Autoradiographic characterisation of [35S]GTPgammaS binding stimulation mediated by 5-HT1B receptor in postmortem human brain

G-protein activation mediated by 5-HT1B receptors was studied in human brain by [35S]GTPgammaS autoradiographic methods. 5-HT (10 microM) increased [35S]GTPgammaS binding in caudate-putamen nucleus, globus pallidus, dentate gyrus, CA1, entorhinal cortex and substantia nigra. In basal ganglia and midbrain, this effect was blocked by GR 127935 (5-HT(1B/1D) antagonist). In contrast, WAY 100635 (selective 5-HT1A antagonist) reversed the effect of 5-HT in hippocampus and entorhinal cortex. Therefore, a detailed pharmacological study was carried out in basal ganglia and substantia nigra using 5-HT and the 5-HT(1B/1D) agonists GTI and CP 93129. In these areas, these agonists stimulated [35S]GTPgammaS binding in a concentration-dependent manner, with no significant differences in the potency for a given structure. Furthermore, GTI was more potent in the putamen than in globus pallidus. In caudate-putamen, the three agonists showed the same efficacy, while in globus pallidus and substantia nigra the efficacy of 5-HT was higher than GTI and CP 93129. The selective 5-HT1B antagonist SB-224289 inhibited GTI- and CP 93129-stimulated [35S]GTPgammaS binding in basal ganglia and substantia nigra, while coincubation with BRL 15572 (selective 5-HT1D antagonist) did not result in any significant change. Here we report the anatomical pattern of distribution of 5-HT1B-dependent functionality by using specific pharmacological tools in human brain sections.     

[3H]sumatriptan labels both 5-HT1D and 5-HT1F receptor binding sites in the guinea pig brain: an autoradiographic study

We have used in vitro autoradiography to visualize [³H]sumatriptan binding sites in sections of guinea-pig and rat brain. In saturation studies, this ligand recognized a single saturable population of high affinity binding sites in all regions examined (pK_D = 8.3–9.3). While 5-HT and the sumatriptan derivative CP-122,288 (5-methyl-aminosulfonylmethyl-3-(N-methylpyrrolidin-2R-yl-methyl)-1H-indole) competed for [³H]sumatriptan binding sites with a high affinity and monophasic profile, displacement experiments with 5-carboxamidotryptamine revealed the existence of 2 classes of binding sites. The high affinity component (pKD = 9.2–9.9) probably corresponded to 5-HT_1B (rat) or 5-HT_1D (guinea-pig) receptors. The intermediate affinity (pK_D = 5.7–7.3) of the other component, taken together with their high affinity for [³H]sumatriptan, was similar to that of the cloned 5-HT_1F receptor. The regional distribution of the 5-HT_1B/1D [³H]sumatriptan binding sites was in agreement with previously published studies (striatonigral system, hypothalamus, central gray, superficial layer of the superior colliculus) and corresponded to the pattern of serotonin-5-O-carboxymethyl-glycyl [¹²⁵I]tyrosinamide labeling in consecutive sections. [³H]sumatriptan binding sites with a low affinity for 5-CT predominated in the intermediate neocortical layers, the claustrum (in the guinea-pig only), the mammillary nuclei, most of the thalamic nuclei and the principal oculomotor nucleus (in the guinea-pig only). This distribution is very similar to that of 5-HT_1F mRNA, indicating further the identity of these sites with 5-HT_1F receptors. Very high densities of 5-HT_1F sites were also found in the rat parafascicular nucleus.Some regions, such as the caudate/nucleus, the lateral geniculate nuclei and the spinal trigeminal nucleus appeared to contain both 5-HT_1B/1D and 5-HT_1F binding sites. Ketanserin had a low affinity for [³H]sumatriptan binding sites in all guinea-pig brain regions, compatible with the presence of the 5-HT_1D\ subtype. An exception was the substantia nigra, where a significant proportion of sites displayed an intermediate affinity for this compound, suggesting the presence of 5-HT_1D receptors. [³H]5-HT labeled 5-HT_1F sites in the claustrum and intermediate cortical layers in the guinea-pig. However these data show that [³H]sumatriptan, in the presence of 10 nM 5-carboxamidotryptamine, is a more suitable radioligand to study the distribution of 5-HT_1F binding sites.     

