were resistant to blockade by BRL-15572 (500 nM) but antagonized by SB-224289 (20, 200 nM).
4. The potency of 5-HT (EC₅₀) was estimated to be 94 nM for the ketanserin-sensitive receptor and 34 nM for the SB-224289-sensitive receptor. The fraction of maximal 5-HT response mediated through SB-224289-sensitive receptors was 0.20–0.67, the remainder being mediated through ketanserin-sensitive receptors.
5. We detected arterial receptor mRNA for the following receptors (incidence): 5-HT_1B (8/8), 5-HT_1D (2/8), 5-HT_1F (0/4), 5-HT_2A (0/8), 5-HT_2B (0/8), 5-HT_2C (0/8), 5-HT₄ (4/8) and 5-HT₇ (4/8).
6. We conclude that the ketanserin-resistant fraction of the 5-HT effects and the effects of sumatriptan are mediated by 5-HT_1B receptors. The lack of antagonism by BRL-15572 rules out 5-HT_1D receptors as mediators of the contractile effects of 5-HT and sumatriptan.

     

5-hydroxytryptamine induced relaxation in the pig urinary bladder neck

Background and purpose

5-Hydroxytryptamine (5-HT) is one of the inhibitory mediators in the urinary bladder outlet region. Here we investigated mechanisms involved in 5-HT-induced relaxations of the pig bladder neck.

Experimental approach

Urothelium-denuded strips of pig bladder were mounted in organ baths for isometric force recordings of responses to 5-HT and electrical field stimulation (EFS).

Key results

After phenylephrine-induced contraction, 5-HT and 5-HT receptor agonists concentration-dependently relaxed the preparations, with the potency order: 5-carboxamidotryptamine (5-CT) > 5-HT = RS67333 > (±)-8-hydroxy-2-dipropylaminotetralinhydrobromide > m-chlorophenylbiguanide > α-methyl-5-HT > ergotamine. 5-HT and 5-CT relaxations were reduced by the 5-HT₇ receptor antagonist (2R)-1-[(3-hydroxyphenyl)sulphonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine hydrochloride and potentiated by (S)-N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide dihydrochloride (WAY 100135) and cyanopindolol, 5-HT_1A and 5-HT_1A/1B receptor antagonists respectively. Inhibitors of 5-HT_1B/1D, 5-HT₂, 5-HT_2B/2C, 5-HT₃, 5-HT₄, 5-HT_5A and 5-HT₆ receptors failed to modify 5-HT responses. Blockade of monoamine oxidase A/B, noradrenergic neurotransmission, α-adrenoceptors, muscarinic and purinergic receptors, nitric oxide synthase, guanylate cyclase and prostanoid synthesis did not alter relaxations to 5-HT. Inhibitors of Ca²⁺-activated K⁺ and ATP-dependent K⁺ channels failed to modify 5-HT responses but blockade of neuronal voltage-gated Na⁺-, Ca²⁺-and voltage-gated K⁺ (K_v)-channels potentiated these relaxations. Adenylyl cyclase activation and cAMP-dependent protein kinase (PKA) inhibition potentiated and reduced, respectively, 5-HT-induced responses. Under non-adrenergic, non-cholinergic, non-nitrergic conditions, EFS induced neurogenic, frequency-dependent, relaxations which were resistant to WAY 100135 and cyanopindolol.

Conclusions and implications

5-HT relaxed the pig urinary bladder neck through muscle 5-HT₇ receptors linked to the cAMP-PKA pathway. Prejunctional 5-HT_1A receptors and K_v channels modulated 5-HT-induced relaxations whereas postjunctional K⁺ channels were not involved in such responses. 5-HT₇ receptor antagonists could be useful in the therapy of urinary incontinence produced by intrinsic sphincter deficiency.     

