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Expression of mineralocorticoid Type I and glucocorticoid Type II receptors in astrocyte glia as a function of time in culture
Developmental Brain Research, 61 (1991) 55-61 © 1991 Elsevier Science Publishers B.V. 0165-3806/91/$03.50 A DONIS 016538069151284M
55
BRESD 51284
Expression of mineralocorticoid Type I and glucocorticoid Type II receptors in astrocyte glia as a function of time in culture Yun-Chia
Chou 1, William G. Luttge 1 and Colin Sumners 1'2
Departments of 1Neuroscience and 2physiology, University of Florida College of Medicine, Gainesville, FL 32610 (U.S.A.) (Accepted 5 March 1991)
Key words: Brain cell culture; Astrocyte; Mineralocorticoid receptor; Glucocorticoid receptor; Glia
In the present study we have examined the expression of mineralocorticoid Type I and glucocorticoid Type II receptors in astrocyte glia maintained in culture for different periods of time. Cytosolic mineralocorticoid Type I receptors were labeled with [SH]aldosterone (ALDO) in the presence of a 500-fold molar excess of the potent Type II receptor ligand RU 28362. [3H]Dexamethasone (DEX) was used to label cytosolic Type II receptors. Both Type I and Type II receptor binding was saturable in astrocyte glia that had been maintained in culture for 20 and 30 days following final plating (i.e. 20- and 30-day-old cultures). Scatchard analysis of [aH]ALDO binding revealed a single class of Type I receptors, with dissociation constants (Kd) of 0.45 + 0.13 nM and 0.53 + 0.07 nM, respectively, in 20- and 30-day-old cultures. The number of Type I receptors in 30-day-old cultures was nearly half that found in 20-day-old cultures (22.06 vs 42.64 fmol/mg protein). Linear Scatchard plots were also obtained for [SH]DEX binding to cytosol prepared from 20- and 30-day-old cultures. There were no significant differences in the Ka or B ~ values for [3H]DEX binding in 20- or 30-day-old cultures, i.e. 2.06 + 0.15 nM and 247.36 + 18.16 fmol/mg protein for 20-day-old cells and 2.3 + 0.74 nM and 261.02 + 3.08 fmol/mg protein for 30-day-old ceils. These Bm~x values are more than double the Bmax value for [aH]DEX binding observed in our previous studies in 10-day-old astrocyte glial cultures. Switching cultured astrocyte glial from serum-supplemented to serum-free medium had no significant effects on the K d values of Type I or Type II receptors in all the cultures tested. However, treatment with serum-free medium increased the number of Type I receptors in 30-day-old cultures to a level similar to that found in 20-day-old cultures. Taken together, these binding data suggest that Type I and Type II receptors are expressed differently in astrocyte glia as a function of time in culture. INTRODUCTION It is now widely accepted that astrocyte glia are essential for n o r m a l brain function 15'17. T h e r e is extensive evidence that in addition to their K+-buffering capacity, astrocyte glia also interact with various neurotransmitters and n e u r o m o d u l a t o r s and m a y actively m o d u l a t e the process of neurotransmission in the brain is' 17. M a n y studies have shown that adrenocorticoids have p r o f o u n d influences on the growth, differentiation, morp h o l o g y and function of astrocyte glia is. F o r instance, it has b e e n d e m o n s t r a t e d that glucocorticoids increase the activity of glutamine synthetase in astrocyte glia via their stimulatory effect on the expression of the gene for this enzyme 16A8"32'33. In view of the fact that astrocyte glia constitute a p p r o x i m a t e l y one-third of the total cell volume of the c e r e b r a l cortex 34, it is very likely that these cells play a significant role in the adrenocorticoid effects o b s e r v e d in the brain. Much evidence has shown that the effects of adrenocorticoids on gene regulation are m e d i a t e d through a
stereospecific binding with their receptors 3'1°'25,29. Results from a u t o r a d i o g r a p h i c and ligand binding studies have d e m o n s t r a t e d that m i n e r a l o c o r t i c o i d Type I and glucocorticoid Type II r e c e p t o r s are p r e s e n t on glial cells in the central nervous system 1,12A3,23,24,37,45. N e v e r t h e less, due to the n u m b e r of different cell types p r e s e n t in the brain, in vivo studies of a d r e n o c o r t i c o i d actions in astrocyte glia have b e e n h a m p e r e d by n u m e r o u s interferences from o t h e r types of cells. W e are able to p r e p a r e p r i m a r y cell cultures that are enriched with approximately 98% astrocyte glia 38, and have used these cultures as a m o d e l system to study a d r e n o c o r t i c o i d actions in the astrocyte glia. Previous studies from this l a b o r a t o r y have d e m o n s t r a t e d the presence of b o t h Type I and Type II receptors in astrocyte glial cultures which have been m a i n t a i n e d in culture for 10 days 6'7. T h e results from ligand binding and i m m u n o c y t o c h e m i c a l staining experiments suggest that these astrocyte glial r e c e p t o r s display binding p r o p e r t i e s and structures similar to those found in the brain and o t h e r target organs 2"4'9"11'23"28. A c c o r d ingly, it is conceivable to use these cultures to investigate
Correspondence: C. Sumners, Department of Physiology, University of Florida College of Medicine, Box J-274, J.H.M. Health Science Center, GainesviUe, FL 32610-0274, U.S.A.
56 the p r o p e r t i e s and regulation o f adrenocorticoid receptors in the astrocyte glia without the complications seen in vivo and in t u m o r cell lines. It is known that the biological effects of adrenocorticoids are d e p e n d e n t on the concentration of their receptors present in target tissues 5'14'26. Therefore, it is o f particular i m p o r t a n c e to know how the receptors are r e g u l a t e d in the target cells. Much evidence has shown that adrenocorticoids are able to regulate their own receptors (i.e. autoregulation) in the brain and peripheral target tissues of adult rats 2°'3°. H o w e v e r , autoregulation of Type II receptors by corticosterone ( C O R T ) is lacking in the n e o n a t e brain 21'42. The reason for this inability of C O R T to regulate its own receptors in the neonate brain remains unclear at the present time. In view of the fact that o u r astrocyte glial cultures are p r e p a r e d from o n e - d a y - o l d rat brains, it is interesting to first examine changes in the expression of Type I and Type II receptors in these cells as a function of time in culture. The results should provide valuable information for subsequent research on the regulation of receptors in these cells. In the p r e s e n t study, we e x a m i n e d the specific binding of [3H]aldosterone ( A L D O ) and [3H]dexamethasone ( D E X ) to Type I and Type II receptors, respectively, in astrocyte glial cells m a i n t a i n e d in culture for different p e r i o d s of time (20 and 30 days) following final plating. It was found that the K d values for Type I and Type II r e c e p t o r s in these cells did not change significantly with time in culture. H o w e v e r , significant changes in the binding capacity of Type I receptors were observed in astrocyte glia cultured for 20 or 30 days. In addition, comparison of the present results with our previous studies shows that 20- and 30-day-old astrocytes contain m o r e than d o u b l e the n u m b e r of Type II receptors c o m p a r e d with 10-day-old astrocytes 7.
chemicals were of reagent grade quality and were obtained from Fisher Scientific.
Preparation of brain astrocyte glial cultures Astrocyte glial cultures were prepared from the brains of one-day-old rats as described previouslyTM. In brief, whole brains were dissected from one-day-old SD rats and placed in an isotonic salt solution containing 100 units of penicillin G, 100/~g streptomycin and 0.25/~g amphotericin B per ml, pH 7.4. After the removal of blood vessels and pia mater, brains were minced and the tissue chunks were incubated with 0.25% trypsin (w/v) at 37 °C for 5 min. After this time, 160/~g DNase I was added and the incubation continued for an additional 5 min. The dissociated cells were then suspended in 50 ml DMEM containing 10% FBS (10% FBS/ DMEM) pelleted by centrifugation at 1,000 g for 10 min, and then resuspended in 10% FBS/DMEM. Lastly, cells were plated in 100-mm diameter Falcon tissue culture dishes precoated with poly-L-lysine at a density of 18 x 106 cells/dish and incubated at 37 °C in a humidified incubator with 10% CO2/90% air. After 3 days in culture the medium was replaced with fresh 10% FBS/DMEM and cells were maintained at 37 °C for an additional 3 days. At this time cells were washed with DMEM, dissociated with 0.25% trypsin (3 ml/dish), resuspended in 10% FBS/DMEM and plated in 100-ram culture dishes at a density of 1 x 106 cells/dish. Cultures were grown for 20 or 30 days at 37 °C prior to use, at which time they consisted of >98% astrocyte glia, as evidenced by immunofluorescent staining with an antibody against glial fibrillary acidic protein (GFAP)3s. The 10% FBS/DMEM was replaced every 10 days.
