2-Nitroimipramine: A selective irreversible inhibitor of [3H] serotonin uptake and [3H] imipramine binding in platelets

The effect(s) of a new imipramine analogue, 2-nitroimipramine, on high affinity [³H] imipramine binding and [³H] serotonin uptake in human platelets were studied. 2-Nitroimipramine was found not only to be a very potent inhibitor of [³H] imipramine binding and [³H] serotonin uptake but was found to irreversibly inhibit binding and uptake simultaneously. This finding supports previous observations from our laboratory and others that high affinity imipramine binding labels serotonin uptake or transport sites. 2-Nitroimipramine should prove an important tool for subsequent studies of the molecular mechanism(s) involved in the transport of serotonin and the binding of imipramine to platelet and brain membranes.     

"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.     

Two Affinity States for [3H]Imipramine Binding to the Human Platelet 5-Hydroxytryptamine Carrier: An Explanation for the Allosteric Interaction Between 5-Hydroxytryptamine and Imipramine

5-Hydroxytryptamine (5-HT) showed a biphasic effect on the dissociation rate of [3H]imipramine from human platelet membranes: At low concentrations (EC50, approximately 2.5 microM), 5-HT stimulated the rate, as expected for mutually exclusive binding of 5-HT and imipramine; at higher concentrations (EC50, approximately 40 microM), 5-HT reduced this stimulated rate, a result consistent with 5-HT binding at a site that is physically distinct from both the imipramine binding site and the 5-HT transport recognition site of the 5-HT carrier. This modulatory effect could be mimicked by tryptamine, was saturable and independent of Na+ concentration, and could also be demonstrated for detergent-solubilized carriers. Monophasic association kinetics for [3H]imipramine binding were found. Heat stability experiments showed biphasic thermal inactivation curves. These results are consistent with [3H]imipramine binding to two classes of binding sites at the 5-HT carrier on human platelet membranes, with affinities three- to fivefold different. 5-HT can convert the lower-affinity state into the higher-affinity state.     

Sodium-dependent [3H]imipramine binding in rat hippocampus and its relationship to serotonin uptake

The relationship of [3H]imipramine recognition sites and serotonergic function was investigated by simultaneously determining the desipramine-defined and sodium-dependent components of [3H]imipramine binding and the serotonin levels and uptake in hippocampus of rats without and with selective lesion of serotonergic neurons with 5,7-dihydroxytryptamine. In control rats, the desipramine-defined [3H]imipramine binding to hippocampal membranes showed a high affinity (Kd = 2 nM) and low affinity (Kd = 31 nM) component. In contrast, the Scatchard analysis of sodium-dependent binding revealed a single class of sites of high affinity (Kd = 1.5 nM). Displacement of sodium-dependent [3H]imipramine binding by cold imipramine resulted in a steep curve best fitted to a one-site model. Sodium-dependent binding of [3H]imipramine at 4 nM concentration represented only about 38% of desipramine-defined binding. 5,7-Dihydroxytryptamine treatment resulted in marked reduction of hippocampal serotonin concentration and uptake without any changes in norepinephrine levels. Virtually only the low affinity component of desipramine-defined [3H]imipramine binding was detected by Scatchard analysis in 5,7-dihydroxytryptamine lesioned rats. The desipramine-defined "specific" [3H]imipramine binding in hippocampi of lesioned rats was decreased by 46%, whereas the sodium-dependent binding was only 18% of that seen in controls. Desipramine-defined specific binding in absence of sodium was not altered by lesion to serotonergic neurons. The results suggest that desipramine-defined specific [3H]imipramine binding may not be appropriate for studying the role of imipramine sites in relation to serotonin neuronal uptake and that determination of sodium-dependent binding components of both [3H]imipramine binding and serotonin uptake should be used in future studies.     

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.     

Demonstration of specific high affinity binding sites for [3H] imipramine in human brain

High affinity binding sites (Kd = 1.7 nM) for [³H] imipramine have been characterized in membranes prepared from human brain. The binding of [³H] imipramine was found to be saturable, reversible, and inhibited by pharmacologically active tricyclic antidepressants. Other psychoactive compounds as well as most neurotransmitter substances were ineffective in inhibiting [³H] imipramine binding at concentrations up to 10 μM. The hypothalamus was found to contain a relatively high density of these binding sites and is enriched approximately 4-fold when compared to cerebral and cerebellar cortex. A very good correlation (r = 0.97) p < 0.001 was found between the abilities of a series of clinically active tricyclic antidepressants in displacing specifically bound [³H] imipramine from human brain and platelet membranes, suggesting that the binding sites from these two tissues are very similar.     

Binding of [3H]Imipramine to Mouse Cerebrocortical Membranes and to Glass Fiber Filters

It was suggested in a recent report by Phillips et al. [J. Neurochem. 43, 479-486 (1984)] that the low-affinity binding of [3H]imipramine in the mouse cerebral cortex could in fact represent binding of [3H]imipramine to the GF/B glass fiber filters used to terminate the assays. The present study demonstrates that this is not the case and advances two lines of evidence: (a) For saturation analysis, mouse cerebrocortical membranes were incubated with [3H]imipramine concentrations between 0.8 nM and 3.6 microM, and parallel incubations were carried out with buffer replacing the brain membranes. The same low-affinity component, in addition to the high-affinity component, was present in the binding of [3H]imipramine to brain membranes plus GF/B filters (uncorrected data), and in that to brain membranes alone (corrected data). (b) Dissociation experiments, in which filter binding is equal for all samples and dissociation time is the only variable, clearly indicated the nonhomogeneity of [3H]imipramine binding. Our results, however, do show that binding to recently purchased GF/B filters is not a negligible phenomenon in saturation experiments. Relatively lower binding was found to GF/C, GF/F, Gelman A/E, and Reeves Angel 934 AH filters; pretreatment of GF/B filters with polyethyleneimine (PEI) reduced binding to a greater extent in the single manifold than in the cell harvester.     

