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Platelet [3H]imipramine and [3H]paroxetine binding in depressed patients
Journal of Affective Disorders 44 (1997) 79–85
Research report
Platelet [ 3 H]imipramine and [ 3 H]paroxetine binding in depressed patients a, ´ a , J. Vallejo a , B. Arranz a , b , M.A. Navarro a , F. Liron ´ a, P. Rosel *, J.M. Menchon P. Alvarez a a
Hormone Unit and Department of Psychiatry, Hospital Princeps d’ Espanya, Feixa Llarga s /n, Hospitalet de Llobregat, Barcelona Spain b ´ Department of Psychiatry. Institut Municipal d’ Investigacio´ Medica ( IMIM), Barcelona Spain Received 23 October 1996; accepted 3 March 1997
Abstract [ 3 H]Paroxetine and [ 3 H]imipramine binding to blood platelet membranes was simultaneously measured in 63 control subjects and 18 patients with DSM-III-R criteria for major depression with melancholia. Both binding sites showed significantly different ( p , 0.001) maximum binding (Bmax ) and equilibrium dissociation constant (Kd ) values. Age was not correlated with either [ 3 H]imipramine Bmax or Kd values, but a negative correlation was found between [ 3 H]paroxetine Bmax and age in healthy controls. Furthermore, depressed patients showed significantly lower [ 3 H]imipramine Bmax values ( p , 0.001) and higher Kd values ( p , 0.001) in comparison to the control group. No differences were observed in [ 3 H]paroxetine Bmax and Kd values between the two groups. 1997 Elsevier Science B.V. Keywords: Imipramine binding; Paroxetine binding; Endogenous depression
1. Introduction [ 3 H]Imipramine was the first antidepressant reported to have a high affinity binding site located both in brain and platelet membranes and associated with the serotonin (5-HT) uptake mechanism (Briley et al., 1979; Paul et al., 1980; Langer et al., 1980). Since then, several reports indicating a decreased number of [ 3 H]imipramine binding sites in depressed patients have suggested the possibility of its being a *Corresponding author.
biological marker of depression (Langer et al., 1987), although the results reported so far are very conflicting (Suranyi-Cadotte et al., 1984; Nankai et al., 1986; Kanof et al., 1987). Recently, specific high affinity binding of [ 3 H]paroxetine to both human brain and platelet membranes has also been found (Mellerup and Plenge, 1986; Habert et al., 1985; Plenge et al., 1990, 1991; Arranz and Marcusson, 1994). These studies suggest that both radioligands bind to the same 5-HT transporter complex, although their binding sites could be located on different subunits. Accordingly,
0165-0327 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved PII S0165-0327( 97 )00033-5
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it seems that paroxetine binding sites are identical in neural and platelet membranes (Klompenhouwer et al., 1990; Mellerup and Langer, 1985; Mellerup and Plenge, 1986) whereas it seems that only part of the imipramine binding can be attributed to this 5-HT reuptake complex (Briley and Langer, 1981; Wennogle and Meyerson, 1985; Sette et al., 1983; Plenge et al., 1991). In this respect, previous reports have found that the affinity of [ 3 H]paroxetine binding to human brain and platelet membranes is higher than that reported for [ 3 H]imipramine, whereas the maximum number of binding sites is comparable (Mellerup and Langer, 1985; Mellerup et al., 1993; Arranz and Marcusson, 1994; Rosel et al., 1997). In the present study, the 5-HT uptake capacity has been examined by determining simultaneously both [ 3 H]imipramine and [ 3 H]paroxetine binding in platelet membranes from a large number of healthy controls (n 5 63). Furthermore, binding experiments with these two radioligands have been performed in a group of melancholic depressed patients so as to determine the state of the 5-HT uptake mechanism.
study was approved by the local ethical committee and by the National Research Committee.
2.2. Platelet membrane preparation A 40 ml volume of blood was drawn by antecubital venopuncture and collected into plastic tubes containing 1 ml anticoagulant (2.5% trisodium citrate, 0.8% citric acid, 2.5% glucose). Each tube was centrifuged at 200 g for 30 min at room temperature. The platelet rich plasma obtained was collected and centrifuged at 10 000 g (48, 10 min). The supernatant was discarded and the platelets were washed and centrifuged twice at 10 000 g (48, 10 min) in 8 ml of buffer (50 mM Tris–HCl, 150 mM NaCl, 20 mM EDTA, pH 7.4). The supernatant was also discarded and the platelet membranes were lysed with an hypotonic buffer (5 mM Tris–HCl, 5 mM EDTA). After homogenizing the samples with a Polytron PT 3000, they were centrifuged twice at 20 000 g (48, 10 min). The pellet thus obtained was finally suspended in incubation buffer (50 mM Tris–HCl, 3 mM KCl, 120 mM NaCl, pH 7.4) to a protein concentration of about 0.5 mg / ml.
2. Material and methods
2.3. Binding assays 2.1. Subjects The control group included 63 healthy individuals (31 men and 32 women) well known to the investigators, with a mean age of 43.8613 years (range 24–70 years). A clinical interview was carried out in order to rule out any psychiatric disorder. Individuals with a 21-item Hamilton Depression Rating Scale (HDRS) score over 6, consuming drugs known to interfere with the serotonergic system, and a family history of psychiatric disorder were excluded from the study. The patient group included 18 (6 male and 12 female) inpatients with a mean age of 59.4610 years (range 38–75 years) meeting DSM-III-R criteria for major depression with melancholia, either with (n 5 10) or without (n 5 8) mood congruent psychotic features. The 21-item HDRS score from this group was 33.165.6 points. All patients were drug free for at least 2 weeks before the sample collection. None of the patients had ever received lithium therapy. Informed consent was obtained in all cases. This
For the [ 3 H]paroxetine binding assay, 0.750 ml of membrane suspensions (0.02 mg / ml of protein) were incubated with six different concentrations of [ 3 H]paroxetine (0.0075 to 0.5 nM) during 60 min at room temperature. Total incubation volume was 1.6 ml. Specific [ 3 H]paroxetine binding was determined as the difference between the binding in the absence and presence of 100 mM 5-HT (Arranz and Marcusson, 1994). The [ 3 H]imipramine binding assays were performed by incubating 0.1 ml of each membrane suspension (0.5 mg / ml of protein) with 0.1 ml of [ 3 H]imipramine at six different concentrations (0.25 to 10 nM). Incubation was performed in an ice tray inside a refrigerated room so as to prevent dissociation from the receptor. Total incubation volume was 0.5 ml. Non-specific binding was also determined with 100 mM 5-HT (Rosel et al., 1995). Samples were analysed blind to subject classification. After incubation, 5 ml of ice-cold incubation buffer was added to each tube, and samples were rapidly filtered through Whatman GF / C glass fiber
P. Rosel et al. / Journal of Affective Disorders 44 (1997) 79 – 85
filters using a 24-channel cell Harvester (Brandell, Gaithersburg, MD, USA). Filters were then washed, placed in vials with scintillation liquid, and the radioactivity trapped in each filter counted according to conventional scintillation techniques. Protein concentrations were determined by a modification of Lowry’s method. Corrections were made for the contribution of Tris–HCl to the optical density (Rej and Richards, 1974; Peterson, 1977; Markwell et al., 1987).