Preparation of cytosol Cytosol was prepared as described previously7. In brief, astrocyte giia maintained in culture for 20 (i.e. 20-day-old) or 30 (i.e. 30-day-old) days following the final plating were removed from culture dishes and washed twice with ice-cold HGEM (consisting of 20 mM HEPES, 10% glycerol (w/v), 1 mM EDTA and 5 mM Na2MoO 4, pH 7.4 at 0-4 °C) for the [3H]ALDO binding experiments or with HGEMD buffer (consisting of 20 mM HEPES, 10% glycerol (w/v) 1 mM EDTA, 20 mM Na2MoO4, and 2 mM DTF, pH 7.4 at 0--4 °C) for the [3H]DEX binding experiments. Next, cells were homogenized in HGEM or HGEMD buffer with a Dounce homogenizer followed by centrifugation at 100,000 g for 60 min at 0 °C to generate the cytosol (i.e. supernatant) fraction. During these and all other procedures, the cytosol was kept at 0-2 °C to minimize degradation of cytosolic receptors. Final protein concentrations were determined by the method of Lowry 19, and were typically in the range of 4-5 mg/ml cytosol.
[3H]ALDO labeling of mineralocorticoid Type I receptors MATERIALS AND METHODS
Materials One-day-old Sprague-Dawley (SD) rats were obtained from our breeding stock, which originated from Charles River Farms (Wilmington, MA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and amphotericin B (Fungizone) were purchased from GIBCO (Grand Island, NY). Trypsin was obtained from Worthington Biochemicals (Freehold, NJ). Penicillin G and streptomycin were from Pfizer, Inc. (New York, NY). [1,2,6,7-3H]Aldosterone ([aH]ALDO) (82 Ci/mmol) and [6,7-3H]dexamethasone ([3H]DEX) were purchased from Dupont New England Nuclear (Boston, MA) and checked for purity by thin-layer silica gel chromatography using a chloroform-methanol (9:1, v/v) solvent system. RU 28362 (llb,17b-dihydroxy-6-methyl-17a-(1propionyl)androsta-l,4,6-trien-3-one) was a gift from Dr. J.P. Raynaud (Roussel-UCLAF, France). DNase I, poly-L-lysine, unlabeled ALDO ([1H]ALDO), dexamethasone (DEX), EDTA, glycerol, HEPES, Sephadex G-25 (fine), dithiothreitol (DTT) and sodium molybdate (Na2MoO4) were purchased from Sigma Chemical Co. (St. Louis, MO). Scinti-Verse II scintillation fluor was purchased from Fisher Scientific (Pittsburgh, PA). All other
Cytosol was incubated at 0 °C for 24 h with [3H]ALDO and a 500-fold molar excess of RU 28362 in the presence (i.e. non-specific binding, Br~s) or absence (i.e. total binding, B-r) of a 200-fold molar excess of unlabeled ALDO. Concentrations of [3H]ALDO ranging from 0.5 to 20 nM were used for saturation analyses of receptor binding. In all experiments, macromolecule-bound [3H]ALDO was separated from free steroids at 4 °C on Sephadex G-25 columns (0.6 x 14 cm) as described previously7. Specific binding (Bsp) for each sample was determined by subtracting non-specific from total binding (i.e. Bsp = BT-Br~s).
[3H]DEX labeling of glucocorticoid Type H receptors The binding assay for [3H]DEX labeling of cytosolic Type II receptors was similar to that described above for ~ I receptors except that cytosol was incubated with [3I-I]DEX in the presence (i.e. BNS) or absence (i.e. BT) of a 200-fold molar excess of unlabeled DEX. In a previous study, use of the specific Type II receptor ligand RU 28362 as the unlabeled displacer for [3H]DEX binding yielded Kd and Bm~ values that were similar to those found when DEX itself was included as the unlabeled displacer. Thus, DEX was included in the non-specific binding tubes in the current experiments. After a 24-h incubation, maeromolecule-bound ste-
57 roids were separated from free steroids at 4 °C on Sephadex G-25 columns (0.6 x 14 cm) as detailed previously 6.
RESULTS
Serum-depletion treatment of astrocyte glial cultures
As shown in Fig. 1A, the binding of [3H]ALDO to cytosolic Type I receptors in 20- and 30-day-old astrocyte glial cultures was saturable. The maximal binding was obtained at 10 nM [3H]ALDO for both 20- and 30day-old cultures. Scatchard analyses44 of the specific [3H]ALDO binding data from the saturation experiments revealed linear plots for both 20- and 30-day-old cultures (Fig. 1 B ) . Therefore, these results suggest that [3H]ALDO binds to a single class of binding sites or a population of binding sites with equal affinity. The linearity of these Scatchard plots also eliminated the possibility that [3H]ALDO was binding to Type II receptors under the current assay conditions. No signifi-
The effect of serum-containing steroids on Type I and Type II receptor binding was investigated in this study by incubating the astrocyte glial cultures with serum-free medium as detailed previously 6'7. In brief, 10% FBS/DMEM was removed from 20- and 30-day-old astrocyte glial cultures, and replaced with fresh DMEM. Cells were then incubated at 37 °C for 12 h followed by the preparation of cytosol.
Data analysis Data were expressed as means + S.E.M. Statistical significance was tested using Student's t-test for paired observations. Comparisons of multiple means were made with a two-way analysis of variance (ANOVA) followed by Duncan's multiple range test to compare individual means. Differences between groups were considered significant at P < 0.05.
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Fig. 1. A: saturation analyses of [3H]ALDO binding to cytosolic Type I receptors in 20- and 30-day-old astrocyte glial cultures. The binding of [3H]ALDO (ranging from 0.5 to 20 nM) to cytosolic Type I receptors was analyzed as detailed in Materials and Methods. The data are expressed as percentages of the specific binding of 20 nM [3H]ALDO. Values represent means + S.E.M. of 3 independent experiments. B: representative Scatchard analyses of [3H]ALDO binding to cytosolic Type I receptors in 20- and 30-day-old astrocyte glial cultures. Bsv/F is the ratio of specific macromolecule-bound [3H]ALDO to free [3H]ALDO. K d and Br, ax values were determined by linear regression with correlation coefficients ranging from 0.96 to 0.99. For these individual experiments, the K d values were 0.34 and 0.41 nM and the B,,ax values were 33.46 and 27.97 fmol/mg protein, respectively, for 20- and 30-day-old astrocyte glial cultures, maintained in 10% FBS/DMEM
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Fig. 2. A: saturation analyses of [3H]DEX binding to cytosolic Type II receptors in 20- and 30-day-old astrocyte glial cultures. The binding of [3H]DEX (ranging from 0.5 to 20 nM) to cytosolic Type II receptors was analyzed as detailed in Materials and Methods. The data are expressed as percentages of the specific binding of 20 nM [3H]DEX. Values represent means _+ S.E.M. of 3 independent experiments. B: representative Scatchard analyses of [3H]DEX binding to cytosolic Type II receptors in 20- and 30-day-old astrocyte glial cultures. BstJF is the ratio of specific macromolecule-bound [3H]DEX to free [3H]DEX. K d and Bmax values were determined by linear regression with correlation coefficients ranging from 0.96 to 0.98. For these individual experiments, the K a values were 2.20 and 1.33 nM, and the Bmax values were 211.07 and 263.10 fmol/mg protein, respectively, for 20- and 30-day-old astrocyte glial cultures maintained in 10% FBS/DMEM.
