Unaltered 5-HT- and desipramine-sensitive [3H]imipramine binding and [3H]5-HT uptake in rat brain after chronic imipramine and norzimeldine treatment

Several reports have shown heterogeneity of [³H]imipramine binding to brain membranes. Recently, a high affinity and 5-HT sensitive [³H]imipramine binding site of protein nature, that was suggested to be identical to the substrate recognition site for 5-HT uptake, was demonstrated. Since most studies on the regulation of the [³H]imipramine binding sites by antidepressants have used desipramine displaceable binding, which is heterogenous in nature and contains binding not related to 5-HT uptake sites, the present report studies the possible effects of chronic (3 weeks) administration of imipramine or norzimeldine (10 mg/kg intraperitoneally twice daily) on 5-HT sensitive [³H]imipramine binding sites. For comparison, desipramine sensitive binding was also studied, as well as the physiological correlate 5-HT uptake. There were no changes in either [³H]imipramine binding or 5-HT uptake after the antidepressant treatment.Supported by the Swedish Medical Research Council Offprint requests to: J. Marcusson at Dept. of Geriatric Medicine     

Binding of some antidepressants to the 5-hydroxytryptamine transporter in brain and platelets

Antidepressant agents with properties to inhibit 5-hydroxytryptamine (5-HT, serotonin) uptake in brain tissue and platelets bind with high affinities to neuronal and platelet membranes. [³H]Imipramine, [³H]paroxetine and [³H]citalopram label specific binding sites related to the 5-HT transporter. [³H]Paroxetine and [³H]citalopram appear to be better ligands than [³H]imipramine. The former label a homogenous population of binding sites, whereas the displaceable binding of [³H]imipramine is heterogenous. Recent observations in several laboratories, which have taken the heterogeneity of [³H]imipramine binding into account, indicate that the binding of antidepressants to the 5-HT transporter probably occurs to the same site that binds 5-HT for transport and not to a separate site as previously suggested. Additional bonds to subsites in close vicinity to the 5-HT recognition site may contribute to the binding. No convincing evidence has been presented of the existence of an endogenous ligand other than 5-HT itself that binds to the [³H]imipramine binding site. Recent studies also suggest that repeated treatment of rats with antidepressant agents does not produce any alterations of the binding of [³H]imipramine or [³H]paroxetine to membranes of cerebral cortex. It is also doubtful whether the density of the 5-HT uptake site in platelets measured with these ligands is decreased in affective disorders as first reported.     

Selective labelling of high affinity 5-hydroxytryptamine1 receptors in whole rat brain

In synaptic membranes prepared from whole rat brain, cinanserin produced a relatively shallow inhibition curve of [³H] 5-HT binding with Hill slope of 0.68, as compared with that observed in the [³H]spiperone binding (Hill slope = 0.99). Furthermore, nonlinear regression analysis demonstrated that the former inhibition curve is best fitted by a two-site model. Regional and Scatchard analyses both clearly revealed that, in rat brain, at least two populations of binding sites were labelled by [³H]5-HT; however, high affinity binding sites (k_d = 1.90 nM, Bmax = 0.188 moles/mg protein) could apparently be differentiated from the others by the action of 5 nM cinanserin.     

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

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

Binding and uptake studies with [3H]CP-93, 129, a radiolabeled selective 5-HT1B receptor ligand

3-(1,2,5,6-Tetrahydro-4-pyridyl)-5-pyrrolo[3,2-b]pyridone, CP-93, 129, is a selective agonist ligand for 5-HT_1B receptors. High affinity binding sites of [³H]CP-93, 129 were found in rat whole brain membranes, which showed K_D and B_max values similar to those for 5-HT_1B sites labeled by [³H]5-HT. Uptake of [³H]CP-93, 129 in crude rat synaptosomes was also observed, which was potently inhibited by 5-HT uptake blockers and 5-HT but not by desipramine (NE uptake blocker) or tametraline (NE and DA uptake blocker). Because of this sensitivity to 5-HT uptake inhibitors and the structural similarity of CP-93, 129 to serotonin, [³H]CP-93, 129 uptake probably occurred in 5-HT neurons.     

