Mesulergine, a selective serotonin-2 ligand in the rat cortex, does not label these receptors in porcine and human cortex: evidence for species differences in brain serotonin-2 receptors

The kinetic and pharmacological characteristics of the binding of [3H]ketanserin and [3H]mesulergine to frontal cortical brain membranes from rat, pig and human were studied. In the 3 species [3H]ketanserin labeled sites with the characteristics of the 5-HT2 receptors previously described in the rat. In contrast, [3H]mesulergine labeled 5-HT2 receptors in rat, but not in pig and human cortices. The characteristics of the sites labeled by [3H]mesulergine in pig cortex were similar to those of sites in the choroid plexus of rats, pigs and humans. While several reputed 5-HT2 ligands presented a similar affinity for the [3H]ketanserin binding sites in the 3 species, other such ligands, e.g. mesulergine, methysergide, cinanserin and LSD which displaced these sites with high affinity in rat brain, had lower affinities in pig and human brain. These results indicate that 5-HT2 receptors show different pharmacological profiles in different species. Caution should thus be exerted in extrapolating data from laboratory animals to humans.     

Molecular pharmacology of 5-HT1 and 5-HT2 recognition sites in rat and pig brain membranes: radioligand binding studies with [3H]5-HT, [3H]8-OH-DPAT, (-)[125I]iodocyanopindolol, [3H]mesulergine and [3H]ketanserin

The pharmacological characteristics of the binding of [3H]8-OH-DPAT ([3H]8-hydroxy-2(di-n-propylamino)tetralin, [125I]CYP ((-)[125I]iodocyanopindolol) (in the presence of 30 microM (-)isoprenaline) and [3H]mesulergine to 5-HT1 recognition sites were studied in rat and pig brain membranes. [3H]8-OH-DPAT bound in rat and pig cortex to the 5-HT1A recognition site characterized by high affinity for 5-CT (5-carboxamido-tryptamine), 8-OH-DPAT, 5-HT (5-hydroxytryptamine, serotonin) and low affinity for pirenperone, ketanserin and mesulergine. [125I]CYP bound in rat but not in pig cortex to the 5-HT1B site which shows high affinity for (-)21-009 (4[3-ter-butyl-amino-2-hydroxy-propoxy]indol-2-carbonic acid isopropyl ester), (+/-)ICYP (3-I-cyanopindolol), 5-HT, RU 24969 (5-methoxy-3-[1,2,3,6-tetrahydropyridon-4-yl]1H-indole) and low affinity for 8-OH-DPAT, mesulergine and pirenperone. [3H]Mesulergine bound in pig choroid plexus and in rat cortex (besides binding to 5-HT2 sites in rat cortex) to the 5-HT1C recognition site characterized by high affinity for metergoline, mesulergine, 5-HT and methergine and low affinity for (-)21-009, ICYP, 8-OH-DPAT and spiroperidol. The pharmacological profile of 5-HT1A sites in rat and pig cortex appears to be identical; 5-HT1C sites in pig choroid plexus and rat cortex show no differences. In contrast, it was not possible to label 5-HT1B sites with [125I]CYP in pig brain membranes indicating that like 5-HT2 receptors, 5-HT1 recognition sites show species differences. The pharmacological profiles of the three 5-HT1 recognition sites are clearly different from one another. Furthermore, the pharmacological profile of each individual 5-HT1 recognition site is also different from that of the 5-HT2 receptors labelled with [3H]ketanserin in rat cortex membranes although some similarities exist between 5-HT2 and 5-HT1C sites. Finally, the beta-adrenoceptor antagonist (-)21-009 which has different affinities for 5-HT1A, 5-HT1B and 5-HT1C recognition sites, yielded triphasic competition curves for [3H]5-HT binding in rat cortex membranes providing evidence that [3H]5-HT labels three distinct 5-HT1 sites in these membranes.     