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Fig. 3. A: saturation analyses of [3H]ALDO binding to cytosolic Type I receptors in 20- and 30-day-old astrocyte glial cultures treated with serum-free medium. The binding Of [3H]ALDO (ranging from 0.5 to 20 nM) to cytosolic Type I receptors was analyzed as detailed in Materials and Methods. The data are expressed as percentages of the specific binding of 20 nM [3H]ALDO. Values represent means + S.E.M. of 3 independent experiments. B: representative Scatchard analyses of [3H]ALDO binding to cytosolic Type I receptors in 20- and 30-day-old astrocyte glial cultures treated with serum-free medium. Bse/F is the ratio of specific macromolecule-bound [3H]ALDO to free [3H]ALDO. Kd and B=,= values were determined by linear regression with correlation coefficients ranging from 0.96 to 0.99. For these individual experiments, the Kd values were 0.34 and 0.38 nM and the Bin,x values were 34.81 and 42.43 fmol/mg protein, respectively, for 20- and 30-day-old cultures incubated with serum-free medium.
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Fig. 4. A: saturation analyses of [3H]DEX binding to cytosolic Type II receptors in 20- and 30-day-old astrocyte glial cultures treated with serum-free medium. The binding of [3H]DEX (ranging from 0.5 to 20 nM) to cytosolic Type II receptors was analyzed as detailed in Materials and Methods. The data are expressed as percentages of the specific binding of 20 nM [3H]DEX. Values represent means + S.E.M. of 3 independent experiments. B: representative Scatchard analyses of [3H]DEX binding to cytosolic Type II receptors in astrueyte cultures treated with serum-free medium. Bsp/F is the ratio of specific macromolecule-bound [3H]DEX to free [3H]DEX. Kd and Bmax values were determined by finear regression with correlation coefficients ranging from 0.96 to 0.98. For these individual experiments, the K d values were 3.22 and 2.09 nM, and the Bm~ values were 199.39 and 250.46 fmol/mg protein, respectively, for 20- and 30-day-old cultures, incubated with serum-free medium.
TABLE I
[3H]ALDO-Type 1 receptor binding constants for cytosol prepared from 20- and 30-day-old astrocyte glial cultures Binding of [3H]ALDO to "laypeI receptors in cytosol prepared from astrcg3rte glial cultures was measured as described in Materials and Methods. Kd and B ~ values were calculated from Scat.chard analyses of the [3H]ALDO binding data. Each value represents the mean + S.E.M. of 3 independent experiments. In all binding assays, the correlation coefficients as applied to linear regression analyses of Scatchard plots ranged from 0.91 to 0.99. a Significantly different from 20-day-old cultures, b Significantlydifferent from 0% FBS.
Age
K d (nM)
B,,~ (fraolgmgprotein)
0% FBS 10% FBS 0% FBS 10% FBS 20-day-old 0.43+0.09 0.45-1-0.13 47.99+7.23 42.64+4,84 30-day-old 0.45_+0.09 0.53+0.07 33.37-+4.8 22.00_+3.18 ~,b
TABLE II
[3H]DEX-Type I1 receptor binding constants for cytosol prepared from 20- and 30-day-old astrocyte gird cultures Binding of [3H]DEX to Type II receptors in cytosoi prepared from astrocyte giial cultures was determined as des~bed ~ Materialsand Methods. Kd and Bea,x values were calculated from Scatchard analyses of the [3I-I]DEX binding data. Each value represents the mean + S.E.M. of 3 independent experiments. In all binding assays, the correlation coefficients as applied to linear regression analyses of Scatchard plots ranged from 0.95. to 1.00.
Age
rd (nM)
0% FBS 20-day-old 2.92+0.81 30-day-old 2.32+0.34
B ~ (fmoUmgp,ntein) 10% FBS 2.06+0.15 2.30+0.74
O% FBS t0% FBS 254.70+27.68 247.36_+18.16 241,09_+ 9.55 261.02_+ 3.08
59 cant difference was found in the apparent dissociation constants (Kd) for [3H]ALDO binding to Type I receptors between 20- and 30-day-old cultures (Table I). However, the number of binding sites (Bm~) found in 20-day-old cultures was nearly double the value observed in 30day-old cultures (Table I). Previous studies from this laboratory have demonstrated [3H]DEX binding to specific Type II receptors in astrocyte glia maintained in culture for 10 days after the final plating (i.e. 10-day-old) 7. In the present study, binding experiments with [3H]DEX concentrations ranging from 0.5 to 20 nM were performed in 20- and 30-day-old cultures. As shown in Fig. 2A, the binding of [3H]DEX to cytosolic Type II receptors was saturable in both 20- and 30-day-old cultures. The linearity of the Scatchard plots shown in Fig. 2B suggests that [3H]DEX bound to a single class of receptors in both 20- and 30-day-old cultures. The affinities of [3H]DEX for cytosolic Type II receptors, as determined by the K d values, were similar in 20- and 30-day-old cultures (Table II), and also were indistinguishable from the Ks value for [3H]DEX binding in 10-day-old astrocyte glial cultures 7. Although no significant difference was found in the number of [3H]DEX binding sites (Bm~x) between 20and 30-day-old cultures (Table II), these cells contained twice the number of binding sites found in 10-day-old cultures 7. It is known that the FBS used to supplement growth media contains low levels of steroids, and this may interfere with the accuracy of ligand binding assays27. In previous studies we have used a serum-depletion strategy to examine the possibility that the steroids present in serum compete with radioactive ligand for the binding sites in 10-day-old cultures 6"7. The results from these studies indicated that the serum-depletion treatment had no significant effect on the K d or Bm~x values for both Type I and Type II receptor binding. The present study also investigated the effect of serum-containing steroids on Type I and Type II receptor binding in 20- and 30-day-old astrocyte cultures by incubating the cells with serum-free medium for 12 h prior to cytosol preparation. As shown in Fig. 3A, [3H]ALDO binding to cytosolic Type I receptors in both 20- and 30-day-old cultures was saturable and maximal binding was obtained at 10 nM [3H]ALDO in both cases. Scatchard analyses of the specific [3H]ALDO binding data from the saturation experiments revealed linear plots for both 20- and 30-day-old cultures (Fig. 3B). Similar findings were also observed in the studies of [3H]DEX binding to Type II receptors (Fig. 4). The serum-depletion treatment had no significant effect on the Kd values of [3H]ALDO and [3H]DEX binding or on the Bmu values of [3H]DEX binding in 20- and 30-day-old astrocyte glial cultures
(Tables I and II). However, it was found that the serum-depletion treatment increased the number of Type I receptors in 30-day-old cultures to a level similar to that observed in 20-day-old cultures (Table I). DISCUSSION The binding characteristics of cytosolic Type I receptors for [3H]ALDO are very similar in 20- and 30-day-old astrocyte glial cultures(Table I). Moreover, there was no significant difference in the affinity of [3H]ALDO for Type I receptors between 20- and 30-day-old cultures (Table I). In fact, the Kd values obtained from 20- and 30-day-old cultures are quite comparable to those seen previously in 10-day-old cultures 6, suggesting that [3H]ALDO binds to Type I receptors with similar affinities in 10-, 20- and 30-day-old astrocyte glial cultures. Likewise, no significant differences were observed in the affinity of Type II receptors for [3H]DEX in the 20- or 30-day-old (Table II) or 10-day-old7 astrocyte glial cultures. We have previously demonstrated that for 10-day-old astrocyte glial cultures, treatment with serum-free medium did not change the Kd values of either Type I or Type II receptors for [3H]ALDO or [3H]DEX, respectively6"7. Similar findings were also observed in 20- and 30-day-old cultures treated with serum-free medium, and this eliminated the possibility that serum-containing steroids compete with the radioactive ligand for the cytosolic receptors in these cells (Tables I and II). In contrast to the Ks values, the numbers of Type I and Type II receptors in astrocyte glial cultures somewhat varied with the length of time in culture. The maximal binding capacity of Type I receptors in 20-day-old cultures is not significantly different from the values observed previously in 10-day-old cultures 6. However, the number of Type I receptors in 30-day-old cultures is nearly half that found in 20-day-old cultures (Table I). Interestingly, when the 30-day-old astrocyte glial cultures were treated with serum-free medium, the number of Type I receptors in these cultures was increased to a level that was not significantly different from that seen in 20-day-old cultures (Table I). It is possible that the astrocyte glia in 30-day-old cultures may degrade the serum components faster and produce more metabolites than those in 20-day-old cultures. In view of the known instability of Type I receptors 35, it is possible that the excess of metabolites may result in severe degradation of Type I receptors in 30-day-old cultures. However, removal of the metabolites may allow the ceils to synthesize new receptors during the 12-h incubation with serum-free medium. Another explanation for the reduced number of Type I receptors in 30-day-old cultures may be the
