Peripheral-type benzodiazepine receptors on human blood mononuclear cells are not regulated by ovarian steroids

Psychoneuroendocrinology, Vol.

19, No. 1, pp. 65-78, 1994

0306-4530/94 $6.00 + .00 ©1993 Pergamon Press Ltd.

Printed in the U.S.A.

PERIPHERAL-TYPE BENZODIAZEPINE RECEPTORS ON HUMAN BLOOD MONONUCLEAR CELLS ARE NOT R E G U L A T E D BY O V A R I A N S T E R O I D S P. FERRERO, 1 P. ROCCA, 2 E. MONTALENTI, l P. BENNA, 1 A. M I L A N I , 2 L . RAVIZZA, 2 and B. BERGAMASCO 1 Departments of ~Neurology and ZPsychiatry, University of Turin, Turin, Italy

(Received 6 January 1993; in final form 12 July 1993)

SUMMARY Peripheral-type benzodiazepine receptors (pBZr) have been shown to be sensitive to hormonal perturbations, including changes in ovarian steroids. This study examines whether estradiol and progesterone modulate pBZr binding in membranes of human blood mononuclear cells, as measured by binding of both 3H-PK 11195 and 3H-Ro 5-4864. Our findings were negative. There was no steroidal modulation of pBZr binding to these membrane preparations in vivo in normal women studied at different sex-steroid phases of the menstrual cycle, or during 8-30 weeks of pregnancy. There was also no effect of hormones on the binding sites in cultures of mononuclear cells treated with estradiol or progesterone (up to 10-5 M) over a period between 2 and 72 h. Further, we performed in vitro competition experiments, which showed that both steroids are not active at the pBZr. Our data suggest that pBZr located in human blood mononuclear cells are insensitive to the physiological variations of circulating female hormones.

INTRODUCTION EFFECTSOF ovarian steroids include alterations in seizure susceptibility, anxiety, mood patterns, and sedation (Maggi & Perez, 1985; Newmark & Penry, 1980; Selye, 1942). One of the possible mechanisms mediating these actions is the regulation of subsynaptic gamma-aminobutyric acid (GABA) type A receptors functions (Maggi & Perez, 1986; Majewska et al., 1986; McEwen, 1991). In the mammalian brain, GABAA receptors are the major site of action of benzodiazepines (BZ) (Delorey & Olsen, 1992). However, certain BZ also bind to the so-called peripheral-type BZ receptors (pBZr) present in the central nervous system as well as in other tissues (Drugan & Holmes, 1991; Krueger & Papadopoulos, 1992). It has been recently shown that cholesterol influx into the inner mitochondrial membrane, the rate-limiting step in steroid synthesis, is regulated by a pBZr present in the outer mitochondrial membrane (Mukhin et al., 1989; Papadopoulos et al., 1990; Yanagibashi et al., 1989). Thus, mitochondrial pBZr was suggested to represent an important link between the endocrine system and nervous system. Endogenous steroids production in the central nervous system via glial pBZr (Guarneri et al., 1992; Hu et al., NON-REPRODUCTIVE

A d d r e s s c o r r e s p o n d e n c e a n d r e p r i n t r e q u e s t s to: Dr. Patrizia F e r r e r o , N e u r o l o g i c a l Clinic, Via C h e r a s c o 15, 10126 T o r i n o , Italy. 65

66

P. FERRERO et al.

1987; Jung-Testas et al., 1989) might provide the basis for an interaction with the GABAA receptors. Alternative possibilities include effects of pBZr in the periphery with subsequent central effects, perhaps mediated by steroids (Paul & Purdy, 1992), or a link through the endogenous peptide diazepam binding inhibitor (DBI), which can affect both GABA A receptors function and steroid biosynthesis via binding to central or peripheral BZr (Besman et al., 1989; Papadopoulos et al., 1992). There is further evidence from animal studies that regulation of pBZr is under both hormonal and neural control. Various hormonal manipulations, sympathetic denervation, and certain forms of stress can modify pBZr sensitivity in several peripheral organs and also in brain (Anholt et al., 1985; Drugan & Holmes, 1991 ; Gavish et al., 1992). Alterations in pBZr density as a result of normal stress or pathologic anxiety have also been reported in distinct blood elements of humans (i.e., platelets or mononuclear cells) (Dar et al., 1991; Ferrarese et al., 1990; Karp et al., 1989; Rocca et al., 1991; Weizman et al., 1987). However, except for an earlier study comparing platelet 3H-PK 11195 binding in a group of anovulatory women before and after treatment with human menopausal gonadotropin, which reports no effect on this binding (Diorio et al., 1990), the potential interaction between these pBZr systems and hormones has not been described in humans. The present study was aimed at determining whether ovarian steroids modify pBZr binding to membranes of human peripheral blood mononuclear cells (PBMC). The objectives were 2-fold. First, we examined whether changes in pBZr binding occur in normal women at different sex-steroid phases of the menstrual cycle or during pregnancy. Second, we investigated the effects of hormones in cultures of mononuclear cells to evaluate whether under this condition and in vivo the receptors are regulated similarly. The experiments compared the binding profile of 3H-Ro 5-4864 (4'-chlorodiazepam) and that of the isoquinoline derivative 3H-PK 11195. These compounds are known to bind specifically to pBZr but probably to different domains or conformational states of the receptor, because some differences have been observed among species and tissues, including the human PBMC (Berkovich et al., 1993; Ferrarese et al., 1990; Ferrero et al., 1991; Marangos et al., 1983). Thus, our comparison might reveal an heterogeneous effect of female hormones on the binding responses. MATERIALS AND METHODS

