BIOL PSYCHIATRY 1991;29:789-798
789
13-Adrenoceptor Density of Intact Mononuclear Leukocytes in Subgroups of Depressive Disorders R. Jeanningros, P. Mazzola, J. M. Azorin, C. Samuelian-Massa, and R. Tissot
Binding parameters of (-)-iodovindoiol to [3z-adrenoceptors were determined on intact mononuclear cells in 41 untreated patients with different DSM-HI subtypes of depression. Both maximal fl-receptor density (B,,~) and dissociation constant ( K ~ were not significantly different between control and all depressed subjects. However, B , ~ was significantly decreased in unipolar patients as compared to controls (p < 0.001) whereas no significant difference was found in bipolar or dysthymic patients. In unipolar patients, a very strong association was found between B,,~ values and the severity of the depression as assessed by the Hamilton Depression Rating Scale score (r = - 0 . 7 5 ; p < 0.005). This correlation was also highly significant in the entire depressed population (r = - 0 . 5 8 ; p < 0.0009). These results suggest that the lower number of [3-adrenoceptors in intact leukocyte cells of depressed patients is related to the depression severity.
Introduction Several obse,-vations have suggested that abnormalities of catecholamine function in depressive illness could result from alterations in the sensitivity of noradrenergic receptors (Ashcroft et al 1972; Vetulani et al 1976; Sulser et al 1978). In the absence of direct methods for studying [3-adrenergic receptor function in the central nervous system, it has been emphasized that leukocytes provide a useful and easily accessible peripheral tissue model for studying [3-adrenergic receptors in depressive illness (Bunney and Murphy 19/:); Landmann et al i983; Pandey et m i98/). A decreased ~-adrenergic receptor responsiveness in the leukocytes of depressed patients has been shown using agoniststimulated cyclic adenosine monophosphate (AMP) productioe (Extein et al 1979; Pandey et al 1979; Mann et al 1985; Halper et al 1988). Receptor studies conducted in order to fi,A out an altered number of [3-adrenergic sites iv._depressed patients have been inconclusive in their findings. A decrease in [5-adrenoceptor number of lymphocyte membrane homogenates, as compared to normal controls, has been demonstrated in untreated depressed patients (Extein et al 1979; Pandey et al i985, 1987; Carstens et al 1987; Magliozzi
From the Unit/de Psychiatrie Biologique, C.N.R.S., Facult6 de M~ecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France (ILl, PM, RT) and *d'JeClinique de PsychiaUie et de Psychologie M(:dicale, CHU Timone, Rue Saint-Pierre, 13385 Marseille Cedex 5, France (JMA, CS-M). Address reprint requests to R. Jeanningros, Unit~ de Psychiatrie Biologique, CNRS, Faculr~ de M&lecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. Received April 25, 1990; revised July 26, 1990.
© 1991 Society of Biological Psychiatry
0006-3223/91/$03.50
790
BIOL PSYCHIATRY 1991;29:789-798
R. Jeanningros et al
et al 1989), in euthymic lithium-treated patients with recurrent unipolar or bipolar illness (Wood et al 1986), and in lymphoblastoid cell lines from families with manic-depressive disorder (Wright et al 1984). In contrast, Heady et al (1983) have shown an increased ~adrenoceptor density in a ~agnostically heterogeneous population of depressed patients. Mann et al (1985) could find no significant differences between depressed patients and normal controls with respect to 13-adrenergic receptor density of intact lymphocytes. These discrepancies have been attributed to differences in experimental procedures. For example, various radioligands with different specific activity, receptor specificities, and degrees of nonspecific binding have beea used in distinct cell preparations. Frozen membranes, which can be stored and studied at the convenience of the investigator, have been used more often than intact cells. When either the racemic isomer or a high concentration of propranolol is used for defining nonspecific binding, the number of physiologically relevant receptors may be overestimated. The cell isolation procedure may also influence experimental observations, particularly when membranes are used (Szefler et al 1987). Intact cells can yield an abnormally high receptor density, depending on the radioligand and the methodology used. In whole cells it has recently been shown that (-)-[nSl]iodocyanopindolol, which has a higher specific activity than (-)-[3H]dihydmalprenolol, labels a heterogeneous population of binding sites due to a high degree of nonspecific binding (Landmann et al 1983). In addition, in some studies, the depressed populations were not always diagnostically homogenous. We demonstrate the binding properties of intact mononuc!ear leukocytes (MNLs) in various subgroups of depressed patients using (-)-['2Sl]iodopindolol (IPlN). This radioligand is characterized by (1) high affinity and high specific activity towards [~-adrenergic receptors, (2) minimal nonspecific binding and (3) an ability to give linear Scatchard plots, which are indicative of only one population of specific binding sites. The binding procedure was performed on living mononuclear cells in order to accurately reproduce the in vivo receptor binding characteristics.