Chronic effects of imipramine and lithium on 5-HT receptor subtypes in rat frontal cortex, hippocampus and choroid plexus: quantitative receptor autoradiographic analysis

The effects of chronic treatment with imipramine or lithium on serotonin (5-HT) receptor subtypes were analyzed in the frontal cortex, hippocampus and choroid plexus of rat brain by quantitative receptor autoradiographic procedures, using radioligands [3H]-5-HT, [3H]-8-hydroxy-2-(di-n-propylamino)tetralin ([3H]-8-OH-DPAT), [125I]-iodocyanopindolol ([125I]-CYP), [3H]-mesulergine and [125I]-7-amino-8-iodo-ketanserin ([125I]-ketanserin) or [3H]-spiperone. Chronic i.p. administration of imipramine (20 mg/kg/day for 21 days) decreased the densities of 5-HT1, 5-HT1A, 5-HT1C and 5-HT2 sites in the frontal cortex, hippocampus and choroid plexus. Lithium (2 mEq/kg/day for 21 days) also decreased the densities of 5-HT1, 5-HT1C and 5-HT2 sites in the frontal cortex, and the densities of those including 5-HT1A sites in the hippocampus and choroid plexus. Imipramine and lithium very markedly decreased the density of 5-HT1C sites in the choroid plexus. We propose that methods employing quantitative receptor autoradiographic analysis can be used to characterize and understand the local effects of these drugs on 5-HT receptor subtypes.     

5-HT1A-receptors mediate stimulation of adenylate cyclase in rat hippocampus

Serotonin (5-HT) stimulated adenylate cyclase activity in homogenates of rat hippocampus. This effect was pharmacologically characterised with a series of agonists and antagonists of various structural classes. These compounds where also tested in radioligand binding studies using selective ligands for the various subtypes of 5-HT1 and 5-HT2 receptors. 5-HT1A, 5-HT1B and 5-HT1C recognition sites were labelled with [3H]8-OH-DPAT([3H]8-hydroxy-2-(di-n-propylamino)-tetralin) in pig cortex membranes, [125I]CYP([125I]iodocyanopindolol) in rat cortex and [3H]mesulergine in pig choroid plexus membranes, respectively. The rank order of potency of 13 agonists stimulating adenylate cyclase activity in homogenates of rat hippocampus was in good agreement with the rank order of affinity of these agonists for the 5-HT1A binding site: N,N-dipropyl-5-carboxamidotryptamine (DP-5-CT) greater than 5-carboxamidotryptamine (5-CT) greater than 8-OH-DPAT greater than 5-HT greater than 5-methoxytryptamine (5-OCH3T) greater than d-LSD greater than 5-methoxy-3-(1,2,3,6-tetrahydro-4-pyridinyl)-1H-indole (RU 24969) greater than alpha-methylserotonin (alpha-CH3-5-HT) greater than dopamine greater than 2-methylserotonin (2-CH3-5-HT). The correlation between the respective potencies and affinities of these agonists was r = 0.934, P less than 0.001. There was no correlation between stimulation of adenylate cyclase activity by these agonists and their affinity for 5-HT1B, 5-HT1C or 5-HT2 binding sites. r = 0.381-0.108, P less than 0.20-0.73.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular pharmacology of 5-HT1D recognition sites: Radioligand binding studies in human,pig and calf brain membranes

1) The binding characteristics of [3H]5-HT (5-hydroxytryptamine, serotonin) were investigated in membrane preparations of several regions from calf, pig and human brain in the presence of 100 nmol/l 8-OH-DPAT (8-hydroxy-2[di-n-dipropylamino]tetralin) and 100 nmol/l mesulergine in order to mask 5-HT1A and 5-HT1C sites. 2) [3H]5-HT bound rapidly, reversibly and stereo-selectively to a population of high affinity recognition sites in membranes from pig caudate, calf caudate and human cortex, caudate and substantia nigra. 3) Saturation experiments carried out with [3H]5-HT in the presence of 100 nmol/l 8-OH-DPAT and 100 nmol/l mesulergine revealed that non-5-HT1A non-5-HT1C sites represented from 50 to more than 90% of the total 5-HT1 sites (determined with [3H]5-HT in the absence of 8-OH-DPAT and mesulergine), depending on the tissue source. 4) The pharmacological profile of these sites was characterized in competition experiments performed with a variety of ligands in membranes of calf, pig and human caudate membranes. Under these conditions, [3H]5-HT labelled a population of "5-HT1-like" sites which display nanomolar affinity for tryptamines (5-carboxamidotryptamine greater than 5-HT greater than or equal to 5-methoxytryptamine greater than tryptamine) and some ergolines (metergoline greater than methysergide). In contrast, these sites showed low affinity for drugs with high affinity and/or selectivity for 5-HT1A (8-OH-DPAT, buspirone), 5-HT1B (21-009, RU 24969), 5-HT1C (mesulergine, mianserin) and 5-HT2 sites (ketanserin, cinanserin).(ABSTRACT TRUNCATED AT 250 WORDS)     

