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3H-Imipramine binding in platelets: Influence of varying proportions of intact platelets in membrane preparations on binding

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Abstract

Human platelets possess high-affinity 3H-imipramine binding sites. A study in ten healthy volunteers showed that platelet preparations produced by mechanical disruption contained varying proportions of intact platelets, as measured by the cytoplasmic marker enzyme LDH. This may invalidate the protein reference. Higher proportions of membranes in the preparations lead to higher B max values of imipramine binding (fmol/mg protein). The use of intact platelets in imipramine binding studies is therefore preferable to membrane preparations, particularly in studies where the interindividual variation of binding parameters is of interest.

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Biological research in the field of suicidology is strictly linked to biological aspects of depressive illness. About half of suicides are retrospectively diagnosed as having suffered from depression, while most of them had had depressive symptoms prior to death (Beskow 1979; Van Praag 1982). The biochemical mechanisms at the basis of depressive illness are not yet fully understood. Several studies suggest an involvement of noradrenaline and serotonin. Low levels of serotonin, or its metabolite 5-hydroxyindoleacetic acid (5-HIAA), have been observed in the brain of suicides (Lloyd et al. 1974). Van Praag (1977) suggested that low concentrations of 5-HIAA in the CNS could be a marker for the vulnerability to depression. Several groups have studied the relationship between suicidal behavior and the concentrations of 5-HIAA in the CNS (Åsberg et al. 1976; Träskman et al. 1981; Van Praag 1982; Roy et al. 1984). Many, but not all of the investigators, observed a correlation between low levels of 5-HIAA and suicidally. Considering that 2% of those who attempt suicide are successful, it is important to predict the risk of suicide to prevent it.
Article
Platelet 3H-imipramine binding was investigated in 31 control subjects and 19 hospitalized bipolar patients, either in the hypomanic or the depressed phase of illness. The mean Bmax value in the bipolar depressed patients did not differ significantly from that in the control subjects or the hypomanic patients. Differences in timing of the assay after blood collection, membrane preparation, protein content used in the assay, or binding of radioactive ligand to the equipment do not appear to explain the discrepancy between these results and previous findings.
Article
The densities of platelet 3H-imipramine sites were determined by repetitive measures of 11 normal controls over the course of 1 year. A significant seasonal variation was found, with a circannual peak on February 17 and a nadir on August 18. The estimated amplitude of this rhythm was +/- 599.54 fmol/mg, which fluctuated about a yearly mean of 2647.5 fmol/mg. The present results underscore the importance of including seasonally matched controls in the evaluation of potential patient differences in platelet binding.
Article
One month of imipramine treatment increased both the Kd and Bmax of platelet 3H-imipramine binding in 11 endogenous unipolar depressed patients. Continued treatment (13 weeks) of 5 patients subsequently lowered the Bmax values of 2 patients who had initially shown the largest increases, so that binding was no longer significantly elevated after 13 weeks. The observed changes in Kd but not in Bmax, could be explained by the carryover of tightly bound drug to the binding assay, although neither of the measures were correlated with plasma imipramine levels. Posttreatment Bmax (4 weeks) values were inversely related to plasma cortisol levels, although a weak but positive correlation was found before treatment. No significant change was found in plasma cortisol with treatment. Clinical responses were not related to cortisol or Bmax changes, although optimal improvement was associated with extreme values (high and low) of pretreatment Bmax. The present results, obtained with imipramine, and similar results obtained after nortriptyline and electroconvulsive shock by others, suggest that at least some antidepressants may induce transient changes in the Bmax of platelet binding that are independent of affective state.
Article
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The platelet is one of the most researched biological markers in psychiatry. Characteristics of MAO activity, 5-HT uptake, imipramine andα 2-adrenergic receptor binding, for example, are similar in platelet and CNS. Methodological factors are not negligible, and range from diagnostic specificity and drug effects to the normal physiological variability of age and hormone-related changes, circadian and seasonal rhythms. As yet, there are no clear state or trait platelet markers in affective disorders and schizophrenia that can be unequivocally used to detect vulnerability to the illness, predict therapeutic response, define clinical diagnostic entities or follow the course of the illness. However, platelet markers are increasingly being used in careful studies to monitor psychopharmacological effects (an in vivo assay of all active metabolites), different ligands can be specific markers for certain aspects of a psychiatric illness (e.g.α 2-adrenergic receptors and weight loss), and this homogeneous preparation of human cells is an increasingly important tool in studying mechanisms in pathophysiology. More longitudinal studies are required to establish functional relationships between platelet variables and psychopathology.
Article
Most investigators have measured binding of 3H-imipramine to platelet membranes, but some of the preparations may have contained varying proportions of intracellular protein because of contamination with incompletely lysed platelets. Since binding to membranes has been expressed with reference to the amount of protein in the membrane preparation, it is not surprising that there are discrepancies and a wide range of reported values for platelet imipramine binding in the literature. We have now completed a 9-month study of the binding of 3H-imipramine to intact platelets obtained monthly from 12 normal subjects. Using the intact platelet assay, as described by Friedl and Propping, we found that both Bmax and Kd for 3H-imipramine binding exhibited large month-to-month variations and no consistent seasonal trend was observed. The substantial variation in both Bmax and Kd among normal subjects and among samples obtained from the same individual at different times may limit the clinical usefulness of these measurements. A single blood sample is unlikely to give reliable values for Bmax and Kd for comparison purposes.
