[3H]Citalopram Binding to Brain and Platelet Membranes of Human and Rat

Citalopram, a selective serotonin (5-HT) uptake inhibitor with antidepressant properties, was found to bind with high affinity to the 5-HT transporter from human neuronal and platelet membranes. At 20 degrees C, KD was about 1.5 nM in both tissues. [3H]Citalopram bound to rat neuronal membranes with higher affinity than to human neuronal and platelet membranes; at 20 degrees C KD was about 0.7 nM. The Bmax value for the binding of [3H]citalopram to platelet membranes was close to that found using the 5-HT uptake inhibitors [3H]imipramine and [3H]paroxetine, suggesting that all three 5-HT uptake inhibitors bind to the 5-HT transporter. The dissociation rate of [3H]citalopram increased twofold with each 4-5 degree C increase in temperature in both human and rat membranes, although at any given temperature, the dissociation rate was about four times faster in the human neuronal and platelet membranes than in rat neuronal membranes.     

Characterization of [3H]Paroxetine Binding in Rat Brain

The binding of the 5-hydroxytryptamine (5-HT, serotonin) uptake inhibitor [3H]paroxetine to rat cortical homogenates has been characterized. The effect of tissue concentration was examined and, with 0.75 mg wet weight tissue/ml in a total volume of 1,600 microliter, the binding was optimized with an apparent dissociation constant (KD) of 0.03-0.05 nM. Competition experiments with 5-HT, citalopram, norzimeldine, and desipramine revealed a high (90%) proportion of displaceable binding that fitted a single-site binding model. Fluoxetine and imipramine revealed, in addition to a high-affinity (nanomolar) site, also a low-affinity (micromolar) site representing approximately 10% of the displaceable binding. The specificity of the [3H]paroxetine binding was emphasized by the fact that 5-HT was the only active neurotransmitter bound and that the serotonin S1 and S2 antagonist methysergide was without effect on the binding. Both 5-HT- and fluoxetine-sensitive [3H]paroxetine binding was completely abolished after protease treatment, suggesting that the binding site is of protein nature. Saturation studies with 5-HT (100 microM) sensitive [3H]paroxetine binding were also consistent with a single-site binding model, and the binding was competitively inhibited by 5-HT and imipramine. The number of binding sites (Bmax) for 5-HT-sensitive [3H]paroxetine and [3H]imipramine binding was the same, indicating that the radioligands bind to the same sites. Lesion experiments with p-chloroamphetamine resulted in a binding in frontal and parietal cortices becoming undetectable and a greater than 60% reduction in the striatum and hypothalamus, indicating a selective localization on 5-HT terminals. Together these findings suggest that [3H]paroxetine specifically and selectively labels the substrate recognition site for 5-HT uptake in rat brain.     

Characterization of Serotonin Transporter in Goldfish Retina by the Binding of [3H]Paroxetine and the Uptake of [3H]Serotonin: Modulation by Light

Abstract: The serotonin (5-HT) uptake system of goldfish retina was evaluated by the binding of [³H]paroxetine to membrane preparations and the uptake of [³H]5-HT into isolated cells from goldfish retina. The order of potency of inhibitors of [³H]paroxetine binding was imipramine > 5-methoxy- N,N -dimethyltryptamine > desipramine > fluoxetine > citalopram > 5-HT. The saturation experiments indicated a high-affinity binding site, and positive cooperativity with Hill coefficient higher than unity. The association reached equilibrium at about one hour of incubation and was efficiently displaced by imipramine. The equilibrium dissociation constants calculated by the antilog of the log concentration of ligand giving 50% of occupation, and by the ratio of dissociation/association constants, were similar: 5.84 and 2.34 n M , respectively. The binding was not significantly reduced by decreasing the temperature of incubation and was sodium dependent. The lesion with 5,7-dihydroxytryptamine reduced the binding to 60%. The uptake of [³H]5-HT into isolated cells also showed positive cooperativity. The order of potency of inhibitors was similar to the one obtained for the binding of [³H]paroxetine. Darkness increased the uptake of 5-HT. The allosteric regulation of the 5-HT transporter and the modulation by light could be related to the physiological role of the monoamine, as a neurotransmitter and as a precursor of melatonin synthesis in the retina.     

