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Tricyclic antidepressant binding to lymphocyte membranes and changes during depression
European Journal of Pharmacology,
149 (1988) 357-361
357
Elsevier EJP 50260
Tricyclic antidepressant binding to lymphocyte membranes and changes during depression R a d o s l a v K r u l i k *, D a n i e l Sliva, J a n Sikora * *, I r e n a Farskfi a n d K v 6 t a Fuksovfi
* * *
Psvchiatric Research Unit, Faculty of Medicine, Charles University, Prague, ** Mental Hospital, Horni Be~kovice, and * * * Institute of Nuclear Biology and Radiochemistry, Czechoslovak Academy of Sciences, Prague, Czechoslovakia
Received 17 November 1987, revised MS received11 February 1988, accepted 23 February 1988
The binding of [3H]imipramine, its 2- and 4-nitroderivatives and [3H]desmethylimipramine to lymphocyte membranes was determined. IC50 values for drugs and neurotransmitters to inhibit [3H]imipramine binding to lymphocyte membranes were comparable with those for brain and thrombocyte membranes. The number of [3H]imipramine and [3H]desmethylimipramine binding sites increased in depressive patients, whereas the dissociation constants remained unchanged. [3H]Imipramine binding; [3H]Desmethylimipramine binding; Lymphocytes; (Depression)
1. I n t r o d u c t i o n
High-affinity binding sites for [3H]imipramine have been described in brain synaptic plasma membranes (Raisman et al., 1979) and in thrombocytes (Briley et al., 1979), and it has been reported that [3H]desmethylimipramine (DMI) also binds to synaptic plasma membranes (Rehavi et al., 1981). Some studies have demonstrated that there are two different binding sites: the [3H]imipramine binding site which is closely related to 5-HT uptake (Langer et al., 1980b) and the [3H]DMI site, which is associated with noradrenaline uptake (Langer et al., 1981). Several authors have demonstrated that depressive patients have a decreased number of [3H]imipramine binding sites (Briley et al., 1980; Asarch et al., 1980; Paul et al., 1981), but these results have not always been confirmed by others (Mellerup et al., 1982; * To whom all correspondenceshould be addressed: Psychiatric Research Unit, Faculty of Medicine, Charles University, Ke Karlovu 11, 12821 Praha 2, Czechoslovakia.
Muscettola et al., 1984). [3H]Imipramine inhibition studies have shown that imipramine has a specific binding site which is unrelated to any neurotransmitter receptor site (Sette et al., 1983; Whitaker et al., 1983; Raisman et al., 1980) and that the repeated administration of imipramine leads to a decrease in the number of binding sites (Arbilla et al., 1981). Certain nitro-derivatives of imipramine have been synthesized, labelled and used to study [3H]imipramine binding sites further (Rehavi et al., 1982); they have also been used to label the binding site in histochemical studies (Rotman and Pribluda, 1982). Previous studies demonstrated that lung membranes, as well as those of brain tissue and thrombocytes, have a high binding affinity for [3H]imipramine (Morion et al., 1984). In our study, we focused on the problem of whether lymphocytes can bind [3H]imipramine as well as [3H]DMI and we used the nitroderivatives of imipramine to test this. In addition, we studied changes in the binding of these tricyclic antidepressants in psychiatric patients.
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (BiomedicalDivision)
358 TABLE 1
2. Materials and methods Experimental studies were carried out with lymphocyte membranes from h u m a n blood taken from healthy volunteers. In the study of [3H]imipramine binding 11 patients, 9 with unipolar and 2 with bipolar depression (6 w o m e n and 5 men aged 43 _+ 11.1 years) who fulfilled the D S M III criteria (American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Third Edition. Washington, D.C.: APA, 1980, p. 213) and 10 controls (7 w o m e n and 3 men aged 35 _+ 4.0) were included. Seven patients, 6 unipolar and 1 bipolar (3 w o m e n and 4 men aged 40.0 _+ 3.3), were included in the study of [ 3 H ] D M I binding. The control group comprised 6 people (5 w o m e n and 1 man) aged 37.3_+ 4.6 years. The patients had not received any medications 14 days before their blood was taken. Blood was taken between 6 and 7 a.m. The lymphocytes were isolated by the m e t h o d of Otto and Schmid 119701. L y m p h o c y t e membranes were isolated by h o m o g e nizing the lymphocytes for 5 s in an Ultra Turax apparatus followed by centrifugation of the h o m o genate at 42000 × g. The m e t h o d of Raisman et al. (1980; 1982) was used for the [3H]imiprarnine and [ 3 H ] D M I binding studies; the total incubation volume was 250 /~1 and the protein content was 100 ~g. Non-specific binding in the [3H]imipramine binding assay was determined in the presence of 100 ~ M D M I and in the presence of 100 /xM nortriptyline for the [ 3 H ] D M I binding assay. [3H]2- and -4-imipramine nitro-derivatives were prepared from [3 H]imipramine by the m e t h o d of Rehavi et al. (1982); their specific activity was 25 C i / m m o l . The binding assay for these derivatives was the same as that for [3 H]imipramine and non-specific binding was determined in the presence of 50 /~M chlorimipramine. Protein was determined by the m e t h o d of L o w r y et al. (1951).
