Brain Research, 581 (1992) 229-236 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00
229
BRES 17762
Sex differences in the binding of Type I and Type II corticosteroid receptors in rat hippocampus Barbara B. Turner Department of Physiology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614 (USA)
(Accepted 7 January 1992) Key words: Corticosteroid receptor; Hippocampus; Sex difference; Mineralocorticoid receptor; Glucocorticoid receptor; Corticosteroid binding globulin; Estrogen; Rat
Binding parameters of soluble Type I and Type II receptors were assessed in hippocampus of adult, adrenalectomized, male and female rats. No sex differences in the number of either Type I or Type II receptors could be demonstrated between gonadally intact animals. When females treated with 17fl-estradiol benzoate (10/tg/day) were compared with males, a statistically significant reduction in Type II receptors was observed in the females; progesterone produced no further decrease in receptor numbers. The amount of tissue-associated corticosteroid-binding globulin in gonadally intact animals (perfused with dextran-saline) was twice as great in females as males. Sex-dependent differences in these gonadally intact rats were found in the affinity, measured as the dissociation constant (Kd), of both the Type I and Type II receptors. For both receptors, affinity in cytosols from females was reduced. The difference for the Type II receptor was slight, but the Kd value of the Type I receptor was several-fold higher in females. The difference in affinity was evident with both natural and synthetic steroid ligands. There appears to be little, if any, difference in affinity between the hippocampal Type I and the Type II receptors in females. This suggests that the occupancy of Type I receptors in females is substantially less than that of males at low circulating concentrations of corticosteroids. INTRODUCTION Pronounced sex differences exist in several aspects of hypothalamic-pituitary-adrenal ( H P A ) function in rats. Some of these differences, such as the higher secretion rate 16 and plasma concentration of corticosterone in females 6 are attributable to the differential influence of estrogen and androgen on hepatic corticosteroid metabolism 14 and corticosteroid-binding globulin (CBG) levels 13'21. Other sex-dependent differences suggest a dimorphism at one or more levels of negative-feedback within the axis. The greater magnitude and duration of the pituitary-adrenal response to stress in females 16 and the greater diurnal variation in plasma corticosterone in females 6 suggest that negative-feedback may be less effective in females. Both Type I and Type II corticosteroid receptors exist in neurons of several limbic structures implicated in the negative feedback of glucocorticoids, particularly the hippocampus 7'25'26. Based on estimates of receptor occupancy, it was originally suggested that Type I receptors do not contribute to the regulation of A C T H secretion 24. However, it now appears that Type I (mineralocorticoid) receptors as well as Type II (gluco-
corticoid) receptors may participate in the process of glucocorticoid negative-feedback in the central nervous system s'a°. Basal A C T H levels are likely regulated by the higher affinity Type I receptors while the lower affinity Type II receptors are involved in regulating stress responses 4. If negative-feedback is less robust in females than males, one might expect reduced corticosteroid receptor numbers in sites such as the hippocampus, hypothalamus and pituitary. Ovariectomy has been shown to increase by several-fold the Type II (glucocorticoid) receptor m R N A content of the pituitary while administration of 17fl-estradiol reverses the increase 22'23. A n up-regulation of Type II receptor m R N A following ovariectomy has also been reported in brain 1. The sex-related difference in pituitary receptor m R N A levels is reflected in the concentration of functional receptors. Our laboratory has found that glucocorticoid binding is less in pituitary cytosols from females than from males; ovariectomy eliminates this difference and the administration of estrogen restores the difference 33. Estrogen-induced changes in glucocorticoid binding in the opposite direction have recently been reported in brain tissues. Ferrini and DeNicola n found that 17fl-es-
Correspondence: B.B. Turner, Dept. of Physiology, Box 19780A, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA.
230 tradiol markedly increased the n u m b e r of Type I receptors in several brain regions, including the hippocampus. This finding is in apparent contradiction to an earlier report by the same investigators that estrogen is without effect on either corticosteroid receptor type in the hippocampus 12. In their more recent paper, Ferrini and DeNicola 11 also observed that estrogen treatment similarly increased the n u m b e r of Type II receptors in the hypothalamus and septum, but not the hippocampus. These observations are not in agreement with our previous report which found that ovariectomy increased binding in the hippocampus and hypothalamus of females 34. Other workers have found that ovariectomy increases, and estrogen suppresses, m R N A concentrations of both the Type II receptor ~ and the Type I receptor 3 in hypothalamus and hippocampus, findings consistent with our earlier results. The purpose of the present work was to compare the binding parameters of the Type I and Type II receptors in the hippocampus of gonadally intact male and female rats. The effect of estrogen on receptor n u m b e r , possi-
RU 28362 used for the determination of glucocorticoid (Type II corticosteroid) binding, was either in 100- or 250-fold excess as specified in each experiment. Aliquots were incubated for 4 h at 4°C. Duplicate aliquots were taken from each vial for the determination of molarity. Separation of bound from Iree steroid by gel exclusion chromatography on LH-20 mini-columns9 was performed in duplicate using 50/A aliquots of cytosol. In the experiment in which dextrancoated charcoal (DCC) was used to separate bound steroid, DCC was added 1:6 (v/v) to cytosol and incubated for 10 min at 4°C before centrifugation2 in a Beckman Microfuge 11. Aliquots were taken in triplicate from each vial and counted. In those experiments in which hydroxylapatite (HAP) was used to separate bound from free steroid, the protocol of Krozowski and Funder17was followed. Protein was determined by a modification of the Coomassie blue method 29. Binding parameters of saturation curves were calculated from double reciprocal plots of the data according to Lineweaver-Burk 19. Student's t-test was used for the comparison of two means. Dunnett's test for multiple comparisons with a control x°, or 2-way analysis of variance was used to test for significance in other experiments as indicated. RESULTS
Saturation binding using [3H]corticosterone
ble sex-differences in receptor stability and the effect of
The first study verified our earlier results 34 on sex dif-
tissue-associated, CBG-like binding proteins on apparent receptor binding parameters are also addressed.
ferences in glucocorticoid binding obtained with saturation plots. Individual hippocampi from gonadally intact rats were homogenized in the buffer used in the previous study: 5 m M TRIS [Tris(hydroxymethyl) aminomethane] containing 1 m M E D T A , 1 m M dithiothreitol and 10% glycerol, p H 7.4, at 4°C. Aliquots of cytosol
MATERIALS AND METHODS Sexually mature Sprague-Dawley rats, virus antibody free, (Charles River, Wilmington, MA) were housed 2-4 per cage under a 12:12 h light-dark cycle. Gonadectomies were performed under Metofane anesthesia (Methoxyflurane, Pitman-Moore, Washington Crossing, NJ) at least 10 days prior to sacrifice. Gonadal steroids were injected s.c. in 0.2 ml of peanut oil. All animals were adrenalectomized under Metofane anesthesia 12 h before sacrifice (except in one experiment where noted). At sacrifice, rats were anesthetized with Metofane and killed by intracardiac perfusion (90 ml of room temperature saline followed by 60 ml of ice-cold 6% dextran in saline). Brains were removed quickly and dissected on a chilled glass plate; the hippocampi were then frozen on dry ice before storage at -80°C. Tritiated steroids were obtained from New England Nuclear Corp. (Boston, MA): 1,2,6,7-[3H]corticosterone (spec. act. 85.8, 101.6 and 112 Ci/mmol); 6,7-[3H]dexamethasone (spec. act. 37.3, 45.8 and 49.9 Ci/mmol); 1,2,6,7-[3H]aldosterone (spec. act. 82 Ci/ mmol); 6,7-[3H]triamcinolone acetonide (spec. act. 43.6 Ci/mmol); 6-methyl-[3H]RU 28-362 (spec. act. 77.8 and 77.9 Ci/mmol). Thin layer chromatography was used to verify the purity of radiolabelled steroids. Unlabelled corticosterone, dexamethasone and aldosterone were purchased from Sigma Chemicals (St. Louis, MO). Unlabelled RU 28-362 was a gift from Roussel-UCLAF. All chemicals used were reagent grade. Hippocampi were homogenized 1:10 (w/v) in HEPES buffer (25 mM) containing 0.25 M sucrose, 10 mM sodium molybdate and 5 mM dithiothreitol, pH 7.4 at 4°C, unless otherwise specified. Homogenates were centrifuged at 105,000 × g for 1 h at 2°C. Incubations consisted of 140/A aliquots of cytosol to which 2.8/A of ethanol containing the steroid was added. In some experiments, soluble receptor binding was measured by saturation plots; in other experiments binding was determined by a single, saturating concentration of tritiated steroid (25 nM). Non-specific binding was determined using a concentration of 500-fold excess of unlabelled steroid unless otherwise indicated. The concentration of unlabelled
were incubated in varying concentrations of [3H]corticosterone (0.75-24 nM). Significant differences were found between the binding parameters of males and females as shown in Fig. 1. The Bmax of males was significantly less than that of females (223 + 11 vs. 276 + 17 fmol/mg protein; P < 0.05). These data are in good agreement with those obtained previously34. The K a of males was also markedly less than that of females (1.20 + 0.08 vs, 2.11 + 0.39 nM; P < 0.05).