Subjects The study was performed in two parts. In part one, 15 healthy women gave informed consent to participate in a longitudinal study over the menstrual cycle. They were recruited on a voluntary basis among hospital staff (medical doctors, nurses, and students). Their mean age (--- SD) was 29.8 -+ 5.2 yr (range 21-39 yr). Seven out of the 15 women were taking oral contraceptives (o.c., 30/zg ethinylestradiol plus 50/xg gestodene) for at least 1 year prior to experimentation, and the other eight had spontaneous menstrual cycles. The inclusion criteria were: (1) absence of moderate to severe clinical history of somatic and psychological symptoms, reported with a timing that appeared to be linked to the menstrual cycle; (2) reports of regular cycles with a usual cycle length of 26-32 days; (3) no current, past, or family related psychiatric diagnoses; (4) normal physical examination and routine blood laboratory profile; and (5) absence of medications, and particularly no history of psychoactive or hormonal drug intake. Before entering the study, all participants were interviewed by the same psychiatrist (P.R.), who completed the Hamilton Rating Scale for Depression (Hamilton, 1960) to exclude a mood disorder.

LYMPHOCYTE PBZR AND OVARIAN STEROIDS

67

At the same visit, as well as at the timing of each blood sampling, the subjects also received the Hamilton Rating Scale for Anxiety (HARS) (Hamilton, 1959) and the Moos Menstrual Distress Questionnaire (MDQ) (Moos, 1968), in the version composed of eight symptom clusters (pain, concentration, arousal, negative affect, behavioral change, autonomic reactions, water retention, and control). The 47 items were rated on a 1-6 point scale, where 1 = no experience of a symptom and 6 = disabling experience. In the second part of the study, eight pregnant women, age 23-39 (mean 31) year, and eight normal cycling women, age 25-39 (mean 33) year, were enrolled. All of the pregnant women were volunteers drawn from the hospital staff or contacted by personal knowledge (three subjects). Informed consent was obtained. They all had regular pregnancies with no history of current or past medical or psychiatric illness. None was taking any medication. The nonpregnant women were a subgroup from those entering part one of the study, who agreed to donate an additional blood sample and served as a control group. Each participant was seen on a single occasion during which clinical data were collected and depression and anxiety were rated by HDRS and HARS to exclude a mood or an anxiety disorder. Pregnancies were dated by history of last menstrual period, early ultrasound evaluation, and obstetrical examination in all cases. Two out of the eight women were studied at early stages of pregnancy (8-10 weeks) and the other six in the second half of gestation (24-30 weeks). In women studied with menstrual cycles, the study was conducted in the early follicular phase of the cycle.

Procedures In study one, volunteers presented for testing on days 1-2, days 6-8, days 13-16, and days 24-26 during one menstrual cycle. Blood samples (80-100 ml) were collected by antecubital venipuncture to heparinized (10 UI/ml blood) glass tubes at 0800-0900 h and then processed for PBMC purification not later than 2 h. Aliquots of blood were examined for determination of female hormones levels, white cell counts, and T lymphocyte subpopulations. These analyses were performed routinely at the General Laboratory of our hospital (San Giovanni Battista, Turin). Progesterone and 17/~-estradiol were assayed by commercial kits (provided by Sorin Biomedica, Italy). Total T lymphocytes and T lymphocytes subsets were analyzed by direct immunofluorescence on an automated laser flow cytometry system using monoclonal antibodies of the OKT (OKT3 +, OKT4 +, OKT8+, Ortho-Immune) series. In the second part of the study, all participants were seen once with blood taken from an antecubital vein between 0900 and 1000 h at the time of each clinic visit. The blood samples were collected into heparinized glass tubes and then processed for PBMC purification as those in study one. As the amount of blood that could be obtained from each pregnant women was low (30-40 ml), the serum levels of hormones and white cells counts were not analyzed. Cell Preparations and SH-PK 11195 and 3H-Ro 5-4864 Binding Assays After removal of the platelet rich plasma, the PBMC (85-90% lymphocytes, 10-15% monocytes with over 95% viability as assessed by trypan blue exclusion) were isolated on Ficoll-sodium metrizoate solution (Lymphoprep-Nycomed, Oslo, Norway) gradient centrifugation. The preparation of PBMC crude membranes or intact cells and pBZr binding assays were conducted according to previous described methods, with minor modifications (Ferrero et al., 1991; Moingeon et al., 1983; Rocca et al., 1993). The binding assays were carried out by incubating at 4°C during 45 min aliquots in duplicate or

