- Email: [email protected]
Effect of endogenous corticosterone on the determination of dexamethasone receptor levels in rat liver cytosol
51
EFFECT OF ENDOGENOUS CORTICOSTERONE ON THE DETERMINATION OF DEXAMETHASONE RECEPTOR LEVELS IN RAT LIVER CYTOSOL George Giannopoulos Department of Experimental Medicine, McGill University and the University clinic, Royal Victoria Hospital, Montreal, Canada H3A 1Al Received: :/X:/7’; ABSTRACT The effect of endogenous corticosterone on the quantitative measurement of dexamethasone receptors in liver cytosols from developLiver cytosols from adrenalectomized rats ing rats has been studied. were preincubated with increasing concentrations of nonlabeled corticosterone and the levels of detectable dexamethasone receptors were subsequently determined either directly or after removal of W-Ibound corticosterone. Corticosterone concentrations of 50 nM or lower had no significant effect on the specific binding of labeled dexamethasone. Higher concentrations of corticosterone resulted in underestimation of dexamethasone receptor levels. The mean levels of endogenous corticosterone in liver cytosols from 19.5- to 21.5- day fetuses, 22-day fetuses, 6-day-old immature rats and adult rats were 27.40, 11.91, 0.81 and 4.05 nM, respectively. It is concluded that variations in the levels of circulating corticosterone in the rat under normal physiological conditions have no significant effect on the quantitative measurement of total (occupied and unoccupied) receptor sites for dexamethasone in liver cytosol. This is supported by the finding that prior treatment of liver cytosols, from rats at different stages of development, with charcoal to remove unbound steroids has no effect on the amount of detectable dexamethasone receptors. INTRODUCTION In a previous communication cytoplasmic dexamethasone a charcoal assay
(1) we reported that the levels of
(2) receptors in rat liver, detectable by
(3), rise markedly above fetal levels shortly after
birth followed by a moderate decline at the adult stage. levels of circulating corticosterone reverse pattern
Since the
in the developing rat follow a
(4,5), it is possible that the observed variations
in the concentration
of hepatic cytoplasmic receptors are not real
but reflect limitations of the charcoal assay, detecting only unoccupied binding sites, and/or changes in the distribution of the receptors between cytoplasmic and nuclear compartments brought about by the association of endogenous corticosterone with cytoplasmic receptors
(6).
In the present studies we have attempted to determine
VoZwne 28, !?w&er
ii
S
TEIROIDS
JuZy,
2976
the effect of endogenous corticosterone on the detection of the total (occupied and unoccupied) receptor sites for dexamethasone cytosols from rats at different stages of development.
in liver
The effect of
endogenous hormone on the nuclear localization of the receptors will be reported in a separate communication. EXPERIMENTAL Materials: 1,2-3H-Dexamethasone (29 Ci/mmol) , 1,2-3H-cortisol (52 Ci/mmol), 1,2-3H-corticosterone (53 Ci/mmol) and Aguasol scintillation fluid were obtained from New England Nuclear Corporation. Dexamethasone was a gift from the Upjohn Co. Corticosterone was purchased from Ikapharm, bovine serum albumin Fraction V from Sigma, Sephadex LH-20 and Dextran T-70 from Pharmacia Fine Chemicals, and Norit A neutral charcoal from Fisher Scientific Co. All glass redistilled cyclohexane, benzene, methanol and dichloromethane were obtained from A & C American Chemicals, Montreal. Pregnant and nonpregnant rats (Sprague-Dawley) were purchased from the Canadian Breeding Farm and Laboratories Ltd. Adrenalectomized rats were obtained from the supplier and maintained on Purina Chow and 0.9% NaCl solution ad libitum for 1 week prior to the beginning of the experiments. Preparation of cytosol: Rats were killed by decapitation and, when pregnant, the fetuses were delivered by laparotomy and decapitated. The livers were excised, minced, and washed three times with 0.01 M Tris-HCl buffer, pH 7.4, containing 0.25 M sucrose and 0.003 M MgC12 to remove excess blood. The minced livers were blotted, weighed and homogenized in batches of 1 g in 1 ml of 0.01 M TrisHCl buffer, pH 7.4, containing 0.0015 M Na2EDTA (Tris/EDTA buffer) at a temperature close to OOC. The homogenate was centrifuged for 30 min at 224,000 x g at 2oC to obtain the cytoplasmic supernatant fraction (cytosol). The cytosol was adjusted to the desired protein concentration by dilution with Tris/EDTA buffer. 