Allosteric Interaction Between the Site Labeled by [3H]Imipramine and the Serotonin Transporter in Human Platelets

The nature of interaction between the site labeled by [3H]imipramine (IMI) and the 5-hydroxytryptamine (5-HT, serotonin) transporter in human platelets was examined. The sulfhydryl characterizing agent N-ethylmaleimide (NEM) differentially affected [3H]5-HT uptake and [3H]IMI binding in human platelet preparations. Concentrations of NEM that completely abolished [3H]5-HT uptake only minimally reduced [3H]IMI binding. Examining the effect of IMI on the kinetics of human platelet [3H]5-HT uptake revealed significant reductions in maximal velocity (Vmax) without altering affinity (Km). IC50 values for selected uptake blockers on [3H]IMI binding and [3H]5-HT uptake were determined. IC50 values of these compounds for uptake and binding revealed that agents such as IMI, chlorpromazine, amitriptyline, and nisoxetine were preferential inhibitors of [3H]IMI binding whereas fluoxetine, CL 216, 303, pyrilamine, and bicifadine were preferential [3H]5-HT uptake blockers. 5-HT was a weak displacer of [3H]IMI binding (IC25 = 3.0 microM) and exhibited a rather low Hill coefficient (nH app = 0.46). Results reported herein support the notion of an allosteric interaction between the [3H]IMI binding site and the 5-HT transporter complex in human platelets.     

In Vivo Labeling of Serotonin Uptake Sites with [3H]Paroxetine

Previous work has shown that [3H]paroxetine is a potent and selective in vitro label for serotonin uptake sites in the mammalian brain. In the present study, [3H]paroxetine was tested in mice as an in vivo label for serotonin uptake sites. Maximum tritium concentration in the whole brain (1.4% of the intravenous dose) was reached 1 h after injection into a tail vein. Distribution of the tracer at 3 h after injection followed the distribution of serotonin uptake sites known from previous in vitro binding studies (r = 0.85). The areas of highest [3H]paroxetine concentration, in decreasing order, were: hypothalamus greater than frontal cortex greater than olfactory tubercles greater than thalamus greater than upper colliculi greater than brainstem greater than hippocampus greater than striatum greater than cerebellum. Preinjection of carrier paroxetine (1 mg/kg) significantly decreased [3H]paroxetine concentration in all areas except in the cerebellum, which is known to contain a relatively low number of specific binding sites. Kinetic studies showed highest specific [3H]paroxetine binding (tissue minus cerebellum) at 2 h after injection and slow clearance of activity thereafter (half-time of dissociation from the hypothalamus, 215 min). The specificity of in vivo [3H]paroxetine binding was studied by preinjecting monoamine uptake blockers or receptor antagonists 5 min before administration of [3H]paroxetine. Serotonergic or muscarinic cholinergic receptor antagonists and dopamine or norepinephrine uptake blockers did not reduce the in vivo binding of [3H]paroxetine. In contrast, there was an excellent correlation (r = 0.99) between the in vivo inhibitory potencies of serotonin uptake blockers in this study and previously published in vitro data on inhibition of [3H] serotonin uptake in brain synaptosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative Autoradiography of [3H]Indalpine Binding Sites in the Rat Brain: I. Pharmacological Characterization

The binding of [3H]indalpine (4-[2-(3-indolyl)]ethyl piperidine) to slide-mounted sections of rat brain has been characterized. This 5-hydroxytryptamine (5-HT) uptake blocker binds to sections with high affinity (KD approximately 1 nM). The binding is saturable, and can be displaced by the addition of clomipramine (1 microM). Other drugs inhibiting the uptake of 5-HT also have the capacity to inhibit the binding of [3H]indalpine. A significant correlation (r = 0.86) was found between the capacity of these compounds to inhibit the uptake of 5-HT and their potencies as inhibitors of [3H]indalpine binding. Binding was Na+ - and Cl- -dependent and was inhibited competitively by 5-HT. Furthermore, electrolytic lesions of the dorsal raphe or medial forebrain bundle, which cause a degeneration of 5-HT cell bodies and fibers, respectively, resulted in a 30-40% reduction in the binding of [3H]indalpine. [3H]Indalpine binds to the 5-HT uptake recognition sites in a different manner from imipramine-like compounds.     