60 presence of low levels of steroids (e.g. cortisol) in the FBS, which pre-occupy the Type I receptors and reduce [3H]ALDO binding. Removal of the FBS and incubation with DMEM may result in increased [3H]ALDO binding. This explanation seems unlikely, however, because in the 20-day-old cultures there was no difference in the numbers of Type I receptors in the presence or absence of 10% FBS (Table I). The temporal pattern of Type II receptor expression in astrocyte glial cultures is different from that of Type I receptors. The number of Type II receptors in 20-day-old cultures is twice that seen in our previous studies in 10-day-old cultures 7. This may suggest that the 10-day-old astrocyte glia are able to synthesize more Type II, but not Type I, receptors over the next 10 days in culture. Nevertheless, the turnover rate of Type II receptors in these astrocyte glial cultures becomes steady at 20 days, since no significant difference is found in the number of receptors between 20- and 30-day-old cultures. The postnatal development of Type I and Type II receptors in the brain has been extensively studied by using ligand binding assays 8'21"31'39'43, in vivo autoradiography 22'41, and immunocytochemical staining techniques 4°. The data obtained from [3H]CORT binding experiments indicated that Type I receptors were undetectable in the cytosol until 8 days after birth, at which time an adult level was reached 39. Nevertheless, the in vivo autoradiographic studies showed specific [3H]CORT binding to Type I receptors at 2 days of age 41. The reason for this discrepancy is unclear, and further investigation is required. However, it was suggested that in 2-day-old rats the Type I receptors exist mainly in the nuclei and thus were not detected by the cytosolic receptor binding
assay 41. With regard to Type II receptors, numerous studies have shown that the levels of these receptors are low in the first week of life, and that this is followed by an increase toward the adult level s'21,31,39'4°,43. It was also found that the level of circulating CORT increases in a time course that parallels the increase in Type II receptors between postnatal days 3 and 3521. At the present time, it is still not known what factor(s) contribute(s) to the absence of autoregulation of Type II receptors in the neonates. Our previous studies and the present findings show that these receptors are expressed differently in astrocyte glial cells as a function of time in culture 6"7 (Tables I and II). While the level of Type I receptor binding is significantly reduced in 30-day-old cultures, the number of Type II receptors doubles after 20 days in culture and becomes steady at this point. Due to the absence of endogenous adrenocorticoids and a functional hypothalamic-pituitary-adrenal system in these cell cultures, it is likely that the temporal changes in the expression of these receptors that are observed in the present study may be determined by the intrinsic properties of the cultured astrocyte glia. It is not the purpose of the current study to correlate the temporal changes in the expression of Type I and Type II receptors in astrocyte glial cultures with their counterparts in vivo. Instead, the findings in the present study will provide useful information for further studies on the regulation of these receptors in the astrocyte glial cultures.
REFERENCES
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1 Agnati, L.E, Fuxe, K., Yu, Z.-Y., Harfstrand, A., Okret, S., Wikstrom, M., Goldstein, M., Zoll, W., Vale, W. and Gustafsson J.-A., Morphometrical analysis of the distribution of corticotrophin-releasing factor, glucocorticoid receptor and phenylethanolamine-N-methyltransferase-immunoreactive structures in the paraventricular hypothalamic nucleus of the rat, Neurosci. Lett., 54 (1985) 147-152. 2 Alexis, M.N., Stylianopoulou,E, Kitraki, E. and Sekeris, C.E., The distribution and properties of the glucocorticoid receptor from rat brain and pituitary, J. Biol. Chem., 258 (1983) 4710-4714. 3 Arriza, J.L., Simerly, R.B., Swanson, L.W. and Evans, R.M., The neuronal mineralocorticoid receptor as a mediator of glucocorticoid response, Neuron, 1 (1985) 887-900. 4 Beaumont, K., Rat C6 glioma cells contain Type I as well as Type II corticosteroidreceptors, Brain Res., 342 (1985) 252-258. 5 Bourgeois, S. and Newby, R.E, Correlation between glucocorticoid receptor and cytolytic response of murine lymphoid cell lines, Cancer Res., 39 (1979) 4749-4751. 6 Chou, Y.-C., Luttge, W.G. and Sumners, C., Characterization of mineraiocorticoid Type I receptors in astrocyte cultures from rat brain, Endocrine Soc. Abstr., 1440 (1989). 7 Chou, Y.-C., Luttge, W.G. and Sumners, C., Characterization
Acknowledgements. The authors wish to thank Tammy Gault for excellent technical assistance. We also thank Pia Jacobs and Kevin Fortin for help with the preparation of this manuscript. This work was supported by PHS Grants NS-19441, HL-36645, and NS-24404.
61 antibody against rat liver glucocorticoid receptor, Endocrinology, 117 (1985) 1803-1812. 14 Gehring, U., Mugele, K. and Ulrich, J., Cellular receptor levels and glucocorticoid responsiveness of lymphoma cells, Mol. Cell. Endocrinol., 36 (1984) 107-113. 15 Hansson, E., Astroglia from defined brain regions as studied with primary cultures, Prog. Neurobiol., 30 (1988) 369-397. 16 Juurlink, B.H., Schousboe, J.A., Jorgensen, O.S. and Hertz, L., Induction by hydrocortisone of glutamine synthetase in mouse primary astrocyte cultures, J. Neurochem., 36 (1981) 136-142. 17 Kimelberg, H.K., Glial Cell Receptors, Raven Press, New York, 1988. 18 Kumar, S. and De Vellis, J., Glucocorticoid-mediated functions in glial cells. In H.K. Kimelberg (Ed.), Glial Cell Receptors, Raven Press, New York, 1988, pp. 243-264. 19 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., Protein measurement with the Folin phenol reagent, J. Biol. Chem., 193 (1951) 265-275. 20 Luttge, W.G., Rupp, M.E. and Davda, M.M., Aldosteronestimulated down-regulation of both Type I and Type II adrenocorticosteroid receptors in mouse brain is mediated via Type I receptors, Endocrinology, 125 (1989) 817-824. 21 Meaney, M.J., Sapolsky, R.M. and McEwen, B.S., The development of the glucocorticoid receptor system in the rat limbic brain. I. Ontogeny and autoregulation, Dev. Brain Res., 18 (1985) 159-164. 22 Meaney, M.J., Sapolsky, R.M. and McEwen, B.S., The development of the glucocorticoid receptor system in the rat limbic brain, II. An autoradiographic study, Dev. Brain Res., 18 (1985) 165-168. 23 Meyer, J. and McEwen, B.S., Evidence for glucocorticoid target cells in the rat optic nerve: physicochemical characterization of cytosol binding sites, J. Neurochem., 39 (1982) 435-442. 24 Meyer, J., Leveille, P.J., De Vellis, J., Gerlach, L. and McEwen, B.S., Evidence for glucocorticoid target cells in the rat optic nerve: hormone binding and glycerophosphate dehydrogenase induction, J. Neurochem., 39 (1982) 423-434. 25 Miesfeld, R.L., Molecular genetics of corticosteroid action, Am. Rev. Respir. Dis., 141 (1990) Sll-S17. 26 Miesfeld, R., Rusconi, S., Godowski, P.J., Baler, B.M., Okret, S., Wikstrom, A.-C., Gustafsson, J.-A. and Yamamoto, K.R., Genetic complementation of a glucocorticoid receptor deficiency by expression of cloned receptor cDNA, Cell, 46 (1986) 389-399. 27 Mitchell, J.B., Rowe, W., Boksa, P. and Meaney, M.J., Serotonin regulates Type II corticosteroid receptor binding in hippocampal cell cultures, J. Neurosci., 10 (1990) 1745-1752. 28 Moguilewsky, M. and Raynaud, J.P., Evidence for a specific mineralocorticoid receptor in rat pituitary and brain, J. Steroid Biochem., 12 (1980) 309-314. 29 Munck, A., Mendel, D.B., Smith, L.I. and Orti, E., Glucocorticoid receptors and actions, Am. Rev. Respir. Dis., 141 (1990) $2-S10. 30 Okret, S., Poellinger, L., Do, Y. and Gustafsson, J.-A., Downregulation of glucocorticoid receptor mRNA by glucocorticoid hormones and recognition by the receptor of a specific
