Identity of inhibitory presynaptic 5-hydroxytryptamine (5-HT) autoreceptors in the rat brain cortex with 5-HT1B binding sites

In rat brain cortex slices preincubated with [3H]5-HT, the potencies of 17 5-HT receptor agonists to inhibit the electrically evoked 3H overflow and the affinities of 13 antagonists (including several beta-adrenoceptor blocking agents) to antagonize competitively the inhibitory effect of unlabelled 5-HT on evoked 3H overflow were determined. The affinities of the compounds for 5-HT1B and 5-HT2 binding sites in rat brain cortex membranes (labelled by [125I]cyanopindolol = [125I]-CYP in the presence of 30 mumol/l isoprenaline and [3H]ketanserin, respectively), for 5-HT1A binding sites in pig and rat brain cortex membranes (labelled by [3H]8-hydroxy-2-(di-n-propylamino)tetralin = [3H]8-OH-DPAT) and for 5-HT1C binding sites in pig choroid plexus membranes (labelled by [3H]mesulergine) were also determined. The affinities of the drugs for the various 5-HT recognition sites ranged over 4-5 log units (the functional experiments revealed the same range of differences between the drugs). There were no significant correlations between the affinities of the drugs at 5-HT1C and 5-HT2 binding sites and their potencies or affinities, determined for the 5-HT autoreceptors. In contrast, significant correlations were found between the potencies or affinities of the drugs for the autoreceptors and their affinities at 5-HT1A or 5-HT1B binding sites; the best correlations were obtained with the 5-HT1B binding site. Some of the drugs investigated were not included in the correlation since their agonistic or antagonistic effects on the autoreceptors were weak and pEC30 or apparent pA2 values could not be determined (less than 5.5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for mediation by 5-HT2 receptors of 5-hydroxytryptamine-induced contraction of canine basilar artery

Summary The agonist potencies of 8 indole derivatives and the potencies of 19 recognized antagonists to inhibit constrictor responses to 5-hydroxytryptamine (5-HT) of canine basilar artery were established. In addition the affinities of the indole derivatives for [³H]5-hydroxytryptamine ([³H]5-HT) binding sites and the affinities of the antagonists for [¹²⁵Iodo]LSD ([¹²⁵I]LSD) binding sites in rat brain cortex membranes were determined. Comparison was also made between the potencies of the antagonists on canine basilar artery and the K _D values published for displacement of [³H]ketanserin binding (Leysen et al. 1982).There was a good correlation between the affinities of the antagonists for 5-HT₂ binding sites labelled by both [¹²⁵I]LSD and [³H]ketanserin and the affinity parameters calculated for inhibition of constrictor responses to 5-HT of canine basilar artery. No correlation could be found between the affinities of the indole derivatives for 5-HT₁ binding sites labelled by [³H]5-HT and their potencies to constrict canine basilar artery.It is concluded that constrictor responses to 5-HT of canine basilar artery are mediated by 5-HT₂-like receptors.     

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.     

Inhibitory presynaptic 5-hydroxytryptamine (5-HT) receptors on the sympathetic nerves of the human saphenous vein

Superfused strips of the human saphenous vein preincubated with 3H-noradrenaline were used to investigate the influences of serotonin (5-HT) receptor agonists and antagonists on the electrically evoked tritium overflow. 5-HT and the preferential 5-HT1A receptor agonist 8-OH-DPAT [8-hydroxy-2-(di-n-propylamino)tetralin] concentration-dependently inhibited the evoked 3H overflow. The evoked 3H overflow was not affected by 0.1 or 1 mumol/l TVX Q 7821 (2-(4-[4-(2-pyrimidinyl)-1-piperazinyl]-butyl)-1,2-benzoisothiazol -3(2H)one-1,1-dioxide), which selectively binds to 5-HT1A sites; TVX Q 7821 10 mumol/l produced an increase in overflow. The inhibitory effect of 5-HT on the impulse-evoked 3H overflow was abolished by the nonselective 5-HT receptor antagonist metitepin, but was not attenuated by propranolol. Metitepin also abolished the inhibitory effect of 8-OH-DPAT on evoked 3H overflow, whereas the 5-HT2 receptor antagonist ketanserin was inactive in this respect. There was also no antagonism of the effect of 8-OH-DPAT by the alpha 2-adrenoceptor antagonist rauwolscine or the dopamine receptor antagonist flupenthixol. These results suggest that both 5-HT and 8-OH-DPAT inhibit noradrenaline release by activating inhibitory 5-HT receptors on the sympathetic nerves of the human saphenous vein. These receptors possess similarities to 5-HT1 recognition sites, but a further subclassification is not yet possible on the basis of the available data.     