3. Results The K d and Bmax values for [3H]imipramine, [ 3 H ] D M I and nitro-derivatives of imipramine membranes are shown in table 1.
the the to The
binding of 2- and 4lymphocyte dissociation
Binding of [3H]imipramine, its nitroderivativcs and [~H]desmethylimipramine to human lymphocyte membranes (" P < 0.05); for comparison, [3H]imipramine binding to lymphocyte and thrombocyte membranes of rats (b P<0.05) was determined ( _+S.E.M.) Membranes
Ligand
Human lymphocytes
[ ~H]Imipramine (n = 101 [ 3H]2-Nitroimipramine (n = 4) [3H]4-Nitroimipramine (n = 4) [ 3H]Desmethylimipramine (n = 6)
Rat lymphocytes
[3H]Imipramine (n = 5)
Rat thrombo- [3H]Imipramine cytes (n = 7)
Bm,,~ (fmol/mg protein)
Kd (nMt
21/9+ 12.9
6.1 -+0.48
237 +_27.4
4.6 _+0.32 ~'
247 + 13.0
4.1 + 1/.64
195 + 12.1
4.9+_0.40
163_+11.3 3.3_+0.39 213+ 13.7 h 2.1 _+1/.26 b
constants are comparable, and the n u m b e r of binding sites for the two nitro-derivatives are similar. The ICs0 values, determined for both drugs and neurotransmitters for the inhibition of [3H]imipramine binding (table 2) are comparable with those for brain synaptic plasma membranes or for t h r o m b o c y t e s (Raisman et al., 1980; Briley et al., 1979). These results also confirm the existence of specific high-affinity binding sites for imipramine. The n u m b e r of binding sites for both [3H]imipramine and [ 3 H ] D M I were found to be higher in depressive patients than in the control group (figs. TABLE 2 Inhibition of specific [3H]imipramine binding to lymphocytes by a variety of active compounds. The IC~0value was the drug concentration required to inhibit the specific binding of [3H]imipramine (2 nM) by 50%. Imipramine 2-Nitroimipramine Amitriptyline Desmethylimipramine Serotonin Noradrenaline
IC50 (nM) 7.5 5.0 20.0 32.0 2500.0 2000.0
359 900-
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700"
o
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o
._= Q.
54)0-
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200" o •
uptake (Raisman et al., 1982). It is assumed that the [3H]imipramine binding sites forms part of the 5-HT uptake complex, which can influence it, but is not identical with it. So far, however, little attention has been paid to imipramine binding to cell membranes of other tissues. In our laboratory, we compared binding to membranes from several organs and found that [3H]imipramine binds to lung and liver membranes, as has been described by others (Morion et al., 1984), and to heart, where other authors have failed to find binding (Raisman et al., 1980; Kinnier et al., 1981); however, we did not find binding to kidney membranes (unpublished results). We also studied binding to erythrocyte membranes and found that the Bmax-Kd values in euthymic patients treated
o
100-
900-
0 CONTROL PATIENTS N KD Bm~x
10 b,l +.0 , 4 7 209+- 12,9
80O.