3H]5-hydroxytryptamine binding sites in postmortem human brain

Binding sites for [3H]5-hydroxytryptamine (5-HT) in postmortem human frontal cortex, hippocampus and amygdala were studied by displacement with 5-HT selective drugs. The results demonstrated the selective labelling of 5-HT1-like sites by [3H]5-HT in the cortex, with little or no labelling of 5-HT2 or 5-HT3 sites. Self-displacement of the binding of [3H]5-HT is consistent with the presence of a single population of sites, indicating that 5-HT is non-selective for the 5-HT1 subtypes. Around 40% of the 5-HT1 sites in the frontal cortex and amygdala were of the 5-HT1A subtype, in contrast to 60% in the hippocampus. The drug RU 24969 consistently displaced with, a high affinity, a greater proportion of [3H]5-HT sites than did 8-OH-DPAT in all three regions of the brain. The nature of these additional sites was not established. A small proportion (less than 10%) of [3H]5-HT sites in the frontal cortex appeared to be of the 5-HT1C subtype, as these sites were displaced with high affinity by mianserin.     

125I]SCH 23982, a 'selective' D-1 receptor antagonist, labels with high affinity 5-HT1C sites in pig choroid plexus

[125I]SCH 23982, a ligand reported to be very selective for dopamine D-1 receptors, binds with high affinity to membranes of pig choroid plexus (KD = 186 pM, Bmax = 142 fmol/mg protein). Saturation and competition experiments suggested that [125I]SCH 23982 labels a homogeneous population of sites. The rank order for affinity of agonists, 5-HT greater than or equal to DOI (1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane) much greater than dopamine greater than fenoldopam, and antagonists, metergoline greater than mesulergine = mianserin greater than SCH 23390 greater than methiothepin greater than ketanserin greater than fluphenazine much greater than (-)-sulpiride greater than (+)-sulpiride, was compatible with labelling of 5-HT1C receptors by [125I]SCH 23982. It also correlates very significantly with [3H]mesulergine binding to pig choroid plexus membranes. The effects of SCH 23390 and its analogues should not be systematically attributed to an interaction with D-1 receptors.     

Distribution and characterization of [3H]mesulergine binding in human brain postmortem.

Much interest is currently being directed towards serotonin (5-HT) receptors of type 2C (5-HT2C) because of their possible involvement in the control of different activities, such as the composition of the cerebrospinal fluid, locomotion, feeding, neuronal excitability and anxiety. The limited information regarding their distribution in the human brain prompted us to investigate, and to characterize the binding of [3H]mesulergine, a HT2C antagonist, in autopsy samples from 24 subjects. The results showed that the [3H]mesulergine binding represented 95% of the total binding and equilibrium saturation binding experiments resulted in a single straight line, consistent with the presence of one site only. The area with the highest density of [3H]mesulergine binding was the choroid plexus, followed at a significantly lower level by the hippocampus, substantia nigra, basal ganglia, amygdala, hypothalamus and prefrontal cortex. The pharmacological profile of the [3H]mesulergine binding was consistent with that of 5-HT2C receptors, since the most effective displacers were ritanserin, mesulergine and mianserine, followed by clozapine, ketanserine and m-CPP, while other compounds had a negligible or no effect. These findings, showing a wide distribution of [3H]mesulergine binding sites in the human brain, could provide anatomical bases for the different functions attributable to 5-HT2C receptors in humans.     

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

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

[3H]Ketanserin labels 5-HT2 receptors and α1-adrenoceptors in human and pig brain membranes

The binding characteristics of [3H]ketanserin (a reported selective radioligand for serotonin 5-HT2 receptors) and [125I]BE 2254 (which labels selectively alpha 1-adrenoceptors) were characterized in brain frontal cortex membranes of pig and man. Saturation experiments indicated that both radioligands label apparently a homogeneous class of binding sites in human and pig fontal cortex membranes. Competition experiments with [125I]BE 2254 using 17 agonists and antagonists showed monophasic and steep curves in human and pig frontal cortex membranes. The pharmacological profile of these sites is typical of alpha 1-adrenoceptors. In competition experiments with [3H]ketanserin, most of the tested compounds displayed shallow or biphasic curves. In particular, alpha 1-adrenoceptor-selective antagonists (prazosin, WB 4101, BE 2254...) displaced with nanomolar affinity about 15 and 40% of the specific [3H]ketanserin binding in human and pig frontal cortex membranes, respectively. The minor component of [3H]ketanserin binding correlated highly significantly with [125I]BE 2254 binding in both membrane preparations. The major component of [3H]ketanserin binding to pig and human frontal cortex membranes correlated significantly with [3H]ketanserin binding in rat brain cortex membranes (which is essentially to 5-HT2 receptors). The present data demonstrate that [3H]ketanserin in nanomolar concentrations binds significantly to alpha 1-adrenoceptors in human and pig frontal cortex membranes; this suggests a rather limited degree of selectivity of ketanserin for 5-HT2 receptors in pig and human tissues.     