68

P. FERREROet al.

triplicate of the PBMC membranes (30-40/zg protein) or the intact cells (0.8-1 × 10-6) in 50 mM tris HC1 buffer pH 7.4 or RPMI-1640 medium containing 2 mM glutamine and 10 mM Hepes pH 7.4 with 8-10 concentrations of 3H-PK 11195 (1-(2-chlorophenyl)-Nmethyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide) (New England Nuclear, Specific Activity 86.9 Ci/mmole, 1-50 nM), or 3H-Ro 5-4864 (4'-chlorodiazepam) (New England Nuclear, Specific Activity 85.4 Ci/mmole, 1-100 nM) in a final volume of 500 or 125/zl for 3H-PK 11195 or 3H-Ro 5-4864 binding, respectively. The reactions were terminated by the addition of 3 ml ice cold buffer and rapid vacuum filtration over Whatman GF/C filters, followed by two washes of 3 ml ice cold buffer. Nonspecific binding was obtained by incubating the PBMC preparations with the 3H-ligands and 1/zM PK I 1195 or 10/xM Ro 5-4864. For competition experiments, the final concentrations of radioligand used were 5 nM for 3H-PK 11195 and 10 nM for 3H-Ro 5-4864. Displacement curves were obtained using four (17/3-estradiol and progesterone) and six (PK 11195, 4'-chlorodiazepam, diazepam, and clonazepam) concentrations of each drug tested (from 10-9 to 10-5 M).

Culture Conditions For these experiments, PBMC were isolated from whole blood buffy coats kindly provided by the Transfusional Service of our hospital. After separation by centrifugation on a Lymphoprep gradient, the PBMC were washed four times with Hank's balanced salt solution and then resuspended at I × 107 cells/ml in RPMI-1640 medium supplemented with 2 mM glutamine, 20 mM HEPES (pH 7.4), 10% fetal calf serum, and antibiotics (I00 units/ml penicillin and 100/.~g/ml streptomycin). Cells were cultured at 37°C under an atmosphere of 5% COz/95% air in 50 cm 2 tissue cultures flasks as indicated by the experimental protocols. At the end of incubation, the nonadherent cells (> 99% pure lymphocytes with viability more than 85%, as determined by trypan blue exclusion) were collected, washed four times in Hank's balanced salt solution, and then processed for the membrane preparations and binding assays as described above. The steroids 17/3estradiol or progesterone (Sigma) were dissolved and stored in dimethyl sulfoxide (DMSO) at 10-2 M and subsequently diluted in medium at the indicated concentrations and added in the cultures. DMSO controls were performed in all experiments. DMSO did not alter PBMC survival or pBZr binding in the presence of control medium (data not shown). Analyses of Data The data from saturation and competition binding experiments were analyzed by nonlinear least squares regression using the computer program LIGAND (Version 3, Munson & Rodbard, 1980). The binding parameters (maximal binding capacities,Bmax, and apparent binding affinities, Ka), hormone levels and psychological variables at various time test points were analyzed by one-way analysis of variance (ANOVA) and Student's t-test when appropriate. Correlation coefficients were determined by linear regression analysis. Proteins were measured by the method of Lowry et al. (1951). Data in the figure and tables refer to the mean --- SEM. RESULTS

The Effect of Ovarian Steroids During the Menstrual Cycle The levels of estradiol and progesterone in serum and binding of 3H-PK 11195 and 3H-Ro 5-4864 to PBMC membranes at four different phases of menstrual cycle were compared in spontaneous and o.c. controlled cycling normal women. The results

LYMPHOCYTE PBZR AND OVARIAN STEROIDS

69

T A B L E I . S P E C I F I C B I N D I N G OF 3H-PK1 1195 A N D 3H-RO 5 - 4 8 6 4 TO MEMBRANES FROM H U M A N BLOOD M O N O N U C L E A R CELLS AT D I F F E R E N T PHASES OF T H E M E N S T R U A L CYCLE W I T H C O R R E S P O N D I N G ESTRADIOL A N D PROGESTERONE SERUM C O N C E N T R A T I O N S

Parameters

Groups

Menstrual

Follicular

Ovulatory

Luteal

3H-PKI 1195 S.B. Bmax (pmol/mg prot)

s o.c.

7.99 - 0.5 8.65 -+ 0.5

8.28 +-- 0.6 7.92 --- 0.6

8.48 - 0.7 9.23 - 1.0

7.92 --- 0.4 8.63 --- 0.6

Kd (nM)

s o.c.

7.7 --- 0.4 7.9 -+ 0.3

8.1 --- 0.3 8.0 -+ 0.2

8.2 + 0.3 7.8 -+ 0.2

8.3 --- 0.4 7.9 - 0.2

3H-Ro 5-4864 S.B. Bmax

(pmol/mg prot) Ka

(nM)

o.c.