Article
Most investigations dealing with the pharmacological characterization of (3H)imipramine binding sites focus on tricyclic antidepressants (TCA). This approach seemed to be justified since imipramine belongs to that chemical group. Langer and coworkers, however, introduced a tetrahydro-beta-carboline (TH beta C) as a possible endogenous ligand. Thus, the high affinity of imipramine towards the binding sites might not be due to its special chemical structure but due to its tricyclic nature. In the present paper the structure-activity-relationships of neuroleptics and beta-carbolines were investigated and compared with that of tricyclic antidepressants. Among the tricyclic neuroleptics those with an electron attracting substituent (-Cl) exerted highest affinity. The effect was attenuated by a long, cyclic side chain. The affinity of tricyclic neuroleptics was only slightly weaker than that of 6-Meo-TH beta C the suggested endogenous ligand. The experiments with other TH beta Cs supported the observation that an electron attracting substituent increases the affinity of a compound to the (3H)imipramine binding sites. Comparison of the binding characteristics of (3H)imipramine to membranes of human brain and thrombocytes as well as those of rat brain and thrombocytes revealed no differences among both species. Furthermore, the displacing potencies of neuroleptics were very similar with only slightly more activity in human tissue. As a methodological aspect the applicability of the "Lowry" method to determine the protein concentration is discussed.
Article
3H-imipramine binding was studied in the blood platelets of 80 normal controls and 50 depressed (psychotic and nonpsychotic) patients. Protein concentration in the incubation mixture, and Kd and Bmax values were correlated in normal controls and depressed patients. A small but significant correlation between protein in the incubation mixture and Bmax was observed in normal controls and depressed patients. There was also a significant correlation between protein and Kd of imipramine binding in the blood platelets of unipolar psychotic depressed patients. However, analysis of covariance to remove the effect of protein on Kd and Bmax did not change the basic finding of decreased Bmax in the blood platelets of psychotic depressed patients.
Article
The density of platelet 3H-imipramine binding sites has been proposed as a biological marker in psychiatry. We report the range of platelet 3H-imipramine binding in 55 psychiatric patients and 11 control subjects. All blood samples were withdrawn at 2300 h (on the day of hospital admission for patients). With the use of a slight modification of a previously described 3H-imipramine binding method, a mean B max of 1,510 fmole/mg protein (range: 390-5,560; median: 1,450) and a mean Kd of 2.0 nM (range: 0.6-17.0; median: 1.4) were determined for psychiatric patients. For the controls, a mean B max of 1,590 fmole/mg protein (range: 870-2,570; median: 1,440) and a mean Kd of 1.4 nM (range 0.8-2.4; median 1.4) were determined. When patients were subdivided based on ICD-9 psychiatric diagnoses, no significant differences between distinct subgroups of psychiatric patients with respect to B max or Kd values for platelet 3H-imipramine binding could be established. Similarly, no significant difference between psychiatric patients and controls was obtained.
Article
3H-Imipramine (3H-IMI) binding to platelet membranes was measured in 19 patients with agoraphobia with panic attacks, 9 patients with major depression, and 22 healthy subjects. In comparison to healthy subjects, the maximal number of binding sites (Bmax) was significantly decreased in depressed patients but not in panic disorder patients, and the apparent affinity of binding was slightly decreased in depressed patients but not in panic disorder patients. The Bmax and Kd of 3H-IMI platelet binding did not differ between panic disorder patients with and without a history of a major depressive episode. Thus, 3H-IMI platelet binding is clearly different in patients with panic disorder compared to those with an active depression. Because 3H-IMI binding is associated with the serotonin reuptake site in platelet and brain membranes, these findings give further support to abnormalities in serotonergic function in patients with major depression.
Article
Measurements of sodium-dependent [3H]imipramine binding to intact human platelets from 20 human volunteers were made and compared to desipramine-defined binding, a method commonly employed in population studies of platelet [3H]imipramine sites. The density (Bmax) of sodium-dependent [3H]imipramine sides in platelets was significantly lower (449 +/- 36 sites/platelet) and the affinity (Kd) significantly higher (1.15 +/- 0.12 nM) than those obtained when excess desipramine was used to define specific binding (Bmax 654 +/- 33 sites/platelet, p less than 0.001; Kd 1.52 +/- 0.11 nM, p less than 0.001). There was no significant correlation between the density (Bmax) of sodium-dependent and desipramine-defined binding in individual subjects, suggesting that a different proportion of sites are labeled under the two assay conditions. No age-dependent variation was found in either Kd or Bmax values of sodium-dependent or desipramine-defined [3H]imipramine binding. The results suggest determination of sodium-dependent [3H]imipramine binding to intact platelets may be a useful measure for the estimation of [3H]imipramine recognition sites relevant to the serotonin uptake in studies of patients with affective disorders.
Article
[3H]-Imipramine and [3H]-paroxetine label with high affinity a site which is associated with the serotonergic transporter in brain and platelets. The pharmacological profile of inhibition by drugs of [3H]-imipramine and [3H]-paroxetine binding is highly correlated with the potency of the drugs to inhibit the uptake of 5HT. Denervation of serotonergic neurons by electrolytic lesions or with 5,7-dihydroxytryptamine produces marked decreases in the density of [3H]-imipramine as well as [3H]-paroxetine binding. Dissociation kinetic experiments support the view that the substrate recognition site for 5HT is different from the modulatory site which is labelled by [3H]-imipramine or [3H]-paroxetine. The existence of an endogenous ligand acting on the [3H]-imipramine recognition site to modulate the 5HT transporter was proposed by several laboratories. [3H]-Imipramine binding in platelets appears to be a biological marker in depression. Studies carried out in several laboratories report a significant decrease in the Bmax of platelet [3H]-imipramine binding without changes in Kd, when severely depressed untreated patients are compared with healthy volunteers matched for age and sex. The Bmax of platelet [3H]-imipramine binding appears to be a state-dependent biological marker in depression. It is tempting to speculate that the endocoid of the [3H]-imipramine recognition site may play a role in the pathogenesis of depression.
Article
Platelet 3H-imipramine binding values from 45 normal controls and 20 depressed subjects were collected over a 2-year period. During this time, wide inter-individual variations in the affinity constant (Kd) and maximal number of binding sites (Bmax) were observed in the control population; however, we failed to observe a seasonal change in platelet 3H-imipramine binding. The Kd and Bmax values of depressed subjects were not significantly different from those of controls.
