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.     

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     

Interaction between 5-HT uptake inhibition and activation of 5-HT autoreceptors by exogenous agonists in rat cerebral cortex slices and synaptosomes

Summary Rat cerebral cortex slices or synaptosomes were labelled with ³H-5-hydroxytryptamine (³H-5-HT) and subsequently superfused. They were depolarized by electrical stimulation (slices) or with high K⁺ (slices and synaptosomes). Continuous electrical stimulation (2 Hz, 24 mA, 2 ms) and continuous or discontinuous K⁺ depolarization (15–25 mM) were used. 1. Continuous electrical stimulation or continuous K⁺-depolarization of slices evoked a steady overflow of tritium that slowly decayed with time. 2. Exposure to increasing concentrations of 5-methoxy-3(1,2,3,6-tetrahydropyridin-4-yl)-1H-indole succinate (RU 24969) (0.001–0.1 M) during continuous electrical stimulation produced a concentration-dependent decrease in tritium overflow. Citalopram (1 M) counteracted the effect of RU 24969. 3. RU 24969 inhibited the evoked ³H-overflow and citalopram reduced the effect of RU 24969 also during continuous depolarization of slices with 20 mM K⁺. Similar results were obtained by using 5-methoxytryptamine or LSD. 4. In slices 1 M citalopram increased significantly the tritium overflow evoked by electrical stimulation or by 20 mM K⁺-depolarization. 5. Increasing the K⁺ concentration from 20 mM to 25 mM mimicked the effects of 1 M citalopram both on the RU 24969 activity and on the evoked tritium overflow. 6. RU 24969 (0.001–0.1 M) decreased in a concentration-dependent way the release of tritium from cortical synaptosomes depolarized with K⁺ (15–20 mM). The presence of 1 M citalopram did not modify significantly the effect of the agonist. Citalopram was ineffective also when the serotonin uptake carrier in superfused synaptosomes was activated by tryptamine. In conclusion, in slices of rat cerebral cortex, the action of exogenous 5-HT autoreceptor agonists is inhibited by 5-HT uptake blockers independently of the depolarizing agent (electrical stimulation or high-K⁺) used to elicit ³H-5-HT release. Increasing K⁺-concentration, which probably increases serotonin in the biophase, mimics the presence of the reuptake inhibitor. These data together with the finding that, in superfused synaptosomes, 5-HT uptake inhibition did not affect the potency of autoreceptor agonists, favours the idea that, in cerebral cortex slices, inhibitors of 5-HT reuptake prevent activation of autoreceptors by exogenous agonists by increasing the concentration of 5-HT in the autoreceptor biophase. Send offprint requests to M. Raiteri at the above address     

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     

Effect of antidepressant and neuroleptic drugs on the electrically evoked release of serotonin from rat cerebral cortex

The effect of antidepressant and neuroleptic drugs on the electrically evoked release of serotonin (5-HT) was investigated in rat brain cortical slices preincubated with 0.1 mol/l ³H-5-HT. Zimelidine, trazodone, clomipramine, doxepin, and viloxazine (1 mol/l each) enhanced the electrically-induced ³H overflow by 20–44%. Six other antidepressants and five neuroleptics did not increase the evoked transmitter release. Only trazodone and viloxazine also increased the ³H overflow in experiments in which neuronal 5-HT reuptake was already blocked by 6-nitroquipazine. 5-HT and clonidine inhibited the electrically-induced ³H-5-HT release by stimulation of presynaptic 5-HT autoreceptors and ₂-adrenoceptors, respectively; trazodone and viloxazine had no effect on the concentration-response curves of 5-HT and clonidine. Other psychotropic agents with well known antiserotonergic activities also failed to block presynaptic 5-HT autoreceptors. It is concluded that zimelidine, clomipramine, and doxepin enhanced the ³H-5-HT overflow by inhibition of neuronal 5-HT uptake, whereas the increase produced by trazodone and viloxazine cannot be explained by reuptake inhibition or interaction with presynaptic receptors.     