Receptor stability and tissue CBG The possible contribution of several technical factors to the apparent sex-related differences in corticosteriod binding was considered. The question of whether the observed disparity in receptor n u m b e r of affinity could be due to a sex-dependent difference in receptor stability was addressed. To determine the stability of corticoid receptors, male and female hippocampi were homogenized 1:5 (w/v) in the absence of sodium molybdate and incubated at 25°C for 0, 15 or 60 min before being incubated with [3H]steroid for 4 h at 4°C. Cytosols prepared from 3 tissue pools of each sex were assayed using [3H]dexamethasone; the n u m b e r of receptors at 0 time was taken as 100%. The percentage of receptors remaining after 15 min (47 + 3 vs. 57 + 3 fmol/mg pro-
231 •
Males
--4--
Females
6O o
~,
-)(-
300
0
T T ~
/I'~
I.- ~'~
200
T
T
~ 4o
/
-
~-
30 2O 100
T
8 10
io rn
0
I
E
10
20
3H-Corticosterone
Concentration (riM}
O)
0 Males
30
Fig. 1. Direct plots of soluble binding experiments using hippocampi from gonadally intact rats (12 h ADX). Tissues were homogenized in TRIS buffer in the absence of sodium molybdate. Each point represents the mean of 6 separate experiments; the error bars indicate the S.E.M. Data from males is indicated by circles, that from females by triangles. Comparison of the LineweaverBurk plots of the data show that females have significantly greater binding capacity but lower affinity (P < 0.05, Student's t-test, twotailed).
tein) or 60 min (3 + 2 vs. 2 + 1 fmol/mg protein) at 25°C in males vs. females, respectively, was not significantly different. Similar results were obtained with [3H]corticosterone. The naturally occurring ligand in rats, corticosterone, binds not only to corticoid receptors, but also to CBG. Although animals are well peffused at sacrifice, small but measurable amounts of CBG remain in hippocampal tissue 32. In order to determine the possible contribution of CBG to the binding differences observed with [3H]corticosterone, binding was measured in cytosols incubated with a single, near-saturating concentration of [3H]corticosterone (24 nM) in the presence of a 100-fold excess dexamethasone. Hippocampi from seven animals of each sex were assayed. CBG-like binding (Fig. 2) was significantly greater in females (47.5 + 4.8 fmol/mg protein) than in males (21.4 _+ 2.5 fmol/mg protein; P < 0.001). This difference in binding, 26 frnol/mg protein, accounts for over half of the observed difference in Bmax (43 fmol/mg protein) found in the saturation study.
Type I and Type H receptor number Does a sex difference in receptor number exist when steroids that have very low, or negligible affinity for CBG are used as ligands? In order to assess the influence of gonadal steroids, animals were either gonadectomized or sham-operated 3 weeks prior to sacrifice. [3H]Triamcinolone was used to determine total receptor binding; [3H]aldosterone was used both alone and in the presence of RU 28362 (a selective Type II agonist) to
Females
Fig. 2. 'CBG-like' binding present in hippocampal cytosols from perfused male and female rats (12 h ADX). Binding was measured using a single, saturating concentration of [3H]corticosterone in the presence of a 100x excess of dexamethasone. Bars indicate the mean + S.E.M. of 7 animals, individually assayed. Females (hatched bar) had significantly more binding, measured as fmol/mg protein, than did males (open bar; *P < 0.001, Student's t-test,
two-tailed).
measure the number of Type I receptors. A single, saturating concentration of each steroid was used; treatment with DCC was used to separate bound from free steroid. Binding obtained with [3H]aldosterone was substantially decreased by the presence of 100x excess RU 28362 (Table I). With both [3H]ligands, hippocampi from females showed less, not greater binding, than did those from males. However, no significant, sex-dependent differences in binding were found by 2-way ANOVA. The possibility that methodological differences might be responsible for the lack of agreement between the two studies was evaluated. (In the second experiment described above, dextran-coated charcoal was used rather than LH-20 and the high affinity ligand, triamcinolone, was employed.) In the next study, using gonadally intact rats and a single concentration of [3H]ligand, bound steroid was separated from free by chromatography on LH-20 mini-columns. [3H]Dexamethasone in the presence and absence of excess RU 28362 was used to determine the total number of receptors and the number of Type I receptors, respectively. The number of Type II receptors was calculated by subtraction of Type I from the total number of receptors. Again, no sex difference was found in the number of corticosteroid receptors, either Type I or Type II (Table II). In the previous studies, in which gonadally intact animals were used, females were found to have somewhat fewer receptors than males although the difference was not statistically significant. The next experiment asked if administration of exogenous steroids might enhance this difference. Gonadally intact males were compared with ovariectomized females treated with either 10 Hg of 17fl-
232
--I
3H-DEX
Males
•
3H-CORT +RU 28362
--A---
Females
75 400
0
0 0 D. t~
60 "7"
T
300
45 m
"0
200
30
m 0
100
tO I
I m
MALES
OVX
D +
E
®
15 ×
0 03
0
O V X + E/P
Fig. 3. Corticosteroid-binding proteins in cytosols prepared from hippocampi of gonadally intact males and ovariectomized (OVX) females treated with estradiol benzoate (E; 10 ~g/rat/day) or estradiol benzoate plus progesterone (EP; 500/~g/rat/day) for 10 days prior to sacrifice. Animals were ADX 12 h prior to sacrifice. Binding was determined by a single point assay and is expressed as fmol/mg protein. Values are the mean of at least 7 animals, assayed individually. *P < 0.05 vs. male value.
I
I
I
10
15
20
25
3H-Dexarnethasone Concentration (nM) •
Males
Females
--&--
360 0
300 240
estradiol b e n z o a t e / r a t / d a y o r estradiol b e n z o a t e t o g e t h e r with 500/~g o f p r o g e s t e r o n e for 10 days p r i o r to sacrifice. Total binding was m e a s u r e d with [ 3 H ] d e x a m e t h a -
I
5
m ~o o3 co
180 120
sone, and [3H]corticosterone with excess R U 28362 was
6O
used to e s t i m a t e Type I binding. A s s h o w n in Fig. 3, est r o g e n a d m i n i s t r a t i o n significantly d e c r e a s e d total r e c e p -
m
tor binding ( [ 3 H ] d e x a m e t h a s o n e ( P < 0.05)). Since estrogen
had
no d i s c e r n a b l e
[3H]corticosterone
O= o
r e c e p t o r s i n v o l v e d a d e c r e a s e in Type II r e c e p t o r s .
Receptor affinity studies Since the initial study with [3H]corticosterone indic a t e d a s e x - r e l a t e d d i f f e r e n c e in r e c e p t o r affinity as well as r e c e p t o r n u m b e r , we also e x a m i n e d the a p p a r e n t dif-
i
I
I
10
15
20
3H-RU 28362
effect on the binding of
+ R U 28362, the d e c r e a s e in total
,
5
25
Concentration (riM)
Fig. 4. Direct plots of soluble receptor experiments using hippocampi from gonadally intact rats (12 h ADX). Tissues were homogenized in HEPES buffer in the presence of sodium molybdate. Each point represents the mean of 5 separate experiments; the error bars indicate the S.E.M. Data from males is indicated by circles, that from females by triangles. Binding parameters were determined by Lineweaver-Burk plots of the data. A: Type I binding was determined by [3H]dexamethasone in the presence of a 250fold excess of RU 28362. B: Type II binding was determined using [3H]RU 28362 as ligand.