Article
3H-Imipramine binding in platelets was measured in 63 severely depressed hospitalized patients, who had been drug free (with the exception of moderate doses of benzodiazepines) for at least 1 month, and in 53 healthy control subjects of comparable age and sex distribution. Bmax of 3H-imipramine binding was significantly lower in the depressed subjects (1012 +/- SD 295 vs. 1123 +/- SD 178 fmole/mg protein). Depressed patients who had attempted suicide by violent means tended to have higher Bmax than nonviolent attempters.
Article
It was examined whether genetic factors are involved in the expression of alpha-2 adrenergic receptors on human platelets, as measured with 3H-yohimbine as ligand. The twin series comprised 17 monozygotic and 15 dizygotic adult, healthy, male, drug-free twin pairs. For control of intraindividual and interassay variation, 13 unrelated pairs of subjects were examined. Bmax values for 3H-yohimbine binding (range 91-305 fmol/mg protein) proved to be under considerable genetic control; this was not the case for KD values. Different possible genetic mechanisms are discussed.
Article
Receptor binding of imipramine in human platelets was assessed by filtration through glass-fiber filters and by equilibrium dialysis. Both methods yield drug-receptor dissociation constants of similar magnitude (10(-9) M) to literature values. However, the density of binding sites (Bmax) was fivefold lower by filtration (473 +/- 92 fmol/mg protein) compared to equilibrium dialysis (2652 +/- 765 fmol/mg protein). Dialysis allows direct assessment of free imipramine and avoids drug loss during the separation step of the filtration assay. Additional advantages were found for computer nonlinear regression analysis of untransformed data to eliminate errors owing to linear transformation in the Scatchard analysis and for simultaneous quantitation of nonspecific and total drug binding.
Article
3H-imipramine binding was determined in freshly prepared intact platelets from 17 monozygotic (MZ) and 15 dizygotic (DZ) twin pairs, all of them male, adult, drug-free, and healthy volunteers. Sixteen males served as controls for determination of intraindividual variation of binding parameters. Both MZ and DZ twin pairs exhibited high intraclass correlations of Bmax values, but DZ twins were nearly as similar as MZ pairs. Interindividual variation of binding parameters is not large enough to reveal a significant genetic control.
Article
3H-imipramine and 3H-cyano-imipramine binding was determined in brain homogenates of rats which had been treated for 21 days with imipramine or desimipramine. When compared to control animals, long-term administration of these antidepressants did not induce any alteration in the maximal number of 3H-imipramine or 3H-cyano-imipramine binding sites. However, a transient increase in the apparent dissociation constant was observed. Such findings are discussed in respect to previous studies, which have been highly contraversial.
Article
Some investigators suggest that the number of platelet 3H-imipramine binding sites is genetically determined and that the reduced 3H-imipramine binding observed in some studies of depression represents a trait marker. Others believe that 3H-imipramine binding is state-dependent because 3H-imipramine binding has been reported to normalize after clinical recovery. We used a genetic selection paradigm in rats to approach this question from another direction. While breeding 10 generations of rats, we used either extremely high or extremely low density of platelet 3H-imipramine binding sites as a selection criterion. We were unsuccessful in producing two distinct rat sublines. These data do not support a predominant genetic influence on 3H-imipramine binding.
Article
1. The effects of protein concentration in the assay mixture on platelet 3H-imipramine binding were studied in normal controls. 2. Increasing protein concentrations (76-926 micrograms/ml) were found to alter estimates of binding affinity (Kd) but not the number of binding sites (Bmax). 3. Increasing Kd estimates were protein dependent at concentrations in excess of ca. 200 micrograms/ml but were not protein dependent at lower concentrations. 4. A comparison of the present results with previous reports suggests that widespread interlaboratory differences in reported Bmax values for normal controls cannot be attributed to differences in the protein content of incubated samples. Rather these differences may be due to the inclusion of non-membrane protein in the assayed material.
Chapter
This chapter discusses the peripheral markers on affective disorders. The major questions that must be addressed when investigating a peripheral model as a biochemical marker for affective disorders are: (1) Within which particular sub-categories of affective disorder is the model reliable? (2) Does the model represent a state or trait marker and to what extent is it effective in diagnosis? (3) Does the model reflect similar changes within the brain; hence, does it represent a primary or secondary biochemical marker? (4) How does the observed difference between healthy and depressed subjects arise and can this information improve our understanding of the biochemical changes associated with affective disorder? None of the peripheral models proposed till date can be considered reliable enough to act as a biochemical marker for affective disorder. The trend in recent years has been to subcategorize the patient group to identify localized markers, such as β-adrenoceptor changes in depressed patients with psychomotor agitation and [³H] Imipramine binding decrease in depressed patients with obsessive compulsive symptoms. The biochemical basis of affective disorders will depend upon parallel developments in studies using animal models, in categorization of the disorder by means of psychiatric assessment and in the gradual amalgamation of biochemical, physiological, and genetic studies carried out in man.
Article
Discrepant results have been reported from at least ten laboratories regarding the status of platelet alpha2 adrenoreceptors in depressed patients. Using a statistical test to combine those studies which utilized radioligand binding techniques, we find the overall data support an elevation in density of platelet alpha2 adrenoreceptors from drug-free depressed patients (p < 0.05) and suggest a normalization to lower binding values following antidepressant drug treatment (0.05 < p < 0.10). However, these positive results are attributable to highly significant findings by only three laboratories. Much of the discrepancy may be attributable to numerous methodological variables which distinguish the studies. Foremost amongst these variables are (A) the use of different platelet size populations, (B) the use of different medium, and (C) the choice of radioactive ligand and competitor (non-radioactive ligand) in the assay. We present a rationale for the proper choice of each methodological condition used in the clinical assessment of platelet alpha2 adrenoreceptor status, hoping that improved experimental designs will resolve the current controversy.