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     

Inhibition of serotonin release in the mouse brain via presynaptic cannabinoid CB1 receptors

We studied whether serotonin release in the CNS is inhibited via cannabinoid receptors. In mouse brain cortex slices preincubated with [3H]serotonin and superfused with medium containing indalpine and metitepine, tritium overflow was evoked either electrically (3 Hz) or by introduction of Ca2+ (1.3 mM) into Ca2+-free K+-rich (25 mM) medium containing tetrodotoxin. The effects of cannabinoid receptor ligands on the electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]choline and on the binding of [3H]WIN 55,212-2 and [35S]GTPgammaS to mouse brain cortex membranes were examined as well. In superfused mouse cortex membranes preincubated with [3H]serotonin, the electrically evoked tritium overflow was inhibited by the cannabinoid receptor agonist WIN 55,212-2 (maximum effect of 20%, obtained at 1 microM; pEC50=7.11) and this effect was counteracted by the CB1 receptor antagonist SR 141716 (apparent pA2=8.02), which did not affect the evoked tritium overflow by itself. The effect of WIN 55,212-2 was not shared by its enantiomer WIN 55,212-3 but was mimicked by another cannabinoid receptor agonist, CP-55,940. WIN 55,212-2 also inhibited the Ca2+-evoked tritium overflow and this effect was antagonized by SR 141716. Concentrations of histamine, prostaglandin E2 and neuropeptide Y, causing the maximum effect at their respective receptors, inhibited the electrically evoked tritium overflow by 33, 69 and 73%, respectively. WIN 55,212-2 (1 microM) inhibited the electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]choline by 49%. [3H]WIN 55,212-2 binding to mouse cortex membranes was inhibited by CP-55,940, SR 141716 and WIN 55,212-2 (pKi=9.30, 8.70 and 8.19, respectively) but not by the auxiliary drugs indalpine, metitepine and tetrodotoxin (pKi<4.5). [35S]GTPgammaS binding was increased by WIN 55,212-2 (maximum effect of 80%, pEC50=6.94) but not affected by WIN 55,212-3. In conclusion, serotonin release in the mouse brain cortex is inhibited via CB1 receptors, which may be located presynaptically and are not activated by endogenous cannabinoids. The extent of inhibition is smaller than that obtained (1) via another three presynaptic receptors on serotoninergic neurones and (2) via CB1 receptors on cholinergic neurones in the same tissue.     

Anpirtoline, a novel, highly potent 5-HT1B receptor agonist with antinociceptive/antidepressant-like actions in rodents.

1. The purpose of the present study was to relate the effects of the novel drug, anpirtoline, on 5-hydroxytryptamine (5-HT) receptor subtypes to its antinociceptive and antidepressant-like actions in rodents. 2. Binding assays with rat brain membranes have shown that anpirtoline bound with a much higher affinity to 5-HT1B receptor (Ki = 28 nM) than to 5-HT1A (Ki = 150 nM) and 5-HT2 (Ki = 1.49 microM) receptors. 3. Like 5-HT, anpirtoline concentration-dependently inhibited forskolin-stimulated adenylate cyclase activity in homogenates from the rat substantia nigra. Both effects were not additive, and could be prevented by 5-HT1B receptor antagonists such as propranolol and penbutolol. 4. In superfused rat and pig brain cortex slices preincubated with [3H]-5-HT, the electrically evoked tritium overflow was inhibited by anpirtoline and 5-HT. Whereas 5-HT was equipotent in both tissues (EC50 = 69 nM), anpirtoline was markedly less potent in pig brain cortex slices (EC50 = 1190 nM) than in rat brain cortex slices (EC50 = 55 nM). The concentration-response curve for anpirtoline was shifted to the right by metitepine in both preparations. 5. In the social behaviour deficit test, anpirtoline and trifluoromethylphenyl-piperazine were effective in reversing the isolation-induced impairments in mice, an effect shown only by compounds with agonist properties at the 5-HT1B receptor. 6. In the electrostimulated pain test using mice, anpirtoline dose-dependently increased the pain threshold with an ED50 of 0.52 mg kg-1, i.p. The antinociceptive activity of anpirtoline was abolished by pretreatment with cyproheptadine or propranolol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel histamine H3 receptor antagonists: affinities in an H3 receptor binding assay and potencies in two functional H3 receptor models.