Methods Patients The study group consisted of forty-one hospitalized depressed patients (23 men and 18 women) with a mean + SEM age of 50.0 + 2.2 years ~range 28-,., years) who consented to take part and were diagnosed using the DSM-III-R classification (American Psychiatric Association 1980) and subtyped into the following groups: unipolar major depressive episode (n = 12), bipolar depression (n = 6), and dysthymic disorder (n = 23). Subsequent to hospitalization, patients had not received antidepressive drugs for at least 10 days prior to the beginning of the study except benzodiazepines (equivalent to 5--20 mg diazepam) or sedative neuroleptic (equivalent to 20-200 mg levomepromazine). Before hospital admission, 11 patients had not received psychotropic drugs, 11 had received an antidepressant drug (chlomipramine, 5; mianserin, 3; amitriptyline, 2; amineptine, 1), 4 had received a mood-stabilizing drug (lithium, 2; carbamazepine, 1; and valpromide, 1), 12 had received neuroleptics (aliphatic phenothiazine), and 22 had received benzodiazepines (di~zepam). Exclusion criteria were as follows: medical illness such as hypertension, atopic dermatitis, organic cerebral affections, asthma, and signs or symptoms of disorders of the cardiopulmonary system of any kind as well as the use of drugs known to affect 13-receptors (13-agonists, {3-antagonists, and steroids). The severity of depression
Leukocyte ~-Adrenoceptors in Depression Subtypes
BIOL PSYCHIATRY 199 i ~9:'/g9-'/98
"79I
was assessed by the Hamilton Rating Scale for Depression (HDRS) (National Institute of l~%r,tal Health version, 1967, 25 items). The patients had a HDRS score greater than I l with a m e a n ( _+ SEM) of 24.8 +_ 1.0. The control group consisted of forty-four volunteers (27 men and 17 women) mean -+ SEM age of 31. l _+ 1.5 years (range 19-60 years), who were free of medical illness and personal or family history of psychiatric disorders.
Preparation of Mononuclear Leukocytes Blood samples (25 ml) were collected by venipuncture in the morning (between 7:30 and 8:00 AM) into a heparinized glass tube and processed immediately. Subjects were restricted to bed rest for 30 min prior to blood drawing. Blood was centrifuged at 215 g for l0 min at room temperature. The pellet was preserved and the platelet-rich plasma collected and centrifuged at 2000 g for 15 rain at room temperature. The supernatant was pooled with the first pellet and diluted with 20 mmol/L Hepes buffer, 154 mmol/L NaCl, pH 7.5. The MNLs were isolated from other cells following the method of Boyum (1968) by centrifugation through a Ficoll-Hypaque gradient (density 1.077 g/ml; Sigma). Finally, the cells were washed twice with the same buffer and processed at once. The preparation yielded a heterogeneous cell population that contains a mixture of monocytes, 85% or more lymphocytes, less than 3.5 platelets per MNL, and negligible red cell contamination. Cell viability determined by trypan blue exclusion was greater than 95%.