Subdivision of Mouse Brain [3H]Imipramine Binding Based on Ion Dependence and Serotonin Sensitivity

The specific binding of [3H]imipramine to mouse brain membranes in an assay containing 120 mM NaCl and 5 mM KCl was similar in regional distribution and pharmacological specificity to that reported previously in rat and human brain. However, the absence of ions decreased the density of the specific binding of [3H]imipramine and did not affect the equilibrium dissociation constant. Sodium was the only cation, and halides were the only anions tested that enhanced the specific binding of [3H]imipramine. Chloride did not increase the density of binding in the absence of sodium. The ion-sensitive binding of [3H]imipramine was regionally dependent and was highly correlated with the uptake of 5-hydroxytryptamine (5-HT, serotonin) into synaptosomes from brain regions. 5-HT did not inhibit the binding of [3H]imipramine in the absence of ions. Antidepressants inhibited binding in the absence and presence of ions, but in the presence of ions inhibition curves were shifted to the left and the apparent complexity of inhibition was increased. Quantitative analysis of the inhibition of [3H]imipramine binding by antidepressants conducted in the presence of ions was consistent with two binding sites. Lesion of the serotonergic input to the cerebral cortex by 5,7-dihydroxytryptamine suggested that both the 5-HT-sensitive and ion-sensitive binding of [3H]imipramine were associated with serotonergic nerve terminals. [3H]Imipramine binding displaced by desipramine, but insensitive to 5-HT and ions, was not affected by the lesion. Thus, the binding of [3H]imipramine that is displaced by desipramine, the most common assay for [3H]imipramine binding, includes a component that is not associated with brain serotonergic nerve terminals and 5-HT uptake, and, in addition, a separable component that is highly correlated with serotonergic function. These data have important implications for studies of serotonergic neurons and for the interpretation of imipramine binding data.     

5-Methoxytryptoline, a Competitive Endocoid Acting at [3H]Imipramine Recognition Sites in Human Platelets

5-Methoxytryptoline potently inhibits [3H]imipramine binding to membranes from the cerebral cortex and platelets. Since 5-methoxytryptoline, which appears to occur endogenously with particularly high levels in the human pineal gland, also inhibits 5-hydroxytryptamine (5-HT, serotonin) uptake, it should be considered as a putative endogenous ligand modulating 5-HT transport. As the 5-HT transporter complex comprises the imipramine and the substrate recognition sites, which interact allosterically, it was essential to define the mechanism of inhibition of [3H]imipramine binding by 5-methoxytryptoline. Human platelets show an active and saturable uptake of 5-HT and tryptamine. The uptake of both substrates appears to be mediated by the same carrier and it is inhibited by 5-methoxytryptoline at submicromolar concentrations. 5-HT and tryptamine inhibit [3H]imipramine binding in human platelets with a Hill slope for inhibition close to unity and IC50 values of 3,265 and 3,475 nM, respectively. This inhibition is, however, not competitive because both 5-HT and tryptamine significantly decrease the rate of [3H]imipramine-receptor dissociation. Although 5-methoxytryptoline potently inhibits [3H]imipramine binding (IC50 = 44 nM) in human platelets with a Hill slope of unity, it does not affect the receptor-ligand dissociation rate of [3H]imipramine even at concentrations up to 100 microM. The present experiments show that 5-methoxytryptoline, in spite of its chemical similarity to the indoleamine transporter substrates, interacts with the imipramine receptor through a mechanism of competitive inhibition. This conclusion is supported by a selective effect of 5-methoxytryptoline on the Kd of [3H]imipramine binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ascorbate Modulates 5-[3H]Hydroxytryptamine Binding to Central 5-HT3 Sites in Bovine Frontal Cortex

Ascorbate is present in millimolar concentrations in mammalian brain and can be released from cellular stores by membrane depolarization. We report here that physiologically relevant concentrations of ascorbate modulate 5-[3H]hydroxytryptamine ([3H]5-HT) binding to bovine frontal cortex membranes. Under conditions where [3H]5-HT binding is reversible and saturable, ascorbate causes a concentration-dependent increase in the affinity of [3H]5-HT for central 5-HT3 binding sites. At pH 7.4, increasing ascorbate from 0 to 5.7 mM changes the equilibrium affinity constant (KD) of binding to 5-HT3 sites from 125 nM to 30 nM, without affecting binding site number. These ascorbate-induced effects are pH dependent. At pH 7.1 binding to central 5-HT3 sites is essentially eliminated in the presence of ascorbate. These studies suggest that ascorbate and hydrogen ion concentration interactions may modulate serotonergic function.     

