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.     

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

Summary Experiments have been carried out to provide direct evidence for the proposed presynaptic 5-HT autoreceptor agonist activity of 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) a compound with selectivity for the 5-HT_1A subtype of the 5-HT₁ binding site. Rat brain frontal cortex slices were preincubated with [³H] 5-hydroxytryptamine and continuously stimulated with Krebs solution containing paroxetine and elevated K⁺ ions (25 mmol/l). The elevated efflux of tritium caused by exposure to K⁺ Krebs was inhibited in a dose related manner by 5-hydroxytryptamine and this inhibition was attenuated in the presence of quipazine and methiothepin.In slices of the rat frontal cortex, 8-OH-DPAT was without agonist or antagonist activity at the 5-HT autoreceptor at concentrations up to 1 mol/l. Higher concentrations caused an increase in basal efflux of tritium. 8-OH-DPAT (1 mol/l) was also without inhibitory activity in the piriform cortex, striatum and the hippocampus.These experiments have therefore failed to provide direct evidence for agonist activity of 8-OH-DPAT at the 5-HT autoreceptor and alternative explanations must be sought for its biochemical and behavioural effects in vivo. Moreover, the fact that 8-OH-DPAT is inactive at the autoreceptor at concentrations selective for the 5-HT_1A recognition site suggests that this subtype of the 5-HT_1A binding site may not correspond to the 5-HT autoreceptor.Part of this work was presented at the Joint Meeting of the French and German Pharmacological and Toxicological Societies, Freiburg, September 1983     

Characterization of an endothelial 5-hydroxytryptamine (5-HT) receptor mediating relaxation of the porcine coronary artery

Summary The pharmacological properties of the endothelial 5-hydroxytryptamine (5-HT) receptors involved in relaxation of vascular smooth muscle were determined in rings of pig coronary artery contracted with 10 nmol/1 of the thromboxane A₂ receptor agonist 9,11-dideoxy-11,9-epoxy-methano-prostaglandin F₂ (U 46619).(1) In the presence of 10 mol/l ketanserin, relaxation was obtained with: 5-HT (apparent pD₂ value 7.00), 5-carboxamidotryptamine (5-CONH₂-T; 6.42), 5-aminotryptamine (5-NH₂-T; 5.96), 5-methoxytryptamine (5-OCH₃-T; 5.92), tryptamine, 7-trifluoromethyl-4(4-methyl-1-piperazinyl)-pyrrolo(1,2-a)quinoxaline maleate (CGS 12066 A) and 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole succinate (RU 24969). The maximum relaxation obtainable with the agonists was about 40–60% of the U 46619-induced contraction and the concentration-response curves for 5-HT, 5-NH₂-T and 5-OCH₃-T were bell-shaped. The endothelium-dependence of this effect (i. e. the failure to relax the artery in endothelium-denuded preparations) was demonstrated for 5-HT, 5-CONH₂-T, RU 24969, CGS 12066A and tryptamine.(2) 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), 4-hydroxytryptamine, quipazine and yohimbine were ineffective in decreasing the tension of arteries with or without endothelium. Ipsapirone elicited full relaxation of U 46619-induced contraction, but this effect was not endothelium-dependent.(3) Metitepine (0.03-1 mol/l), 6-chloro-2-(1-piperazinyl)pyrazine (MK 212; 10 mol/l), methysergide (1 gmol/l) and cyanopindolol (0.1 mol/l) antagonized the relaxing effect of 5-HT in a non-surmountable manner, whereas metergoline (0.1 mol/l), quipazine (10 mol/l), yohimbine (1 mol/l), propranolol (1 mol/l) and (3-tropanyl)-1H-indole-3-carboxylic acid ester (ICS 205-930; 0.1 mol/l) did not. However, spiroxatrine (0.1 mol/l) and mesulergine (10 mol/l) enhanced the 5-HT-induced relaxation. The endothelium-dependent relaxation induced by 5-CONH₂-T was also inhibited by metitepine 1 gmol/l.(4) The 5-HT-induced relaxation was probably mediated by release of an endothelium-derived relaxing factor (EDRF). Gossypol, an inhibitor of EDRF, virtually abolished the 5-HT-induced relaxation while indometacin, an inhibitor of cyclooxygenase and accordingly of PGI₂ formation, did not.In conclusion, the failure of ketanserin and ICS 205–930 to counteract the relaxant effect of 5-HT receptor agonists excludes the involvement of 5-HT₂ and 5-HT₃ receptors, respectively, in the endothelium-dependent relaxation of the porcine coronary artery. The rather high potency of 5-CONH₂-T and the ability of certain 5-HT receptor antagonists, such as metitepine, methysergide and cyanopindolol, to counteract the effect of 5-HT are compatible with a 5-HT₁ character of the endothelial receptor. However, on the basis of the present data, no final classification, in particular with respect to the known 5-HT₁ receptor subtypes, is possible. Classification is also hampered by the bell-shaped character of the concentration-response curves for 5-HT receptor agonists and by their property to produce only partial relaxation. Send offprint requests to M. Gothert at the above address     