TABLE I
Binding of [3H]aldosterone and [3H]triamcinolone acetonide to hippocampal cytosols of gonadally intact and castrated male and ovariectomized female rats Gonadectomies (or sham operations) were performed 3 weeks prior to sacrifice. Animals were killed 12 h after ADX. Binding was measured at a single, saturating concentration (25 nM) of [3H]ligand; bound steroid was separated from free by treatment with DCC. Values represent the mean _+ S.E.M. of 7-8 experiments in which individual hippocampi were used. No significant differences were found using 2-way ANOVA.
Binding (fmol/mg protein)
Intact male Gonadectomized male Intact female Ovariectomized female
[3]Aldosterone
[3]Aldosterone + RU 28362
13H]Triamcinolone acetonide
261 241 230 227
105 103 84 101
359 352 332 329
+ 12 + 11 _+ 15 + 19
+ + + +
10 7 7 8
+ + + +
11 21 23 20
233 TABLE II Corticosteroid receptor concentration in cytosols prepared from hippocampi of gonadally intact male and female rats
60
Binding in ADX rats (12 h) was measured at a single, saturating concentration of [3H]dexamethasone in the presence and absence of a 100-fold excess of RU 28362. Type II was calculated as the difference between total receptor binding ([3H]dexamethasone alone; n - 5) and Type I binding ([3H]dexamethasone in the presence of RU 28362; n = 10). Binding is expressed as fmol/mg protein. Values given are the mean + S.E.M.
Male Female
Type I
Type H
Total
85 _+ 15 73 _+ 15
246 + 17 225 _+ 10
336 + 24 295 _+ 12
•
Males
--
- -
20
JrO
O 0
2
4
3H-Corticosterone
ference in corticosteroid r e c e p t o r affinity. Saturation plot experiments were p e r f o r m e d on h i p p o c a m p i from gonadally intact male and female rats. [3H]RU 28362 was used as the selective ligand for Type II receptors and [3H]dexamethasone plus 250-fold excess unlabelled R U 28362 for Type I receptors. H i p p o c a m p i from 2 - 3 rats of each sex were p o o l e d for each assay. Binding values were averaged from 5 s e p a r a t e experiments and are shown in Fig. 4. F o r the Type I receptors, no difference was found b e t w e e n the Bmax of tissues from male and female rats (48.9 + 3.6 and 44.7 + 2.7 fmols/mg protein, respectively). H o w e v e r , the K d for the Type I receptors was several-fold lower in males than females (0.53 + 0.05 vs. 1.30 + 0.25 nM, P < 0.02). No difference was found in the Bmax of males (361 + 16 fmols/mg protein) c o m p a r e d with that of females (347 + 15 fmols/mg protein). The numerically small difference in the K d value of males and females for the Type II rec e p t o r was statistically significant (1.41 + 0.03 nM and 1.30 + 0.04 nM, respectively; P < 0.025). Since corticosterone would be expected to have con-
TABLE III Estimation of the affinity of Type I binding in hippocampal cytosols with [3H]corticosterone in the presence of excess RU 28362 as ligand
Hippocampal tissues were taken from gonadaUy intact rats that had been ADX 12 h prior to sacrifice. Bound steroid was separated from free by passage through LH-20 mini-columns. Maximal binding (Bmax), dissociation constant (Kd) and the regression coefficient (r) are derived from Lineweaver-Burk plots of the saturation curves. Values represent the mean + S.E.M. of 4 separate experiments. *Significantly different from male value (P < 0.01, Student's t-test, two-tailed).
Male Female
B,,~.~ (fmol/mg protein)
Ka (nM)
r
76.1 + 9.4 91.6 + 6.0
0.21 + 0.01 0.76 + 0.20*
0.989 0.995
Females
--A--
t
t
6
8
Concen~'ation
10
(rtvl)
Fig. 5. Binding experiments using hydroxylapatite to measure the Type I receptor in gonadally intact male and female rats. [3H]Corticosterone was used as ligand in the presence of a 100x excess of RU 28362. Each point represents the mean of 4 separate experiments; the error bars indicate the S.E.M. Data from males is indicated by circles, that from females by triangles. Comparison of the Lineweaver-Burk plots of the data show that females have significantly lower affinity (P < 0.05, Student's t-test, one-tailed).
siderably greater affinity for the Type I r e c e p t o r than d e x a m e t h a s o n e , we asked whether the sex-related difference in affinity is e n h a n c e d when [3H]corticosterone is used as the ligand. Table I I I shows the results of 4 saturation plots using [3H]corticosterone in the presence of a 100-fold excess of R U 28362 to estimate the affinity of Type I receptors in gonadaUy intact males vs. females. According to L i n e w e a v e r - B u r k plots of the data, binding in cytosols from males had a 3.5-fold greater affinity than did that from females ( P < 0.01). The Bmax values for males and females were not statistically different. The p r o b a b l e endogenous ligand of the Type I receptor in hippocampus, corticosterone, has greater affinity for the r e c e p t o r than do synthetic c o m p o u n d s 34. However, in o r d e r to use this ligand, its binding to the C B G present in the cytosol needs to be eliminated because [3H]corticosterone binds to C B G as well as to the Type I receptor. The a m o u n t of C B G present in tissue m a y well affect the dissociation constant. H y d r o x y l a p a t i t e , which has affinity for the r e c e p t o r but not C B G , was therefore used. Using the batch p r o c e d u r e of Krozowski and F u n d e r t7, we first verified that in our hands the assay adequately s e p a r a t e d b o u n d from free steroid and that the hydroxylapatite b o u n d negligible amounts of C B G . Cytosol was p r e p a r e d from cerebral cortex to which 1 0 / 4 of undiluted plasma was a d d e d p e r ml. Aliquots (in quadruplicate) were incubated either with [3H]dexamethasone or with [3H]corticosterone in the presence of a 100-fold excess of R U 28362; non-specific binding was also deter-
234 mined for each ligand. Binding in parallel incubates was compared using LH-20 and hydroxylapatite procedures with [3H]dexamethasone as ligand. We found that the hydroxylapatite procedure adequately separated bound from free steroid, but that recovery of receptor was reduced while non-specific binding was increased. Hydroxylapatite bound less than 0.5% of the CBG measured by LH-20. Is the binding affinity of hippocampal Type I receptors in females less than that of males when the naturally-occurring ligand is used? As illustrated in Fig. 5, four saturation experiments of cytosols prepared from each sex were run on hydroxylapatite using 6 steroid concentrations (0.2-8.0 nM) of [3H]corticosterone in the presence of excess RU 28362. Females were found to have a higher K a (1.67 nM) than did males (0.39 nM; P < 0.05, one-tailed). Bmax was similar for males (56 + 4 fmol/mg protein) and females (61 + 12 fmol/mg protein).
DISCUSSION
Findings of present studies In gonadally intact rats, no significant difference in the number of Type I or Type II receptors could be demonstrated between males and females. However, males did consistently tend to have a slightly greater number of Type I and Type II receptors. The difference between males and females became statistically significant when estrogen-treated females were compared with males. The reduced binding observed in estrogen-treated females is primarily attributable to fewer Type II receptors. Progesterone treatment did not effect receptor number. The major finding of this study is the difference in receptor affinity between gonadally intact males and females. In cytosols from males the K d of the Type I receptor was several-fold lower than that found in cytosols from females. The lower affinity in females was not due to sex-dependent differences in tissue-associated transcortin or receptor stability. A small, but statistically significant, difference in affinity was also found in the Type II receptor, with males again having the higher affinity. These findings of lower affinity in females relative to males are in agreement with our previous report in which the affinity of total [3H]corticosteroid binding between males and females was compared in the hippocampus 34. In contrast to the present findings, an earlier report from this laboratory concluded that the Bmax in hippocampal cytosols from females was greater (by about 40 fmol/mg protein) than from males 34. The saturation experiments in that study used primarily [3H]corticosterone
which is now known to bind to both Type I and Type II receptors as well as CBG. The present studies show that two factors contributed to the incorrect earlier interpretation: first, the presence in females of significantly greater amounts of tissue-associated, CBG-like binding accounted for approximately one-half of the reported difference; and second, the marked, sex-dependent difference in the K d of the higher affinity Type I receptor. The greater affinity of the Type I receptor in males would have the effect of lowering the calculated Bmax for males in saturation curves involving heterogenous populations of Type I and Type II receptors.
