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     

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     

Autoradiographic characterisation and localisation of 5-HT1D compared to 5-HT1B binding sites in rat brain

Summary The regional distribution and the pharmacology of the binding sites labelled with the novel 5-hydroxytryptamine (serotonin) 5-HT_1B/1D selective radioligand serotonin-O-carboxy-methyl-glycyl-[¹²⁵I]tyrosinamide (abbreviated [¹²⁵I]GTI for the sake of simplicity) was determined using quantitative autoradiography in rat brain. The distribution of [¹²⁵I]GTI binding sites was largely comparable to that of [¹²⁵I] iodocyanopindolol ([¹²⁵I] ICYP) which labels 5-HT_1B binding sites (in the presence of 8-OH-DPAT (8-hydroxy-[2N-dipropylamino]tetralin) and isoprenaline, to prevent binding to 5-HT_1A and -adrenoceptor binding sites), although a detailed analysis revealed differences.The pharmacology of the [¹²⁵I]GTI binding sites was analysed using compounds known to display high affinity for and/or distinguish between 5-HT_1B and 5-HT_1D sites: 5-carboxamidotryptamine (5-CT), sumatriptan, CP 93129 (5-hydroxy-3(4-1,2,5,6-tetrahydropyridyl)-4-azaindole), (–)pindolol, PAPP (4[2-[4-[3-(trifluoromethyl)phenyl]-1-piperazinyl]ethyl] benzeneamine), rauwolscine, and 8-OH-DPAT. The displacement of [¹²⁵I]GTI by 5-CT was monophasic. By contrast, the selective 5-HT_1B compound CP 93129 and (–)pindolol produced biphasic curves showing a majority of high affinity sites in the globus pallidus and the substantia nigra, whereas PAPP and sumatriptan (which are somewhat 5-HT_1D selective) produced biphasic curves indicating a minority of high affinity sites in these areas. In addition, by blocking the 5-HT_1B sites with 100 nM CP 93129, the remaining population of [¹²⁵I]GTI binding sites could be studied and was found to have high affinity for PAPP, rauwolscine and 8-OH-DPAT. The pharmacological profile of the major binding component was typical of the 5-HT_1B type: 5-CT > CP 93129 (–)pindolol > sumatriptan >/ PAPP > rauwolscine. The profile of the minor component of [¹²⁵I] GTI binding is best characterised as that of a 5-HTID site: 5-CT > PAPP sumatriptan > rauwolscine > (–)pindolol CP 93129.The localisation of the non 5-HT_1B [¹²⁵I]GTI binding sites was characterised by blocking the 5-HT_1B receptors with 100 nM CP 93129. Low densities of the 5-HT_1D recognition sites were found to be present in globus pallidus, ventral pallidum, caudate-putamen, subthalamic nucleus, entopeduncular nucleus, substantia nigra (reticular part), nuclei of the (normal and accessory) optic tract, different nuclei of the geniculate body and frontoparietal cortex, although higher densities of 5-HT_1B sites were always observed in the same structures. Thus, in agreement with the recent cloning of a rat 5-HT_1D receptor cDNA, the presence and the distribution of 5-HT_1D sites could be documented in rat brain. However, when compared to 5-HT_1B sites, 5-HT_1D sites represent only a minor component of the [¹²⁵I]GTI binding in the rat brain structures studied.Correspondence to: D. Hoyer at the above address     

An inhibitory prejunctional 5-HT1-like receptor in the isolated perfused rat kidney