1.50 -+ 0.3 1.66 -+ 0.1

1.46 --- 0.2 1.34 --+ 0.1

1.30 -+ 0.2 1.58 --- 0.1

1.51 -+ 0.3 1.43 --+ 0.2

s o.c.

21 --- 2.9 20 -+ 3.4

20 -+ 3.2 21 - 3.0

19 + 2.7 21 -+ 3.1

21 - 2.3 19 -+ 2.3

s

Hormones Estradiol (pmol/l)

s o.c.

201 -+ 20 152 -+ 8.8

354 -+ 94 173 - 8.5

"901 --- 82 184 -+ 8.6

"631 --- 49 166 -+ 7.7

Progesterone (nmol/1)

s o.c.

0.97 -+ 0.1 2.41 _+ 0.2

1.18 --- 0.1 2.24 -+ 0.2

4.30 --- 0.3 2.76 _+ 0.2

**35.0 --- 2.0 3.07 --_ 0.3

Data shown are the mean -+ SEM of eight women with spontaneous cycle(s) and seven under oral contraceptives (o.c.). The preparation of PBMC membranes and binding assays were performed as described in Methods. *p < .001 if compared with the values in menstrual and follicular period **p < .001 if compared with the values in menstrual, follicular and ovulatory period

o f this s t u d y are s h o w n in Table I, w h e r e a s s c a t t e r g r a m s o f the individual binding p a r a m e t e r s (Bronx and/Ca) are illustrated in Fig. 1. W h e n analyzing d a t a f r o m binding e x p e r i m e n t s b y the L I G A N D p r o g r a m , one population o f binding sites f o r 3H-PK 11195 o r 3H-Ro 5-4864 w a s found. H o w e v e r , consistent differences w e r e o b s e r v e d b e t w e e n the equilibrium binding c o n s t a n t s (nmax o r gd) o f these t w o ligands. T h e density o f binding sites for 3H-Ro 5-4864 was a p p r o x i m a t e l y 5-fold less than that for 3H-PK 11195. Also, the a p p a r e n t affinity for 3H-Ro 5-4864 was l o w e r than that o f 3H-PK- 11195. As e x p e c t e d , a o n e - w a y A N O V A r e v e a l e d significant variations o f s e r u m h o r m o n e s levels in the g r o u p with s p o n t a n e o u s m e n s e s , but not in the one u n d e r o.c. controlled cycle. Estradiol levels p e a k e d at mid-cycle, w e r e l o w e r in the menstrual and follicular p h a s e s , and intermediate at the luteal period (F: 36.2, p < .001). P r o g e s t e r o n e values i n c r e a s e d f r o m early t h r o u g h to late c y c l e (F: 26.5, p < .001). H o w e v e r , no significant

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Fr6. 1: Scattergrams of 3H-PK 11195 (circles) and 3H-Ro 5-4864 (squares) binding in blood mononuclear cell membranes from normal women during the menstrual cycle. Each point represents an individual subject. Open symbols indicate women under oral contraceptives. Solid lines and hatched lines represent the means -+ SEM of each group.

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LYMPHOCYTE PBZR AND OVARIAN STEROIDS

71

T A B L E I I . T H E E F F E C T S OF M E N S T R U A L C Y C L E ON T H E MOOS M E N S T R U A L DISTRESS Q U E S T I O N N A I R E A N D T H E H A M I L T O N R A T I N G SCALE F O R A N X I E T Y

Cycle phases Variable

Groups

Menstrual

Follicular

Ovulatory

Luteal

F values

Pain

s o.c.

7.8 7.1

7.8 8.1

9.3 8.4

9.1 9.3

1.001 ns 2.741 ns

Concentration

s o.c.

8.6 8.7

8.6 8.6

9.1 9.4

9.8 10

1.675 ns 1.904 ns

Behavioral changes

s o.c.

5.0 4.8

4.9 5.0

5.6 5.3

6.0 5.9

2.355 ns 1.211 ns

Autonomic reactions

s o.c.

4.6 4.6

4.5 4.7

5.3 4.7

6 5.6

2.333 ns 1.922 ns

Water retention

s o.c.

5.1 5.1

5.1 5.3

5.6 5.0

6.3 6.0

1.979 ns 0.866 ns

Negative affect

s o.c.

9.0 9.0

9.5 8.7

10.0 8.9

10.5 9.9

1.867 ns 1.422 ns

Arousal

s o.c.

6.9 6.4

7.3 5.9

7.4 6.1

7.4 6.9

0.157 ns 1.081 ns

Control

s o.c.

6.4 6.3

6.6 6.7

6.8 6.4

7 7

0.995 ns 1.595 ns

HRSA

s o.c.

