Antidepressant drugs given repeatedly increase binding to alpha 1-adrenoceptors in the rat cortex

The effect of antidepressant drugs, administered repeatedly, on binding to alpha 1-adrenoceptors in the rat cerebral cortex was studied using [3H]prazosin as a ligand. Imipramine, amitriptyline, citalopram and mianserin increased the binding (Bmax) assessed at 8:00 h. At 20:00 h such an effect was produced by imipramine, citalopram and mianserin. [3H]Prazosin binding in control rats was higher at 20:00 h than at 8:00 h. An increase in the density of alpha 1-adrenoceptors may be associated with the therapeutic activity of antidepressant drugs.     

The effect of imipramine and lithium on "learned helplessness" and acetylcholinesterase in rat brain

The effect of short- and long-term treatment with imipramine and lithium on shock stress-induced escape failures in a shuttlebox (the "learned helplessness" model of depression) was investigated in rats. Acetylcholinesterase (AChE) activity was measured in the frontal cortex, hippocampus and striatum after the shuttlebox test. Imipramine was found to normalize escape behavior, whereas lithium further aggravated escape behavior. No correlation was found between escape behavior and AChE activity in the three brain areas investigated. However, a significant decrease in AChE activity in striatum was found in rats exposed either to shock stress and no drug treatment or to drug treatment and no shock stress. In rats exposed to the combination of shock stress and drug (imipramine or lithium), a slight or no decrease of AChE activity occurred. Exposure to shock stress alone produced no changes in AChE activity in the hippocampus and frontal cortex. In conclusion, lithium did not have an antidepressant effect on "learned helplessness" and AChE activity was not correlated to escape behavior. However, both imipramine and lithium normalized the decreased level of AChE activity in striatum in rats exposed to shock stress.     

Examination of the relationship between the uptake site for 5-hydroxytryptamine and the high affinity binding site for [3H]imipramine. II. The role of sodium ions

Exogenous sodium ions stimulated both the high affinity binding of [3H]5-imipramine to membranes from the cortex of the rat and the high affinity accumulation of [3H]5-hydroxytryptamine (5-HT) into synaptosomes from the cortex of the rat with similar potencies. Imipramine and zimelidine inhibited synaptosomal uptake of [3H]5-HT potently in standard Tris-Krebs medium, but in a low-sodium medium their inhibitory potencies were significantly attenuated. The inhibitory potencies of panuramine and exogenous 5-HT on the uptake of [3H]5-HT were not significantly affected whether the uptake was measured in a normal or low-sodium Tris-Krebs. Imipramine and zimelidine were potent blockers of high affinity binding of [3H]imipramine whereas panuramine and 5-HT only inhibited the binding of [3H]imipramine at concentrations in excess of those required to inhibit the uptake of [3H]5-HT. It is suggested that imipramine inhibits the uptake of 5-HT by a sodium-dependent action probably at the high affinity binding site for [3H]imipramine, whereas panuramine and 5-HT inhibit the uptake of 5-HT by a sodium-independent mechanism at a site other than the binding site for [3H]imipramine.     

Effect of adrenalectomy and corticosterone on [3H]imipramine binding in rat blood platelets and brain

The effect of adrenalectomy and administration of glucocorticoids on [3H]imipramine binding (IB) of rat blood platelets and brain was investigated. Adrenalectomy significantly increased both Kd and Bmax of IB in the blood platelets but not the brain of male Sprague-Dawley rats. Administration of corticosterone acetate, 1 mg/kg i.p. for 7 days, decreased both Kd and Bmax in the blood platelets of sham-operated rats, but only Bmax in adrenalectomized rats. Corticosterone administration also decreased Bmax in frontal cortex and hypothalamus of sham and adrenalectomized rats but had no effect on Kd. These results suggest that glucocorticoids may modulate imipramine binding.     