Desensitization of serotonin-stimulated adenylate cyclase in the liver fluke Fasciola hepatica

Incubation of the intact liver fluke Fasciola hepatica with serotonin (5-HT) resulted in a time-dependent decrease in both 5-HT-stimulated adenylate cyclase activity and specific [³H]LSD binding in the subsequently prepared cell-free fluke particles. In control fluke particles, the activation of adenylate cyclase by 5-HT was biphasic, indicating a high and low affinity form of the 5-HT receptor with half-maximal activation constants (K_A) of 0.35 and 2.8 μM respectively. In contrast, 5-HT activation of desensitized particles occurred through a single set of low affinity sites having a K_A value of 6.3 μM. The maximal level of 5-HT activation of adenylate cyclase was also reduced in the desensitized particles. Lysergic acid diethylamide (LSD)-stimulated adenylate cyclase activity was also less in the desensitized particles. However, unlike with 5-HT, activation by LSD occurred through a single set of sites for both control and desensitized particles. [³H]LSD binding studies showed that the affinity of LSD for the 5-HT receptors in the control and desensitized particles was similar. Thus, the decrease in [³H]LSD binding and serotonin-stimulated adenylate cyclase activity in the desensitized particles appeared to be due to a preferential loss or inactivation of the high affinity form of the 5-HT receptor. A similar time-dependent loss in 5-HT-stimulated adenylate cyclase and in [³H]LSD binding occurred in cell-free fluke particles upon the addition of 5-HT or LSD. These effects were not due to protein denaturation or metabolism of the ligand during the incubation procedure. This cell-free desensitization was reversible and temperature dependent, and was not affected by ATP or other nucleotides.     

Transitional states of the neuronal serotonergic site

Binding of [³H]5-HT was studied wuing purified neuronal membranes isolted frrom horse brain striatum. It is shown that the preincubation of these membranes with an agonist (5-HT, bufotenin) followed by washibng, modifined the binding of [³H]5-GT from a low affinity to a high affinity constant. This effect was blocked by previous pretreatment of the membranes with a sulfhydryl group reagent (N-ethylmaleimide) and was reversed by guanoside 5′-triphosphate.     

Inhibition by arylsulphatase A of Na-independent [3H]-GABA and [3H]-muscimol binding to bovine cerebellar synaptic membranes

Over an identical concentration range, both [³H]-GABA and [³H]-muscimol were bound to bovine cerebellar synaptic membranes through a sodium-independent and high-affinity process. Preincubating the synaptic membrane suspensions with arylsulphatase A inhibited [³H]-GABA and [³H]-muscimol binding. Furthermore, by using 4-nitrocatechol sulphate as a substrate for arylsulphatase A, a direct correlation appeared to exist between the amounts of SO^2?in4 released and the degree of inhibition of [³H]-GABA binding to synaptic membranes. The inhibitory effect of arylsulphatase A was blocked by known inhibitors of arylsulphatase such as AgNO₃. Scatchard analysis of specific [³H]-GABA binding to synaptic membranes showed that arylsulphatase decreased the number of [³H]-GABA binding sites from 2.65 to 1.75 pmol mg proteins without changing appreciably the affinity of [³H]-GABA binding to cerebellar synaptic membranes. These observations are interpreted to indicate that some components of GABA recognition sites may also contain sulpholipid(s) in addition to phospholipid(s).     

Unusual binding of [3H] MDL 72222 in guinea pig hippocampus

[³H] MDL 72222 labeled a non-homogeneous population of sites in guinea pig hippocampal membranes (K_d1 = 1 nM; K_d2 = 60 nM). The binding was not sodium dependent. Competition studies with a variety of characterizing agents showed displacement of [³H] MDL 72222 binding by 5-HT uptake inhibitors. [³H] MDL 72222 binding was not effectively displaced by established 5-HT₃ antagonists. MDL 72222, fluoxetine, fluvoxamine and citalopram competitively inhibited the uptake of [³H] 5-HT into guinea pig hippocampal synaptosomes with K_i values of 1.97, 0.02, 0.023, 0.049 μM, respectively. The results demonstrate that [³H] MDL 72222 labels a non-homogeneous population of sites in guinea pig brain, as well as inhibiting 5-HT uptake into synaptosomal preparations.     