Involvement of adenylate cyclase and protein kinase C in the α2-adrenoceptor-mediated inhibition of noradrenaline and 5-hydroxytryptamine release in rat hypothalamic slices

Summary In superfused rat hypothalamic slices prelabelled with [³H]-noradrenaline, the ₂-adrenoceptor agonist UK 14304 inhibited in a concentration-dependent manner the electrically-evoked release of tritium. This inhibition was antagonized by the ₂-adrenoceptor blocking agent idazoxan, which by itself increased the electrically-evoked tritium overflow. Exposure to forskolin, an adenylate cyclase activator, increased the electrically-evoked release of [³H]-noradrenaline. In the presence of forskolin (1 mol/l), both the inhibitory effect of UK 14304 and the increasing effect of idazoxan on the electrically-evoked release of [³H]-noradrenaline were less pronounced than in the absence of the adenylate cyclase activator. Exposure to forskolin and to the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine shifted to the right the concentration-effect curve for UK 14304 in a similar manner as that observed in the presence of forskolin alone. Exposure to phorbol-12,13-dibutyrate (0.01–10 mol/l), a drug which activates protein kinase C, increased the electrically-evoked release of [³H]-noradrenaline. In the presence of phorbol-12,13-dibutyrate (0.1 and 1 mol/l), the concentration effect curve for UK 14304 on tritium overflow was significantly shifted to the right. The increasing effect of idazoxan on tritium overflow was significantly less pronounced in the presence of 1 mol/l phorbol-12,13-dibutyrate.In superfused rat hypothalamic slices prelabelled with [³H]-5-hydroxytryptamine, the ₂-adrenoceptor agonist UK 14304 significantly inhibited the electrically-evoked release of tritium. Exposure to forskolin increased in a concentration-dependent manner [³H]-5-hydroxytryptamine overflow, but did not modify the UK 14304-mediated inhibition. Exposure to 3-isobutyl-1-methylxanthine enhanced the electrically-evoked release of [³H]-5-hydroxytryptamine. In the presence of both forskolin (1 mol/l) and 3-isobutyl-l-methylxanthine (1 mmol/l), the concentration-response curve for UK 14304 was significantly shifted to the right. Exposure to phorbol-12,13-dibutyrate (0.01–10 mol/l) enhanced in a concentration-dependent manner the electrically-evoked overflow of [³H]-5-hydroxytryptamine. In the presence of phorbol-12,13-dibutyrate (0.1 and 1 mol/l), UK 14304 was significantly less potent to inhibit tritium release than in the absence of the protein kinase C activator.It is concluded that both cyclic AMP and phosphoinositide turnover are involved in the modulation of noradrenaline and 5-hydroxytryptamine release by presynaptic ₂-adrenoceptors in rat hypothalamic slices. However, these interactions do not represent definitive proof for a cause-effect relationship for the second messengers mediating the ₂-adrenoceptor induced inhibition of transmitter release either as autoreceptor or as heteroreceptor.Send offprint requests to S. Z. Langer at the above address     

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.     

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     

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     

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     

Interactions of methylenedioxymethamphetamine with monoamine transmitter release mechanisms in rat brain slices