DEPRESSIVE PATIENTS 11 5,2 ." 0 , 3 5 404!4q, b
Fig. 1. [3H]Imipramine binding to lymphocyte membranes in maniodepressive patients. The choice of patients, as well as the preparation of lymphocyte membranes, have been described in the Methods. Binding was determined in duplicate at 0 o C for 60 min. The concentration of protein was 100 ~g in 250 /~1. The control group included 10 volunteers and 11 patients matched in age and sex. The Bm~ value of the control group was 209 ± 12.9 f m o l / m g protein and the K a value was 6.1 ± 0.47 nM. In the group of depressive patients, a Bmax value of 404±49.6 f m o l / m g protein and a K a value of 5.2±0.35 nM were found. The data are means ± S.E. The differences between depressive patients and controls are marked by (P < 0.01).
._c
700-
()
bOOClb
E
500-
0
E 40OoG
E m
30O"
o o
200$ 100-
1 and 2): imipramine binding was 95% higher and DMI binding was 102% higher.
4. D i s c u s s i o n
Previous studies have shown that [3H]imipramine can bind to brain synaptic plasma membranes and to thrombocytes (Raisman et al., 1979; Briley et al., 1979). Such binding is associated with tissues that have a high turnover of serotonin and is considered to be related directly with serotonin
OCONTROL PATIENTS N b Ko 4,9 +.0,40 Bmax 19S -+'12,'1
DEPRESSIVE PATIENTS 7 4,7T 0 , 4 9 431.+ 73,3 *
Fig. 2. [3 H]Desmethylimipramine binding to lymphocyte membranes in maniodepressive patients. Binding was determined in duplicate at 2 3 ° C for 30 min. The Bma~ value of the control group was 195 ± 12.1 f m o l / m g protein and the K d value was 4.9 + 0.40 nM. In the group of depressive patients, the value for Bn~~ was 431 ± 73.3 f m o l / m g protein and for K d was 4.7±0.49 nM. The data are m e a n s + S.E. The differences between depressive patients and controls are marked by (P < 0.01).
360 with lithium were no different from those in the control group (Sou~ek and Krulik, 1985). Since lymphocytes are used for studying catecholamines, in particular/~-adrenoceptors, we were interested in whether they would bind tricyclic antidepressants. We found high-affinity binding of [3H]DMI (which is associated with noradrenaline uptake) and [3H]imipramine binding (which is associated with 5-HT uptake). Speculation on the inhibition of uptake does not, however, take sufficiently into account the fact that imipramine acts on catecholamines just as strongly as it does on indolamines (Laduron, 1984). The results obtained for imipramine and DMI with lymphocyte membranes are comparable with those obtained with brain synaptic plasma membranes (Raisman et al., 1980; Langer et al., 1980a). However, lymphocytes are not a homogenous cell population; in addition to type B and T cells there is a series of subtypes and the same problems are encountered with thrombocytes (Corash et al., 1977). At present, we do not know whether these various types participate in imipramine binding in different ways. The specificity of binding was also shown by binding studies with [3H]2-nitroimipramine, which have a longer association and dissociation half-time than imipramine itself (Davis, 1983). Drug and neurotransmitter inhibition of imipramine binding show that imipramine was displaced from its binding site in lymphocyte membranes by tricyclic antidepressants, as also occurs in brain and thrombocyte membranes, and that 5-HT and NA have little effect (Raisman et al., 1980; Briley et al., 1979). Our results indicate that it is not bound to the receptor sites of these neurotransmitters. The ICs0 study of imipramine, DMI and 2nitroimipramine for [3H]2-NO2- and [3H]4NO2-imipramine binding to synaptic plasma membranes shows that the displacement of [3H]4-nitroimipramine requires higher imipramine and DMI concentrations than those needed in the case of [3H]2-NOz-imipramine. Recent results on imipramine binding in depressive patients show either a decreased binding (Briley et al., 1980; Paul et al., 1981; Asarch et al., 1980), an increased binding (Mellerup et al., 1982) or no effect (Muscettola et al., 1984). We demon-
strated an increase in both imipramine and DMI binding to lymphocyte membranes from depressive patients. In evaluation of the results, knowledge of imipramine binding in relation to age, sex and seasonal variation was taken into account (Langer et al., 1980; Egrise et al., 1983; Whitaker et al., 1984). Differences between our results and those published can be chiefly ascribed to methodological differences. This could influence the results considerably, since in addition to the choice of patients, results depend on the size of the sample, the quantity of membranes and the concentration of ligand and unlabelled substance used for the determination of nonspecific binding. Last, but not least, the type and size of the glass microfibre filter, the filtration rate and whether substances that reduce the binding of the ligand to the glass microfibre filters (e.g. polyethylenimine) are used, also play an important role. Further studies of tricyclic antidepressant binding to different cell structures from patients with depression and other psychiatric disorders will evidently be necessary. Some studies show that the thrombocytes of schizophrenics, as well as those of patients with depression, have a smaller number of binding sites (Rotman et al., 1982). A thorough study is needed to determine whether imipramine binding is always associated with 5-HT uptake. In our opinion, this is not necessarily the case, since the concentration of 5-HT and the number of its binding sites on lymphocytes are low (Eliseyeva and Stefanovich, 1984; Meyer et al., 1974; Eliseyeva et al., 1982), whereas there are more imipramine binding sites on the same membrane. The ratio of binding sites for tricyclic antidepressant drugs to the sites of serotonin uptake, needs to be examined.