Characterization of Two [3H]Glutamate Binding Sites in Rat Hippocampal Membranes

The specific binding of L-[3H]glutamate was investigated in the presence and the absence of sodium ions in freshly prepared membranes from rat hippocampus. Sodium ions were found to have a biphasic effect; low concentrations induced a marked inhibition of the binding (in the range 0.5-5.0 mM), whereas higher concentrations resulted in a dose-dependent stimulation of binding (in the range 10-150 mM). These results permit the discrimination of two binding sites in hippocampal membranes. Both Na+-independent and Na+-dependent binding sites were saturable, exhibiting dissociation constants at 30 degrees C of 750 nM and 2.4 microM, respectively, with Hill coefficients not significantly different from unity, and maximal number of sites of 6.5 and 75 pmol/mg protein, respectively. [3H]Glutamate binding to both sites reached equilibrium between 5 and 10 min and was reversible. The relative potencies of a wide range of compounds, with known pharmacological activities, to inhibit [3H]glutamate binding were very different for the Na+-independent and Na+-dependent binding and suggested that the former sites were related to post-synaptic glutamate receptors, whereas the latter were related to high-affinity uptake sites. This conclusion was also supported by the considerable variation in the regional distribution of the Na+-dependent binding site, which paralleled that of the high-affinity glutamate uptake; the Na+-independent binding exhibited less regional variation.     

Artifactual High-Affinity and Saturable Binding of [3H]5-Hydroxytryptamine Induced by Radioligand Oxidation

The binding of [3H]5-hydroxytryptamine (5-HT, serotonin) to cerebellar membranes was examined after preincubation of [3H]5-HT in the presence or absence of ascorbate. The tissue preparation was identical in all experiments and consisted of rat cerebellar homogenates in Tris-HCl buffer with 0.1% ascorbate. Cerebellar membranes were used because of their low density of 5-HT1 binding sites. In the presence of ascorbate during a 4-h preincubation period, minimal specific binding of 2 nM [3H]5-HT is detected. Similar results are obtained with equimolar concentrations of other antioxidants (butylated hydroxytoluene, sodium dithionite, and sodium metabisulfite). Apparent specific binding increases 14-fold following a 4-h preincubation of [3H]5-HT in the absence of ascorbate. The increase in apparent specific [3H]5-HT binding is time-dependent and plateaus after 4-6 h of preincubation. When ascorbate is present during the 4-h preincubation, Scatchard analysis of [3H]5-HT binding reveals a KD value of 3.0 +/- 0.3 nM and a Bmax value of 1.9 +/- 0.2 pmol/g tissue. When ascorbate is absent during the preincubation, the KD is essentially unchanged at 3.6 +/- 0.1 nM but the Bmax is significantly increased to 36.5 +/- 7 pmol/g tissue. Drug competition studies reveal that the apparent specific "[3H]5-HT binding" in the absence of ascorbate appears to be displaced by nanomolar concentrations of hydroxylated tryptamines (5-HT, bufotenine) but not by nonhydroxylated tryptamines (5-methoxytryptamine, tryptamine). HPLC analysis demonstrates that [3H]5-HT is essentially destroyed by a 4-h incubation at 22 degrees C in the absence of ascorbate.(ABSTRACT TRUNCATED AT 250 WORDS)     

[3H]Paroxetine Binding Is Altered in the Hippocampus but Not the Frontal Cortex or Caudate Nucleus from Subjects with Schizophrenia

Abstract: [³H]Paroxetine binding to particulate membrane from tissue, obtained at autopsy, from the hippocampus, frontal cortex, and caudate nucleus from subjects who had or had not had schizophrenia was measured. The density of [³H]paroxetine binding to membranes from subjects who had or had not had schizophrenia did not differ. Similarly, the affinity of [³H]paroxetine binding in the frontal cortex and caudate nucleus was not different. By contrast, the affinity of [³H]paroxetine binding to hippocampal membrane from subjects who had schizophrenia was significantly lower than the affinity of binding for the nonschizophrenic subjects (0.40 ± 0.06 vs. 0.26 ± 0.02; p < 0.05). As [³H]paroxetine binds to the serotonin transporter, these data suggest that the serotonin transporter is altered in the hippocampus in subjects with schizophrenia.     