5-HT1D binding sites in various species: similar pharmacological profile in dog,monkey, calf,guinea-pig and human brain membranes

Summary Radioligand binding studies were performed in membranes of calf caudate, guinea-pig cortex, dog caudate and whole brain, monkey caudate and whole brain, and human caudate using the novel iodinated radioligand, Serotonin-5-O-Carboxymethyl-Glycyl[¹²⁵I] Tyrosinamide (abbreviated [¹²⁵I]GTI for the sake of simplicity), a ligand known to label 5-HT _1B and 5-HT _1D sites.In all membrane preparations tested, [¹²⁵I]GTI labelled high affinity sites with the following rank order of affinity: 5-carboxamidotryptamine > 5-HT = DHE = ergotamine >- sumatriptan >- metergoline = CGS 12066 >- yohimbine = methysergide >- methiothepin > 8-OHDPAT >_ mianserin >- CP 93129 >- (–)pindolol = ketanserin >_ isamoltane = mesulergine >- corynanthine = spiperone > MDL 72222. The affinity profiles were very similar in the membranes of the different species, especially in dog, monkey and human brain. The pharmacological profile of [1251]GTI binding (determined with up to 25 different drugs) was fully comparable to the binding profile reported previously in human substantia nigra (using [1251]GTI) or in a variety of brain preparations known to contain 5-HTID sites using [³H]5-HT as a radioligand.Although, the affinity profiles obtained in the various preparations displayed statistically highly significant correlations with slope values close to one, some drugs displayed slight species-related variations in affinity, as already reported in rabbit brain (see Xiong and Nelson 1989; Hoyer et al. 1992, accompanying report).The present report 1) establishes for the first time the pharmacological profile of 5-HT1D sites in dog and monkey brain, 2) shows that the pharmacological characteristics of these sites is indeed very similar in the brain of a variety of species including man, and 3) demonstrates the advantageous features of [1251]GTI as an iodinated 5-HT_1D radioligand which can be used without the need to mask the binding to other 5-HT receptor subtypes. Correspondence to D. Hoyer at the above address     

Autoradiographic mapping of 5-HT1 receptors in the guinea-pig brain with particular reference to the 5-HT1D receptor sites

Summary The anatomical distribution of 5-HT₁ receptors in the guinea-pig brain was studied by means of in vitro quantitative autoradiography using [³H]-5-HT as ligand. The relative presence of the subtypes of the 5-HT₁ binding site was investigated by adding selective concentrations of 8-OH-DPAT, (-)21009, mesulergine and 5-CT In addition, differentiation of 5-HT_1D receptors was achieved by incubation of the tissues with [³H]-5-HT in the presence of 100 nmol/1 8-OH-DPAT together with 100 nmol/1 mesulergine. Areas presenting high densities of 5-HT_1A receptors included the neocortex (internal layers), hippocampal formation (dentate gyrus, CA₁ field), septum and raphe nuclei, while 5-HT_1C sites accounted for most of the [³H]-5-HT binding to the choroid plexus. Non 5-HT_1A-non 5-HT_1C sites (mainly 5-HT_1D and, also probably, 5-HT_1E receptors) were clearly predominant in the guinea-pig brain. These sites were mainly present in the neocortex (external layers), basal ganglia, hypothalamus and midbrain (substantia nigra, superior colliculus). As previously described, sites with the properties of 5-HT_1B receptors could not be clearly identified in the guinea-pig brain. The present results, in addition to providing a detailed map of the 5-HT₁ receptors in the guinea-pig brain, indicate that the guinea-pig is a useful laboratory animal for the study of 5-HT_1D receptors. Correspondence to A. Pazos 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     

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.     