Corticosteroid receptor regulation by estrogen These results suggest that, in the hippocampus, estrogen down-regulates glucocorticoid receptors in a manner similar to that seen in the pituitary 33, but that the effect of endogenous estrogen in the normally cycling rat is relatively small, resulting in only non-significant differences between males and females. In contrast, two recent papers report enhancement of binding in the hippocampus of estrogen-treated rats. In the first study, Burgess and Handa 3 noted an estrogen-induced increase in Type I binding in hippocampus (using [3H]corticosterone as ligand) although they observed a decrease in Type I receptor mRNA. The second paper by Ferrini and De Nicola 11 reported estrogen up-regulation of Type I, but not Type II receptors in hippocampus. The bases of these discrepancies are not readily apparent. However, in the latter study, the number of Type I receptors in the hippocampus was found to be similar to the number of Type II receptors; other investigators have reported the concentration of Type II receptors to be severaltimes that of the Type I receptor in the hippocampus 25' 30
Regulation of Type I receptor affinity The markedly reduced affinity of the Type I receptor observed in females could be due to the induction of an inhibitory factor by either estrogen or progesterone; it could also be the direct result of progesterone binding to the receptor. However, progesterone has virtually the same relative affinity for both the Type I and the Type II receptors in vitro 35. It seems unlikely, therefore, that the presence of progesterone would reduce the affinity of the Type I receptor selectively. Changes in the K d of corticosteroid receptors has has been reported in other circumstances. The type II receptor has a decreased affinity in obese Zucker rats 36, and an increased affinity in spontaneously hypertensive rats TM. Also, the transformed form of the Type II receptor has a lower steroid-binding affinity than the non-transformed receptor 2°.
235 Role o f ovarian steroids in H P A function E s t r o g e n is known to be involved in the m e d i a t i o n of s e x - d e p e n d e n t differences in the circadian patterns of corticosteroids and the response of the adrenocortical system to stress. The corticosteroid response of rats to restraint stress 15 and to footshock stress 3 is increased by estrogen in ovariectomized animals. In rhesus macaques, ovariectomy dramatically r e d u c e d plasma cortisol 2a. O n e mechanism by which estrogen might cause increased basal a d r e n a l steroid levels and exaggerated stress responses would be through a reduction in r e c e p t o r numb e r at f e e d b a c k sites such as the hippocampus. T h e antiglucocorticoid action of p r o g e s t e r o n e in increasing the rate of dissociation of agonists from Type II receptors is well d o c u m e n t e d 5'31'37. Considerably less is known regarding the effect of p l a s m a p r o g e s t e r o n e concentrations on p i t u i t a r y - a d r e n a l f e e d b a c k in rats. D a t a o b t a i n e d in rhesus m o n k e y s showed no differences in circadian cortisol concentrations b e t w e e n animals in the late follicular phase c o m p a r e d to the those in the midluteal phases of their cycles, suggesting that progesterone does not have a m o d u l a t o r y effect 2s. In w o m e n , however, the morning p e a k concentrations of aldosterone and corticosterone (but not cortisol) have b e e n found to be significantly greater during the luteal phase than the follicular phase of the menstrual cycle 27.
REFERENCES 1 Barden, N. and Pfeiffer, A., Regulation of glucocorticoid receptor messenger RNA concentrations in rat brain regions, Soc. Neurosci. Abstr., 13 (1987) 1085. 2 Beato, M. and Feigelson, P., Glucocorticoid-bindingproteins of rat liver cytosol: separation and identification of the binding proteins, J. BioL Chem., 247 (1972) 7890-7896. 3 Burgess, L.H. and Handa, R.J., Ovariectomy (Ovx) and estrogen (E) replacement in female rats: effects on ACTH, corticosterone (Cort), mineraiocorticoid receptor (MR), and MR mRNA, Soc. Neurosci. Abstr., 16 (1990) 1070. 4 Canny, B.J., Hippocampal glucocorticoid receptors and the regulation of ACTH secretion, Mol. Cell. Endocrinol., 71 (1990) C35-C38. 5 Chou, Y.-C. and Luttge, W.G., Activated Type II receptors in brain cannot rebind glucocorticoids: relationship to progesterone's antiglucocorticoid actions, Brain Res., 440 (1988) 67-78. 6 Critchlow, V., Liebelt, R.A., Bar-Sela, M., Mountcastle, W. and Lipscomb, H.S., Sex difference in resting pituitary-adrenal function in the rat, Am. J. Physiol., 205 (1963) 807-815. 7 Dallman, M.E, Akana, S.E, Cascio, C.S., Darlington, D.N., Jacobson, L. and Levin, N., Regulation of ACTH secretion: variations on a theme of B, Rec. Prog. Horm. Res., 43 (1987) 113-173. 8 Dallman M.E, Levin, N., Cascio, C.S., Akana, S.F., Jacobson, L. and Kuhn, R.W., Pharmacological evidence that the inhibition of diurnal adrenocorticotropin secretion by corticosteroids is mediated via Type I corticosterone-preferring receptors, Endocrinology, 124 (1989) 2844-2850. 9 De Kloet, R., Wallach, G. and McEwen, B.S., Differences in corticosterone and dexamethasone binding to rat brain and pituitary, Endocrinology, 96 (1975) 598-609.
Physiological significance If the h i p p o c a m p a l Type I receptor plays a major role in setting the basal level of activity in the h y p o t h a l a m i c p i t u i t a r y - a d r e n a l axis, then any reduction in affinity of this r e c e p t o r would be expected to raise the basal levels of circulating A C T H as well as corticosteroids. Should Type I r e c e p t o r function be related primarily to attention, m e m o r y , or m o o d , then only an indirect effect would be expected on stress-induced corticosteroid feedback via the Type II receptors. Since in females the affinity of the Type II r e c e p t o r is similar to that of the Type I receptor, Type II receptors in females would be occupied to a somewhat greater extent at low steroid concentrations than would be the case in males. T h e present results have possible implications for the m o d u l a t i o n of h i p p o c a m p u s - m e d i a t e d processes and pit u i t a r y - a d r e n a l function by the h o r m o n a l changes of the estrus cycle, pregnancy and ovarian failure. H o w e v e r , the current d a t a were o b t a i n e d in in vitro assays and need to be confirmed within an in vivo context. Acknowledgements. This work was supported in part by National Institute of Neurological and Communicative Disorders and Stroke Grant NS 22158 and by a Research Development Grant from East Tennessee State University. The author gratefully acknowledges Mr. M.S. Ansari for his excellent technical assistance, and Ms. Jean Kintzel for her administrative support.
10 Dunnett, C.W., New tables for multiple comparisons with a control, Biometrics, 20 (1964) 482-491. 11 Ferrini, M. and De Nicola, A.E, Estrogens up-regulate Type I and Type II glucocorticoid receptors in brain regions from ovariectomized rats, Life Sci., 48 (1991) 2593-2601. 12 Ferrini, M., Magarinos, A.M. and De Nicola, A.E, Oestrogens down-regulate Type I but not Type II adrenal corticoid receptors in rat anterior pituitary, J. Steroid Biochem., 35 (1990) 671-677. 13 Gala, R.R. and Westphal, U., Corticosteroid-binding globulin in the rat: studies on the sex difference, Endocrinology, 77 (1965) 841-851. 14 Glenister, D.W., Yates, EE., Sex difference in the rate of disappearance of corticosterone-4-CTM from plasma of intact rats: further evidence for the influence of hepatic A4-steroid hydrogenase activity on adrenal cortical function, Endocrinology, 68 (1961) 747-758. 15 Grandberry, A.A., Bunnell, B.N., Mougey, E.H. and Meyerhoff, J.L., Estrogen mediates sex differences in adrenal cortical responses to stress, Soc. Neurosci. Abstr., 15 (1989) 716. 16 Kitay, J.I., Sex differences in adrenal cortical secretion in the rat, Endocrinology, 68 (1961) 818-824. 17 Krozowski, Z.S. and Funder, J.W., Renal mineralocorticoid receptors and hippocampal binding species have identical intrinsic steroid specificity, Proc. Natl. Acad. Sci. USA, 80 (1983) 6056-6060. 18 Laux, M., Bauer, H., Ganten, U., Zimmeran, F. and Vecsei, P., Glucocorticoid receptors and dissociation constant (Kd) are decreased in mononuclear leukocytes of spontaneously hypertensive rats (SHR-SP) as compared to normotensive WistarKyoto rats (WKY), J. Steroid Biochem., 34 (1989) 331-332. 19 Lineweaver, H. and Burk, D., The determination of enzyme dissociation constants, J. Am. Chem. Soc., 56 (1934) 658-666.