Saturation Binding Assays Radioligand binding studies were performed with ( - )-[~251]iodoi,L,dolol (specific activity 2200 Ci/mmol) purchased from New England Nuclear (Paris, France). Binding assays were conducted in duplicate in siliconized polypropylene tubes. A tom of 2 x 105 to 6 x l05 cells, corresponding to 15-45 ~g of proteins, were incubateu at 37°C for 30 min in 187 p,l of 8 mmol/L Hepes, 62 mmol/L NaCl, pH 7.35 containing nine different concentrations of IPIN (12-380 pmol/L) with or without l i~mol/L (-)propranonol to define nonspecific binding. Incubation was terminated with 3 ml of ice-cold buffer (154 mmol/L NaCl, l0 mmol/L Tris HCI, pH 7.4) and then filtered immediately through GF/C glass fiber filters (Whatman) using a Millipore filtration unit. The incubation tubes were rinsed with 3 m~ of the same buffer, and poured over the filters. The filters were washed twice __...L _,,I .,._ . .~.-.U U I!.~ a ClLJUll[ei,lt . . . . + a ~.. irn ~altwc~ma ,.~ Specifi c WIHI 6 ~ U UI~; :I U U k..¢¢ U U l l l .~ .l . .~lllU I l l a l~J.ll.'~.ll~ counter, binding was at least 75% of the total binding at the K,t value. We checked that the addition of 10-5 mol/L chloroquine in the incubation mixtmae (Marinetti et al 1983) did not affect the determination of specific binding parameters. Bm~ was expressed as sites per cell (10 a sites per cell correspond to 1.66 fmol per 106 cells).
Data Analysis All the Scatchard plots were monophasic, with a correlation coefficient for linear regression of at least 0.90. When data were analyzed by a weighted, nonlinear, least-squares, curve-fitting program (Munson and Rodbard 1980), the affinity and the number of binding sites were equivalent to those derived by Scatchard transformation. Statistical analysis were performed using the X 2 t e s t for categorical data, and analysis of vaxiance (ANOVA) for continuous data differences. Student's t-test was used for comparisons between groups of par~e~'ic data and the Mann-Whitney U test was used
792
R. Jcanningros et al
BIOL PSYCHLATRY 1991 ;29:789-798
m spEc~c
"
A ~os sm:,Rc
4
0,06~ a
~"
TOTAL BOUND A
B/F
,
0,05
~
(1) 0
0.04
0,03
¢,0
o
3 •
E
2
o Q3
1
o
,..-,, ¢-
_ . _._..~
~
i
~
%~
0.02
II -
0,01 0.00
0
1
2
3
Specific bound (fmol/lO 6cells) I
0
100
•
!
200
"
!
300
"
!
400
[l 125]-iodopindolol Figure 1. [~251]iodopindolol binding to intact mononuclear leukocytes in a control subject. Left: total, specific, nonspecific binding is presented as a function of iodopindolol concentration (in pmol/L). Right:. graphic representation of corresponding Scatchard analysis indicating the presence of only one binding site (r 0.98).
for nonparametric data. The relationship between B,,~ values and either age or HDRS scores was evaluated by the Fisher test for statistical significance of the Pearson's correlation coefficient.