binding sequence within a receptor cDNA clone, Proc. Natl. Acad. Sci. U.S.A., 83 (1980) 5899-5903. 31 Olpe, H.R. and McEwen, B.S., Glucocorticoid binding to receptor-like protein in rat brain and pituitary: ontogenetic and experimentally induced changes, Brain Res., 105 (1976) 121128. 32 Patel, A.J. and Hunt, A., Observations on cell growth and regulation of glutamine synthetase by dexamethasone in primary cultures of forebrain and cerebellar astrocytes, Dev. Brain Res., 18 (1985) 175-184. 33 Patel, A.J., Hunt, A. and Tahourdin, S.M., Regulation of in vivo glutamine synthetase activity by glucocorticoids in the developing rat brain, Dev. Brain Res., 10 (1983) 83-91. 34 Pope, A., Neuroglia: quantitative aspects. In E. Schoffeniels, C. Franck, D.B. Towers and L. Hertz (Eds.), Dynamic Properties of Glial Cells, Pergamon Press, London, 1978, pp. 13-20. 35 Rafestin-Oblin, M.-E., Michaud, A., Claire, M. and Corvol, P., Dramatic protective effect of ligand against thermal degradation on mineralo- and glucocorticoid receptors of rat kidney, J. Steroid Biochem., 8 (1977) 19-23. 36 Raizada, M.K., Phillips, M.I., Crews, F.T. and Sumners, C., Distinct angiotensin II receptor in primary cultures of glial cells from rat brain, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 4655-4659. 37 Rees, H.D., Stumpf, W.E. and Sar, M., Autoradiographic studies with [3H]dexamethasone in the rat brain and pituitary. In W.E. Stumpf and L.D. Grand (Eds.), Anatomical Neuroendocrinology, Karger, Basel, 1975, pp. 262-269. 38 Richards, E.M., Sumners, C., Chou, Y.-C., Raizada, M.K. and Phillips, M.I., a2-Adrenergic receptors in neuronal and glial cultures: characterization and comparison, J. Neurochem., 53 (1989) 287-296. 39 Rosenfeld, P., Sutanto, W., Levine, S. and De Kloet, E.R., Ontogeny of Type I and Type II corticosteroid receptors in the rat hippocampus, Dev. Brain Res., 42 (1988) 113-118. 40 Rosenfeld, P., Van Eekelen, J.A.M., Levine, S. and De Kloet, E.R., Ontogeny of the Type 2 glucocorticoid receptor in discrete rat brain regions: an immunocytochemical study, Dev. Brain Res., 42 (1988) 119-127. 41 Rosenfeld, P., Sutanto, W., Levine, S. and De Kloet, E.R., Ontogeny of mineralocorticoid (Type I) receptors in brain and pituitary: an in vivo autoradiographical study, Dev. Brain Res., 52 (1990) 57-62. 42 Sapolsky R.M. and Meaney, M.J., Maturation of the adrenocortical stress response: neuroendocrine control mechanisms and the stress hyporesponsive period, Brain Res. Rev., 11 (1986) 65-76. 43 Sarrieau, A., Sharma, S. and Meaney, M.J., Postnatal development and environmental regulation of hippocampal glucocorticoid and mineralocorticoid receptors, Dev. Brain Res., 43 (1988) 158-162. 44 Scatchard, G., The attraction of proteins for small molecules and ions, Ann. N.Y. Acad. Sci., 51 (1949) 660-672. 45 Warembourg, M., Radioautographic study of the brain and pituitary after injection of [3H]dexamethasone, Cell Tissue Res., 161 (1975) 183-191.
55
BRESD 51284
Expression of mineralocorticoid Type I and glucocorticoid Type II receptors in astrocyte glia as a function of time in culture Yun-Chia
Chou 1, William G. Luttge 1 and Colin Sumners 1'2
Departments of 1Neuroscience and 2physiology, University of Florida College of Medicine, Gainesville, FL 32610 (U.S.A.) (Accepted 5 March 1991)
Key words: Brain cell culture; Astrocyte; Mineralocorticoid receptor; Glucocorticoid receptor; Glia
In the present study we have examined the expression of mineralocorticoid Type I and glucocorticoid Type II receptors in astrocyte glia maintained in culture for different periods of time. Cytosolic mineralocorticoid Type I receptors were labeled with [SH]aldosterone (ALDO) in the presence of a 500-fold molar excess of the potent Type II receptor ligand RU 28362. [3H]Dexamethasone (DEX) was used to label cytosolic Type II receptors. Both Type I and Type II receptor binding was saturable in astrocyte glia that had been maintained in culture for 20 and 30 days following final plating (i.e. 20- and 30-day-old cultures). Scatchard analysis of [aH]ALDO binding revealed a single class of Type I receptors, with dissociation constants (Kd) of 0.45 + 0.13 nM and 0.53 + 0.07 nM, respectively, in 20- and 30-day-old cultures. The number of Type I receptors in 30-day-old cultures was nearly half that found in 20-day-old cultures (22.06 vs 42.64 fmol/mg protein). Linear Scatchard plots were also obtained for [SH]DEX binding to cytosol prepared from 20- and 30-day-old cultures. There were no significant differences in the Ka or B ~ values for [3H]DEX binding in 20- or 30-day-old cultures, i.e. 2.06 + 0.15 nM and 247.36 + 18.16 fmol/mg protein for 20-day-old cells and 2.3 + 0.74 nM and 261.02 + 3.08 fmol/mg protein for 30-day-old ceils. These Bm~x values are more than double the Bmax value for [aH]DEX binding observed in our previous studies in 10-day-old astrocyte glial cultures. Switching cultured astrocyte glial from serum-supplemented to serum-free medium had no significant effects on the K d values of Type I or Type II receptors in all the cultures tested. However, treatment with serum-free medium increased the number of Type I receptors in 30-day-old cultures to a level similar to that found in 20-day-old cultures. Taken together, these binding data suggest that Type I and Type II receptors are expressed differently in astrocyte glia as a function of time in culture. INTRODUCTION It is now widely accepted that astrocyte glia are essential for n o r m a l brain function 15'17. T h e r e is extensive evidence that in addition to their K+-buffering capacity, astrocyte glia also interact with various neurotransmitters and n e u r o m o d u l a t o r s and m a y actively m o d u l a t e the process of neurotransmission in the brain is' 17. M a n y studies have shown that adrenocorticoids have p r o f o u n d influences on the growth, differentiation, morp h o l o g y and function of astrocyte glia is. F o r instance, it has b e e n d e m o n s t r a t e d that glucocorticoids increase the activity of glutamine synthetase in astrocyte glia via their stimulatory effect on the expression of the gene for this enzyme 16A8"32'33. In view of the fact that astrocyte glia constitute a p p r o x i m a t e l y one-third of the total cell volume of the c e r e b r a l cortex 34, it is very likely that these cells play a significant role in the adrenocorticoid effects o b s e r v e d in the brain. Much evidence has shown that the effects of adrenocorticoids on gene regulation are m e d i a t e d through a
stereospecific binding with their receptors 3'1°'25,29. Results from a u t o r a d i o g r a p h i c and ligand binding studies have d e m o n s t r a t e d that m i n e r a l o c o r t i c o i d Type I and glucocorticoid Type II r e c e p t o r s are p r e s e n t on glial cells in the central nervous system 1,12A3,23,24,37,45. N e v e r t h e less, due to the n u m b e r of different cell types p r e s e n t in the brain, in vivo studies of a d r e n o c o r t i c o i d actions in astrocyte glia have b e e n h a m p e r e d by n u m e r o u s interferences from o t h e r types of cells. W e are able to p r e p a r e p r i m a r y cell cultures that are enriched with approximately 98% astrocyte glia 38, and have used these cultures as a m o d e l system to study a d r e n o c o r t i c o i d actions in the astrocyte glia. Previous studies from this l a b o r a t o r y have d e m o n s t r a t e d the presence of b o t h Type I and Type II receptors in astrocyte glial cultures which have been m a i n t a i n e d in culture for 10 days 6'7. T h e results from ligand binding and i m m u n o c y t o c h e m i c a l staining experiments suggest that these astrocyte glial r e c e p t o r s display binding p r o p e r t i e s and structures similar to those found in the brain and o t h e r target organs 2"4'9"11'23"28. A c c o r d ingly, it is conceivable to use these cultures to investigate
Correspondence: C. Sumners, Department of Physiology, University of Florida College of Medicine, Box J-274, J.H.M. Health Science Center, GainesviUe, FL 32610-0274, U.S.A.