236 20 Nemoto, T., Ohara-Nemoto, Y., Denis, M. and Gustafsson, J.A., The transformed glucocorticoid receptor has a lower steroid-binding affinity than the non-transformed receptor, Biochemistry, 29 (1990) 1880-1886. 21 Ottenweller, J.E., Meier, A.H., Russo, A.C. and Frenzke, M.E., Circadian rhythms of plasma corticosterone binding activity in the rat and mouse, Acta Endocrinol., 91 (1979) 150157. 22 Pfeiffer, A. and Barden, N., Glucocorticoid receptor gene expression in rat pituitary gland intermediate lobe following ovariectomy, Mol. Cell. Endocrinol., 55 (1988) 115-120. 23 Pfeiffer, A. and Barden, N., Estrogen-induced decrease of glucocorticoid receptor messenger ribonucleic acid concentration in rat anterior pituitary gland, Mol. Endocrinol., 1 (1987) 435440. 24 Reul, J.M.H.M. and de Kloet, E.R., Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation, Endocrinology, 117 (1985) 2505-2511. 25 Reul, J.M.H.M., van den Bosch, F.R. and de Kloet, E.R., Relative occupation of Type I and Type II corticosteroid receptors in rat brain following stress and dexamethasone treatment: functional implications, J. Endocrinol., 115 (1987) 459-467. 26 Sapolsky, R.M., Krey L.C. and McEwen, B.S., Glucocorticoidsensitive hippocampal neurons are involved in terminating the adrenocortical stress response, Proc. Natl. Acad. Sci. USA, 81 (1984) 6174-6177. 27 Schoneshofer, M. and Wagner, G.G., Sex differences in corticosteroids in man, J. Clin. Endocrinol. Metab., 45 (1977) 814817.
28 Smith, C.J. and Norman, R.L., Influence of the gonads on cortisol secretion in female rhesus macaques, Endocrinology, 121 (1987) 2192-2198. 29 Spector, T., Refinement of the Coomassie blue method of protein quantitation, Anal. Biochem., 86 (1976) 260-269. 30 Spencer, R.L., Young, E.A., Choo, P.H. and McEwen, B.S., Adrenal steroid Type I and Type II receptor binding: estimates of in vivo receptor number, occupancy and activation with varying level of steroid, Brain Res., 514 (1990) 37-48. 31 Suthers, M.B., Pressley, L.A. and Funder, J.W., Glucocorticoid receptors: evidence for a second non-glucocorticoid binding site, Endocrinology, 99 (1976) 260-269. 32 Turner, B.B., Tissue differences in the up-regulation of glucocorticoid-binding proteins in the rat, Endocrinology, 118 (1986) 1211-1216. 33 Turner, B.B., Sex difference in glucocorticoid binding in rat pituitary is estrogen dependent, Life Sci., 46 (1990) 1399-1406. 34 Turner, B.B. and Weaver D.A., Sexual dimorphism of glucocorticoid binding in rat brain, Brain Res., 343 (1985) 16-23. 35 Veldhuis, H.D., van Koppen, C., van Ittersum, M. and de Kloet, E.R., Specificity of the adrenal steroid receptor system in rat hippocampus, Endocrinology, 110 (1982) 2044-2051. 36 White, B.D. and Martin, R.J., Alterations in the binding characteristics of glucocorticoid receptors from obese Zucker rats, J. Steroid Biochem., 36 (1990) 681-686. 37 Xu, X., Hoebeke, J. and Bjorntorp, P., Progestin binds to the glucocorticoid receptor and mediates antiglucocorticoid effect in rat adipose precursor cells, J. Steroid Biochem., 36 (1990) 465471.
229
BRES 17762
Sex differences in the binding of Type I and Type II corticosteroid receptors in rat hippocampus Barbara B. Turner Department of Physiology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614 (USA)
(Accepted 7 January 1992) Key words: Corticosteroid receptor; Hippocampus; Sex difference; Mineralocorticoid receptor; Glucocorticoid receptor; Corticosteroid binding globulin; Estrogen; Rat
Binding parameters of soluble Type I and Type II receptors were assessed in hippocampus of adult, adrenalectomized, male and female rats. No sex differences in the number of either Type I or Type II receptors could be demonstrated between gonadally intact animals. When females treated with 17fl-estradiol benzoate (10/tg/day) were compared with males, a statistically significant reduction in Type II receptors was observed in the females; progesterone produced no further decrease in receptor numbers. The amount of tissue-associated corticosteroid-binding globulin in gonadally intact animals (perfused with dextran-saline) was twice as great in females as males. Sex-dependent differences in these gonadally intact rats were found in the affinity, measured as the dissociation constant (Kd), of both the Type I and Type II receptors. For both receptors, affinity in cytosols from females was reduced. The difference for the Type II receptor was slight, but the Kd value of the Type I receptor was several-fold higher in females. The difference in affinity was evident with both natural and synthetic steroid ligands. There appears to be little, if any, difference in affinity between the hippocampal Type I and the Type II receptors in females. This suggests that the occupancy of Type I receptors in females is substantially less than that of males at low circulating concentrations of corticosteroids. INTRODUCTION Pronounced sex differences exist in several aspects of hypothalamic-pituitary-adrenal ( H P A ) function in rats. Some of these differences, such as the higher secretion rate 16 and plasma concentration of corticosterone in females 6 are attributable to the differential influence of estrogen and androgen on hepatic corticosteroid metabolism 14 and corticosteroid-binding globulin (CBG) levels 13'21. Other sex-dependent differences suggest a dimorphism at one or more levels of negative-feedback within the axis. The greater magnitude and duration of the pituitary-adrenal response to stress in females 16 and the greater diurnal variation in plasma corticosterone in females 6 suggest that negative-feedback may be less effective in females. Both Type I and Type II corticosteroid receptors exist in neurons of several limbic structures implicated in the negative feedback of glucocorticoids, particularly the hippocampus 7'25'26. Based on estimates of receptor occupancy, it was originally suggested that Type I receptors do not contribute to the regulation of A C T H secretion 24. However, it now appears that Type I (mineralocorticoid) receptors as well as Type II (gluco-
corticoid) receptors may participate in the process of glucocorticoid negative-feedback in the central nervous system s'a°. Basal A C T H levels are likely regulated by the higher affinity Type I receptors while the lower affinity Type II receptors are involved in regulating stress responses 4. If negative-feedback is less robust in females than males, one might expect reduced corticosteroid receptor numbers in sites such as the hippocampus, hypothalamus and pituitary. Ovariectomy has been shown to increase by several-fold the Type II (glucocorticoid) receptor m R N A content of the pituitary while administration of 17fl-estradiol reverses the increase 22'23. A n up-regulation of Type II receptor m R N A following ovariectomy has also been reported in brain 1. The sex-related difference in pituitary receptor m R N A levels is reflected in the concentration of functional receptors. Our laboratory has found that glucocorticoid binding is less in pituitary cytosols from females than from males; ovariectomy eliminates this difference and the administration of estrogen restores the difference 33. Estrogen-induced changes in glucocorticoid binding in the opposite direction have recently been reported in brain tissues. Ferrini and DeNicola n found that 17fl-es-
Correspondence: B.B. Turner, Dept. of Physiology, Box 19780A, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA.