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     

Brain 5-HT1 binding sites in depressed suicides

5-HT₁ and 5-HT_1A binding sites were measured in brain tissue obtained at postmortem from 19 suicides, with definite evidence of depression, and 19 sex and age-matched controls. Thirteen of the depressed suicides had not been prescribed psychoactive drugs recently (drug-free suicides); six had been receiving antidepressant drugs, alone or in combination with other drugs (antidepressant-treated suicides). No significant differences were found in the number or affinity of 5-HT₁ and 5-HT_1A binding sites in frontal or temporal cortex between drug-free suicides and controls. The number of 5-HT₁ sites was significantly lower (by 20%), affinity unaltered, in hippocampus and the affinity significantly lower (by 33%), number unaltered, in amygdala of drug-free suicides than controls. The number of 5-HT₁ binding sites tended to be higher and the affinity lower in the antidepressant-treated compared to drug-free suicides, and significantly so in hippocampus. The present results, together with our previous studies, provide no evidence of altered cortical 5-HT markers in depressed suicides, but further emphasise abnormalities in the hippocampus.     

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.     

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).     

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     

Pharmacology of cloned human 5-HT1D receptor-mediated functional responses in stably transfected rat C6-glial cell lines: further evidence differentiating human 5-HT1D and 5-HT1B receptors

This study was undertaken to investigate the pharmacology of human serotonin (5-HT)_1D receptor sites by measuring two functional cellular responses, inhibition of forskolin-stimulated cAMP formation and promotion of cell growth, using transfected rat C6-glial cell lines and a broad series of 5-HT receptor agonists. Stable and separate transfection of a pcDNA3 or pRcRSV plasmid, each containing a cloned human 5-HT_1D receptor gene, in rat C6-glial cells was confirmed with RT PCR of 5-HT_1D receptor mRNA and radioligand binding with [³H] 5-carboxamidotryptamine (5-CT) and [³H] sumatriptan. The 5-HT_1D receptor density was 350 and 1050 fmol/mg protein for the C6-glial/pcDNA3/5-HT_1D and C6-glial/pRcRSV/5-HT_1D cell line, and forskolin (100 M)-induced cAMP formation was inhibited by 45 and 78% in the presence of 1 M 5-HT, respectively. A comparison of the intrinsic agonist activities for sixteen 5-HT receptor ligands with their corresponding binding affinities for the human 5-HT_1D receptor site showed similar results for both cell lines with the exception of the partial agonist m-trifluoro-phenyl-piperazine (TFMPP). Three classes of compounds were observed: 1. efficacious agonists, such as 5-CT, 5-methoxytryptamine, 5-HT, sumatriptan, bufotenine, 5-methoxy-3(1,2,3,6-tetrahydro-4-pyridinyl)1H-indole (RU 24,969), tryptamine and 8-hydroxy-2(di-n-propilamino)tetralin (8-OH-DPAT), with agonist potency close to their binding affinity; 2. the partial agonists metergoline, 7-trifluoromethyl-4(4-methyl-l-piperazinyl)-pyrolo-(1,2-a) quinoxaline (CGS 12066B), 1-naphthylpiperazine and 2-methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methylpiperazin-1-yl)-phenyl]-amide (GR 127,935) with marked intrinsic agonist activity but at concentrations higher than their binding affinity; and 3. the silent antagonists ritanserin, ketanserin and methiothepin, apparently free of intrinsic agonist activity, with antagonist potency close to their binding affinity. The cAMP data were further supported by the observed promotion of cell growth by stimulation of both transfected cell lines with sumatriptan under serum-free conditions; half-maximal stimulation was obtained at 4.4 nM (C6-glial/pcDNA3/5-HT_1D) fully in agreement with its EC₅₀-value (5.7 nM) for inhibition of cAMP formation. This growth promoting effect was antagonised by 1 M methiothepin and not observed in pcDNA3-plasmid-transfected and non-transfected C6-glial cells. A comparative study with a C6-glial/pcDNA3/5-HT_1B cell line expressing a similar amount of cloned human 5-HT_1B receptors (B _max: 360 fmol/mg protein) showed almost no intrinsic agonist activity for metergoline, 1-naphtylpiperazine and GR 127,935. Together with the 5-HT_1D receptor binding selectivity and antagonist activity of ketanserin and ritanserin, the findings define important pharmacological differences between cloned human 5-HT_1D and 5-HT_1B receptor sites.     