References
Arbilla, S., M. Briley, F. Cathala, S.Z. Langer, C. Pornin and R. Raisman, 1981, Parallel changes in [3H]imipramine binding sites in cat brain and platelets following chronic treatment with imipramine, Br. J. Pharmacol. 72, 154. Asarch, K.B., J.C. Shih and A. Kulesar, 1980, Decreased [3H]imipramine binding in depressed males and females. Commun. Psychopharmacol.4, 425. Briley, M.S., S.Z. Langer, R. Raisman, D. Schechter and E.
361 Zarifian, 1980, Tritiated imipramine binding sites are decreased in platelets of untreated depressed patients, Science 29, 303. Briley, M.S., R. Raisman and S.Z. Langer, 1979, Human platelets posses high-affinity binding sites for [3H]imipramine, European J. Pharmacol. 58, 347. Corash, L., H. Tau and H.R. Gralnick, 1977, Heterogeneity of human whole blood platelet subpopulations. I. Relationship between bouyant density, cell volume and ultrastructure, Blood 49, 71. Davis, A., 1983, Kinetic properties of [3H]2-nitroimipramine binding to human platelets, European J. Pharmacol. 96, 105. Egrise, D., D. Desmedt, A. Schoutens and J. Mendlewicz, 1983, Circannual variations in the density of tritiated imipramine binding sites on blood platelets in man, Neuropsychobiology 10, 101. Eliseyeva, L.S. and L.E. Stefanovich, 1982, Specific binding of serotonin by blood leukocytes and peritoneal cells of mice, Biochimia 47, 810. Eliseyeva, L.S., L.E. Stefanovich and V.S. Popova, 1984, Serotonin binding by separate populations of peritoneal cells and blood leukocytes of intact and thymectomized mice, Tsitologia 26, 1174. Kinnier, W.J., D.M. Chuang, G. Gwynn and E. Costa, 1981, Characteristic and regulation of high affinity [3H]imipramine binding to rat hippocampal membranes, Neuropharmacology 20, 411. Laduron, P.M., 1984, [3H]Imipramine and [3H]desipramine binding: non-specific displaceable sites or physiologically relevant sites associated with the uptake of serotonin and noradrenaline?, Trends Pharmacol. Sci. February, 52. Langer, S.Z., M.S. Briley, R. Raisman, J.F. Henry and P.L. Morselli, 1980a, Specific [3H]imipramine binding in human platelets: Influence of age and sex, Naunyn-Schmiedeb. Arch. Pharmacol. 313, 189. Langer, S.Z., C. Motet, R. Raisman, M.L. Dubocovick and M.S. Briley, 1980b, High-affinity [3H]imipramine binding in rat hypothalamus is associated with the uptake of serotonin but not norepinephrine, Science 210, 1133. Langer, S.Z., R. Raisman and M. Briley, 1981, High affinity [~H]DMI binding is associated with neuronal noradrenaline uptake in the periphery and the central nervous system, European J. Pharmacol. 72, 423. Lowry, C.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Mellerup, E.T., R. Plenge and R. Rosenberg, 1982, [~H]hnipramine binding sites in platelets from psychiatric patients, Psychiat. Res. 7, 221. Meyer, D.C. and P.D. Sturkie, 1974, Distribution of serotonin among blood cells of the domestic fowl, Proc. Soc. Exp. Biol. Med. 147, 382. Morion, D., R. Zini, J. Lange and J.P. Tillement, 1984, Evi-