Results Determination of the binding characteristics of iodinated iodopindolol on intact mononuclear cells was performed on 44 control subjects. The saturation curves were hyperbofic and the Scatchard analysis was monophasic, indicating the presence of only one binding site at the ligand concenwations used (Figure 1). The coefficient of variation of Bm~ as measured repeatedly from the same sample varied from 4% to 15%, indicating a high assay reliability. When three subjects were tested four times at intervals several mouths apart, the variation coefficient was of the same magnitude, indicating the proba'uic absence of seasonal influence on i3-adrenoceptor number. In the controls, the mean K4 value 4. SEM was 53.3 +_ 4.3 pmol/L and mean Bm~, value -+ SEM was 2033 4. 92 sites per cell. There was no correlation between age and Bm~ values (r - 0.007, NS). Mean Bm~ values for women and men were not significantly different (2020 4- 181 sites per cell, n - 17, and 204I _+ 100 sites per cell, n - 27, respectively). Forty-one depressed pati=nts were studied and compared to control subject~ Thr. ~ex ratio between these two groups was not significantly different (X2 - 2.9, NS). When the entire depressed population was considered, neither mean Kd value (+_ SEM) nor me~_q Br,~ value (_+ SEM) of depressed patients was significantly different from control mean values (62.4 _ 7.1 pmol/L, n = 41, and 53.3 4. 4.3 pmoFL, n -- 44, respectively, p - 0.27, NS, for Kd; and for B,~,~ 1892 _+ 86 sites per cell, n - 41, and 2033 _+ 92 sites per cell, n - a4, respectively, p = 0.27, NS). However, there was a significant decrease in mean Bm~ value (4. SEM) in the unipolar depressed patients compared to controls (1460 +_ 69 sites ~ r celi, t: -- 12, and 2033 4. 92 sites per cell, n = 44,
Leukocyte [$-Adrenoceptors in Depression Subtypes
BtOt. ps¥c'mA~Y
793
1991 ;29:789-798
Table 1. HDRS Rating Scores and Characteristics of [ml]lodopindolol B i n ~ g Sites on Mononuclear Leukocytes in Different DSM-m Subtypes of Depressed Patients and in Controls~ Group
Age (yr~'
Control (n
=
31.1
± 1.5
HDRS score~
Bm~ (sites per ceH~
K~ (pmol/L)"
--
2033 ± 92
53.3 ± 4 . 3
44)
Unipolar ( n - - 12)
5 9 . 0 ± 3.3
28.4 ± 2.0
1460 _ 6 9 /
6 8 . 6 ± 16.2
Bipolar (n = 6)
52.5 -4- 6 . 0
23.7 ± 3.4
1941 ± 129'
80.5 ± 31.0
Dyslhymic (n = 23)
4 4 . 7 ± 2.8
23.3 _ 1.2 s
2104 _ 124 t
54.4 ± 5.5
°All values are given as mean _ SEM. bAnalysis o f v a r i ~ c e (ANOVA): F = 22.2, df = 3, p = 0 . ~ 0 1 . CANOVA: F = 2.6, df = 2, p = 0.09. ~ANOVA: F = 3.9, df = 3, p = 0.01. cANOVA: F - 1.31, df = 3, p = 0.25. < 0.001 as compared to controls. ~p < 0.01 as compared to unipolar patients, Mann-Whitney U test. hp < 0.03 as compmed to unipolar patients, Mann-Whitney U test.
respectively; p < 0.001), with no change in mean Kd value (68.6 _+ 16.2 versus 53.3 --+ 4.3 pmol/L; NS). The difference was still significant when these patients were compared to a smaller number of 12 randomly sampled controls (p < 0.04). In the two other diagnostic subgroups, m , a n Bm~ value did not differ significantly from the control group (1941 _+ 129 sites per cell, n = 6 , for bipolar depressed subjects, and 2104 +_ 124 sites per cell, n = 23, for dysthymic disorders versus 2033 _+ 92 sites per cell for controls; NS). These results are summarized in Table 1. The distribution of Bm~ values for the control group and the different diagnostic subgroups is shown in Figure 2. Seven of the 12 unipolar depressed patients (58%), 1 of the 6 bipolar patients (16.6%), and 3 of the
4° t
&
3500
A
® u it}
3(L00 &
2500 2000-
v
x
E
1500 -
O3
I0(0 500
-
&
A
A
Control
•
Bipolar
¢
[ rnipolar
&
Dysthymic
0 Figure 2. Scattergram of [3-adrenoceptor maximal density (Bm~) on intact mononuclea: leul,ocytes in controls (n = 44) and in the different DSM-Ill diagnostic subgroups: bipolar depressio~ (n = 6), unipo!ar depression (n= 12), and dysthymic disorder (n = 23). - - , mean; . . . . , SEAM. * significantly different from controls, p < 0.001, Mann-Whitney U test.
