Steroid induction of low-affinity glucocorticoid binding sites in rat liver microsomes

J..~ier~jdRjoehe~. Vol.34.Nos I-6,pp.97-105.1989

0022-4731:89 53.00i-O.00 Copyright

Printed in Great Britain. All rights raewed

;,c~1989 Pergamon

Press plc

STEROID INDUCTION OF LOW-AFFINITY GLUCOCORTIC~ID BINDING SITES IN RAT LIVER MICROSOMES R. CHIRINO, A. LOPEZ,D. NAVARRO,J. J. CABRERA,J. F. RIVEROand B. N. D~Az-CHICO* Department of Physiology, Colegio Universitario de Las Palmas. P.O. Box 550. Las Palmas de Gran Canaria, 35080 Spain Summary-Rat liver contains two glucocorticoid binding sites: the high-affinity or glucocorticoid receptor (GR) and the low-affinity glucocorticoid binding sites, or LAGS. The Kd of LAGS predicts that they can be half-saturated by plasma corticosteroids in some physiological circumstances and, therefore, that they can play relevant roles in the rat liver. i3Hfdexamethasone was used as a ligand in exchange assays, to study the relative abundance of GR and LAGS in cell fractions of rat liver. GR were found in the cytosol, but not in the purified nuclei, the mitochondria, or the microsomes. LAGS were found in all the particulate fractions, being more abundant in the smooth-surfaced microsomes, but they were not found in the cytosol. The LAGS of microsomes and purified nuclei showed the same Kd and also the same broad range of steroid competition with [‘Hldexamethasone (cortisol = progesterone > dexamethasone > corticosterone > R5020 > DHEA > testosterone = estradiol). LAGS were found in liver, placenta and kidney. but not in other GR-containing organs. This suggests that the LAGS could be involved in physiological functions related to the metabolism of steroid hormones. The liver microsome LAGS were undetectable at rat birth, and became present in the 25-day-old rat. The level of LAGS then increased progressively, reaching its maximum level in the 2-3-month-old rats (10 pmol/mg protein), and declining afterwards to reach the adulthood level (5 pmol/mg protein) in 6-month-oId rats. LAGS are mainly controiled by the corticoadrenal steroids. which is shown by their dramatic decrease after adrenalectomy, and especially after hypophysectomy. Many steroid hormones, like estradiol, testosterone, and corticosterone (but not progesterone) induce LAGS, estradiol being the most effective. A combination of T4 and corticosterone was more effective in inducing LAGS than when the two hormones were injected separately. It is possible to conclude that rat liver LAGS are mainly microsomal proteins, whose concentration is regulated by a multihormone system under pituitary control.

INTRODUCTION

that rat liver cells contain low-affinity glucocorticoid binding sites (LAGS) in the nuclei [7-i I]. In the early seventies it was thought that LAGS could be the nuclear form of GR [7, 81. However, Parchman et al. [9] found that LAGS are very rare in the nucleus of the liver cells before the rats are weaned. This clearly contrasts with the ontogeny of GR [12, 131, which is present in important amounts in the fetal liver cytosol and which does not suffer important variations during the postnatal stage. These data, together with a number of other properties of LAGS, namely their affinity for progesterone, higher than for dexamethasone, and also the fact that they do not bind triamcinolone acetonide [lo], disproved the hypothesis that nuclear LAGS could be the nuclear form of GR. Little has been published about rat liver LAGS since then, However, the Kd of LAGS for glucocorticoids predicts that they can be half saturated in some

In recent years much discussion has taken place about the precise location of the steroid receptors inside the cells [ 11.In adrenalectomized rat liver cells, as well as in cultured cells, it has been shown that the glucocorticoid receptor (GR) is located in the cytoplasm 1241. It is widely accepted that after glucocorticoid binding the GR is activated, attaining a greater affinity for DNA (for review see Ref. [5]). In fact, after hormone treatment, the GR was located in the nucleus, where it carries out its functions by modulating gene expression [24,6]. Besides the GR, several authors have reported Proceedings of the 9th International Symposium of the Journal of Steroid Biochemistry, Recent Advances in Steroid Biochemistry, Las Palmas, Canary Islands, Spain, 28.-31 May 1989. *To whom correspondence should be addressed. 97

R.

98

CHIRINO et

physiological circumstances [&IO]. It is therefore possible that they can play an unknown role in the intracellular action of the steroid hormones. In this article we describe the characterization of a type of LAGS which are present in the rat liver microsomes and have similar properties to the nuclear LAGS, although they are more abundant. We also suggest an approach to the hormone regulation of LAGS and to their possible physiological role.