Article
Platelet 5-HT uptake rate, 3H-imipramine binding and monoamine oxidase activity are reviewed and compared with those of preparations of nerve endings (synaptosomes). The properties of the 5-HT uptake mechanisms in platelet and brain seem to be very similar in most respects and the platelets therefore provide a useful model for brain 5-HT uptake. There is evidence both of genetic, stable control of the uptake and of a state-dependent modifying influence. The findings of some recent studies regarding the platelet 5-HT uptake rates in various psychiatric disorders are reviewed. A consistent finding seems to be a lower Vmax in patients with unipolar depression than in controls. The 3H-imtpramine binding also shows characteristics very similar to that of brain synaptosomes. Although this receptor is functionally linked to 5-HT uptake the density of imipramine binding (Bmax). is obviously independent of 5-HT uptake rates. In the case of imipramine binding the evidence for some form of constitutional (trait-dependent) control is even weaker than for 5-HT uptake. The review of findings in patients suffering from psychiatric disorders shows a confusing situation without a firmly established pattern. Platelet monoamine oxidase activity (MAO) seems to be a stable constitutional factor with a clear correlation to personality traits such as sensation-seeking, extraversion and impulsiveness, which might also explain connections with disorders such as alcoholism, psychopathy and suicidal behaviour. There are several findings which support the hypothesis that platelet MAO activity is a marker for the density or capacity of the central serotonin system. □ Blood platelets, serotonin, 5-HT3H-imipramine binding, MAO.
Article
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Article
In receptors, as in enzymes, quantitative as well as qualitative genetic variation may exist. Studies in inbred strains of mice have shown for various receptors that the receptor density as determined by Bmax values is under genetic control. In healthy adult twins we have shown that the density of α-adrenoceptors on platelets is also influenced by genetic factors, since monozygotic twins were much more similar to one another than dizygotic twins. However, Bmax values are up-regulated and down-regulated by endogenous neurotransmitters and pharmacologically active agents. Thus, receptor densities are under considerable regulatory influences. Bmax values therefore reflect regulatory mechanisms rather than innate characteristics of the receptor protein. In another twin study we failed to find evidence for a genetic influence on the density of imipramine-binding sites on platelets.Since qualitative variation (polymorphism) is well known in enzymes, it may also apply to receptors. Qualitative differences in the receptor protein within one species would be of particular interest because of possible functional implications. As a first approach we examined central benzodiazepine receptors by photoaffinity labelling and sodium dodecyl sulphate–polyacrylamide gel electrophoresis. A comparison of fish, frog, chicken, mouse, rat and calf led to the detection of variation between species. Investigations in five inbred mouse and rat strains have not so far revealed genetic variation in benzodiazepine receptors. Nevertheless variation may be detectable by more sensitive methods such as peptide mapping after limited proteolysis or two-dimensional electrophoresis.
Chapter
Although antidepressant drugs have been successfully used in the treatment of depression for more than two decades, the mechanism(s) by which these drugs produce their therapeutic effect is not understood. Tricyclic antidepressants have been shown to inhibit the neuronal uptake of norepinephrine (noradrenaline; NA) and serotonin, (5-hydroxytryptamine; 5-HT) (Glowinski and Axelrod, 1964) and their therapeutic effect has been related to this activity (Schildkraut, 1965). However, the effect of these drugs on the uptake of biogenic amines occurs immediately and does not correlate with the time course of the therapeutic response that usually sets in after only 1–3 wk of treatment. Moreover, there are other antidepressants, such as iprindole and mianserin, that do not block 5-HT and/or NA reuptake, but have been shown to lessen the symptoms of depression. Other drugs, such as cocaine, block the uptake of biogenic amines, but are not clinically effective antidepressants (Post et al., 1974). Some tricyclic antidepressants have also been shown to interact with the in vitro binding of specific radioactive ligands to several classes of neurotransmitter receptors in brain membrane preparations: (α-and γ-adrenergic (U’Prichard et al., 1978), serotonergic (Peroutka and Snyder, 1980), H1-histaminergic (Tran et al., 1981), and muscarinic cholinergic (Snyder and Yamamura, 1977) receptors.
Article
3H-Clonidine and 3H-yohimbine were observed to bind to glass fiber filters. The binding was displaced by co-filtration with the corresponding non-radioactive ligand. Phentolamine and (–)-norepinephrine were ineffective in displacing either 3H-clonidine or 3H-yohimbine bound to filters. Failure to correct for filter binding resulted in an over-estimation of specific binding to platelet membranes. Certain published methodologies may have consequently misidentified up to 20% of the specific binding to platelets, that was actually due to displaced filter binding. Experimental conditions are described which eliminate filter binding. These results are significant for the interpretation of data from studies of platelet binding in depressed patients.