2.4. Data and statistical analysis The binding data was analysed using the iterative non-linear least squares curve fitting program EBDA, and values for maximum binding (Bmax ) and dissociation constant (Kd ) were obtained. As all values were normally distributed (Kolmogorov–Smirnov test), the Student’s t-test for unpaired data was used to test differences in Bmax and Kd between the two groups of patients (controls versus depressed). A paired t-test was used to test differences in Bmax and Kd obtained for both radioligands ([ 3 H]imipramine versus [ 3 H]paroxetine) within the control group. Pearson’s correlation coefficient was used to test associations. The influence of age in the binding parameters was tested through a analysis of covariance.
2.5. Materials [ 3 H]Paroxetine (17.8 Ci / mmol) and [ H]imipramine (24 Ci / mmol) were obtained from New England Nuclear (Boston, MA, USA). 5-Hydroxytryptamine creatinine sulphate (serotonin) was obtained from Sigma. All other chemicals were standard laboratory reagents. 3
3. Results [ 3 H]Paroxetine and [ 3 H]imipramine binding parameters obtained in the 63 healthy controls under study are indicated in Table 1. Significant differences between both radioligands were found for both Bmax ( p , 0.001) and Kd ( p , 0.001), with [ 3 H]paroxetine showing lower Bmax and lower Kd (better affinity) than [ 3 H]imipramine. Bmax values determined with
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Table 1 [ 3 H]Imipramine and [ 3 H]paroxetine binding parameters in platelet membranes from 63 healthy controls
[ 3 H]Imipramine [ 3 H]Paroxetine
Bmax (fmol / mg)
Kd (nM)
1842660 (860–2750) 1505666 (466–2656) p , 0.001
1.1660.03 (0.62–1.89) 0.0760.003 (0.04–0.13) p , 0.001
Both Bmax and Kd are expressed as mean6S.E.M., with range values indicated in parenthesis. A paired t-test was used for statistical comparison between [ 3 H]imipramine and 3 [ H]paroxetine binding.
both radioligands were significantly correlated (r 5 0.26; p 5 0.04, Pearson’s correlation coefficient), whereas no correlation was noted for Kd values (r 5 0.03; p . 0.05). When [ 3 H]imipramine binding parameters were plotted with regard to the age of the individual, no significant correlation was obtained for either Bmax (r 5 0.16; p . 0.05) or Kd (r 5 0.07; p . 0.05). However, when [ 3 H]paroxetine was the radioligand used to label the 5-HT uptake site, a significant negative correlation was observed between its Bmax and the control age (r 5 2 0.35; p 5 0.005). These results are reflected in Fig. 1. The maximum number of [ 3 H]imipramine binding sites was significantly ( p , 0.001) lower in depressed patients than in healthy individuals (10426149 fmol / mg versus 1842660 fmol / mg respectively). Significant differences ( p , 0.001) were also found for [ 3 H]imipramine Kd values, being 2.1560.44 nM (mean6S.E.M. value) in the depressed group and 1.1960.05 nM in control individuals. [ 3 H]Paroxetine binding parameters did not show any significant differences between the two groups (Table 2). These same results were obtained when an analysis of covariance, with age as covariant, was used instead of Student’s t-test.
4. Discussion The Bmax and Kd values obtained in this study for 3 3 the radioligands [ H]paroxetine and [ H]imipramine are in agreement with those reported by other authors
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Fig. 1. Correlation between age and [ 3 H]paroxetine (A) or [ 3 H]imipramine (B) Bmax in healthy individuals. A significant negative correlation (r 5 2 0.35; p 5 0.005) was noted between [ 3 H]paroxetine Bmax and age (Pearson’s correlation coefficient).
Table 2 [ 3 H]Imipramine and [ 3 H]paroxetine binding parameters in depressed patients with respect to healthy controls
[ 3 H]Imipramine
Controls Patients
[ 3 H]Paroxetine
Controls
Bmax (fmol / mg)
Kd (nM)
1842660 10426149 ( p , 0.001) 1505666 12986122 N.S
1.1660.03 2.1560.44 ( p 5 0.04) 0.07460.003 0.08060.009 N.S.
Bmax and Kd values are expressed as mean6S.E.M.. Student’s t-test for unpaired data was used to test differences. N.S.: non-significant.
(D’Haenen et al., 1988; Nankai et al., 1993; Mellerup and Plenge, 1986; Mellerup et al., 1993; Klompenhouwer et al., 1990; Andersson and Marcusson, 1990; Arranz and Marcusson, 1994). [ 3 H]Paroxetine seems to label a significantly lower number of binding sites than [ 3 H]imipramine, while at the same time showing a significantly higher affinity (lower Kd ). These results do not completely agree with those reported by Mellerup et al. (1993) who found similar Bmax values for both radioligands, probably due to the existence of methodological differences in their use of unlabelled imipramine to determine the amount of non-specific binding. Despite the evidence of the binding protein labelled by these two radioligands being closely related to the 5-HT uptake mechanism located either in neural or platelet membranes (Langer et al., 1987; Plenge et al., 1990, 1991), several studies on brain tissue have shown that [ 3 H]paroxetine has a more selective distribution than [ 3 H]imipramine in serotonergic regions (Hrdina, 1984; Hrdina et al., 1990a,b). In the present study, the finding of a poor correlation between the maximum number of sites labelled with both radioligands and the absence of correlation between their respective Kd values further supports the suggestion that both antidepressants bind to different subunits on the same macromolecular complex of the 5-HT reuptake mechanism (Plenge et al., 1991). This hypothesis is further supported by our finding of a higher number of binding sites labelled with [ 3 H]imipramine as compared with [ 3 H]paroxetine, together with their different Kd values. In this respect, similar results concerning the lack of correlation between both radioligands have also been reported by other researchers (Mellerup and Langer, 1985; Habert et al., 1985; Suranyi-Cadotte et al., 1989). Age is an important confounding variable when studies of [ 3 H]imipramine binding are performed, as the relationship between this variable and the serotonergic function is still unclear. A certain controversy exists between studies about the variations in [ 3 H]imipramine binding with increased age. Thus, while some authors have reported increased [ 3 H]imipramine Bmax in blood platelets from elderly controls (Schneider et al., 1985; Suranyi-Cadotte et al., 1985), others have indicated the existence of a negative correlation between both variables in con-
P. Rosel et al. / Journal of Affective Disorders 44 (1997) 79 – 85