EXPERIMENTAL

Animals

was resuspended and used for experimentation as a mitochondrial fraction. Nuclei were purified as previously described by other authors [15]. Briefly, the pellets of the 1500 g centrifugation were homogenized in TMMDS buffer, and a solution of 2.3 M sucrose was added until the whole suspension reached 1.6 M sucrose. This suspension was layered onto a cushion of 2.3 M sucrose prepared in TMMDS buffer and centrifuged at 124,OOOg for 30 min. The purified nuclear pellet which was obtained was resuspended in the same buffer and then used for experimentation. Measurement

Male and female Sprague-Dawley rats of different ages were used throughout these experiments. Both intact and hypophysectomized rats were purchased from Charles River (France). Adrenalectomy, orchiectomy, or both, were performed under ether anesthesia. The estrous cycle was staged by smear examination of the vaginal fluid under light microscopy. All the animals were killed by decapitation. Their livers were removed, washed in ice-cold saline and stored in liquid nitrogen until used for experimentation. Preparation of cytosol and microsome fractions All the steps were carried out at &4”C. Liver samples were homogenized in a teflon-glass PotterElvejhem homogenizer in TMMDS buffer (50mM Tris-HCI, 10 mM sodium molybdate, 5 mM magnesium chloride, 2 mM dithiothreitol and 0.25 M sucrose, pH 7.5). The homogenates were centrifuged at 17,000g for 15 min to separate the nuclear and mitochondrial fractions. The supernatant was centrifuged at 105,OOOg for 1 h to obtain the cytosol. The 105,OOOg pellet was resuspended in TMMDS buffer and centrifuged again at 105,OOOg. The supernatant was discarded, and the pellet resuspended and used as a microsomal suspension for binding assays. Rough and smooth surfaced microsomes were prepared as previously described by other authors [14]. Microsomal suspensions were layered over a gradient consisting of 3.5 ml of 1.35 M sucrose and 3.5 ml of 2 M sucrose, both in TMMDS buffer. Centrifugation at 90,OOOg for 3 h produced two bands: the upper band consisted principally of smooth microsomes, and the lower band of rough microsomes. The bands were collected separately mixed with TMMDS buffer and centrifuged at 105,OOOg for 1 h. The pellets were then resuspended in TMMDS buffer and used for exchange assays. Preparation

al.

of mitochondria and purified nuclei

Tissues were homogenized in TMMDS buffer and centrifuged at 15OOg. The supernatant was resuspended by homogenization in TMMDS buffer and centrifuged again at 17,OOOg for 15 min. The resultant pellet (crude mitochondria) was resuspended and centrifuged again at 17,000g. The precipitate

of microsomal LAGS

Microsomal LAGS were measured by incubating aliquots of microsomal suspensions with 2&l 50 nM of [‘Hldexamethasone (final concentration) with or without a 200-fold excess of unlabeled dexamethasone, overnight at o-4 C. Then a suspension of dextran-coated charcoal (DCC 0.08~.8%, final concentration) was added and the samples were shaken, incubated for 1Omin and centrifuged. The radioactivity of the supernatant was counted and the results were plotted as described by Scatchard [16]. Nuclear LAGS were measured by incubaitng ahquots of nuclear suspensions with [‘HIdexamethasone (20-150 nM, final concentration), with or without a 200-fold excess of unlabeled dexamethasone, overnight at &4”C. Then the nuclei were washed three times with buffer, and the remaining radioactivity was extracted with ethanol and counted. Cytosolic GR were measured by incubating aliquots of cytosol with 0.5550 nM of [‘Hldexamethasone (final concentration) with or without a 200-fold excess of unlabeled dexamethasone. The rest of the steps were carried out as for microsomal LAGS. Protein and DNA measurement Microsomal, nuclear and cytosolic proteins were measured as described by Lowry et al. [ 171. DNA was measured as described by Burton [ 181. Statistics Comparison between groups was made by means of the ANOVA and Scheffe tests, included in the SSPS computer program. RESULTS

Steroid binding capacity of cell ,jiactions

qf rat liver

Table 1 shows the [‘Hldexamethasone binding to cell fractions obtained as described above. Only the cytosol fractions contain high-affinity GR (&= 2.1 nM). All the other fractions contain saturable low-affinity binding sites, LAGS, which have approx. 50 times less affinity for [3H]dexamethasone than GR. The microsomal fractions, especially the smooth surfaced microsome fraction, were the richest