dence for high affinity [3H]imipramine binding sites in human lung, Biochem. Biophys. Res. Commun. 120, 926. Muscettola, G., A. Ditauro and C.P. Giannini, 1984, Blood cells as biological trait markers in affective disorders, J. Psychiat. Res. 18, 447. Otto, F. and D.O. Schmid, 1970, Lymphozytenisolierung aus dem Blur des Menchen und der Tiere, Blur 21, 118. Paul, S.M., M. Rehavi, P. Skolnick, J.C. Ballenger and K. Goodwin, 1981, Depressed patients have decreased binding of tritiated imipramine to platelet serotonin 'transporter', Arch. Gen. Psychiat. 38, 1315. Raisman, R., M.S. Briley and S.Z. Eanger, 1979, Specific tricyclic antidepressant binding sites in rat brain, Nature 281, 148. Raisman, R., M.S. Briley and S.Z. Langer, 1980, Specific tricyclic antidepressant binding sites in rat brain characterised by high-affinity [3H]imipramine binding, European J. Pharmacol. 61, 373. Raisman, R.~ M. Sette, C. Pimoule, M. Briley and S.Z. Langer, 1982, High-affinity [3H]desmethylimipramine binding in the peripheral and central nervous system: a specific site associated with the neuronal uptake of noradrenaline, European J. Pharmacol. 78, 345. Rehavi. M., Y. htah, P. Skolnick, K.C. Rice and S.M. Paul, 1982, Nitroimipramines. Synthesis and pharmacological effects of potent long-acting inhibitors of [3H]serotonin uptake and [3H]imipramine binding, Naunyn-Schmiedeb. Arch. Pharmacol. 320, 45. Rehavi, M., P. Skolnick, B. Huliban and S.M. Paul, 1981, High affinity binding of [3H]desipramine to rat cerebral cortex: relationship to tricyclic antidepressant-induced inhibition of norepinephrine uptake, European J. Pharmacol. 70, 597. Rotman, A. and V. Pribluda, 1982, Photoaffinity labelling of the serotonin carrier protein in platelets and brain synaptosomes, Biochim. Biophys. Acta 714, 173. Rotman, A., A. Shatz and G.A. Szekely, 1982, Correlation between serotonin uptake and imipramine binding in schizophrenic patients, Prog. Neuro-Psychopharmacol. Biol. Psychiat. 6, 57. Sette, M., M.S. Briley and S.Z. Langer, 1983, Complex inhibition of [3H]imipramine binding by serotonin and noncyclic serotonin uptake blockers, J. Neurochem. 40, 622. Sou~ek, K. and R. Krulik, 1985, [~H]Desmethylimipramine and [3H]imipramine bindings in erythrocyte and thrombocyte membranes of maniodepressive patients, 9th Czechoslovak-Yugosl. Psychiatric Congress, No. 97, 81. Whitaker, P.M., C.K. Virat and R. Morin, 1983, [3H]Imipramine labels sites on brain astroglial cells not related to serotonin uptake, J. Neurochem. 41. 1319. Whitaker, P.M., J.J. Warsh, H.C. Stancer, E. Persad and C.K. Vim, 1984, Seasonal variation in platelet [3H]imipramine binding. Comparable values in control and depressed populations, Psychiat. Res. 1l, 127.
