Aldosterone modulates glucocorticoid receptor binding in hippocampal cell cultures via the mineralocorticoid receptor

BRAIN RESEARCH ELSEVIER

Brain Research 636 (1994) 49-54

Research Report

Aldosterone modulates glucocorticoid receptor binding in hippocampal cell cultures via the mineralocorticoid receptor Dajan O'Donnell *, Michael J. Meaney Developmental Neuroendocrinology Laboratory, Douglas Hospital Research Center, Departments of Psychiatry and Neurology & Neurosurgery, McGill University, 6875 Boul. LaSalle, Montreal, Que. H4H 1R3, Canada

(Accepted 21 September 1993)

Abstract

The regulation of corticosteroid receptor expression in the rat brain by adrenal steroids remains controversial. The results of in vivo studies [Brinton and McEwen, 1988; Luttge et al., 1989] suggest that activation of type I receptors can modulate both mineralocorticoid (MR; type I) and glucocorticoid (GR; type II) receptor binding in selected brain regions. The present study utilized primary hippocampal cell cultures from rats sacrificed at E19-20 days of gestation to examine the effects of RU 28362, corticosterone (CORT) and aldosterone (ALDO) on GR binding ([3H]dexamethasone + RU 28362). Four days of exposure to 10 nM RU 28362, a highly selective GR agonist, resulted in a robust (~ 70%) decrease in GR binding. Similar exposure to 10 nM of either CORT or ALDO also produced a significant (50-55%) decrease in GR binding capacity. Scatchard analyses confirmed that the diminished GR binding capacity in response to ALDO was due to a decrease in total number of binding sites (Bma x for Control = 112 + 25 fmol/mg vs. ALDO = 48 + 12 fmol/mg) with no significant change in the affinity constant. The calculated ECs0 from the ALDO concentration response curve was 3.5 nM. Competition studies demonstrated that such low nM concentrations of ALDO were unable to displace specific [3H]dexamethasone + RU 28362 binding. Spironolactone, a highly specific MR antagonist, inhibited the ALDO-induced down-regulation of GR binding. These findings support the hypothesis that MR activation can modulate GR binding in hippocampal cells. Key words: Corticosterone receptor; Primary culture; Hippocampus; Glucocorticoid; Mineralocorticoid

I. Introduction

The existence of two distinct corticosteroid receptor systems in the rat brain is now well established [22,31,42]. Although mineralocorticoid (MR; type I) and glucocorticoid (GR; type II) receptors are differentially distributed in the brain [22,31,43], both receptors are highly expressed in the hippocampus. The two receptor types may be distinguished by their relative affinities for ligands in vitro. Hippocampal G R s bind the synthetic glucocorticoid agonist, R U 28362, with high affinity ( K d ~ 0.5-1 nM) [13,42] whereas the M R binds aldosterone ( A L D O ) with high affinity ( K d ~ 0.5-1.5 nM) [4,42]. Both receptors bind corticosterone (CORT), the principal endogenous glucocorticoid in the rat; however, the M R binds to C O R T with slightly

* Corresponding author. Fax: (1) (514) 766-9460. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(93)E1311-P

higher affinity ( K a ~ 0.5-1 nM) than does the G R (Ka ~ 2 - 5 nM) [42,55]. Intracellular G R concentration is a major factor in determining the extent of biological response in t~rget cells [7,8,24,29]. Corticosteroids are known to modulate the expression of their own receptors as evidenced by several in vitro studies demonstrating G R autoregulation in a wide variety of cell culture systems [5,12,15,32,51]. In vivo, circulating plasma steroid levels are potent modulators of corticosteroid receptor expression in the brain. Removal of endogenous steroid production by adrenalectomy produces an increase in G R levels in the hippocampus which is restored to control levels by administration of corticosteroids [45,46,52]. In contrast, elevated plasma C O R T levels, resulting from either chronic stress or exogenous administration, down-regulate corticosteroid receptors in a site-specific manner in the brain [44]. In these studies, C O R T decreases hippocampal G R levels~ however,