3 to liver Measurement of specific binding of H-dexamethasone ---3H-dexamethasone alone or in comcytosol: Appropriate aliquots of bination with a lOOO-fold amount of nonlabeled dexamethasone, dissolved in redistilled ehtanol, were pipetted into tubes and the ethanol was evaporated under nitrogen. Aliquots of cytosol were then added, mixed in a vortex, and the mixtures were incubated. Incubation temperatures, duration of incubations, concentrations of hormones employed, and special treatment of cytosols before or after incubation will be described in individual experiments. Specifically bound 3H-dexamethasone was measured by the charcoal assay described earlier (3). The apparent dissociation constant (Kd) of the binding reaction and the concentration of specific binding sites for dexamethasone (n) were estimated by plotting the data according to Scatchard (7). In certain cases the concentration
of receptor sites was measured by incubating the cytosol with a single, saturating, concentration of 3H-dexasnethasone for time periods required to obtain optimal binding (single point analysis). Determination of endogenous corticosterone in liver cytosol by radioimmunoassay: About 1000 cpm of 3H-corticosterone dissolved in 0.1 ml water was added to 0.5 - 1.9 ml of cytosol to monitor procedural losses, All samples were diluted to a final volume of 2 ml with water. The mixture was extracted with 10 ml dichloromethane, centrifuged at 800 x g for 10 min, and the dichloromethane extract was evaporated to dryness under a stream of nitrogen, Corticosterone in the dichloromethane extract was purified and assayed according to the procedures recommended by New England Nuclear Corporation, manufacturer of the rabbit antiserum against cortisol-21-succinyl-bovine serum albumin, fox the determination of cortisol. Standard curves were prepared using 3H-cortisol (3000-3500 cpm) as the labeled antigen and corticosterone (O-2.5 ng) as the nonlabeled antigen. The cross-reactivity of corticosterone with the antiserum, furnished by the manufacturer and confirmed in our laboratory, was 24%. Other steroids cross-reacting with the antiserum axe cortisone (4%), Il.-deoxycortisol (4%), androstenedione (4%), progesterone (2%), deoxycorticosterone (2%), testosterone (1%) and aldosterone (1%). Most of these steroids were either completely or predominantly eliminated by chromatography on a Sephadex LH-20 microcolumn (0.85 g) using the solvent system cyclohexane:benzene:methanol (60:40:10, This v/v) * Cortisol was also well separated from corticosterone. chromatographic step, however, did not eliminate ll-deoxycortisol which co-eluted with corticosterone. Thus the corticosterone values obtained in the cytosol preparations may be clightly overestimated. The recovery of 3H-corticosterone adaed to cytosol and carried through the extraction and chromatography steps ranged between 48.0 and 82.0% with a mean of 61.0 + (S-E.) 1.1 (n = 156). An aliguot of the corticosterone fraction elutad from the column was removed for counting and the remaining was used for assay. For most s:~mples, two different amounts of the eluted corticosterone fraction were assayed In order to test the parallelism between various levels in duplicate. of endogenous and authentic hormone, increasing amounts of cytosol from a homogeneous cytosol pool were assayed. For each amount 3 replicate measurements were carried out and the results, expressed as logits, were compare: with the logits resulting from the assay of 3 increasing amounts (in triplicate) of authentic corticosterone. A complete analysis of variance (8) indicated that the relationship between increasing amount F of cytosol and authentic hormone was parallel and linear in all insV?nces. The presence of blank was estimated by processing water (2 ml) in the same manrcr as the cytosol samples. The cpm resulting from the radioimmunoassay of the blank samples were compared with the cpm of those tubes of the standard curve which did not contain any nonlabeled steroid. The difference between means of both series of cpm was evaluated by the t-test. No significant difference could be detected at the 95% probability level. Thus the presence of blank in the assay was negligible, if any.