230 tradiol markedly increased the n u m b e r of Type I receptors in several brain regions, including the hippocampus. This finding is in apparent contradiction to an earlier report by the same investigators that estrogen is without effect on either corticosteroid receptor type in the hippocampus 12. In their more recent paper, Ferrini and DeNicola 11 also observed that estrogen treatment similarly increased the n u m b e r of Type II receptors in the hypothalamus and septum, but not the hippocampus. These observations are not in agreement with our previous report which found that ovariectomy increased binding in the hippocampus and hypothalamus of females 34. Other workers have found that ovariectomy increases, and estrogen suppresses, m R N A concentrations of both the Type II receptor ~ and the Type I receptor 3 in hypothalamus and hippocampus, findings consistent with our earlier results. The purpose of the present work was to compare the binding parameters of the Type I and Type II receptors in the hippocampus of gonadally intact male and female rats. The effect of estrogen on receptor n u m b e r , possi-
RU 28362 used for the determination of glucocorticoid (Type II corticosteroid) binding, was either in 100- or 250-fold excess as specified in each experiment. Aliquots were incubated for 4 h at 4°C. Duplicate aliquots were taken from each vial for the determination of molarity. Separation of bound from Iree steroid by gel exclusion chromatography on LH-20 mini-columns9 was performed in duplicate using 50/A aliquots of cytosol. In the experiment in which dextrancoated charcoal (DCC) was used to separate bound steroid, DCC was added 1:6 (v/v) to cytosol and incubated for 10 min at 4°C before centrifugation2 in a Beckman Microfuge 11. Aliquots were taken in triplicate from each vial and counted. In those experiments in which hydroxylapatite (HAP) was used to separate bound from free steroid, the protocol of Krozowski and Funder17was followed. Protein was determined by a modification of the Coomassie blue method 29. Binding parameters of saturation curves were calculated from double reciprocal plots of the data according to Lineweaver-Burk 19. Student's t-test was used for the comparison of two means. Dunnett's test for multiple comparisons with a control x°, or 2-way analysis of variance was used to test for significance in other experiments as indicated. RESULTS
Saturation binding using [3H]corticosterone
ble sex-differences in receptor stability and the effect of
The first study verified our earlier results 34 on sex dif-
tissue-associated, CBG-like binding proteins on apparent receptor binding parameters are also addressed.
ferences in glucocorticoid binding obtained with saturation plots. Individual hippocampi from gonadally intact rats were homogenized in the buffer used in the previous study: 5 m M TRIS [Tris(hydroxymethyl) aminomethane] containing 1 m M E D T A , 1 m M dithiothreitol and 10% glycerol, p H 7.4, at 4°C. Aliquots of cytosol
MATERIALS AND METHODS Sexually mature Sprague-Dawley rats, virus antibody free, (Charles River, Wilmington, MA) were housed 2-4 per cage under a 12:12 h light-dark cycle. Gonadectomies were performed under Metofane anesthesia (Methoxyflurane, Pitman-Moore, Washington Crossing, NJ) at least 10 days prior to sacrifice. Gonadal steroids were injected s.c. in 0.2 ml of peanut oil. All animals were adrenalectomized under Metofane anesthesia 12 h before sacrifice (except in one experiment where noted). At sacrifice, rats were anesthetized with Metofane and killed by intracardiac perfusion (90 ml of room temperature saline followed by 60 ml of ice-cold 6% dextran in saline). Brains were removed quickly and dissected on a chilled glass plate; the hippocampi were then frozen on dry ice before storage at -80°C. Tritiated steroids were obtained from New England Nuclear Corp. (Boston, MA): 1,2,6,7-[3H]corticosterone (spec. act. 85.8, 101.6 and 112 Ci/mmol); 6,7-[3H]dexamethasone (spec. act. 37.3, 45.8 and 49.9 Ci/mmol); 1,2,6,7-[3H]aldosterone (spec. act. 82 Ci/ mmol); 6,7-[3H]triamcinolone acetonide (spec. act. 43.6 Ci/mmol); 6-methyl-[3H]RU 28-362 (spec. act. 77.8 and 77.9 Ci/mmol). Thin layer chromatography was used to verify the purity of radiolabelled steroids. Unlabelled corticosterone, dexamethasone and aldosterone were purchased from Sigma Chemicals (St. Louis, MO). Unlabelled RU 28-362 was a gift from Roussel-UCLAF. All chemicals used were reagent grade. Hippocampi were homogenized 1:10 (w/v) in HEPES buffer (25 mM) containing 0.25 M sucrose, 10 mM sodium molybdate and 5 mM dithiothreitol, pH 7.4 at 4°C, unless otherwise specified. Homogenates were centrifuged at 105,000 × g for 1 h at 2°C. Incubations consisted of 140/A aliquots of cytosol to which 2.8/A of ethanol containing the steroid was added. In some experiments, soluble receptor binding was measured by saturation plots; in other experiments binding was determined by a single, saturating concentration of tritiated steroid (25 nM). Non-specific binding was determined using a concentration of 500-fold excess of unlabelled steroid unless otherwise indicated. The concentration of unlabelled
were incubated in varying concentrations of [3H]corticosterone (0.75-24 nM). Significant differences were found between the binding parameters of males and females as shown in Fig. 1. The Bmax of males was significantly less than that of females (223 + 11 vs. 276 + 17 fmol/mg protein; P < 0.05). These data are in good agreement with those obtained previously34. The K a of males was also markedly less than that of females (1.20 + 0.08 vs, 2.11 + 0.39 nM; P < 0.05).
Receptor stability and tissue CBG The possible contribution of several technical factors to the apparent sex-related differences in corticosteriod binding was considered. The question of whether the observed disparity in receptor n u m b e r of affinity could be due to a sex-dependent difference in receptor stability was addressed. To determine the stability of corticoid receptors, male and female hippocampi were homogenized 1:5 (w/v) in the absence of sodium molybdate and incubated at 25°C for 0, 15 or 60 min before being incubated with [3H]steroid for 4 h at 4°C. Cytosols prepared from 3 tissue pools of each sex were assayed using [3H]dexamethasone; the n u m b e r of receptors at 0 time was taken as 100%. The percentage of receptors remaining after 15 min (47 + 3 vs. 57 + 3 fmol/mg pro-
231 •
Males
--4--
Females
6O o
~,
-)(-
300
0
T T ~
/I'~
I.- ~'~
200
T
T
~ 4o
/
-
~-
30 2O 100
T
8 10
io rn
0
I
E
10
20
3H-Corticosterone
Concentration (riM}
O)
0 Males
30
Fig. 1. Direct plots of soluble binding experiments using hippocampi from gonadally intact rats (12 h ADX). Tissues were homogenized in TRIS buffer in the absence of sodium molybdate. Each point represents the mean of 6 separate experiments; the error bars indicate the S.E.M. Data from males is indicated by circles, that from females by triangles. Comparison of the LineweaverBurk plots of the data show that females have significantly greater binding capacity but lower affinity (P < 0.05, Student's t-test, twotailed).
tein) or 60 min (3 + 2 vs. 2 + 1 fmol/mg protein) at 25°C in males vs. females, respectively, was not significantly different. Similar results were obtained with [3H]corticosterone. The naturally occurring ligand in rats, corticosterone, binds not only to corticoid receptors, but also to CBG. Although animals are well peffused at sacrifice, small but measurable amounts of CBG remain in hippocampal tissue 32. In order to determine the possible contribution of CBG to the binding differences observed with [3H]corticosterone, binding was measured in cytosols incubated with a single, near-saturating concentration of [3H]corticosterone (24 nM) in the presence of a 100-fold excess dexamethasone. Hippocampi from seven animals of each sex were assayed. CBG-like binding (Fig. 2) was significantly greater in females (47.5 + 4.8 fmol/mg protein) than in males (21.4 _+ 2.5 fmol/mg protein; P < 0.001). This difference in binding, 26 frnol/mg protein, accounts for over half of the observed difference in Bmax (43 fmol/mg protein) found in the saturation study.