50

Dajan O'DonneU, Michael J. Meaney / Brain Research 636 (1994) 49-54

it is not known whether this action is mediated by mineralocorticoid or glucocorticoid receptors. Pioneering studies by McEwen and co-workers [10,30] demonstrated that very low doses of corticosterone, administered via Alzet minipumps, reduce both MRs and GRs in the hippocampus of the rat. Since the concentration of CORT used in these studies was not expected to bind to GRs, the authors postulated that MRs could modulate hippocampal GR numbers. Luttge and co-workers [28] confirmed and extended these findings by showing that systemic administration of ALDO to adrenalectomized-ovariectomized mice produced significant region-specific decreases in both MR and GR binding which could be blocked in most instances by co-administration with the MR antagonist, RU 26752. However, it is not clear whether the effect of ALDO is exerted directly on hippocampal cells. In the present study, we addressed this issue by examining the regulation of GR binding capacity by various steroids in primary cultures of dispersed hippocampal cells.

2. Materials and methods Z 1. Cell cultures Primary hippocampal cell cultures were prepared using a procedure based on the studies by Banker and Cowan [2] and previously described by Mitchell et al. [33]. Hippocampi were dissected from fetuses sacrificed at 19-20 days of gestation and cells mechanically dissociated by trituration. Cells were plated at a density of 1 × 106 cells/ml and maintained in medium consisting of Dulbecco's Modified Eagle Medium (Gibco BRL, Burlington, Ontario) supplemented with 10% fetal bovine serum, 10 m M HEPES, 5.2 m g / 1 0 0 ml gentomycin, 10,000 IU penicillin/100 ml, 10 m g / 1 0 0 ml streptomycin, with glucose and glutamine concentrations increased to 0.2% and 0.8%, respectively. The presence of trace levels of adrenal steroids in the serum ( ~ 50-100 pM) have previously been shown not to interfere with the corticosteroid receptor binding assays [33]. Three days after plating, medium was replaced with fresh medium containing 3 m g / 1 0 0 ml uridine and 1.5 m g / 1 0 0 ml flurodeoxyuridine (Sigma, St. Louis, MO) in order to prevent proliferation of glial cells and thus provide a predominantly neuronal culture ( ~ 80% neurons), established using immunocytohistochemistry for glial fibrillary acidic protein (GFAP) and neuron-specific enolase.

2.2. Treatment with steroids Cultures were exposed to various steroids starting on the third day after plating for a period of 4 days after which time cells were harvested for binding assays. A concentration response study was performed by incubating cultures for 4 days with increasing concentrations of A L D O (Sigma). In the antagonist experiments, 20 nM of either the highly specific M R (spironolactone; Sigma) or G R ( R U 38486; RousseI-UCLAF, France) antagonists were co-administered with 10 nM A L D O to the cultures. Steroids were dissolved in 100% ethanol and stored at 4°C as a 1 m M stock solution for up to one month. Serial dilutions were performed in medium. Control cultures were treated with the appropriate vehicle (0.001-0.003% ethanol).

2.3. Glucocorticoid receptor binding assay Two hours prior to harvesting, medium was replaced with control medium in order to remove steroids administered during treatment of the cultures and thus prevent any interference with the binding assay. Betito et al. [6] have previously established that this wash-out period permits the recovery of chromatin-bound receptors following steroid treatment in cultured chromaffin cells. Cultures were then rinsed twice with isotonic buffer and the cells scraped in T E G M D buffer (20 mM Tris, 1 m M EDTA, 10% glycerol, 10 m M molybdate, and 1 m M dithiothreitrol, pH 7.4). Samples were centrifuged at 2°C for 45 min at 105,000× g in a Beckman L 8 - 8 0 ultracentrifuge; supernatant was collected and stored at - 8 0 ° C . Receptor binding assays were performed according to a method previously described [33]. Soluble fractions were assayed in triplicate for G R binding capacity by single-point method using a saturating concentration (10 nM) of [3H]dexamethasone (87.8 C i / m m o l ; New England Nuclear, Boston, MA) _+200-fold excess unlabeled R U 28362 (RousseI-UCLAF). R U 28362 is a highly specific ligand for the G R with little or no affinity for MRs and thus its use ensures that the assay measures only G R sites [16,39,42]. A theoretical maximal number of binding sites (B ..... ) and dissociation constant ( K d) were derived from saturation experiments with the soluble fraction of cultured hippocampal cells according to the method of Scatchard [47]. Protein content was determined by the method of Bradford [9], and results were expressed as f m o l / m g protein. Since G R binding in control cultures varied from ~ 100-180 f m o l / m g protein over the course of this study, data were expressed as % control. Individual experiments used cultures (n = 4-10) from at least 2 separate preparations. Competition studies were performed by incubating cytosolic preparations from control hippocampal cultures with 10 nM [3H]dexamethasone _+200-fold R U 28362 alone or in the presence of increasing concentrations of A L D O . The results are expressed as the percentage of maximum specific G R binding in the absence of ALDO.