3.5 2.7

3.8 2.l

3.1 1.9

3.5 1.4

0.038 ns 1.257 ns

The data are shown in the form of means and ANOVA F values for clarity. S denotes spontaneous cycle and o.c. denotes oral contraceptives, n = 8 and 7, respectively.

differences between the untreated and treated w o m e n were o b s e r v e d with respect to the equilibrium binding constants of 3H-PK 11195 or 3H-Ro 5-4864 for any of the four test points in the cycle. S e r u m concentrations of the h o r m o n e s did not correlate significantly with the individual binding values, further indicating the lack of effect of ovarian steroids on the pBZr. Table II summarizes the results of an A N O V A performed on the factors of M D Q and on H A R S . F values of main effects due to menstrual phases are also showed. The analysis shows no significant cycle-related effect for all M D Q factors, although inspection of the data indicated that there were increases in concentration, behavioral changes, w a t e r retention, and negative affects in the luteal phase. Anxiety, rated by H A R S , was absent in all w o m e n and did not change during the study periods. The l y m p h o c y t e counts and percentages of T cells (total C D 3 + or after separation into C D 4 + and C D 8 + subsets) did not reveal any significant cycle-related variation, either in the spontaneously cycling group or in the one under o.c. (data not shown). The difference in binding profile between 3H-PK 11195 and 3H-Ro 5-4864 was not related to the P B M C m e m b r a n e preparations used for these experiments. Table I I I shows the binding characteristics of 3H-PK 11195 and 3H-Ro 5-4864 to intact and disrupted m o n o n u c l e a r

72

P. FERRERO et al.

T A B L E I I I . B I N D I N G PROPERTIES OF 3H-PK11195 OR 3H-RO 5 - 4 8 6 4 TO H U M A N P E R I P H E R A L BLOOD M O N O N U C L E A R CELLS. C O M P A R I S O N OF I N T A C T CELLS A N D CRUDE CELL MEMBRANES

3H-Ro 5-4864 S.B.

3H.PKU195 S.B.

nm&g Cell preparations

nmllg

pmol/mg pt (fmol/10 6 cells)

Kd nM

pmoi/mg pt (fmol/10 6 cells)

11.3 --+ 1.1 (405 -+ 40)

6.9 - 0.4

2.63 --- 0.5 (94 --- 19)

26 --+ 5.0

9.6 --- 0.4

7.5 -+ 0.6

2.07 -+ 0.4

16 - 2.0

Intact cells Crude membranes

Kd

nM

Mononuclear cells were obtained freshly from six normal women. 3H-PK11195 or 3H-Ro 5-4864 were incubated with preparations of intact mononuclear cells (1.6-2 × 106/ml) or crude membranes (20000 g, 20 min, 60-80 ,u.g protein/ml), as described in Methods. Data shown are the mean -+ SEM.

cells, freshly isolated f r o m the same individuals. 3 H - R o 5-4864 had consistent smaller values and also a lower affinity than 3H-PK 11195 in both cell preparations.

Bma x

Effect of Pregnancy Table IV presents 3H-PK 11195 binding p a r a m e t e r s to PBMC m e m b r a n e s obtained in nonpregnant control subjects and in pregnant w o m e n during 8 - 3 0 weeks of gestation. N o significant differences in Bmax or g d values were found between the groups, indicating the absence of an effect of pregnancy on the pBZr. Further, a m o n g the pregnant w o m e n , the binding values f r o m two w o m e n who were studied at early stages of pregnancy ( 8 - I 0

T A B L E I V . P R E G N A N C Y DOES NOT ALTER T H E SPECIFIC B I N D I N G OF 3H-PK11195 TO MEMBRANES FROM H U M A N BLOOD M O N O N U C L E A R CELLS

3H-PKll195 S.B. Bmax

Kd

No.

(pmol/mg prot)

(nM)

Nonpregnant

8

8.78 -+ 0.4

8.7 -+ 0.4

Pregnant 8-10 weeks 24-30 weeks Total

2 6 8

8.40 8.87 8.75 -+ 0.5

8.6 8.8 8.7 -+ 0.7

Groups

The preparation of mononuclear cell membranes and binding assays were performed as described in Methods. Data shown represent the mean -+ SEM.

73

LYMPHOCYTE PBZR AND OVARIAN STEROIDS

TABLE V. OVARIAN STEROID TREATMENT OF HUMAN MONONUCLEAR CELLS AT DIFFERENT CULTURE TIME PERIODS DOES NOT ALTER THE SPECIFIC BINDING OF 3H-PK11195 OR 3H-RO 5-4864 TO LYMPHOCYTE MEMBRANES Time in culture (h)

Treatment

3

24

48

72

3

SH-PKlI195 S.B. B ~ (pmol/mg prot) K d (nM)

None

24

48

72

SH-Ro 5-4864 S.B. B..~ (pmol/mg prot) K d (DAM)