Table

I. [3H]Dexamethasone

binding

sites in subcellular

fractions

of

the rat liver [‘Hldexamethasone

Subcellular

binding

fraction

pmol/mg protein

Pmol/g tissue

Cytosol

0.29

34

Nuclei

I.311

Mitochondria

1.8

83

102

10.5

209

105

G

(nM) 2.1

2.8

II5

Microsomes: Crude Smooth

surfaced

Rough

I I.4

‘Nuclear

binding

94

9.3

surfaced

= 3 pmol/mg

99

in rat liver microsomes

LAGS

85 DNA

LAGS content. The presence of LAGS in the mitochondrial fraction is probably an artefact due to contamination with microsomes. Crude nuclei assayed for LAGS show a much higher amount of LAGS than purified nuclei, probably due also to contamination with microsomes (data not shown). in

LAGS showed no affinity for the potent glucocorticoid triamcinolone acetonide, which contrasts with the high affinity of the GR for that glucocorticoid. The affinity of LAGS for progesterone was higher than for the synthetic progestin R5020, which contrasts with the binding properties of the progesterone receptor. The exact Kd of LAGS for cortisol, progesterone and dexamethasone was calculated by incubating microsomal suspensions with these compounds, labeled with ‘H. Figure 2 summarizes the obtained results. The similarity of the Kd obtained for cortisol and progesterone (68 and 62 nM, respectively) is noteworthy. The Kd of LAGS for dexamethasone in this particular experiment was 115 nM, almost twice as high as for the other two hormones. The average Kd of LAGS for dexamethasone in the whole experiment was 99.5 nM (SE = 24). Association between LAGS and steroid hormones

Steroid hormone binding specificity of microsomal LAGS The specificity of steroid hormone binding to the LAGS was studied by incubating aliquots of microsomal suspensions with 50 nM [‘Hldexamethasone, and performing displacement reactions by adding a series of increasing concentrations (10 nM to 50 p M) of steroid hormones. Parallel experiments using cytosol instead of microsomal suspensions were performed to compare the binding specificities of GR and LAGS. The results are shown in Fig. 1. The GR are specific for glucocorticoids, with little affinity for other steroid hormones [Fig. l(A)]. LAGS are much less specific for any hormone group, showing their highest affinity for progesterone and cortisol [Fig. l(B)].

The interaction of LAGS with [‘Hldexamethasone was studied by incubating the hormone with microsomal suspensions during increasing periods of time, both at 04 and 25‘C. To compare GR and LAGS, parallel incubations were performed using cytosol instead of microsomal suspensions. The obtained results are shown in Fig. 3. At both studied temperatures [Fig. 3(A), WC; Fig. 3(B), 25”C], the [‘Hldexamethasone binding to GR was faster than to LAGS. The LAGS were more stable than the GR in the conditions used in this assay, remaining unaltered for 48 h, whereas the GR were stable for 24 h only. The association of microsomal LAGS to other steroid hormones was also investigated, the [3H]progesterone and [3H]cortisol being able to saturate LAGS in 4 h at WC (results not shown).

(A)

GR

0 Progesterone 0 Dexomethosone -

20-

20

x Estradiol

-

O-

0 Dexamethosone X Estrodiol

A Corticosterona

A Corticosterone

A Triamcinolone ocetonide

D -

6

7 -Log

6 [steroid

5

A Triamcinolone

I 8

4

1

acetonide

I 7 -Log

I

I

6

5

[steroid]

Fig. 1. Displacement of [3H]dexamethasone from LAGS and GR by various steroid hormones. Cytosol (A) or microsomal suspensions (B) from adult male rat liver were incubated with 50nM [3H]dexamethasone, alone (100% binding), or in the presence of increasing concentrations of competitors from 10 nM to 50 PM of final concentration. Binding assays were performed as described in Experimental section. sa

1461%

H

’ /F

4

R. CHIRINO ef al.

100

placenta and kidney. all of them also containing GR. However. LAGS are not detectable in other tissues which are rich in GR, such as the spleen. LAGS were not present in the uterus either. even after the rats were injected with estrogens to increase the concentrations of PR.

LAGS o Dexameth, n

Cortisol

Q Progester.

