Imipramine binding site. Temperature dependence of the binding of 3H-labeled imipramine and 3H-labeled paroxetine to human platelet membrane.

The characteristics of 3H-labeled imipramine and 3H-labeled paroxetine binding to human platelet membranes were determined at various temperatures between 0 and 37 degrees C. Both paroxetine and imipramine probably bind to the same molecular complex in the platelet membrane, but the binding characteristics are different for the two molecules. The dissociation constant (Kd) for imipramine increases from 0.3 nM to 7.0 nM with increasing incubation temperature in a continuous way, whereas Kd for paroxetine is almost constant, about 0.05 nM, between 0 and 19 degrees C, and first begins to increase from 0.06 nM to 0.16 nM between 20 and 37 degrees C. This suggests that the binding of paroxetine to the binding site induces a conformational change in the molecular complex of the binding site, whereas the binding of imipramine takes place without conformational changes in the binding site.

Brain 5-HT1A, 5-HT1D, and 5-HT2 receptors in suicide victims.

We report on 5-HT1A, 5-HT1D, and 5-HT2 binding sites in 23 control subjects and 18 suicide victims subdivided according to the method of death and the previous existence of depressive symptoms. No difference in maximum binding (Bmax) or binding affinity (Kd) was found between the control and overall suicide groups for the binding sites studied. The drug overdose subgroup showed, however, a significant decrease in the 5-HT1A binding affinity, probably explained by the higher sensitivity of this binding site to the acute administration of tricyclic antidepressants. A significant decrease in 5-HT1D binding affinity was also found in the depressed suicides, together with a significant decrease in the number of 5-HT1D binding sites in the nondepressed suicides. Further studies should be carried out on the 5-HT1D binding site as it might represent a new tool in the understanding of the depressive illness.

24-hour lithium concentration in human brain studied by Li-7 magnetic resonance spectroscopy.

Brain and serum lithium concentrations were measured every second hour during a 24-hr period following lithium intake, and again 48-hr later in two normal subjects in steady state lithium treatment receiving lithium carbonate (Priadel Synthelabo) once every evening. The brain-lithium concentration was measured by 7Li magnetic resonance spectroscopy (MRS). The brain lithium level was found to undulate in a peak-trough pattern that followed the serum lithium profile, although in an attenuated form. The brain/serum lithium concentration ratio varied considerably during the 48-hr period, ranging from 0.5 to 1.3, but the ratio was independent of the serum-lithium concentration. The median half-life for lithium was 28 hr in the brain, and 16 hr in serum. The brain lithium concentration in the morning was about 75% of the clinically relevant standard 12-hr serum lithium concentration. The finding that brain lithium undulates during the day means that MRS measurements of brain lithium can only be compared if carried out under standard conditions that include a fixed interval following lithium intake and an identical treatment regimen.

[3H]imipramine binding in idiopathic pain syndromes. Basal values and changes after treatment with antidepressants.

The binding affinity (Bmax, Kd) of [3H]imipramine to platelet membranes was investigated in 43 patients with a diagnosis of idiopathic pain syndrome. Measurements were carried out prior to and after 6 weeks' treatment with the antidepressants clomipramine or maprotiline (randomized double-blind trial). Before treatment the Bmax was 11% lower (1109 +/- 237 fmol/mg protein) compared to historical control subjects (n = 21, 1240 +/- 390) and comparable to patients with psychogenic pain with affective symptoms (n = 62, 1110 +/- 360) previously measured at the same laboratory. The Kd was 0.5 +/- 0.3 nM comparable to the Kd of the controls (0.6 +/- 0.2). After 6 weeks of treatment the clomipramine group (n = 11) had significantly lower Bmax = 208.5 +/- 286 (P less than 0.001). In the maprotiline-treated patients (n = 18) the Bmax values (1221 +/- 258) tended to increase towards the values of the controls but without statistical significance. No significant statistical correlation was found between initial Bmax values and clinical outcome.

Lithium effects on rat brain glucose metabolism in long-term lithium-treated rats studied in vivo.

