3H]zacopride: ligand for the identification of 5-HT3 recognition sites

[3H]Zacopride displayed saturable binding to homogenates of the rat entorhinal cortex as measured by the inclusion of the 5-HT3 receptor antagonist BRL43694 in the incubation media. Scatchard analysis indicated a single high affinity binding site (KD 0.76 +/- 0.08 nM, Bmax 77.5 +/- 6.5 fmol (mg protein)-1) with a Hill slope close to unity. Other 5-HT3 receptor antagonists (zacopride, ICS 205-930, GR38032F, GR65630, metoclopramide and cocaine) also competed for the binding site displacing 60% of the total [3H]zacopride binding. 5-HT and 2-methyl-5-HT also were competitive antagonists for [3H]zacopride binding whereas 5-HT1/5-HT2 agonists and antagonists, and agents acting on other neurotransmitter receptors had Ki values greater than 10(-5) M. It is concluded that [3H]zacopride may prove a useful ligand for the study of 5-HT3 recognition sites.     

Antagonism of [3H]zacopride binding to 5-HT3 recognition sites by its (R) and (S) enantiomers

[3H]Zacopride exhibits high affinity (Kd less than or equal to 1 nM) for 5-HT3 binding sites (inhibited by ICS 205-930) in rabbit intestinal muscularis and vagus nerve, human jejunum, rat intestinal muscularis and rat brain cortex. Its binding was inhibited by several 5-HT3 antagonists that displayed similar rank orders of potency in the tissues examined. Zacopride's (S) enantiomer was significantly more potent than its (R) enantiomer (21- to 42-fold in rabbit and human; 8- to 12-fold in rat) as an inhibitor of [3H]zacopride binding. These studies indicate that the utility of [3H]zacopride as a high affinity 5-HT3 ligand resides with the (S) enantiomer.     

Evidence that the atypical 5-HT3 receptor ligand, [3H]-BRL46470, labels additional 5-HT3 binding sites compared to [3H]-granisetron.

1. The radioligand binding characteristics of the 3H-derivative of the novel 5-HT3 receptor antagonist BRL46470 were investigated and directly compared to the well characterized 5-HT3 receptor radioligand [3H]-granisetron, in tissue homogenates prepared from rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen. 2. In rat cerebral cortex/hippocampus, rat ileum, NG108-15 cell and HEK-5-HT3As cell homogenates, [3H]-BRL46470 bound with high affinity (Kd (nM): 1.57 +/- 0.18, 2.49 +/- 0.30, 1.84 +/- 0.27, 3.46 +/- 0.36, respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 102 +/- 16, 44 +/- 4, 968 +/- 32 and 2055 +/- 105, respectively; mean +/- s.e. mean, n = 3-4) but failed to display specific binding in human putamen homogenates. 3. In the same homogenates of rat cerebral cortex/hippocampus, rat ileum, NG108-15 cells, HEK-5-HT3As cells and human putamen as used for the [3H]-BRL46470 studies, [3H]-granisetron also bound with high affinity (Kd (nM): 1.55 +/- 0.61, 2.31 +/- 0.44, 1.89 +/- 0.36, 2.03 +/- 0.42 and 6.46 +/- 2.58 respectively; mean +/- s.e. mean, n = 3-4) to an apparently homogeneous saturable population of sites (Bmax (fmol mg-1 protein): 39 +/- 4, 20 +/- 2, 521 +/- 47, 870 +/- 69 and 18 +/- 2, respectively; mean +/- s.e. mean, n = 3-4).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

[3H]-5-carboxamidotryptamine labels 5-HT1D binding sites in bovine substantia nigra.

1. [3H]-5-hydroxytryptamine (5-HT) has been shown to radiolabel at least five types of 5-HT binding sites in mammalian brain tissue, 5-HT1A, 5-HT1C, 5-HT1D and 5-HT1D and 5-HT1E (Frazer et al., 1990). Selective masking of 5-HT1A and 5-HT1C receptors, has uncovered binding sites which display both high (5-HT1D) and low (5-HT1E) affinity for 5-carboxamidotryptamine (5-CT). By utilizing [3H]-5-CT we have eliminated a portion of the complex binding (5-HT1E) seen when [3H]-5-HT is used as a radioligand. 2. [3H]-5-CT binding to 5-HT1D sites in bovine substantia nigra was rapid, reversible and saturable, displaying high affinity (Kd = 0.38 +/- 0.04 nM) and low non-specific binding (> 90% specific binding). 3. In bovine substantia nigra, [3H]-5-CT labelled an equivalent number of binding sites to [3H]-5-CT (403 +/- 18 and 362 +/- 20 fmol mg-1 protein, respectively) and binding was sensitive to guanine nucleotides. 4. A linear correlation (r2 = 0.99) existed between the potency of compounds to displace [3H]-5-HT and [3H]-5-CT in bovine substantia nigra. 5. Therefore, [3H]-5-CT is a novel radioligand for the examination of 5-HT1-like binding sites, which under proper experimental conditions can be used to radiolabel selectively 5-HT-1D-like binding sites.     