2.4. Statistical analysis All data are expressed as mean + S.E.M. and represent the resuits from at least 2 separate experiments with different cell preparations. The data were analyzed with one-way analysis of variance followed by the Student's t-test. Significance was set at P < 0.05.

3. Results

In the first set of experiments (Fig. 1) GR binding capacity was determined in cultured hippocampal neurons following 4-day treatment with a 10 nM concentration of various steroids. Exposure to the synthetic glucocorticoid agonist, RU 28362, as well as the naturally-occurring glucocorticoid, CORT, produced a significant (P < 0.05) reduction in GR binding capacity (~ 70% and 55%, respectively). The classical mineralocorticoid, ALDO, also elicited a decrease in GR binding capacity in roughly the same magnitude as CORT ( ~ 50% vs. 55%, respectively). Scatchard analyses (see Fig. 2) revealed that the ALDO-induced decrease in GR binding was due to a decrease in total number of binding sites and not a change in the affinity constant (Ka). The average of 3 separate experiments yielded a

Dajan O'Donnell, Michael J. Meaney / Brain Research 636 (1994) 49-54

51

I00

100'

z

z~ ~

50

25 t~ 0

CONT

RU28362

CORT

ALDO

25

i

-

!

20

TREATMENT

Fig. 1. Effects of various steroids on GR binding capacity in cultured hippocampal cells. Mean +_S.E.M. (% control) specific GR binding as measured by [3H]DEX+RU 28362 in controls (CONT) and hippocampal cell cultures exposed to a 10 nM concentration of either R U 28362, CORT, or A L D O for 4 days (n = 8). * P < 0.05; significant difference,

•

40

[ALDO]

|

i

60

80

•

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Fig. 3. ALDO concentration-response curve in cultured hippocampal neurons. Mean + S.E.M. (% control) specific GR binding (measured by [3H]DEX+RU 28362) in hippocampal cell cultures exposed to control conditions or 1, 2, 5, 10, 25, 50 or 100 nM ALDO for 4 days starting on the third day after plating. The calculated ECs0 is 3.5 nM (n = 6-8).

Bma x value for control cultures of 112 + 25 f m o l / m g

protein versus that of 48 + 12 f m o l / m g protein for ALDO-treated cultures. The K d for both conditions was not found to be statistically different ( K d for C O N T = 1.7 + 0.4 nM and for A L D O = 1.5 + 0.3 nM). The results from a concentration-response study of A L D O on G R density are illustrated in Fig. 3. The calculated ECs0 was 3.5 nM and suggested the involvement of mineralocorticoid receptors since the K d of the glucocorticoid receptor for A L D O is in the range of 20-25 nM [42,55]. In order to verify that A L D O concentrations in the low nanomolar range were indeed binding to the MR and not the GR, we examined the ability of A L D O to compete for specific [3H]DEX + RU 28362 binding sites in hippocampal cell ho-

mogenates from control cultures. As depicted by the competition curve in Fig. 4, only higher concentrations of A L D O (i.e. 50 and 100 nM) competed for specific G R binding sites. To confirm that A L D O modulated G R binding capacity in cultured hippocampal cells via the MR, cells were exposed to A L D O either in the absence or presence of MR or G R antagonists. Spironolactone, a highly specific MR antagonist [36,50], successfully blocked the ALDO-induced decline in G R binding (Fig. 5A). The potent antiglucocorticoid RU 38486 [34,35] was not expected to block the effect; however, surprisingly, on its own evoked a significant decrease in specific [3H]DEX _ RU 28362 binding (Fig. 5B). This