S
TlOROIDS
The sensitivity of the method, defined as the mass equal to twice the standard deviation of the zero binding, was approximately 25 pg. The accuracy of the assay was assessed by the addition of 5,10, 20 and 40 ng of corticosterone to 2 ml cytosol. After correction for recovery and endogenous corticosterone, the mean values C S.E. (n - 5) were 5.20 + 0.34, 9.30 t- 0.95, 20.16 C 2.15 and 38.37 -t 5.50 ng, respectively. The reproducibility of the method was evaluated in studies of the intra-assay and interassay variations carried out on a homogeneous cytosol pool. The intra-assay coefficient of variation in replicate assays (n = 8) of this pool was 12.0%. The interassay coefficient of variation in assays of the same cytosol pool performed on 5 occasions was 16.6%. Measurement of radioactivity: Radioactivity was measured in a Packard Tri-Carb model 3002 spectrometer. In studies of 3H-dexamethasone binding, aqueous samples (0.5 ml) were mixed with 10 ml Omnifluor in toluene and counted after a pre-equilibration period of at least 2 hrs at 45% efficiency for 3H. In the corticosterone radioinununoassay, aqueous samples (1.1 ml) were counted in 10 ml Aquasol at 25% efficiency for 3H.
&.
Protein assay: Protein was determined by the method of Lowry -et (9) using bovine serum albumin as standard. RESULTS AND DISCUSSION Since the binding of steroid hormones to their intracellular
receptors is a reversible reaction, it should be theoretically
feasible
to detect occupied receptor sites by exchange of the receptor-bound endogenous steroid with labeled hormone. the rate of dissociation
Because in some instances
of stc: i-7 from its receptor is very slow at
O°C, the use of higher temperatures may be necessary for complete equilibration of endogenous and labeled steroid.
Most steroid hormone
receptors, however, have a tendency to be degraded or inactivated upon exposure to higher temperatures or even at O°C for prolonged periods of time.
Thus experimental conditions of temperature and length of
incubation must be carefully standardized to ensure equilibration of endogenous steroid with the labeled hormone in the absence of inactivation of occupied or unoccupied receptor sites. criteria are met, the total number
Once these
(occupied and unoccupied) receptor
S
TDROIDS
sites can be quantitatively measured by incubating aliquots of cytosol with increasing concentrations according to Scatchard
(7).
of labeled steroid and plotting the data
Such quantitative assays have already been
developed for the measurement of receptor sites for estradiol-175 and progesterone
(10)
(11) in the uterus.
To test the stability of
H-dexamethasone-receptor
complexes,
liver cytosol was first incubated for 2 hr at O°C with a saturating concentration
(50 nM) of
at 15OC or 25OC.
3 H-dexamethasone
followed by further incubation
After various time intervals of exposure to 15OC
or 25OC, aliquots of the cytosol were transferred to O°C and assayed after 1 hr for specifically bound The 'H-dexamethasone-receptor
3 H-dexamethasone by the charcoal assay.
complexes appear to undergo rapid degrad-
ation at 25OC and slower but significant degradation at 15OC (Fig.1).
I
30
60
90
Incubation Fig-l.
120 150 180 210 240 Time
(
min.)
3 Stability of H-dexamethasone-receptor complexes at 15Oc or 25OC. Incubation details are given in the text.
Obviously if degradation of the receptor occurs during incubation at 15OC or 25OC, these temperatures cannot be used for a quantitative measurement of the receptor sites. At O°C, the
3 H-dexamethasone-receptor
least 25 hr (Fig.2).
complexes are stable for at
Unoccupied receptor sites are stable at O°C
for 5 hr but undergo significant degradation during longer exposure (Fig.2).
Since non-saturating
concentrations of
3 H-dexamethasone
are
used for the measurement of the number of receptor sites by Scatchard analysis, incubation times longer than 5 hr should not be used in this method.
75
2 ._ > ._
50
a F ._
25
0
‘5 m ._ :
0
0
Steroid - Receptor Free Receptor
5
10
15
Incubation Time Fig.2.
Complex
20
25
(Hours)
3 Stability of H-dexamethasone-recepgor complexes and unoccupied receptor sites (free receptor) at 0 C. The sta$ility of free receptors was tested by incubating cytosol at 0 C for the time periods indicated and then assaying for specific binding after a further 2-hr incubation at O°C with 50 nM 3H-dexamethasone.