Multiple [3H]imipramine binding sites in brains of male and female Fawn-Hooded and Long-Evans rats

Comparisons of high- and low-affinity [3H]imipramine binding to whole brain homogenates from adult male and female rats of the Fawn-Hooded and Long-Evans strains were performed. Most strikingly, no significant differences were observed between the two strains in any of the binding parameters, indicating that brain [3H]imipramine binding sites, which may be related to the serotonergic uptake process, appear normal in a strain of rats with serotonin platelet storage pool disease. However, a significant sex difference in high- but not low-affinity whole brain [3H]imipramine Bmax values was observed, with females of both strains having higher densities than males. The observed sex difference in densities of high-affinity [3H]imipramine binding sites necessitates further research into possible sex hormone interactions with this binding site and serotonergic transmitter systems.     

Examination of the relationship between the uptake system for 5-hydroxytryptamine and the high-affinity [3H]imipramine binding site--I. Inhibition by drugs

The relationship between the binding site for imipramine and the uptake system for 5-hydroxytryptamine was examined. This was determined from the interaction between various drugs (including tricyclic antidepressants) and the high affinity accumulation of [3H]5-hydroxytryptamine in cortical synaptosomes from the rat, and with the high affinity binding of [3H]imipramine to cortical membranes of the rat. Imipramine and clomipramine, but not desipramine, were potent inhibitors of both binding of [3H]imipramine and the uptake of [3H]5-hydroxytryptamine. However, ouabain, panuramine and 5-hydroxytryptamine itself, all inhibited the binding of [3H]imipramine only at concentrations greater than those required to inhibit the uptake of [3H]5-hydroxytryptamine. Kinetic analysis revealed that inhibitors of the uptake system for 5-hydroxytryptamine produced inhibition by different mechanisms, but this did not account for their differential potency against uptake and binding. It is concluded that the binding site for [3H]imipramine and the uptake site for 5-HT are not directly linked and that drugs may inhibit the uptake of 5-HT at sites other than the binding site for [3H]imipramine.     

A two-week treatment with antidepressants does not increase the density of 3H-prazosin binding sites in the cerebral cortex of elderly rats

The effect of repeated administration of antidepressants (imipramine, amitriptyline, citalopram) on the 3H-prazosin binding to alpha 1-adrenoceptors in the cerebral cortex of elderly (12-14-month old) rats was studied. Additionally, the effect evoked by imipramine was also studied in their young (2-3-month old) counterparts. Imipramine increased the Bmax value by 27% in young animals, while imipramine, amitriptyline and citalopram did not change the 3H-prazosin binding (Bmax, KD) in elderly rats. The obtained results demonstrate that the ability of antidepressants to increase the number of alpha 1-adrenoceptors in the rat cerebral cortex disappears with age.     

Size determination of binding polymers for [3H]imipramine and [3H]paroxetine in human platelet membranes

Imipramine and paroxetine both inhibit the transport of serotonin in serotonergic neurons and in platelets; furthermore specific high affinity binding sites for [3H]imipramine and [3H]paroxetine are located in these two cell types, probably on the serotonin transport mechanism. However, previous studies indicated that the binding site for [3H]imipramine was different from the binding site for [3H]paroxetine. We now report that the polymers on which the two binding sites are located have different molecular weights.     

Binding of some antidepressants to the 5-hydroxytryptamine transporter in brain and platelets

Antidepressant agents with properties to inhibit 5-hydroxytryptamine (5-HT, serotonin) uptake in brain tissue and platelets bind with high affinities to neuronal and platelet membranes. [³H]Imipramine, [³H]paroxetine and [³H]citalopram label specific binding sites related to the 5-HT transporter. [³H]Paroxetine and [³H]citalopram appear to be better ligands than [³H]imipramine. The former label a homogenous population of binding sites, whereas the displaceable binding of [³H]imipramine is heterogenous. Recent observations in several laboratories, which have taken the heterogeneity of [³H]imipramine binding into account, indicate that the binding of antidepressants to the 5-HT transporter probably occurs to the same site that binds 5-HT for transport and not to a separate site as previously suggested. Additional bonds to subsites in close vicinity to the 5-HT recognition site may contribute to the binding. No convincing evidence has been presented of the existence of an endogenous ligand other than 5-HT itself that binds to the [³H]imipramine binding site. Recent studies also suggest that repeated treatment of rats with antidepressant agents does not produce any alterations of the binding of [³H]imipramine or [³H]paroxetine to membranes of cerebral cortex. It is also doubtful whether the density of the 5-HT uptake site in platelets measured with these ligands is decreased in affective disorders as first reported.     