Implication of 5-HT(2B) receptors in the serotonin syndrome

The serotonin (5-HT) syndrome occurs in humans after antidepressant overdose or combination of drugs inducing a massive increase in extracellular 5-HT. Several 5-HT receptors are known to participate in this syndrome in humans and animal models. The 5-HT_2B receptor has been proposed as a positive modulator of serotonergic activity, but whether it is involved in 5-HT syndrome has not yet been studied.We analyzed here, a putative role of 5-HT_2B receptors in this disorder by forced swimming test (FST) and behavioral assessment in the open field. In FST, genetic (5-HT_2B^−/− mice) or pharmacological (antagonist RS127445 at 0.5 mg/kg) ablation of 5-HT_2B receptors facilitated selective 5-HT reuptake inhibitors (SSRI)-induced increase of immobility time as well as expression of other symptoms related to 5-HT syndrome like hind limb abduction and Straub tail. Increase in immobility was also developed in FST by both wild type (WT) and 5-HT_2B^−/− mice after the administration of 5-HT_1A, 5-HT_2A or 5-HT_2C receptor agonists, 8-OH-DPAT (5 mg/kg), DOI (1 mg/kg), or WAY161503 (5 mg/kg), respectively. In contrast, the 5-HT_2B receptor agonist BW723C86 (3 mg/kg) or 5-HT_1B receptor agonist CGS12066A (2 mg/kg) decreased immobility time in both genotypes. The 5-HT syndrome induced by fluoxetine at high doses was blocked in WT and 5-HT_2B^−/− mice by administration of 5-HT_1A and 5-HT_2C receptor antagonists (WAY100635 0.5 mg/kg and SB242084 0.5 mg/kg) but not by the 5-HT_2A receptor antagonist MDL100907 (1 mg/kg). By behavioral assessment, we confirmed that 5-HT_2B^−/− mice were more prone to develop 5-HT syndrome symptoms after administration of high dose of SSRIs or the 5-HT precursor 5-Hydroxytryptophan, 5-HTP, even if increases in 5-HT plasma levels were similar in both genotypes.This evidence suggests that the presence of 5-HT_2B receptors hinders acute 5-HT toxicity once high levels of 5-HT are attained. Therefore, differential agonism/antagonism of 5-HT receptors should be considered in the search of therapeutic targets for treating this serious disorder.     

Functional 5-HT receptors in human occipital artery

5-HT receptors were studied in human occipital arteries, obtained from patients during neurosurgery. We detected mRNA for the following receptors (incidence): 5-HT_1B (14/18), 5-HT_1D (15/18), 5-HT_2A (16/18), 5-HT_2B (8/8), 5-HT_4(a) (13/18), 5-HT_4(b) (5/18), 5-HT_4(g) (7/18), 5-HT_4(i) (1/18), 5-HT_7(a/b) (10/18) and 5-HT_7(d) (12/18). 5-HT contracted and relaxed arterial rings at low (–logEC₅₀ M=7.0) and high (–logEC₅₀ M=4.2) concentrations, respectively. 5-HT-evoked contractions were antagonized partially by both 5-HT_1B-selective SB224289 (200 nM) and 5-HT_2A-selective ketanserin (1 M) but not by 5-HT_1D-selective BRL15572 (500 nM) or prazosin (1 M). Sumatriptan caused contractions (–logEC₅₀ M=6.8, intrinsic activity with respect to 5-HT=0.3). Sumatriptan-evoked contractions were antagonized by SB224289 with high potency (pK_B=9.4) but not by BRL15572. 5-HT-induced relaxations were resistant to blockade by 5-HT_1B-selective SB224289 (1 M), 5-HT_1D-selective BRL15572, 5-HT_2B-selective SB204741 (1 M), 5-HT₄-selective GR113808 (100 nM) and 5-HT₇-selective SB269970 (1 M), and a combination of SB204741 and SB269970, inconsistent with an involvement of 5-HT_1B, 5-HT_1D, 5-HT_2B, 5-HT₄ and 5-HT₇ receptors. Triton X-100 treatment of the arteries abolished acetylcholine-induced relaxations of rings precontracted by prostaglandin F₂, but a reduction of the relaxant effects of 5-HT did not reach significance. Nitro-L-arginine (1 mM) reduced 5-HT-induced relaxations, suggesting a contribution of nitric oxide released from endothelial cells. Ketanserin (1 M) prevented the relaxant effects of 5-HT. We conclude that 5-HT contracts human occipital artery through 5-HT_1B receptors at low concentrations and through 5-HT_2A receptors at high concentrations. Sumatriptan contracts mostly through 5-HT_1B receptors. These results are consistent with the 5-HT_1B and 5-HT_2A mRNA data. 5-HT-induced relaxation is mediated, in part, through ketanserin-sensitive receptors, but 5-HT_1B, 5-HT_1D, 5-HT_2B, 5-HT₄ and 5-HT₇ receptors appear not to be involved.     