Serotonin autoreceptor in rat hippocampus: Pharmacological characterization as a subtype of the 5-HT1 receptor

Summary The 5-hydroxytryptamine (5-HT) autoreceptors mediating inhibition of [³H]5-HT release in rat hippocampus have been characterized pharmacologically in terms of 5-HT receptor subtype by using superfused synaptosomes depolarized with 15 mM KCl. Exogenous 5-HT inhibited in a concentration-dependent way (pEC₃₀=8.74) the K⁺-evoked release of [³H]5-HT. Methiothepin shifted the concentration-response curve of 5-HT to the right (pA₂=8.62). The 5-HT₂ receptor antagonists, ketanserin, methysergide or spiperone were ineffective against 5-HT. The 5-HT₁ receptor agonist, 5-methoxy-3-[1,2,3,6-tetra-hydropyridin-4-yl]-1H-indole (RU 24969) mimicked 5-HT and was equipotent as an inhibitor of the release of [³H]5-HT. In contrast, the putative 5-HT_1A receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) was almost ineffective at 1 M. Finally, (–)propranolol, used as a non-selective 5-HT_1A/5-HT_1B receptor antagonist, shifted to the right (pA₂=7.91) the concentration-response curve of 5-HT whereas the 5-HT_1C receptor antagonist mesulergine was ineffective. In conclusion, 5-HT nerve terminals of rat hippocampus possess autoreceptors which appear to belong to the 5-HT_1B subtype.     

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     

Inhibition of 5-hydroxytryptamine accumulation and deamination by substituted phenylalkylamines in hypothalamic synaptosomes from normal and reserpine-pretreated rats

Summary 1. In the present study the abilities of different compounds to inhibit MAO inside and outside the serotonergic neurons, to inhibit the accumulation of 5-HT and to release 5-HT were separated by using different in vitro techniques. With these methods a number of substituted phenylalkylamines, which are reversible inhibitors of monoamine oxidase (MAO) type A, were characterized. 2. The compounds were examined regarding their ability to inhibit the accumulation of 5-HT and to inhibit MAO in the same synaptosomal preparation of hypothalamus from normal and reserpine-pretreated rats. The difference in the uptake of ¹⁴C-5-HT (0.1 mol/l) in the absence and presence of citalopram (0.25 mol/l) was taken as a measure of the accumulation into the serotonergic synaptosomes. The deamination of ¹⁴C-5-HT (0.1 mol/l) in the presence of citalopram (0.25 mol/l) was considered as that brought about outside the serotonergic synaptosomes, whereas the difference between the deamination in the absence and presence of citalopram was taken as the MAO activity inside the serotonergic synaptosomes. 3. Most of the phenylalkylamines were slightly more potent as MAO inhibitors outside serotonergic synaptosomes than as inhibitors of 5-HT accumulation in normal rats. The most potent MAO inhibitors, both in absolute terms and in comparison with uptake inhibitory potency, were the 2,6-dichloro-(FLA 365) and the phenylpropylene-(FLA 417) derivatives. 4. A difference in potency on the accumulation in synaptosomes from normal and reserpine-pretreated rats was found for many of the phenylalkylamines with the exception of FLA 365, FLA 417 and the 2,5-dimethyl derivative RAN 113. The compounds shared this difference with the 5-HT releasers p-chloroamphetamine (pCA) and H75/12, but not with the uptake inhibitors citalopram, cocaine, alaproclate, norzimeldine and fluoxetine. 5. For most of the phenylalkylamines, the MAO inhibition obtained within serotonergic synaptosomes was not higher than that obtained outside these. This was in contrast to previously shown in vivo results were a preference for inhibiting MAO inside the serotonergic neurons, due to a transport into the neurons by the amine carrier, was found. For the uptake inhibitors, the inhibition of 5-HT deamination intrasynaptosomally was a more sensitive measure of inhibition of accumulation than to determine this accumulation directly. 6. It is concluded that a classification of uptake inhibitors, releasing compounds, and MAO inhibitors that are and that are not transported by the 5-HT carrier may be performed by measuring the inhibition of the uptake and the deamination in synaptosomes from normal and reserpine-pretreated rats.Parts of this work were presented at the 14th CINP Congress, Florence, Italy, June 19–23, 1984 and the VIIIth International Symposium on Medicinal Chemistry, Uppsala, Sweden, August 27–31, 1984 Send offprint requests to A.-L. Ask     

5-Hydroxytryptamine 5-HT1D receptors mediating inhibition of cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells

5-Hydroxytryptamine 5-HT_1B/5-HT_1D receptors are members of the same receptor subfamily, but display a different pharmacology (Hartig et al. (1992) Trends Pharmacol Set 13:152–159). Whereas several cell lines have been reported to contain 5-HT_1B receptors, none has been described, however, that endogenously expresses well-characterized 5-HT_1D receptors. The present study deals with the identification of 5-HT_1D receptors inhibiting cyclic AMP accumulation in Madin-Darby canine kidney (MDCK) cells. 5-HT (1 nM– 10 M) induced a concentration-dependent inhibition of the cyclic AMP accumulation stimulated by prostaglandin E₁ (1 M) in MDCK cells. The maximal effect of 5-HT averaged 50% inhibition and was abolished after a pre-treatment of the cells with pertussis toxin. Other agonists mimicked the effects of 5-HT, with the following rank order of potency (pEC₅₀ ± SEM, n 3): 5-carboxamidotryptamine (8.36 ± 0.48) > PAPP (p-aminophenylethyl-m-trifluoromethylphenyl piperazine, 7.89 ± 0.23) > 5-HT (7.35 ± 0.05) > sumatriptan (6.65 ± 0.27). PAPP behaved as a partial agonist. 8-OH-DPAT (8-hydroxy-2(di-n-propylamino)tetralin) was less potent, its maximal effect being not reached at 0.1 mM. Methiothepin, GR127935, (–)propranolol, rauwolscine and ketanserin were all devoid of intrinsic activity (up to 10 M or 0.1 mM). Methiothepin (10 nM, 0.1 M and 1 M) antagonized 5-HT effect (pA₂ 8.57 ± 0.44, Schild slope 1.17 ± 0.21, n = 3). GR127935 (1 nM, 10 nM and 0.1 M) shifted the curve of 5-HT to the right, but the antagonism was not fully surmountable (apparent pK_B value, 9.80 ± 0.16, n = 9). From the shifts obtained with rauwolscine (1 M) and (–)propranolol (10 M), respective pK_B values were estimated 6.68 ± 0.30 and 5.4 (n = 3 each). PAPP, when tested as an antagonist at 1 M, also shifted the curve of 5-HT to the right, with a pK_B of 8.27 ± 0.16 (n = 3). Finally, ketanserin (10 M) also antagonized the effects of 5-HT, the pK_B being 6.54 ± 0.16 (n = 9). The rank orders of agonist and antagonist potencies strongly suggest 5-HT receptors mediating inhibition of cyclic AMP accumulation in MDCK cells to be 5-HT_1D receptors. This is the first report of a cell line expressing endogenous, well-characterized, 5-HT_1D receptors. With regard to the 5-HT_1D receptor subtype involved, the relatively high potency of ketanserin would suggest it to be a 5-HT_1D subtype or a mixture of 5-HT_1D/5-HT_1D\ subtypes. However, caution must be exercised here, owing to the poor knowledge of canine 5-HT_1D receptor subtypes.     

Endothelium-dependent relaxation of porcine pulmonary arteries via 5-HT1C-like receptors

Summary In PGF₂-precontracted pulmonary arteries with intact endothelium, 5-hydroxytryptamine (5-HT, 1.0-100 nmol/l) caused a concentration-dependent reversible relaxation, at higher concentrations the contractile response prevailed. In endothelium-denuded vessels relaxation was absent. 5-HT-induced relaxation of precontracted pulmonary arteries was probably mediated by release of an endothelium-derived relaxing factor (EDRF). Preincubation of the arteries with methylene blue or N^G-nitro-Lrarginine (200 mol/l) attenuated the relaxant effect. The 5-HT-induced relaxation was accompanied by an increase in cGMP. Indomethacin (3 mol/l) did not influence the 5-HT-induced relaxation indicating that eicosanoids are not involved in the relaxant response to 5-HT.The 5-HT_1C and 5-HT₂ receptor agonist -methyl-5HT was as potent as 5-HT in inducing relaxation. The rank order of relaxant potency of the agonists investigated was -methyl-5-HT > 5-HT > 5-methoxytryptamine > tryptamine > -methyl-5-HT > 5-carboxamidotryptamine >2-methyl-5-HT > 5,6-dihydroxytryptamine > m-chlorophenylpiperazine >sumatriptan > 8-OH-DPAT.Phentolamine, pindolol and ICS 205-930 did not interfere with the relaxant effect. The 5-HT₂ receptor antagonist ketanserin (1 mol/l) inhibited the contractile response but did not alter vasodilatation. Apart from the blockade of the contractile effects, mesulergine, cyproheptadine and mianserin (0.1-3.0 mol/l, each) induced a parallel shift to the right of the concentration-response curve for the relaxation induced by a-methyl-5-HT or 5-HT. Spiperone (0.3 mol/l) exerted weak inhibitory effects on relaxation and contraction. The most potent (noncompetitive) antagonist against relaxant responses was metitepine (0.1-1.0 mol/l) which markedly depressed the relaxant maximum effect of the agonists.The failure of ketanserin and ICS 205-930 to inhibit the relaxant effect of 5-HT receptor agonists suggests that classical 5-HT₂ and 5-HT₃ receptors are not involved in the endothelium-dependent relaxation. Comparison of the rank order of potencies of agonists and antagonists with their affinities for brain binding sites revealed that the endothelial 5-HT receptors are similar to the 5-HT_1C receptor subtype. Furthermore, the endothelial receptors exhibit marked similarity to the recently cloned 5-HT receptor mediating contraction of the rat stomach fundus. Correspondence to E. Glusa at the above address     