Chlorimipramine--but not imipramine--rapidly reduces [3H]imipramine binding in human platelet membranes

A single dose of 50 mg chlorimipramine was followed by a rapid and pronounced decrease in [3H]imipramine binding to platelet membranes. Incubation of human platelets or platelet membranes with 25 nM chlorimipramine similarly reduced [3H]imipramine binding. Imipramine, desmethylchlorimipramine, chlorpromazine and some serotonin uptake inhibitors did not have this effect. The effect was not due to chlorimipramine remaining in the membranes during the binding analysis.     

High affinity binding of [3H]paroxetine and [3H]imipramine to human platelet membranes

Paroxetine, one of the most potent and specific serotonin uptake inhibitors, was tritiated and used for binding studies with human platelet membranes. Specific, high affinity binding was demonstrated. The binding was compared with [3H]imipramine binding; it was found that the maximal binding (Bmax) was the same for [3H]paroxetine and [3H]imipramine, whereas the affinity was much higher for [3H]paroxetine (KD 0.08 nM and 0.56 nM for paroxetine and imipramine binding, respectively). IC50 was calculated for the inhibition of [3H]paroxetine and [3H]imipramine binding by a number of antidepressants; the corresponding Hill coefficients were also calculated.     

Evidence for the existence of at least two different binding sites for 5HT-reuptake inhibitors within the 5HT-reuptake system from human platelets

Chemical modification procedures have been used to study the interaction of tricyclic and non-tricyclic 5HT-reuptake inhibitors with the [3H]imipramine binding site (IBS). N-Ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) induced a pronounced loss in [3H]imipramine binding due to a reduction in Bmax. Preincubation with reuptake inhibitors and subsequent inactivation by EEDQ revealed that imipramine and 5HT prevented the EEDQ-induced inhibition, but citalopram and fluoxetine did not. Thiol modification studies demonstrated that reduction by dithiothreitol (DTT) enhanced the binding of [3H]imipramine by increasing the Bmax. The thioselective reagents 1,1-diazobis- (N,N-dimethylformamide) (diamide), phenyl-arsineoxide (PAO) and N-ethylmaleimide (NEM) attenuated the binding capacity by lowering the Bmax. PAO, a reversible thiol reagent, prevented NEM alkylation indicating that dithiols are involved in the NEM-induced inactivation. Binding of tricyclics or non-tricyclics prior to PAO inactivation revealed that tricyclics provide complete protection against thiol modification, while the non-tricyclics do not. The results support the hypothesis that the 5HT-reuptake system of human platelets possesses at least two distinguishable binding sites.     

Effect of chronic treatment with selective monoamine oxidase inhibitors and specific 5-hydroxytryptamine uptake inhibitors on [3H]paroxetine binding to cerebral cortical membranes of the rat

[3H]Paroxetine is a highly selective ligand for the 5-hydroxytryptamine transporter complex and the specific binding of this ligand to membrane fractions from cerebral cortex or hippocampus was studied in rats treated with specific inhibitors of the uptake of 5-hydroxytryptamine and monoamine oxidase inhibitors. The Kd and Bmax of the binding of [3H]paroxetine to cerebral cortical membranes of the rat was unaffected, compared to sham controls, by either acute or chronic administration with citalopram or chlorimipramine. Also, chronic treatment with chlorimipramine did not alter the parameters of the binding of [3H]paroxetine to hippocampal membranes from the rat compared to sham controls. Furthermore, chronic and acute treatments with clorgyline or deprenyl did not produce any significant changes in the Kd and Bmax of the binding of [3H]paroxetine to cerebral cortical membranes in the rat. These findings on the binding of [3H]paroxetine are discussed in light of previous equivocal results on the plasticity of neuronal binding sites for [3H]imipramine after various pharmacological treatments.     