Identification of presynaptic 5-HT1 autoreceptors in pig brain cortex synaptosomes and slices

Summary Pig brain cortex synaptosomes and slices preincubated with ³H-5-hydroxytryptamine (³H-5-HT) were superfused with physiological salt solution containing citalopram (an inhibitor of 5-HT uptake), and the effects of indolethylamines and 5-HT receptor antagonists on the potassium- or electrically evoked ³H overflow were determine. The potassium (25 mmol/l)-evoked tritium overflow from cortex synaptosomes was inhibited by 5-HT; the inhibitory effect of 5-HT was counteracted by metitepine, which, by itself, did not affect the evoked overflow. 5-Methoxytryptamine (examined in the absence of citalopram) also produced an inhibition of the evoked overflow. In cortex slices, the electrically (3 Hz) evoked overflow was inhibited by 5-HT and 5-carboxamidotryptamine. The inhibitory effect of 5-HT was antagonized by metitepine, which, given alone, increased the evoked overflow, but was not attenuated by ketanserin and ICS 205-930 ([³-tropanyl]-1H-indole-3-carboxylic acid ester), which, by themselves, did not influence the evoked overflow. The present results suggest that the serotoninergic nerve fibres of the pig brain cortex are endowed with presynaptic 5-HT₁ receptors, which can be activated by endogenous and exogenous 5-HT.Send offprint requests to E. Schlicker at the above address     

The 5-HT3 receptor agonist 1-(m-chlorophenyl)-biguanide facilitates noradrenaline release by blockade of α2-adrenoceptors in the mouse brain cortex

We analyzed the facilitatory effect of the 5-HT₃ receptor agonist 1-(m-chlorophenyl)-biguanide (mCPBG) on the electrically evoked noradrenaline release in superfused mouse brain tissue. In addition, we determined the affinities of mCPBG and two other 5-HT receptor ligands, namely, 2-methyl-5-hydroxytryptamine (2-methyl-5-HT; also a 5-HT₃ receptor agonist) and 5-carboxamidotryptamine (5-CT; a 5-HT₁ receptor agonist) for ₂ binding sites. The latter two 5-HT receptor agonists were included because of the claimed involvement of ₂-adrenoceptors in their effects on noradrenaline release.In superfusion experiments on mouse brain cortex slices preincubated with ³H-noradrenaline, tritium overflow evoked by 2-min periods of electrical field stimula tion (3 Hz) was facilitated by mCPBG and, in addition, by rauwolscine (₂-adrenoceptor antagonist) and tetraethylammonium (K⁺ channel blocker) (which were examined for comparison). The effect of mCPBG was not affected by the 5-HT3 receptor antagonist tropisetron or by desipramine but was abolished by rauwolscine. In slices superfused with medium containing desipramine, the concentration-response curve of unlabelled noradrenaline for its inhibitory effect on the electrically (0.3 Hz) evoked overflow was shifted to the right by mCPBG and rauwolscine (apparent pA₂ 5.35 and 7.88, respectively). In another series of superfusion experiments, 4 electrical pulses, administered at 100 Hz, were used to evoke tritium overflow. Tritium overflow evoked by this stimulation procedure (under which an endogenous tone of noradrenaline does not develop) was not affected by mCPBG and rauwolscine but still increased by tetraethylammonium. The specific binding of 3H-rauwolscine to rat brain cortex homogenates was displaced monophasically by unlabelled rauwolscine, mCPBG, 2-methyl-5-HT and 5-CT (pK_i 8.59, 5.84, 5.05 and 5.86, respectively).In conclusion the present results indicate that mCPBG acts as a low-affinity antagonist at ₂-adrenoceptors. This property has to be considered in functional studies of 5-HT₃ receptor-mediated effects in tissues containing ₂-adrenoceptors as well.Abbreviations mCPBG 1-(m-chlorophenyl)-biguanide - 5-CT 5-carboxamidotryptamine - 2-methyl-5-HT 2-methyl-5-hydroxytryptamine - POP pseudo-one-pulse - TEA tetraethylammonium Correspondence to: E. Schlicker at the above address     

Radioligand binding evidence implicates the brain 5-HT2 receptor as a site of action for LSD and phenylisopropylamine hallucinogens