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

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

3H]WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

In the presence of a 30 nM prazosin mask, [3H]-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ([3H]WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for [3H] WB4101 binding in cerebral cortex. Furthermore, we have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at [3H]WB4101-binding sites in the presence of 30 nM prazosin and [3H] lysergic acid diethylamide ([3H]LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5 mM MgSO4, the Bmax of [3H]WB4101 is significantly lower than the Bmax of [3H]LSD in various brain regions. WB4101 competition for [3H] LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of [3H]WB4101 binding derived from saturation experiments. This suggests that [3H]WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by [3H]LSD. Interestingly, the selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for [3H]WB4101 but compete for multiple [3H]LSD 5-HT1 binding sites. These data indicate that [3H]WB4101 selectively labels the 5-HT1A serotonin receptor, whereas [3H] LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of [3H]WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of [3H]WB4101 binding. These characteristics are typical of agonists interacting with receptors which modulate cellular function via a guanine nucleotide-regulatory subunit.     

Differential inactivation and G protein reconstitution of subtypes of [3H]5-hydroxytryptamine binding sites in brain

The sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide (NEM) inactivate high affinity [3H]serotonin [( 3H]5-HT) binding to bovine and rat brain membranes in a concentration-dependent manner. In both species, 15-25% of total specific high affinity [3H]5-HT binding is relatively insensitive to NEM. This study examines the NEM sensitivity of the various high affinity [3H]5-HT binding subtypes, using selective ligands, tissues, and pharmacological masks to study each subtype. Reconstitution of NEM-inactivated binding by addition of GTP-binding proteins (G proteins, Gi and Go) is also described. Agonist binding to 5-HT1A, 5-HT1B, and 5-HT1D sites in rat brain and to 5-HT1A and 5-HT1D sites in bovine brain is sensitive to NEM. Binding of [3H]dihydroergotamine and [125I]iodocyanopindolol, both of which are weak partial agonists to 5-HT1B sites is relatively insensitive to NEM. Binding of [3H]5-HT to 5-HT1C sites in rat and bovine brain and choroid plexus is relatively insensitive to NEM. In the presence of spiperone to mask binding of 5-HT2 sites, binding of antagonist [( 3H]mesulergine) to 5-HT1C sites is also insensitive to NEM. Likewise, binding of the agonist [3H]4-bromo-2,5-dimethoxyphenylisopropylamine and of the antagonist [3H]ketanserin to 5-HT2 sites is not inhibited by NEM treatment of membranes. These findings suggest that agonist binding to 5-HT1A, 5-HT1B, and 5-HT1D sites is sensitive to NEM alkylation. Binding of neither agonist nor antagonist to 5-HT1C and 5-HT2 sites is sensitive to NEM. Inability of high concentrations of a variety of ligands to protect the sensitive binding sites against NEM inactivation indicates that the critical sulfhydryl group(s) are not located in the ligand binding domain of the NEM-sensitive binding sites. When membranes are treated with NEM, displacement of [125I]iodocyanopindolol by 5-HT is no longer sensitive to 5'-guanylyl imidodiphosphate (Gpp(NH)p). Gpp(NH)p sensitivity of agonist displacement of 5-HT1B binding to NEM-treated membranes is restored by addition of purified guanine nucleotide binding proteins (Gi plus Go). In addition, NEM-inactivated binding to 5-HT1A and 5-HT1D sites can be restored by addition of Gi plus Go. These data suggest that NEM exerts its effects on 5-HT1A, 5-HT1B, and 5-HT1D binding sites by inactivating the G protein(s) associated with the 5-HT receptor subtypes.     

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-HT1C receptors mediate phosphoinositide turnover activation in the immature rat hippocampus.