794
R. Jeanningros et al
BIOL PSYCHIATRY I~1 ;29:789-798
4500
!
r = - 0.58 4000
p
789
13-Adrenoceptor Density of Intact Mononuclear Leukocytes in Subgroups of Depressive Disorders R. Jeanningros, P. Mazzola, J. M. Azorin, C. Samuelian-Massa, and R. Tissot
Binding parameters of (-)-iodovindoiol to [3z-adrenoceptors were determined on intact mononuclear cells in 41 untreated patients with different DSM-HI subtypes of depression. Both maximal fl-receptor density (B,,~) and dissociation constant ( K ~ were not significantly different between control and all depressed subjects. However, B , ~ was significantly decreased in unipolar patients as compared to controls (p < 0.001) whereas no significant difference was found in bipolar or dysthymic patients. In unipolar patients, a very strong association was found between B,,~ values and the severity of the depression as assessed by the Hamilton Depression Rating Scale score (r = - 0 . 7 5 ; p < 0.005). This correlation was also highly significant in the entire depressed population (r = - 0 . 5 8 ; p < 0.0009). These results suggest that the lower number of [3-adrenoceptors in intact leukocyte cells of depressed patients is related to the depression severity.
Introduction Several obse,-vations have suggested that abnormalities of catecholamine function in depressive illness could result from alterations in the sensitivity of noradrenergic receptors (Ashcroft et al 1972; Vetulani et al 1976; Sulser et al 1978). In the absence of direct methods for studying [3-adrenergic receptor function in the central nervous system, it has been emphasized that leukocytes provide a useful and easily accessible peripheral tissue model for studying [3-adrenergic receptors in depressive illness (Bunney and Murphy 19/:); Landmann et al i983; Pandey et m i98/). A decreased ~-adrenergic receptor responsiveness in the leukocytes of depressed patients has been shown using agoniststimulated cyclic adenosine monophosphate (AMP) productioe (Extein et al 1979; Pandey et al 1979; Mann et al 1985; Halper et al 1988). Receptor studies conducted in order to fi,A out an altered number of [3-adrenergic sites iv._depressed patients have been inconclusive in their findings. A decrease in [5-adrenoceptor number of lymphocyte membrane homogenates, as compared to normal controls, has been demonstrated in untreated depressed patients (Extein et al 1979; Pandey et al i985, 1987; Carstens et al 1987; Magliozzi
From the Unit/de Psychiatrie Biologique, C.N.R.S., Facult6 de M~ecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France (ILl, PM, RT) and *d'JeClinique de PsychiaUie et de Psychologie M(:dicale, CHU Timone, Rue Saint-Pierre, 13385 Marseille Cedex 5, France (JMA, CS-M). Address reprint requests to R. Jeanningros, Unit~ de Psychiatrie Biologique, CNRS, Faculr~ de M&lecine, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. Received April 25, 1990; revised July 26, 1990.
© 1991 Society of Biological Psychiatry
0006-3223/91/$03.50
790
BIOL PSYCHIATRY 1991;29:789-798
R. Jeanningros et al
et al 1989), in euthymic lithium-treated patients with recurrent unipolar or bipolar illness (Wood et al 1986), and in lymphoblastoid cell lines from families with manic-depressive disorder (Wright et al 1984). In contrast, Heady et al (1983) have shown an increased ~adrenoceptor density in a ~agnostically heterogeneous population of depressed patients. Mann et al (1985) could find no significant differences between depressed patients and normal controls with respect to 13-adrenergic receptor density of intact lymphocytes. These discrepancies have been attributed to differences in experimental procedures. For example, various radioligands with different specific activity, receptor specificities, and degrees of nonspecific binding have beea used in distinct cell preparations. Frozen membranes, which can be stored and studied at the convenience of the investigator, have been used more often than intact cells. When either the racemic isomer or a high concentration of propranolol is used for defining nonspecific binding, the number of physiologically relevant receptors may be overestimated. The cell isolation procedure may also influence experimental observations, particularly when membranes are used (Szefler et al 1987). Intact cells can yield an abnormally high receptor density, depending on the radioligand and the methodology used. In whole cells it has recently been shown that (-)-[nSl]iodocyanopindolol, which has a higher specific activity than (-)-[3H]dihydmalprenolol, labels a heterogeneous population of binding sites due to a high degree of nonspecific binding (Landmann et al 1983). In addition, in some studies, the depressed populations were not always diagnostically homogenous. We demonstrate the binding properties of intact mononuc!ear leukocytes (MNLs) in various subgroups of depressed patients using (-)-['2Sl]iodopindolol (IPlN). This radioligand is characterized by (1) high affinity and high specific activity towards [~-adrenergic receptors, (2) minimal nonspecific binding and (3) an ability to give linear Scatchard plots, which are indicative of only one population of specific binding sites. The binding procedure was performed on living mononuclear cells in order to accurately reproduce the in vivo receptor binding characteristics.