10.7 ± 1.6 18 ± 1.2

14.3 --- 2.3 19 ± 2.5

10.2 ± 1.3 18 --- 0.6

15.3 -+ 4.3 18 ± 1.8

1.82 ± 0.2 29 ± 3.5

2.06 ± 0.4 35 -+ 9.1

1.57 ± 0.4 34 ± 3.8

1.90 ± 0.5 35 ± 5.1

10 -9

9.2 ± 1.4 19 ± 2.0

13.7 -+ 3.9 19 ± 2.5

10.0 ± 1.8 17 ± 3.4

12.0 --- 3.5 19 ± 1.8

1.93 ± 0.1 34 ± 5.0

2.10 + 0.6 39 ± 4.2

1.67 ± 0.5 33 ± 6.5

2.05 ± 0.6 32 ± 5.9

10 7

10.3 ± 1.5 17 ± 1.0

15.4 ± 3.8 18 ± 1.5

10.7 ± 1.2 19 ± 1.7

13.5 -+ 3.8 17 ± 2.4

1.80 ± 0.1 35 -+ 7.5

2.00 ± 0.7 33 -+ 8.5

1.70 ± 0.6 33 ± 2.5

1.60 ± 0.6 34 ± 4.9

10 -5

10.7 ± 1.6 18 ± 1.3

13.1 -+ 2.4 17 ± 2.1

11.0 ± 2.1 16 -+ 1.5

13.5 ± 3.2 18 "4- 1.9

1.80 --- 0.2 29 ± 1.5

2.01 ± 0.5 37 -+ 7.2

1.70 ± 0.7 32 ± 4.3

1.70 ± 0.5 36 ± 5.5

10 9

12.7 ± 1.6 18 ± 1.7

13.1 -+ 2.9 17 -+ 1.3

11.1 ± 2.0 18 ± 2.2

12.3 ± 0.7 18 ± 1.6

1.91 ± 0.1 32 - 5.6

1.68 ± 0.5 33 ± 4.4

1.80 - 0.5 35 ± 3.7

1.94 ± 0.3 30 ± 3.3

10 7

11.8 ± 1.2 19 ± 3.2

14.1 ± 3.5 18 ± 0.9

10.7 ± 1.0 18 ± 1.9

11.8 ± 0.2 18 ± 3.1

1.90 ± 0.2 36 ± 5.5

2.19 ± 0.7 31 ± 6.9

1.73 ± 0.4 34 ± 3.0

2.00 -+ 0.4 29 ± 0.6

10 5

10.7 ± 1.1 18 ± 1.5

14.0 -+- 3.1 20 ± 1.8

9.9 ± 1.7 17 ± 1.8

11.8 -+ 0.1 18 ± 3.2

1.99 -_+ 0.2 37 ± 4.5

2.25 -+ 0.4 30 ± 10

1.88 ± 0.6 32 ± 3.2

1.79 ± 0.3 32 ± 2.3

Estradiol

Progesterone

Mononuclear cells from whole blood buffy coats were cultured in the presence or absence of the indicated concentrations of sex steroids for up to 72 h, after which the nonadherent cells were collected and then homogenized. Following washing, the membranes were tested for binding as described in Methods. Data shown represent the mean -+ S E N of 3-5 and 6-8 separate series of experiments for each steroid and controls respectively. F values (p n.s.) are not indicated for clarity.

weeks) were similar to the values obtained from another six women, each tested between 24 and 30 weeks of gestation.

The Effect of Treatment with Ovarian Steroids on Mononuclear Cell in Culture pBZr were reliably measured in crude membranes prepared from the nonadherent mononuclear cells (> 99% pure lymphocytes) in culture. Initial preliminary time course experiments indicated no significant changes in binding parameters of 3H-PK 11195 or 3H-Ro 5-4864 to the lymphocyte membranes after culture of the mononuclear cells over a period between 3 and 72 h. The results from a representative series of experiments are shown in Table V. The displacement by various BZ and PK 11195 of 3H-PK 11195 and 3H-Ro 5-4864 bound to the lymphocyte membranes after 24 h in culture displayed a typical peripheralBZr pharmacology. As shown in Table VI, the calculated IC50 values of the tested inhibitors were very similar for both radioligands. PK 11195 was always the most potent displacer, followed by 4'-chlorodiazepam and diazepam, clonazepam being inactive.

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T A B L E V I . I N H I B I T I O N OF 3 H - P K 1 1 1 9 5 A N D 3H-RO 5 - 4 8 6 4 S P E C I F I C B I N D I N G TO L Y M P H O C Y T E M E M B R A N E S P R E P A R E D FROM M O N O N U C L E A R CELLS AFTER l DAY CULTURE

Drugs

3H-PKlU95 S.B. ICso (nM)

3H-Ro5-4864 S.B. ICso (nM)

PK 11195 4' Chlorodiazepam Diazepam Clonazepam Estradiol Progesterone

30-+ 3 73 -+ 5 237 -+ 17 > 10000 inactive inactive

34_+ 3 70 + 4 226 -+ 12 > 10000 inactive inactive

Crude membranes prepared from the cultured mononuclear cells obtained from whole blood buffy coats were incubated with 3H-PK11195 (5 nM) and 3H-Ro 5-4864 (10 nM) in the presence of the indicated drugs as described in methods. Drug concentrations required to inhibit binding by 50% (IC50) were determined by nonlinear least squares regression analysis. Data shown are the mean -+ SEM of 3-4 independent experiments. Estradiol and progesterone were inactive at concentrations up to I0 -5 M.