56 the p r o p e r t i e s and regulation o f adrenocorticoid receptors in the astrocyte glia without the complications seen in vivo and in t u m o r cell lines. It is known that the biological effects of adrenocorticoids are d e p e n d e n t on the concentration of their receptors present in target tissues 5'14'26. Therefore, it is o f particular i m p o r t a n c e to know how the receptors are r e g u l a t e d in the target cells. Much evidence has shown that adrenocorticoids are able to regulate their own receptors (i.e. autoregulation) in the brain and peripheral target tissues of adult rats 2°'3°. H o w e v e r , autoregulation of Type II receptors by corticosterone ( C O R T ) is lacking in the n e o n a t e brain 21'42. The reason for this inability of C O R T to regulate its own receptors in the neonate brain remains unclear at the present time. In view of the fact that o u r astrocyte glial cultures are p r e p a r e d from o n e - d a y - o l d rat brains, it is interesting to first examine changes in the expression of Type I and Type II receptors in these cells as a function of time in culture. The results should provide valuable information for subsequent research on the regulation of receptors in these cells. In the p r e s e n t study, we e x a m i n e d the specific binding of [3H]aldosterone ( A L D O ) and [3H]dexamethasone ( D E X ) to Type I and Type II receptors, respectively, in astrocyte glial cells m a i n t a i n e d in culture for different p e r i o d s of time (20 and 30 days) following final plating. It was found that the K d values for Type I and Type II r e c e p t o r s in these cells did not change significantly with time in culture. H o w e v e r , significant changes in the binding capacity of Type I receptors were observed in astrocyte glia cultured for 20 or 30 days. In addition, comparison of the present results with our previous studies shows that 20- and 30-day-old astrocytes contain m o r e than d o u b l e the n u m b e r of Type II receptors c o m p a r e d with 10-day-old astrocytes 7.
chemicals were of reagent grade quality and were obtained from Fisher Scientific.
Preparation of brain astrocyte glial cultures Astrocyte glial cultures were prepared from the brains of one-day-old rats as described previouslyTM. In brief, whole brains were dissected from one-day-old SD rats and placed in an isotonic salt solution containing 100 units of penicillin G, 100/~g streptomycin and 0.25/~g amphotericin B per ml, pH 7.4. After the removal of blood vessels and pia mater, brains were minced and the tissue chunks were incubated with 0.25% trypsin (w/v) at 37 °C for 5 min. After this time, 160/~g DNase I was added and the incubation continued for an additional 5 min. The dissociated cells were then suspended in 50 ml DMEM containing 10% FBS (10% FBS/ DMEM) pelleted by centrifugation at 1,000 g for 10 min, and then resuspended in 10% FBS/DMEM. Lastly, cells were plated in 100-mm diameter Falcon tissue culture dishes precoated with poly-L-lysine at a density of 18 x 106 cells/dish and incubated at 37 °C in a humidified incubator with 10% CO2/90% air. After 3 days in culture the medium was replaced with fresh 10% FBS/DMEM and cells were maintained at 37 °C for an additional 3 days. At this time cells were washed with DMEM, dissociated with 0.25% trypsin (3 ml/dish), resuspended in 10% FBS/DMEM and plated in 100-ram culture dishes at a density of 1 x 106 cells/dish. Cultures were grown for 20 or 30 days at 37 °C prior to use, at which time they consisted of >98% astrocyte glia, as evidenced by immunofluorescent staining with an antibody against glial fibrillary acidic protein (GFAP)3s. The 10% FBS/DMEM was replaced every 10 days.
Preparation of cytosol Cytosol was prepared as described previously7. In brief, astrocyte giia maintained in culture for 20 (i.e. 20-day-old) or 30 (i.e. 30-day-old) days following the final plating were removed from culture dishes and washed twice with ice-cold HGEM (consisting of 20 mM HEPES, 10% glycerol (w/v), 1 mM EDTA and 5 mM Na2MoO 4, pH 7.4 at 0-4 °C) for the [3H]ALDO binding experiments or with HGEMD buffer (consisting of 20 mM HEPES, 10% glycerol (w/v) 1 mM EDTA, 20 mM Na2MoO4, and 2 mM DTF, pH 7.4 at 0--4 °C) for the [3H]DEX binding experiments. Next, cells were homogenized in HGEM or HGEMD buffer with a Dounce homogenizer followed by centrifugation at 100,000 g for 60 min at 0 °C to generate the cytosol (i.e. supernatant) fraction. During these and all other procedures, the cytosol was kept at 0-2 °C to minimize degradation of cytosolic receptors. Final protein concentrations were determined by the method of Lowry 19, and were typically in the range of 4-5 mg/ml cytosol.
[3H]ALDO labeling of mineralocorticoid Type I receptors MATERIALS AND METHODS
Materials One-day-old Sprague-Dawley (SD) rats were obtained from our breeding stock, which originated from Charles River Farms (Wilmington, MA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS) and amphotericin B (Fungizone) were purchased from GIBCO (Grand Island, NY). Trypsin was obtained from Worthington Biochemicals (Freehold, NJ). Penicillin G and streptomycin were from Pfizer, Inc. (New York, NY). [1,2,6,7-3H]Aldosterone ([aH]ALDO) (82 Ci/mmol) and [6,7-3H]dexamethasone ([3H]DEX) were purchased from Dupont New England Nuclear (Boston, MA) and checked for purity by thin-layer silica gel chromatography using a chloroform-methanol (9:1, v/v) solvent system. RU 28362 (llb,17b-dihydroxy-6-methyl-17a-(1propionyl)androsta-l,4,6-trien-3-one) was a gift from Dr. J.P. Raynaud (Roussel-UCLAF, France). DNase I, poly-L-lysine, unlabeled ALDO ([1H]ALDO), dexamethasone (DEX), EDTA, glycerol, HEPES, Sephadex G-25 (fine), dithiothreitol (DTT) and sodium molybdate (Na2MoO4) were purchased from Sigma Chemical Co. (St. Louis, MO). Scinti-Verse II scintillation fluor was purchased from Fisher Scientific (Pittsburgh, PA). All other
Cytosol was incubated at 0 °C for 24 h with [3H]ALDO and a 500-fold molar excess of RU 28362 in the presence (i.e. non-specific binding, Br~s) or absence (i.e. total binding, B-r) of a 200-fold molar excess of unlabeled ALDO. Concentrations of [3H]ALDO ranging from 0.5 to 20 nM were used for saturation analyses of receptor binding. In all experiments, macromolecule-bound [3H]ALDO was separated from free steroids at 4 °C on Sephadex G-25 columns (0.6 x 14 cm) as described previously7. Specific binding (Bsp) for each sample was determined by subtracting non-specific from total binding (i.e. Bsp = BT-Br~s).