trol subjects (Langer et al., 1980; Wagner et al., 1985; Halbreich et al., 1991). The lack of correlation found in this study between age and either [ 3 H]imipramine Bmax or Kd , is in agreement with other reports (Marcusson et al., 1987; Baron et al., 1984). Regarding the relationship between [ 3 H]paroxetine binding sites and age, the small number of studies performed so far also show some discrepancies. Thus, our finding of a reduced number of [ 3 H]paroxetine binding sites with increased age is in agreement with the study of Nankai et al. (1993), and not with the results reported by Andersson et al. (1992), probably due to the smaller size of the control group used in their study. Another important source of discrepancy is the choice of the drug used to define the non-specific binding. In this respect, the vast majority of studies on [ 3 H]imipramine binding have used desipramine to define specific binding. However, this compound has been reported to include a significant proportion (up to 30%) of binding not related to the 5-HT uptake site (Marcusson et al., 1985, Marcusson et al., 1986, ¨ Marcusson et al., 1987; Backstrom and Marcusson, 1987; Hrdina, 1989). Regarding the [ 3 H]paroxetine binding assay, 5-HT (Arranz and Marcusson, 1994), fluoxetine (Nankai et al., 1993; Moret and Brley, 1991), citalopram (Andersson and Marcusson, 1990), and clomipramine (Klompenhouwer et al., 1990) have been used at varying concentrations to define non-specific binding. In our binding assays, 100 mM 5-HT was used to define non-specific binding, as it has been demonstrated that 5-HT concentrations between 100 mM and 1 mM do not include the low affinity non-serotonergic component of the binding (Arranz and Marcusson, 1994). In the present study, depressed patients showed significant lower number of [ 3 H]imipramine binding sites, together with a significantly lower affinity constant (higher Kd ) than healthy controls. However, no significant variations were found in the [ 3 H]paroxetine binding parameters. These results are in agreement with those published by other researchers (Iny et al., 1989; Suranyi-Cadotte et al., 1989; Nankai et al., 1993; D’Haenen et al., 1988), although none of them have simultaneously compared both radioligands in the same population. Since our patients had been drug-free for at least two weeks before the blood sample collection, the reported Kd
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increase is not likely to be due to pharmacological interferences, nor to methodological flaws, as samples were analysed blind to subject classification. Nevertheless, although a four-week wash-out period would undoubtedly have been much better to exclude any pharmacological interferences, our inpatients could not be maintained without treatment for such a long period. Furthermore, the binding assays used in this study have been previously standardized with respect to external variables such as membrane preparation, concentration of the displacing compound, incubation time and temperature, and protein concentration (Arranz and Marcusson, 1994; Rosel et al., 1995). Moreover, our patients were diagnostically homogeneous, since all of them suffered from severe major depression with melancholic features, their age and sex distribution was similar to that of the control group, and the sampling procedure was performed in the same year period (autumn–winter) so as to avoid seasonal variations (Rosel et al., 1996a). The present results may have clinical correlates. Thus, while imipramine treatment has been proved to be effective in severe melancholic and delusional hospitalized patients, there is a lack of clinical evidence on the efficacy of paroxetine (or other selective serotonin reuptake inhibitors) in this subgroup of depressed patients (Osser, 1993). Furthermore, there is a trend in clinical settings to use tricyclic antidepressants in this particular group of psychiatric patients. There is no doubt that this clinical evidence may parallel the findings of the present study on a decreased number of [ 3 H]imipramine but not [ 3 H]paroxetine binding sites in severe melancholic and delusional hospitalized patients. Despite the higher potency and affinity of the selective 5-HT uptake blocker paroxetine, the present study demonstrates the better diagnostic sensitivity and specificity displayed by [ 3 H]imipramine in blood platelets from depressed patients.
Acknowledgments The authors wish to thank Pilar Alastruey and Elena Arnaiz from the technical staff of the Hormone
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Unit for their expert assistance. This work has been supported by a FIS grant (93 / 0318).
References 3
Andersson, A. and Marcusson, J. (1990) [ H]Paroxetine binding in human platelets in relation to age and sex. Neurobiol. Aging 11, 615–618. Andersson, A., Sundman, I. and Marcusson, J. (1992) Age stability of human brain 5-HT terminals studied with [ 3 H]paroxetine binding. Gerontology 38, 127–132. 3 Arranz, B. and Marcusson, J. (1994) [ H]Paroxetine and [ 3 H]citalopram as markers of the human brain 5-HT uptake site: a comparison study. J. Neural Transm. (Gensect) 97, 27–40. ¨ Backstrom, I.T. and Marcusson, J. (1987) 5-Hydroxytryptamine3 sensible [ H]imipramine binding of protein nature in the human brain, I. Characteristics. Brain Res. 425, 128–136. Baron, M., Kowalik, S., Barkai, A. and Gruen, R. (1984) High 3 affinity [ H]imipramine binding in human platelets: age and sex effects. Neuropsychobiology 11, 178–180. Briley, M.S., Raisman, R. and Langer, S.Z. (1979) Human platelets possess high affinity binding sites for [ 3 H]imipramine. Eur. J. Pharmacol. 58, 347–348. Briley, M.S. and Langer, S.Z. (1981) Sodium dependency of [ 3 H]imipramine binding in rat cerebral cortex. Eur. J. Pharmacol. 72, 377–380. D’Haenen, H., De Weale, M. and Leysen, J.E. (1988) Platelet [ 3 H]paroxetine binding in depressed patients. Psychiatry Res. 26, 11–17. Habert, E., Grahan, D., Tahraoni, L., Claustre, Y. and Langer, S.Z. (1985) Characterization of [ 3 H]paroxetine binding to rat cortical membranes. Eur. J. Pharmacol. 118, 107–114. Halbreich, U., Rojansky, N., Zander, K.J. and Barkai, A. (1991) Influence of age, sex and diurnal variability on impramine receptor binding and serotonin uptake in platelets of normal subjects. J. Psychiatry Res. 25, 7–18. Hrdina, P.D. (1984) Differentiation of two components of specific [ 3 H]imipramine binding in rat brain. Eur. J. Pharmacol. 102, 481–488. Hrdina, P.D. (1989) Differences between sodium dependent and desipramine defined [ 3 H]imipramine binding in intact human platelets. Biol. Psychiatry 20, 576–584. Hrdina, P.D., Foy, B., Hepher, A. and Summer, R.J. (1990a) Antidepressant binding sites in brain: autoradiographic comparison of [ 3 H]paroxetine and [ 3 H]imipramine localization and relation to serotonin transporter. J. Pharmacol. Exp. Ther. 252, 410–418. Hrdina, P.D., Foy, B. and Summer, R.J. (1990b) 2D imaging of serotonin uptake sites in brain with [ 3 H]paroxetine: comparison with [ 3 H]imipramine. In: B. Leonard and P. Spenser (Eds.), Antidepressants: Thirty Years On. pp. 54–59. Iny, L., Meaney, M., Desjardins, P. and Nair, N.P.V. (1989) [ 3 H]Imipramine and [ 3 H]paroxetine binding in blood platelets