The effect of endogenous corticosterone on the quantitative detection of the total dexamethasone preincubating
liver cytosol from adrenalectomized
O°C with nonlabeled corticosterone 0 to 300 nM.
receptor sites was evaluated by rats for 1 hr at
at concentrations ranging from
Following the incubation, the specific binding of
3 H-dexamethasone
was measured either directly or after brief treat-
ment with charcoal to remove excess
(unbound) corticosterone.
In
control experiments using buffer or heated cytosol to inactivate the receptor, it was established that a charcoal concentration of 3-4 mg/ml was sufficient to absorb corticosterone at concentrations up to 1 MM almost quantitatively.
This amount of charcoal also absorbed
3-5% of cytosol protein in a non-specific manner so that the levels of receptor detectable after charcoal treatment remained unaltered. Pre-treatment
of cytosol with higher amounts of charcoal resulted in
detection of somewhat reduced concentrations of the receptor.
A
linear relationship between receptor and protein concentration was found at least over the range of 5-30 mg of protein per ml of cytosol. In all assays the cytosol was adjusted to a protein concentration of 12-15 mg/ml. The number of dexamethasone
receptor sites in cytosols treated
as described above was measured by two methods.
In the first method,
aliquots of the cytosol were incubated for 5 hr at O°C with increasing concentrations
of
3 H-dexamethasone
amount of nonlabeled dexamethasone
with or without a lOO-fold
and the concentration of the
receptors was determined by plotting the data according to Scatchard (7) (Scatchard analsis).
In the second procedure, the cytosol was
incubated at O°C with a saturating concentration
(50 nM) of 3H-dexa-
metfiasone
in the presence
or absence of 5
j_iM af
nonlabeLed dexameth-
asone and aliquots wexe assayed at various time intervals until maximum specific binding was obtained
(single point analysis).
Preincubation of cytosol with increasing amounts of corticosterone resulted in a gradual decrease of the number of detectable dexamethasone
3.5
3.0
_____.
- Charcoal + Charcoal Control 0.05 pM Corticosterone 0.10 p M Cor ticosterone 0.30 pM Corticosterone
2.5
Fig. 3.
Effects of cytosoi preincubation with corticostesone and of remwval of unbound carticosterone by brief treatment with charcoal on the estimation of the number of specific 3Hdexamethasone binding sites by Scatchard analysis. Experimental details are given in the text.
receptor
sites (Rn) and an increase of the apparent dissociation constant
(Kd) of the binding reaction as determined by Scatchard analysis (Figs.3 and 4).
The effect of corticosterone on both Rn and Kd was diminished
30
25
0
-
Charcoal
0
+
Charcoal
T
20
5 - 15 y” 10
5
50 100 [ Fig.4.
Corticosterone
300
1(nM)
Effects of cytosol preincubation with corticosterone and of removal of unbound corticosterone by brief treatment with charcoal on the apparent dissociation constant (Kd) of 3Hdexamethasone binding determined by Scatchard analysis. Experimental details are given in the text.
S
TIIROIDS
but not completely eliminated by brief treatment of the cytosol with charcoal to remove excess hormone prior to the assay.
Both binding parameters
3 H-dexamethasone binding
(En and Kd), however, were not signif-
icantly affected by corticosterone
concentrations up to 50 nM (Figs. 3
and 4). The effect of corticosterone on the detection of Rn by single point analysis is shown in Fig.5.
In control samples, not preincubated with
corticosterone, maximum specific binding of
3 H-dexamethasone was ob-
tained by 5 hr of incubation and remained unchanged up to 30 hr.
0.6
.---___. A
a
5
Fig.5.
10 Incubation :i”,e
Control 0.05 pM 0.10 pM 0.30 pM
(l&s)
- Charcoal + Charcoal Corticosterone Corticosterone Corticosterone
25
3o
Effects of cytosol preincubation with corticosterone and of removal of unbound corticosterone by brief treatment with charcoal on the estimation of the number of specific 3Hdexamethasone binding sites by single point analysis. Conditions of incubation are given in the text.