149 (1988) 357-361
357
Elsevier EJP 50260
Tricyclic antidepressant binding to lymphocyte membranes and changes during depression R a d o s l a v K r u l i k *, D a n i e l Sliva, J a n Sikora * *, I r e n a Farskfi a n d K v 6 t a Fuksovfi
* * *
Psvchiatric Research Unit, Faculty of Medicine, Charles University, Prague, ** Mental Hospital, Horni Be~kovice, and * * * Institute of Nuclear Biology and Radiochemistry, Czechoslovak Academy of Sciences, Prague, Czechoslovakia
Received 17 November 1987, revised MS received11 February 1988, accepted 23 February 1988
The binding of [3H]imipramine, its 2- and 4-nitroderivatives and [3H]desmethylimipramine to lymphocyte membranes was determined. IC50 values for drugs and neurotransmitters to inhibit [3H]imipramine binding to lymphocyte membranes were comparable with those for brain and thrombocyte membranes. The number of [3H]imipramine and [3H]desmethylimipramine binding sites increased in depressive patients, whereas the dissociation constants remained unchanged. [3H]Imipramine binding; [3H]Desmethylimipramine binding; Lymphocytes; (Depression)
1. I n t r o d u c t i o n
High-affinity binding sites for [3H]imipramine have been described in brain synaptic plasma membranes (Raisman et al., 1979) and in thrombocytes (Briley et al., 1979), and it has been reported that [3H]desmethylimipramine (DMI) also binds to synaptic plasma membranes (Rehavi et al., 1981). Some studies have demonstrated that there are two different binding sites: the [3H]imipramine binding site which is closely related to 5-HT uptake (Langer et al., 1980b) and the [3H]DMI site, which is associated with noradrenaline uptake (Langer et al., 1981). Several authors have demonstrated that depressive patients have a decreased number of [3H]imipramine binding sites (Briley et al., 1980; Asarch et al., 1980; Paul et al., 1981), but these results have not always been confirmed by others (Mellerup et al., 1982; * To whom all correspondenceshould be addressed: Psychiatric Research Unit, Faculty of Medicine, Charles University, Ke Karlovu 11, 12821 Praha 2, Czechoslovakia.
Muscettola et al., 1984). [3H]Imipramine inhibition studies have shown that imipramine has a specific binding site which is unrelated to any neurotransmitter receptor site (Sette et al., 1983; Whitaker et al., 1983; Raisman et al., 1980) and that the repeated administration of imipramine leads to a decrease in the number of binding sites (Arbilla et al., 1981). Certain nitro-derivatives of imipramine have been synthesized, labelled and used to study [3H]imipramine binding sites further (Rehavi et al., 1982); they have also been used to label the binding site in histochemical studies (Rotman and Pribluda, 1982). Previous studies demonstrated that lung membranes, as well as those of brain tissue and thrombocytes, have a high binding affinity for [3H]imipramine (Morion et al., 1984). In our study, we focused on the problem of whether lymphocytes can bind [3H]imipramine as well as [3H]DMI and we used the nitroderivatives of imipramine to test this. In addition, we studied changes in the binding of these tricyclic antidepressants in psychiatric patients.
0014-2999/88/$03.50 © 1988 Elsevier Science Publishers B.V. (BiomedicalDivision)
358 TABLE 1
2. Materials and methods Experimental studies were carried out with lymphocyte membranes from h u m a n blood taken from healthy volunteers. In the study of [3H]imipramine binding 11 patients, 9 with unipolar and 2 with bipolar depression (6 w o m e n and 5 men aged 43 _+ 11.1 years) who fulfilled the D S M III criteria (American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders, Third Edition. Washington, D.C.: APA, 1980, p. 213) and 10 controls (7 w o m e n and 3 men aged 35 _+ 4.0) were included. Seven patients, 6 unipolar and 1 bipolar (3 w o m e n and 4 men aged 40.0 _+ 3.3), were included in the study of [ 3 H ] D M I binding. The control group comprised 6 people (5 w o m e n and 1 man) aged 37.3_+ 4.6 years. The patients had not received any medications 14 days before their blood was taken. Blood was taken between 6 and 7 a.m. The lymphocytes were isolated by the m e t h o d of Otto and Schmid 119701. L y m p h o c y t e membranes were isolated by h o m o g e nizing the lymphocytes for 5 s in an Ultra Turax apparatus followed by centrifugation of the h o m o genate at 42000 × g. The m e t h o d of Raisman et al. (1980; 1982) was used for the [3H]imiprarnine and [ 3 H ] D M I binding studies; the total incubation volume was 250 /~1 and the protein content was 100 ~g. Non-specific binding in the [3H]imipramine binding assay was determined in the presence of 100 ~ M D M I and in the presence of 100 /xM nortriptyline for the [ 3 H ] D M I binding assay. [3H]2- and -4-imipramine nitro-derivatives were prepared from [3 H]imipramine by the m e t h o d of Rehavi et al. (1982); their specific activity was 25 C i / m m o l . The binding assay for these derivatives was the same as that for [3 H]imipramine and non-specific binding was determined in the presence of 50 /~M chlorimipramine. Protein was determined by the m e t h o d of L o w r y et al. (1951).
