[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.     

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)     

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.     

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.     

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.     

[3H]Imipramine Labels Sites on Brain Astroglial Cells Not Related to Serotonin Uptake

Brain astroglial cells, whether from a bulk isolated preparation or in culture, have been shown to take up serotonin actively. [3H]imipramine has been proposed as a specific label for serotonin uptake sites in brain. We therefore studied the binding of [3H]imipramine to C6 astroglial cells in culture to determine if some of the binding of this radioligand in brain homogenates is actually to serotonin transporting sites on glia. [3H]Imipramine binds saturably (Bmax = 202 fmol/mg protein) and with high affinity (KD = 1.72 nM) to C6 cells. This binding is competitively inhibited by other tricyclic antidepressants. The C6 cells actively transport [3H]serotonin with a Km of 2 microM and a Vmax of 1080 fmol/10(6) cells/min. However, the pharmacological profile for inhibition of serotonin uptake does not correlate with the pharmacological profile for inhibition of [3H]imipramine binding. These results suggest that the binding of [3H]imipramine to astroglial cells is not related to their capacity for active uptake of serotonin. Further, in brain homogenates, some of the binding of [3H]imipramine may not be to neuronal uptake sites but rather may be to sites on astroglial cells.     

Tryptamine, a Substrate for the Serotonin Transporter in Human Platelets, Modifies the Dissociation Kinetics of [3H]Imipramine Binding: Possible Allosteric Interaction

Tricyclic antidepressants and nontricyclic serotonin (5-hydroxytryptamine) uptake blockers monophasically inhibit [3H]imipramine binding in human platelets. Similarly, serotonin and tryptamine inhibit the binding of [3H]imipramine in the low micromolar range and with a pseudo-Hill coefficient near unity. Dissociation of the [3H]imipramine receptor complex in the presence of uptake inhibitors follows first-order kinetics with a half-life of approximately 60 min. Although serotonin and tryptamine do not decrease [3H]imipramine binding when added under equilibrium conditions, simultaneous addition of serotonin or tryptamine with serotonin uptake inhibitors decreases the rate of ligand-receptor dissociation in a concentration-dependent manner. These data suggest a common site of action for serotonin, which is the substrate of the transporter system, and of tryptamine, its nonhydroxylated analog. This hypothesis is supported by the identification of a high-affinity (Km = 0.55 microM), saturable, and temperature-dependent uptake of [3H]tryptamine in human platelets. Uptake of [3H]tryptamine was inhibited potently by imipramine and nontricyclic serotonin uptake inhibitors with a potency similar to that observed for [3H]serotonin uptake. These data support the hypothesis that in platelets, [3H]imipramine, tricyclic, and nontricyclic serotonin uptake inhibitors bind to a common recognition site that is associated with the serotonin transporter but that differs from the substrate recognition site of the carrier through which serotonin and tryptamine exert a heterotropic allosteric modulation on [3H]imipramine binding.     

Comparison of the In Vitro Receptor Binding Properties of N-[3H]Methylspiperone and [3H]Raclopride to Rat and Human Brain Membranes

The aim of the present investigation was to study and compare the in vitro binding properties of the two radioligands N-[3H]methylspiperone ([3H]NMSP) and [3H]raclopride. These compounds, labeled with 11C, have been extensively used in positron emission tomography studies on central dopamine D2 receptors in schizophrenic patients, although with diverging results. One study (using [11C]NMSP) showed an increased dopamine receptor density in drug-naive schizophrenic patients, whereas in another study (using [11C]raclopride) the density in schizophrenic patients was no different from that in healthy controls. In the present study, using in vitro binding techniques, the density of the binding sites was found to be similar irrespective of which of the two radioligands was used (20 fmol/mg wet weight in rat striatum and 10 fmol/mg in human putamen; the 5-hydroxytryptamine 2 receptors were blocked with 40 nM ketanserin). [3H]NMSP had a 10-fold higher affinity (KD, 0.3 nM in rat striatum and 0.2 nM in human putamen) than [3H]raclopride (KD, 2.1 nM in rat striatum and 3.9 nM in human putamen), which was consistent with the longer dissociation half-life of [3H]NMSP compared with [3H]raclopride (14.8 and 1.19 min, respectively). There was an approximate overall similarity between the inhibition constants for five dopamine antagonists, chlorpromazine, haloperidol, raclopride, remoxipride, and NMSP, when using either radioligand. The Ki values were, however, two- to four-fold higher when using [3H]NMSP as the radioligand, irrespective of inhibiting compound, except for chlorpromazine (and haloperidol in human putamen). NMSP was found to inhibit the binding of [3H]raclopride competitively, whereas raclopride inhibited the binding of [3H]NMSP both competitively and noncompetitively. This difference suggests that part of the binding site is exclusively used by NMSP and can only be allosterically interfered with by raclopride. It is proposed that [3H]NMSP binds to an additional set of accessory binding sites, presumably located more distantly from the agonist binding active site than the sites to which [3H]raclopride binds.     