Alterations in brain serotonergic function have been implicated in the mechanism of action of LSD, mescaline, and other similarly acting hallucinogenic drugs of abuse such as STP (2,5-dimethoxyphenylisopropylamine; DOM). In order to test the hypothesis that the mechanism of action of LSD and phenylisopropylamine hallucinogens is through stimulation of a specific brain serotonin receptor sub-type, the affinities of these compounds for radiolabelled 5-HT₂, 5-HT_1A, 5-HT_1B, and 5-HT_1C receptors have been determined using recently developed in vitro radioligand binding methodologies. The 5-HT₂ receptor was labelled with the agonist/hallucinogen radioligand ³H-DOB (4-bromo-2,5-dimethoxyphenylisopropylamine). The 5-HT_1A, 5-HT_1B, and 5-HT_1C receptors were labelled with ³H-OH-DPAT, ³H-5-HT, and ³H-mesulergine, respectively. In general, the phenylisopropylamines displayed 10–100 fold higher affinities for the 5-HT₂ receptor than for the 5-HT_1C receptor and 100–1000 fold higher affinities for the 5-HT₂ receptor than for the 5-HT_1A or 5-HT_1B receptor. There was a strong correlation between hallucinogenic potencies and 5-HT₂ receptor affinities of the phenylisopropylamines (r=0.90); the correlation coefficients for the 5-HT_1A, 5-HT_1B, and 5-HT_1C were 0.73, 0.85, and 0.78, respectively. Because there is no evidence that 5-HT_1A-selective or 5-HT_1B-selective agonists are hallucinogenic and because the phenylisopropylamines are potent hallucinogens, a 5-HT₂ receptor interaction is implicated and supports our previous suggestions to this effect. A secondary role for 5-HT_1C receptors cannot be discounted at this time. These results, when integrated with other receptor pharmacological information, indicate that an important component of the mechanism of action of LSD and the phenylisopropylamine hallucinogens is through stimulation of brain 5-HT₂ receptors. Offprint requests to: M. Titeler     

5-Hydroxytryptamine receptors with a 5-HT6 receptor-like profile stimulating adenylyl cyclase activity in pig caudate membranes

This study deals with the characterization of 5-hydroxytryptamine (5-HT, serotonin) receptors positively linked to adenylyl cyclase in membranes from pig brain caudate. 5-HT and related agonists induced a concentration-dependent stimulation of adenylyl cyclase activity in pig caudate membranes, with the following rank order of potency (mean pEC₅₀ values): 5-HT (7.1) 5-methoxytryptamine (6.9) > 5-carboxamidotryptamine (5.6) > sumatriptan (<5). Maximal stimulation by 5-HT averaged 35 pmol cyclic AMP/min/mg protein over a basal activity of 159 pmol cyclic AMP/min/mg protein. 5-Methoxytryptamine and 5-carboxamidotryptamine had similar efficacies to that of 5-HT, whereas sumatriptan was about half efficacious. Other compounds known as agonists at some 5-HT receptors were weakly potent (mean pEC₅₀ values <5). They include the 5-HT_1A receptor agonist, 8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OH-DPAT), the 5-HT₄ receptor agonist, renzapride and the 5-HT₂ receptor agonist, (1-(2,5-dimethoxy-4-iodophenyl)-2 aminopropane) (DOI). In antagonist studies, methiothepin (0.1 and 1 mol/l) shifted the 5-HT curve to the right with no depression of the E_max, yielding pK_B values of 7.4–8.0. Clozapine (1 mol/l) also produced surmountable antagonism of 5-HT-induced effects (pK_B 6.9). Ketanserin (10 mol/l) weakly antagonized 5-HT (pK_B 5.0). The 5-HT₄ receptor antagonists, tropisetron (ICS 205–930) and SDZ 205–557 (2-methoxy-4-amino-5-chloro-benzoic acid 2-(diethylamino) ethyl ester), each at 1 mol/l, did not significantly alter the concentration-response curve of 5-HT. The present receptor shares some characteristics of the recently cloned 5-HT₆ receptor (Monsma et al. (1993) Mol Pharmacol 43:320–327): similar pharmacological profile, location (striatum) and ability to stimulate adenylyl cyclase. It may thus represent the functional 5-HT₆ receptor in its natural environment. Correspondence to: P. Schoeffter at the above address     

Biochemical and behavioural effects of isamoltane,a β-adrenoceptor antagonist with affinity for the 5-HT1B receptor of rat brain