Species differences in presynaptic serotonin autoreceptors: mainly 5-HT1B but possibly in addition 5-HT1D in the rat, 5-HT1D in the rabbit and guinea-pig brain cortex

Summary The pharmacological properties of presynaptic serotonin autoreceptors were compared in slices of rat, rabbit, and guinea-pig brain cortex. The slices were preincubated with ³H-serotonin and then superfused with medium containing fluvoxamine 3 mol/l and stimulated four times by trains of four pulses delivered at 100 Hz. Cumulative concentration-response curves were determined and used for the calculation of agonist EC₅₀ values and maximal effects and antagonist K _B values.Unlabelled serotonin itself and the serotonin receptor agonists 5-carboxamidotryptamine (5-CT), 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) and (±)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) reduced the stimulation-evoked overflow of tritium with a rank order of potency 5-CT = RU 24969 > serotonin > 8-OH-DPAT in the rat and 5-CT > serotonin > RU 24969 > 8-OH-DPAT in the rabbit and guinea-pig. Ipsapirone caused no change. Metitepine and metergoline antagonized the effect of 5-CT; the K _B values were lower in the rabbit and guinea-pig than in the rat. Yohimbine at up to 1 mol/1 did not reduce the evoked overflow of tritium and did not antagonize the inhibitory effect of 5-CT in the rat but reduced the evoked overflow in the rabbit and counteracted the effect of 5-CT in the guinea-pig. (–)-Propranolol, conversely, reduced the evoked overflow of tritium in the rat but neither reduced the evoked overflow nor antagonized the effect of 5-CT in the rabbit and guinea-pig. Isamoltane did not significantly change the effect of 5-CT in any species. In the rat, it also failed to antagonize the inhibitory effect of 8-OH-DPAT but did antagonize the effect of RU 24969. The inhibition caused by 8-OH-DPAT persisted in the presence of idazoxan but was attenuated by metitepine in all species.The experimental conditions used permit the determination of the constants of agonist and antagonist action undistorted by autoinhibition. The results confirm the view that the serotonin axons of rat brain possess 5-HT_1B autoreceptors. They show by direct comparison under identical conditions that the autoreceptors in rabbit and guinea-pig are very similar to each other but differ markedly from those in the rat. The results give additional credence to previous suggestions that, in the rabbit and guinea-pig, the autoreceptors are 5-HT_1D. The serotonin axons of rat brain cortex may possess 5-_1D in addition to 5-HT_1B autoreceptors. In many previous studies agonist potencies at, and antagonist affinities for, presynaptic serotonin autoreceptors have been underestimated due to the use of too intense stimuli to elicit serotonin release. Send offprint requests to N. Limberger at the above address     

In vitro and in vivo activity of 1-(1-naphthyl)piperazine at terminal 5-HT autoreceptors in guinea-pig brain

The effect of 1-(I-naphthyl)piperazine (NP) on the 5-HT terminal autoreceptor modulating 5-HT release was investigated in vitro and in vivo. In vitro 5-HT release was measured in slices of guinea-pig substantia nigra and hypothalamus prelabelled with ³H-5-HT, superfused with Krebs solution and depolarized electrically. NP, at 0.1 and 1 mol/l, did not modify the calcium-dependent release of ³H-5-HT elicited by electrical stimulation using a frequency of 5 Hz, however at 0.1 mol/l NP shifted to the right the inhibition curve of the non-selective autoreceptor agonist, 5-carboxamidotryptamine, in both regions. In hypothalamus when using lower frequencies (1 Hz or 0.2 Hz) or under pseudo-one-pulse stimulation, NP decreased the release of ³H-5-HT at 1 mol/l. In vivo microdialysis was used to measure extracellular levels of endogenous 5-HT in the substantia nigra of freely moving guinea-pigs. The endogenous release of 5-HT was tetrodotoxin (TTX)-sensitive, indicating a neuronal origin of this efflux. NP, administered through the microdialysis probe (1–100 mol/1), increased the levels of extracellular 5-HT in concentration-dependent and TTX-sensitive manner. These results suggest that in vitro NP acts as a 5-HT autoreceptor partial (ant)agonist in the substantia nigra and hypothalamus of guinea-pigs, and as a full antagonist in vivo. However, NP administered systemically at 10 mg/kg i.p., did not modify the levels of extracellular 5-HT in the substantia nigra. This lack of systemic effect of NP probably results from its interaction at other receptors that modify 5-HT neurotransmission. In particular, NP is an agonist at 5-HT_1A somatodendritic receptors in the raphe nucleus, an action which would decrease the release of 5-HT.     