Human platelets possess multiple [3H]imipramine binding sites

Scatchard analysis of [3H]imipramine binding to human platelets over the concentration range of 0.1-250 nM revealed a biphasic concave upward curve. Computer-assisted analysis indicated the best fit of the data by a two-site model with apparent Kd values of 0.68 and 293 nM and apparent Bmax values of 802 fmol/mg protein and 12.72 pmol/mg protein for the high- and low-affinity components, respectively. These results demonstrate the complex manner in which [3H]imipramine binding to human platelets, suggesting a reason for inconsistencies in Bmax values reported in the literature. Further studies of these low- as well as high-affinity sites may help elucidate the functional role of [3H]imipramine binding sites in affective disorders.     

Parallel changes in the sensitivity of gamma-aminobutyric acid and noradrenergic receptors following chronic administration of antidepressant and GABAergic drugs. A possible role in affective disorders

Chronic treatment with the antidepressants imipramine or nomifensine, or the gamma-aminobutyric acid (GABAergic) agents, baclofen or THIP, produced a decrease in the Bmax for binding sites of GABAergic and noradrenergic receptors. Chronic treatment with imipramine or nomifensine produced a decrease in the Bmax of both binding of [3H]dihydroalprenolol and [3H]GABA receptors in the cerebral cortex and hippocampus. Chronic treatment with baclofen or THIP also produced a decrease in the Bmax of binding of [3H]dihydroalprenolol receptor in the cerebral cortex and hippocampus. These results suggest a possible link between the GABAergic and noradrenergic systems, which may be important in understanding the mechanism of action of antidepressant drugs, and suggests a possible role for GABA in affective disorders.     

Influence of gonadotropin-releasing hormone on castration-induced 'depression' in mice: a behavioral and binding study

Long-term (33-35 days) castration caused a significant increase in the duration of immobility of male and female mice in the tail suspension test (an animal model of depression), and a significant decrease in the maximum number (Bmax) of [3H]imipramine binding sites in the cerebral cortex of male mice. In the tail suspension test, gonadotropin-releasing hormone (GnRH), s.c. injected 3 times at 3-h intervals at doses of 0.2, 2 or 20 micrograms/kg, did not significantly modify the duration of immobility of castrated animals and did not reduce that of sham-operated ones, while desipramine (20 mg/kg s.c. 1 h before testing) restored immobility to normal in castrated animals and reduced it significantly in sham-operated ones. The same treatment schedule with GnRH produced an increase in the number of [3H]imipramine Bmax in cortical membranes that was statistically significant at the dose of 2 micrograms/kg. It is concluded that the castration-induced depression-like behavior in mice seems not to be due to the decreased levels and release of GnRH, and that GnRH has no antidepressant-like effect in mice, at least at our dose levels; however, GnRH seems to increase the number of cortical [3H]imipramine binding sites.     

Single-site model of the neuronal 5-hydroxytryptamine uptake and imipramine-binding site