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.     

Elevated Density of [3H]Imipramine Binding in Aged Human Brain

Aging was associated with an increase in the density of specific binding sites for [3H]imipramine in postmortem specimens of human hypothalamus, frontal cortex, and parietal cortex. In general, [3H]imipramine binding was not affected by factors considered difficult to control in postmortem studies, i.e., time from death to autopsy and cause of death. The in vitro regulation of [3H]imipramine binding by sodium was impaired with age in hypothalamic homogenates. In vitro regulation of [3H]imipramine binding by chloride was intact. Determination of the concentrations of 5-hydroxytryptamine (serotonin) and 5-hydroxyindoleacetic acid in hypothalamus and frontal cortex indicated no apparent age-related changes in indole metabolism. The age-related increase in brain [3H]imipramine binding and impairment in the in vitro regulation of binding by ions are similar to changes observed previously in aged mouse brain. The increase in brain antidepressant binding sites is discussed in relationship to other indices of brain serotonergic function in aging and to the relationship of [3H]imipramine binding and depression.     

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)     

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.     

[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.     

Binding of [3H]Imipramine to Human Platelet Membranes with Compensation for Saturable Binding to Filters and Its Implication for Binding Studies with Brain Membranes

Abstract: Apparent specific binding of [³H]imipramine to human platelet membranes at high concentrations of imipramine showed deviation from that expected of a single binding site, a result consistent with a low-affinity binding site. The deviation was due to displaceable, saturable binding to the glass fibre filters used in the assays. Imipramine, chloripramine, desipramine, and fluoxetine inhibited binding to filters whereas 5-hydroxytryptamine and ethanol were ineffective. Experimental conditions were developed that eliminated filter binding, allowing assay of high and low-affinity binding to membranes. Failure to correct for filter binding may lead to overestimation of binding parameters, B_max and K_D for high-affinity binding to membranes, and may also be misinterpreted as indicating a low-affinity binding component in both platelet and brain membranes. Low-affinity binding ( K_D < 2 μ M ) of imipramine to human platelet membranes was demonstrated and its significance discussed.     

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.     

Distribution of Specific High-Affinity Binding Sites for [3H]Imipramine in Human Brain

Abstract: [³H]Imipramine binds with high affinity to membranes from different regions of the human brain. The highest density of binding sites was observed in the hypothalamus and substantia nigra and the lowest density in the white matter and cerebellum. As found in rat brain, tricyclic antidepressant drugs are potent inhibitors of [³H]imipramine binding. Atypical antidepressants are, however, much weaker at inhibiting the specific binding. The [³H]imipramine binding site in human cortex is apparently identical to the site already described in the rat brain and in human platelets.     

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.     

[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.     

Imipramine inhibition of transient K+ current: an external open channel blocker preventing fast inactivation.

Rapidly inactivating K+ current (KA current) is recorded from rat hippocampal neurons by whole-cell patch-clamp technique and suitable voltage protocols. It is found that imipramine, a commonly prescribed tricyclic antidepressant, is an open KA channel blocker with a binding rate constant of 5.6 x 10(6) M-1 s-1 and an apparent dissociation constant of no more than 6 microM if applied extracellularly in pH 7.4. The inhibitory effect is more pronounced in more alkaline extracellular solution, suggesting that the neutral form of imipramine is much more active than the charged form. In contrast, intracellular imipramine shows no inhibitory effect. Furthermore, the inhibitory effect of imipramine is antagonized by external but not internal K+. These findings suggest an imipramine binding site located close to the external pore mouth. It is also found that the inactivation curve of KA current is not changed by imipramine. Moreover, the recovery of KA current after a step depolarization is accelerated in the presence of imipramine. These findings suggest insignificant binding of imipramine to the fast inactivated KA channel. The selective binding of imipramine to only the activated but not the deactivated or inactivated states seems to suggest continual gating conformational changes in the external pore mouth of these neuronal KA channels during membrane depolarization.     

Localization of [3H]-imipramine binding sites in rat brain by light microscopic autoradiography

The binding characteristics of [³H]-imipramine in slide mounted tissue sections of rat forebrain have been studied to ascertain the optimal binding conditions for labeling the sites prior to autoradiographic localization. The conditions for the experiments and the kinetics of the imipramine binding correspond reasonably well with those used in membrane preparations to initially define the imipramine binding site. Subsequent labeling of sections, using these parameters, allowed the autoradiographic localization of high concentrations of imipramine binding sites in such areas as the cerebral cortex, striatum, and several limbic and visual system structures. In addition, there was a marked overlap between regions demonstrating imipramine binding and areas known to be innervated by serotonergic neurons. This study outlines the potential sites of action of imipramine in the brain and defines areas for future investigations which attempt to localize brain regions involved in the etiology of depression and areas involved in the side effects of antidepressant drug therapy.