[3H]DEX labeling of glucocorticoid Type H receptors The binding assay for [3H]DEX labeling of cytosolic Type II receptors was similar to that described above for ~ I receptors except that cytosol was incubated with [3I-I]DEX in the presence (i.e. BNS) or absence (i.e. BT) of a 200-fold molar excess of unlabeled DEX. In a previous study, use of the specific Type II receptor ligand RU 28362 as the unlabeled displacer for [3H]DEX binding yielded Kd and Bm~ values that were similar to those found when DEX itself was included as the unlabeled displacer. Thus, DEX was included in the non-specific binding tubes in the current experiments. After a 24-h incubation, maeromolecule-bound ste-
57 roids were separated from free steroids at 4 °C on Sephadex G-25 columns (0.6 x 14 cm) as detailed previously 6.
RESULTS
Serum-depletion treatment of astrocyte glial cultures
As shown in Fig. 1A, the binding of [3H]ALDO to cytosolic Type I receptors in 20- and 30-day-old astrocyte glial cultures was saturable. The maximal binding was obtained at 10 nM [3H]ALDO for both 20- and 30day-old cultures. Scatchard analyses44 of the specific [3H]ALDO binding data from the saturation experiments revealed linear plots for both 20- and 30-day-old cultures (Fig. 1 B ) . Therefore, these results suggest that [3H]ALDO binds to a single class of binding sites or a population of binding sites with equal affinity. The linearity of these Scatchard plots also eliminated the possibility that [3H]ALDO was binding to Type II receptors under the current assay conditions. No signifi-
The effect of serum-containing steroids on Type I and Type II receptor binding was investigated in this study by incubating the astrocyte glial cultures with serum-free medium as detailed previously 6'7. In brief, 10% FBS/DMEM was removed from 20- and 30-day-old astrocyte glial cultures, and replaced with fresh DMEM. Cells were then incubated at 37 °C for 12 h followed by the preparation of cytosol.
Data analysis Data were expressed as means + S.E.M. Statistical significance was tested using Student's t-test for paired observations. Comparisons of multiple means were made with a two-way analysis of variance (ANOVA) followed by Duncan's multiple range test to compare individual means. Differences between groups were considered significant at P < 0.05.
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Fig. 1. A: saturation analyses of [3H]ALDO binding to cytosolic Type I receptors in 20- and 30-day-old astrocyte glial cultures. The binding of [3H]ALDO (ranging from 0.5 to 20 nM) to cytosolic Type I receptors was analyzed as detailed in Materials and Methods. The data are expressed as percentages of the specific binding of 20 nM [3H]ALDO. Values represent means + S.E.M. of 3 independent experiments. B: representative Scatchard analyses of [3H]ALDO binding to cytosolic Type I receptors in 20- and 30-day-old astrocyte glial cultures. Bsv/F is the ratio of specific macromolecule-bound [3H]ALDO to free [3H]ALDO. K d and Br, ax values were determined by linear regression with correlation coefficients ranging from 0.96 to 0.99. For these individual experiments, the K d values were 0.34 and 0.41 nM and the B,,ax values were 33.46 and 27.97 fmol/mg protein, respectively, for 20- and 30-day-old astrocyte glial cultures, maintained in 10% FBS/DMEM
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Fig. 2. A: saturation analyses of [3H]DEX binding to cytosolic Type II receptors in 20- and 30-day-old astrocyte glial cultures. The binding of [3H]DEX (ranging from 0.5 to 20 nM) to cytosolic Type II receptors was analyzed as detailed in Materials and Methods. The data are expressed as percentages of the specific binding of 20 nM [3H]DEX. Values represent means _+ S.E.M. of 3 independent experiments. B: representative Scatchard analyses of [3H]DEX binding to cytosolic Type II receptors in 20- and 30-day-old astrocyte glial cultures. BstJF is the ratio of specific macromolecule-bound [3H]DEX to free [3H]DEX. K d and Bmax values were determined by linear regression with correlation coefficients ranging from 0.96 to 0.98. For these individual experiments, the K a values were 2.20 and 1.33 nM, and the Bmax values were 211.07 and 263.10 fmol/mg protein, respectively, for 20- and 30-day-old astrocyte glial cultures maintained in 10% FBS/DMEM.
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0.140
0.160
Fig. 3. A: saturation analyses of [3H]ALDO binding to cytosolic Type I receptors in 20- and 30-day-old astrocyte glial cultures treated with serum-free medium. The binding Of [3H]ALDO (ranging from 0.5 to 20 nM) to cytosolic Type I receptors was analyzed as detailed in Materials and Methods. The data are expressed as percentages of the specific binding of 20 nM [3H]ALDO. Values represent means + S.E.M. of 3 independent experiments. B: representative Scatchard analyses of [3H]ALDO binding to cytosolic Type I receptors in 20- and 30-day-old astrocyte glial cultures treated with serum-free medium. Bse/F is the ratio of specific macromolecule-bound [3H]ALDO to free [3H]ALDO. Kd and B=,= values were determined by linear regression with correlation coefficients ranging from 0.96 to 0.99. For these individual experiments, the Kd values were 0.34 and 0.38 nM and the Bin,x values were 34.81 and 42.43 fmol/mg protein, respectively, for 20- and 30-day-old cultures incubated with serum-free medium.
0.000 0.000
0.200
0.400 B=p (nM)
0.600
0.800
Fig. 4. A: saturation analyses of [3H]DEX binding to cytosolic Type II receptors in 20- and 30-day-old astrocyte glial cultures treated with serum-free medium. The binding of [3H]DEX (ranging from 0.5 to 20 nM) to cytosolic Type II receptors was analyzed as detailed in Materials and Methods. The data are expressed as percentages of the specific binding of 20 nM [3H]DEX. Values represent means + S.E.M. of 3 independent experiments. B: representative Scatchard analyses of [3H]DEX binding to cytosolic Type II receptors in astrueyte cultures treated with serum-free medium. Bsp/F is the ratio of specific macromolecule-bound [3H]DEX to free [3H]DEX. Kd and Bmax values were determined by finear regression with correlation coefficients ranging from 0.96 to 0.98. For these individual experiments, the K d values were 3.22 and 2.09 nM, and the Bm~ values were 199.39 and 250.46 fmol/mg protein, respectively, for 20- and 30-day-old cultures, incubated with serum-free medium.
TABLE I
[3H]ALDO-Type 1 receptor binding constants for cytosol prepared from 20- and 30-day-old astrocyte glial cultures Binding of [3H]ALDO to "laypeI receptors in cytosol prepared from astrcg3rte glial cultures was measured as described in Materials and Methods. Kd and B ~ values were calculated from Scat.chard analyses of the [3H]ALDO binding data. Each value represents the mean + S.E.M. of 3 independent experiments. In all binding assays, the correlation coefficients as applied to linear regression analyses of Scatchard plots ranged from 0.91 to 0.99. a Significantly different from 20-day-old cultures, b Significantlydifferent from 0% FBS.
Age
K d (nM)
B,,~ (fraolgmgprotein)
0% FBS 10% FBS 0% FBS 10% FBS 20-day-old 0.43+0.09 0.45-1-0.13 47.99+7.23 42.64+4,84 30-day-old 0.45_+0.09 0.53+0.07 33.37-+4.8 22.00_+3.18 ~,b
TABLE II
[3H]DEX-Type I1 receptor binding constants for cytosol prepared from 20- and 30-day-old astrocyte gird cultures Binding of [3H]DEX to Type II receptors in cytosoi prepared from astrocyte giial cultures was determined as des~bed ~ Materialsand Methods. Kd and Bea,x values were calculated from Scatchard analyses of the [3I-I]DEX binding data. Each value represents the mean + S.E.M. of 3 independent experiments. In all binding assays, the correlation coefficients as applied to linear regression analyses of Scatchard plots ranged from 0.95. to 1.00.