"Specific" Binding of [3H]Imipramine to Protease-Sensitive and Protease-Resistant Sites

A number of 5-hydroxytryptamine (5-HT) uptake inhibitors have been shown to displace the binding of [3H]imipramine to rat cortical membranes in a complex manner with Hill slopes less than unity. Norzimeldine displaced the binding of [3H]imipramine in a biphasic manner with IC50 values for the two components of about 30 nM and 30 microM. This latter site alone was found in tissues that had been treated with a protease. Binding to both of these sites was displaced by 10 microM desipramine. The protease-sensitive [3H]imipramine binding sites were found to be saturable, high-affinity binding sites with a KD of 8 nM. The number of these sites varied between brain regions and was positively correlated with the regional distribution of [14C]5-HT but not [3H]noradrenaline uptake. This was not the case however for the protease-resistant but desipramine-displaceable binding sites. Since most previous [3H]imipramine binding studies have been performed with high concentrations of desipramine (10 microM) to define "specific binding," these data would suggest that either protease-sensitivity or displacability by 1 microM norzimeldine would give more reliable estimates of the specific binding.     

One-step purification of the serotonin transporter located at the human platelet plasma membrane.

A 68-kDa glycoprotein bearing the biological activity of the plasma membrane serotonin (5-hydroxytryptamine, 5-HT) transporter has been purified from human blood platelets, a classical cell model for the study of 5-HT uptake. After treatment of the whole platelet population or its plasma membrane fraction by sulfhydryl-dependent bacterial protein toxins or by digitonin, purification was reproducibly obtained by a one-step affinity chromatography using two different columns with 5-HT or 6-fluorotryptamine as ligands and elution by 5-HT or Na(+)-free buffer. The purified fraction migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band with an apparent molecular mass of 68 kDa and exhibited an apparent isoelectric point of 5.6-6.2. Two sialic acid residues were detected in the purified material. The purified glycoprotein bound the 5-HT uptake blocker [3H]paroxetine with a Kd (0.25 nM) similar to the one observed for intact human platelets. It also bound [3H] 5-HT but neither [3H]hydroxytetrabenazine nor [3H] ouabain, the respective markers of the granular monoamine transporter and of the Na+,K(+)-ATPase associated to the plasma membrane 5-HT transporter. 5-HT derivatives and 5-HT uptake inhibitors exhibited similar Ki values for 5-HT uptake and paroxetine binding in intact human platelets and in the purified glycoprotein. Under laser UV irradiation, 40% of this purified glycoprotein could be labeled by either [3H]paroxetine or [3H]cyanoimipramine. No labeling was detected with either [3H] gamma-aminobutyric acid or [3H]GBR 12783, the respective markers of gamma-aminobutyric acid and dopamine carriers. The purified 68-kDa protein is therefore likely to correspond at least to the binding domain of the 5-HT transporter located at the human platelet plasma membrane.     

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.     

[3H]Imipramine Binding of Protein Nature in Human Platelets: Inhibition by 5-Hydroxytryptamine and 5-Hydroxytryptamine Uptake Inhibitors

Abstract: The nature of [³H]imipramine binding to human platelets was investigated. Desipramine and 5-hydroxytryptamine (5-HT) displaced the same amount of binding and the binding was sensitive to protease treatment. The nature of pharmacological inhibition of [³H]imipramine binding was investigated in saturation experiments. Increases in K d without changes in B _max were noted with the addition of 5-HT, desipramine, norzimeldine, or 5-methoxytryptoline. Reductions in B _max without alterations in K _D were obtained when citalopram or clomipramine was added. It is concluded that the [³H]imipramine binding site in human platelets is of protein nature and that this binding site contains the substrate recognition site for 5-HT uptake. In addition, [³H]imipramine and other 5-HT uptake inhibitors have bonds to other parts of the 5-HT uptake carrier or to the surrounding lipid membrane. This additional binding outside the substrate recognition site is not one single site but most likely represents sites that are specific for the chemical structure of each uptake inhibitor, respectively.     

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.     