Long-term 5-HT reuptake blockade,but not monoamine oxidase inhibition,decreases the function of terminal 5-HT autoreceptors: an electrophysiological study in the rat brain

Summary 5-HT-containing terminals possess autoreceptors which modulate the release of 5-HT into the synaptic cleft. Tritiated imipramine ([³H]IMI), and more specifically [³H]citalopram and [³H]paroxetine, bind to a site associated with the 5-HT reuptake carrier on the 5-HT terminals. The function of terminal 5-HT autoreceptors is decreased following long-term treatment with the 5-HT reuptake blocker citalopram. The present study was undertaken to determine whether an increased synaptic availability of 5-HT or, the occupation of the [³H]IMI site, were responsible for this modification. Unitary extracellular recordings were obtained from CA₃ dorsal hippocampus pyramidal neurons under chloral hydrate anesthesia in rats treated daily with fluoxetine (10 mg/kg/day × 14 days), a selective 5-HT reuptake blocker, or clorgyline (1 mg/kg/day × 21 days), an inhibitor of type A monoamine oxidase. The function of the terminal 5-HT autoreceptors was assessed by comparing the effectiveness of the electrical stimulation of the ascending 5-HT pathway on the firing activity of hippocampus pyramidal neurons prior to, and following, the administration of methiothepin, an antagonist of the terminal 5-HT autoreceptor, and, by determining the ratio of effectiveness of 0.8 Hz (S1) and 5 Hz (S2) stimulations. Long-term administration of fluoxetine or clorgyline both increased the efficacy of the stimulation of the 5-HT pathway. However, the enhancing effect of methiothepin on the efficacy of the stimulation was attenuated by the fluoxetine, but not by the clorgyline, treatment. The reduction of the function of the terminal 5-HT autoreceptor by the long-term fluoxetine treatment was further indicated by the decreased ratio of effectiveness of the 0.8 and 5 Hz stimulations as compared to that of control rats. To verify that the reuptake blockade per se could not account for the increased synaptic efficacy of 5-HT projections following long-term fluoxetine, the drug was administered acutely to naive rats. It did not increase the efficacy of the stimulation of the 5-HT pathway. Two conclusions are drawn from these results: 1) the increased efficacy of 5-HT synaptic transmission by long-term treatment with antidepressant 5-HT reuptake blockers is not directly due to 5-HT reuptake blockade, but rather, to a reduced function of the terminal 5-HT autoreceptor; 2) the latter phenomenon cannot be ascribed to an increased availability of 5-HT in the synaptic cleft as it was not produced by long-term clorgyline treatment. Hence, these results suggest that long-term occupation of the [³H]IMI site might result in a decreased function of terminal 5-HT autoreceptors.Send offprint requests to Claude de Montigny     

Novel serotonin receptors in Fasciola: Characterization by studies on adenylate cyclase activation and [3H]LSD binding

Serotonin (5-HT) receptors coupled to adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] in the liver fluke Fasciola hepatica have been characterized by adenylate cyclase activation studies and by direct binding studies using $\text{[}{}^3\text{H}\text{]-d-}\text{lysergic acid}$ diethylamide ([³H]LSD) as a radioligand. Inhibition of 5-HT stimulation of adenylate cyclase by a series of 5-HT antagonists revealed a potency order of LSD = 2-bromo-LSD > methiothepin > metergoline = cyproheptadine > methysergide > spiroperidol. [³H]LSD binding to a cell-free fluke particle preparation was rapid, stereospecific, and proportional to protein concentration. Scatchard analysis indicated multiple binding sites which, when resolved into two components, gave for the high affinity site an apparent dissociation constant of 25 nM and a receptor concentration of 160 fmoles/mg protein. The ability of a series of compounds to compete for [³H]LSD binding sites correlated closely with their ability to inhibit 5-HT stimulation of adenylate cyclase. [³H]LSD binding sites were most concentrated in the anterior region of the fluke which was consistent with the higher levels of 5-HT activated adenylate cyclase found in this region. GTP and 5′-guanylyl imidophosphate, a poorly hydrolyzable GTP analog, decreased the affinity of the agonist 5-HT for the binding sites but had little effect on the affinity of the antagonist 2-bromo-LSD. Calcium at concentrations above 300 μM significantly reduced both [³H]LSD binding and 5-HT activation of adenylate cyclase. The results indicate that [³H]LSD can be used to label the 5-HT receptors coupled to adenylate cyclase activity. The pharmacological specificity and other characteristics of the fluke receptors appear to differ from the properties of reported mammalian 5-HT receptors. As a result, serotonin receptors in the flukes represent sites that may be amenable to selective manipulation by new chemotherapeutic agents useful in the treatment of these parasite infections.     