Type I and Type H receptor number Does a sex difference in receptor number exist when steroids that have very low, or negligible affinity for CBG are used as ligands? In order to assess the influence of gonadal steroids, animals were either gonadectomized or sham-operated 3 weeks prior to sacrifice. [3H]Triamcinolone was used to determine total receptor binding; [3H]aldosterone was used both alone and in the presence of RU 28362 (a selective Type II agonist) to
Females
Fig. 2. 'CBG-like' binding present in hippocampal cytosols from perfused male and female rats (12 h ADX). Binding was measured using a single, saturating concentration of [3H]corticosterone in the presence of a 100x excess of dexamethasone. Bars indicate the mean + S.E.M. of 7 animals, individually assayed. Females (hatched bar) had significantly more binding, measured as fmol/mg protein, than did males (open bar; *P < 0.001, Student's t-test,
two-tailed).
measure the number of Type I receptors. A single, saturating concentration of each steroid was used; treatment with DCC was used to separate bound from free steroid. Binding obtained with [3H]aldosterone was substantially decreased by the presence of 100x excess RU 28362 (Table I). With both [3H]ligands, hippocampi from females showed less, not greater binding, than did those from males. However, no significant, sex-dependent differences in binding were found by 2-way ANOVA. The possibility that methodological differences might be responsible for the lack of agreement between the two studies was evaluated. (In the second experiment described above, dextran-coated charcoal was used rather than LH-20 and the high affinity ligand, triamcinolone, was employed.) In the next study, using gonadally intact rats and a single concentration of [3H]ligand, bound steroid was separated from free by chromatography on LH-20 mini-columns. [3H]Dexamethasone in the presence and absence of excess RU 28362 was used to determine the total number of receptors and the number of Type I receptors, respectively. The number of Type II receptors was calculated by subtraction of Type I from the total number of receptors. Again, no sex difference was found in the number of corticosteroid receptors, either Type I or Type II (Table II). In the previous studies, in which gonadally intact animals were used, females were found to have somewhat fewer receptors than males although the difference was not statistically significant. The next experiment asked if administration of exogenous steroids might enhance this difference. Gonadally intact males were compared with ovariectomized females treated with either 10 Hg of 17fl-
232
--I
3H-DEX
Males
•
3H-CORT +RU 28362
--A---
Females
75 400
0
0 0 D. t~
60 "7"
T
300
45 m
"0
200
30
m 0
100
tO I
I m
MALES
OVX
D +
E
®
15 ×
0 03
0
O V X + E/P
Fig. 3. Corticosteroid-binding proteins in cytosols prepared from hippocampi of gonadally intact males and ovariectomized (OVX) females treated with estradiol benzoate (E; 10 ~g/rat/day) or estradiol benzoate plus progesterone (EP; 500/~g/rat/day) for 10 days prior to sacrifice. Animals were ADX 12 h prior to sacrifice. Binding was determined by a single point assay and is expressed as fmol/mg protein. Values are the mean of at least 7 animals, assayed individually. *P < 0.05 vs. male value.
I
I
I
10
15
20
25
3H-Dexarnethasone Concentration (nM) •
Males
Females
--&--
360 0
300 240
estradiol b e n z o a t e / r a t / d a y o r estradiol b e n z o a t e t o g e t h e r with 500/~g o f p r o g e s t e r o n e for 10 days p r i o r to sacrifice. Total binding was m e a s u r e d with [ 3 H ] d e x a m e t h a -
I
5
m ~o o3 co
180 120
sone, and [3H]corticosterone with excess R U 28362 was
6O
used to e s t i m a t e Type I binding. A s s h o w n in Fig. 3, est r o g e n a d m i n i s t r a t i o n significantly d e c r e a s e d total r e c e p -
m
tor binding ( [ 3 H ] d e x a m e t h a s o n e ( P < 0.05)). Since estrogen
had
no d i s c e r n a b l e
[3H]corticosterone
O= o
r e c e p t o r s i n v o l v e d a d e c r e a s e in Type II r e c e p t o r s .
Receptor affinity studies Since the initial study with [3H]corticosterone indic a t e d a s e x - r e l a t e d d i f f e r e n c e in r e c e p t o r affinity as well as r e c e p t o r n u m b e r , we also e x a m i n e d the a p p a r e n t dif-
i
I
I
10
15
20
3H-RU 28362
effect on the binding of
+ R U 28362, the d e c r e a s e in total
,
5
25
Concentration (riM)
Fig. 4. Direct plots of soluble receptor experiments using hippocampi from gonadally intact rats (12 h ADX). Tissues were homogenized in HEPES buffer in the presence of sodium molybdate. Each point represents the mean of 5 separate experiments; the error bars indicate the S.E.M. Data from males is indicated by circles, that from females by triangles. Binding parameters were determined by Lineweaver-Burk plots of the data. A: Type I binding was determined by [3H]dexamethasone in the presence of a 250fold excess of RU 28362. B: Type II binding was determined using [3H]RU 28362 as ligand.
TABLE I
Binding of [3H]aldosterone and [3H]triamcinolone acetonide to hippocampal cytosols of gonadally intact and castrated male and ovariectomized female rats Gonadectomies (or sham operations) were performed 3 weeks prior to sacrifice. Animals were killed 12 h after ADX. Binding was measured at a single, saturating concentration (25 nM) of [3H]ligand; bound steroid was separated from free by treatment with DCC. Values represent the mean _+ S.E.M. of 7-8 experiments in which individual hippocampi were used. No significant differences were found using 2-way ANOVA.
Binding (fmol/mg protein)
Intact male Gonadectomized male Intact female Ovariectomized female
[3]Aldosterone
[3]Aldosterone + RU 28362
13H]Triamcinolone acetonide
261 241 230 227
105 103 84 101
359 352 332 329
+ 12 + 11 _+ 15 + 19
+ + + +
10 7 7 8
+ + + +
11 21 23 20
233 TABLE II Corticosteroid receptor concentration in cytosols prepared from hippocampi of gonadally intact male and female rats
60
Binding in ADX rats (12 h) was measured at a single, saturating concentration of [3H]dexamethasone in the presence and absence of a 100-fold excess of RU 28362. Type II was calculated as the difference between total receptor binding ([3H]dexamethasone alone; n - 5) and Type I binding ([3H]dexamethasone in the presence of RU 28362; n = 10). Binding is expressed as fmol/mg protein. Values given are the mean + S.E.M.
Male Female
Type I
Type H
Total
85 _+ 15 73 _+ 15
246 + 17 225 _+ 10
336 + 24 295 _+ 12
•
Males
--
- -
20
JrO
O 0
2
4
3H-Corticosterone
ference in corticosteroid r e c e p t o r affinity. Saturation plot experiments were p e r f o r m e d on h i p p o c a m p i from gonadally intact male and female rats. [3H]RU 28362 was used as the selective ligand for Type II receptors and [3H]dexamethasone plus 250-fold excess unlabelled R U 28362 for Type I receptors. H i p p o c a m p i from 2 - 3 rats of each sex were p o o l e d for each assay. Binding values were averaged from 5 s e p a r a t e experiments and are shown in Fig. 4. F o r the Type I receptors, no difference was found b e t w e e n the Bmax of tissues from male and female rats (48.9 + 3.6 and 44.7 + 2.7 fmols/mg protein, respectively). H o w e v e r , the K d for the Type I receptors was several-fold lower in males than females (0.53 + 0.05 vs. 1.30 + 0.25 nM, P < 0.02). No difference was found in the Bmax of males (361 + 16 fmols/mg protein) c o m p a r e d with that of females (347 + 15 fmols/mg protein). The numerically small difference in the K d value of males and females for the Type II rec e p t o r was statistically significant (1.41 + 0.03 nM and 1.30 + 0.04 nM, respectively; P < 0.025). Since corticosterone would be expected to have con-
TABLE III Estimation of the affinity of Type I binding in hippocampal cytosols with [3H]corticosterone in the presence of excess RU 28362 as ligand
Hippocampal tissues were taken from gonadaUy intact rats that had been ADX 12 h prior to sacrifice. Bound steroid was separated from free by passage through LH-20 mini-columns. Maximal binding (Bmax), dissociation constant (Kd) and the regression coefficient (r) are derived from Lineweaver-Burk plots of the saturation curves. Values represent the mean + S.E.M. of 4 separate experiments. *Significantly different from male value (P < 0.01, Student's t-test, two-tailed).