The activation of phosphoinositide turnover in rat cerebral cortex and choroid plexus is triggered by the stimulation of 5-HT2 and 5-HT1C receptors, respectively. To characterize the 5-HT receptor subtype mediating the activation of phosphoinositide turnover in the hippocampus, the potency of several 5-HT agonists and antagonists on total [3H]inositol phosphate formation has been compared in the hippocampus, cerebral cortex and choroid plexus of immature rats. 5-HT, alpha-methyl-5-HT, quipazine, MK-212, mCPP (m-chlorophenylpiperazine) and TFMPP (m-trifluoromethylphenylpiperazine) are less potent and efficient in stimulating phosphoinositide turnover in the hippocampus and cerebral cortex than in the choroid plexus. However, for a number of 5-HT receptor antagonists (ketanserin, spiperone, ritanserin, pizotifen, cyproheptadine, mesulergine, mianserin, methiothepin, methysergide) there is a good correlation (r = 0.82) between their antagonistic potency in the hippocampus and choroid plexus while such correlation is not observed for the hippocampus and cerebral cortex. The specific 5-HT2 receptor antagonist spiperone only partially antagonizes (37% inhibition at 1 microM) the stimulation by 5-HT of phosphoinositide turnover in the hippocampus. These results suggest that in the immature rat hippocampus the activation of phosphoinositide turnover by 5-HT is mainly mediated by the 5-HT1C receptor subtype.     

Multiple serotonin receptors: Regional distribution and effect of raphe lesions

These studies confirm and extend the recent work suggesting that [³H]lysergic acid dielhylamide (LSD) labels two distinct binding sites in rat brain resembling serotonin (5HT) receptors. Although Scatchard analyses of [³H]LSD binding to membranes prepared from cortex/hippocampus were linear, the heterogeneity of the [³H]LSD binding sites was clearly demonstrated in displacement studies. The displacement curves for both 5HT and spiperone were bisigmoidal with the concentration required to saturate the high affinity components nearly 3 orders of magnitude lower than the concentrations necessary to saturate the low affinity components. Additivity studies suggested that the sites with high affinity for 5HT and spiperone are different, independent sites. These sites are referred to as 5HT₁ and 5HT₂, respectively. Regional analyses showed, that in the frontal cortex, the density of the 5HT₂ site was slightly greater than the 5HT₁ site, whereas the 5HT₁, site was predominant in all other brain areas, including the spinal cord. The pharmacological properties of the two sites have features in common with 5HT receptors; however, electrolytic lesions of the midbrain raphe nuclei did not change the densities or binding constants of the two apparent 5HT receptor subtypes, even though the number of high affinity 5HT uptake sites was markedly reduced.     

Distribution and characterization of [3H]mesulergine binding in human brain postmortem

Much interest is currently being directed towards serotonin (5-HT) receptors of type 2C (5-HT_2C) because of their possible involvement in the control of different activities, such as the composition of the cerebrospinal fluid, locomotion, feeding, neuronal excitability and anxiety. The limited information regarding their distribution in the human brain prompted us to investigate, and to characterize the binding of [³H]mesulergine, a HT_2C antagonist, in autopsy samples from 24 subjects.The results showed that the [³H]mesulergine binding represented 95% of the total binding and equilibrium saturation binding experiments resulted in a single straight line, consistent with the presence of one site only. The area with the highest density of [³H]mesulergine binding was the choroid plexus, followed at a significantly lower level by the hippocampus, substantia nigra, basal ganglia, amygdala, hypothalamus and prefrontal cortex. The pharmacological profile of the [³H]mesulergine binding was consistent with that of 5-HT_2C receptors, since the most effective displacers were ritanserin, mesulergine and mianserine, followed by clozapine, ketanserine and m-CPP, while other compounds had a negligible or no effect.These findings, showing a wide distribution of [³H]mesulergine binding sites in the human brain, could provide anatomical bases for the different functions attributable to 5-HT_2C receptors in humans.     

5-HTIC receptors mediate phosphoinositide turnover activation in the immature rat hippocampus