D CONTROL 0.6

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Fig. 2. Representative Scatchard plot of G R binding ([3H]DEX + R U 28362) in cultures from dispersed hippocampal cells. Cultures were exposed to control conditions or 10 nM A L D O for 4 days starting on the third day after plating. Scatchard analysis revealed a decrease in G R density, as observed by a change in the Bmax (CONT = 90 f m o l / m g vs. A L D O = 40 f m o l / m g ) , with no change in the the affinity constant ( K d = 1.4 nM for both C O N T and ALDO).

25

......

i 10 [ALDOI

..... 100 aM

Fig. 4. In vitro competition of ALDO for specific binding of [3H]DEX+ RU 28362. Cytosol preparations of control hippocampal cell cultures were assayed for GR binding capacity with either 0, 1, 2.5, 5, 10, 25, 50, or 100 nM ALDO. Only high concentrations of ALDO (50 and 100 nM) were able to significantly compete for specific GR binding sites.

Dajan O'Donnell, Michael J. Meaney / Brain Research 636 (1994) 49-54

52

A

100

Z x Z~

~et

75

50

25

0

CONT

ALDO

ALDO

+ SPIRO

SPIRO

CONT

ALDO

ALDO + RU486

RU486

100 z

r,

0

TREATMENT

Fig. 5. Effects of M R and G R antagonists on the A L D O - m e d i a t e d decrease in G R binding capacity in cultured hippocampal cells. Mean+_S.E.M. (% control) specific G R binding (as measured by [3H]DEX_+ R U 28362) in hippocampal cell cultures following 4 days of exposure to: (A) either control conditions, 10 nM A L D O , 10 nM A L D O in the presence of 20 nM spironolactone (SPIRO), or 20 nM SPIRO alone; (B) either control conditions, 10 nM A L D O , 10 nM A L D O in the presence of 20 nM R U 38486 ( R U 486), or 20 nM R U 486 alone (n = 8-10). * P < 0.05; significant difference.

observed decrease in GR binding capacity can be attributed to RU 38486's ability to prevent recycling of the receptor [41].

4. D i s c u s s i o n

In the present study, we have demonstrated that a 4-day treatment with either C O R T or RU 28362 decreased G R binding in primary cultures of hippocampal neurons. These findings are consistent with the potent glucocorticoid effects of R U 28362 and confirm the homologous down-regulation of GRs by glucocorticoids previously reported in a variety of other cell culture systems [5,12,15,32,51]. Moreover, these find-

ings indicate that glucocorticoids can act directly on hippocampal cells to regulate G R density and corroborate a recent study demonstrating a dose-dependent regulation of GRs by C O R T in a similar hippocampal cell culture system [53]. Original in vivo studies by McEwen's group [30] demonstrated that very low doses of CORT, administered to adrenalectomized rats, reduce both MRs and GRs in the rat hippocampus. The doses used in their studies reflected concentrations believed to occupy only MRs. However, given the close affinity of MR and G R for CORT, it was not known whether this effect was mediated by MR or GR. One approach to distinguish between MR- vs. GRmediated actions is to use ALDO, since MR binds A L D O with approximately a 25-fold greater affinity than does GR. Exposure of hippocampal cultures to A L D O resulted in a decrease in G R binding capacity equivalent to that seen after C O R T treatment. Scatchard analyses confirmed that the ALDO-induced decrease in G R binding was due to a decrease in total number of binding sites (nmax) with no change in the affinity constant (Kd). The calculated ECs0 from the A L D O concentration-response curve was in the low nanomolar range and is consistent with concentrations known to elicit an MR response [1]. Furthermore, we demonstrated in competition studies that such low nanomolar concentrations of A L D O were unable to compete for specific G R binding sites in homogenates of cultured hippocampal cells (Fig. 4) suggesting that the concentration of A L D O used in our experiments did not bind to GR. These findings agree with data from in vitro assays for hMR and hGR function which show that transcriptional activation of GR-regulated genes by A L D O is only observed at concentrations exceeding 20 nM whereas maximal response of MRregulated genes was achieved at ~ 1 nM A L D O [1]. Finally, the ALDO-induced decrease in G R binding capacity was fully blocked by co-treatment with spironolactone, a highly specific MR antagonist [36,50]. It is unlikely that the concentration of spironolactone used here might act via the G R since the ICs0 of spironolactone for MR is 200 times greater than that for G R [14,50] and this compound was shown to behave as an antiglucocorticoid only at concentrations exceeding 1000 nM [14]. Taken together, the data presented herein confirm earlier findings demonstrating selective in vivo effects of A L D O on G R levels in specific brain regions in the rat [10] and in the mouse [28] and strengthen the hypothesis that adrenal steroids can act via the MR to regulate G R binding in hippocampal cells. It is interesting to note that G R activation has been shown to increase MR levels in vivo and thus MR and G R action form a coordinate cycle of regulation maintaining an M R / G R balance (see [16]). Steroid receptors act as ligand-dependent transcription factors [3,20] and therefore a plausible mechanism