Summary This study investigates the effects of methylenedioxymethamphetamine (MDMA) and amphetamine on monoamine release from rat superfused brain slices in both the presence and absence of vesicular stores of transmitter. MDMA caused the release of radioactivity from slices incubated with [³H]5-hydroxytryptamine, [³H]noradrenaline or [³H]dopamine with EC₅₀ values of 1.9 mol/l (95% confidence limits 1.5–2.3 mol/l), 4.5 mol/l (2.3–8.7 mol/l), and greater than 30 mol/l, respectively. In contrast, amphetamine (0.1–300 mol/l) was more effective in releasing radioactivity from slices incubated with [³H]dopamine than [³H]noradrenaline or [³H]5-hydroxytryptamine. When Ca²⁺ was excluded from the superfusion fluid, the MDMA induced release of radioactivity from slices incubated with [³H]dopamine was unaltered, but that from slices incubated with [³H]noradrenaline or [³H]5-hydroxytryptamine was enhanced. MDMA (10 mol/l) facilitated the stimulation-induced (5 Hz, 1 min) outflow of radioactivity from slices incubated with [³H]noradrenaline or [³H]5-hydroxytryptamine to 7.5-fold and 2.1-fold of control values, respectively, but had no effect on that from slices incubated with [³H]dopamine. Amphetamine (1 mol/l) increased the stimulation-induced outflow from slices incubated with [³H]noradrenaline, but not that from slices incubated with [³H]5-hydroxytryptamine or [³H]dopamine.Inhibition of monoamine oxidase by a 30-min incubation with pargyline (100 mol/l) enhanced the releasing action of MDMA on all three monoamines. Pargyline (100 mol/l) also enhanced the facilitation caused by MDMA, of the stimulation-induced outflow of radioactivity from slices incubated with [³H]noradrenaline, [³H]5-hydroxytryptamine or [³H]dopamine.In some experiments, slices were obtained from reserpinised rats (2.5 mg/kg s.c. 24 h prior) and pre-exposed for 30 min to the monoamine oxidase inhibitor pargyline (100 mol/l). Under these conditions, electrical stimulation evoked a small residual stimulation-induced outflow of radioactivity from slices incubated with [³H]noradrenaline, and failed to evoke an outflow of radioactivity from slices incubated with [³H]5-hydroxytryptamine or [³H]dopamine. However, a Ca²⁺-dependent stimulation-induced outflow of radioactivity was evoked in the presence of either MDMA (10 mol/l) or amphetamine (1 mol/l) from slices incubated with either [³H]dopamine or [³H]noradrenaline, but not from slices incubated with [³H]5-hydroxytryptamine. The stimulation-induced outflow of radioactivity from slices incubated with [³H]noradrenaline was enhanced in the presence of desipramine (1 mol/l), however this enhancement was less than that caused by 10 mol/l MDMA or 1 mol/l amphetamine. The Ca²⁺-dependent response to electrical stimulation in the presence of MDMA from slices incubated with [³H]noradrenaline was greatly reduced when rats were pretreated with a higher dose of reserpine (10 mg/kg s.c.).This study demonstrates that MDMA and amphetamine release radioactivity from brain slices incubated with [³H]noradrenaline, [³H]dopamine and [³H]5-hydroxytryptamine, but their order or potency as releasers of brain monoamines differs. The results also suggest that MDMA and amphetamine have significant effects on the exocytotic release of monoamines and may interact with both vesicular and cytoplasmic monoamines. Correspondence to J. J. Reid at the above address     

Releasing activities of d-fenfluramine and fluoxetine and fluoxetine on rat hippocampal synaptosomes preloaded with [3H]serotonin

Summary Rat hippocampal synaptosomes preloaded with [³H]serotonin and maintained in a superfusion apparatus were exposed for 3 min to d-fenfluramine or fluoxetine. Both drugs evoked a tritium overflow which was reserpine-sensitive requiring the presence of intact synaptic vesicles. However the two drugs displayed different characteristics: 1) the overflow was immediate with dfenfluramine whereas the releasing activity of fluoxetine showed a delay of about 2 min; 2) d-fenfluramine-induced overflow was already apparent at 0.15 mol/l whereas the minimal effective concentration of fluoxetine was 2.5 mol/l. Their concentration-effect curves were differently shaped, the effect of d-fenfluramine being saturable at 5–20 mol/l (EC₅₀ about 1 gmol/l) while no saturation was observed with fluoxetine up to 10 mol/l; 3) only 1907o of the tritium overflow evoked by fluoxetine (2.5–10 mol/l) consisted of true [³H]serotonin, compared with 7001o when 0.5 mol/l d-fenfluramine was used; 4) the releasing action of 0.5 mol/l d-fenfluramine was completely Ca⁺⁺-dependent, while at higher dfenfluramine concentrations the Ca⁺⁺-independent overflow became more important. The fluoxetine induced overflow was mainly. (70010) Ca⁺⁺-independent; 5) the releasing acitvity of d-fenfluramine was mainly (80%) blocked by the serotonin uptake blockers indalpine, midalcipram and also fluoxetine whereas fluoxetine-induced overflow was insensitive to inhibition of the serotonin carrier.In conclusion, the releasing activity of d-fenfluramine is already present at a very low concentration (0.5 mol/l) and at this concentration its mechanism of action was Ca⁺⁺-dependent, together with the requirement of a functional serotonin carrier. These data therefore do not support the hypothesis of a simple. displacement of 5-HT from its storage vesicles but suggest an exocytotic release possibly triggered by interaction of d-fenfluramine with intracellular receptors. A direct releasing activity is also shown for fluoxetine, very marked at 5–10 mol/l; such effect is different from that of d-fenfluramine and is probably due to the overflow of 5-hydroxyindoleacetic acid, formed in the synaptosomes after the fluoxetine-induced displacement of serotonin from its storage vesicles. The active concentrations of fluoxetine on serotonin release are compatible with those found in rat brain at doses inducing an anorectic activity. Send offprint requests to M. Gobbi at the above address     