3. Results The K d and Bmax values for [3H]imipramine, [ 3 H ] D M I and nitro-derivatives of imipramine membranes are shown in table 1.
the the to The
binding of 2- and 4lymphocyte dissociation
Binding of [3H]imipramine, its nitroderivativcs and [~H]desmethylimipramine to human lymphocyte membranes (" P < 0.05); for comparison, [3H]imipramine binding to lymphocyte and thrombocyte membranes of rats (b P<0.05) was determined ( _+S.E.M.) Membranes
Ligand
Human lymphocytes
[ ~H]Imipramine (n = 101 [ 3H]2-Nitroimipramine (n = 4) [3H]4-Nitroimipramine (n = 4) [ 3H]Desmethylimipramine (n = 6)
Rat lymphocytes
[3H]Imipramine (n = 5)
Rat thrombo- [3H]Imipramine cytes (n = 7)
Bm,,~ (fmol/mg protein)
Kd (nMt
21/9+ 12.9
6.1 -+0.48
237 +_27.4
4.6 _+0.32 ~'
247 + 13.0
4.1 + 1/.64
195 + 12.1
4.9+_0.40
163_+11.3 3.3_+0.39 213+ 13.7 h 2.1 _+1/.26 b
constants are comparable, and the n u m b e r of binding sites for the two nitro-derivatives are similar. The ICs0 values, determined for both drugs and neurotransmitters for the inhibition of [3H]imipramine binding (table 2) are comparable with those for brain synaptic plasma membranes or for t h r o m b o c y t e s (Raisman et al., 1980; Briley et al., 1979). These results also confirm the existence of specific high-affinity binding sites for imipramine. The n u m b e r of binding sites for both [3H]imipramine and [ 3 H ] D M I were found to be higher in depressive patients than in the control group (figs. TABLE 2 Inhibition of specific [3H]imipramine binding to lymphocytes by a variety of active compounds. The IC~0value was the drug concentration required to inhibit the specific binding of [3H]imipramine (2 nM) by 50%. Imipramine 2-Nitroimipramine Amitriptyline Desmethylimipramine Serotonin Noradrenaline
IC50 (nM) 7.5 5.0 20.0 32.0 2500.0 2000.0
359 900-
BOO.
700"
o
bOO.
o
._= Q.
54)0-
o o
E o
400-
~v
E oo
300 -
l
E m
200" o •
uptake (Raisman et al., 1982). It is assumed that the [3H]imipramine binding sites forms part of the 5-HT uptake complex, which can influence it, but is not identical with it. So far, however, little attention has been paid to imipramine binding to cell membranes of other tissues. In our laboratory, we compared binding to membranes from several organs and found that [3H]imipramine binds to lung and liver membranes, as has been described by others (Morion et al., 1984), and to heart, where other authors have failed to find binding (Raisman et al., 1980; Kinnier et al., 1981); however, we did not find binding to kidney membranes (unpublished results). We also studied binding to erythrocyte membranes and found that the Bmax-Kd values in euthymic patients treated
o
100-
900-
0 CONTROL PATIENTS N KD Bm~x
10 b,l +.0 , 4 7 209+- 12,9
80O.
DEPRESSIVE PATIENTS 11 5,2 ." 0 , 3 5 404!4q, b
Fig. 1. [3H]Imipramine binding to lymphocyte membranes in maniodepressive patients. The choice of patients, as well as the preparation of lymphocyte membranes, have been described in the Methods. Binding was determined in duplicate at 0 o C for 60 min. The concentration of protein was 100 ~g in 250 /~1. The control group included 10 volunteers and 11 patients matched in age and sex. The Bm~ value of the control group was 209 ± 12.9 f m o l / m g protein and the K a value was 6.1 ± 0.47 nM. In the group of depressive patients, a Bmax value of 404±49.6 f m o l / m g protein and a K a value of 5.2±0.35 nM were found. The data are means ± S.E. The differences between depressive patients and controls are marked by (P < 0.01).
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1 and 2): imipramine binding was 95% higher and DMI binding was 102% higher.