Summary The present study has identified a receptor for 5-hydroxytryptamine (5-HT) which functions to inhibit the stimulus-induced release of [³H] noradrenaline following sympathetic periarterial nerve stimulation to the isolated perfused rat kidney. In addition to 5-HT (IC₃₀=4.5×10^–8 mol/l), both 5-carboxamidotryptamine (IC₃₀=8×10^–9 mol/l) and 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl) indole (RU-24969, IC₃₀=2.5×10^–7 mol/l) acted as agonists whereas 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) was inactive. The inhibitory effect of 5-HT on the electrically-evoked release of tritium was antagonized in a concentration-dependent manner by methiothepin (IC₅₀=4×10^–9 mol/l), metergoline (IC₅₀=4×10^–8 mol/l) and methysergide (IC₅₀=1.3×10^–7 mol/l) but not by cyproheptadine, ketanserin, mesulergine, (–)-propranolol, (±)-pindolol, (±)-cyanopindolol, metoclopramide or phentolamine. It is concluded that the receptor to 5-HT conforms to general criteria defining 5-HT₁-like receptors but at the present time the receptor site cannot be fitted to the designated 5-HT_1A, 5-HT_1B or 5-HT_1C subtypes.Preliminary accounts of this work were presented to the British Pharmacological Society in London (December, 1984) and Southampton (July, 1985)     

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.     

5-HT1D receptors in guinea-pig and pigeon brain

Summary The characteristics of high affinity [³H]5-HT (5-hydroxytryptamine) binding to non 5-HTIA non 5-HT_1A sites were examined in crude membranes prepared from different regions of guinea-pig and pigeon brains. The coupling of these sites to adenylate cyclase was examined, and its pharmacological profile investigated. In the presence of 100 nmol/1 8-OH-DPAT (8-hydroxy-2-(di-n-propylamino)tetralin) and 100 nmol/l mesulergine, [³H]5-HT labelled with nanomolar affinity an apparently homogeneous population of recognition sites in guinea-pig and pigeon brain membranes. The rank order of affinities of agonists and antagonists (5-CT (5-carboxamidotryptamine) > 5-HT > RU 24969 pyridinyl)-1H indole succinate) > yohimbine rauwolscine > DP-5-CT (N,N dipropyl-5-carboxamidotryptamine) mianserin > 8-OH-DPAT > mesulergine > SDZ 21-009 ((±)-4(3-tert-butyl-amino-2-hydroxypropoxy)-in-dol-2 carbonic acid isopropyl ester) > (-)propranolol), as well as their individual pKD values, were very similar to those at porcine caudate 5-HT_1D sites and clearly different from those at rat cortex 5-HT_1B sites. In the substantia nigra of the guinea-pig the 5-HT receptor-mediated inhibition of forskolin-stimulated adenylate cyclase had a pharmacological profile fully comparable to that of 5-HT_1D binding sites (5-CT > 5-HT > yohimbine > RU 24969 > 8-OH-DPAT > SDZ 21-009 = isamoltane > (–)pindolol > (–)propranolol). The rank order of potency of agonists and antagonists in this system closely paralleled their corresponding rank order of potency in the calf substantia nigra (5-HT_1D), but was clearly different from that in rat substantia nigra (5-HT_1B) These results demonstrate the existence of 5-HT_1D recognition sites in the guinea-pig and pigeon brain and their similarity to 5-HT_1D sites of higher mammals, in terms of both drug affinity profile and second messenger coupling. No evidence of the presence of 5-HTIB sites was obtained. The present findings also suggest that 5-HT_1D sites may be present in the brain of the majority of vertebrate species located higher than the sauropside-mammalian divergence in the phylogenic tree, whereas 5-HT_1B sites are only found in some (e.g., mouse, rat, hamster) but not in other rodents (e.g. guinea-pig).     