10

20 B

40

30

(pmol/ml.)

of [“Hlcortisol, [‘Hjprogesterone or Fig. 2. Binding [” Hldexamethasone to rat liver microsomes, Microsomal suspensions from adult male rat liver were incubated with increasing concentrations of the ligands, in a range of 1-I 50 nM. alone or with 200-fold excess of unlabeled ligand. Incubations were performed as described in Experimental section. and the results are shown in Scatchard plots. o-4

‘c

2466

I

25 “C

Many enzymes and proteins related to glucocorticoids undergo changes in their expression during the life of the rat. Therefore. a follow-up experiment was carried out to determine the c(~ncentration of LAGS in the rat liver at various stages from the fetus up to old age. Figure 4(A) shows the lifetime changes of both CR and LAGS in male rats. LAGS are not present in the rat liver during fetal life. whereas GR are quite abundant. During the first 3 weeks of life, GR have reached the same level as in the adult animals, but LAGS are indetectable. During the fourth week of lift the LAGS start to bc dctectahle, and in the following weeks the concentration of LAGS rises linearly until puberty. The LAGS remain stable for a few weeks. and then, during the fourth month of life, the LAGS decrease until reaching the characteristic level of adulthood. They then remain stable until old age. Female rats show a delayed expression of LAGS in comparison with male rats. the LAGS becoming detectable only after the fourth week of life [Fig. 4(B)]. The level of LAGS then rises abruptly until puberty. During the period of increase in the level of LAGS, the male rats showed a level significantly higher than the female rats of the same age. After puberty the female rats. in contrast to the males, displayed quite an erratic expression of LAGS, a few of them having very low levels and the rest showing a great disparity of concentration. In order to address this question. the influence of the sex cycle on the concentration of LAGS was investigated. The results are summarized in Fig. 4(C). In all phases of the sex cycle an average of 20% of

123456 Hrs

3. Association curves of [‘Hldexamethasone with microsomal LAGS and GR. Microsomal suspensions or cytosol from adult male rat liver were incubated with 50nM [3H]dexamethasone at O-4 or 25’C. The reaction was stopped at the indicated time by the addition of DCC and further centrifugation. Results are expressed as a percentage of the maximum binding which was 4 pmol/mg protein for LAGS and 278 fmol/mg protein for GR.

Table 2. Relative abundance

Fig.

Demonstration of LAGS in tissues other than liver The binding properties of the LAGS indicate that they could be related to both PR and GR. The obvious way to find out more about the relationship between these three steroid binding entities was to investigate their tissue expression. Table 2 shows that the LAGS are present only in three tissues: liver,

Oman Liver Kidney Pl~CenlZ Spleen lJ1erlls BWtl Small inlestine MUSCIC Prostate Tests Lung

01‘LAGS and GR m VXIOUS TPI organs I.AC;S

C;R

~fmol:me nrolrln)

(fmol’mr nrokin)

9513 473 471 IND IND IND IND IND IND IND IND

291

IO2 63

1h3 i 5
Tissue fracti~l~~~io1~ and ~H]de~meth~~s~n~ binding assays arc described in Experimental section. PlaCenlas uere obtained from “at-term” pregnant rus. The uterus were obtained from two 2-3-month-old female rats injected 24 h prevmusly %ith I mg!kg rstradiol. The other tissues were from :! -3.month-old male rats. Results are the mean of two separate experiments. IND = indetectable.

LAGS in rat liver microsomes

101

I -..-.-.' Ii

I

-2 t 2 10

Birth

II

I

I

2227

37

50

I, 2

Me 3

Birth

.DoysAMonths-Rat

-D,,ys

-Months-

we

6r

Rat we

(0

I E

Fig. 4. Ontogeny of the microsomal LAGS and of the GR. Intact male or female rats at the age indicated were used for experimentation. Fetuses were obtained from rats at the twentieth day of pregnancy. (A) Shows the evolution of the GR and LAGS levels in male rats during their life. (B) Shows a comparison of the LAGS ontogeny in male and female rats. (C) Shows the concentration of LAGS in the rat liver throughout the sex cycle. D = diestrous; P = proestrous; E = estrous.

the rats had very low levels of LAGS (less than 250 fmol/mg protein instead of a minimum of 4000 fmol/mg protein in all the other female rats). No significant differences were found between the three phases of the cycle. Adrenal influence on microsomal