Chapter
Although antidepressant drug treatment is a well-established therapeutic approach in manic-depressive disorders, the mechanism by which such drugs ameliorate the depressive syndromes remains an area of active research. The mechanism of action of antidepressants most likely has two components. The clinical manifestation of their therapeutic effects generally is seen after a l-to 2-wk treatment, thus implying that a degree of neuronal adaptation under the effect of antidepressants is required for clinical efficacy. The development of neuronal sub-or supersensitivity following subchronic antidepressant treatment, however, is thought to be secondary to the direct interaction of these drugs with specific receptors and/or enzymes in the brain. To characterize this interaction of antidepressants with such receptors and/or enzymes, radioligand binding studies using mostly 3H-labeled antidepressants have been used extensively. Foremost among these is [3H]-imipramine, a tricyclic antidepressant that inhibits neuronal serotonin (5-hydroxytryptamine, 5-HT) and norepinephrine (noradrenaline, NA) transport. Other radiolabeled antidepressants whose high-affinity binding sites have been studied in the brain and peripheral tissues include [3H]-desipramine, [3H]-nomifensine, [3H]-indalpine, [3H]-paroxetine, [3H]-cyanoimipramine, [3H]-amitriptyline, [3H]-doxepin, [3H]-mianserin, and [3H]-rolipram (Table 1 Table 1 Antidepressants as High-Affinity Radiohgands [3H]-AntidepressantPutative identity of its high-affinity recognition site[3H]-Imipramine [3H]-Cyanoimipramine [3H]-IndalpineSerotonergic transporter[3H]-Paroxetine [3H]-Norzimelidine [3H]-DesipramineNoradrenergic transporter[3H]-Nomifensine [3H]-NomifensineDopammergic transporter[3H]-DesipramineAdrenergic transporter[3H]-AmitriptylineMuscarmic and histamine (H1) receptor[3H]-DoxepinHistamine (H1) receptor[3H]-MiansennHistamine (H1) and serotonin (5-HT2) re- ceptors[3H]-RolipramcAMP phosphodiesterase
Article
Most investigations dealing with the pharmacological characterization of (3H)imipramine binding sites focus on tricyclic antidepressants (TCA). This approach seemed to be justified since imipramine belongs to that chemical group.Langer and coworkers, however, introduced a tetrahydro-β-carboline (THβC) as a possible endogenous ligand. Thus, the high affinity of imipramine towards the binding sites might not be due to its special chemical structure but due to its tricyclic nature. In the present paper the structure-activity-relationships of neuroleptics andβ-carbolines were investigated and compared with that of tricyclic antidepressants. Among the tricyclic neuroleptics those with an electron attracting substituent (-Cl) exerted highest affinity. The effect was attenuated by a long, cyclic side chain. The affinity of tricyclic neuroleptics was only slightly weaker than that of 6-Meo-THβC the suggested endogenous ligand. The experiments with other THβCs supported the observation that an electron attracting substituent increases the affinity of a compound to the (3H)imipramine binding sites. Comparison of the binding characteristics of (3H)imipramine to membranes of human brain and thrombocytes as well as those of rat brain and thrombocytes revealed no differences among both species. Furthermore, the displacing potencies of neuroleptics were very similar with only slightly more activity in human tissue. As a methodological aspect the applicability of the “Lowry” method to determine the protein concentration is discussed.
Article
3H-Imipramine binding (IB) was studied in the blood platelets of 13 pairs of monozygotic (MZ) and dizygotic (DZ) twins, and 15 pairs of unrelated normal volunteers, to determine if IB is under genetic control. The intrapair variance of Bmax (the maximum number of 3H-IB sites) was significantly smaller in MZ twins and unrelated control pairs than in DZ twins. The intraclass correlations (ICC) of Bmax were significant for all the pairs with no difference between these correlations. The ICC of the Kd (inversely related to the affinity for 3H-imipramine) of IB was significant only for the normal control pairs and the DZ twins. These results suggest that the kinetic constants of IB in blood platelets are not under genetic control and that interassay variance significantly affects the absolute values for Kd and Bmax of 3H-IB. The environmental and assay factors that influence 3H-IB may account for the numerous discrepancies in platelet 3H-IB between various research reports.
Article
1. The relationship between 14C-serotonin uptake, 3H-imipramine binding and surface area was studied in blood platelets. 2. Platelet rich plasma from 5 normal subjects was divided into 5 size subfractions by differential centrifugation and the total surface areas computed from platelet size profiles. 3. Linear relationships were found between both uptake and binding as a function of surface area regardless of platelet size. 4. The results suggest that platelet heterogeneity may contribute to the variability of uptake measurements which are commonly expressed on a per platelet basis. In contrast, the importance of heterogeneity to binding measurements, which are expressed per unit protein, may have been previously overestimated.
Article
In order to examine whether species differences of benzodiazepine receptor subunits exist, we compared the fluorographic pattern of photoaffinity labelled subunits after SDS-PAGE in five species: fish, frog, chicken, mouse and calf. Each species showed a distinct pattern of specifically labelled proteins. We conclude that species variation of benzodiazepine receptor does indeed exist.
Article
Previous reports of elevated platelet serotonin (5-HT) concentrations in autistic subjects suggest that platelet 5-HT uptake might be altered in autism. Parameters of 3H-imipramine (IMI) binding were measured in 11 drug-free autistic subjects and 10 normal volunteers. Similar means (±SD) for Bmax(autistics, 1350 ± 171 fmole / mg protein; normals, 1590 ± 206 fmole / mg protein) and Kd(autistics, 0.98 ± 0.10 nM; normals, 0.94 ± 0.13 nM) were found in the two groups. The normal number (Bmax) and affinity (Kd) of the IMI binding site in autistic subjects suggest that the regulation of 5-HT uptake is not different in autism.
Article
Ten patients with DSM-III diagnoses of nonbipolar recurrent major depression were studied in an attempt to assess the relationship between 3H- imipramine binding site density and clinical depressive state. They were compared with eight healthy controls who had no past or family history of affective disorders. Evaluations with the Hamilton Rating Scale for Depression and the Self-Rated Scale for Depression were done on the same day as platelet collection at baseline, and also at 2 and 5 weeks after the beginning of treatment with tricyclic antidepressants. The number (Bmax) and the affinity (Kd) of platelet 3H-imipramine binding sites were highly correlated with the improvement of the clinical depression. These results raise the interesting possibility that a decrease in 3H-imipramine binding sites may be a state-dependent marker in patients suffering from nonbipolar recurrent major depression.