The time course of lithium effects on several brain energy metabolites has been investigated in rats. The rats were injected once daily with lithium chloride and killed by freezing in liquid nitrogen 1--8 h after the last injection. The effect of lithium was most marked in the period in which the brain lithium concentration was increasing, whereas the effect was wearing off when the brain lithium concentration had stabilized, even though the lithium concentration was higher. These results led to the hypothesis that the effect of lithium on several parameters depends on the increase in lithium concentration following the administration of lithium, rather than on the absolute concentration of lithium.

Lithium effects on rat brain glucose metabolism in vivo. Effects after administration of lithium by various routes.

The effects of lithium on several brain energy metabolites were investigated in rats. Lithium was administered by three alternative routes: 1) in food, 2) via IP injection, or 3) intracisternally via the suboccipital route. Lithium given in food induced permanent changes, mainly in glycolytic processes and in glycogen content. Lithium injected IP induced, in addition, several changes which depended on the increase in brain lithium concentration following injection of lithium. These changes in brain metabolites disappeared as brain lithium concentration stabilized. Intracisternal injection of lithium produced brain lithium concentrations between 1 and 2 mmoles/kg wet wt., with a mean of about 1.6 mmoles/kg wet wt. Lithium concentrations below about 1.6 mmoles/kg wet wt. induced changes in brain metabolites which were similar to the changes seen after IP injection of lithium. Lithium concentrations above about 1.6 mmoles/kg wet wt. induced changes in several brain metabolites which were at variance with the changes induced by lower lithium concentrations. These changes were in many respects similar to changes in brain metabolites seen in rats exposed to convulsive treatment. It is hypothesized that such metabolic changes during lithium treatment, in discrete areas of the brain with higher concentration of lithium, e.g., hypothalamus, might be related to the prophylactic effect of lithium treatment in man.

3H-Imipramine high-affinity binding sites in rat brain. Effects of imipramine and lithium.

The specific high-affinity binding of 3H-imipramine to rat brain membranes was investigated. Five weeks of lithium treatment decreased the number of binding sites, but had no effect on the affinity constants. Long-term imipramine treatment had no effect on the number of binding sites but apparently decreased the affinity. The latter effect was probably due to imipramine remaining in the membrane preparation.

High affinity binding of 3H-paroxetine and 3H-imipramine to rat neuronal membranes.

Paroxetine is the most potent and one of the most specific serotonin uptake inhibitors. High-affinity 3H-paroxetine and 3H-imipramine binding was compared in rat neuronal membranes. The KD value for 3H-paroxetine binding to neuronal membranes was 0.08 nM, which is exactly the same value as with platelet membranes. The KD value for 3H-imipramine binding to neuronal membranes was about 4 nM, which is higher than the KD value for 3H-imipramine binding to platelet membranes (0.5 nM). The results indicated that the 3H-paroxetine binding site is identical in neuronal membranes and in platelet membranes; this binding site is probably located on the serotonin transport mechanism. In addition, part of the high-affinity 3H-imipramine binding to neuronal membranes is probably located on the serotonin transport mechanism, but another part is located elsewhere. Furthermore the polypeptides containing the 3H-imipramine binding sites may not be identical in neuronal and platelet membranes.

Lithium effects on serum calcium, magnesium and phosphate, in rats.

LiCl was injected daily to rats in a dose of 3 mmol/kg. The rats were, in different experiments, unoperated rats, parathyroidectomized rats and thyroparathyroidectomized rats. Serum concentrations of calcium, magnesium, and phosphate were measured 2 h after a lithium injection. Serum calcium was unaffected by lithium in unoperated and parathyroidectomized rats. In thyroparathyroidectomized rats lithium increased the serum calcium concentration. Serum magnesium was increased by lithium in all 3 groups of rats. Serum phosphate was slightly decreased by lithium in all 3 groups of rats. It is concluded that lithium increases both serum calcium and magnesium, but in the intact organism only a slight or no increase in serum calcium is seem after lithium due to physiological control mechanisms. After removal of the calcitonin producing C-cells in the thyroid gland the organism is unable to produce a fast decrease in serum calcium, and lithium is then able to increase the serum calcium concentration.

Lithium treatment regimens induce different changes in [3H]paroxetine binding protein and other rat brain proteins.