of psychiatric patients and healthy volunteers. J. Clin. Psychopharmacol. 3, 78–82. Kanof, P.D., Coccaro, E.F., Johns, C.A., Siever, L.J. and Davis, K.L. (1987) Platelet [ 3 H]imipramine binding in psychiatric disorders. Biol. Psychiatry 22, 278–286. Klompenhouwer, J.L., Fekkes, D. and Van Hults, A.M. (1990) Seasonal variation in binding of [ 3 H]paroxetine to blood platelets in healthy volunteers: indications for a gender differences. Biol. Psychiatry 28, 509–517. Marcusson, J., Fowler, C.J., Hall, H., Ross, S.B. and Winblad, B. (1985) Specific binding of [ 3 H]imipramine to protease-sensitive and protease-resistent sites. J. Neurochem. 44, 705–711. ¨ Marcusson, J., Backstrom, J.T. and Ross, S.B. (1986) Single-site model of the neuronal 5-hydroxytryptamine uptake and imipramine binding site. Mol. Pharmacol. 30, 121–154. ¨ Marcusson, J., Alafozoff, I., Backstrom, I.T., Eriksson, E., Gottfries, C.G. and Winblad, B. (1987) 5-Hydroxytryptaminesensitive [ 3 H]imipramine binding of protein nature in the human brain. II. Effect of normal aging and dementia disorders. Brain Res. 425, 137–145. Markwell, M.A., Haas, S.M., Bieber, L.L. and Tolbert, N.E. (1987) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87, 203–225. Mellerup, E.T. and Langer, S.Z. (1985) Size determination of binding polymers for [ 3 H]imipramine and [ 3 H]paroxetine in human membranes. Eur. J. Pharmacol. 106, 411–413. Mellerup, E.T. and Plenge, P. (1986) High affinity binding of [ 3 H]paroxetine and [ 3 H]imipramine to rat neuronal membranes. Psychopharmacology 89, 436–439. Mellerup, E.T., Errebo, I., Molin, J., Plenge, P. and Dan, H. (1993) Platelet paroxetine binding and light therapy in winter depression. J. Affect. Dis. 22, 11–15. Moret, C. and Brley, M. (1991) Platelet [ 3 H]paroxetine binding to the serotonin transporter is insensitive to changes in the central serotonergic innervation in the rat. Psychiatry Res. 38, 97–104. Nankai, M., Joshimoto, S., Narita, K. and Takahashi, R. (1986) Platelet [ 3 H]imipramine binding in depressed patients and its circadian variations in healthy controls. J. Affective Disord. 11, 207–212. Nankai, M., Jamada, S. and Joshimoto, S. (1993) Platelet [ 3 H]paroxetine binding in subjects and depressed patients: Relationship to serotonin uptake and age. Psychiatry Res. 51, 147–155. Langer, S.Z., Moret, C., Raisman, R., Dobocovich, M.L. and Briley, M.S. (1980) High affinity [ 3 H]imipramine binding in rat hypothalamus: association with uptake of serotonin but not norepinephrine. Science 210, 1133–1135. Langer, S.Z., Galzin, A.M., Poirier, M.F., Loo, H., Sechter, D. and Zarifian, E. (1987) Association of [ 3 H]imipramine and [ 3 H]paroxetine binding with the 5-HT transporter in brain and platelets: relevance to studies in depression. J. Receptor Res. 7, 499–521. Osser, D.N. (1993) A systematic approach to the classification and pharmacotherapy of nonpsychotic major depression and dysthymia. J. Clin. Psychopharmacol. 13, 133–144. Paul, S.M., Rehavi, M., Kolnick, P. and Goodwin, F.K. (1980)
P. Rosel et al. / Journal of Affective Disorders 44 (1997) 79 – 85 Demostration of specific ‘‘high affinity’’ binding sites for [ 3 H]imipramine on human platelets. Life Sci. 26, 953–959. Plenge, P., Mellerup, E.T. and Laursen, H. (1990) Regional distribution of the serotonin transport complex in human brain, identified with [ 3 H]paroxetine, [ 3 H]citalopram and 3 [ H]imipramine. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 14, 61–72. Plenge, P., Mellerup, E.T. and Laursen, H. (1991) Affinity modulation of [ 3 H]imipramine, [ 3 H]paroxetine and 3 [ H]citalopram binding to the 5-HT transporter from brain and platelets. Eur. J. Pharmacol. 206, 243–250. Peterson, G.L. (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83, 346–356. Rej., Richards, A.H. (1974) Interference by Tris buffer in the estimation of protein by the Lowry procedure. Anal. Biochem. 62, 240–247. ´ J.M. and Rosel, P., Vallejo, J., Oros, M., Serrallonga, J., Menchon, Navarro, M.A. (1995) Changes in platelet [ 3 H]imipramine binding: influences of protein concentration of varying proportions of cytosol or intact platelets and displacing agents used. Biol. Psychiatry 38, 464–470. ´ J.M., Cortadellas, T., Rosel, P., Vallejo, J., Oros, M., Menchon, Navarro, M.A. and Urretavizcaya, M. (1996a) Seasonal variability in blood platelet [ 3 H]imipramine binding in healthy controls: age and gender effects. Biol. Psychiatry 40, 981–985. ´ J.M., Oros, M., Vallejo, J., Cortadellas, T., Rosel, P., Menchon, Arranz, B., Alvarez, P. and Navarro, M.A. (1997) Regional
85
distribution of specific high affinity binding sites for [ 3 H]imipramine and [ 3 H]paroxetine in human brain. J. Neural Transm. (Gensect) 104, 89–96. Schneider, L.S., Severson, J.A. and Sloane, B.B. (1985) Platelet [ 3 H]imipramine binding in depressed elderly patients. Biol. Psychiatry 20, 1232–1234. Sette, M., Briley, S. and Langer, S.Z. (1983) Complex inhibition of [ 3 H]imipramine binding by serotonin and nontricyclic serotonin uptake blockers. J. Neurochem. 40, 622–628. Suranyi-Cadotte, B., Quirion, R. and McQuade, P. (1984) Platelet [ 3 H]imipramine binding: a state dependent marker in depression. Neuropsychopharmacol. 8, 737–741. Suranyi-Cadotte, B., Gauthiers, S. and Lafaille, F. (1985) Platelet [ 3 H]imipramine binding distinguishes depression from Alzheimer dementia. Life Sci. 37, 2305–2323. Suranyi-Cadotte, B., Iny, L., Desjardins, P., Jassa, R. and Welner, P. (1989) Decreased density of platelet [ 3 H]imipramine but not [ 3 H]paroxetine binding sites in major depression. Soc. Neurosci. 15, 673 (Abst. 274.4) Wagner, A., Aberg-Wistedt, A., Asberg, M., Ekqvist, B., Martensson, B. and Montero, D. (1985) Lower [ 3 H]imipramine binding in platelets from untreated depressed patients compared to healthy controls. Psychiatry Res. 16, 131–139. Wennogle, L.P. and Meyerson, L.R. (1985) Serotonin uptake inhibitors differentially modulate high affinity imipramine dissociation in human platelet membranes, Life Sci. 36, 1541– 1550.