Methods Patients The study group consisted of forty-one hospitalized depressed patients (23 men and 18 women) with a mean + SEM age of 50.0 + 2.2 years ~range 28-,., years) who consented to take part and were diagnosed using the DSM-III-R classification (American Psychiatric Association 1980) and subtyped into the following groups: unipolar major depressive episode (n = 12), bipolar depression (n = 6), and dysthymic disorder (n = 23). Subsequent to hospitalization, patients had not received antidepressive drugs for at least 10 days prior to the beginning of the study except benzodiazepines (equivalent to 5--20 mg diazepam) or sedative neuroleptic (equivalent to 20-200 mg levomepromazine). Before hospital admission, 11 patients had not received psychotropic drugs, 11 had received an antidepressant drug (chlomipramine, 5; mianserin, 3; amitriptyline, 2; amineptine, 1), 4 had received a mood-stabilizing drug (lithium, 2; carbamazepine, 1; and valpromide, 1), 12 had received neuroleptics (aliphatic phenothiazine), and 22 had received benzodiazepines (di~zepam). Exclusion criteria were as follows: medical illness such as hypertension, atopic dermatitis, organic cerebral affections, asthma, and signs or symptoms of disorders of the cardiopulmonary system of any kind as well as the use of drugs known to affect 13-receptors (13-agonists, {3-antagonists, and steroids). The severity of depression
Leukocyte ~-Adrenoceptors in Depression Subtypes
BIOL PSYCHIATRY 199 i ~9:'/g9-'/98
"79I
was assessed by the Hamilton Rating Scale for Depression (HDRS) (National Institute of l~%r,tal Health version, 1967, 25 items). The patients had a HDRS score greater than I l with a m e a n ( _+ SEM) of 24.8 +_ 1.0. The control group consisted of forty-four volunteers (27 men and 17 women) mean -+ SEM age of 31. l _+ 1.5 years (range 19-60 years), who were free of medical illness and personal or family history of psychiatric disorders.
Preparation of Mononuclear Leukocytes Blood samples (25 ml) were collected by venipuncture in the morning (between 7:30 and 8:00 AM) into a heparinized glass tube and processed immediately. Subjects were restricted to bed rest for 30 min prior to blood drawing. Blood was centrifuged at 215 g for l0 min at room temperature. The pellet was preserved and the platelet-rich plasma collected and centrifuged at 2000 g for 15 rain at room temperature. The supernatant was pooled with the first pellet and diluted with 20 mmol/L Hepes buffer, 154 mmol/L NaCl, pH 7.5. The MNLs were isolated from other cells following the method of Boyum (1968) by centrifugation through a Ficoll-Hypaque gradient (density 1.077 g/ml; Sigma). Finally, the cells were washed twice with the same buffer and processed at once. The preparation yielded a heterogeneous cell population that contains a mixture of monocytes, 85% or more lymphocytes, less than 3.5 platelets per MNL, and negligible red cell contamination. Cell viability determined by trypan blue exclusion was greater than 95%.