Article
Treatment of human platelets with activators of protein kinase C (PKC) for 5-20 min resulted in substantial reductions in the rate of platelet serotonin (5-HT) transport. The mean Vmax observed after 5 min treatment with 1 microM 4-beta-12-tetradecanoylphorbol 13-acetate (beta-TPA) was 66% (n = 16, P = 0.0001) of the control value. 5 min of treatment with 1 microM mezerein reduced uptake to 78% (n = 3, P = 0.01) of control. Both beta-TPA and mezerein had little effect on the Km of transport and had EC50 values of approx. 100 mM when a 20-min treatment period was used. The maximum effects of both were reached at approx. 20 min and could be blocked with staurospine. The beta-TPA effect was stereospecific, as alpha-TPA did not alter platelet 5-HT uptake. Although the PKC activators may have altered transmembrane ion-gradients for Na+ and Cl-, which are co-transported with 5-HT, minimizing ion-gradient changes had little effect on the observed reductions in transport. The PKC activators also had little or no effect on platelet 5-HT release or on the number (Bmax) of 5-HT transporters expressed at the platelet surface. The data indicate that PKC activation may down-regulate the activity of the 5-HT transporter in platelets. Apparently, most of this effect is mediated through mechanisms other than changes in ion-gradients, reductions in the number of available transporters, or increased 5-HT release. The apparent regulation of 5-HT transport by PKC may have important implications in platelet and neuronal functioning.
Article
14C-Serotonin (5-HT) uptake and 3H-imipramine binding (IB) were studied in the blood platelets of 20 obsessive-compulsive disorder (OCD) patients, 53 normal controls (5-HT uptake) and 32 normal controls (IB binding). The maximum number of binding sites (Bmax) was significantly decreased in OCD patients compared to normal controls, but there was no difference in the affinity for 3H-imipramine (Kd). The affinity for 5-HT uptake (Km) was also decreased in the OCD patients but the maximum velocity of 5-HT uptake sites (Vmax) was not significantly different in OCD patients and normal volunteers. There were trends for the Slowness Subscale of the Maudsley Obsessional-Compulsive Inventory (MOCI) to be positively correlated with the Km of 5-HT uptake (p = 0.094), whereas the Global Scale, Checking Subscale, and Doubting Conscientiousness Subscale of MOCI were negatively correlated with the Kd of IB (p = 0.066, p = 0.08, and p = 0.062, respectively). The results provide further evidence for the dysfunction of the serotonergic system in OCD.
Article
We measured platelet 3H-imipramine binding parameters in 16 subjects affected by different types of mental deficiency, all characterized by hyperactive and/or aggressive behaviour, and in 16 healthy controls. The patients had a lower maximum binding capacity than the controls, with no difference in Kd, irrespectively of the type of mental disorder. These findings suggest a link between 5-HT disturbances, reflected by reduced imipramine binding sites, and behavioural dyscontrol, expressed as hyperactivity and aggression.
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Several methodological factors in the preparation of platelets and the determination of platelet 3H-imipramine (3H-IMI) binding were examined. The ionic composition of the assay significantly affected platelet 3H-IMI binding. Approximately 25% of the specific binding of 3H-IMI to intact platelet preparations was retained in the absence of sodium and chloride ions. The addition of sodium ions enhanced the specific binding of 3H-IMI, but the addition of chloride in the presence of sodium had a more pronounced effect, enhancing binding approximately five-fold over that observed with the addition of sodium. Sodium was the only cation tested that enhanced binding. Only halides enhanced binding in the presence of sodium with the following order of potency: Cl- greater than Br- greater than I- = F-. Ions increased the density of binding sites (Bmax) and did not affect the affinity of the binding sites for 3H-IMI. In the presence of sodium and chloride, the use of serotonin (5HT) to define nonspecific binding in saturation experiments resulted in lower binding densities (Bmax) than when desipramine was used to define nonspecific binding. The component of binding that was insensitive to 5HT was roughly equal to the Bmax of 3H-IMI binding obtained in the absence of sodium and chloride using desipramine to define nonspecific binding. Overall, these data suggest that not all 3H-IMI binding that is displaced by desipramine is related to serotonergic mechanisms, and suggest that 5HT is a better choice than desipramine for the determination of the nonspecific binding of 3H-IMI. In addition, the binding of 3H-IMI to different platelet preparations was compared. The binding of 3H-IMI to intact platelets was less than that obtained using lysed platelet membranes when data were expressed per mg protein. The Coomassie Blue dye-binding method to determine platelet protein resulted in greater Bmax values than were obtained with the Folin phenol reagent method. The method of platelet preparation that is commonly used to prepare platelets for 3H-IMI binding resulted in similar binding values when compared to a method that prepares the entire platelet population. The results suggest that some, but not all, variations in laboratory methods used to prepare platelets and assay for platelet 3H-IMI binding may affect clinical studies examining this measure.
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A decreased density of platelet 3H-imipramine (3H-IMI) binding sites has been proposed as a putative trait marker of major depressive illness. However, subsequent studies have demonstrated that the number of such sites is increased so as to be more like normal controls upon chronic treatment with antidepressant drugs. In addition, there is some evidence to suggest that altered 3H-IMI binding may be secondary to elevated plasma cortisol levels which are common in depressed patients and which normalize with remission. The present study compares platelet 3H-imipramine binding, plasma cortisol levels, and clinical improvement of 10 endogenous depressed patients before and after 6 weeks of treatment with imipramine-HCl. Total high affinity 3H-IMI binding sites were further differentiated into two subclasses on the basis of their relative sensitivities to cyanoimipramine (CNIMI) inhibition. Treatment was associated with a significant increase (134%) in CNIMI resistant binding but a decrease (45.2%) in CNIMI sensitive binding. While the former was significantly correlated with posttreatment cortisol levels, no significant correlation was found between cortisol and CNIMI specific binding. Neither site appeared to be directly related to mood state. The significance of these findings to the evaluation of platelet binding as a trait dependent marker is discussed.