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     

Mu and kappa opioid receptor modulation of 5-HT3 and NK-3 receptor-evoked release of acetylcholine from the guinea-pig ileum myenteric plexus

Summary The effects of three different opioid agonists on contractions and [³H]-acetylcholine (ACh) release evoked by 5-hydroxytryptamine₃ (5-HT₃) and neurokinin-3 (NK-3) receptor activation were examined in the guinea-pig ileum longitudinal muscle-myenteric plexus strip (LMMP) preparation. The selective mu ()-opioid receptor agonist (d-Ala²,NMe-Phe⁴,Gly-ol]-enkephalin) (DAMGO; 1 nM–100 nM) and the selective kappa ()-opioid receptor agonist U50488 (10 nM -1 M) inhibited contractile responses to 5-HT and to the selective NK-3 receptor agonist senktide, producing a concentration-related progressive flattening of their concentration-response curves. IC₅₀ estimates for DAMGO and U50488 were somewhat higher for inhibition of 5-HT-evoked as compared to senktide-evoked contractions, and overall lay in the range 6 nM – 51 nM. The selective delta ()-opioid receptor agonist [d-Pen^2,5]-enkephalin (DPDPE) inhibited contractile responses only at the highest concentration used (1 M). ³H-overflow from LMMP preparations preincubated with [³H]-choline was measured as an indicator of [³H]-ACh release. DAMGO (1 nM –100 nM) and U50488 (10 nM -1 M) inhibited the increases in release of [³H]-ACh evoked by 5-HT (10 M) and by senktide (10 nM) in a concentration-dependant manner. IC₅₀ estimates for DAMGO and U50488 were not significantly different for inhibition of 5-HT as compared to senktide-evoked increases in [³H]-ACh release and lay in the range 6 nM –23 nM. DPDPE again only inhibited these responses at the maximum concentration used (1 M). The inhibitory effects of DAMGO, U50488 and DPDPE on contractions and [³H]-ACh release evoked by 5-HT and senktide were completely reversed by naloxone (10 M).These results show that ACh release in the guinea-pig ileum evoked by 5-HT and senktide can be modulated to a similar extent by the opioid agonists DAMGO and U50488, but not by DPDPE. This suggests that the pathways of excitation for 5-HT₃ and NK-3 receptors converge at some level susceptible to opioid inhibition, which may be mediated by - and -, but not -, opioid receptors.     

α2-Adrenoceptor mediated inhibition of the release of radiolabelled 5-hydroxytryptamine and noradrenaline from slices of the dorsal region of the rat brain

Summary Possible local interactions between noradrenergic and serotonergic systems in the dorsal raphe region of the rat were investigated by studying the effects of various drugs on depolarization (20 mmol/l K⁺)-induced release of [³H]5-hydroxytryptamine (5-HT) and [³H]noradrenaline (NA) in vitro using a superfusion method. Exogenous 5-HT did not influence the release of [³H]NA. However, NA (in the presence of 10 mol/l desipramine) as well as the selective ₂-adrenoceptor agonists clonidine and oxymetazoline strongly inhibited [³H]5-HT release. The selective ₁-adrenoceptor agonists phenylephrine and methoxamine did not affect the release of either [³H]5-HT or [⁵H]NA. The inhibition by NA of both [³H]5-HT and [⁵H]NA release was not affected by the -adrenoceptor antagonist sotalol nor by the selective ₁-adrenoceptor antagonist prazosin. However, phentolamine and the selective ₂-adrenoceptor antagonists yohimbine and rauwolscine competitively antagonized the inhibitory effect of NA on [³H]NA release (respective pA₂-values 7.5 and 8.3) and on [³H]5-HT release (respective pA₂-values 7.7 and 8.2). Moreover, the release of [³H]NA and also, but to a lesser extent, that of [³H]5-HT were increased by the antagonists. It is concluded that the release of both 5-HT and NA in the dorsal raphe region may be subject to presynaptic inhibition by NA via activation of ₂-adrenoceptors.Send offprint requests to A. L. Frankhuijzen     

Interactions of isamoltane (CGP 361A), an anxiolytic phenoxypropanolamine derivative,with 5-HT1, receptor subtypes in the rat brain