LAGS

In order to investigate whether adrenal secretion has any influence on the microsomal LAGS, adrenalectomy was performed on adult male rats, and the animals were killed either 3 days or 1 week later. Sham-operated animals showed the same concentration of LAGS as intact animals, but the adrenalectomized rats showed a significant decrease in LAGS, with levels becoming as low as 50% of those of the intact animals [Fig. 5(A)]. In another set of experiments, the effects of both orchiectomy and adrenalectomy on LAGS were tested in adult rats. Figure 5(B) shows how orchiectomy alone does not significantly decrease the level of LAGS after a week. The effect of combined adrenalectomy and orchiectomy was not stronger than the effect of adrenalectomy alone. One week after adrenalectomy the level of LAGS was still high, which suggests that LAGS could be a long-lived protein. To test this possibility, male rats were injected with 2 mg cycloheximide/kg, every 8 h

for 24 h. Figure 6 shows that cycloheximide lowers the level of LAGS to 15% of the controls (P = O.OOl), whereas the level of GR is 85% of the control (NS). The half life of LAGS is, therefore, shorter than the half-life of the GR. To try to find out whether adrenal catecholamines have an affect on LAGS, an experiment was performed to block the adrenal influence on liver, by means of injecting a mixture of alpha and beta blocking agents, propranolol and phentolamine, in doses capable of completely blocking the action of catecholamines (1.25 and 7 mg/kg respectively, every 8 h). Figure 7 shows that neither GR nor LAGS are affected by the adrenergic blocking agents. The effect of adrenalectomy on LAGS must, therefore, be derived from the lack of corticosteroids, and not the adrenal catecholamines. Unfortunately, none of the corticosteroid substitute protocols so far tried was effective in terms of completely restoring the normal level of LAGS (results not shown).

Steroid rats

induction

of microsomal

LAGS

in immature

In order to test the possible hormone induction of LAGS, the effect of pharmacological doses of a set of

I02

R.

A)

&IRINO

et ai.

LAGS

GR

10

T

Days

Intact

after operation

SHAM OPRT

OX

ADX &ZX

Fig. 5. Effect of orchiectomy (OX), adrenalectomy (ADX) or combined OX and ADX on the concentration of microsomai LAGS. (A) Three-month-old male rats were adrenalectomzied and used for expe~mentation at the indicated times. (B) Three-month-old male rats were either orchi~tomi~d, or adrena~ectomized, or both, under ether anesthesia and used for ex~rimentation I week later. Animals of the same age were sham-operated and used as a control. Each point is the mean k SE of four separate determinations.

hormones was tested on 22-day-old rats, an age at which male rats show no detectable microsomal LAGS. The protocol of injection was as follows: 1 mg of hormone was injected subcutaneously every 12 h, for 4 days. The animals were killed 2 h after the last injection. Figure 8 shows the obtained effects of the assayed hormones on LAGS. The experiment shows that many steroid hormones are able to induce LAGS in this protocol. The most effective was estradiol, even in male rats. The difference between intact rats and vehicle injected rats is probably due to the stress induction of LAGS caused by repeated injection. It is interesting that the thyroid hormone T4 in combination with corticosterone was also effective in inducing LAGS. These data suggest that many hormones may be involved in the regulation of LAGS.

Efect of hypoph.v~ectonl?~on LAGS

In order to try to understand whether any hormones other than adrenal and sexual are involved in the regulation of LAGS, the classical physiological approach of measuring the level of LAGS in hypophysectomized adult male rat was taken. Figure 9 shows how the hypophysectomy of adult male rats provoked an increase in the level of GR, which is the expected result after glucocorticoid suppression. Figure 9 also shows the dramatic dependence of LAGS on the hypophyseal hormones: they became almost indetectable 2 weeks after hypophysectomy. Since some steroid hormone treatments were effective for the induction of LAGS in immature rats, the same approach was tried to induce LAGS in LAGS

GR GR

LAGS

300

t

75

controt

CHX

l-7 L_controt CHX

Fig. 6. Effect of cyciohexidmide on GR and microsomal LAGS. Intact male rats, 3 months old, were injected with 2 mg cycloheximide/kg, every 8 h, for 24 h. Control animals received the vehicle only. Each value is the mean + SE of four separate experiments.

Controt

-

~ KG

Fig. 7. Effect of alpha- and beta-blocking agents on GR and microsomal LAGS. Three-month-old male rats were injected with vehicle (control or I.25 mg propranolol plus 7 mg phentolamine/kg body weight (BLKG), every 8 h for 24 h. The results represent the mean of four separate determinations & SE.