Dajan O'Donnell, Michael J. Meaney / Brain Research 636 (1994) 49-54

for the MR-mediated down-regulation of hippocampal GRs is at the genomic level. Rat brain hippocampal neurons have been shown to encode for and contain both the MR and GR [1,23,25,42]. Likewise, the presence of GR and MR binding has been established in primary hippocampal cells cultures prepared from fetal rat tissue [33,53]. Activated MRs have been demonstrated to regulate the transcription of GR responsive genes, presumably by binding to identical regulatory elements known as hormone response elements (HREs) [1,3,20]. Since the GR gene possesses an up-stream HRE [11,38], MR may act directly at the genomic level to inhibit transcriptional activation of GR. The functional implications of a mechanism whereby MRs regulate GR numbers in the hippocampus remain unclear. However, the findings of this paper are in general agreement with some authors' contention that mineralocorticoids play a role in opposing GR actions. As early as 1950, Hans Selye proposed the 'pendulum hypothesis' on antagonizing mineralocorticoid and glucocorticoid actions [49]. Subsequently, in view of the strong evidence implying the co-expression of both receptor types in neuronal cells, De Kloet postulated the 'corticosteroid receptor balance hypothesis' proposing that MR- and GR-mediated effects function oppositely to regulate the set-point for homeostatic control in individual neurons [16]. The idea that MR activation counters GR actions is supported by several behavioral and biochemical studies. Veldhuis and De Kloet [54] have reported that administration of ALDO 30 min. prior to CORT prevented the restoration of exploratory behavior by CORT in adrenalectomized animals. In another study, similar pretreatment with ALDO blocked the CORT-induced increase in serotonin turnover in the raphe-hippocampal system [17]. Finally, cellular excitability in the hippocampal CA1 neurons is modulated by coordinative, antagonistic MRand GR-mediated effects on electrical activity [26,27]. In a very convincing fashion, Jofils and De Kloet demonstrated that low doses (1 nM) of CORT or ALDO, operating via MR, decreased the spike frequency accommodation and afterhyperpolarization elicited by a depolarizing current pulse whereas higher concentrations of CORT ( ~ 30 nM) or selective glucocorticoid agonists produced an increase in accommodation and a m p l i t u d e of the afterhyperpolarization [26,27]. The acute nature of these studies most likely precludes a mechanism which involves down-regulation of GRs; however, the ALDO-mediated MR effects might antagonize second messenger systems [19,37] or immediate early gene products [18,21,40,48,56] with which the GR interacts. Our demonstration of an MR-induced down-regulation of GRs in hippocampal cells could suggest a more long-term method of countering GR actions which may come into play under conditions of chronic low glucocorticoid levels.

53

Acknowledgements. This research was supported by a research grant from the Medical Research Council of Canada (MRCC) to M.J.M., who holds an MRCC Scientist career award. D.O.D. is supported by a grant from ALCAN Canada Ltd. to the Aging Research Program of the Douglas Hospital Research Center.

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