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.     

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     

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.     

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.     

Modifications of central 5-hydroxytryptamine binding sites in synaptic membranes from rat brain after long-term administration of tricyclic antidepressants.

The effects of single and long-term administration of tricylic antidepressants, imipramine, desmethylimipramine (desipramine), amitriptyline and dimetacrine on 5-hydroxytrptamine (5-HT) binding sites in synaptic membranes from rat brain were studied. There were two types of specific [³H]-5-HT binding sites; one with high affinity and the other with low affinity. Intraventricular injection of 5,6-dihydroxytryptamine did not modify the binding parameters. In in vitro experiments, 5-HT, tryptamine derivatives and 5-HT antagonists were found to be effective displacers of [³H]-5-HT binding while all antidepressants studied were ineffective in displacing the binding at 10^?6 M. Single i.p. administration of antidepressants did not affect significantly the specific binding. In contrast, administration of antidepressants for 3 weeks produced a significant decrease in the maximal numbers of binding sites, without affecting the dissociation constants of the specific binding. It is suggested that the modification of central 5-HT binding sites caused by long-term administration of tricylclic antidepressants is due to persistent exposure of the binding sites to elevated concentrations of 5-HT, a consequence of the 5-HT uptake inhibition by the antidepressants. These observations could have some implication for the new theory proposed recently that in depression, there is an involvement of synaptic 5-HT with a hypersensitive receptor in the postsynaptic membranes.     

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.     

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.     

[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)     

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.     

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     

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.     

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.     

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.     

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.     

Different effects of serotonin (5-HT) uptake blockers in caudate nucleus and hippocampus of the rabbit: Role of monoamine oxidase in dopaminergic terminals

Slices of rabbit hippocampus or caudate nucleus were incubated with [³H]-5-HT (0.1 µM, 60 min) or with [³H]-DA. In hippocampal tissue, the 5-HT uptake blockers chlorimipramine, fluvoxamine, and 6-nitroquipazine (0.1, 1, 10 µM) reduced the percentage content of [³H]-5-HT in a concentration dependent manner. The degree of inhibition of [³H]-5-HT content produced by the 5-HT uptake inhibitors was not affected by the MAO inhibitors pargyline or amezinium (which by themselves enhanced [³H] loading) or the catecholamine uptake inhibitor nomifensine (which by itself did not affect [³H] loading). In caudate nucleus tissue, however, the [³H]-5-HT accumulation was reduced only at the highest concentration of the 5-HT uptake blockers (10 µM). In the additional presence of the MAO inhibitors or nomifensine (which by themselves increased or diminished, respectively, the [³H] labelling) the 5-HT uptake inhibitors became more potent in reducing the percentage [³H]-5-HT accumulation of caudate nucleus slices. These results indicate (1) that a false labelling of [³H]-5-HT into dopaminergic terminals in the caudate nucleus can be prevented by nomifensine, (2) that the 5-HT uptake blockers seem to accumulate within the dopaminergic terminals, where they may display a MAO inhibitory property. The 5-HT uptake blockers were ineffective on the percentage tritium accumulation of caudate nucleus slices incubated with [³H]-DA, regardless of the presence of pargyline or nomifensine. Tritiated DA and deaminated [³H]-metabolites were separated in the superfusate of [³H]-DA-release experiments in caudate nucleus tissue. In the presence of 6-nitroquipazine the percentage efflux of unmetabolized [³H]-DA was significantly enhanced in a concentration and time dependent manner. In comparison to 6-nitroquipazine, fluvoxamine was less potent in that respect. 6-Nitroquipazine inhibited the electrically evoked [³H]-DA and [³H]-ACh release from caudate nucleus slices in a concentration dependent manner. The effects on [³H]-DA release were abolished in the presence of pargyline. The inhibition of [³H]-ACh release was significantly diminished by the D₂-receptor antagonist domperidone. In conclusion, some 5-HT-related drugs may diminish the release of ACh from caudate nucleus slices via an enhanced dopaminergic transmission due to inhibition of MAO within the dopaminergic terminals.     

α-Flupenthixyl chloride: Binding profile to dopaminergic,serotonergic, adrenergic,and cholinergic neuro-receptors

The binding of the putative affinity ligand α-flupenthixyl chloride (FPT-Cl), a derivative of the neuroleptic α-flupenthixol (FPT), to rat striatal dopamine, α₁-adrenergic, and muscarinic cholinergic recognition sites, as well as to serotonin (5-HT) receptors and to the presynaptic 5-HT transport complex, was examined in vitro. FPT and FPT-Cl were relatively potent at inhibiting the binding of [³H]-flupenthixol and [³H]-spiroperidol in striatal tissue preparations as well as [³H]/spiroperidol and [³H]-WB-4101 in cortex with K_i values in the 10–20 nM range. With the exception of the muscarinic cholinergic sites (³H-quinuclidinyl-benzilate [³H-QNB]), where FPT-Cl was more potent than FPT, the K_i values for FPT-Cl were, in general, slightly greater than those for FPT for other neuro-receptors tested. FPT-Cl was found to be essentially inactive at 5-HT binding sites labeled with [³H]-LSD and at the 5-HT transport complex labeled with [³H]-imipramine, with lC₅₀ values for both exceeding 5μM. It is concluded that the transformation of FPT to FPT-Cl is not severely detrimental to its dopamine receptor binding characteristics, suggesting that in the presence of appropriate antagonists for masking other receptors, FPT-Cl may represent a useful affinity ligand to label dopamine receptors.     