LAGS

103

in rat liver microsomes

All these binding sites have the same high-affinities and specificities as the cytosolic receptor for their respective ligands, which contrasts with the data reported here about microsomal LAGS. The low-affinity nuclear glucocorticoid binding sites previously described, have the same Kd and binding

properties

as those

described

here

for micro-

LAGS [&lo]. The nuclear binding sites are in a very low concentration in newborn animals, increasing abruptly after weaning [9], as do the LAGS. The low-affinity nuclear binding sites are very much restricted to the nuclear envelope [lo], so it is possible to conclude that microsomal and nuclear LAGS are the same molecular entity. LAGS are probably mostly microsomal and, because of that, they are also found in the outer nuclear envelope. Since glucocorticoids and progesterone can be bound with high affinity by GR, CBG and PR, it is necessary to compare the properties of LAGS with these molecules. On the basis of the steroid hormone binding properties, the LAGS found in the microsomal fraction of rat liver seem to be a molecular entity separate from CBG, GR and PR. In contrast to CBG, the LAGS bind [3H]dexamethasone; in contrast to GR, LAGS do not bind triamcinolone acetonide at all; and, in contrast to PR, LAGS possess almost the same affinity for cortisol as for progesterone, and they have more affinity for progesterone than for R5020. In addition to the binding properties, there is further evidence which points to the fact that the LAGS are not related to the GR. The LAGS are not present in some tissues where the GR are abundant (Table 2). The LAGS display a totally different ontogeny in liver to the GR (Fig. 4). The LAGS decreased after adrenalectomy and, dramatically, after hypophysectomy, both of which increased the concentration of GR. Moreover, the LAGS have a somal

Fig. 8. Effect of diverse hormones on the induction of microsomal LAGS. Twenty-four-day-old male rats were injected intraperitoneally with one of the following hormones: progesterone, estradiol, testosterone, DHEA, corticosterone or T4. Other groups of animals received a combination of corticosterone + T4 or corticosterone + DHEA. The doses were 1 mg steroid/kg every 12 h, or 20 pg T4/kg every 24 h. The animals were killed 4 days later. Control animals received the vehicle only. Intact animals did not receive any treatment. Results represent the mean f SE of four separate experiments. LAGS

GR

_f zi h

-

10

400

-

6

300

-6k

200

-43

100

-2,

e a

b E 2

2

.g

c=l

L

Control

Hypox

Contmt

d E

Hypox

Fig. 9. Effect of hypophysectomy (HYPOX) on microsomal LAGS and on GR. Three-month-old male rats were hypophysectomized 2 weeks before they were used for experimentation. Control animals were sham-operated. Each value is the mean + SE of three separate experiments.

11

c

LAGS

T I-

adult-hypophysectomized rats. Figure 10 shows that the effect of steroid hormone treatment was quite similar in adult-hypophysectomized rats to that recorded in prepuber rats, estradiol being the most effective steroid for the induction of LAGS. DISCUSSION

LAGS were first described in rat liver nuclear fractions [S-lo]. In this article we describe the presence of a high concentration of LAGS in rat liver microsomes, and also their hormone regulation. This provides new insights, and gives rise to a discussion about the nature and function of LAGS. Microsomal binding sites have been described for estradiol [19,201, progesterone [2 1,221 and androgens [23] in their respective hormone-responsive tissues.

Fig. 10. Effect of steroid hormone treatment on the microsomal LAGS or hypophysectomized (HYPOX) rats. Threemonth-old male rats were hypophysectomized 2 weeks before they were used for experimentation. These rats were treated with E2, testosterone or corticosterone, at a dose of 1 mg/kg every 12 h for 4 days. Control hypophysectomized rats received the vehicle only. The results are the mean & SE of three separate experiments.

104

R. CHIRINO et al.

half-life shorter than GR, and also LAGS are far more stable than GR under the experimental conditions used. In addition to the different steroid hormone binding properties of LAGS and CBG, the presence of LAGS in kidney and placenta rules out the possibility of any relationship with CBG. The possibility of LAGS being related to PR is in contradiction to the data obtained on the tissue distribution of LAGS. Besides, PR is not expressed by liver cells [24]. The experiments conducted to find clues to the regulation of LAGS are still inconclusive, but they have given some new insights. The peculiar ontogeny of LAGS would indicate that they are related to sex maturation. However, immature male rats that were orchiectomized at 21 days, an age at which LAGS are still either very low or undetectable, showed the same amount of LAGS at adulthood as the intact animals (results not shown). In addition, orchiectomy did not affect the concentration of LAGS in mature animals. Testosterone was not very effective in the induction of LAGS in immature rats. These data indicate that testis are not decisive for either the ontogenesis of the LAGS or their expression in adults. Since estradiol was the most effective steroid for the induction of LAGS in the pharmacological treatment of prepuberal rats, it was expected that physiological situations that imply variations of circulating estrogens (i.e. pregnancy or the sex cycle) should show important changes in the level of LAGS. However, no significant changes in the level of LAGS was observed during the sex cycle, the estrous phase showing the higher level. Pregnant rats showed a level of LAGS comparable to the average level of LAGS during the sex cycle. It seems clear that the observed effects of pharmacological doses of estradiol are not reproducible under physiological conditions. Besides, the reproducible presence of a very low concentration of LAGS in some 20% of the adult female rats challenges any rational interpretation. Therefore, it is possible to conclude that neither testis nor ovaries, by themselves, play a determinant role in the regulation of LAGS. but they may still play an adjuvant role. The adrenals play a major role on the regulation of LAGS: I -week adrenalectomized adult rats showed a 50% decrease in the level of LAGS. However, none of the assayed trials of glucocorticoid administration completely restored the level of LAGS, and the injection of glucocorticoids did not provoke any change in the level of LAGS. Since alpha and beta blockers do not affect the level of LAGS, it seems to be clear that adrenal steroids must carry out the adrenal role in the regulation of LAGS. However, in prepuber animals, neither corticosterone nor DHEA were particularly