The 5-HT1A receptor probe [3H]8-OH-DPAT labels the 5-HT transporter in human platelets

The present study characterizes a serotonin (5-HT) binding site on human platelet membranes, using [3H]8-OH-DPAT as the radioligand. [3H]8-OH-DPAT binds specifically and saturably to a site on human platelet membranes with an average KD of 43 nM and Bmax of 1078 fmol/mg protein. Determinations of IC50 values for various serotonergic characterizing agents in platelets for displacement of [3H]8-OH-DPAT were performed. For example, 8-OH-DPAT 5HT1A had an IC50 of 117 nM; TFMPP 5HT1B (2.3 microM0 and PAPP 1A + 5HT2 (9 microM); ipsapirone 5HT1A (21.1 microM) and buspirone 5HT1A (greater than 100 microM); ketanserin 5HT2 (greater than 100 microM); 5-HT uptake inhibitors: paroxetine (13 nM); chlorimipramine (73 nM) and fluoxetine (653 nM). The pharmacological inhibitory profile of the platelet 8-OH-DPAT site is not consistent with profiles reported for brain. 8-OH-DPAT does not inhibit [3H]imipramine binding, however, it does inhibit [3H]5-HT uptake in human platelets near 5-HT's Km value (IC50 = 2-4 microM). These results suggest that the human platelet site labeled by [3H]8-OH-DPAT is pharmacologically different from the neuronal site and probably is a component of the 5-HT transporter.     

Solubilization and characterization of [3H]Imipramine and [3H]Paroxetine binding sites from calf striatum

The serotonin (5-HT) transporter from calf striatum cerebral membranes was solubilized with digitonin and characterized by gel exclusion chromatography. [³H]Imipramine and [³H]paroxetine were utilized as markers for labeling it.³H-imipramine labels a high- and a low-affinity site on striaturn membranes, whereas it binds to a single high-affinity site on the solubilized fraction. [³H]Paroxetine binds with the same affinity to a single site on both membranes and solubilized preparations. After gel exclusion chromatography of the solubilizate both [³H]imipramine and [³H]paroxetine bind on an identical fraction of 205 kDa molecular weight, with a similar maximum number of binding sites (Bmax). Our results suggest that both³H-imipramine and [³H]paroxetine bind to a common site on the 5-HT transporter.     

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)     

Diurnal Variation in the Function of Serotonin Terminals in the Rat Hypothalamus

The high-affinity binding of [3H]imipramine is associated with the serotonin (5-hydroxytryptamine; 5-HT) transporter in the brain and in platelets. In the rat hypothalamus it has been reported that the density of these sites is increased in the dark period of the day, and this could result in an alteration in the release of 5-HT. The electrically evoked release of [3H]5-HT was thus studied in preloaded hypothalamic slices prepared from rats kept under 12:12 h light/dark or dark/light schedules. The fractional release of [3H]5-HT evoked by electrical stimulation, but not by the 5-HT releasing agent fenfluramine, was significantly decreased during the dark period when compared with the light period. The effects of the 5-HT reuptake blocker citalopram, of the two 5-HT autoreceptor agonists 5-methoxytryptamine and RU 24969, and of the 5-HT autoreceptor antagonist methiothepin on the release of [3H]5-HT were the same in both groups of rats. In conclusion, the release of [3H]5-HT from prelabelled rat hypothalamic slices is decreased during the dark period of the day. This modification is not reflected by changes in the effects of citalopram, an inhibitor of 5-HT reuptake, to modify the overflow of [3H]5-HT. The sensitivity and efficacy of agonists of the 5-HT autoreceptor are the same during the light and dark periods of the day.     

High- and Low-Affinity Binding Components for [3H]Imipramine in Rat Cerebral Cortex

The present study demonstrates that [3H]imipramine binds to both high- and low-affinity imipramine binding components on membranes prepared from rat cerebral cortex. Scatchard and computer analyses of saturation experiments using a wide range of [3H]imipramine concentrations (0.5 nM-50 nM) revealed the presence of two binding components. Inhibition experiments in which membranes were incubated with [3H]imipramine and various concentrations of unlabelled imipramine gave shallow inhibition curves with a Hill coefficient of 0.60 +/- 0.04. When dissociation rates of imipramine were studied, biphasic dissociation curves were obtained with apparent half-times of dissociation of 2.5 +/- 0.4 min and 18.5 +/- 2.5 min. Thus analysis of saturation, competition, and dissociation experiments indicate that [3H]imipramine binds to low as well as high-affinity binding sites in rat cortex.     

[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.     

High- and Low-Affinity Binding of [3H]Imipramine in Rat Hypothalamus

In the rat hypothalamus [3H]imipramine binding is inhibited by tricyclic and nontricyclic antidepressant drugs in a complex manner, with biphasic curves and Hill coefficients less than 1.0. 5-Hydroxytryptamine (serotonin) inhibited with high affinity a decreasing proportion of the [3H]imipramine binding sites as the [3H]imipramine concentration was raised. In the absence of sodium ions, IC50 values for the inhibition by tricyclic and nontricyclic antidepressants were increased by approximately 1,000-fold, and the inhibition curves became classically monophasic with Hill coefficients close to 1.0. These data are interpreted as suggesting that [3H]imipramine binds to two independent sites in the rat hypothalamus. One site is sodium-dependent with a high affinity for the drugs tested; the other is sodium-independent and has a low affinity for these drugs.