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     

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

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

5.HT1 receptors in the vertebrate brain

Summary The regional distribution of high affinity [³3H]5-HT recognition sites in the brain of several vertebrates (pigeon, rat, mouse, guinea-pig, cat, dog, monkey and human) was analyzed using in vitro autoradiography. The presence of subtypes of 5-HT₁ binding sites was investigated by selective displacements with 8-OH-DPAT, mesulergine and (±)SDZ 21-009 at appropriate concentrations to block 5-HT_1A, 5-HT_1c and 5-HT_1B sites respectively. In addition, 5-HT_1A, and 5-HT_1c sites were directly visualized with the more selective radioligands [³H]8-OH-DPAT and [³H]mesulergine, respectively. In the pigeon brain, total [³H]5-HT binding sites were enriched in all telencephalic areas. Densely labelled regions were also present in the optic tectum and the brainstem. No binding was observed in the cerebellum. 8-OH-DPAT and mesulergine only displaced a small proportion of [³H]5-HT binding in most of the areas where high concentrations of 5-HT₁ sites were found. (±)SDZ 21-009 did not affect [³H]5-HT binding in the regions examined. Taking into account our pharmacological studies, these results suggest that the majority of 5-HT₁ sites belong to the 5-HT_1D subtype in the pigeon brain. In the mammalian species investigated high levels of [³H]5-HT binding were found in the neo-cortex, hippocampal formation, basal ganglia and related structures (substantia nigra), raphe dorsalis, nucleus superior colliculus and choroid plexus. However, these brain areas were differentially enriched in subtypes of 5-HT₁ recognition sites. 5-HT_1A sites were observed in the neo-cortex, the hippocampal formation and the raphe nucleus, whereas 5-HT_1C sites accounted for all 5-HT₁ binding in the choroid plexus. In the mouse and rat brain, 5-HT_1B binding sites were enriched in the basal ganglia and associated regions (substantia nigra). These areas were enriched in 5-HT_1D sites in the brain of the other mammals studied. In these animals, no site with a 5-HT_1B pharmacological profile were detected.These results indicate that 5-HT_1A 5-HT_1c and 5-HT_1D sites are present already in the lower vertebrate species investigated and that 5-HT_1B appear to be exclusive of the myomorph rodents (mouse, rat). Furthermore, the different subtypes of the 5-HT₁, receptors present a conserved regional distribution with the 5-HT_1D sites being enriched in the basal ganglia and the 5-HT_1A sites predominating in the hippocampal formation.     

Piglet sinoatrial 5-HT receptors resemble human atrial 5-HT4-like receptors

Summary 5-Hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT) and the gastrointestinal kinetic benzamides renzapride and cisapride caused tachycardia in spontaneously beating right atria of piglet in the presence of 400 nmol/l(±)-propranolol and 6 mol/l cocaine. The maximum tachycardia caused by agonists, compared to that evoked by 200 mol/l(–)-isoprenaline, was 63% for 5-HT, 50% for 5-CT, 50% for renzapride and 28% for cisapride. The rank order of potency was 5-HT > renzapride > cisapride > 5-CT. The effects of the agonists, but not those of (–)-isoprenaline, were antagonised by 3-tropanyl-1H-indole-3-carboxylic acid (ICS 205930); the pK_B of ICS 205930 (vs 5-HT) was 6.9. These characteristics suggest that piglet sinoatrial 5-HT receptors are similar to so-called 5-HT₄ receptors previously described in mouse colliculi neurons. Piglet sinoatrial 5-HT₄-like receptors resemble the human atrial 5-HT receptors that mediate positive isotropic effects of 5-HT.Send of fprint requests to A. J. Kaumann at the above address     

Evidence that 5-HT agonist-induced rotational behaviour in the rat is mediated via 5-HT1 receptors

The rotational behaviour induced by 5-HT agonists has been investigated in rats with lesions of the dorsal raphe' nucleus (DRN). We have previously reported that 5-methyoxy-N,N-dimethyl-tryptamine (5-MeODMT) caused dose-related contralateral rotation in rats with 5,7-dihydroxytryptamine (5,7-DHT) lesions of the DRN. Similar findings are now presented for the 5-HT₁ agonists 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) and 5-methoxy-3 (1,2,3,6-tetrahydropyridin-4-yl) (1H indole) (RU24969). In this model, in agreement with the behavioural studies, both agonists were shown to have a greater affinity for the 5-HT₁ binding site when compared with the 5-HT₂ binding site. Antagonist studies using selective 5-HT₂ antagonists (ketanserin and pirenperone) at non-sedative doses failed to inhibit this behaviour. In contrast, the classical 5-HT antagonist methysergide caused significant inhibition of the rotational behaviour. These results suggest that 5-HT agonist-induced rotation in the rat is mediated via 5-HT₁ receptors, probably located in the substantia nigra.     