Age
rd (nM)
0% FBS 20-day-old 2.92+0.81 30-day-old 2.32+0.34
B ~ (fmoUmgp,ntein) 10% FBS 2.06+0.15 2.30+0.74
O% FBS t0% FBS 254.70+27.68 247.36_+18.16 241,09_+ 9.55 261.02_+ 3.08
59 cant difference was found in the apparent dissociation constants (Kd) for [3H]ALDO binding to Type I receptors between 20- and 30-day-old cultures (Table I). However, the number of binding sites (Bm~) found in 20-day-old cultures was nearly double the value observed in 30day-old cultures (Table I). Previous studies from this laboratory have demonstrated [3H]DEX binding to specific Type II receptors in astrocyte glia maintained in culture for 10 days after the final plating (i.e. 10-day-old) 7. In the present study, binding experiments with [3H]DEX concentrations ranging from 0.5 to 20 nM were performed in 20- and 30-day-old cultures. As shown in Fig. 2A, the binding of [3H]DEX to cytosolic Type II receptors was saturable in both 20- and 30-day-old cultures. The linearity of the Scatchard plots shown in Fig. 2B suggests that [3H]DEX bound to a single class of receptors in both 20- and 30-day-old cultures. The affinities of [3H]DEX for cytosolic Type II receptors, as determined by the K d values, were similar in 20- and 30-day-old cultures (Table II), and also were indistinguishable from the Ks value for [3H]DEX binding in 10-day-old astrocyte glial cultures 7. Although no significant difference was found in the number of [3H]DEX binding sites (Bm~x) between 20and 30-day-old cultures (Table II), these cells contained twice the number of binding sites found in 10-day-old cultures 7. It is known that the FBS used to supplement growth media contains low levels of steroids, and this may interfere with the accuracy of ligand binding assays27. In previous studies we have used a serum-depletion strategy to examine the possibility that the steroids present in serum compete with radioactive ligand for the binding sites in 10-day-old cultures 6"7. The results from these studies indicated that the serum-depletion treatment had no significant effect on the K d or Bm~x values for both Type I and Type II receptor binding. The present study also investigated the effect of serum-containing steroids on Type I and Type II receptor binding in 20- and 30-day-old astrocyte cultures by incubating the cells with serum-free medium for 12 h prior to cytosol preparation. As shown in Fig. 3A, [3H]ALDO binding to cytosolic Type I receptors in both 20- and 30-day-old cultures was saturable and maximal binding was obtained at 10 nM [3H]ALDO in both cases. Scatchard analyses of the specific [3H]ALDO binding data from the saturation experiments revealed linear plots for both 20- and 30-day-old cultures (Fig. 3B). Similar findings were also observed in the studies of [3H]DEX binding to Type II receptors (Fig. 4). The serum-depletion treatment had no significant effect on the Kd values of [3H]ALDO and [3H]DEX binding or on the Bmu values of [3H]DEX binding in 20- and 30-day-old astrocyte glial cultures
(Tables I and II). However, it was found that the serum-depletion treatment increased the number of Type I receptors in 30-day-old cultures to a level similar to that observed in 20-day-old cultures (Table I). DISCUSSION The binding characteristics of cytosolic Type I receptors for [3H]ALDO are very similar in 20- and 30-day-old astrocyte glial cultures(Table I). Moreover, there was no significant difference in the affinity of [3H]ALDO for Type I receptors between 20- and 30-day-old cultures (Table I). In fact, the Kd values obtained from 20- and 30-day-old cultures are quite comparable to those seen previously in 10-day-old cultures 6, suggesting that [3H]ALDO binds to Type I receptors with similar affinities in 10-, 20- and 30-day-old astrocyte glial cultures. Likewise, no significant differences were observed in the affinity of Type II receptors for [3H]DEX in the 20- or 30-day-old (Table II) or 10-day-old7 astrocyte glial cultures. We have previously demonstrated that for 10-day-old astrocyte glial cultures, treatment with serum-free medium did not change the Kd values of either Type I or Type II receptors for [3H]ALDO or [3H]DEX, respectively6"7. Similar findings were also observed in 20- and 30-day-old cultures treated with serum-free medium, and this eliminated the possibility that serum-containing steroids compete with the radioactive ligand for the cytosolic receptors in these cells (Tables I and II). In contrast to the Ks values, the numbers of Type I and Type II receptors in astrocyte glial cultures somewhat varied with the length of time in culture. The maximal binding capacity of Type I receptors in 20-day-old cultures is not significantly different from the values observed previously in 10-day-old cultures 6. However, the number of Type I receptors in 30-day-old cultures is nearly half that found in 20-day-old cultures (Table I). Interestingly, when the 30-day-old astrocyte glial cultures were treated with serum-free medium, the number of Type I receptors in these cultures was increased to a level that was not significantly different from that seen in 20-day-old cultures (Table I). It is possible that the astrocyte glia in 30-day-old cultures may degrade the serum components faster and produce more metabolites than those in 20-day-old cultures. In view of the known instability of Type I receptors 35, it is possible that the excess of metabolites may result in severe degradation of Type I receptors in 30-day-old cultures. However, removal of the metabolites may allow the ceils to synthesize new receptors during the 12-h incubation with serum-free medium. Another explanation for the reduced number of Type I receptors in 30-day-old cultures may be the
60 presence of low levels of steroids (e.g. cortisol) in the FBS, which pre-occupy the Type I receptors and reduce [3H]ALDO binding. Removal of the FBS and incubation with DMEM may result in increased [3H]ALDO binding. This explanation seems unlikely, however, because in the 20-day-old cultures there was no difference in the numbers of Type I receptors in the presence or absence of 10% FBS (Table I). The temporal pattern of Type II receptor expression in astrocyte glial cultures is different from that of Type I receptors. The number of Type II receptors in 20-day-old cultures is twice that seen in our previous studies in 10-day-old cultures 7. This may suggest that the 10-day-old astrocyte glia are able to synthesize more Type II, but not Type I, receptors over the next 10 days in culture. Nevertheless, the turnover rate of Type II receptors in these astrocyte glial cultures becomes steady at 20 days, since no significant difference is found in the number of receptors between 20- and 30-day-old cultures. The postnatal development of Type I and Type II receptors in the brain has been extensively studied by using ligand binding assays 8'21"31'39'43, in vivo autoradiography 22'41, and immunocytochemical staining techniques 4°. The data obtained from [3H]CORT binding experiments indicated that Type I receptors were undetectable in the cytosol until 8 days after birth, at which time an adult level was reached 39. Nevertheless, the in vivo autoradiographic studies showed specific [3H]CORT binding to Type I receptors at 2 days of age 41. The reason for this discrepancy is unclear, and further investigation is required. However, it was suggested that in 2-day-old rats the Type I receptors exist mainly in the nuclei and thus were not detected by the cytosolic receptor binding
assay 41. With regard to Type II receptors, numerous studies have shown that the levels of these receptors are low in the first week of life, and that this is followed by an increase toward the adult level s'21,31,39'4°,43. It was also found that the level of circulating CORT increases in a time course that parallels the increase in Type II receptors between postnatal days 3 and 3521. At the present time, it is still not known what factor(s) contribute(s) to the absence of autoregulation of Type II receptors in the neonates. Our previous studies and the present findings show that these receptors are expressed differently in astrocyte glial cells as a function of time in culture 6"7 (Tables I and II). While the level of Type I receptor binding is significantly reduced in 30-day-old cultures, the number of Type II receptors doubles after 20 days in culture and becomes steady at this point. Due to the absence of endogenous adrenocorticoids and a functional hypothalamic-pituitary-adrenal system in these cell cultures, it is likely that the temporal changes in the expression of these receptors that are observed in the present study may be determined by the intrinsic properties of the cultured astrocyte glia. It is not the purpose of the current study to correlate the temporal changes in the expression of Type I and Type II receptors in astrocyte glial cultures with their counterparts in vivo. Instead, the findings in the present study will provide useful information for further studies on the regulation of these receptors in the astrocyte glial cultures.
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Acknowledgements. The authors wish to thank Tammy Gault for excellent technical assistance. We also thank Pia Jacobs and Kevin Fortin for help with the preparation of this manuscript. This work was supported by PHS Grants NS-19441, HL-36645, and NS-24404.
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