Agonistic properties of alniditan,sumatriptan and dihydroergotamine on human 5-HT1B and 5-HT1D receptors expressed in various mammalian cell lines

1. Alniditan, a novel migraine abortive agent, is a potent 5-HT_1B/5-HT_1D receptor agonist of nM affinity. We compared the agonistic properties of alniditan, sumatriptan and dihydroergotamine on the cloned human 5-HT_1B receptor expressed at 200 fmol mg⁻¹ protein (B_max) in non-induced L929sA cells, at 740 fmol mg⁻¹ protein in HEK 293 and at 2300 fmol mg⁻¹ protein in mIFNβ-induced L929sA cells, and on the human cloned 5-HT_1D receptor expressed in C6 glioma cells (B_max 780 fmol mg⁻¹ protein).
2. Sodium butyrate treatment increased the expression level of human (h)5-HT_1B receptors in HEK 293 cells and h5-HT_1D receptors in C6 glioma cells approximately 3 fold, the binding affinities of [³H]-5-HT and [³H]-alniditan were unaffected.
3. Agonistic properties were evaluated based on inhibition of cyclic AMP accumulation in the cells after stimulation of adenylyl cyclase by forskolin or isoproterenol. Alniditan, sumatriptan and dihydroergotamine were full agonists at the h5-HT_1B receptor (IC₅₀ values were 1.7, 20 and 2 nM, respectively in HEK 293 cells) and h5-HT_1D receptors (IC₅₀ values of 1.3, 2.6 and 2.2 nM, respectively). At the h5-HT_1B receptor the agonist potency of the compounds slightly increased with higher receptor density. The opposite was seen for antagonists (ocaperidone, risperidone and ritanserin).
4. This comparative study demonstrated that alniditan was 10 times more potent than sumatriptan at the h5-HT_1B receptor, and twice as potent at the h5-HT_1D receptor. Dihydroergotamine was more potent an agonist at the h5-HT_1B receptor when expressed at high and low level in L929sA cells (but not in HEK 293 cells), and was less potent at the h5-HT_1D receptor.

     

An examination of the behavioural specificity of hypophagia induced by 5-HT1B, 5-HT1C and 5-HT2 receptor agonists using the post-prandial satiety sequence in rats

Previous studies have shown that administration of 5-HT_1B, 5-HT_1C or 5-HT₂ agonists decreases food intake in rats. However, it has not been established whether these drugs induce satiety or decrease feeding by a non-specific mechanism. In the present study the post-prandial satiety sequence was used to characterise the actions of the 5-HT₂ receptor agonist, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), the 5-HT_1B/5-HT_1C receptor agonists, 1-(3-chorophenyl) piperazine (mCPP) and 1-[3-(trifluoromethyl)phenyl] piperazine (TFMPP), and the 5-HT_1B agonist, 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)H-indole (RU 24969), on feeding in rats. All four compounds reduced food intake in rats that had been food deprived overnight. The 5-HT_1B/5-HT_1C agonists, TFMPP (at a dose of 1.0 mg/kg) and mCPP (at a dose of 3.0 mg/kg), appeared to produce satiety as their effects on the satiety sequence were similar to those induced by a food pre-load. In contrast, the 5-HT_1B agonist RU 24969 and the 5-HT₂ agonist DOI did not produce behavioural profiles that resembled satiety. Thus, RU 24969 elevated active behaviours and did not accelerate resting whereas DOI appeared to induce hypophagia by a non-specific fragmentation of behaviour. The results suggest that simultaneous activation of 5-HT_1B and 5-HT_1C receptors may be sufficient to elicit behaviourally specific satiety in the rat. In contrast, selective activation of 5-HT₂ receptors does not induce satiety but elicits active behaviours and decreases feeding by response competition.     