Saturation Binding Assays Radioligand binding studies were performed with ( - )-[~251]iodoi,L,dolol (specific activity 2200 Ci/mmol) purchased from New England Nuclear (Paris, France). Binding assays were conducted in duplicate in siliconized polypropylene tubes. A tom of 2 x 105 to 6 x l05 cells, corresponding to 15-45 ~g of proteins, were incubateu at 37°C for 30 min in 187 p,l of 8 mmol/L Hepes, 62 mmol/L NaCl, pH 7.35 containing nine different concentrations of IPIN (12-380 pmol/L) with or without l i~mol/L (-)propranonol to define nonspecific binding. Incubation was terminated with 3 ml of ice-cold buffer (154 mmol/L NaCl, l0 mmol/L Tris HCI, pH 7.4) and then filtered immediately through GF/C glass fiber filters (Whatman) using a Millipore filtration unit. The incubation tubes were rinsed with 3 m~ of the same buffer, and poured over the filters. The filters were washed twice __...L _,,I .,._ . .~.-.U U I!.~ a ClLJUll[ei,lt . . . . + a ~.. irn ~altwc~ma ,.~ Specifi c WIHI 6 ~ U UI~; :I U U k..¢¢ U U l l l .~ .l . .~lllU I l l a l~J.ll.'~.ll~ counter, binding was at least 75% of the total binding at the K,t value. We checked that the addition of 10-5 mol/L chloroquine in the incubation mixtmae (Marinetti et al 1983) did not affect the determination of specific binding parameters. Bm~ was expressed as sites per cell (10 a sites per cell correspond to 1.66 fmol per 106 cells).
Data Analysis All the Scatchard plots were monophasic, with a correlation coefficient for linear regression of at least 0.90. When data were analyzed by a weighted, nonlinear, least-squares, curve-fitting program (Munson and Rodbard 1980), the affinity and the number of binding sites were equivalent to those derived by Scatchard transformation. Statistical analysis were performed using the X 2 t e s t for categorical data, and analysis of vaxiance (ANOVA) for continuous data differences. Student's t-test was used for comparisons between groups of par~e~'ic data and the Mann-Whitney U test was used
792
R. Jcanningros et al
BIOL PSYCHLATRY 1991 ;29:789-798
m spEc~c
"
A ~os sm:,Rc
4
0,06~ a
~"
TOTAL BOUND A
B/F
,
0,05
~
(1) 0
0.04
0,03
¢,0
o
3 •
E
2
o Q3
1
o
,..-,, ¢-
_ . _._..~
~
i
~
%~
0.02
II -
0,01 0.00
0
1
2
3
Specific bound (fmol/lO 6cells) I
0
100
•
!
200
"
!
300
"
!
400
[l 125]-iodopindolol Figure 1. [~251]iodopindolol binding to intact mononuclear leukocytes in a control subject. Left: total, specific, nonspecific binding is presented as a function of iodopindolol concentration (in pmol/L). Right:. graphic representation of corresponding Scatchard analysis indicating the presence of only one binding site (r 0.98).
for nonparametric data. The relationship between B,,~ values and either age or HDRS scores was evaluated by the Fisher test for statistical significance of the Pearson's correlation coefficient.