effective in the induction of LAGS. Therefore, from the described experiments it is not possible to conclude which one of the adrenal steroids is important in the regulation of LAGS. The thyroid hormone, T4, alone or in combination with corticosterone, was also effective in inducing LAGS in pharmacological doses applied to prepuber male rats. An adjuvant role of T4 on LAGS regulation cannot be ruled out. The most important evidence obtained on LAGS regulation was the spectacular decrease provoked by hypophysectomy. This experiment indicates that LAGS are completely under the regulation of pituitary hormones. These data allow us to speculate about which pituitary hormones are involved, but the experimental approaches in aiuo used so far cannot give an appopriate answer. Investigation using other experimental models is under way. It was previously suggested by other authors that nuclear low-affinity binding sites may act as a “steroid buffer” in the intracellular action of glucocorticoids, but no conclusive proof was reported [9, lo]. Our experiments to discover the possible physiological role of the microsomal LAGS are still in their early stages and, at present, any comment on this role is mere speculation. However, the expression of LAGS only in liver, kidney and placenta, all of which are capable of metabolizing steroid hormones, suggests that LAGS may be involved in processes related to steroid transformation. It has been reported that many liver steroid metabolizing enzymes are increased by phenobarbital [25]. Since LAGS are microsomal proteins, a preliminary question is whether phenobarbital is also able to induce the LAGS. Hence, a daily dose of 5 mg phenobarbital/rat was injected into immature animals for 5 days, a protocol capable of inducing microsomal enzymes [25], but the concentration of both GR and LAGS remained unchanged (results not shown). Castration has been shown to decrease the level of microsomal enzymes [25], but LAGS are not affected by this operation. In addition, hypophysectomy has been reported as capable of inducing some other steroid metabolizing enzymes [26], which contrasts with the profound suppression that it causes on LAGS. Taking into account all the available data, it is possible to conclude that: (1) the microsomal LAGS seem to be a molecular entity different from any other steroid binding sites; (2) the concentration of LAGS is regulated by the hypothesis, throughout various periferic hormones, mainly, but not only, by the corticosteroids; and (3) their possible physiological role is, at present, unknown.

Acknowledgements-We wish to thank Mr Antonio Lleb Mart4 for his technical assistance, and MS Susan Cranfield for her help in preparing the manuscript.