Effects of triiodothyronine and imipramine on basal 5-HT levels and 5-HT(1) autoreceptor activity in rat cortex

Clinical studies have shown that triiodothyronine (T3) both augments and accelerates the therapeutic response to antidepressant drugs, particularly tricyclics. There is evidence that this effect is mediated by the serotonergic system. We show here that T3 administered daily for 7 days over the range 0.02-0.5 mg/kg increases basal serotonin (5-hydroxytryptamine, 5-HT) levels, as measured by in vivo microdialysis in rat cortex, in a dose-dependent fashion. All the doses of T3 examined reduced 5-HT(1A) autoreceptor activity, as measured by the effect of 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 0.05 mg/kg s.c.) to decrease 5-HT levels in frontal cortex. T3 administered daily for 14 days at 0.02 mg/kg also reduced 5-HT(1B) autoreceptor activity, as measured by the effect of locally administered 3-(1,2,5,6-tetrahydropyrid-4-yl)pyrrolo[3,2-b]pyrid-5-one (CP 93129, 10 microM) to decrease 5-HT levels. In animals administered imipramine (10 mg/kg/day by osmotic minipump) concurrently with T3 injections, no further changes in either 5-HT(1A) or 5-HT(1B) autoreceptor activity were seen. We suggest that the effect of T3 to accelerate the therapeutic actions of antidepressant drugs may be due to a combination of the actions of T3 at autoreceptors and the actions of the drugs at postsynaptic 5-HT(1A) receptors.     

Recombinant saphenous vein 5-HT1B receptors of the rabbit: comparative pharmacology with human 5-HT1B receptors

1. The rabbit recombinant saphenous vein 5-hydroxytryptamine_1B (rb 5-HT_1B) receptor stably transfected in rat C6-glial cells was characterized by measuring adenosine 3′:5′-cyclic monophosphate (cyclic AMP) formation upon exposure to various 5-HT receptor ligands. The effects of agonists and antagonists were compared with their effects determined previously at the human cloned 5-HT_1B (h 5-HT_1B) receptor under similar experimental conditions.
2. Intact C6-glial cells expressing rb 5-HT_1B receptors exhibited [³H]-5-carboxamidotryptamine (5-CT) binding sites with a K_d of 0.80±0.13 nM and a B_max between 225 to 570 fmol mg⁻¹ protein. The binding affinities of a series of 5-HT receptor ligands determined in a membrane preparation with [³H]-5-CT or [³H]-N-[4-methoxy-3-(4-methylpiperazin-1-yl)phenyl]-3-methyl-4-(4-pyridyl)benzamide (GR 125,743) were similar. With the exception of ketanserin, ligand affinities were comparable to those determined at the cloned h 5-HT_1B receptor site.
3. rb 5-HT_1B receptors were negatively coupled to cyclic AMP formation upon stimulation with 5-HT agonists. Of the several 5-HT agonists tested, 5-CT was the most potent, the potency rank order being: 5-CT>5-HT>zolmitriptan>naratriptan>rizatriptan>sumatriptan>R(+)-8-(hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). The maximal responses of these agonists were similar to those induced by 5-HT. The potency of these agonists showed a positive correlation (r²=0.87; P<0.002) with their potency at the cloned h 5-HT_1B receptor subtype.
4. 2′-Methyl-4-(5-methyl-[1,2,4]oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide (GR 127,935), methiothepin and ketanserin each behaved as silent, competitive antagonists at rb 5-HT_1B receptors; pK_B values were 8.41, 8.32 and 7.05, respectively when naratriptan was used as an agonist. These estimates accorded with their binding affinities and the potencies found on 5-HT and/or sumatriptan-mediated contraction of isolated rabbit saphenous vein segments.
5. In conclusion, the recombinant saphenous vein 5-HT_1B receptor of the rabbit shares important pharmacological similarities with the cloned h 5-HT_1B receptor. However, ketanserin is a more potent antagonist of rb 5-HT_1B receptors.