Pindolol-insensitive [3H]-5-hydroxytryptamine binding in the rat hypothalamus; identity with 5-hydroxytryptamine7 receptors.

Pindolol-insensitive [3H]-5-hydroxytryptamine ([3H]-5-HT) binding to rat hypothalamic membranes was pharmacologically and functionally characterized to resolve whether this procedure selectively labels 5-HT7 receptors. Consistent with a previous report, 3 microM and not 100 nM pindolol was required to occupy fully 5-HT1A and 5-HT1B receptors. Remaining [3H]-5-HT binding was saturable (KD, 1.59+/-0.21 nM; Bmax, 53.8+/-3.1 fmol x mg protein(-1)). Displacement of [3H]-5-HT with metergoline and 5-CT revealed shallow Hill slopes (<0.5) but seven other compounds had slopes >0.8 and pKi values and the rank order of affinity were significantly correlated (r = 0.81 and 0.93, respectively) with published [3H]-5-HT binding to rat recombinant 5-HT7 receptors. In the presence of pindolol, 5-HT-enhanced accumulation of [32P]-cyclic AMP was unaffected by the 5-HT4 antagonist RS39604 (0.1 microM) or the 5-ht6 antagonist Ro 04-6790 (1 microM) but significantly attenuated by mesulergine (250 nM), ritanserin (450 nM) or methiothepin (200 nM) which have high affinity for the 5-HT7 receptor. Intracerebroventricular pretreatment with the serotonergic neurotoxin 5,7-dihydroxytryptamine, 5,7-DHT, elevated the [3H]-5-HT Bmax 2 fold, indicating that the hypothalamic 5-HT7 receptor is post-synaptic to 5-HT nerve terminals and regulated by synaptic 5-HT levels. These results suggest that, in the presence of 3 microM pindolol, [3H]-5-HT selectively labels hypothalamic binding sites consistent with functional 5-HT7 receptors.     

Examination of the relationship between the uptake site for 5-hydroxytryptamine and the high affinity binding site for [3H]imipramine. II. The role of sodium ions

Exogenous sodium ions stimulated both the high affinity binding of [3H]5-imipramine to membranes from the cortex of the rat and the high affinity accumulation of [3H]5-hydroxytryptamine (5-HT) into synaptosomes from the cortex of the rat with similar potencies. Imipramine and zimelidine inhibited synaptosomal uptake of [3H]5-HT potently in standard Tris-Krebs medium, but in a low-sodium medium their inhibitory potencies were significantly attenuated. The inhibitory potencies of panuramine and exogenous 5-HT on the uptake of [3H]5-HT were not significantly affected whether the uptake was measured in a normal or low-sodium Tris-Krebs. Imipramine and zimelidine were potent blockers of high affinity binding of [3H]imipramine whereas panuramine and 5-HT only inhibited the binding of [3H]imipramine at concentrations in excess of those required to inhibit the uptake of [3H]5-HT. It is suggested that imipramine inhibits the uptake of 5-HT by a sodium-dependent action probably at the high affinity binding site for [3H]imipramine, whereas panuramine and 5-HT inhibit the uptake of 5-HT by a sodium-independent mechanism at a site other than the binding site for [3H]imipramine.     

Binding studies with the 5-HT1B receptor agonist [3H]CP-96,501 in brain tissues

Binding studies in rat brain membranes have shown that [³H]CP-96,501 is a selective, high affinity radioligand for 5-HT_1B receptors. Additional studies with [³H]CP-96,501 in brain tissues of several other species were undertaken to investigate its utility in detecting this receptor subtype. The presence of 5-HT_1B receptors in brain membranes of the hamster and gerbil was demonstrated with [³H]CP-96,501. This finding was confirmed by [³H]5-HT binding experiments that also indicated the presence of other 5-HT₁ subtypes (possibly 5-HT_1D, 5-HT_1E) in these species as well as the rat. Negligible specific binding of [³H]CP-96,501 was found in membranes of guinea pig brain, dog hypothalamus, bovine caudate, pig choroid plexus, and several human brain tissues, consistent with the reported absence of the 5-HT_1B receptor subtype in these species. Autoradiographic examination of rat brain sections labeled by [³H]CP-96,501 showed a dense localization in areas known to be enriched in 5-HT_1B receptors (e.g., globus pallidus, substantia nigra, superior colliculus, subiculum). On the other hand, brain sections of dog and guinea pig appeared to show no specific binding of [³H]CP-96,501 by autoradiography, in agreement with the lack of brain 5-HT_1B receptors in these tissues. © 1992 Wiley-Liss, Inc.