Preincubation of cytosol with 50 nM corticosterone had no significant effect on the detection of E&Iand on the time required to obtain maximum binding, indicating that bound corticosterone exchanges with 'H-dexamethasone within 5 hr.
That such an exchange is taking place at O°C is
further supported by the observation that maximum binding of 'H-dexamethasone in the presence of higher concentrations (100-300 nM) of corticosterone was not observed until after about 20 hr.
In the presence
of 100 or 300 nM corticosterone, however, the maximum detectable P.nwere below control levels and were not affected by further incubation suggesting that a fraction of the receptor sites are occupied with corticosterone which either does not exchange with exchanges at an extremely slow rate.
3 H-dexamethasone or it
This results in an underestimation
of in at corticosterone concentrations higher than 50 nM. As in the case of Scatchard analysis (Fig.3), pretreatment with charcoal results in a modest increase of detectable Rn but it does not abolish the corticosterone effect (Fig.5). Fig.6 summarizes the effects of cytosol preincubation with corticosterone and of removal of excess hormone by subsequent charcoal treatment on the Pn detectable by Scatchard and single point analyses. Both methods detect virtually 100% of the total Rn in the presence of corticosterone concentrations up to 50 nM.
Charcoal pretreatment is
not necessary at these levels of corticosterone. The presence of higher concentrations of corticosterone results in underestimation of Rn by both methods. Charcoal pretreatment in the latter cases improves the assays but does not eliminate the corticosterone effect. Thus in the presence of 100 XI!+ corticosterone, 62% of Pn are detected by Scatchard analysis and 82% by single point analysis. After charcoal
treatment, the corresponding values are 76 and 86%. of 300 nM corticosterone,
0 0
Fig-G.
In the presence
Scatchard analysis detects 35% and single
Scatchard analysis Single point analysis - Charcoal + Charcoal
Percentage of the receptor sites for 'H-dexamethasone detectable by Scatchard or single point analysis following preincubation of liver cytosol from adrenalectomized rats with various amounts of corticosterone with or without charcoal pretreatment. Values were estimated from data shown in Figs. 3 and 5.
point analysis detects 62% of R.n. The latter values increase to 52 and 70%,
respectively,
after charcoal treatment.
It therefore appears that
when the cytosol contains corticosterone at concentrations
higher than
50 nM charcoal treatment followed by single point analysis gives the best quantitative
estimation of the total Rn.
To assess the effect of endogenous corticosterone on the quantitative detection of Rn, the levels of corticosterone from fetal, immature and adult rats were measured. the endogenous corticosterone
concentration
in liver cytosols
As shown in Table 1,
in liver cytosols ranged
between 0.8 nM in immature rats and 27 nM in fetal rats.
These values
TABLE 1. Levels of endogenous corticosterone from developing rats
in liver cytosols
Corticosterone ng/ml
Age of rats
Fetal (19.5 - 21.5 days) Fetal (22 days) Immature (6-day-old) Adult
9.48 4.12 0.28 1.40
Concentration nM
_+ 1.45a(10)b 3~ 0.52 (10) + 0.06 ( 7) ? 0.28 ( 6)
27.40 11.91 0.81 4.05
k + + +
4.19 1.50 0.17 0.81
aMean j: S.E. b
Number in parentheses
indicates number of determinations
are below the minimum corticosterone
concentrations
(50 nM) required
to produce any significant effect on the quantitative measurement of Pn by Scatchard or single point analysis
(Fig.6).
is supported by the data shown in Table 2.
This conclusion
Similar concentrations
of
receptor were detected in liver cytosols from fetal or adult rats by both methods.