Results Determination of the binding characteristics of iodinated iodopindolol on intact mononuclear cells was performed on 44 control subjects. The saturation curves were hyperbofic and the Scatchard analysis was monophasic, indicating the presence of only one binding site at the ligand concenwations used (Figure 1). The coefficient of variation of Bm~ as measured repeatedly from the same sample varied from 4% to 15%, indicating a high assay reliability. When three subjects were tested four times at intervals several mouths apart, the variation coefficient was of the same magnitude, indicating the proba'uic absence of seasonal influence on i3-adrenoceptor number. In the controls, the mean K4 value 4. SEM was 53.3 +_ 4.3 pmol/L and mean Bm~, value -+ SEM was 2033 4. 92 sites per cell. There was no correlation between age and Bm~ values (r - 0.007, NS). Mean Bm~ values for women and men were not significantly different (2020 4- 181 sites per cell, n - 17, and 204I _+ 100 sites per cell, n - 27, respectively). Forty-one depressed pati=nts were studied and compared to control subject~ Thr. ~ex ratio between these two groups was not significantly different (X2 - 2.9, NS). When the entire depressed population was considered, neither mean Kd value (+_ SEM) nor me~_q Br,~ value (_+ SEM) of depressed patients was significantly different from control mean values (62.4 _ 7.1 pmol/L, n = 41, and 53.3 4. 4.3 pmoFL, n -- 44, respectively, p - 0.27, NS, for Kd; and for B,~,~ 1892 _+ 86 sites per cell, n - 41, and 2033 _+ 92 sites per cell, n - a4, respectively, p = 0.27, NS). However, there was a significant decrease in mean Bm~ value (4. SEM) in the unipolar depressed patients compared to controls (1460 +_ 69 sites ~ r celi, t: -- 12, and 2033 4. 92 sites per cell, n = 44,
Leukocyte [$-Adrenoceptors in Depression Subtypes
BtOt. ps¥c'mA~Y
793
1991 ;29:789-798
Table 1. HDRS Rating Scores and Characteristics of [ml]lodopindolol B i n ~ g Sites on Mononuclear Leukocytes in Different DSM-m Subtypes of Depressed Patients and in Controls~ Group
Age (yr~'
Control (n
=
31.1
± 1.5
HDRS score~
Bm~ (sites per ceH~
K~ (pmol/L)"
--
2033 ± 92
53.3 ± 4 . 3
44)
Unipolar ( n - - 12)
5 9 . 0 ± 3.3
28.4 ± 2.0
1460 _ 6 9 /
6 8 . 6 ± 16.2
Bipolar (n = 6)
52.5 -4- 6 . 0
23.7 ± 3.4
1941 ± 129'
80.5 ± 31.0
Dyslhymic (n = 23)
4 4 . 7 ± 2.8
23.3 _ 1.2 s
2104 _ 124 t
54.4 ± 5.5
°All values are given as mean _ SEM. bAnalysis o f v a r i ~ c e (ANOVA): F = 22.2, df = 3, p = 0 . ~ 0 1 . CANOVA: F = 2.6, df = 2, p = 0.09. ~ANOVA: F = 3.9, df = 3, p = 0.01. cANOVA: F - 1.31, df = 3, p = 0.25. < 0.001 as compared to controls. ~p < 0.01 as compared to unipolar patients, Mann-Whitney U test. hp < 0.03 as compmed to unipolar patients, Mann-Whitney U test.
respectively; p < 0.001), with no change in mean Kd value (68.6 _+ 16.2 versus 53.3 --+ 4.3 pmol/L; NS). The difference was still significant when these patients were compared to a smaller number of 12 randomly sampled controls (p < 0.04). In the two other diagnostic subgroups, m , a n Bm~ value did not differ significantly from the control group (1941 _+ 129 sites per cell, n = 6 , for bipolar depressed subjects, and 2104 +_ 124 sites per cell, n = 23, for dysthymic disorders versus 2033 _+ 92 sites per cell for controls; NS). These results are summarized in Table 1. The distribution of Bm~ values for the control group and the different diagnostic subgroups is shown in Figure 2. Seven of the 12 unipolar depressed patients (58%), 1 of the 6 bipolar patients (16.6%), and 3 of the
4° t
&
3500
A
® u it}
3(L00 &
2500 2000-
v
x
E
1500 -
O3
I0(0 500
-
&
A
A
Control
•
Bipolar
¢
[ rnipolar
&
Dysthymic
0 Figure 2. Scattergram of [3-adrenoceptor maximal density (Bm~) on intact mononuclea: leul,ocytes in controls (n = 44) and in the different DSM-Ill diagnostic subgroups: bipolar depressio~ (n = 6), unipo!ar depression (n= 12), and dysthymic disorder (n = 23). - - , mean; . . . . , SEAM. * significantly different from controls, p < 0.001, Mann-Whitney U test.
794
R. Jeanningros et al
BIOL PSYCHIATRY I~1 ;29:789-798
4500
!
r = - 0.58 4000
p