5-Hydroxytryptamine (5-HT) contracts the guinea-pig isolated iliac artery via 5-HT1-like and 5-HT2 receptors

Summary The characterization of 5-hydroxytryptamine (5-HT) receptors mediating contractions of the guinea-pig isolated iliac artery was studied when the basal tone was slightly increased by prostaglandin F₂ (PGF₂).In the presence of ketanserin (1 mol/l), 5-HT and several 5-HT receptor agonists induced contractile responses with the rank order of agonist potency: 5-HT = 5-carboxamidotryptamine (5-CT) = lysergol > ergometrine = methylergometrine > RU 24969 5-methoxytryptamine (5-MeOT) > methysergide > sumatriptan > tryptamine. Concentration-effect curves to the ergot alkaloids, lysergol, ergometrine, methylergometrine and methysergide, were biphasic.In the presence of ketanserin (1 mol/l), contractile responses to 5-HT, 5-CT, RU 24969, 5-MeOT, sumatriptan and tryptamine were antagonized by methiothepin (30 nmol/l) and flesinoxan (3 mol/l) with approximate pK_B values of 8.5–9.0 and 6.0–6.3, respectively. The first phase of contraction produced by the ergot alkaloids, lysergol, ergometrine, methylergometrine and methysergide, were blocked by methiothepin (30 nmol/l) and flesinoxan (3 mol/l), respectively, with approximate pK_B values about 8.4–8.7 and 6.2–6.4, respectively. The mechanism underlying the second phase of contraction remains to be established.Maximum responses of the concentration-effect curves to 5-HT (1 nmol/l-1 mol/l) were concentration-dependently depressed by ketanserin (1 nmol/l-1 mol) and spiperone (30 nmol/l-0.3 mol/l) and reached approximately 60% of the 5-HT maximum response in the presence of ketanserin (1 mol/l) and spiperone (0.1 mol/l), respectively. Agonist potency of 5-HT was not affected by the antagonists. 5-HT (1 nmol/l-1 mmol) produced biphasic concentration-effect curves (first phase: 1 nmol/l-1 gmol/l; second phase: 1 mol/l-1 mmol/l) in the presence of ketanserin (100 and 300 nmol/l), spiperone (100 and 300 nmol/l), (R)--methylketanserin (3 mol/l) and (S)--methylketanserin (10 nmol/l). Contractions mediating the first phase of the effects of 5-HT accounted for approximately 60% of the 5-HT maximum response and were resistant to blockade by the antagonists. pK_B values at the receptor mediating the second phase of the effects of 5-HT were 9.2–9.3 for ketanserin, 9.2–9.6 for spiperone, 10.5 for (S)--methylketanserin and 7.2 for (R)--methylketanserin.It is concluded that 5-HT contracts the guinea-pig isolated iliac artery via a mixture of 5-HT₁-like receptors and 5-HT₂ receptors. At low concentrations contractions are mediated via 5-HT₁-like receptors which accounted for approximately 60% of the 5-HT maximum response. At higher concentrations 5-HT-induced contractions are mediated via 5-HT₂ receptors.     

Effects of 5-HT4 receptor stimulation on basal and electrically evoked release of acetylcholine from guinea-pig myenteric plexus

Summary 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 mol/1) did not affect the stimulatory response of 5-methoxytryptamine but ICS 205-930 (1 and 3 mol/1) produced parallel rightward displacements of the concentration-response curve to 5-methoxytryptamine. The PK_B 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 mol/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 mol/1) reinforced the inhibitory effect of 5-methoxytryptamine but not that of 5-HT. In the presence of methiothepine (0.1 mol/1) 5-methoxytryptamine enhanced the evoked outflow of [³H]acetylcholine, an effect which was attenuated by 3 mol/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. Send offprint requests to H. Kilbinger at the above address     

Serotonin type-2 receptor mediated regulation of substance P release in the ventral spinal cord and the effects of chronic antidepressant treatment