Dissociation Kinetics of [3H]Paroxetine Binding to Rat Brain Consistent with a Single-Site Model of the Antidepressant Binding/5-Hydroxytryptamine Uptake Site

The effects of 5-hydroxytryptamine (5-HT) and 5-HT uptake inhibitors on the dissociation of [3H]paroxetine from rat brain membrane binding sites have been investigated. The dissociation induced by 5-HT (100 microM), paroxetine (0.15 microM), clomipramine (1 microM), citalopram (1 microM), imipramine (1 microM), or norzimeldine (1 microM) was consistent with first-order dissociation kinetics with half-life values of dissociation (t1/2) between 130 and 140 min. The dissociation induced by the combination of 5-HT (100 microM) with either citalopram (1 microM) or imipramine (1 microM) was not different from that initiated by either agent alone. These dissociation data, which are at variance with previous data on the 5-HT transporter labeled with [3H]imipramine, support a single-site model of the antidepressant binding/5-HT uptake site.     

Modulation of [3H]Paroxetine Binding to the 5-Hydroxytryptamine Uptake Site in an Animal Model of Depression

The effects of learned helplessness on the 5-hydroxytryptamine (5-HT) uptake site were studied in rats using [3H]paroxetine binding. This ligand was chosen because it was demonstrated to label directly the 5-HT uptake site whereas the [3H]imipramine binding site has been demonstrated to be heterogeneous in nature. Moreover, [3H]imipramine appears to bind to a presynaptic recognition site different from the uptake site. Exposure to uncontrollable shock training and testing resulted in an overall increase in [3H]paroxetine binding in all the groups studied [nonhelpless (NLH), learned helpless (LH), spontaneously helpless (SPLH)] as compared to naive controls (NC). However, the increase in [3H]paroxetine binding was significantly higher in the LH and SPLH groups. The maximum number of [3H]paroxetine binding sites in the rat hippocampus was increased significantly in learned helpless rats (LH and SPLH) at day 4 and day 30 after the shock escape test as compared to NC and NLH rats. By contrast, in the rat hypothalamus the maximum number of [3H]paroxetine binding sites was reduced significantly in the LH rats as compared to naive controls and NLH rats during the same time course. There was no change in [3H]paroxetine binding sites in any other brain regions examined in LH, NLH, and NC rats. The results suggest that a hippocampal hypothalamic connection might play a role in the serotonergic mediation of learned helpless behavior.     

Comparison of (R)-[3H] Tomoxetine and (R/S)-[3H] Nisoxetine Binding in Rat Brain

Abstract: ( R )-[³H]Tomoxetine is a radioligand that binds to the norepinephrine (NE) uptake site with high affinity but also binds to a second, lower-affinity site. The goal of the present study was to identify the nature of this low-affinity site by comparing the binding properties of ( R )-[³H]tomoxetine with those of ( R/S )-[³H]nisoxetine, a highly selective ligand for the NE uptake site. In homogenate binding studies, both radioligands bound to the NE uptake site with high affinity, whereas ( R )-[³H]tomoxetine also bound to a second, lower-affinity site. The autoradiographic distribution of binding sites for both radioligands is consistent with the known distribution of NE-containing neurons. However, low levels of ( R )-[³H]-tomoxetine binding were seen in the caudate-putamen, globus pallidus, olfactory tubercle, and zona reticulata of the substantia nigra, where ( R/S )-[³H]nisoxetine binding was almost absent. In homogenates of the caudate-putamen, the NE uptake inhibitors desipramine and ( R )-nisoxetine and the serotonin (5-HT) uptake inhibitor citalopram produced biphasic displacement curves. Autoradiographic studies using 10 n M ( R )-nisoxetine to mask the binding of ( R )-[³H]tomoxetine to the NE uptake site produced autoradiograms that were similar to those produced by [³H]citalopram. Therefore, ( R )-[³H]tomoxetine binds to the NE uptake site with high affinity and the 5-HT uptake site with somewhat lower affinity.     