1. We determined the affinities of ten novel H3 receptor antagonists in an H3 receptor binding assay and their potencies in two functional H3 receptor models. The novel compounds differ from histamine in that the aminoethyl side chain is replaced by a propyl or butyl chain linked to a polar group (amide, thioamide, ester, guanidine, guanidine ester or urea) which, in turn, is connected to a hexocyclic ring or to an alicyclic ring-containing alkyl residue [corrected]. 2. The specific binding of [3H]-N alpha-methylhistamine to rat brain cortex membranes was monophasically displaced by each of the ten compounds at pKi values ranging from 7.56 to 8.68. 3. Inhibition by histamine of the electrically evoked tritium overflow from mouse brain cortex slices preincubated with [3H]-noradrenaline was antagonized by the ten compounds and the concentration-response curve was shifted to the right with apparent pA2 values ranging from 7.07 to 9.20. 4. The electrically induced contraction in guinea-pig ileum strips (which was abolished by atropine) was inhibited by the H3 receptor agonists R-(-)-alpha-methylhistamine (pEC50 7.76), N alpha-methylhistamine (7.90) and imetit (8.18). The concentration-response curve of R-(-)-alpha-methylhistamine was shifted to the right by thioperamide (apparent pA2 8.79) and by the ten novel compounds (range of pA2 values 6.64-8.81). 5. The affinities and potencies were compared by linear regression analysis. This analysis was extended to thioperamide, the standard H3 receptor antagonist, which is also capable of differentiating between H3A and H3B sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

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     

Acetylcholine release from rat hippocampal slices is modulated by 5-hydroxytryptamine

Experiments were performed with slices of rat hippocampus in order to investigate whether the release of acetylcholine in this area is modulated through 5-hydroxytryptamine (5-HT) receptors. The slices were prelabeled with [3H]choline then stimulated electrically twice for 4 min each at a frequency of 3 Hz. The overflow of tritium evoked was inhibited by exogenous 5-HT in a concentration-dependent manner. The 5-HT2 receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HC1 ((+/-)-DOI), did not mimic 5-HT. The effect of 5-HT was antagonized by methiothepin but not by the 5-HT2 antagonist, ketanserin. The 5-HT1 agonist, 5-methoxy-3-[1,2,3,6-tetrahydropyridin-4-yl]-1H-indole (RU 24969), inhibited the electrically evoked overflow of tritium, whereas the 5-HT1A-selective agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), was ineffective. Methiothepin itself, but not ketanserin, increased the evoked overflow of tritium. In contrast, the overflow was inhibited by the 5-HT uptake blocker, 6-nitroquipazine. The evoked overflow was also reduced by d-fenfluramine, a serotonin releaser. The concentration-inhibition curve for d-fenfluramine was shifted to the right by methiothepin. It is concluded that the release of ACh in rat hippocampus may be tonically inhibited by 5-HT through the activation of receptors, possibly belonging to the 5-HT1B subtype.     

Release studies with rat brain cortical synaptosomes indicate that tramadol is a 5-hydroxytryptamine uptake blocker and not a 5-hydroxytryptamine releaser.

Tramadol is a centrally acting opioid analgesic whose mechanism of action could also involve an increase in central serotoninergic transmission. Thus, tramadol inhibits synaptosomal serotonin (5-hydroxytryptamine, 5-HT) reuptake and induces tritium release from [3H]5-HT-preloaded slices. We investigated the effect of (+/-)-tramadol in release studies with superfused rat brain cortex synaptosomes preloaded with [3H]5-HT. Tramadol had no releasing effect up to 30 microM, whereas at 10 microM tramadol significantly inhibited by 45% D-fenfluramine-induced [3H]5-HT release. At 100 microM, tramadol showed a slight releasing effect in the absence or in the presence of pargyline, which was not augmented in synaptosomes pre-exposed to Ro 04-1284 (2-ethyl-1,3,4,6,7,11b-hexahydro-3-isobutyl-9,10-dimethoxy-2H-benzo [a]quinolizin-2-ol hydrochloride), a reserpine-like compound that enhances cytoplasmic 5-HT levels. In summary, (+/-)-tramadol behaved as a classical 5-HT uptake blocker (like citalopram) and not as a substrate of the 5-HT carrier with indirect 5-HT mimetic properties (like D-fenfluramine).     