LAGS

in rat liver microsomes

REFERENCES

I. Clark C. R.: Intracellular localization of steroid receptors. In Inferaction qf .S/eroid Hormone Receptors with DN.4 (Edited by M. Sluyser). Ellis Horwood, Chichester (1985) pp. 7-56. 2. Fuxe K.. Wikstrom A.-C., Okret S., Agnatti L. F.. Harfstrand A.. Yu Z. Y.. Granholm L.. Zoli M.. Vale W. and GustaRson J. A.: Mapping of glucocorticoid receptor immunoreactive neurons in the rat tel- and dicncephalon using a monoclonal antibody against rat liver glucocorticoid receptor. Em/ocrirzolog~ 117 (1985) 1803~ 1811. 3. Antakly T. and Eisen H. J.: lmmunocytochemical localization of glucocorticoid receptor in target cells. Endocrinologja 115 (1984) 1984 -1989. 4. Picard D. and Yamamoto K.: Two signals mediate hormone-dependent nuclear localization of the glucocorticoid receptor. EMBO J. 6 (1987) 3333-3340. 5. Schmidt T. and Litwack G.: Activation of the glucocorticoid receptor complex. Ph~siol. Rec. 62 (1982) II31 1192. 6. Gustafsson J. A., Carlstedt-Duke J., Poellinger L.. Okret S.. Wikstrom A.-C., Bronnegard M., Glillner M., Dong Y., Fuxe K.. Cintra A.. Harfstrand A. and Agnati L.: Biochemistry, molecular biology and physiology of the glucocorticoid receptor. Endocr. Rec. 8 (1987) 185-234. 7. Kalimi M., Beato M. and Feigelson P.: Interaction of glucocorticoids with rat liver nuclei. I. Role of the cytosol proteins. Biochrmistr~~ 12 (1973) 336553371. 8. Beato M., Kalimi M., Beato W. and Feigelson P.: Interaction of glucocorticoids with rat liver nuclei. Effect of adrenalectomy and cortisol administration. E~r~k,c,rirlolog~, 94 ( 1974) 377-387. 9. Parchman L. G.. Cake M. H. and Litwack G.: Functionality of the liver glucocorticoid receptor during the life cycle and development of a low affinity mcmbrdnc binding site. Me&. Ageing De?. 7 (1978) 227 240. 10. Kaufmann S. H. and Shaper J. H.: Binding of dexamethasone to rat liver nuclei in viro and in vitro. Evidence for two distinct binding sites. J. steroid Biochem. 20 (1984) 699 708. II. Giannopoulos G.: Binding of glucocorticoids to liver nuclei and chromatin of fetal, immature and adult rats. Steroi& 29 (1977) 309-329. 12. Giannopoulos G.: Ontogeny of glucocorticoid receptors in rat livjcr. J. &I/. C/rem. 250 (1975) 584775851. 13. Feldman D.: Ontogeny of rat hepatic glucocorticoid receptors. Endocrinologj~ 95 (1974) 1219. 1227.

105

R. B. and Rabin B. R.: The 14. Blyth C. A., Freedman effects of aflatoxin Bl on the sex-specific binding of steroid hormones to microsomal membranes of rat liver. Eur. J. Biochem. 20 (1971) 580-586. 15. Lawson G. M., Tsai M. J., Tsai S. Y., Mingheti P. P., McClure M. E. and O’Malley B. W.: Nuclei and chromatin. Isolation, characterization and structure. In Laboratory Methods Manual for Hormone Action and Molecular Endocrinology (Edited by W. T. Schrader and B. W. O’Malley). Houston Biological Association Texas (1986). Vol. 7, pp. l-52. G.: The attraction of proteins for small 16. Scatchard molecules and ions. Ann. N.Y. Acad. Sci. 51 (1949) 660-672. 17 Lowry 0. H., Rosebrough N. J., Farr A. I. and Randall R. J.: Protein measurement with the folin-phenol reagent. J. biol. Chem. 193 (1951) 265-275. and mechanisms I8 Burton K.: A study of the conditions of the diphenilamine reaction for the calorimetric stimulation of deoxyribonucleic acid. Biochem J. 62 (1956) 315-323. W. and Neame P.: Identification 19 Parikh I., Anderson of high affinity estrogen binding sites in calf uterine microsome membranes. J. biol. Chem. 255 (1980) 10,26610,270. 20. Muldoon T. G., Warson G. H., Evans C. and Steinsapir J.: Microsomal receptors for steroid hormones: functional implications for nuclear activity. J. s/eroid Biochem. 30 (1988) 23-31. M., Nienala A. and Tuohimaa P.: 21 Haukkamaa Progesterone-binding properties of the microsomal fraction from chick oviduct. Molec. Endocr. 19 (1980) 123-130. M. and Laukkainen T.: Progesterone 22 Haukkamaa binding properties of microsomes from pregnant rat uterus. J. steroid Biochem. 6 (1975) 134-141. 23 Steinsapir J., Evans A. C., Bryhan M. and Muldoon T. G.: Androgen receptor dynamics in the rat ventral prostate. Biochim. biophys. Acta 842 (1985) I-1 I. G., Klock G., Stewart F. and 24 Strahle U., Boshart Schulz G.: Glucocorticoidand progesterone-specific effects are determined by differential expression of the responsive hormone receptors. Nature 339 (1989) 629-632. 25 Berg A. and Gustafsson J. A.: Regulation of hydroxylation of 5cc-androstane-3a,l7B_diol in liver microsomes from male and female rats. J. biol. Chem. 248 (1973) 6559-6567. 26 Gustafsson J. A. and Stenberg A.: Masculinization of rat liver enzymes activities following hypophysectomy. Endocrinology 95 (1974) 891-896.