The (S)-isomer of [3H]zacopride labels 5-HT3 receptors with high affinity in rat brain

The distribution of 5-HT3 receptor sites was examined in rat brain by autoradiography using 3H-enantiomers of zacopride. The (S)-3H-isomer labelled high densities of binding sites in the hippocampus, amygdala and cortex. The (R)-3H-isomer labelled considerably fewer sites than the (S)-isomer in nuclei of the lower medulla and did not exhibit any specific binding in the forebrain. These differences confirm that the (S)-isomer is specific for 5-HT3 binding sites and that it has a higher affinity than the (R)-isomer at these sites. These results are not consistent with the notion that 5-HT3 antagonist activity explains the anxiolytic effects of zacopride.     

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.     

The selective labelling of central 5-HT1A receptor binding sites by [3H]5-methoxy-3-(di-n-propylamino)chroman

Investigations on the pharmacological properties of a series of chroman derivatives indicated that 5-methoxy-3-(di-n-propylamino)chroman (5-MeO-DPAC) acts in the nM range on 5-HT1A sites but recognizes very poorly other 5-HT sites and D2 sites in rat brain membranes. As expected from these observations, the tritiated derivative [3H]5-MeO-DPAC bound to a single class of specific sites which exhibited the same pharmacological properties as 5-HT1A sites labelled by [3H]8-OH-DPAT in hippocampal and cortical membranes. In contrast to [3H]8-OH-DPAT, [3H]5-MeO-DPAC did not bind to presynaptic striatal sites (possibly associated with 5-HT reuptake in serotoninergic terminals), which indicated that this new chroman derivative was even more selective than the [3H]tetralin ligand for the in vitro labelling of 5-HT1A sites. Comparison of the chemical structures of 5-MeO-DPAC and other 5-HT1A ligands suggests that electronic enrichment due to isosteric O-substitution in the chroman derivative may play an important role in the highly selective recognition of the 5-HT1A receptor by this drug.     

Characterization of binding sites for [3H]spiroperidol

Experiments were designed to investigate the biochemical properties of binding sites for [3H]spiroperidol ([3H]SPD) solubilized from canine caudate and to define the effect of detergent on the binding of the radioligand. Extraction of canine caudate with 0.75-1.0% digitonin was found to generate the maximum yield of binding sites for [3H]SPD while minimizing extraction of membrane proteins. Although binding sites were solubilized with 1.0% digitonin, a 10-fold reduction in detergent concentration was necessary to achieve maximal binding of [3H]SPD. The rank order of affinity for agonists and antagonists was consistent with the pharmacologic properties of the D2 subtype of the dopamine receptor. However, the binding of antagonists was found to be complex. Studies with some preparations of pooled canine caudate resulted in competition curves for the D2-selective antagonists domperidone and sulpiride that best fit a single-site model. Other preparations exhibited biphasic inhibition curves with these antagonists. The class of binding sites for [3H]SPD with low affinity for D2-selective antagonists constituted as much as 30-40% of the binding sites. Enrichment of solubilized binding sites for [3H]SPD was achieved by size exclusion HPLC followed by adsorption to DEAE-Sephadex and elution with buffer of increasing ionic strength. Enrichment of binding sites was accompanied by a decrease in the affinity of solubilized sites for [3H]SPD.     

Affinity modulation of [3H]imipramine, [3H]paroxetine and [3H]citalopram binding to the 5-HT transporter from brain and platelets.

The dissociations of [3H]imipramine, [3H]paroxetine and [3H]citalopram from the 5-HT (serotonin 5-hydroxytryptamine) transporter were found to be markedly influenced by several drugs, although concentrations in the microM range were needed. Most of these drugs attenuated the dissociation rate, i.e. increased the affinity between the ligand and the binding site. A few increased the dissociation rate however. The binding of drugs to the affinity-modulating site was specific, although of low affinity and probably changing the conformation of the high-affinity binding site, thereby changing the fit between the ligand and the interacting amino acid side-chains. Although the drugs usually affected the dissociation rates of the three ligands in the same manner, there were some which had different effects on [3H]imipramine, [3H]paroxetine and [3H]citalopram. For example, 5-HT markedly attenuated the dissociation of [3H]imipramine, had a moderate effect on [3H]paroxetine and very little effect on [3H]citalopram dissociation. This indicates that the three ligands are bound to different domains on the 5-HT transporter. [3H]Citalopram dissociation from human brain and rat brain were differently affected by several drugs. Indalpine augmented the dissociation rate of the [3H]citalopram 5-HT transport complex in human brain but attenuated it in rat brain, thus revealing a species difference of the 5-HT transporter.     

Characteristics of 5-HT3 binding sites in NG108-15, NCB-20 neuroblastoma cells and rat cerebral cortex using [3H]-quipazine and [3H]-GR65630 binding.