Interaction of arylpiperazines with 5-HT1A, 5-HT1B, 5-HT1C and 5-HT1D receptors: do discriminatory 5-HT1B receptor ligands exist?

Summary The effects of several putative 5-HT₁ receptorsubtype selective ligands were investigated in biochemical models for 5-HT_1A, 5-HT_1B, and 5-HT_1D receptors (inhibition of forskolin-stimulated adenylate cyclase activity in calf hippocampus, rat and calf substantia nigra, respectively) and 5-HT_1C receptors (stimulation of inositol phosphates production in pig choroid plexus). Following compounds were studied: 5-HT (5-hydroxytryptamine), TFMPP (1-(mtrifluoromethylphenyl)piperazine), mCPP (1-m-chlorophe-nyl)piperazine, 1 CGS 12066 (7-trifluoromethyl-4-(4-methyl1-piperazinyl)-pyrrolo[1,2-a]quinoxaline 1), isamoltane (CGP 361A, 1-(2-(1-pyrrolyl)-phenoxy)-3-isopropylamino-2-propranol), quipazine, 1-NP (1-(1-naphthyl)piperazine), and PAPP (LY165163, 1-[2-(4-aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)-piperazine). Among reported 5-HT_1B receptor selective drugs, TFMPP had similar potency at 5HT_1A, 5-HT_1B and 5-HT_1C receptors, mCPP did not separate between 5-HT_1B and 5-HT_1C receptors, CGS 12066 was equipotent at 5-HT_1B and 5-HT_1D receptors, and isamoltane was only slightly 5-HTIB versus 5-HT_1A selective. Quipazine showed equal potency at 5-HTIB and 5-HT_1C receptors and 1-NP did not discriminate between the four receptor subtypes. PAPP described as 5-HT_1A receptor selective, was equally potent at 5-HT_1A and 5-HT_1D receptors. The potencies determined in second messenger studies were in good agreement with the affinity values determined in radioligand binding studies. Thus 5-HT_1A, 5-HT_1B, 5-HT_1C and 5-HT_1D receptors have different pharmacological profiles as predicted from radioligand binding studies. Despite claims to the contrary, none of the tested compounds had actual selectivity for a given 5-HT₁ receptor subtype. Of interest were the properties of several of these drugs, which behaved as agonists at some receptors and as antagonists at others (e. g. quipazine, 1-NP, PAPP and isamoltane). Send offprint requests to D. Hoyer at the above address     

5-Hydroxytryptamine 5-HT1B and 5-HT1D receptors mediating inhibition of adenylate cyclase activity

Summary 5-Hydroxytryptamine_1B (5-HT_1B) receptor mediated-inhibition of forskolin-stimulated adenylate cyclase activity in rat substantia nigra was characterized pharmacologically and compared to 5-HT_1D receptor mediated-inhibition of forskolin-stimulated adenylate cyclase activity in calf substantia nigra. Special attention was paid to the effects of drugs known to bind with high affinity to 5-HT_1B (pindolol, propranolol, cyanopindolol, SDZ 21-009, isamoltane) or 5-HT_1D recognition sites (yohimbine, rauwolscine).PEC₅₀ or pK _B values of a variety of 5-HT-receptor ligands (6 agonists including 5-HT, and 12 antagonists) for the inhibition of adenylate cyclase activity in rat substantia nigra, correlated significantly to the corresponding pK _D values at 5-HT_1B binding sites (r = 0.90, P = 0.0001). Amongst the ₂- and -adrenoceptor antagonists tested, none of the drugs expressed more than 35% of the intrinsic activity of 5-HT at 5-HT_1B receptors. When tested as antagonists, their pK _B values were in good agreement with their pK _D values for 5-HT_1B sites. By contrast, these drugs displayed marked intrinsic activity at 5-HT_1D receptors: their pEC₅₀ values were close to their pK _D values for 5-HT_1D sites and their effects could be potently antagonized by methiothepin. The rank orders of potency of the tested compounds at 5-HT_1B and 5-HT_1D were markedly different.The results strengthen the identity between 5-HT receptors mediating inhibition of adenylate cyclase activity in rat and calf substantia nigra and 5-HT_1B and 5-HT_1D binding sites, respectively. They underline the differences between these receptors in terms of intrinsic activities and potencies of drugs. Send offprint requests to: D. Hoyer 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.     