Summary Regulation of the release of substance P (SP) by the coexisting neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) in the ventral spinal cord and the effects of chronic antidepressant treatment mediated changes in serotonin metabolism on the regulation, were examined.The K⁺ (40 mmol/l) evoked release of (SP) from slices of the ventral spinal cord of the rat was potentiated by (5-HT) applied to 100 mol/l concentration. This effect was blocked by the serotoninergic antagonists methysergide (10 mol/l), methiotepin (10 mol/l) and fully blocked by ketanserin (10 mol/l). Thus the 5-HT receptor which regulates the release of SP appears to belong to the type-2 5-HT receptors. Chronic treatment with the selective serotonin uptake inhibitor zimelidine (14 days, 2×10 mol/kg/day, p.o.) lowered the tissue levels of the 5-HT metabolite: 5-hydroxyindol acetic acid (5-HIAA) and elevated the tissue levels of SP in both the ventral and dorsal spinal cord as compared to that in the vehicle treated group (14 days, 2×5 ml saline/kg/day, p.o.). The decrease in the 5-HIAA levels after chronic zimelidine treatment was quantitatively similar in the dorsal (33%,p<0.01) and ventral (31%,p<0.05) spinal cord. The increase in SP levels after chronic zimelidine treatment was more pronounced in the ventral cord (80%,p<0.01) where the majority of the SP containing nerve endings also contain 5-HT, than in the dorsal spinal cord (22% increase in SP,p<0.05), where only a minor fraction of the SP-containing nerve endings shows a 5-HT/SP coexistence. Chronic treatment with imipramine (14 days, 2×10 mol/kg/day, p.o.) gave qualitatively similar results to those obtained by zimelidine treatment, but increases in SP levels, and decreases in 5-HT and 5-HIAA levels in ventral and dorsal spinal cord, were less pronounced. The K⁺ (40 mmol/l) evoked release of SP was studied in a slice preparation of the ventral spinal cord, from rats chronically treated with imipramine, zimelidine and saline. In the zimelidine treated group the amount of SP released (pmol/g tissue) and the fractional SP release upon K⁺ (40 mmol/l) stimulation was increased by 53% (p<0.01) and 42% (p<0.01) respectively, when compared to the control group. No significant changes in the amount of SP released or fractional SP release were observed when tissue preparations from rats treated with imipramine were used. Thus, it seems that treatment with specific serotoninergic or monoaminergic antidepressant drugs does not only change the tissue levels of the monoamine and its metabolite but also affects the coexisting peptidergic transmitter; SP in the ventral spinal cord. This change is also reflected in the size of the releaseable pool of SP.     

Paradoxical decrease of brain 5-HT turnover by metergoline,a central 5-HT receptor blocker

Summary Since metergoline (1-methyl-8-beta-carbobenzyloxy-aminomethyl-10-alpha-ergoline) is a potent 5-HT antagonist in peripheral organs, its possible blocking effects on 5-HT receptors in the rat brain were investigated. In vitro, metergoline inhibited both the specific high affinity binding of ³H-5-HT onto synaptosomal membranes (IC 50=18 nM) and the stimulating effect of 10 M 5-HT on the adenylate cyclase activity in colliculi homogenates from newborn rats (IC 50=12 M). In vivo, the administration of metergoline (10 mg/kg i.p., 60 min before death) resulted in a significant decrease in the ³H-5-HT binding capacity of synaptosomal membranes from the forebrain of adult rats. Taken together, these data clearly indicated that metergoline is a potent blocker of some serotoninergic receptors in the rat brain. Surprisingly, the changes in 5-HT turnover occurring in the brainstem and in the forebrain 1 h after metergoline (2–10 mg/kg) treatment were similar to those normally induced by a central 5-HT agonist: both the rate of 5-HT utilisation and that of 5-HT synthesis were significantly decreased. These changes were in contrast to the acceleration of 5-HT turnover induced by the administration of another potent central 5-HT antagonist, methiothepin. These results are discussed in relation to the possible existence of several types of serotoninergic receptors in the rat brain. It is possible that the positive feedback regulation of 5-HT turnover is triggered by the blockade of serotoninergic receptors sensitive to methiothepin, but not to metergoline.     

5-Hydroxytryptamine-induced contractions of the human isolated saphenous vein: involvement of 5-HT2 and 5-HT1D-like receptors,and a comparison with grafted veins