Research report
Platelet [ 3 H]imipramine and [ 3 H]paroxetine binding in depressed patients a, ´ a , J. Vallejo a , B. Arranz a , b , M.A. Navarro a , F. Liron ´ a, P. Rosel *, J.M. Menchon P. Alvarez a a
Hormone Unit and Department of Psychiatry, Hospital Princeps d’ Espanya, Feixa Llarga s /n, Hospitalet de Llobregat, Barcelona Spain b ´ Department of Psychiatry. Institut Municipal d’ Investigacio´ Medica ( IMIM), Barcelona Spain Received 23 October 1996; accepted 3 March 1997
Abstract [ 3 H]Paroxetine and [ 3 H]imipramine binding to blood platelet membranes was simultaneously measured in 63 control subjects and 18 patients with DSM-III-R criteria for major depression with melancholia. Both binding sites showed significantly different ( p , 0.001) maximum binding (Bmax ) and equilibrium dissociation constant (Kd ) values. Age was not correlated with either [ 3 H]imipramine Bmax or Kd values, but a negative correlation was found between [ 3 H]paroxetine Bmax and age in healthy controls. Furthermore, depressed patients showed significantly lower [ 3 H]imipramine Bmax values ( p , 0.001) and higher Kd values ( p , 0.001) in comparison to the control group. No differences were observed in [ 3 H]paroxetine Bmax and Kd values between the two groups. 1997 Elsevier Science B.V. Keywords: Imipramine binding; Paroxetine binding; Endogenous depression
1. Introduction [ 3 H]Imipramine was the first antidepressant reported to have a high affinity binding site located both in brain and platelet membranes and associated with the serotonin (5-HT) uptake mechanism (Briley et al., 1979; Paul et al., 1980; Langer et al., 1980). Since then, several reports indicating a decreased number of [ 3 H]imipramine binding sites in depressed patients have suggested the possibility of its being a *Corresponding author.
biological marker of depression (Langer et al., 1987), although the results reported so far are very conflicting (Suranyi-Cadotte et al., 1984; Nankai et al., 1986; Kanof et al., 1987). Recently, specific high affinity binding of [ 3 H]paroxetine to both human brain and platelet membranes has also been found (Mellerup and Plenge, 1986; Habert et al., 1985; Plenge et al., 1990, 1991; Arranz and Marcusson, 1994). These studies suggest that both radioligands bind to the same 5-HT transporter complex, although their binding sites could be located on different subunits. Accordingly,
0165-0327 / 97 / $17.00 1997 Elsevier Science B.V. All rights reserved PII S0165-0327( 97 )00033-5
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it seems that paroxetine binding sites are identical in neural and platelet membranes (Klompenhouwer et al., 1990; Mellerup and Langer, 1985; Mellerup and Plenge, 1986) whereas it seems that only part of the imipramine binding can be attributed to this 5-HT reuptake complex (Briley and Langer, 1981; Wennogle and Meyerson, 1985; Sette et al., 1983; Plenge et al., 1991). In this respect, previous reports have found that the affinity of [ 3 H]paroxetine binding to human brain and platelet membranes is higher than that reported for [ 3 H]imipramine, whereas the maximum number of binding sites is comparable (Mellerup and Langer, 1985; Mellerup et al., 1993; Arranz and Marcusson, 1994; Rosel et al., 1997). In the present study, the 5-HT uptake capacity has been examined by determining simultaneously both [ 3 H]imipramine and [ 3 H]paroxetine binding in platelet membranes from a large number of healthy controls (n 5 63). Furthermore, binding experiments with these two radioligands have been performed in a group of melancholic depressed patients so as to determine the state of the 5-HT uptake mechanism.
study was approved by the local ethical committee and by the National Research Committee.
2.2. Platelet membrane preparation A 40 ml volume of blood was drawn by antecubital venopuncture and collected into plastic tubes containing 1 ml anticoagulant (2.5% trisodium citrate, 0.8% citric acid, 2.5% glucose). Each tube was centrifuged at 200 g for 30 min at room temperature. The platelet rich plasma obtained was collected and centrifuged at 10 000 g (48, 10 min). The supernatant was discarded and the platelets were washed and centrifuged twice at 10 000 g (48, 10 min) in 8 ml of buffer (50 mM Tris–HCl, 150 mM NaCl, 20 mM EDTA, pH 7.4). The supernatant was also discarded and the platelet membranes were lysed with an hypotonic buffer (5 mM Tris–HCl, 5 mM EDTA). After homogenizing the samples with a Polytron PT 3000, they were centrifuged twice at 20 000 g (48, 10 min). The pellet thus obtained was finally suspended in incubation buffer (50 mM Tris–HCl, 3 mM KCl, 120 mM NaCl, pH 7.4) to a protein concentration of about 0.5 mg / ml.
2. Material and methods
2.3. Binding assays 2.1. Subjects The control group included 63 healthy individuals (31 men and 32 women) well known to the investigators, with a mean age of 43.8613 years (range 24–70 years). A clinical interview was carried out in order to rule out any psychiatric disorder. Individuals with a 21-item Hamilton Depression Rating Scale (HDRS) score over 6, consuming drugs known to interfere with the serotonergic system, and a family history of psychiatric disorder were excluded from the study. The patient group included 18 (6 male and 12 female) inpatients with a mean age of 59.4610 years (range 38–75 years) meeting DSM-III-R criteria for major depression with melancholia, either with (n 5 10) or without (n 5 8) mood congruent psychotic features. The 21-item HDRS score from this group was 33.165.6 points. All patients were drug free for at least 2 weeks before the sample collection. None of the patients had ever received lithium therapy. Informed consent was obtained in all cases. This
For the [ 3 H]paroxetine binding assay, 0.750 ml of membrane suspensions (0.02 mg / ml of protein) were incubated with six different concentrations of [ 3 H]paroxetine (0.0075 to 0.5 nM) during 60 min at room temperature. Total incubation volume was 1.6 ml. Specific [ 3 H]paroxetine binding was determined as the difference between the binding in the absence and presence of 100 mM 5-HT (Arranz and Marcusson, 1994). The [ 3 H]imipramine binding assays were performed by incubating 0.1 ml of each membrane suspension (0.5 mg / ml of protein) with 0.1 ml of [ 3 H]imipramine at six different concentrations (0.25 to 10 nM). Incubation was performed in an ice tray inside a refrigerated room so as to prevent dissociation from the receptor. Total incubation volume was 0.5 ml. Non-specific binding was also determined with 100 mM 5-HT (Rosel et al., 1995). Samples were analysed blind to subject classification. After incubation, 5 ml of ice-cold incubation buffer was added to each tube, and samples were rapidly filtered through Whatman GF / C glass fiber
P. Rosel et al. / Journal of Affective Disorders 44 (1997) 79 – 85
filters using a 24-channel cell Harvester (Brandell, Gaithersburg, MD, USA). Filters were then washed, placed in vials with scintillation liquid, and the radioactivity trapped in each filter counted according to conventional scintillation techniques. Protein concentrations were determined by a modification of Lowry’s method. Corrections were made for the contribution of Tris–HCl to the optical density (Rej and Richards, 1974; Peterson, 1977; Markwell et al., 1987).