Characterization of serotonin receptors and lack of effect of antidepressant therapy on monoamine functions in various regions of the rabbit brain

The effects of single and long-term administration of the antidepressants imipramine, desimipramine, amitriptyline, zimelidine and maprotiline were studied in the rabbit brain. Special attention was given to the brain serotonin (5-HT) receptors. Our results show that in different areas of the rabbit brain, the binding sites for 5-HT display pharmacological characteristics very similar to those of the 5-HT1 and 5-HT2 receptors described for the rat brain. No significant correlation could be shown between the distribution of either of the receptors and the distribution of serotonergic nerve terminals (as measured by the 5-HT content and the [3H]5-HT accumulation). Addition of antidepressants to rabbit brain slices, in vitro, caused an inhibition of the [3H]5-HT accumulation. The compounds only weakly inhibited the binding of [3H]5-HT and [3H]ketanserin as compared to the inhibition caused by serotonergic agonists and antagonists. The [3H]5-HT accumulation in brain slices was markedly reduced 2 h after a single i.p. injection of imipramine. After a two-week administration of the antidepressants, the specific binding of neither [3H]5-HT nor [3H]ketanserin was significantly altered. The simultaneous determination of monoamine metabolites and of dopamine-beta-hydroxylase in the cerebrospinal fluid of these treated rabbits did not reveal any significant difference from the control animals.     

Serotonin-sensitive adenylate cyclase and [3H]serotonin binding sites in the CNS of the rat—II: Respective regional and subcellular distributions and ontogenetic developments

The 5-HT receptor linked to adenylate cyclase and the high affinity binding site of [³H]5-HT were compared on the basis of their localization and ontogenetic changes in the CNS of the rat. Subcellular fractionation of cerebral tissues from newborn rats showed a good correlation between the distributions of 5-HT-sensitive cyclase and [³H]5-HT binding sites, with the mitochondrial fraction exhibiting the highest specific adenylate cyclase activity and density of binding sites. There was also a good correlation between the regional distributions of the cyclase and the binding in the CNS of newborn rats. However, when the regional distribution of [³H]5-HT binding in newborns was compared to that of adults, no correlation was found, showing that large changes were occurring during ontogenesis. In the cortex and hippocampus, there was little change in the amount of 5-HT-sensitive adenylate cyclase during development whereas [³H]5-HT binding increased approximately 7-fold from birth to adulthood. Only in the striatum was there a positive correlation between the changes in the cyclase and the binding. The injection of kainic acid into the striatum of 10-day-old rats produced large decreases in both DA-and 5-HT-sensitive adenylate cyclase activities. The specific binding of [³H]5-HT was also reduced in the injected striatum but to a smaller extent. Therefore, the 5-HT-sensitive adenylate cyclase but not the [³H]5-HT-high affinity binding site appeared to be preferentially associated with neurons destroyed by kainic acid, i.e. neurons intrinsic to the striatum. The findings that the 5-HT-sensitive adenylate cyclase and the [³H]5-HT binding sites can develop independently and are localized, at least partly, on different types of cells provide additional evidence for the existence of multiple types of 5-HT receptors in the CNS of the rat.     

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.     

Platelet 5-HT uptake sites in depression: three concurrent measures using [3H] imipramine and [3H] paroxetine

Platelet 5-HT uptake sites were measured in 40 depressed patients and 40 controls using [³H] imipramine binding, defined with desmethylimipramine (DMI) and Na⁺ dependence, and [³H] paroxetine binding. In control subjects the B_max of DMI defined [³H] imipramine binding was significantly higher than both Na⁺ dependent [³H] imipramine (by 30%) and [³H] paroxetine binding (by 22%). The B_max of Na⁺ dependent [³H] imipramine and [³H] paroxetine binding did not differ significantly. The K_d of Na⁺ dependent [³H] imipramine binding was significantly lower than the K_d of DMI defined [³H] imipramine binding. The binding of DMI defined and Na⁺ dependent [³H] imipramine and [³H] paroxetine did not differ significantly between depressed patients and controls in the total group, in those depressed patients who had never taken antidepressants or in those depressed patients who had been recently with-drawn from antidepressants. This study provides no support for the view that the number of platelet 5-HT uptake sites are reduced in depression.     

Changes in the rat brain 5-HT1A and 5-HT2 receptors after chronic administration of levoprotiline, (+)-oxaprotiline and other antidepressant drugs.