1. The biochemical and pharmacological properties of 5-HT3 receptors in homogenates of NG108-15 and NCB-20 neuroblastoma cells and rat cerebral cortex have been ascertained by the use of [3H]-quipazine and [3H]-GR65630 binding. 2. In NG108-15 and NCB-20 cell homogenates, [3H]-quipazine bound to a single class of high affinity (NG108-15: Kd = 6.2 +/- 1.1 nM, n = 4; NCB-20: Kd = 3.0 +/- 0.9 nM, n = 4; means +/- s.e.means) saturable (NG108-15: Bmax = 1340 +/- 220 fmol mg-1 protein; NCB-20: Bmax = 2300 +/- 200 fmol mg-1 protein) binding sites. In rat cortical homogenates, [3H]-quipazine bound to two populations of binding sites in the absence of the 5-hydroxytryptamine (5-HT) uptake inhibitor, paroxetine (Kd1 = 1.6 +/- 0.5 nM, Bmax1 = 75 +/- 14 fmol mg-1 protein; Kd2 = 500 +/- 300 nM, Bmax2 = 1840 +/- 1040 fmol mg-1 protein, n = 3), and to a single class of high affinity binding sites (Kd = 2.0 +/- 0.5 nM, n = 3; Bmax = 73 +/- 6 fmol mg-1 protein) in the presence of paroxetine. The high affinity (nanomolar) component probably represented 5-HT3 binding sites and the low affinity component represented 5-HT uptake sites. 3. [3H]-paroxetine bound with high affinity (Kd = 0.02 +/- 0.003 nM, n = 3) to a site in rat cortical homogenates in a saturable (Bmax = 323 +/- 45 fmol mg-1 protein, n = 3) and reversible manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereoselective blockade of central [3H]5-hydroxytryptamine binding to multiple sites (5-HT1A, 5-HT1B and 5-HT1C) by mianserin and propranolol

The interaction of the enantiomers of mianserin and propranolol with the binding of [3H]5-hydroxytryptamine ([3H]5-HT) to the 5-HT1A, 5-HT1B and 5-HT1C sites, and with the binding of [3H]ketanserin to the 5-HT2 site, has been evaluated in rat brain membranes. A stereoselective interaction at the 5-HT1A, 5-HT1B and 5-HT1C sites was demonstrated for both compounds, with (+)-mianserin being a more potent displacer than (-)-mianserin and (-)-propranolol being more potent than (+)-propranolol. Only mianserin interacted in a stereoselective manner with the 5-HT2 site, (+)-mianserin being the more potent isomer. The stereoselective association of mianserin and propranolol with the 5-HT1A, 5-HT1B and 5-HT1C sites may prove useful in the characterization of these sites.     

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

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

Characterization of [3H]tryptamine binding sites in brain

[3H]Tryptamine binds with high affinity to sites on rat brain membranes. The sites have the characteristics of tryptamine receptor recognition sites. These sites are widely distributed among rat brain regions with the highest density occurring in the cerebral cortex, striatum and hippocampus. The site is also found in human cerebral cortex. The binding site is localized mainly to the synaptosomal fraction. Drug competition studies indicate that the [3H]tryptamine binding site is distinct from serotonin receptors. Drugs that are potent inhibitors of [3H]tryptamine binding include tetrahydro-beta-carboline, quipazine, phenylethylamine, amphetamine, p-chloroamphetamine and methamphetamine.     

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     

Characterisation of the 5-HT receptor binding profile of eletriptan and kinetics of [3H]eletriptan binding at human 5-HT1B and 5-HT1D receptors

The affinity of eletriptan ((R)-3-(1-methyl-2-pyrrolidinylmethyl)-5-[2-(phenylsulphonyl )ethyl]-1H-indole) for a range of 5-HT receptors was compared to values obtained for other 5-HT1B/1D receptor agonists known to be effective in the treatment of migraine. Eletriptan, like sumatriptan, zolmitriptan, naratriptan and rizatriptan had highest affinity for the human 5-HT1B, 5-HT1D and putative 5-ht1f receptor. Kinetic studies comparing the binding of [3H]eletriptan and [3H]sumatriptan to the human recombinant 5-HT1B and 5-HT1D receptors expressed in HeLa cells revealed that both radioligands bound with high specificity (>90%) and reached equilibrium within 10-15 min. However, [3H]eletriptan had over 6-fold higher affinity than [3H]sumatriptan at the 5-HT1D receptor (K(D)): 0.92 and 6.58 nM, respectively) and over 3-fold higher affinity than [3H]sumatriptan at the 5-HT1B receptor (K(D): 3.14 and 11.07 nM, respectively). Association and dissociation rates for both radioligands could only be accurately determined at the 5-HT1D receptor and then only at 4 degrees C. At this temperature, [3H]eletriptan had a significantly (P<0.05) faster association rate (K(on) 0.249 min(-1) nM(-1)) than [3H]sumatriptan (K(on) 0.024 min(-1) nM(-1)) and a significantly (P<0.05) slower off-rate (K(off) 0.027 min(-1) compared to 0.037 min(-1) for [3H]sumatriptan). These data indicate that eletriptan is a potent ligand at the human 5-HT1B, 5-HT1D, and 5-ht1f receptors and are consistent with its potent vasoconstrictor activity and use as a drug for the acute treatment of migraine headache.