2.4. Data and statistical analysis The binding data was analysed using the iterative non-linear least squares curve fitting program EBDA, and values for maximum binding (Bmax ) and dissociation constant (Kd ) were obtained. As all values were normally distributed (Kolmogorov–Smirnov test), the Student’s t-test for unpaired data was used to test differences in Bmax and Kd between the two groups of patients (controls versus depressed). A paired t-test was used to test differences in Bmax and Kd obtained for both radioligands ([ 3 H]imipramine versus [ 3 H]paroxetine) within the control group. Pearson’s correlation coefficient was used to test associations. The influence of age in the binding parameters was tested through a analysis of covariance.
2.5. Materials [ 3 H]Paroxetine (17.8 Ci / mmol) and [ H]imipramine (24 Ci / mmol) were obtained from New England Nuclear (Boston, MA, USA). 5-Hydroxytryptamine creatinine sulphate (serotonin) was obtained from Sigma. All other chemicals were standard laboratory reagents. 3
3. Results [ 3 H]Paroxetine and [ 3 H]imipramine binding parameters obtained in the 63 healthy controls under study are indicated in Table 1. Significant differences between both radioligands were found for both Bmax ( p , 0.001) and Kd ( p , 0.001), with [ 3 H]paroxetine showing lower Bmax and lower Kd (better affinity) than [ 3 H]imipramine. Bmax values determined with
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Table 1 [ 3 H]Imipramine and [ 3 H]paroxetine binding parameters in platelet membranes from 63 healthy controls
[ 3 H]Imipramine [ 3 H]Paroxetine
Bmax (fmol / mg)
Kd (nM)
1842660 (860–2750) 1505666 (466–2656) p , 0.001
1.1660.03 (0.62–1.89) 0.0760.003 (0.04–0.13) p , 0.001
Both Bmax and Kd are expressed as mean6S.E.M., with range values indicated in parenthesis. A paired t-test was used for statistical comparison between [ 3 H]imipramine and 3 [ H]paroxetine binding.
both radioligands were significantly correlated (r 5 0.26; p 5 0.04, Pearson’s correlation coefficient), whereas no correlation was noted for Kd values (r 5 0.03; p . 0.05). When [ 3 H]imipramine binding parameters were plotted with regard to the age of the individual, no significant correlation was obtained for either Bmax (r 5 0.16; p . 0.05) or Kd (r 5 0.07; p . 0.05). However, when [ 3 H]paroxetine was the radioligand used to label the 5-HT uptake site, a significant negative correlation was observed between its Bmax and the control age (r 5 2 0.35; p 5 0.005). These results are reflected in Fig. 1. The maximum number of [ 3 H]imipramine binding sites was significantly ( p , 0.001) lower in depressed patients than in healthy individuals (10426149 fmol / mg versus 1842660 fmol / mg respectively). Significant differences ( p , 0.001) were also found for [ 3 H]imipramine Kd values, being 2.1560.44 nM (mean6S.E.M. value) in the depressed group and 1.1960.05 nM in control individuals. [ 3 H]Paroxetine binding parameters did not show any significant differences between the two groups (Table 2). These same results were obtained when an analysis of covariance, with age as covariant, was used instead of Student’s t-test.
4. Discussion The Bmax and Kd values obtained in this study for 3 3 the radioligands [ H]paroxetine and [ H]imipramine are in agreement with those reported by other authors
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Fig. 1. Correlation between age and [ 3 H]paroxetine (A) or [ 3 H]imipramine (B) Bmax in healthy individuals. A significant negative correlation (r 5 2 0.35; p 5 0.005) was noted between [ 3 H]paroxetine Bmax and age (Pearson’s correlation coefficient).
Table 2 [ 3 H]Imipramine and [ 3 H]paroxetine binding parameters in depressed patients with respect to healthy controls
[ 3 H]Imipramine
Controls Patients
[ 3 H]Paroxetine
Controls
Bmax (fmol / mg)
Kd (nM)
1842660 10426149 ( p , 0.001) 1505666 12986122 N.S
1.1660.03 2.1560.44 ( p 5 0.04) 0.07460.003 0.08060.009 N.S.
Bmax and Kd values are expressed as mean6S.E.M.. Student’s t-test for unpaired data was used to test differences. N.S.: non-significant.
(D’Haenen et al., 1988; Nankai et al., 1993; Mellerup and Plenge, 1986; Mellerup et al., 1993; Klompenhouwer et al., 1990; Andersson and Marcusson, 1990; Arranz and Marcusson, 1994). [ 3 H]Paroxetine seems to label a significantly lower number of binding sites than [ 3 H]imipramine, while at the same time showing a significantly higher affinity (lower Kd ). These results do not completely agree with those reported by Mellerup et al. (1993) who found similar Bmax values for both radioligands, probably due to the existence of methodological differences in their use of unlabelled imipramine to determine the amount of non-specific binding. Despite the evidence of the binding protein labelled by these two radioligands being closely related to the 5-HT uptake mechanism located either in neural or platelet membranes (Langer et al., 1987; Plenge et al., 1990, 1991), several studies on brain tissue have shown that [ 3 H]paroxetine has a more selective distribution than [ 3 H]imipramine in serotonergic regions (Hrdina, 1984; Hrdina et al., 1990a,b). In the present study, the finding of a poor correlation between the maximum number of sites labelled with both radioligands and the absence of correlation between their respective Kd values further supports the suggestion that both antidepressants bind to different subunits on the same macromolecular complex of the 5-HT reuptake mechanism (Plenge et al., 1991). This hypothesis is further supported by our finding of a higher number of binding sites labelled with [ 3 H]imipramine as compared with [ 3 H]paroxetine, together with their different Kd values. In this respect, similar results concerning the lack of correlation between both radioligands have also been reported by other researchers (Mellerup and Langer, 1985; Habert et al., 1985; Suranyi-Cadotte et al., 1989). Age is an important confounding variable when studies of [ 3 H]imipramine binding are performed, as the relationship between this variable and the serotonergic function is still unclear. A certain controversy exists between studies about the variations in [ 3 H]imipramine binding with increased age. Thus, while some authors have reported increased [ 3 H]imipramine Bmax in blood platelets from elderly controls (Schneider et al., 1985; Suranyi-Cadotte et al., 1985), others have indicated the existence of a negative correlation between both variables in con-