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Platelet 3-H-imipramine binding exhibits considerable variation, both interindividually and between several groups. The aim of this study was to measure 3-H-imipramine binding, simultaneously in platelet membranes vs. intact platelets vs. cytosol or intracytosolic protein in order to determine their effect on Bmax and Kd values. 3-H-imipramine binding was carried out at different protein concentrations. Our results indicate that the affinity constant is heavily influenced by the presence of cytosol and intact platelets in membrane preparations. Finally, we demonstrate a negative correlation between Bmax and protein concentration. Only perfect analytical conditions will allow platelet 3-H-imipramine binding to be a biological marker for affective disorders.
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Human platelets were subjected to hypotonic lysis after being loaded intracellularly with glycerol (4.3 m) by centrifugation in a glycerol gradient under controlled conditions. A homogeneous membrane fraction, free of intracellular organelles, was isolated after a single density step centrifugation, while continuous sucrose density gradient centrifugation gave two subfractions with densities of 1.090 and 1.120, and with identical isoelectric points (pI 3.9) as determined by isoelectric focusing. Compositional studies indicated that the platelet membrane was a lipoglycoprotein with a carbohydrate content of approximately 7%. The sugar components were identified as glucose, galactose, mannose, hexosamines (GlcN:GalN = 6:1), sialic acid, and fucose. The chemical composition of the platelet membranes isolated at d, 1.090 was protein, 31.9%; lipid, 55.9%; carbohydrate, 7.3%; RNA, 0.3%; DNA, 0.0%; and for those isolated at d, 1.120, it was protein, 40.1%; lipid, 48.2%; carbohydrate, 6.9%; RNA, 0.4%; DNA, 0.0%. The membranes were characterized by a high molar ratio of cholesterol to phospholipid (0.49 and 0.45) that was similar to that found in liver plasma membranes. Phosphodiesterase, acid phosphatase and ATPase were purified 8-, 4-, and 2-fold, respectively, in the membrane fractions, whereas succinic dehydrogenase, esterase, and a variety of β-glycosidases were present only at very low levels. These results demonstrate the absence of intracellular membranes and, together with the chemical analyses, they suggest that the outer membrane of the platelet is similar to plasma membranes of other cells, and they provide biochemical confirmation of its origin from the plasma membrane of the megakaryocyte. The vesicles of the two membrane bands differed in ultrastructure, the lighter (d, 1.090) having an average diameter of 1750 A with numerous concentric double membrane structures, while the heavier (d, 1.120) consisted of single membranes of diameter 700 A.
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Plasma membrane vesicles isolated from porcine blood platelets take up approximately 8 to 15 pmol of [3H]imipramine per mg of membrane protein. This apparent binding requires Na+ in the external medium and is reversed by 5-hydroxytryptamine and fluoxetine. The apparent KD for imipramine uptake is 23 nM, which agrees well with the KI for competitive inhibition of 5-hydroxytryptamine transport by imipramine. In contrast to 5-hydroxytryptamine transport, imipramine uptake is not dependent on transmembrane Na+ and K+ gradients and is insensitive to ionophores such as nigericin and gramicidin which dissipate these gradients. Although 5-hydroxytryptamine rapidly and competitively displaces imipramine from membrane vesicles, imipramine does not cause 5-hydroxytryptamine efflux and inhibits 5-hydroxytryptamine exchange. These results are consistent with the proposal that imipramine binds to the substrate site of the 5-hydroxytryptamine transporter but cannot be transported.
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The influence of the membrane environment on the integrity of the human platelet [3H]-imipramine recognition site was examined. When platelet membranes were isolated in a buffer containing enzyme inhibitors (EDTA, EGTA and antiprotease) a significantly greater number of high affinity [3H]-imipramine binding sites was observed. A calcium-stimulated degradation of imipramine sites was also demonstrated. This degradation occurred in vitro over physiologically relevant time periods. Furthermore, inactivation of imipramine binding was achieved by very low concentrations (IC50 = 5 microgram/ml) of phospholipase A2. Specific serotonin reuptake inhibitors were potent displacers of [3H]-imipramine binding; histamine (H1), alpha-adrenergic (alpha 1), and muscarinic agents were much less active. The receptor was shown to be proteinaceous in nature due to its sensitivity to protease, heat denaturation and chemical modification with N-ethylmaleimide. From these results it is proposed that membrane lipid perturbations, catalyzed by calcium, may control expression of platelet [3H]-imipramine sites. The relation of this recognition site to aminergic systems and the possible relevancy to the action of antidepressants are addressed.
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• The high-affinity tritiated (3H) imipramine binding sites are functionally (and perhaps structurally) associated with the presynaptic neuronal and platelet uptake sites for serotonin. Since there is an excellent correlation between the relative potencies of a series of antidepressants in displacing 3H-imipramine from binding sites in human brain and platelet, we have examined the binding of 3H-imipramine to platelets from 14 depressed patients and 28 age- and sex-matched controls. A highly significant decrease in the number of 3H-imipramine binding sites, with no significant change in the apparent affinity constants, was observed in platelets from the depressed patients compared with the controls. These results, coupled with previous studies showing a significant decrease in the maximal uptake of serotonin in platelets from depressed patients, suggest that an inherited or acquired deficiency of the serotonin transport protein or proteins may be involved in the pathogenesis of depression.
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The discovery of high-affinity binding sites for psychoactive drugs such as benzodiazepines, opiates and neuroleptics has opened up new approaches to the study of these drugs and their mechanisms of action. Although most tricyclic antidepressants inhibit neuronal uptake of noradrenaline and serotonin, their mechanism of action remains unclear. Changes in the sensitivity of the beta-receptor after chronic tricyclic antidepressant treatment suggest that they modulate noradrenergic neurotransmission. Tricyclic antidepressants also act directly on cholinergic, histaminergic, alpha-adrenergic and serotonergic receptors. It is not clear, however, which, if any, of these effects are related to the primary antidepressant effect or whether they are simply responsible for some of the side effects. We have thus investigated the possibility that specific binding sites for tricyclic antidepressants exist in the central nervous system. So far, binding studies using 3H-labelled tricyclic antidepressant drugs have only detected binding to histaminergic H2 and cholinergic muscarinic receptors and low-affinity binding. We demonstrate here a population of specific high-affinity binding sites for 3H-imipramine on brain membranes which may be responsible for the antidepressant effects of these drugs.