Pretreatment
of the cytosols with charcoal did not
increase the amount of detectable Rn indicating a minimal effect, if any, of endogenous corticosterone on the detection of receptor sites. CONCLUSION Due to instability of the receptor at higher temperatures and the slow rate of dissociation costerone-receptor
of at least a fraction of the corti-
complexes at OOC, we were not successful in devel-
- Charcoal 0.195 f 0,021(5) 0.315 t 0.018(8) 0.483 t 0.016(10)
+ Charcoal 0.187 I!I 0.018(6) 0.280 f 0.006(8) 0.512 f 0.015(16)
0.180 k 0.015C(6)d 0.300 k O.Ol.3(8) 0.534 r 0.012(16)
'Mean k S.E. d Number in parentheses indicates number of determinations
a
0.195 + 0.025(5) 0.308 i- 0.03.9(8) 0.475 r 0.020(10)
+ Charcoal
Single p oint analysis
- Charcoal
Scatchard analysis
tpmol/mg protein)
Untreated (- charcoal) or charcoal-treated (t charcoal) cytosol was incubated for 22 hr at Q°C with a saturating concentration (50 r&I) of 3H-dexamethasone and assayed for specific binding by the charcoal assay. b 20 days of gestation
a
Fetalb (Intact) Adult (Intact) Adult (Adrenaletomized)
Age of rats
Receptor concentration
Concentrations of glucocorticoid receptors in liver cytosols from fetal and adult rats estimated by Scatchard or single point analysis with or without charcoal pretreatment
TABLE 2.
oping a quantitative assay for the measurement of glucocorticoid receptor sites in liver cytosols containing high amounts of endogenous corticosterone, similar to the exchange assays previously described for estradiol-17B (10) and progesterone (11) receptors. However, the assays described in this report measure quantitatively receptor sites in liver cytosols as long as the concentration of endogenous corticosterone does not exceed 50 nM.
Since this value is higher
than the corticosterone levels found in liver cytosols from rats at various developmental stages, the present assays can be used for the quantitative measurement of cytoplasmic receptor sites in rat liver under most physiological conditions. When the levels of corticosterone in cytosol are higher than 50 nM, the assays are only semiquantitative. It is therefore recommended that the assay of glucocorticoid receptor sites in liver cytosol should include measurement of the endogenous corticosterone concentrations in cytosol preparations. The present results indicate that the postnatal increase in the levels of cytoplasmic glucocorticoid receptors in rat liver described earlier (1) is not due to methodological limitations resulting from variations in the concentration of endogenous corticosterone in cytosol preparations. It is, however, possible that the concentration of the receptor in liver cells does not change significantly during development but the observed variations in the levels of receptor sites in the cytosol reflects changes in the distribution of the receptor between cytoplasmic and nuclear compartments. This latter possibility will be examined in a subsequent communication.
66
S
fJ?~EOfDB
This research was supported by Grant MT-4474 from the Medical Research Council of Canada. The author is a Scholar of the Medical Research Council of Canada. PEFERENCES Giannopoulos, G., J. Biol. Chem. 250, 5847(1975). The following trivial names are used: dexamethasone r 118, 17% 21-trihydroxy-9)(-fluoro-~~-methyl-l,4-pregnadiene-3,20-dione; cortisol z corticosterone t 118, 21-dihydroxy-4-pregnene-3,20-dione; Il-deoxycortisol ll$, lP, 2f-trihydroxy-4-pregnene-3,20-dione; 17=, 2f-dihydroxy-4-pregnene-3,20-dione;cortisone = 174, 21dihydroxy-4-pregnene-3,11,20+rione; dexoycorticosterone= Zlhydroxy-4-pregnene-3,20-dione;aldosterone = 118, 21-dihydroxy3,20-dioxo-4-pregnen-18-al;progesterone = 4-pregnene-3,20-dione; androstenedione = 4-androstene-3,20-dione;testosterone = 17Bhydroxy-4-androsten-3-one;estradiol-176 = estra-1,3,5(10)-triene3, 178-dial. Chem. 248, 3876(1973). 3. Giannopoulos, G., J. Sol. 4. Dupouy, J.P., Coffigny, H. and Magre, S., J. Endocr. 65, 347(1975) 5. Malinowska, K.W., Hardy, R.N., and Nathaniel%, P.W., Experientia 28, 1366(1972). 6. Giannopoulos, G., J. Biol. Chem. 250, 2896(1975). 7. Scatchard, G., Ann. N.Y. Acad. Sci. 51, 660(1949). 8. Brenner, P.F., Guerrero, R.# Cekan, Z., and Diczfalusy, E., Steroids 22, 775(1973). 9. Lowry, O-H., Rosebrough, N.J., Farr, A.L., and Randall, R-J., J. Biol. Chem. 193, 265(1951). 10. Katzenellenbogen, J.A., Johnson, H-J., Jr., and Carson, K.E., Biochemistry 12, 4092(1973). 11. Milgrom, E., Perrot, M., Atger, M., and Baulieu, E.-E., Endocrinology 90, 1064(1972). 1.