Estradiol and progesterone (up to 10-5 M) were both ineffective in displacing either 3H-PK 11195 or 3H-Ro 5-4864. To determine whether exposure to ovarian hormones had any effect on pBZr binding, the mononuclear cells were cultured for defined intervals (between 3 and 72 h) in the presence of various concentrations of estradiol or progesterone (ranging from 10-9 to 10-5 M). Unexposed, control cells were treated with medium solution alone for similar times. Table VI shows that the binding of 3H-PK 11195 and that of 3H-Ro 5-4864 to the lymphocyte membranes were both unaffected by the presence of the steroids in the culture medium. Using a one-way ANOVA, no significant hormone-related effects were detected in any of the binding parameters measured at any time. DISCUSSION Demonstration of both estrogen and progesterone-induced regulation of pBZr (Fares et al., 1987, 1988; Gavish et al., 1986, 1987, 1992) are of interest because of the widespread distribution of pBZr in the mammalian tissues, their connections with steroid biosynthesis, and their potential involvement in physio-pathological processes (i.e., stress, anxiety, seizures, and development of tolerance to BZ) (Drugan & Holmes, 1991; Krueger & Papadopoulos, 1992; Miller et al., 1992; Weizman & Gavish, 1989), in which a role for gonadal steroids has been suggested (Maggi & Perez, 1985; Newmark & Penry, 1980; Wilson & Biscardi, 1992). The blood mononuclear cells have been a useful tissue source for the pharmacological characterization of pBZr in humans, their immune-coupled responses, and their variation in diseases (Alexander et al., 1992; Berkovich et al., 1993; Bessler et al., 1992; Canat et al., 1993; Ferrarese et al., 1990; Ferrero et al., 1991; Moingeon et al., 1983; Rocca et al., 1991; Ruffet al., 1985; Zavala et al., 1990). Recent evidence suggests that these local pBZr may be implicated in immune endocrine interactions. This includes (I) the ability of various pBZr ligands, including certain DBI peptide metabolites, to modulate secretion

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of selected cytokines from human monocytes (Taupin et al., 1991a, 1991b, 1993); and (2) the enrichment of pBZr in the mitochondrial fraction of homogenates from PBMC and their coexistence in these cells with a DBI-like immunoreactivity (Rocca et al., 1993). In this study, we tested the hypothesis that the pBZr in human PBMC may be affected by the female sex steroids. Our findings in this respect were negative. We found no significant changes in binding densities and affinities of 3H-PK 11195 and 3H-Ro 5-4864 in vivo in normal women tested at different sex-steroid phases of the menstrual cycle or during 8-30 weeks of pregnancy. We similarly failed to demonstrate an influence of ovarian steroids in cultures of mononuclear cells treated with estradiol or progesterone (up to 10-5 M) over a period between 3 and 72 h. Furthermore, we performed in vitro competition experiments, which showed that both hormones are not active directly on our pBZr binding systems. Taken together, the results reasonably suggest that the female hormones are not physiologically relevant modulators of the pBZr in PBMC. Our current negative results are consistent with and extend limited prior evidence regarding the ineffectiveness of estradiol to modify 3H-PK I1195 binding to pBZr in human platelets (Diorio et al., 1990). Our use of mononuclear cells indicates clearly that the lack of action of estradiol is not due to the peculiar anucleate cellular organization of platelets. Further, we also showed that progesterone is equally ineffective in modulating pBZr binding in our cellular system. The contribution that ovarian steroids may play in the reported alterations of platelet and PBMC pBZr during normal stress or pathologic anxiety remains unknown at the present time. We should emphasize that the plausible influence of anxiety has been excluded in the actual studied population and was not taken into account in Diorio's study. Hence, the possibility that a regulatory effect of hormones may manifest during abnormal emotional situations or pathologic disorders is intriguing. Further investigations on pBZr and female steroids in blood of women with premenstrual syndrome, "maternity blues," and catamenial epilepsy would be important in clarifying this point. Two methodological aspects of our study deserve further comment. Though we did not observe an effect of female hormones, our experiments demonstrate that lymphocytes in culture maintain high levels of pBZr. Thus, they would offer a useful model system for studies of regulation of pBZr in humans in response to various pharmacological, immunological, or pathologic situations. The Kd values for 3H-PK 11195 and 3H-Ro 5-4864 were slightly lower with the membranes from cultures than with those prepared freshly. This may reflect the fact that the cultured membranes did not contain monocytes. However, in our previous studies with intact fresh PBMC, the affinity of 3H-PK 11195 does not vary between monocytes and lymphocytes (Rocca et al., 1993). Alternatively, the difference may depend on the cell source, and it could be related to the fact that the cells from buffy coats displayed a higher degree of mortality, due probably to delay from the time of blood drawing and the time at which PBMC isolation could be initiated. It is noteworthy that under an identical procedure for PBMC purification from single individuals and whole blood buffy coats, we have reported a similar difference in binding characteristics of 3H-N-methyl-scopolamine (Eva et al., 1989). In our experiments with either fresh or cultured mononuclear cells, the heterogeneity in recognition sites for 3H-PK 11195 and 3H-Ro 5-4864 is evident. We report a number of binding sites of 3H-PK 11195 about 4-5-fold larger than that of 3H-Ro 5-4864, both in intact and disrupted cells, and an affinity higher for 3H-PK 11195 than for 3H-Ro 5-4864. The binding characteristics are in accord with those reported by a recent investigation of Berkovich et al. (1993). Differences in binding domains between