Examination of the relationship between the uptake site for 5-hydroxytryptamine and the high affinity binding site for [3H]imipramine. II. The role of sodium ions

Exogenous sodium ions stimulated both the high affinity binding of [3H]5-imipramine to membranes from the cortex of the rat and the high affinity accumulation of [3H]5-hydroxytryptamine (5-HT) into synaptosomes from the cortex of the rat with similar potencies. Imipramine and zimelidine inhibited synaptosomal uptake of [3H]5-HT potently in standard Tris-Krebs medium, but in a low-sodium medium their inhibitory potencies were significantly attenuated. The inhibitory potencies of panuramine and exogenous 5-HT on the uptake of [3H]5-HT were not significantly affected whether the uptake was measured in a normal or low-sodium Tris-Krebs. Imipramine and zimelidine were potent blockers of high affinity binding of [3H]imipramine whereas panuramine and 5-HT only inhibited the binding of [3H]imipramine at concentrations in excess of those required to inhibit the uptake of [3H]5-HT. It is suggested that imipramine inhibits the uptake of 5-HT by a sodium-dependent action probably at the high affinity binding site for [3H]imipramine, whereas panuramine and 5-HT inhibit the uptake of 5-HT by a sodium-independent mechanism at a site other than the binding site for [3H]imipramine.     

Further characterization of the ORL1 receptor-mediated inhibition of noradrenaline release in the mouse brain in vitro.

Mouse brain slices preincubated with [3H]-noradrenaline or [3H]-serotonin were superfused with medium containing naloxone 10 microM; we studied whether nociceptin (the endogenous ligand at ORL1 receptors) affects monoamine release. Furthermore, the affinities of ORL1 ligands were determined using [3H]-nociceptin binding. The electrically (0.3 Hz) evoked tritium overflow in mouse cortex slices preincubated with [3H]-noradrenaline was inhibited by nociceptin and [Tyr14]-nociceptin (maximally by 80%; pEC50 7.52 and 8.28) but not affected by [des-Phe1]-nociceptin (pEC50<6). The ORL1 antagonist naloxone benzoylhydrazone antagonized the effect of nociceptin and [Tyr14]-nociceptin. The effect of nociceptin did not desensitize, was not affected by blockade of NO synthase, cyclooxygenase and P1-purinoceptors and was decreased by the alpha2-adrenoceptor agonist talipexole. Nociceptin also inhibited the evoked overflow in mouse cerebellar, hippocampal and hypothalamic slices in a manner sensitive to naloxone benzoylhydrazone. The electrically (3 Hz) evoked tritium overflow in mouse cortex slices preincubated with [3H]-serotonin was inhibited by nociceptin; naloxone benzoylhydrazone antagonized this effect. The affinities (pKi) for [3H]-nociceptin binding to mouse cortex membranes were: nociceptin, 8.71; [Tyr14]-nociceptin, 9.82; [des-Phe1]-nociceptin, <5.5; naloxone benzoylhydrazone, 5.85; naloxone, <4.5. In conclusion, nociceptin inhibits noradrenaline release in the mouse cortex via ORL1 receptors, which interact with presynaptic alpha2-autoreceptors on noradrenergic neurones. The effect of nociceptin does not desensitize nor does it involve NO, prostanoids or adenosine. Nociceptin also attenuates noradrenaline release from several subcortical regions and serotonin release from cortical slices by a naloxone benzoylhydrazone-sensitive mechanism.     