     

Acute and chronic tryptophan depletion differentially regulate central 5-HT1A and 5-HT2A receptor binding in the rat

Rationale Tryptophan depletion is used to reduce central serotonergic function and to investigate its role in psychiatric illness. Despite widespread clinical use, its effects on serotonin (5-HT) receptors have not been well characterized. Objective The aim of this study was to examine the effect of acute (ATD) and chronic tryptophan depletion (CTD) on free-plasma tryptophan (TRP), central TRP and 5-HT and brain 5-HT_1A and 5-HT_2A receptor binding in the rat. Methods TRP and 5-HT were measured by high-performance liquid chromatography and receptor levels determined by homogenate radioligand binding and in-vitro receptor autoradiography. Results Free-plasma TRP, central TRP and central 5-HT levels were significantly and similarly reduced by ATD and 1- and 3-week CTD compared to controls. ATD significantly reduced 5-HT_1A binding in the dorsal raphe (14%) but did not significantly alter postsynaptic 5-HT_1A binding (frontal cortex, remaining cortex and hippocampus) or 5-HT_2A binding (cortex and striatum). One-week CTD did not significantly alter cortical 5-HT_2A binding or postsynaptic 5-HT_1A binding. Furthermore, 3-week CTD did not significantly alter 5-HT_1A binding but significantly increased cortical 5-HT_2A binding without affecting striatal or hippocampal levels. In the CTD 1 and 3-week groups, rat body weight was significantly decreased as compared to controls. However, weight loss was not a confounding factor for decreased cortical 5-HT_2A-receptor binding. Conclusion ATD-induced reduction in somatodendritic 5-HT_1A autoreceptor binding may represent an intrinsic ‘homeostatic response’ reducing serotonergic feedback in dorsal raphe projection areas. In contrast, the increase in 5-HT_2A receptor after CTD may be a compensatory response to a long-term reduction in 5-HT.     

Effects of acute and chronic desipramine treatment on somatostatin receptors in brain

The effects of acute (5 mg/kg, IP twice daily for 2 days) and chronic (5 mg/kg IP twice daily for 21 days) administration of desipramine (DMI) on [¹²⁵I]-Tyr¹¹-somatostatin binding sites in brain were examined. There was no change in [¹²⁵I]Tyr¹¹-somatostatin binding in membranes prepared from the frontal cortex, striatum, and hippocampus of rats acutely or chronically treated with DMI as compared to non treated animals. [¹²⁵I]Tyr¹¹-Somatostatin binding was increased in membranes prepared from the rat nucleus accumbens only after chronic DMI administration. Scatchard analysis of the binding data from the nucleus accumbens showed that [¹²⁵I]Tyr¹¹-somatostatin labels a single population of somatostatin binding sites with an affinity constant, K_d, of 1.8±0.60 nM and a B_max of 330±90 fmol/mg protein. Chronic treatment with DMI increased the B_max (500±140 fmol/mg protein) but had no effect on the K_d. This finding shows a regional effect of DMI on [¹²⁵I]Tyr¹¹-somatostatin binding sites in rat brain and suggests that somatostatin may play a role in the pathophysiology of depression.     