Rats were treated with lithium administered either via the food or by intraperitoneal injection. Lithium administration via the food results in a rather stable serum lithium concentration, whereas lithium injection results in a varying serum lithium concentration whereby a sharp increase shortly after the injection is followed by an exponential decline until the next injection (Plenge et al. 1981). After 5 months of lithium treatment the 5HT transport protein, the beta-adrenergic receptor and several other brain proteins were determined. The 5HT transport protein, labelled with [3H]paroxetine, was found to be decreased in the lithium-injected rats (Bmax = 347 fmol/mg protein) but was unchanged in the lithium-fed rats (Bmax = 389 fmol/mg protein), as compared with control rats (Bmax = 396 fmol/mg protein), and therefore probably is a specific effect only seen with varying lithium concentration. In contrast, the neuronal membrane marker protein D3 was decreased in the lithium-fed rats (88% of the control value), and showed a trend towards decrease in the lithium-injected rats. The decrease in D3 in the lithium-fed rats may indicate some neuronal damage due to the continuous presence of lithium. This damage may be more pronounced than in rats, where periods of low lithium concentration enable repair to take place. The beta-adrenergic receptor and the neural cell adhesion molecule NCAM were unaffected by the different lithium treatment regimens. Lithium has been reported to inhibit the 5HT1B receptor (the serotonin autoreceptor).(ABSTRACT TRUNCATED AT 250 WORDS)

Twelve-hour brain lithium concentration in lithium maintenance treatment of manic-depressive disorder: daily versus alternate-day dosing schedule.

The 12-h brain lithium concentration was measured by lithium-7 magnetic resonance spectroscopy in ten manic-depressive patients receiving daily or alternate-day lithium carbonate treatment. The median dose of lithium carbonate was 800 mg in the daily treatment group and 1200 mg in the alternate-day group. Median 12-h serum lithium concentration in the two groups was 0.86 mmol l-1 and 0.55 mmol l-1, respectively, while the corresponding concentration in brain was 0.67 mmol l-1 and 0.52 mmol l-1, respectively. The 12-h brain lithium concentration was independent of lithium dosing schedule (multiple linear regression), but correlated significantly with the 12-h serum lithium concentration (P = 0.003; B = 0.53, 95% c.l. 0.24-0.82; beta = 0.83). Thus at identical 12-h serum lithium concentrations the 12-h brain lithium concentration is similar with both treatment regimes. As the risk of manic-depressive relapse during alternate-day lithium treatment is in our experience 3-fold greater than with daily treatment (at similar mean 12-h serum lithium concentration), the findings suggest that the difference in the prophylactic efficacy of the two dosing schedules is unrelated to differences in the 12-h brain lithium concentration.

Lithium treatment regimen and renal water handling: the significance of dosage pattern and tablet type examined through comparison of results from two clinics with different treatment regimens.

For many year two Danish psychiatric hospitals having used different lithium treatment regimens. In one, slow-release tablets were given in two daily doses and, in the other conventional tablets were given in a single daily dose. In both hospitals many patients developed polyuria. Multiple regression analyses with sex, age, treatment duration, serum lithium concentration, and treatment regimen as predictor variables showed that the two treatment regimens did not affect the glomerular filtration rate or the proximal reabsorption differently, but that distal water reabsorption was significantly less affected and polyuria less pronounced in the patients given conventional tablets once daily than in those give slow-release tablets twice daily. The authors are divided among themselves as regards the implications of these findings.

Effect of aging in human cortical pre- and postsynaptic serotonin binding sites.

5-HT1A, 5-HT1D, 5-HT2 binding sites and affinity and 5-HT uptake sites were simultaneously determined in frontal cortex samples from 23 control subjects, aged 16-75 years. A significant reduction in the number of 5-HT1D and 5-HT2 binding sites was found with regard to age, together with a significant decrease in the 5-HT2 binding affinity. It is suggested that the total 5-HT1 age-related loss described in previous studies could be ascribed to the 5-HT2 subtype. Furthermore, aging does not seem to be associated with a reduced cortical serotonergic innervation, as indicated by the stability of the [3H]paroxetine-labeled 5-HT uptake sites.

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.

Antidepressive drugs can change the affinity of [3H]imipramine and [3H]paroxetine binding to platelet and neuronal membranes.