Summary The receptors mediating the contractile effect of 5-hydroxytryptamine (5-HT) on the human isolated saphenous vein, obtained from 42 patients undergoing coronary bypass surgery, have been further characterized using a number of 5-HT-related drugs. The rank order of agonist potency was 5-carboxamidotryptamine (5-CT) 5-HT > methysergide sumatriptan -methyl-5-HT 5-methoxy-3-(1,2,3,6-tetrahydropyridin-4-yl)-1-Hindolesuccinate (RU 24969) 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) > 2-methyl-5-HT > 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT). Flesinoxan was inactive as an agonist. Ketanserin (1 mol/l) hardly affected sumatriptan-induced contractions but it caused a rightward shift of the upper part of the concentration-response curve of 5-HT and 5-CT. The same concentration of ketanserin caused a parallel rightward shift of the concentration-response curves of -methyl-5-HT and DOI with pK_B values of 7. 1 and 7.1, respectively. The responses to sumatriptan were antagonized by methiothepin (0.1 mol/l), metergoline (0.1 and 1 mol/l), rauwolscine (1 mol/l) and cyanopindolol (1 mol/l); the calculated pK_B values were 7.3, 6.9, 7.3, 6.7 and 6.5, respectively. Contractions to 5-HT were antagonized by methysergide (1 mol/l), methiothepin (0.1 mol/l; pKB = 7.1), ICS 205-930 (1 mol/l; pK_B = 5.9) and flesinoxan (30 mol/l; pK_B = 5.3). Remarkably, the contractions elicited by 2-methyl-5-HT were not attenuated by ICS 205-930, but were antagonized by methiothepin (0.1 mol/l) and, more markedly, by ketanserin (1 mol/l).There was a high correlation between the functional pD₂ values of 5-HT₁-like receptor agonists (5-CT, 5-HT, methysergide, sumatriptan, RU 24969 and 8-OH-DPAT) and their reported binding affinities for the 5-HT_1D receptor in human or calf brain membranes. Such a correlation for the antagonism of sumatriptan-induced responses was less marked than for the agonists, but of the 5-HT₁-like receptor subtypes it was the highest for the 5-HT_1D receptor identified in human or calf brain membranes.In 3 patients, undergoing heart transplantation, saphenous vein which had previously functioned as a graft for 6–11 years, was dissected out from the heart. Though the contractions to potassium were significantly smaller in the grafted veins, the pD₂ and E_max values (calculated as percentage of potassium-induced contractions) for 5-HT and sumatriptan were similar to those found in the veins obtained directly from the lower leg.It is concluded that contractions in the human isolated saphenous vein induced by 5-HT are mediated by 5-HT₂ receptors as well as by a 5-HT₁-like receptor resembling the 5-HT_1D subtype found in brain membranes. It is also to be noted that 2-methyl-5-HT, considered selective for the 5-HT₃ receptor, contracts the saphenous vein mainly via 5-HT₂ receptors.This study was supported by the Netherlands Heart Foundation, grant 89.252 Send offprint requests to W. A. Bax at the above address     

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     

Modulation by drugs of the release of total tritium and 3H-5-HT from rat hypothalamic slices

Summary The release of total tritium and ³H-5hydroxytryptamine (5-HT) evoked by electrical stimulation from prelabelled rat hypothalamic slices was studied. Lysergic acid diethylamide (LSD) decreased while methiothepin increased both total tritium and ³H-5-HT overflow. The proportion of total tritium present as ³H-5-HT was equivalent under control conditions and in the presence of methiothepin and slightly increased in the presence of LSD. Whereas the addition of the 5-HT uptake blocker, citalopram, did not modify the evoked release of total tritium, the monoamine oxidase inhibitor, pargyline, decreased it. In the presence of either of these drugs the proportion of ³H-5-HT was increased. In the presence of citalopram, the inhibition by LSD was reduced on both the release of total tritium and of ³H-5HT. It thus appears that changes in electrically evoked total tritium overflow in general reflect changes in ³H-5-HT release. When uptake inhibitors or monoamine oxidase inhibitors are present in the medium, the situation is, however, more complex and results from experiments measuring only the release of total tritium should be interpreted with caution. Send offprint requests to: C. Moret at the above address     

5-HT1-like and 5-HT2A receptors mediate 5-hydroxytryptamine-induced contraction of rabbit isolated mesenteric artery

The contractions induced by 5-hydroxytryptamine (5-HT) and the 5-HT₁-like receptor agonist, sumatriptan, were investigated in the open ring preparations of rabbit mesenteric artery in order to characterize the 5-HT receptors. 5-HT induced concentration-dependent contractions. Sumatriptan did not induce any contraction of unstimulated rings, whereas it elicited concentration-dependent contractions in preparations given a moderate tone by a threshold concentration of prostaglandin F₂ (PGF₂). Pargyline, cocaine or normetanephrine were without significant effect on the contractions induced by 5-HT and sumatripan. The 5-HT concentration-effect curve was clearly biphasic. Methiothepin (0.01 M) shifted the both phases of the concentration-effect curve to the right. Ketanserin (0.1 M) shifted the second, low affinity, phase and prazosin did not alter concentration-effect curve to 5-HT. The sumatriptan concentration-effect curve was shifted by methiothepin (0.01 M) to the right (pK_B = 9.19) but not by ketanserin (1 M). Concentration-effect curves to 5-HT and sumatriptan were not affected by the 5-HT₃ receptor antagonist tropisetron (1 M). These results suggest that 5-HT₁-like type receptors are responsible for the first phase of 5-HT-induced contraction and 5-HT_2A receptor for the second phase, in rabbit mesenteric artery. Sumatriptan-induced contractions appear to be mediated by 5-HT₁-like type receptors in this artery. These results also suggest that this kind of amplification may be a common feature of vascular 5-HT₁-like type receptor as has been shown in other vascular segments such as rabbit femoral, iliac and renal arteries, and guinea-pig iliac artery.     