2.
EFFECT OF ENDOGENOUS CORTICOSTERONE ON THE DETERMINATION OF DEXAMETHASONE RECEPTOR LEVELS IN RAT LIVER CYTOSOL George Giannopoulos Department of Experimental Medicine, McGill University and the University clinic, Royal Victoria Hospital, Montreal, Canada H3A 1Al Received: :/X:/7’; ABSTRACT The effect of endogenous corticosterone on the quantitative measurement of dexamethasone receptors in liver cytosols from developLiver cytosols from adrenalectomized rats ing rats has been studied. were preincubated with increasing concentrations of nonlabeled corticosterone and the levels of detectable dexamethasone receptors were subsequently determined either directly or after removal of W-Ibound corticosterone. Corticosterone concentrations of 50 nM or lower had no significant effect on the specific binding of labeled dexamethasone. Higher concentrations of corticosterone resulted in underestimation of dexamethasone receptor levels. The mean levels of endogenous corticosterone in liver cytosols from 19.5- to 21.5- day fetuses, 22-day fetuses, 6-day-old immature rats and adult rats were 27.40, 11.91, 0.81 and 4.05 nM, respectively. It is concluded that variations in the levels of circulating corticosterone in the rat under normal physiological conditions have no significant effect on the quantitative measurement of total (occupied and unoccupied) receptor sites for dexamethasone in liver cytosol. This is supported by the finding that prior treatment of liver cytosols, from rats at different stages of development, with charcoal to remove unbound steroids has no effect on the amount of detectable dexamethasone receptors. INTRODUCTION In a previous communication cytoplasmic dexamethasone a charcoal assay
(1) we reported that the levels of
(2) receptors in rat liver, detectable by
(3), rise markedly above fetal levels shortly after
birth followed by a moderate decline at the adult stage. levels of circulating corticosterone reverse pattern
Since the
in the developing rat follow a
(4,5), it is possible that the observed variations
in the concentration
of hepatic cytoplasmic receptors are not real
but reflect limitations of the charcoal assay, detecting only unoccupied binding sites, and/or changes in the distribution of the receptors between cytoplasmic and nuclear compartments brought about by the association of endogenous corticosterone with cytoplasmic receptors
(6).
In the present studies we have attempted to determine
VoZwne 28, !?w&er
ii
S
TEIROIDS
JuZy,
2976
the effect of endogenous corticosterone on the detection of the total (occupied and unoccupied) receptor sites for dexamethasone cytosols from rats at different stages of development.
in liver
The effect of
endogenous hormone on the nuclear localization of the receptors will be reported in a separate communication. EXPERIMENTAL Materials: 1,2-3H-Dexamethasone (29 Ci/mmol) , 1,2-3H-cortisol (52 Ci/mmol), 1,2-3H-corticosterone (53 Ci/mmol) and Aguasol scintillation fluid were obtained from New England Nuclear Corporation. Dexamethasone was a gift from the Upjohn Co. Corticosterone was purchased from Ikapharm, bovine serum albumin Fraction V from Sigma, Sephadex LH-20 and Dextran T-70 from Pharmacia Fine Chemicals, and Norit A neutral charcoal from Fisher Scientific Co. All glass redistilled cyclohexane, benzene, methanol and dichloromethane were obtained from A & C American Chemicals, Montreal. Pregnant and nonpregnant rats (Sprague-Dawley) were purchased from the Canadian Breeding Farm and Laboratories Ltd. Adrenalectomized rats were obtained from the supplier and maintained on Purina Chow and 0.9% NaCl solution ad libitum for 1 week prior to the beginning of the experiments. Preparation of cytosol: Rats were killed by decapitation and, when pregnant, the fetuses were delivered by laparotomy and decapitated. The livers were excised, minced, and washed three times with 0.01 M Tris-HCl buffer, pH 7.4, containing 0.25 M sucrose and 0.003 M MgC12 to remove excess blood. The minced livers were blotted, weighed and homogenized in batches of 1 g in 1 ml of 0.01 M TrisHCl buffer, pH 7.4, containing 0.0015 M Na2EDTA (Tris/EDTA buffer) at a temperature close to OOC. The homogenate was centrifuged for 30 min at 224,000 x g at 2oC to obtain the cytoplasmic supernatant fraction (cytosol). The cytosol was adjusted to the desired protein concentration by dilution with Tris/EDTA buffer. 