4. D i s c u s s i o n
Previous studies have shown that [3H]imipramine can bind to brain synaptic plasma membranes and to thrombocytes (Raisman et al., 1979; Briley et al., 1979). Such binding is associated with tissues that have a high turnover of serotonin and is considered to be related directly with serotonin
OCONTROL PATIENTS N b Ko 4,9 +.0,40 Bmax 19S -+'12,'1
DEPRESSIVE PATIENTS 7 4,7T 0 , 4 9 431.+ 73,3 *
Fig. 2. [3 H]Desmethylimipramine binding to lymphocyte membranes in maniodepressive patients. Binding was determined in duplicate at 2 3 ° C for 30 min. The Bma~ value of the control group was 195 ± 12.1 f m o l / m g protein and the K d value was 4.9 + 0.40 nM. In the group of depressive patients, the value for Bn~~ was 431 ± 73.3 f m o l / m g protein and for K d was 4.7±0.49 nM. The data are m e a n s + S.E. The differences between depressive patients and controls are marked by (P < 0.01).
360 with lithium were no different from those in the control group (Sou~ek and Krulik, 1985). Since lymphocytes are used for studying catecholamines, in particular/~-adrenoceptors, we were interested in whether they would bind tricyclic antidepressants. We found high-affinity binding of [3H]DMI (which is associated with noradrenaline uptake) and [3H]imipramine binding (which is associated with 5-HT uptake). Speculation on the inhibition of uptake does not, however, take sufficiently into account the fact that imipramine acts on catecholamines just as strongly as it does on indolamines (Laduron, 1984). The results obtained for imipramine and DMI with lymphocyte membranes are comparable with those obtained with brain synaptic plasma membranes (Raisman et al., 1980; Langer et al., 1980a). However, lymphocytes are not a homogenous cell population; in addition to type B and T cells there is a series of subtypes and the same problems are encountered with thrombocytes (Corash et al., 1977). At present, we do not know whether these various types participate in imipramine binding in different ways. The specificity of binding was also shown by binding studies with [3H]2-nitroimipramine, which have a longer association and dissociation half-time than imipramine itself (Davis, 1983). Drug and neurotransmitter inhibition of imipramine binding show that imipramine was displaced from its binding site in lymphocyte membranes by tricyclic antidepressants, as also occurs in brain and thrombocyte membranes, and that 5-HT and NA have little effect (Raisman et al., 1980; Briley et al., 1979). Our results indicate that it is not bound to the receptor sites of these neurotransmitters. The ICs0 study of imipramine, DMI and 2nitroimipramine for [3H]2-NO2- and [3H]4NO2-imipramine binding to synaptic plasma membranes shows that the displacement of [3H]4-nitroimipramine requires higher imipramine and DMI concentrations than those needed in the case of [3H]2-NOz-imipramine. Recent results on imipramine binding in depressive patients show either a decreased binding (Briley et al., 1980; Paul et al., 1981; Asarch et al., 1980), an increased binding (Mellerup et al., 1982) or no effect (Muscettola et al., 1984). We demon-
strated an increase in both imipramine and DMI binding to lymphocyte membranes from depressive patients. In evaluation of the results, knowledge of imipramine binding in relation to age, sex and seasonal variation was taken into account (Langer et al., 1980; Egrise et al., 1983; Whitaker et al., 1984). Differences between our results and those published can be chiefly ascribed to methodological differences. This could influence the results considerably, since in addition to the choice of patients, results depend on the size of the sample, the quantity of membranes and the concentration of ligand and unlabelled substance used for the determination of nonspecific binding. Last, but not least, the type and size of the glass microfibre filter, the filtration rate and whether substances that reduce the binding of the ligand to the glass microfibre filters (e.g. polyethylenimine) are used, also play an important role. Further studies of tricyclic antidepressant binding to different cell structures from patients with depression and other psychiatric disorders will evidently be necessary. Some studies show that the thrombocytes of schizophrenics, as well as those of patients with depression, have a smaller number of binding sites (Rotman et al., 1982). A thorough study is needed to determine whether imipramine binding is always associated with 5-HT uptake. In our opinion, this is not necessarily the case, since the concentration of 5-HT and the number of its binding sites on lymphocytes are low (Eliseyeva and Stefanovich, 1984; Meyer et al., 1974; Eliseyeva et al., 1982), whereas there are more imipramine binding sites on the same membrane. The ratio of binding sites for tricyclic antidepressant drugs to the sites of serotonin uptake, needs to be examined.
References
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