Recently, a high affinity [3H]imipramine-binding site of protein nature that appeared to be related to the 5-hydroxytryptamine (5-HT, serotonin) uptake mechanism was demonstrated. This binding site was only part of desipramine-displaceable [3H]imipramine binding, which contained a significant amount of additional binding not related to 5-HT uptake. The present study further investigates the [3H]imipramine-binding site of protein nature in the rat brain. Displacement by 5-HT and 6-methoxytetrahydro-beta-carboline (6-MeO-TH beta C) revealed monophasic displacement patterns with 60% displaceable binding. This binding fraction was abolished by protease treatment of the brain tissue prior to binding assay. Saturation studies of [3H]imipramine binding (1-30 nM) in rat cortex showed that the binding displaced by 30 microM 5-HT [Bmax 322 +/- 16 fmol/mg of protein, Kd 4.17 +/- 1.07 nM (means +/- SE)] was not different from the binding displaced by 1.0 microM norzimeldine (Bmax 349 +/- 15 fmol/mg of protein, Kd 4.47 +/- 1.07 nM) or 30 microM 6-MeO-TH beta C (Bmax 439 +/- 28 fmol/mg of protein, Kd 5.49 +/- 1.09 nM). When 100 microM desipramine was used in saturation studies, the binding was different from that displaced by 5-HT with Bmax 608 +/- 42 fmol/mg of protein and Kd 6.68 +/- 1.09 nM. Both displacement and saturation studies in which two displacing agents were combined indicated that most of the binding competed by 5-HT (30 microM) and norzimeldine (1.0 microM) is identical. Similarly, the binding displaced by 5-HT or norzimeldine is subsumed within 6-MeO-TH beta C (30 microM)-displaceable binding. Lesion studies with parachloroamphetamine, a selective toxin for 5-HT terminals, which resulted in a 83% reduction of [3H] 5-HT uptake ( [3H]noradrenaline uptake unaffected), abolished cortical [3H]imipramine binding displaced by 30 microM 5-HT or 1.0 microM norzimeldine. (greater than 80% reduction). However, with 100 microM desipramine as displacer, 40% of the binding remained in lesioned animals. The [3H]imipramine binding displaced by 30 microM 5-HT or 1.0 microM norzimeldine was sodium dependent, and an increase in NaCl concentration from 0 to 120 mM resulted in a 10-fold increase in affinity without effect on Bmax, whereas no change in binding was observed with increasing concentrations of LiCl.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differentiation of two components of specific [3H]imipramine binding in rat brain

Specific binding of [3H]imipramine to membrane preparation from rat cerebral cortex can be resolved in two distinct components: the high-affinity binding with a KD in nanomolar range (6.9 +/- 0.4 nM) and a maximum number of binding sites (Bmax) 285 +/- 19 fmol/mg protein and the low-affinity component with a KD of 292 +/- 45 nM and Bmax of 2459 +/- 428 fmol/mg protein. Tricyclic antidepressants, a non-tricyclic serotonin uptake inhibitor fluoxetine and a tetracyclic antidepressant maprotiline show a markedly different pattern in displacing [3H]imipramine binding at the high- and the low-affinity site. When the high-affinity sites were protected, the differences in potency of tested drugs in displacing specific [3H]imipramine binding, and the correlation with their ability to inhibit serotonin reuptake, were not observed. The Hill coefficients of competing drugs at low-affinity sites were markedly lower (0.45-0.69) and the shape of displacement curves indicated that more than one site may be involved in low affinity binding of [3H]imipramine.     

Strain differences in changes in some parameters of cerebral cortical adrenergic system following chronic imipramine administration to rats.

The characteristics of [3H]prazosin binding sites in the membranes from cerebral cortex, the basal level of formation of cyclic AMP in cortical slices, and the responsiveness of the cyclic AMP generating system to noradrenaline and isoproterenol in this preparation were measured in Long-Evans, Wistar and Sprague-Dawley rats treated chronically with saline or imipramine. No differences between strains and treatments were observed regarding the Bmax and KD of [3H]prazosin binding sites. The basal levels of cyclic AMP formation were similar in control rats of all strains, but imipramine treatment augmented it significantly in Sprague-Dawley rats. The responses of the cyclic AMP generating system to noradrenaline were significantly lower in Long-Evans than in the remaining strains of rats. Only in Sprague-Dawley rats a significant downregulation of response to noradrenaline was observed after imipramine treatment. All three strains of rats differed significantly among themselves in their responsiveness to isoproterenol; only in Sprague-Dawley rats this response was down-regulated significantly (by 80%) by imipramine treatment.     

The (-)-deprenyl actions on beta-adrenergic receptors require the integrity of brain serotonergic axon terminals

In rats receiving (-)-deprenyl (1 mumol/kg, s.c.) twice daily for 3 weeks, the Bmax of imipramine binding sites located in crude synaptic membranes prepared from frontal cortex increases while the NE stimulation of cAMP accumulation in minces prepared from frontal cortex is attenuated. The presence of intact 5HT axon terminals is an absolute requirement for the down-regulation of the beta-adrenergic receptor function by repeated injections of (-)-deprenyl. These and other lines of evidence suggest that the increase in the Bmax of [3H]imipramine binding sites and the attenuation of beta-adrenergic receptor function elicited by (-)-deprenyl might be causally related.