Male Female
B,,~.~ (fmol/mg protein)
Ka (nM)
r
76.1 + 9.4 91.6 + 6.0
0.21 + 0.01 0.76 + 0.20*
0.989 0.995
Females
--A--
t
t
6
8
Concen~'ation
10
(rtvl)
Fig. 5. Binding experiments using hydroxylapatite to measure the Type I receptor in gonadally intact male and female rats. [3H]Corticosterone was used as ligand in the presence of a 100x excess of RU 28362. Each point represents the mean of 4 separate experiments; the error bars indicate the S.E.M. Data from males is indicated by circles, that from females by triangles. Comparison of the Lineweaver-Burk plots of the data show that females have significantly lower affinity (P < 0.05, Student's t-test, one-tailed).
siderably greater affinity for the Type I r e c e p t o r than d e x a m e t h a s o n e , we asked whether the sex-related difference in affinity is e n h a n c e d when [3H]corticosterone is used as the ligand. Table I I I shows the results of 4 saturation plots using [3H]corticosterone in the presence of a 100-fold excess of R U 28362 to estimate the affinity of Type I receptors in gonadaUy intact males vs. females. According to L i n e w e a v e r - B u r k plots of the data, binding in cytosols from males had a 3.5-fold greater affinity than did that from females ( P < 0.01). The Bmax values for males and females were not statistically different. The p r o b a b l e endogenous ligand of the Type I receptor in hippocampus, corticosterone, has greater affinity for the r e c e p t o r than do synthetic c o m p o u n d s 34. However, in o r d e r to use this ligand, its binding to the C B G present in the cytosol needs to be eliminated because [3H]corticosterone binds to C B G as well as to the Type I receptor. The a m o u n t of C B G present in tissue m a y well affect the dissociation constant. H y d r o x y l a p a t i t e , which has affinity for the r e c e p t o r but not C B G , was therefore used. Using the batch p r o c e d u r e of Krozowski and F u n d e r t7, we first verified that in our hands the assay adequately s e p a r a t e d b o u n d from free steroid and that the hydroxylapatite b o u n d negligible amounts of C B G . Cytosol was p r e p a r e d from cerebral cortex to which 1 0 / 4 of undiluted plasma was a d d e d p e r ml. Aliquots (in quadruplicate) were incubated either with [3H]dexamethasone or with [3H]corticosterone in the presence of a 100-fold excess of R U 28362; non-specific binding was also deter-
234 mined for each ligand. Binding in parallel incubates was compared using LH-20 and hydroxylapatite procedures with [3H]dexamethasone as ligand. We found that the hydroxylapatite procedure adequately separated bound from free steroid, but that recovery of receptor was reduced while non-specific binding was increased. Hydroxylapatite bound less than 0.5% of the CBG measured by LH-20. Is the binding affinity of hippocampal Type I receptors in females less than that of males when the naturally-occurring ligand is used? As illustrated in Fig. 5, four saturation experiments of cytosols prepared from each sex were run on hydroxylapatite using 6 steroid concentrations (0.2-8.0 nM) of [3H]corticosterone in the presence of excess RU 28362. Females were found to have a higher K a (1.67 nM) than did males (0.39 nM; P < 0.05, one-tailed). Bmax was similar for males (56 + 4 fmol/mg protein) and females (61 + 12 fmol/mg protein).
DISCUSSION
Findings of present studies In gonadally intact rats, no significant difference in the number of Type I or Type II receptors could be demonstrated between males and females. However, males did consistently tend to have a slightly greater number of Type I and Type II receptors. The difference between males and females became statistically significant when estrogen-treated females were compared with males. The reduced binding observed in estrogen-treated females is primarily attributable to fewer Type II receptors. Progesterone treatment did not effect receptor number. The major finding of this study is the difference in receptor affinity between gonadally intact males and females. In cytosols from males the K d of the Type I receptor was several-fold lower than that found in cytosols from females. The lower affinity in females was not due to sex-dependent differences in tissue-associated transcortin or receptor stability. A small, but statistically significant, difference in affinity was also found in the Type II receptor, with males again having the higher affinity. These findings of lower affinity in females relative to males are in agreement with our previous report in which the affinity of total [3H]corticosteroid binding between males and females was compared in the hippocampus 34. In contrast to the present findings, an earlier report from this laboratory concluded that the Bmax in hippocampal cytosols from females was greater (by about 40 fmol/mg protein) than from males 34. The saturation experiments in that study used primarily [3H]corticosterone
which is now known to bind to both Type I and Type II receptors as well as CBG. The present studies show that two factors contributed to the incorrect earlier interpretation: first, the presence in females of significantly greater amounts of tissue-associated, CBG-like binding accounted for approximately one-half of the reported difference; and second, the marked, sex-dependent difference in the K d of the higher affinity Type I receptor. The greater affinity of the Type I receptor in males would have the effect of lowering the calculated Bmax for males in saturation curves involving heterogenous populations of Type I and Type II receptors.
Corticosteroid receptor regulation by estrogen These results suggest that, in the hippocampus, estrogen down-regulates glucocorticoid receptors in a manner similar to that seen in the pituitary 33, but that the effect of endogenous estrogen in the normally cycling rat is relatively small, resulting in only non-significant differences between males and females. In contrast, two recent papers report enhancement of binding in the hippocampus of estrogen-treated rats. In the first study, Burgess and Handa 3 noted an estrogen-induced increase in Type I binding in hippocampus (using [3H]corticosterone as ligand) although they observed a decrease in Type I receptor mRNA. The second paper by Ferrini and De Nicola 11 reported estrogen up-regulation of Type I, but not Type II receptors in hippocampus. The bases of these discrepancies are not readily apparent. However, in the latter study, the number of Type I receptors in the hippocampus was found to be similar to the number of Type II receptors; other investigators have reported the concentration of Type II receptors to be severaltimes that of the Type I receptor in the hippocampus 25' 30
Regulation of Type I receptor affinity The markedly reduced affinity of the Type I receptor observed in females could be due to the induction of an inhibitory factor by either estrogen or progesterone; it could also be the direct result of progesterone binding to the receptor. However, progesterone has virtually the same relative affinity for both the Type I and the Type II receptors in vitro 35. It seems unlikely, therefore, that the presence of progesterone would reduce the affinity of the Type I receptor selectively. Changes in the K d of corticosteroid receptors has has been reported in other circumstances. The type II receptor has a decreased affinity in obese Zucker rats 36, and an increased affinity in spontaneously hypertensive rats TM. Also, the transformed form of the Type II receptor has a lower steroid-binding affinity than the non-transformed receptor 2°.
235 Role o f ovarian steroids in H P A function E s t r o g e n is known to be involved in the m e d i a t i o n of s e x - d e p e n d e n t differences in the circadian patterns of corticosteroids and the response of the adrenocortical system to stress. The corticosteroid response of rats to restraint stress 15 and to footshock stress 3 is increased by estrogen in ovariectomized animals. In rhesus macaques, ovariectomy dramatically r e d u c e d plasma cortisol 2a. O n e mechanism by which estrogen might cause increased basal a d r e n a l steroid levels and exaggerated stress responses would be through a reduction in r e c e p t o r numb e r at f e e d b a c k sites such as the hippocampus. T h e antiglucocorticoid action of p r o g e s t e r o n e in increasing the rate of dissociation of agonists from Type II receptors is well d o c u m e n t e d 5'31'37. Considerably less is known regarding the effect of p l a s m a p r o g e s t e r o n e concentrations on p i t u i t a r y - a d r e n a l f e e d b a c k in rats. D a t a o b t a i n e d in rhesus m o n k e y s showed no differences in circadian cortisol concentrations b e t w e e n animals in the late follicular phase c o m p a r e d to the those in the midluteal phases of their cycles, suggesting that progesterone does not have a m o d u l a t o r y effect 2s. In w o m e n , however, the morning p e a k concentrations of aldosterone and corticosterone (but not cortisol) have b e e n found to be significantly greater during the luteal phase than the follicular phase of the menstrual cycle 27.
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Physiological significance If the h i p p o c a m p a l Type I receptor plays a major role in setting the basal level of activity in the h y p o t h a l a m i c p i t u i t a r y - a d r e n a l axis, then any reduction in affinity of this r e c e p t o r would be expected to raise the basal levels of circulating A C T H as well as corticosteroids. Should Type I r e c e p t o r function be related primarily to attention, m e m o r y , or m o o d , then only an indirect effect would be expected on stress-induced corticosteroid feedback via the Type II receptors. Since in females the affinity of the Type II r e c e p t o r is similar to that of the Type I receptor, Type II receptors in females would be occupied to a somewhat greater extent at low steroid concentrations than would be the case in males. T h e present results have possible implications for the m o d u l a t i o n of h i p p o c a m p u s - m e d i a t e d processes and pit u i t a r y - a d r e n a l function by the h o r m o n a l changes of the estrus cycle, pregnancy and ovarian failure. H o w e v e r , the current d a t a were o b t a i n e d in in vitro assays and need to be confirmed within an in vivo context. Acknowledgements. This work was supported in part by National Institute of Neurological and Communicative Disorders and Stroke Grant NS 22158 and by a Research Development Grant from East Tennessee State University. The author gratefully acknowledges Mr. M.S. Ansari for his excellent technical assistance, and Ms. Jean Kintzel for her administrative support.
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