The effects of levoprotiline (LEV), a (-)-enantiomer of oxaprotiline (OXA) and a clinically effective antidepressant, on the binding parameters of hippocampal 5-HT1A and cortical 5-HT2 receptors of rats were compared with those of (+)-enantiomer of OXA ((+)-OXA), imipramine and mianserin. Both LEV and (+)-OXA displayed in vitro some affinity for 5-HT1A receptors labelled with [3H]-8-OH-DPAT, and for 5-HT2 receptors labelled with [3H]-ketanserin. Repeated administration of LEV, for 14 days led to a marked increase in the number of 5-HT1A binding sites in the rat hippocampus, with no change in the KD values. (+)-OXA, imipramine and mianserin produced similar effects on 5-HT1A binding parameters. The number of 5-HT2 receptors was increased after two weeks of LEV administration, not altered after (+)-OXA, and decreased after imipramine or mianserin. The number of [3H]-ketanserin binding sites was decreased after four weeks of (+)-OXA administration, but not altered after LEV. The specific binding of [3H]-ketanserin in the rat cerebral cortex was decreased after repeated treatment with LEV and (+)-OXA (ex vivo). In competition studies the affinity of serotonin for [3H]-ketanserin binding sites was decreased in LEV- and increased in (+)-OXA-treated rats. The results suggest that LEV similarly to other antidepressants increases the number of 5-HT1A receptors, however without common alteration in 5-HT2 receptor number and function.     

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     

[3H]Rauwolscine: an antagonist radioligand for the cloned human 5-hydroxytryptamine2B (5-HT2B) receptor

In previous reports, [³H]5-HT has been used to characterize the pharmacology of the rat and human 5-HT_2B receptors. 5-HT, the native agonist for the 5-HT_2B receptor, has a limitation in its usefulness as a radioligand since it is difficult to study the agonist low-affinity state of a G protein-coupled receptor using an agonist radioligand. When using [³H]5-HT as a radioligand, rauwolscine was determined to have relatively high affinity for the human receptor (K_i human = 14.3 ± 1.2 nM, compared to K_i rat = 35.8 ± 3.8 nM). Since no known high affinity antagonist was available as a radioligand, these studies were performed to characterize [³H]rauwolscine as a radioligand for the cloned human 5-HT_2B receptor expressed in AV12 cells. When [³H]rauwolscine was initially tested for its usefulness as a radioligand, complex competition curves were obtained. After testing several α₂-adrenergic ligands, it was determined that there was a component of [³H]rauwolscine binding in the AV12 cell that was due to the presence of an endogenous α₂-adrenergic receptor. The α₂-adrenergic ligand efaroxan was found to block [³H]rauwolscine binding to the α₂-adrenergic receptor without significantly affecting binding to the 5-HT_2B receptor and was therefore included in all subsequent studies. In saturation studies at 37° C, [³H]rauwolscine labeled a single population of binding sites, K_d = 3.75 ± 0.23 nM. In simultaneous experiments using identical tissue samples, [³H]rauwolscine labeled 783 ± 10 fmol of 5-HT_2B receptors/mg of protein, as compared to 733 ± 14 fmol of 5-HT_2B receptors/mg of protein for [³H]5-HT binding. At 0° C, where the conditions for [³H]5-HT binding should label mostly the agonist high affinity state of the human 5-HT_2B receptor, [³H]rauwolscine (B_max = 951 ± 136 fmol/ mg), again labeled significantly more receptors than [³H]5-HT (B_max = 615 ± 34 fmol/mg). The affinity of [³H]rauwolscine for the human 5-HT_2B receptor at 0° C did not change, K_d = 4.93 ± 1.27 nM, while that for [³H]5-HT increased greatly (K_d at 37° C = 7.76 ± 1.06 nM; K_d at 0° C = 0.0735 ± 0.0081 nM). When using [³H]rauwolscine as the radioligand, competition curves for antagonist structures modeled to a single binding site, while agonist competition typically resulted in curves that best fit a two site binding model. In addition, many of the compounds with antagonist structures displayed higher affinity for the 5-HT_2B receptor when [³H]rauwolscine was the radioligand. Typically, ∼ 85% of [³H]rauwolscine binding was specific binding. These studies display the usefulness of [³H]rauwolscine as an antagonist radioligand for the cloned human 5-HT_2B receptor. This should provide a good tool for the study of both the agonist high- and low-affinity states of the human cloned 5-HT_2B receptor. Received: 26 June 1997 / Accepted: 30 August 1997     

Serotonin-sensitive adenylate cyclase and [3]serotonin binding sites in the CNS of the rat—I: Kinetic parameters and pharmacological properties