[3H]Spiroxatrine: A 5-HT1A Radioligand with Agonist Binding Properties

Spiroxatrine has been reported to be a 5-HT1A serotonin receptor antagonist. Therefore [3H]spiroxatrine was synthesized and its 5-HT1A receptor binding properties in homogenates of rat hippocampal membranes were characterized with the expectation that it would be the first 5-HT1A antagonist radioligand. [3H]8-Hydroxydipropylaminotetralin [( 3H]8-OH-DPAT), a well-characterized 5-HT1A agonist radioligand, was studied in parallel for comparative purposes. Scatchard analyses of saturation studies of [3H]spiroxatrine and [3H]8-OH-DPAT binding produced KD values of 0.9 nM and 1.8 nM, with Bmax values of 424 and 360 fmol/mg protein, respectively. A highly significant correlation (r = 0.98; p less than 0.001) exists between Ki values obtained for a series of drugs in competing for [3H]-spiroxatrine and [3H]8-OH-DPAT binding. Of special interest was the observation that 5-HT1A agonists such as serotonin, 8-OH-DPAT, and ipsapirone competed with equal high affinities for [3H]spiroxatrine or [3H]8-OH-DPAT-labelled 5-HT1A receptors. [3H]Spiroxatrine and [3H]8-OH-DPAT binding to 5-HT1A receptors was inhibited by guanosine 5'-(beta,gamma-imido)triphosphate (a nonhydrolyzable analog of GTP) in a concentration-dependent manner whereas adenosine 5'-(beta,gamma-imido)triphosphate (a nonhydrolyzable analog of ATP) had no effect. The similarities in the 5-HT1A receptor radiolabelling properties of [3H]spiroxatrine and [3H]8-OH-DPAT, i.e., the high affinities of agonists and the guanyl nucleotide sensitivity, indicate that [3H]spiroxatrine has "agonist-like" binding properties in its interaction with the 5-HT1A receptor.     

Complex Inhibition of [3H]Imipramine Binding by Serotonin and Nontricyclic Serotonin Uptake Blockers

Tricyclic antidepressant drugs inhibit [3H]imipramine binding to the rat brain cortex in a competitive manner, giving linear Hofstee plots and Hill coefficients of approximately 1.0. Serotonin, the only neurotransmitter to inhibit [3H]imipramine binding, does so in a complex manner, exhibiting a Hill coefficient of 0.40-0.50. Nontricyclic inhibitors of serotonin uptake such as fluoxetine, paroxetine, norzimelidine, and citalopram inhibit [3H]imipramine binding in the same complex manner as serotonin. These results are interpreted as suggesting that [3H]imipramine binds to a site associated with the serotonin uptake system but different from either the substrate recognition site for serotonin or the site of action of the nontricyclic inhibitors of neuronal uptake of serotonin.     

[3H]1-[2-(4-Aminophenyl)Ethyl]-4-(3-Trifluoromethylphenyl)Piperazine: A Selective Radioligand for 5-HT1A Receptors in Rat Brain

1-[2-(4-Aminophenyl)ethyl]-4-(3-trifluoromethylphenyl)piperazine (PAPP) inhibits [3H]5-hydroxytryptamine (5-HT, serotonin) binding to 5-HT1A and 5-HT1B sites in rat brain with apparent equilibrium dissociation constants (KD) of 2.9 and 328 nM, respectively. [3H]PAPP was synthesized, its binding to central serotonin receptors was examined, and its potential usefulness as a 5-HT1A receptor radioligand was evaluated. With either 10 microM 5-HT or 1 microM 8-hydroxy-2-(di-n-propylamino)tetralin to define nonspecific binding, [3H]PAPP bound to a single class of sites in rat cortical membranes with a KD of 1.6 nM and a maximal binding density (Bmax) of 162 fmol/mg of protein. d-Lysergic acid diethylamide and 5-HT, two nonselective inhibitors of [3H]5-HT binding, displaced 1 nM [3H]PAPP with a potency that matched their affinity for 5-HT1 receptors. Spiperone and 8-hydroxy-2-(di-n-propylamino)tetralin, two compounds that discriminate [3H]5-HT binding to 5-HT1A and 5-HT1B sites, inhibited [3H]PAPP binding in accordance with their much higher affinities for the 5-HT1A receptor subtype. Furthermore, the ability of N-(m-trifluoromethylphenyl)piperazine and ketanserin to inhibit [3H]PAPP binding reflected their low affinities for the 5-HT1A receptor. Several nonserotonergic compounds were also found to be relatively poor displacers of [3H]PAPP binding. The regional distribution of serotonin-sensitive [3H]PAPP sites correlated with the densities of 5-HT1A receptors in the cortex, hippocampus, corpus striatum, and cerebellum of the rat. These results indicate that [3H]PAPP binds selectively and with high affinity to 5-HT1A receptor sites in rat brain.     