Role of 5-HT1A and 5-HT1B receptors in the antinociceptive effect of tramadol

Tramadol, (1RS,2RS)-2-[(dimethylamine)-methyl]-1-(3-methoxyphenyl)-cyclohexanol hydrochloride, is an atypical centrally acting analgesic agent with relatively weak opioid receptor affinity and which, like some antidepressants, is able to inhibit the reuptake of serotonin (5-hydroxytryptamine, 5-HT) in the raphe nucleus. We have previously demonstrated that pindolol, a beta-adrenoceptor blocker/5-hydroxytryptamine(1A/1B) receptor antagonist, enhanced tramadol antinociception and that the selective 5-HT1A agonist 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) reduced it. These effects were related to the negative feedback control that regulates raphe region neurones. The current study examines the ability of the selective antagonist at somatodendritic 5-HT1A receptors, N-[2-[4-(2-methoxyphenyl)-1-piperazinyl] ethyl]-N-(2-pyridinyl) cyclohexane carboxamide (WAY100635, 0.8 mg/kg), the selective antagonist at terminal 5-HT1B receptors, N-[3-(2-dimethylamino) ethoxy-4-methoxyphenyl]-2'-methyl-4'-(5-methyl-1,2,4-oxadiazol-3-yl)-(1,1'-biphenyl)-4-carboxamide (SB216641, 0.1-0.8 mg/kg) and the selective agonist at 5-HT1B receptors, 1,4-tDihydro-3-(1,2,3,6-tetrahydro-4-pyridinyl)-5H-pyrrolo[3,2-b] pyridin-5-one (CP93129, 0.2-0.4 mg/kg), to modify the antinociceptive effect of 4-64 mg/kg of tramadol in the hot plate test in mice. The results show that 0.8 mg/kg of WAY100635 enhanced antinociceptive effect of tramadol while neither agonism nor antagonism at the 5-HT1B receptor modifies it significantly at the doses tested. These results account for involvement of the somatodendritic 5-HT1A receptors in the analgesic effect of tramadol and support the supraspinal interaction of serotonin and the opioid system in the regulation of pain.     

Social instigation and aggressive behavior in mice: role of 5-HT1A and 5-HT1B receptors in the prefrontal cortex

Rationale  Social instigation is used in rodents to induce high levels of aggression, a pattern of behavior with certain parallels to that of violent individuals. This procedure consists of a brief exposure to a provocative stimulus male, before direct confrontation with an intruder. Studies using 5-HT_1A and 5-HT_1B receptor agonists show an effective reduction in aggressive behavior. An important site of action for these drugs is the ventral orbitofrontal cortex (VO PFC), an area of the brain which is particularly relevant in the inhibitory control of aggressive and impulsive behavior. Objectives  The objectives of the study are to assess the anti-aggressive effects of 5-HT_1A and 5-HT_1B agonist receptors [8-hydroxy-2-(di-n-propylamino) tetralin hydrobromide (8-OH-DPAT) and CP-93,129] in the VO PFC of socially provoked male mice. To confirm the specificity of the receptor, 5-HT_1A and 5-HT_1B antagonist receptors (WAY-100,635 and SB-224,289) were microinjected into the same area, in order to reverse the agonist effects. Results  8-OH-DPAT (0.56 and 1.0 μg) reduced the frequency of attack bites. The lowest dose of CP-93,129 (0.1 μg) also decreased the number of attack bites and lateral threats. 5-HT1A and 5-HT1B receptor agonists differed in their effects on non-aggressive activities, the former decreasing rearing and grooming, and the latter, increasing these acts. Specific participation of the 1A and 1B receptors was verified by reversal of anti-aggressive effects using selective antagonists WAY-100,635 (10.0 μg) and SB-224,289 (1.0 μg). Conclusions  The decrease in aggressiveness observed with microinjections of 5-HT_1A and 5-HT_1B receptor agonists into the VO PFC of socially provoked mice, supports the hypothesis that activation of these receptors modulates high levels of aggression in a behaviorally specific manner.