Characterization of prejunctional 5-HT1 receptors that mediate the inhibition of pressor effects elicited by sympathetic stimulation in the pithed rat

1. A study was made of the effects of 5-carboxamidotryptamine (5-CT) on pressor responses induced in vivo by electrical stimulation of the sympathetic outflow from the spinal cord of pithed rats. All animals had been pretreated with atropine. Sympathetic stimulation (0.1, 0.5, 1 and 5 Hz) resulted in frequency-dependent increases in blood pressure. Intravenous infusion of 5-CT at doses of 0.01, 0.1 and 1 μg kg⁻¹ min⁻¹ reduced the pressor effects obtained by electrical stimulation. The inhibitory effect of 5-CT was significantly more pronounced at lower frequencies of stimulation. In the present study we characterized the pharmacological profile of the receptors mediating the above inhibitory effect of 5-CT.
2. The inhibition induced by 0.01 μg kg⁻¹ min⁻¹ of 5-CT on sympathetically-induced pressor responses was partially blocked after i.v. treatment with methiothepin (10  μg kg⁻¹), WAY-100,635 (100 μg kg⁻¹) or GR127935T (250 μg kg⁻¹), but was not affected by cyanopindolol (100 μg kg⁻¹).
3. The selective 5-HT_1A receptor agonist 8-OH-DPAT and the selective 5-HT_1B/1D receptor agonists sumatriptan and L-694,247 inhibited the pressor response, whereas the 5-HT_1B receptor agonists CGS-12066B and CP-93,129 and the 5-HT_2C receptor agonist m-CPP did not modify the pressor symapthetic responses.
4. The selective 5-HT_1A receptor antagonist WAY-100,635 (100 μg kg⁻¹) blocked the inhibition induced by 8-OH-DPAT and the selective 5-HT_1B/1D receptor antagonist GR127935T (250 μg kg⁻¹) abolished the inhibition induced either by L-694,247 or sumatriptan.
5. None of the 5-HT receptor agonists used in our experiments modified the pressor responses induced by exogenous noradrenaline (NA).
6. These results suggest that the presynaptic inhibitory action of 5-CT on the electrically-induced pressor response is mediated by both r-5-HT_1D and 5-HT_1A receptors.

     

Inhibition of noradrenaline release via presynaptic 5-HT1B receptors of the rat vena cava