The 5-HT receptor linked to adenylate cyclase and the high affinity binding site for [³H]-5-HT were compared on the basis of their kinetic and pharmacological properties in the CNS of new born rats. Under normal assay conditions, the apparent affinity of 5-HT for its specific binding sites (K_d = 1?2 nM) was much higher than that for the receptor coupled to adenylate cyclase (K_A app = 0.5?1.0 μM). When measured under the conditions of the cyclase assay, the apparent K_d for the binding was increased to 11.9 nM, a value which is still more than 40 times lower than the K_A app characterizing the activation of adenylate cyclase by 5-HT. GTP affected both the binding of [³H]-5-HT and the 5-HT-sensitive adenylate cyclase. Guanyl nucleotides appeared to be essential for the activation of adenylate cyclase by 5-HT as 5-HT was inactive in a preparation of washed membranes unless added in the presence of GTP or GppNHp. In whole homogenates, GTP increased the affinity of 5-HT for the receptor-adenylate cyclase complex (K_A app = 0.33μM in the presence of 10μM GTP). The specific binding of [³H]-5-HT was reduced by GTP and GppNHp but not GMP or ATP. However, the range of concentrations inducing a significant effect (?0.10mM GTP) was far higher than those which increased the 5-HT-induced activation of adenylate cyclase.There was little in common between the pharmacological profiles of the two systems. A group of 5-HT agonists containing a piperazine heterocycle [1- (m-trifluoromethylphenyl) piperazine, quipazine and MK-212] effectively displaced [³H]-5-HT from its binding sites but exerted no action on the 5-HT-sensitive cyclase, affecting neither the basal nor the 5-HT-stimulated cAMP production. Likewise, there was no correlation between the respective potencies of a series of 5-HT antagonists for inhibiting the binding of [³H]-5-HT and the 5-HT-induced cAMP production. These data suggest that the 5-HT receptor linked to adenylate cyclase is not identical with that which is measured by the binding of [³H]-5-HT and, thus, provide evidence for the possible existence of multiple receptors for 5-HT in the rat brain.     

[3H]ICS 205-930 labels 5-HT3 recognition sites in membranes of cat and rabbit vagus nerve and superior cervical ganglion

Summary The binding characteristics of [³H]ICS 205-930, a 5-hydroxytryptamine 5-HT₃ receptor antagonist, were investigated in membranes prepared from cat and rabbit vagus nerve (VN) and superior cervical ganglion (SCG). The autoradiographic localisation of 5-HT₃ recognition sites was also assessed using [³H]ICS 205-930 in slices from cat medulla oblongata, nodose ganglion and vagus nerve.[³H]ICS 205-930 bound to a homogeneous population of high affinity recognition sites in cat VN: B_max = 201 ± 43 fmol/mg protein, pK_D = 9.26 ± 0.17 and SCG: B_max = 291 ± 40 fmol/mg, pK_D = 9.35 ± 0.80 (n = 3). Competition experiments performed in membranes from cat VN and SCG with agonists and antagonists suggested the presence of a homogeneous population of [³H]ICS 205-930 recognition sites. Competition curves were steep and monophasic and were best fitted by a 1 receptor site model. The following rank order of affinity for [³H]ICS 205-930 binding sites was observed with antagonists: SDZ 206-830 = ICS 205-930 > BRL 43694 > SDZ 206–792 > quipazine > MDL 72222 > metoclopramide > mCPP and agonists: 2-methyl-5-HT = 5-HT > phenylbiguanide. A similar profile was observed for a limited series of compounds in rabbit membranes. Drugs acting at 5-HT₁, 5-HT₂ and dopamine receptors (domperidone, spiperone and metergoline) showed very low affinities for [³H]ICS 205-930 recognition sites. The sites labelled with [³H]ICS 205-930 in vagus nerve and superior cervical ganglion of both species displayed the pharmacological profile of a 5-HT₃ receptor. There was a significant correlation between the rank order of affinity of the tested compounds for [³H]ICS 205-930 recognition sites in cat and rabbit membranes and their rank order of affinity for 5-HT₃ receptors from neuroblastoma-glioma NG 108-15 cells. Autoradiographic studies suggest that [³H]ICS 205-930 binding sites are present over and around the nodose ganglion cell somata, along certain fibers of the vagus nerve and in the terminal areas of this nerve in the medullar nucleus of the vagus.The present data demonstrate that [³H]ICS 205-930 identifies 5-HT₃ receptors in preparations of cat and rabbit vagus nerve and superior cervical ganglion.Send offprint requests to D. Hoyer at the above addressThe present results have been presented in part at the Winter Meeting of the British Pharmacological Society, London, December 20–22, 1988 (Hoyer et al. 1989)