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trol subjects (Langer et al., 1980; Wagner et al., 1985; Halbreich et al., 1991). The lack of correlation found in this study between age and either [ 3 H]imipramine Bmax or Kd , is in agreement with other reports (Marcusson et al., 1987; Baron et al., 1984). Regarding the relationship between [ 3 H]paroxetine binding sites and age, the small number of studies performed so far also show some discrepancies. Thus, our finding of a reduced number of [ 3 H]paroxetine binding sites with increased age is in agreement with the study of Nankai et al. (1993), and not with the results reported by Andersson et al. (1992), probably due to the smaller size of the control group used in their study. Another important source of discrepancy is the choice of the drug used to define the non-specific binding. In this respect, the vast majority of studies on [ 3 H]imipramine binding have used desipramine to define specific binding. However, this compound has been reported to include a significant proportion (up to 30%) of binding not related to the 5-HT uptake site (Marcusson et al., 1985, Marcusson et al., 1986, ¨ Marcusson et al., 1987; Backstrom and Marcusson, 1987; Hrdina, 1989). Regarding the [ 3 H]paroxetine binding assay, 5-HT (Arranz and Marcusson, 1994), fluoxetine (Nankai et al., 1993; Moret and Brley, 1991), citalopram (Andersson and Marcusson, 1990), and clomipramine (Klompenhouwer et al., 1990) have been used at varying concentrations to define non-specific binding. In our binding assays, 100 mM 5-HT was used to define non-specific binding, as it has been demonstrated that 5-HT concentrations between 100 mM and 1 mM do not include the low affinity non-serotonergic component of the binding (Arranz and Marcusson, 1994). In the present study, depressed patients showed significant lower number of [ 3 H]imipramine binding sites, together with a significantly lower affinity constant (higher Kd ) than healthy controls. However, no significant variations were found in the [ 3 H]paroxetine binding parameters. These results are in agreement with those published by other researchers (Iny et al., 1989; Suranyi-Cadotte et al., 1989; Nankai et al., 1993; D’Haenen et al., 1988), although none of them have simultaneously compared both radioligands in the same population. Since our patients had been drug-free for at least two weeks before the blood sample collection, the reported Kd
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increase is not likely to be due to pharmacological interferences, nor to methodological flaws, as samples were analysed blind to subject classification. Nevertheless, although a four-week wash-out period would undoubtedly have been much better to exclude any pharmacological interferences, our inpatients could not be maintained without treatment for such a long period. Furthermore, the binding assays used in this study have been previously standardized with respect to external variables such as membrane preparation, concentration of the displacing compound, incubation time and temperature, and protein concentration (Arranz and Marcusson, 1994; Rosel et al., 1995). Moreover, our patients were diagnostically homogeneous, since all of them suffered from severe major depression with melancholic features, their age and sex distribution was similar to that of the control group, and the sampling procedure was performed in the same year period (autumn–winter) so as to avoid seasonal variations (Rosel et al., 1996a). The present results may have clinical correlates. Thus, while imipramine treatment has been proved to be effective in severe melancholic and delusional hospitalized patients, there is a lack of clinical evidence on the efficacy of paroxetine (or other selective serotonin reuptake inhibitors) in this subgroup of depressed patients (Osser, 1993). Furthermore, there is a trend in clinical settings to use tricyclic antidepressants in this particular group of psychiatric patients. There is no doubt that this clinical evidence may parallel the findings of the present study on a decreased number of [ 3 H]imipramine but not [ 3 H]paroxetine binding sites in severe melancholic and delusional hospitalized patients. Despite the higher potency and affinity of the selective 5-HT uptake blocker paroxetine, the present study demonstrates the better diagnostic sensitivity and specificity displayed by [ 3 H]imipramine in blood platelets from depressed patients.
Acknowledgments The authors wish to thank Pilar Alastruey and Elena Arnaiz from the technical staff of the Hormone
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Unit for their expert assistance. This work has been supported by a FIS grant (93 / 0318).
References 3
Andersson, A. and Marcusson, J. (1990) [ H]Paroxetine binding in human platelets in relation to age and sex. Neurobiol. Aging 11, 615–618. Andersson, A., Sundman, I. and Marcusson, J. (1992) Age stability of human brain 5-HT terminals studied with [ 3 H]paroxetine binding. Gerontology 38, 127–132. 3 Arranz, B. and Marcusson, J. (1994) [ H]Paroxetine and [ 3 H]citalopram as markers of the human brain 5-HT uptake site: a comparison study. J. Neural Transm. (Gensect) 97, 27–40. ¨ Backstrom, I.T. and Marcusson, J. (1987) 5-Hydroxytryptamine3 sensible [ H]imipramine binding of protein nature in the human brain, I. Characteristics. Brain Res. 425, 128–136. Baron, M., Kowalik, S., Barkai, A. and Gruen, R. (1984) High 3 affinity [ H]imipramine binding in human platelets: age and sex effects. Neuropsychobiology 11, 178–180. Briley, M.S., Raisman, R. and Langer, S.Z. (1979) Human platelets possess high affinity binding sites for [ 3 H]imipramine. Eur. J. Pharmacol. 58, 347–348. Briley, M.S. and Langer, S.Z. (1981) Sodium dependency of [ 3 H]imipramine binding in rat cerebral cortex. Eur. J. Pharmacol. 72, 377–380. D’Haenen, H., De Weale, M. and Leysen, J.E. (1988) Platelet [ 3 H]paroxetine binding in depressed patients. Psychiatry Res. 26, 11–17. Habert, E., Grahan, D., Tahraoni, L., Claustre, Y. and Langer, S.Z. (1985) Characterization of [ 3 H]paroxetine binding to rat cortical membranes. Eur. J. Pharmacol. 118, 107–114. Halbreich, U., Rojansky, N., Zander, K.J. and Barkai, A. (1991) Influence of age, sex and diurnal variability on impramine receptor binding and serotonin uptake in platelets of normal subjects. J. Psychiatry Res. 25, 7–18. Hrdina, P.D. (1984) Differentiation of two components of specific [ 3 H]imipramine binding in rat brain. Eur. J. Pharmacol. 102, 481–488. Hrdina, P.D. (1989) Differences between sodium dependent and desipramine defined [ 3 H]imipramine binding in intact human platelets. Biol. Psychiatry 20, 576–584. Hrdina, P.D., Foy, B., Hepher, A. and Summer, R.J. (1990a) Antidepressant binding sites in brain: autoradiographic comparison of [ 3 H]paroxetine and [ 3 H]imipramine localization and relation to serotonin transporter. J. Pharmacol. Exp. Ther. 252, 410–418. Hrdina, P.D., Foy, B. and Summer, R.J. (1990b) 2D imaging of serotonin uptake sites in brain with [ 3 H]paroxetine: comparison with [ 3 H]imipramine. In: B. Leonard and P. Spenser (Eds.), Antidepressants: Thirty Years On. pp. 54–59. Iny, L., Meaney, M., Desjardins, P. and Nair, N.P.V. (1989) [ 3 H]Imipramine and [ 3 H]paroxetine binding in blood platelets