Summary In the rat inferior vena cava preincubated with ³H-noradrenaline, the effects of nine serotonin (5-HT) receptor agonists and of eight antagonists (including two -adrenoceptor blocking agents) on the electrically evoked ³H overflow were determined. 1. 5-HT, 5-carboxamidotryptamine, 5-methoxy-3(1,2,3,6-tetrahydropyridine-4-yl)-1H-indole (RU 24969), 5-methoxytryptamine, N,Ndimethyl-5-HT, tryptamine and 5-aminotryptamine inhibited the evoked ³H overflow. The potencies of these agonists in inhibiting overflow were significantly correlated with their affinities for 5-HT_1B binding sites, but not with their affinities for 5-HT_1A, 5-HT_1C or 5-HT₂ binding sites. 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5HT_1A receptor agonist, and ipsapirone, a partial agonist at these receptors, did not inhibit overflow. 2. Cyanopindolol facilitated the evoked ³H overflow, an effect which was abolished by propranolol. The maximum inhibition of overflow obtainable with 5-HT was diminished by cyanopindolol. 3. The concentration-response curve for 5-HT was shifted to the right by metitepine, metergoline, quipazine, 6-chloro-2-(1-piperazinyl)pyrazine (MK 212) and propranolol which, given alone, did not affect ³H overflow. The apparent pA₂ values of these antagonists tended to be correlated with their affinities for 5-HT_1B (but not 5-HT_1A, 5-HT_1c or 5-HT₂) binding sites. Ketanserin, a 5-HT₂ receptor antagonist, and spiperone, which blocks 5-HT₂ and 5-HT_1A but not 5-HT_1B or 5-HT_1C receptors, failed to antagonize the effect of 5-HT. These results suggest that the inhibitory presynaptic 5-HT receptors on the sympathetic nerve terminals of the rat vena cava appear to belong to the 5-HT_1B subtype. Cyanopindolol may act as a partial agonist at these receptors, as it does at the facilitatory prosynaptic -adrenoceptors.This study was supported by a grant of the Deutsche Forschungsgemeinschaft Send offprint requests to M. Göthert     

Actions of non-peptide ergot alkaloids at 5-HT1-like and 5-HT2 receptors mediating vascular smooth muscle contraction

Summary This report describes the actions of the non-peptide ergot alkaloids methysergide, methylergometrine and ergometrine at two types of 5-HT receptor mediating vascular contraction; the well established 5-HT₂ receptor in rabbit aorta and a non-5-HT₂ receptor in rabbit saphenous vein which resembles the 5-HT₁-like receptor in dog saphenous vein.In the rabbit aorta ergometrine (1 mol/l) and methylergometrine (0.3 mol/l), but not methysergide, produced small contractions (14% and 7% respectively of the maximal response to 5-HT). This contraction was not related to activation of 5-HT₂ receptors since it was resistant to blockade by ketanserin (0.3 mol/l). When examined as antagonists of 5-HT-induced contractions of rabbit aorta, each ergot displayed nanomolar affinity at the 5-HT₂ receptor but only methysergide behaved as a simple competitive antagonist (pK_B = 8.25). Methylergometrine and ergometrine produced surmountable blockade which was accompanied by a non-parallel displacement of the 5-HT concentration-effect curves. The selective 5-HT₁-like receptor agonist GR43175 ( 30 mol/l) was devoid of affinity at the 5-HT₂ receptor in rabbit aorta.In the rabbit saphenous vein each of the ergots produced concentration-dependent contractions which resulted in overtly biphasic concentration-effect curves. Only the first phase of contraction mimicked the effects of 5-HT and GR43175 since contractions were not blocked by MDL 72222 (1 mol/l), but were surmountably antagonised by methiothepin (10 nmol/1), ketanserin (0.3 mol/l) and spiperone (0.3 mol/l). These results are expected for interactions at the 5-HT₁-like receptor in this preparation (Martin and MacLennan 1990). The mechanism(s) underlying the second phase of contraction with the ergots remains to be established. Receptor inactivation studies using the alkylating agent benextramine tetrahydrochloride enabled each agonists' affinity and efficacy at the 5-HT₁-like receptor to be estimated. Affinity estimates (pK_A) decreased in the order: methylergo metrine (7.79), ergometrine (7.75), 5-HT (7.19), methysergide (6.76), GR43175 (6.20), whereas efficacies () decreased in the order: 5-HT (3.28), methylergometrine (2.24), GR43175 (2.14), ergometrine (1.94), methysergide (0.99). Of particular interest, methysergide was significantly lower in affinity and efficacy than its primary demethylated metabolite methylergometrine. Evidently, at the 5-HT₁-like receptor mediating vascular contraction the ergots ergometrine and methylergometrine are both higher in affinity than, and comparable in efficacy to, the natural receptor agonist 5-HT. This contrasts with their actions at the 5-HT₂ receptor in rabbit aorta where they demonstrated a higher affinity but much lower intrinsic efficacy than 5-HT. These results favour the view that vascular contraction induced by these ergots is more likely to be mediated by 5-HT₁-like, rather than 5-HT₂ receptors. These results are discussed in relation to the therapeutic applications of these ergots, particularly in obstetrics and in migraine, and to their utility as diagnostic agents in patients with Prinzmetal's variant form of angina.Send offprint requests to S. J. MacLennan 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     