Summary The biochemical and behavioural effects of isamoltane, a \-adrenoceptor and 5-HT_1B receptor antagonist that has higher affinity for 5-HT_1B receptors than for 5-HTIA receptors, on 5-HT neurotransmission in the rat brain were examined. In binding experiments isamoltane was found to be about five times more potent as a ligand for the 5-HT_1B receptor than for the 5-HT_1A receptor (K_i values 21 and 112 nmol/l, respectively). Isamoltane increased the K⁺-evoked overflow of ³H from ³H-5-HT loaded slices of rat occipital cortex at 0.1 mol/l, consistent with inhibition of the terminal 5HT autoreceptor. In vivo, isamoltane significantly increased the concentration of 5-hydroxyindoleacetic acid in hypothalamus and hippocampus indicating an increased 5-HT turnover with a maximal effect at 3 mg/kg s.c. A higher dose produced a less pronounced effect. This effect did not seem to be due to the -adrenoceptor blocking action of isamoltane since the -adrenoceptor antagonists, (–)-alprenolol, betaxolol or ICI 118,551 had no significant effects on 5-HT turnover at 5 mg/kg s.c. Isamoltane at 3 mg/kg s.c. induced the wet-dog shake response which was blocked by the tryptophan hydroxylase inhibitor p-chlorophenylalanine. In contrast, the same response induced by the 5-HT₂ receptor agonist quipazine was not blocked by pretreatment with p-chlorophenylalanine. The wet-dog shakes evoked by isamoltane and quipazine were blocked by ritanserin, which indicates that 5-HT₂ receptors are involved in their expression. These observations indicate that isamoltane, by inhibiting the terminal 5-HT autoreceptors, increased the synaptic concentration of 5-HT to a level that induced a behavioural response. Send of offprint requests to S. B. Ross at the above addressThe present results have been presented in part at the Second IUPHAR Satellite Meeting on Serotonin, Basel, Switzerland, July 11–13, 1990     

Serotonergic modulation of neurotransmission in the rat subicular cortex in vitro: a role for 5-HT1B receptors

Summary We have studied the effect of serotonin on synaptic transmission in rat hippocampal subiculum slices. Electrical stimulation of the alveus induced a field potential in the subiculum. The non-NMDA glutamate receptor antagonist, NBQX (3 × 10^–6 mol/l) suppressed the response by 78%, indicating that the signal involves glutamatergic neurons. Application of serotonin suppressed (EC₅₀ = 3.6 × 10^–6 mol/l) the amplitude of he evoked potentials in a reversible, concentration-dependent manner. The responses to 5-HT were not altered after pretreatment with the 5-HT uptake blocker, fluvoxamine (10^–5 mol/l) or a combination of the MAO inhibitor pargyline (10^–5 mol/l) and ascorbic acid (10^–4 mol/l). The responses to 5-HT were also unaffected by pretreatment with the 5-HT_1A selective antagonist NAN-190 (10^–6 mol/l), the 5-HT_2A antagonist ketanserin (10^–6 mol/l) or the 5-HT₃/5-HT₄ antagonist ICS 205–930 (10^–6 mol/l).The 5-HT_1B selective agonist CP 93,129 mimicked the effects of serotonin, but was more potent (EC₅₀ 4.1 × 10^–7 mol/l). The 5-HT_1B receptor antagonist, (±)21-009 (3 × 10^–7 mol/l), antagonized the response to 5-HT and CP 93,129 with a pK_B value of 7.1 and 7.2, respectively. These results suggest that the effect of 5-HT in the rat subiculum is mediated by 5-HT_1B receptors.Correspondence to: H.W.G.M. Boddeke at the above address     

Modulation by 5-HT3 and 5-HT4 receptors of the release of 5-hydroxytryptamine from the guinea-pig small intestine

Summary The effects of agonists and antagonists of 5-hydroxytryptamine (5-HT) receptors on the release of endogenous 5-HT from enterochromaffin cells were studied in the vascularly perfused isolated guinea-pig small intestine. The experiments were done in the presence of tetrodotoxin in order to exclude a neuronally mediated influence on 5-HT release.The 5-HT₃ receptor agonist 2-methyl-5-HT increased 5-HT release, and this effect was antagonized by 1 nmol/l tropisetron. Nanomolar concentrations of tropisetron, MDL 72 222 and granisetron decreased 5-HT release. Ondansetron (0.1 and 1 mol/1) did not modify 5-HT release.5-Methoxytryptamine, BIMU8 and cisapride concentration-dependently inhibited 5-HT release. BIMU8 was more potent than 5-methoxytryptamine. Micromolar concentrations of tropisetron (1 and 10 mol/1) enhanced the release, whilst methiothepine (0.1 mol/l) did not affect the release of 5-HT.The results suggest that enterochromaffin cells of the guinea-pig ileum do not contain 5-HT₁ and 5-HT₂ receptors, but are endowed with 5-HT₃ and 5-HT₄ autoreceptors. Activation of the 5-HT₃ receptors triggers a positive feedback mechanism leading to an increase of 5-HT release. The 5-HT₃ receptors on the enterochromaffin cell differ from neuronal 5-HT3 receptors on guinea-pig myenteric plexus by their high affinity for tropisetron and MDL 72 222, and their very low affinity for ondansetron. Stimulation of 5-HT₄ receptors causes inhibition of release; the inhibitory 5-HT₄ receptor mechanism appears to predominate.Correspondence to H. Kilbinger at the above address