Characterization of [3H]Quipazine Binding to 5-Hydroxytryptamine3 Receptors in Rat Brain Membranes

[3H]Quipazine was used to label binding sites in rat brain membranes that display characteristics of a 5-hydroxytryptamine3 (5-HT3) receptor. The radioligand binds with high affinity (KD, 1.2 +/- 0.1 nM) to a saturable population of sites (Bmax, 3.0 +/- 0.4 pmol/g of tissue) that are differentially located in the brain. Specific [3H]quipazine binding is not affected by guanine or adenine nucleotides. ICS 205-930, BRL 43964, Lilly 278584, and zacopride display less than nanomolar affinity for these sites whereas MDL 72222 is approximately one order of magnitude less potent. The pharmacological profile of the binding site is in excellent agreement with that of 5-HT3 receptors characterized in peripheral physiological models. We conclude that [3H]quipazine labels a 5-HT3 receptor in the rat CNS.     

Characterization of Multiple [3H]5–Hydroxytryptamine Binding Sites in Rat Spinal Cord Tissue

Abstract: High-affinity [³H]5-hydroxytryptamine ([³H]5-HT) binding in the rat spinal cord is similar to that demonstrated in the frontal cortex. [³H]5-HT binds with nearly the same affinity to sites in both tissues. Furthermore, similar patterns of displacement of [³H]5–HT were seen in both tissues, with either spiperone or LSD as the unlabeled ligand. This high-affinity binding appears to be to multiple sites, since displacement studies using 2 nM [³H]5–HT result in Hill coefficients less than unity for spiperone, LSD, and quipazine [Hill coefficients (n_H): 0.44, 0.39, 0.40, respectively]. These sites apparently have an equal affinity for [³H]5-HT, since unlabeled 5-HT did not discriminate between them. Thus, the high-affinity [³H]5-HT binding in the spinal cord may be analogous to that observed in the frontal cortex, where two populations of sites have previously been described (5-HT_IA, 5-HT_IB). In addition to the multiple high-affinity spinal cord binding sites, a low-affinity [³H]5-HT binding component was also identified. A curvilinear Scatchard plot results from saturation studies using [³H]5-HT (0.5–100 nM) in the spinal cord. The plot can be resolved into sites having apparent dissociation constants of 1.4 nM and 57.8 nM for the high-and low-affinity components, respectively. Additional support for a change in affinity characteristics at higher radioligand concentrations comes from the displacement of 30 nM [³H]5-HT by the unlabeled ligand. A nonparallel shift in the dissociation curve was seen, resulting in a Hill coefficient less than unity (0.32). None of the specifically bound [³H]5-HT in the spinal cord is associated with the 5-HT uptake carrier, since fluoxetine, an inhibitor of 5-HT uptake, does not alter binding characteristics. In addition, a 5-HT binding site analogous to the site designated 5-HT, was not apparent in the spinal cord. Ketanse-rin and cyproheptadine, drugs that are highly selective for 5-HT, sites, did not displace [³H]5-HT from spinal tissue, and [³H]spiperone, a radioligand that binds with high affinity to 5-HT₂ sites, did not exhibit saturable binding in the tissue. Thus, the 5-HT₂ binding site reported in other regions of the central nervous system, and the serotonin uptake carrier do not appear to contribute to the multiple binding sites demonstrated in the spinal cord.