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     

Alpha-adrenoceptor-mediated modulation of 5-hydroxytryptamine release from rat brain cortex slices

Slices of rat brain cortex preincubated with 3H-5-hydroxytryptamine were superfused with physiological salt solution and stimulated electrically, or they were superfused with Ca2+-free solution containing 25 mM K+ and stimulated by introduction of 1.3 mM CaCl2 for 2 min. 1. The electrically evoked 3H overflow was decreased by noradrenaline and increased by phentolamine in a concentration-dependent manner. 2. Phentolamine caused a parallel shift to the right of the concentration-response curve of noradrenaline for its inhibitory effect on impulse-evoked 3H overflow; by contrast, it left the inhibitory effect of unlabelled 5-hydroxytryptamine on the electrically evoked 3H overflow unaffected. 3. Propranolol did not alter the inhibitory effect of noradrenaline on impulse-induced 3H overflow. 4. The increasing effect of phentolamine on the electrically evoked 3H overflow was not modified by paroxetine. 5. Cocaine (at a concentration which almost completely blocks the uptake of 3H-noradrenaline but only partially that of 3H-5-hydroxytryptamine into cortex slices) decreased impulse-evoked 3H overflow. This effect was abolished by phentolamine. 6. In the presence of tetrodotoxin throughout superfusion, the Ca2+-evoked 3H-overflow from slices superfused with Ca2+-free solution was inhibited by noradrenaline and increased by phentolamine. These findings suggest that the terminal serotoninergic fibers of rat brain cortex possess alpha-adrenoceptors, activation of which by exogenous or endogenous noradrenaline leads to inhibition of the release of 5-hydroxytryptamine.     

Binding of [3H]haloperidol to dopamine D2 receptors in the rat striatum.

The present study was designed to examine the properties of [3H]haloperidol binding to dopamine D2-receptors in rat striatum membranes, displacement potencies of various chemicals and differences between the affinities of various chemicals and two new 5-hydroxytryptamine (5-HT2) receptor antagonists, MCI-9042, (+/-)-2-(dimethylamino)-1-[[o-(m-methoxyphenetyl)phenoxy]methyl]et hyl hydrogen succinate hydrochloride and one of its metabolites. The plots of specific binding for the striatum membranes obtained from the Scatchard analysis using [3H]haloperidol were monophasic when non-specific binding was determined with 10 microM chlorpromazine, and the Kd and Bmax values were 7.42 +/- 1.03 nM and 1.58 +/- 0.20 pmol (mg protein)-1 (n = 10), respectively. The displacement potencies of D2 receptor, 5-HT2 receptor, histamine H1-receptor, and adrenoceptor antagonists were characterized by [3H]haloperidol binding to D2 receptors. The pKi values of a new antiplatelet agent, MCI-9042, and its metabolite were 5.02 and 5.53, respectively, and these values were lower than those of the D2-receptor antagonists, fluphenazine, spiperone, haloperidol, prochlorperazine, chlorpromazine, thioridazine, and sulpiride. They were also lower than the pKi values of the 5-HT2-receptor antagonists, pirenperone, ketanserin, methysergide, and mianserin. We conclude that the binding site of [3H]haloperidol in the rat striatum is the D2 receptor, that MCI-9042 and its metabolite have lower affinities for D2 receptors than for 5-HT2 receptors, and that this radioreceptor assay is useful for assessing the affinities of various agents.     

Interaction of the central analgesic, tramadol, with the uptake and release of 5-hydroxytryptamine in the rat brain in vitro.

1. Tramadol is a centrally acting analgesic with low opioid receptor affinity and therefore presumably other mechanisms of analgesic action. Tramadol inhibits noradrenaline uptake but since 5-hydroxytryptamine (5-HT) is also involved in the modulation of pain perception, we tested the effects of tramadol on 5-HT uptake and release in vitro. 2. Tramadol inhibited the uptake of [3H]-5-HT into purified rat frontal cortex synaptosomes with an IC50 of 3.1 microM. The (+)-enantiomer was about four times more potent than the (-)-enantiomer; the main metabolite of tramadol, O-desmethyltramadol, was about ten times less potent. 3. Rat frontal cortex slices were preincubated with [3H]-5-HT, then superfused and stimulated electrically. Tramadol facilitated the basal outflow of [3H]-5-HT, at concentrations greater than 1 microM, while the uptake inhibitor 5-nitroquipazine enhanced both basal and stimulation-evoked overflow. Effects of the (+)-enantiomer were more potent than either the racemate, the (-)-enantiomer or the principal metabolite. 4. The effects of tramadol on the basal outflow of [3H]-5-HT were almost completely abolished when the superfusion medium contained a high concentration of the selective 5-HT uptake blocker, 6-nitroquipazine. 5. The results provide evidence for an interaction of tramadol with the neuronal 5-HT transporter. An intact uptake system is necessary for the enhancement of extraneuronal 5-HT concentrations by tramadol indicating an intraneuronal site of action.     