Operational characteristics of the 5-HT1-like receptors mediating external carotid vasoconstriction in vagosympathectomized dogs

It has recently been shown that the external carotid vasoconstrictor response to 5-HT in the dog is primarily mediated by sumatriptan-sensitive 5-HT₁-like receptors; however, the fact that these receptors are not blocked by metergoline, a 5-HT_1D ligand, raises questions about their possible correlation with the 5-HT_1D receptor subtype. Since a number of drugs display high affinity for the 5-HT_1D (GR127935) and 5-HT_1F (e.g. methysergide and oxymetazoline) receptor subtypes, in this study we have used these drugs to determine whether the above vasoconstrictor 5-HT₁-like receptors correlate with the 5-HT_1D and/or 5-HT_1F receptor subtypes.One-minute intracarotid infusions of 5-HT (0.3–30 g/min), sumatriptan (1–30 g/min), oxymetazoline (0.03–3 g/min) and noradrenaline (0.3–3 g/min) resulted in dose-dependent decreases in external carotid blood flow without changes in arterial blood pressure or heart rate. These vasoconstrictor responses remained unaltered after i.v. administration of physiological saline (0.015, 0.05 and 0.15 ml/kg; n = 4) or ritanserin (1 mg/kg; n = 5). In contrast, GR127935 (1, 3 and 10 g/kg, n = 6) potently blocked the responses to 5-HT (unmasking a dose-dependent vasodilator component) and sumatriptan without affecting those to oxymetazoline or noradrenaline. Interestingly, methysergide (10, 30 and 100 g/kg, n = 5) also blocked the vasoconstrictor responses to 5-HT and sumatriptan, but unlike GR127935, did not revert the vasoconstrictor response to 5-HT; the responses to oxymetazoline remained unaffected, but those to noradrenaline were apparently attenuated by the highest dose.Taken together, the above findings suggest that the sumatriptan-sensitive 5-HT₁-like receptors mediating canine external carotid vasoconstriction resemble 5-HT_1D receptors, probably of the 5-HT_1D subtype on the basis of the resistance to blockade by ritanserin. The pharmacological profile of these receptors could be similar (bovine and human cerebral arteries, porcine carotid arteriovenous anastomoses and human coronary arteries) to other putative 5-HT_1D receptors mediating vascular responses.     

Evidence that blockade of post-synaptic 5-HT1 receptors elicits feeding in satiated rats

The effects of nine central 5-HT antagonists on food intake in free feeding male rats were examined. The 5-HT₂ antagonists ritanserin and ketanserin and the selective 5-HT₃ antagonists ICS 205-930 and MDL 72222 had no effect on food intake. In contrast, the non-selective 5-HT antagonists metergoline, methiothepin, mesulergine, mianserin and methysergide (all of which have high affinity for various 5-HT₁ receptor subtypes), dose-dependently increased food intake during a 4-h daytime test. Furthermore, metergoline dose dependently increased food intake over a 24-h period. Suprisingly, mesulergine decreased food intake over a 24-h period at the same doses that increased daytime food intake. This may indicate that the increase in daytime feeding produced by mesulergine is a non-specific response. Although the antagonists used have varying degrees of selectivity for 5-HT receptor subtypes, the pattern of results suggests that postsynaptic 5-HT₁ receptors (possibly of the 5-HT_1C type) play an important role in the control of feeding in rats.