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Some recent modifications of the protein assay by the method of Lowry, Rosebrough, Farr, and Randall (1951, J. Biol. Chem.193, 265–275) have been reexamined and altered to provide a consolidated method which is simple, rapid, objective, and more generally applicable. A DOC-TCA protein precipitation technique provides for rapid quantitative recovery of soluble and membrane proteins from interfering substances even in very dilute solutions (< 1 μg/ml of protein). SDS is added to alleviate possible nonionic and cationic detergent and lipid interferences, and to provide mild conditions for rapid denaturation of membrane and proteolipid proteins. A simple method based on a linear log-log protein standard curve is presented to permit rapid and totally objective protein analysis using small programmable calculators. The new modification compared favorably with the original method of Lowry et al.
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Four methods of platelet lysis, namely, sonication, nitrogen cavitation in a high pressure bomb, the use of a “no clearance” tissue homogenizer and simple osmotic lysis have been compared in terms of ease, effectiveness and reproducibility in comparison with a method involving the hypotonic lysis of glycerol-loaded platelets ; the most extensive studies have been carried out using sonication and the tissue homogenizer. In order to reduce the degree of fragmentation of the platelet membrane, the use of surface stabilizing agents such as zinc chloride and fluorescein mercuric acetate have also been investigated. Membranes obtained in these different ways have been isolated by centrifugation on continuous or discontinuous sucrose density gradients and the resulting fractions compared by electron microscopy, enzymatic activity, chemical analysis and hemagglutination inhibition. The glycerol-lysis technique was found to be most effective (85%) in untreated platelets and the degree of lysis was considerably reduced in platelets hardened by the zinc chloride method. Ultrastructural studies showed that the largest membrane fragments (175 nm) were obtained by glycerol lysis of untreated platelets and that smaller vesicles (50-100 nm) were obtained by the other techniques even following membrane stabilization. Double membrane structures were obtained in several of these methods.
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High affinity and saturable binding sites for [3H] imipramine have been demonstrated on human platelet membranes. These binding sites appear to be specific for tricyclic antidepressants and their pharmacologically-active metabolites. In contrast, inactive tricyclic compounds such as the parent iminodibenzyl and iminostilbenes do not inhibit [3H] imipramine binding. The binding of [3H] imipramine to human platelets is of high affinity , saturable , and sensitive to proteolytic degradation. The effects of various drugs and neurotransmitter agonists and antagonists suggests that these binding sites are pharmacologically distinct from the previously reported binding of tricyclic antidepressants to alpha-adrenergic, muscarinic-cholinergic, and histaminergic receptors. The binding characteristics of [3H] imipramine to platelets is similar to that in rat and human brain and may thus serve as a useful model in elucidating the pharmacological and physiological significance of these binding sites.
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The high-affinity tritiated (3H) imipramine binding sites are functionally (and perhaps structurally) associated with the presynaptic neuronal and platelet uptake sites for serotonin. Since there is an excellent correlation between the relative potencies of a series of antidepressants in displacing 3H-imipramine from binding sites in human brain and platelet, we have examined the binding of 3H-imipramine to platelets from 14 depressed patients and 28 age- and sex-matched controls. A highly significant decrease in the number of 3H-imipramine binding sites, with no significant change in the apparent affinity constants, was observed in platelets from the depressed patients compared with the controls. These results, coupled with previous studies showing a significant decrease in the maximal uptake of serotonin in platelets from depressed patients, suggest that an inherited or acquired deficiency of the serotonin transport protein or proteins may be involved in the pathogenesis of depression.
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The high-affinity binding of 3H-imipramine to platelet membranes from 23 untreated depressed men and women and 16 normal men and women was studied. The maximal binding (Bmax) in the depressed groups, male as well as female, were significantly lower (20%) than the normal groups, while the dissociation constants (Kd) were not significantly different. Neither Bmax nor Kd differed statistically between the sexes for either the depressed or normal subject groups. No significant correlations were found between age and either Bmax or Kd for any of the four subject subgroups. A statistically significant correlation did exist, though, between the Bmax of all of the subjects and their respective dissociation constants. In addition, 3H-imipramine binding to platelet membranes remained unchanged during two weeks of daily therapy on the monoamine oxidase inhibitor, tranylcypromine.
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The high-affinity binding of triatiated imipramine to platelet membranes was compared in samples from 16 untreated depressed women and 21 age-matched controls of the same sex. The maximal binding in the depressed group was significantly lower than that of the controls, although the affinity constants were similar. These results suggest that binding of tritiated imipramine in human platelets may represent a biochemical index of depression, possibly reflecting similar changes in the brain.
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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.
Article
Human platelets have been shown to possess high-affinity binding sites for 3H-imipramine. These binding sites have a similar affinity and drug specificity to those already described in rat brain. The platelets from healthy volunteers show no difference in 3H-imipramine binding between the sexes but there is a decrease in maximal 3H-imipramine binding with increasing age of the donor.
Depressed patients have decreased binding of tritiated imipramine to platelet serotonin 'transporter' Demonstration of specific "high-affinity" binding sites for 3H-imipramine on human platelets
  • Paui
  • Rehavi M Sm
  • P Skolnick
  • Jc Ballenger
  • Goodwin
Paui SM, Rehavi M, Skolnick P, Ballenger JC, Goodwin FK (1981) Depressed patients have decreased binding of tritiated imipramine to platelet serotonin 'transporter'. Arch Gen Psychiatry 38:1315-Paul SM, Rehavi M, Skolnick P, Goodwin FK (1980) Demonstration of specific "high-affinity" binding sites for 3H-imipramine on human platelets. Life Sci 26:953-959