of psychiatric patients and healthy volunteers. J. Clin. Psychopharmacol. 3, 78–82. Kanof, P.D., Coccaro, E.F., Johns, C.A., Siever, L.J. and Davis, K.L. (1987) Platelet [ 3 H]imipramine binding in psychiatric disorders. Biol. Psychiatry 22, 278–286. Klompenhouwer, J.L., Fekkes, D. and Van Hults, A.M. (1990) Seasonal variation in binding of [ 3 H]paroxetine to blood platelets in healthy volunteers: indications for a gender differences. Biol. Psychiatry 28, 509–517. Marcusson, J., Fowler, C.J., Hall, H., Ross, S.B. and Winblad, B. (1985) Specific binding of [ 3 H]imipramine to protease-sensitive and protease-resistent sites. J. Neurochem. 44, 705–711. ¨ Marcusson, J., Backstrom, J.T. and Ross, S.B. (1986) Single-site model of the neuronal 5-hydroxytryptamine uptake and imipramine binding site. Mol. Pharmacol. 30, 121–154. ¨ Marcusson, J., Alafozoff, I., Backstrom, I.T., Eriksson, E., Gottfries, C.G. and Winblad, B. (1987) 5-Hydroxytryptaminesensitive [ 3 H]imipramine binding of protein nature in the human brain. II. Effect of normal aging and dementia disorders. Brain Res. 425, 137–145. Markwell, M.A., Haas, S.M., Bieber, L.L. and Tolbert, N.E. (1987) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal. Biochem. 87, 203–225. Mellerup, E.T. and Langer, S.Z. (1985) Size determination of binding polymers for [ 3 H]imipramine and [ 3 H]paroxetine in human membranes. Eur. J. Pharmacol. 106, 411–413. Mellerup, E.T. and Plenge, P. (1986) High affinity binding of [ 3 H]paroxetine and [ 3 H]imipramine to rat neuronal membranes. Psychopharmacology 89, 436–439. Mellerup, E.T., Errebo, I., Molin, J., Plenge, P. and Dan, H. (1993) Platelet paroxetine binding and light therapy in winter depression. J. Affect. Dis. 22, 11–15. Moret, C. and Brley, M. (1991) Platelet [ 3 H]paroxetine binding to the serotonin transporter is insensitive to changes in the central serotonergic innervation in the rat. Psychiatry Res. 38, 97–104. Nankai, M., Joshimoto, S., Narita, K. and Takahashi, R. (1986) Platelet [ 3 H]imipramine binding in depressed patients and its circadian variations in healthy controls. J. Affective Disord. 11, 207–212. Nankai, M., Jamada, S. and Joshimoto, S. (1993) Platelet [ 3 H]paroxetine binding in subjects and depressed patients: Relationship to serotonin uptake and age. Psychiatry Res. 51, 147–155. Langer, S.Z., Moret, C., Raisman, R., Dobocovich, M.L. and Briley, M.S. (1980) High affinity [ 3 H]imipramine binding in rat hypothalamus: association with uptake of serotonin but not norepinephrine. Science 210, 1133–1135. Langer, S.Z., Galzin, A.M., Poirier, M.F., Loo, H., Sechter, D. and Zarifian, E. (1987) Association of [ 3 H]imipramine and [ 3 H]paroxetine binding with the 5-HT transporter in brain and platelets: relevance to studies in depression. J. Receptor Res. 7, 499–521. Osser, D.N. (1993) A systematic approach to the classification and pharmacotherapy of nonpsychotic major depression and dysthymia. J. Clin. Psychopharmacol. 13, 133–144. Paul, S.M., Rehavi, M., Kolnick, P. and Goodwin, F.K. (1980)
P. Rosel et al. / Journal of Affective Disorders 44 (1997) 79 – 85 Demostration of specific ‘‘high affinity’’ binding sites for [ 3 H]imipramine on human platelets. Life Sci. 26, 953–959. Plenge, P., Mellerup, E.T. and Laursen, H. (1990) Regional distribution of the serotonin transport complex in human brain, identified with [ 3 H]paroxetine, [ 3 H]citalopram and 3 [ H]imipramine. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 14, 61–72. Plenge, P., Mellerup, E.T. and Laursen, H. (1991) Affinity modulation of [ 3 H]imipramine, [ 3 H]paroxetine and 3 [ H]citalopram binding to the 5-HT transporter from brain and platelets. Eur. J. Pharmacol. 206, 243–250. Peterson, G.L. (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal. Biochem. 83, 346–356. Rej., Richards, A.H. (1974) Interference by Tris buffer in the estimation of protein by the Lowry procedure. Anal. Biochem. 62, 240–247. ´ J.M. and Rosel, P., Vallejo, J., Oros, M., Serrallonga, J., Menchon, Navarro, M.A. (1995) Changes in platelet [ 3 H]imipramine binding: influences of protein concentration of varying proportions of cytosol or intact platelets and displacing agents used. Biol. Psychiatry 38, 464–470. ´ J.M., Cortadellas, T., Rosel, P., Vallejo, J., Oros, M., Menchon, Navarro, M.A. and Urretavizcaya, M. (1996a) Seasonal variability in blood platelet [ 3 H]imipramine binding in healthy controls: age and gender effects. Biol. Psychiatry 40, 981–985. ´ J.M., Oros, M., Vallejo, J., Cortadellas, T., Rosel, P., Menchon, Arranz, B., Alvarez, P. and Navarro, M.A. (1997) Regional
85
distribution of specific high affinity binding sites for [ 3 H]imipramine and [ 3 H]paroxetine in human brain. J. Neural Transm. (Gensect) 104, 89–96. Schneider, L.S., Severson, J.A. and Sloane, B.B. (1985) Platelet [ 3 H]imipramine binding in depressed elderly patients. Biol. Psychiatry 20, 1232–1234. Sette, M., Briley, S. and Langer, S.Z. (1983) Complex inhibition of [ 3 H]imipramine binding by serotonin and nontricyclic serotonin uptake blockers. J. Neurochem. 40, 622–628. Suranyi-Cadotte, B., Quirion, R. and McQuade, P. (1984) Platelet [ 3 H]imipramine binding: a state dependent marker in depression. Neuropsychopharmacol. 8, 737–741. Suranyi-Cadotte, B., Gauthiers, S. and Lafaille, F. (1985) Platelet [ 3 H]imipramine binding distinguishes depression from Alzheimer dementia. Life Sci. 37, 2305–2323. Suranyi-Cadotte, B., Iny, L., Desjardins, P., Jassa, R. and Welner, P. (1989) Decreased density of platelet [ 3 H]imipramine but not [ 3 H]paroxetine binding sites in major depression. Soc. Neurosci. 15, 673 (Abst. 274.4) Wagner, A., Aberg-Wistedt, A., Asberg, M., Ekqvist, B., Martensson, B. and Montero, D. (1985) Lower [ 3 H]imipramine binding in platelets from untreated depressed patients compared to healthy controls. Psychiatry Res. 16, 131–139. Wennogle, L.P. and Meyerson, L.R. (1985) Serotonin uptake inhibitors differentially modulate high affinity imipramine dissociation in human platelet membranes, Life Sci. 36, 1541– 1550.