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.     

Effect of Different Photoperiod Exposure on [3H]Imipramine Binding and Serotonin Uptake in the Rat Brain

Seasonal rhythmicity in the occurrence of acute depressive episodes and the therapeutic efficacy of light exposure suggest the possible involvement of the pineal gland or other biological oscillators in the pathophysiology of depressive illness. We have performed studies to clarify whether different light/dark (LD) cycle schedules may induce changes in the biochemical targets of antidepressants in the rat CNS. In particular, we have investigated the effect of short- (LD 8:16) or long-day (LD 14:10) photoperiods on different biochemical parameters of serotonergic neurons. A significant increase in the density of [3H]imipramine ([3H]IMI) binding and in the Vmax of 5-[3H]hydroxytryptamine (5-[3H]HT) uptake was found in the hypothalamus of LD 8:16-with respect to LD 14:10-exposed rats, whereas no difference was found in the kinetic properties of postsynaptic 5-HT receptors and in 5-HT metabolism in the hypothalami and cerebral cortices of rats exposed to the two different photoperiods. A seasonal rhythm of [3H]IMI binding sites and 5-HT uptake seems to exist only in certain brain areas, such as the hypothalamus, because no differences were found in the cerebral cortex of LD 14:10- and LD 8:16-accustomed rats. [3H]IMI binding and 5-HT uptake were significantly increased in the hypothalamus of rats accustomed to a light/dark-inverted cycle (DL 10:14) and killed 6 h after the stopping of lighting in comparison to rats exposed to normal LD 14:10 cycles and killed 6 h after the beginning of lighting. Therefore, a circadian modification of the serotonergic presynaptic sites seems to be present and related to light/dark exposure. Because the existence of endogenous compounds able to modulate [3H]IMI binding and 5-HT uptake, other than 5-HT, has been postulated in the mammalian brain, the involvement of these substances in the periodic changes observed could be suggested.     

Similarities and Differences Between High-Affinity Binding Sites for Cocaine and Imipramine in Mouse Cerebral Cortex

Previously we found close similarities between high-affinity binding sites for [3H]cocaine and those for [3H]imipramine in the mouse cerebral cortex in regard to their association with neuronal uptake of serotonin. In the present study we investigated whether the two ligands bind to the same site. The two ligands had the following high-affinity binding properties in common: localization in both synaptosomal and microsomal fractions; vulnerability to treatment with N-ethylmaleimide, trypsin, and phospholipase A2; and resistance to exposure to dithiothreitol. In contrast, cocaine binding in the cerebral cortex was more sensitive to heat inactivation than imipramine binding. In addition, the mechanism by which cocaine inhibited [3H]imipramine binding differed from that by which imipramine inhibited [3H]cocaine binding. These data suggest that the high-affinity binding sites for [3H]cocaine and [3H]imipramine in the cerebral cortex are distinct entities.     

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.     

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.     

The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test

Pre-clinical and clinical studies have employed treatment with glucocorticoid receptor (GR) antagonists in an attempt to limit the deleterious behavioral and physiological effects of excess glucocorticoids. Here, we examined the effects of GR antagonists on neuroendocrine and behavioral stress responses, using two compounds: mifepristone, a GR antagonist that is also a progesterone receptor antagonist, and CORT 108297, a specific GR antagonist lacking anti-progestin activity. Given its well-documented impact on neuroendocrine and behavioral stress responses, imipramine (tricyclic antidepressant) served as a positive control. Male rats were treated for five days with mifepristone (10 mg/kg), CORT 108297 (30 mg/kg and 60 mg/kg), imipramine (10 mg/kg) or vehicle and exposed to forced swim test (FST) or restraint stress. Relative to vehicle, imipramine potently suppressed adrenocorticotropin hormone (ACTH) responses to FST and restraint exposure. Imipramine also decreased immobility in the FST, consistent with antidepressant actions. Both doses of CORT 108297 potently suppressed peak corticosterone responses to FST and restraint stress. However, only the higher dose of CORT 108297 (60 mg/kg) significantly decreased immobility in the FST. In contrast, mifepristone induced protracted secretion of corticosterone in response to both stressors, and modestly decreased immobility in the FST. Taken together, the data indicate distinct effects of each compound on neuroendocrine stress responses and also highlight dissociation between corticosterone responses and immobility in the FST. Within the context of the present study, our data suggest that CORT 108297 may be an attractive alternative for mitigating neuroendocrine and behavioral states associated with excess glucocorticoid secretion.     

"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.     

Regulation of β1-Adrenergic Receptor mRNA and Ligand Binding by Antidepressant Treatments and Norepinephrine Depletion in Rat Frontal Cortex

Abstract: The number of β₁-adrenergic receptor (β₁AR) binding sites is decreased by chronic antidepressant treatments, including electroconvulsive seizure (ECS) and imipramine, whereas administration of agents that deplete norepinephrine (NE) increases the number of β₁AR binding sites in cerebral cortex. The present study was carried out to examine the influence of these treatments on levels of β₁ AR mRNA in frontal cortex to study the molecular mechanisms that underlie the regulation of β₁ ARs in brain. Levels of β₁ AR mRNA were measured by RNase protection analysis using a riboprobe derived from rat β₁AR cDNA, and the levels of βAR binding were measured using the nonselective ligand [³H]CGP-12177. Studies to verify the specificity of the RNase protection assay revealed that the distribution of β₁AR mRNA was in agreement with the reported distribution of β₁AR ligand binding: Levels of β₁AR mRNA were highest in cerebral cortex or frontal cortex, intermediate in neostriatum, hippocampus, lung, and heart, and lowest in cerebellum, kidney, and liver. Chronic ECS treatment (once daily for 10 days) significantly decreased levels of βAR ligand binding and resulted in a corresponding, time-dependent down-regulation of β₁AR mRNA levels in frontal cortex. However, imipramine administration regulated levels of β₁AR mRNA in a biphasic manner, with treatments for 7–14 days increasing and treatments for 18–21 days decreasing levels of β₁AR mRNA in frontal cortex. In contrast, levels of [³H]CGP-12177 ligand binding were decreased at all time points examined (3–21 days). The influence of NE depletion, using the neurotoxin 6-hydroxy-dopamine (6-OHDA), on levels of β₁AR mRNA was also examined. Three days after 6-OHDA treatment, levels of [³H]CGP-12177 ligand binding were not altered, but 7–14 days after neurotoxin treatment, levels of ligand binding were significantly increased. In contrast, 3–9 days after 6-OHDA treatment, levels of β₁AR mRNA were significantly decreased, and 14 days after treatment, levels of β₁AR mRNA returned to control values. The results demonstrate that β₁AR mRNA and ligand binding are regulated in parallel by ECS treatment but that levels of receptor mRNA are regulated in a complex manner by imipramine or 6-OHDA treatments, not predicted by changes in ligand binding.     

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.     

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.