Summary Isamoltane (CGP 361A; (1-(2-(1-pyrrolyl)phenoxy)-3-isopropylamino-2-propanol hydrochloride), -adrenoceptor ligand (IC₅₀ = 8.4 nmol/l) which has reported activity as an anxiolytic in man was found to be a reasonably active inhibitor of the binding of [¹²⁵I]ICYP to 5-HT_1B recognition sites in rat brain membranes with 27-fold selectivity (IC₅₀ = 39 nmol/l) as compared to the inhibition of binding of [³H]8-OH-DPAT to 5-HT_1A receptors (IC₅₀ = 1070 nmol/l). This selectivity was considerably greater than that observed for other -adrenoceptor ligands including propranolol (5-HT_1A/5-HT_1B ratio = 2), oxprenolol (3.5) and cyanopindolol (8.7). The 5-HT_1B activity of the compound resided in the (–)-enantiomer. (–)Isamoltane had weak activity (IC₅₀ 3–10 mol/l) at 5-HT₂ and ₁-adrenoceptors. The compound was devoid of activity at a number of other central neurotransmitter recognition sites including the 5-HT_1C site. Isamoltane increased the electrically evoked release of [³H]5-HT from prelabeled rat cortical slices in a manner similar to that of cyanopindolol. While both compounds were similar in potency to methiothepin, they had lower efficacy. Oxprenolol was less potent that both isamoltane and cyanopindolol while propranolol was essentially inactive. The effects of the compounds on 5-HT release appeared to be correlated with their 5-HT_1B rather than 5-HT_1A activity. In vivo, isamoltane increased 5-HTP accumulation in rat cortex following central decarboxylase inhibition at doses of 1 and 3 mg/kg i. p. At higher doses this effect was gradually diminished. Similar, but less clearcut results were obtained with cyanopindolol and oxprenolol, but propranolol was ineffective. No changes in brain tryptophan levels were associated with the isamoltaneevoked changes in brain 5-HTP levels. In reserpinized animals, isamoltane reduced 5-HTP accumulation even at doses which enhanced accumulation of this metabolite when given alone. The effects of the putative 5-HT_1B agonist, m-trifluoromethylphenylpiperazine (TFMPP), the mixed 5-HT autoreceptor agonist/antagonist/ -adrenoceptor antagonist, pindolol, the 5-HT uptake inhibitor, CGP 6085A and the MAO-A inhibitor, brofaromine, were not antagonized by pretreatment with isamoltane. The possibility that isamoltane and the other -adrenoceptor antagonists are antagonists at 5-HT_1B receptors and that their effect on 5-GT synthesis in vivo is the net result of their agonist/antagonist effects at 5-HT_1A and 5-HT_1B receptors is discussed in relation to the potential mechanism of the anxiolytic activity of isamoltane.Abbreviations 5-HT Serotonin - ICYP Iodocyanopindolol - 8-OHDPAT 8-hydroxy-2(di-n-propylamino) tetralin - TFMPP m-trifluoromethylphenylpiperazine - MAO-A monoamine oxidase-A Send offprint requests to P. C. Waldmeier at the above address     

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     

Subclassification of presynaptic 5-HT autoreceptors in the human cerebral cortex as 5-HT1Dβ receptors

Human cerebral cortical synaptosomes were used to determine the 5-hydroxytryptamine (5-HT) receptor subtype to which the inhibitory presynaptic 5-HT autoreceptor belongs. The synaptosomes preincubated with [³H]5-HT were superfused and tritium overflow was stimulated by high K⁺. The K⁺-evoked tritium overflow, which was Ca²⁺-dependent but tetrodotoxin-resistant, was concentration-dependently inhibited by the nonselective 5-HTlD_1D/1D receptor agonist, 5-carboxamidotryptamine. Ketanserin at a concentration which should block the 5-HT_1D but not the 5-HT_1D receptor failed to antagonize the inhibitory effect of 5-carboxamidotryptamine. In contrast, the non-selective 5-HT_1D/1D receptor antagonist, methiothepin, at a concentration which should block both the 5-HT_1D and the 5-HT_1D receptor abolished the effect of 5-carboxamidotryptamine. It is concluded that the presynaptic 5-HT autoreceptor, which has previously been classified as 5-HT_1D, belongs to the 5-HT_1D subtype.     

5-Methoxytryptamine and 2-methyl-5-hydroxytryptamine-induced desensitization as a discriminative tool for the 5-HT3 and putative 5-HT4 receptors in guinea pig ileum

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

Frequency-dependence of serotonin autoreceptor but not α2-adrenoceptor inhibition of [3H]-serotonin release in rat hypothalamic slices

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