Antagonistic properties of RU 24969, a preferential 5-HT1 receptor agonist, at presynaptic alpha 2-adrenoceptors of central and peripheral neurones

The effect of RU 24969 (5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1 H-indole) on the electrically evoked 3H overflow was studied in superfused rat brain cortex slices preincubated with 3H-noradrenaline or 3H-serotonin and in superfused segments of the rat vena cava preincubated with 3H-noradrenaline. In cortex slices preincubated with 3H-noradrenaline, RU 24969 facilitated the electrically (3 Hz) evoked 3H overflow. This effect was abolished by phentolamine but was not affected by desipramine or the 5-HT3 receptor antagonist ICS 205-930. The concentration-response curve of noradrenaline for its inhibitory effect on the evoked overflow (determined in the presence of desipramine) was shifted to the right by RU 24969 32 and 100 mumol/l. In this respect, RU 24969 was about 500 times less potent than phentolamine. In cortex slices preincubated with 3H-serotonin, the inhibitory effect of 3.2 mumol/l RU 24969 on the electrically evoked 3H overflow was increased by phentolamine. In segments of the vena cava, RU 24969 inhibited the electrically (0.66 Hz) evoked 3H overflow. The concentration-response curve of RU 24969 was U-shaped, since at concentrations higher than 0.1 mumol/l the extent of inhibition decreased with increasing concentrations of RU 24969. In the presence of phentolamine, the concentration-dependent attenuation of the RU 24969-induced inhibition of overflow was no longer detectable. The present results suggest that RU 24969 is a weak antagonist at presynaptic alpha 2-adrenoceptors (by more than 2.5 log units less potent than as an agonist at presynaptic 5-HT1B auto- and heteroreceptors).     

The putative 5-HT1 receptor agonist, RU 24969, inhibits the efflux of 5-hydroxytryptamine from rat frontal cortex slices by stimulation of the 5-HT autoreceptor

The putative central 5-HT receptor agonist, 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole succinate (RU 24969), was found to be a potent inhibitor of the continuous K+ evoked efflux of [3H]5-HT from superfused rat frontal cortex slices (pD2 7.45). The effects of RU 24969 were attenuated by the putative 5-HT autoreceptor antagonists, methiothepin, quipazine and (-)-propranolol but not by the alpha 2-adrenoceptor antagonist, idazoxan. It is concluded that RU 24969 inhibits K+ evoked efflux of [3H]5-HT from rat frontal cortex slices by stimulation of the 5-HT autoreceptor. Moreover, since RU 24969 potently displaced ligand binding to the 5-HT1 and 5-HT1B recognition sites but was only weakly active at the 5-HT2 receptor, the results lend support to the claim for a pharmacological resemblance between the 5-HT autoreceptor and the 5-HT1 recognition site and in particular the low affinity 5-HT1B subtype.     

Characterization and radioautography of [3H] LSD binding by rat brain slices in vitro: The effect of 5-hydroxytryptamine

Binding of D-[³H] lysergic acid diethylamide (LSD) to rat coronal brain slices and its blockade by 5-hydroxytryptamine (5-HT) had characteristics similar to those of brain homogenates in respect of K_D, kinetics and reversibility of binding. Radioautography was done on slices that had been incubated in 6 nM [³H] LSD and on adjacent slices incubated in the same concentration of tritiated LSD plus 10^?5 M of 5-HT. Choroid plexus showed densest labeling of [³H] LSD. In neuropil, dense labeling occurred within parts of the hippocampal formation except for fields CA2 and CA3 which were sparsely labeled. All layers of the cortex except the posterior cingulate gyrus were labeled by LSD. 5-HT blocked labeling of choroid plexus, hippocampal formation, septum, pons, medulla and parts of cortex but only reduced labeling of most other structures. LSD binding sites may relate to some of its pharmacological effects.