Serotonin transport in synapses and platelets is inhibited by tricyclic antidepressants as well as by more selective transport inhibitors. This inhibition is hypothesized to be of importance for the psychotropic effect, although it is known that some new antidepressants do not possess this transport inhibitory action. We now report that antidepressive drugs can influence the serotonin transport complex in platelets and brain in other ways: [3H]imipramine and [3H]paroxetine, which bind with high affinity to the serotonin transport complex, can be dissociated from the complex with velocity constants strongly influenced by the different antidepressants. This effect is not correlated to the inhibitory action of the drugs on serotonin transport. Furthermore the effect is seen in the micromolar range in contrast to the high affinity binding process which takes place in the pico- and nanomolar range. The effects of antidepressants on the dissociation rates of bound ligand make it possible to differentiate between serotonin reuptake inhibitors which appear identical in other assays. Antidepressive drugs can thus be divided into groups which differ from the usual classifications.

Affinity modulation of [3H]imipramine, [3H]paroxetine and [3H]citalopram binding to the 5-HT transporter from brain and platelets.

The dissociations of [3H]imipramine, [3H]paroxetine and [3H]citalopram from the 5-HT (serotonin 5-hydroxytryptamine) transporter were found to be markedly influenced by several drugs, although concentrations in the microM range were needed. Most of these drugs attenuated the dissociation rate, i.e. increased the affinity between the ligand and the binding site. A few increased the dissociation rate however. The binding of drugs to the affinity-modulating site was specific, although of low affinity and probably changing the conformation of the high-affinity binding site, thereby changing the fit between the ligand and the interacting amino acid side-chains. Although the drugs usually affected the dissociation rates of the three ligands in the same manner, there were some which had different effects on [3H]imipramine, [3H]paroxetine and [3H]citalopram. For example, 5-HT markedly attenuated the dissociation of [3H]imipramine, had a moderate effect on [3H]paroxetine and very little effect on [3H]citalopram dissociation. This indicates that the three ligands are bound to different domains on the 5-HT transporter. [3H]Citalopram dissociation from human brain and rat brain were differently affected by several drugs. Indalpine augmented the dissociation rate of the [3H]citalopram 5-HT transport complex in human brain but attenuated it in rat brain, thus revealing a species difference of the 5-HT transporter.

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.

Inhibitory and regulatory binding sites on the rat brain serotonin transporter: molecular weight of the [3H]paroxetine and [3H]citalopram binding proteins.

Citalopram binds with high affinity to a specific binding site located on the serotonin (5-HT) transporter in 5-HT neurons. The binding affinity of [3H]citalopram was found to increase with increasing concentration of citalopram. This may be a homotropic positive allosteric effect, thus indicating the presence of an allosteric binding site for citalopram. The molecular weight of the proteins containing the high-affinity binding sites for citalopram and paroxetine, as well as the allosteric binding site for citalopram were determined by the irradiation method. The molecular weights of the three binding site proteins were found to be the same, suggesting that all three binding sites are located on the same protein molecule in the 5-HT transporter. The results support a hypothesis that the binding area for [3H]citalopram is located deeper in the transport channel than the [3H]paroxetine binding area. Thus the two high-affinity binding sites probably cover different, but overlapping, parts of the protein molecule. The allosteric binding site may be located elsewhere on the protein where it induces conformational changes of the 5-HT transporter with the result that high-affinity bound ligands get trapped in the transport channel, thereby explaining the increase in affinity.

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.

Non-acetylcholine displaceable 3[H]epibatidine binding in the rat brain.

Using autoradiography two types of 3[H]epibatidine binding in rat brain was found, the by far most widespread was nicotine displaceable, and represented nicotinic cholinergic receptors, probably the two subtypes alpha4beta2 and alpha3beta4. Using acetylcholine and nicotine in concentrations able to displace 3[H]epibatidine from the alpha4beta2 and alpha3beta4 subunits, in a few brain structures a non-acetylcholine and non-nicotine displaceable 3[H]epibatidine binding was found. The binding was almost exclusively localised to the medial and lateral habenula, interpeduncular nucleus and pineal gland; at 0.1 nM 3[H]epibatidine it represented about 40% of the total 3[H]epibatidine binding. A literature search directed at neurotransmitters in habenula did not demonstrate any neurotransmitter with this anatomical distribution in the brain, suggesting that the non-acetylcholine displaceable 3[H]epibatidine binding represents a binding site, not previously described.