The activation of phosphoinositide turnover in rat cerebral cortex and choroid plexus is triggered by the stimulation of 5-HT₂ and 5-HT_IC receptors, respectively. To characterize the 5-HT receptor subtype mediating the activation of phosphoinositide turnover in the hippocampus, the potency of several 5-HT agonists and antagonists on total [³H]inositol phosphate formation has been compared in the hippocampus, cerebral cortex and choroid plexus of immature rats. 5-HT, α-methyl-5-HT, quipazine, MK-212, mCPP (m-chlorophenylpiperazine) and TFMPP (m-trifluoromethylphenylpiperazine) are less potent and efficient in stimulating phosphoinositide turnover in the hippocampus and cerebral cortex than in the choroid plexus. However, for a number of 5-HT receptor antagonists (ketanserin, spiperone, ritanserin, pizotifen, cyproheptadine, mesulergine, mianserin, methiothepin, methysergide) there is a good correlation (r = 0.82) between their antagonistic potency in the hippocampus and choroid plexus while such correlation is not observed for the hippocampus and cerebral cortex. The specific 5-HT₂ receptor antagonist spiperone only partially antagonizes (37% inhibition at 1 μM) the stimulation by 5-HT of phosphoinositide turnover in the hippocampus. These results suggest that in the immature rat hippocampus the activation of phosphoinositide turnover by 5-HT is mainly mediated by the 5-HT_IC receptor subtype.     

Eltoprazine, a drug which reduces aggressive behaviour, binds selectively to 5-HT1 receptor sites in the rat brain: an autoradiographic study

Eltoprazine, a phenylpiperazine derivative, selectively reduces offensive aggression in animal models. The present study was designed to localize and characterize the binding sites of [3H]eltoprazine in the rat brain and to compare the distribution of these sites with the distribution of [3H]5-HT binding sites. The binding of [3H]eltoprazine to whole tissue sections was saturable and revealed an apparent dissociation constant (Kd) of 11 nM. Autoradiographic studies demonstrated a widespread distribution of [3H]eltoprazine binding sites throughout the brain. Specific [3H]eltoprazine binding was completely displaced by 5-HT; conversely, unlabelled eltoprazine reduced [3H]5-HT binding to the levels of non-specific binding. The overall distribution of [3H]eltoprazine binding sites showed a strong resemblance to the location of 5-HT1 binding sites labelled with [3H]5-HT. Yet, regions enriched in 5-HT1A and 5-HT1C sites (e.g. dentate gyrus and choroid plexus, respectively) revealed relatively more [3H]5-HT binding as compared to [3H]eltoprazine binding, whereas [3H]eltoprazine binding was more pronounced in 5-HT1B receptor dense areas such as the dorsal subiculum, substantia nigra, ventral pallidum and globus pallidus. Displacement of [3H]eltoprazine with various selective serotonergic drugs demonstrated binding of [3H]eltoprazine to 5-HT1 receptor subtypes. The pharmacological and anatomical data indicate that eltoprazine binds to 5-HT1A, 5-HT1B and to a lesser extent to 5-HT1C binding sites in the rat brain. These results emphasize the important role of serotonin in the regulation of offensive aggression and suggest that eltoprazine may serve as a new tool to study the involvement of central 5-HT1 receptors in the expression of this behaviour.     

The anti-aggressive drug eltoprazine preferentially binds to 5-HT1A and 5-HT1B receptor subtypes in rat brain: sensitivity to guanine nucleotides

Eltoprazine (DU 28853) inhibits offensive aggressive behaviour in several animal species. We characterized the binding of radiolabelled eltoprazine in rat brain by autoradiography. [3H]Eltoprazine displayed saturable and high-affinity binding to several brain areas, including the basal ganglia, hippocampal formation and cerebral cortex (Kd values ranging from 4.2 to 9.5 nM). The maximal binding capacities (Bmax) for [3H]eltoprazine were similar to those for [3H]5-HT and were highest in the substantia nigra and subiculum. Competition with eltoprazine for [3H]ligand binding to the various 5-HT1 receptor subtypes revealed preferential binding to 5-HT1A (IC50 values ranging from 42 to 50 nM) and 5-HT1B (IC50 values ranging from 25 to 38 nM) recognition sites. The drug had moderate affinity for 5-HT1C sites (IC50 = 282 nM). Addition of GTP or its stable analogue Gpp(NH)p to the radioligand assay caused a marked reduction (50-90%) in both [3H]eltoprazine and [3H]5-HT binding. These effects were substantially less in the choroid plexus. The binding of the antagonist (-)[125I]Iodocyanopindolol ([125I]ICYP) to 5-HT1B recognition sites, as quantified in the subiculum and substantia nigra, was either unaltered or slightly enhanced by the addition of 10(-3) M GTP. Furthermore, GTP did not affect the competition for [125I]ICYP binding by the 5-HT1-antagonist methiothepin, whereas it did significantly reduce the displacement by eltoprazine, resulting in an almost twofold increase in IC50 values. The data indicate that the anti-aggressive drug eltoprazine preferentially binds to 5-HT1A and 5-HT1B receptor sites and that this interaction is modulated by guanine nucleotides.