Cyclic AMP facilitates the electrically evoked release of radiolabelled noradrenaline,dopamine and 5-hydroxytryptamine from rat brain slices

Summary The adenylate cyclase activator forskolin as well as 8-bromo-cyclic AMP enhanced the electrically evoked release of³H-noradrenaline and³H-5-hydroxytryptamine from superfused rat neocortical slices and that of³H-dopamine from neostriatal slices with comparable EC₅₀'s of about 0.5 and 50 M, respectively, without affecting spontaneous tritium efflux. The phosphodiesterase inhibitor ZK 62771 (3–100 M) also enhanced³H-noradrenaline and³H-dopamine release but slightly reduced³H-5-hydroxytryptamine release. However, this drug profoundly enhanced spontaneous tritium release in the latter case. The facilitatory effect of forskolin (0.3 M) on the release of the amine neurotransmitters was potentiated in the presence of ZK 62771 (30 M). Therefore, cyclic AMP appears to exert a general facilitatory effect on the release of these biogenic amines from central nerve terminals.     

5-HT3 receptor ligands lack modulatory influence on acetycholine release in rat entorhinal cortex

Summary The objective of this study was to explore the role of 5-HT₃ receptors in modulating potassium (K⁺)-evoked release of [³H]-acetylcholine ([³H]-ACh) from superfused slices of rat entorhinal cortex previously loaded with [³H]-choline. Rat entorhinal cortices were cross-chopped into 300 m slices, superfused with oxygenated Krebs buffer containing 2.5 mmol/1 Ca²⁺ and stimulated with two consecutive exposures of 20 mmol/l K⁺ for 4 min (S₁ and S₂, respectively). Compounds were added 20 min before S₂ stimulation and remained in the superfusion buffer for the duration of the experiment. The S₂/S₁ ratio was then calculated.Stimulated release of [³H]-ACh was dependent on extracellular Ca²⁺ and K⁺ concentration. In Sprague Dawley rats, 2-methyl-5-HT (10^-9–10^-6 mol/l), in the presence of 1 mol/l ritanserin or 1 gmmol/l ondansetron, had no influence on K⁺-evoked release of [³H]-ACh. In slices prepared from Hooded Lister rats, 2 mol/l 5-HT but not 2-Me-5-HT significantly (P<0.05) inhibited K⁺-evoked [³H]-ACh release only 17% in the presence of 1 mol/l ritanserin. However, 2 mol/l 2-Me-5-HT plus 1 nmol/l ondansetron had no effect. High performance liquid chromatography coupled to electrochemical detection (HPLC-ECD) was used to monitor endogenous release of ACh in the above conditions to confirm data from the radiolabelled experiments. No significant inhibition or increase in K⁺-evoked ACh release was observed with either 5-HT₃ receptor agonists or antagonists. 2-Me-5-HT (10^–9 – 10^–5 mol/l) or 1-(m-chlorophenyl)-biguanide (10^–9 – 10^–5 mol/l), when added simultaneously at the S₂ stimulation, in the presence of 1 l/l methysergide, also showed no effect on [³H]ACh release.In entorhinal cortex slices from aged Wistar rats, neither 1-(m-chlorophenyl)-biguanide (2 or 10 ol/l) nor 2-Me-5-HT (2 mol/l) in combination with ritanserin (1 mol/l) or ondansetron (1 nmol/l) elicited any effect on K⁺-evoked [³H]-ACh release. However, release of [³H]-ACh was inhibited by carbachol (10 mol/l) and adenosine (10 mol/l). DuP 996 (3,3-bis(4- pyridinyl-methyl)-1-phenylindolin-2-one) (10^–7 – 10^–5 mol/l), a known releaser of ACh, markedly augmented K⁺-evoked [³H]-ACh release.These studies have failed to confirm the postulated role of 5-HT₃ receptors in modulating cortical ACh release in rat entorhinal cortex slices and suggest that a critical reexamination of the interaction of 5-HT₃ receptor and cortical cholinergic function needs to be addressed.Abbreviations 5-HT serotonin - ACh acetylcholine - HPLC-ECD high performance liquid chromatography - electrical chemical detection - EGTA ethylene glycol bis(-aminoethyl ether)-N,N-tetraacetic acid - 2-ME-5-HT 2-methyl-5-hydroxytryptamine - DuP 996 (3,3-bis(4pyrindinylmethyl)-1-phenylindolin-2-one) A preliminary report of this work was presented at the 1992 Federation of American Societies for Experimental Biology, April 6–9, Anaheim, California, USA (The FASEB J 6A1559) Correspondence to R. M. Johnson at the above address