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isoquinoline and BZ ligands have been reported among species and tissues, and are thought to reflect structural variations of proteins assembling to form an heterooligomeric pBZr complex (McEnery et al., 1992; Riond et al., 1991; Sprengel et al., 1989). The pBZr identified on human PBMC may be also heterogeneous. Besides the difference in radioligands binding profiles, recent reports indicate multiple subcellular locations that might correspond to a differential receptor sensitivity to endogenous ligands (i.e., porphyrins vs. DBI or its active metabolites) (Berkovich et al., 1993; Rocca et al., 1993). Whether the presumptive subtypes of pBZr in immune cells may represent functionally and/or pharmacologically distinct entities is as yet unknown. We can exclude, however, a differential effect of ovarian steroids on BZ and isoquinolines recognition sites, based on the failure of hormones to change binding of both 3H-PK 11195 and 3H-Ro 5-4864 to these cells. As outlined above, there is much evidence from animal experiments that ovarian steroids can influence pBZr sensitivity. Further, a regulatory influence of these hormones has recently been suggested to explain the sexual dimorphism in pBZr responsivity after exposure of animals to an acute environmentally induced stress (Drugan et al., 1991). The actual finding that pBZr in human PBMC are unresponsive to female hormones may indicate discrepancies due to species or the examined tissue. Although these possibilities cannot be distinguished at this time, such a tissue specificity has already been demonstrated in animals and correlated in part with atrophic influence exerted by the hormones in that tissue (Gavish et al., 1992). The possibility that pBZr in immune cells are sensitive to other hormones or immune factors (Ferrero et al., 1992; Oh et al., 1992) appears an interesting hypothesis to investigate. REFERENCES Alexander BEE, Roller E, Klotz U (1992) Characterization of peripheral-type benzodiazepine binding sites on human lymphocytes and iymphoma cell lines and their role in cell growth. Biochem Pharmacol 442:269-274. Anholt RRH, De Souza EB, Kuhar JJ, Snyder SH (1985) Depletion of peripheral-type benzodiazepine receptors after hypophysectomy in rat adrenal gland and testis. Eur J Pharmacol 110:41-46. Berkovich A, Ferrarese C, Woodbridge M, Cavallotti G, Alho H, Marzorati C, Bianchi G, Guidotti A, Costa E (1993) Topology of 2 DBI receptors in human lymphocytes. Life Sci 52:1265-1277. Besman MJ, Yanagibashi K, Lee TD, Kawamura M, Hall PF, Shilvey JE (1989) Identification of des-(Gly Ile)-endozepine as an effector of corticotropin-dependent adrenal steroidogenesis: Stimulation of cholesterol delivery is mediated by the peripheral-type benzodiazepine receptor. Proc Natl Acad Sci USA 86:4897-4901. Bessler H, Weizman R, Gavish M, Notti I, Djaldetti M (1992) Immunomodulatory effect of peripheral benzodiazepine receptor ligands on human mononuclear cells. J Neuroimmunol 38:19-26. Canat X, Carayon P, Bova Boula M, Cahard D, Shire D, Roque C, Le Fur G, Casellas P (1993) Distribution profile and properties of peripheral-type benzodiazepine receptors on human hemopoietic Cells. Life Sci 52:107-118. Dar DE, Weizman A, Karp L, Grishpoon A, Bidder M (1991) Platelet peripheral benzodiazepine receptors in repeated stress. Life Sci 44:1077-1082. Delorey TM, Olsen RW (1992) Gamma-aminobutyric acid A receptor structure and function. J Biol Chem 267:16747-16750. Diorio DL, Welner SA, Tulandi T, Samarthji Lal S, Suranyi-Cadotte BE (1990) Effect of increasing estradiol levels on platelet peripheral benzodiazepine binding in women undergoing human menopausal gonadotropin treatment. Neuropsychobiology 23:169-172. Drugan RC, Holmes PV (1991) Central and peripheral benzodiazepine receptors in an organism's response to physical and psychological stress. Neurosci Biobehav Rev 15:277-278. Drugan RC, Holmes PV, Stringier AP (1991) Sexual dimorphism of stress-induced changes in renal peripheral benzodiazepine receptors in rat. Neuropharmacology 30:413-416.

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