3 to liver Measurement of specific binding of H-dexamethasone ---3H-dexamethasone alone or in comcytosol: Appropriate aliquots of bination with a lOOO-fold amount of nonlabeled dexamethasone, dissolved in redistilled ehtanol, were pipetted into tubes and the ethanol was evaporated under nitrogen. Aliquots of cytosol were then added, mixed in a vortex, and the mixtures were incubated. Incubation temperatures, duration of incubations, concentrations of hormones employed, and special treatment of cytosols before or after incubation will be described in individual experiments. Specifically bound 3H-dexamethasone was measured by the charcoal assay described earlier (3). The apparent dissociation constant (Kd) of the binding reaction and the concentration of specific binding sites for dexamethasone (n) were estimated by plotting the data according to Scatchard (7). In certain cases the concentration
of receptor sites was measured by incubating the cytosol with a single, saturating, concentration of 3H-dexasnethasone for time periods required to obtain optimal binding (single point analysis). Determination of endogenous corticosterone in liver cytosol by radioimmunoassay: About 1000 cpm of 3H-corticosterone dissolved in 0.1 ml water was added to 0.5 - 1.9 ml of cytosol to monitor procedural losses, All samples were diluted to a final volume of 2 ml with water. The mixture was extracted with 10 ml dichloromethane, centrifuged at 800 x g for 10 min, and the dichloromethane extract was evaporated to dryness under a stream of nitrogen, Corticosterone in the dichloromethane extract was purified and assayed according to the procedures recommended by New England Nuclear Corporation, manufacturer of the rabbit antiserum against cortisol-21-succinyl-bovine serum albumin, fox the determination of cortisol. Standard curves were prepared using 3H-cortisol (3000-3500 cpm) as the labeled antigen and corticosterone (O-2.5 ng) as the nonlabeled antigen. The cross-reactivity of corticosterone with the antiserum, furnished by the manufacturer and confirmed in our laboratory, was 24%. Other steroids cross-reacting with the antiserum axe cortisone (4%), Il.-deoxycortisol (4%), androstenedione (4%), progesterone (2%), deoxycorticosterone (2%), testosterone (1%) and aldosterone (1%). Most of these steroids were either completely or predominantly eliminated by chromatography on a Sephadex LH-20 microcolumn (0.85 g) using the solvent system cyclohexane:benzene:methanol (60:40:10, This v/v) * Cortisol was also well separated from corticosterone. chromatographic step, however, did not eliminate ll-deoxycortisol which co-eluted with corticosterone. Thus the corticosterone values obtained in the cytosol preparations may be clightly overestimated. The recovery of 3H-corticosterone adaed to cytosol and carried through the extraction and chromatography steps ranged between 48.0 and 82.0% with a mean of 61.0 + (S-E.) 1.1 (n = 156). An aliguot of the corticosterone fraction elutad from the column was removed for counting and the remaining was used for assay. For most s:~mples, two different amounts of the eluted corticosterone fraction were assayed In order to test the parallelism between various levels in duplicate. of endogenous and authentic hormone, increasing amounts of cytosol from a homogeneous cytosol pool were assayed. For each amount 3 replicate measurements were carried out and the results, expressed as logits, were compare: with the logits resulting from the assay of 3 increasing amounts (in triplicate) of authentic corticosterone. A complete analysis of variance (8) indicated that the relationship between increasing amount F of cytosol and authentic hormone was parallel and linear in all insV?nces. The presence of blank was estimated by processing water (2 ml) in the same manrcr as the cytosol samples. The cpm resulting from the radioimmunoassay of the blank samples were compared with the cpm of those tubes of the standard curve which did not contain any nonlabeled steroid. The difference between means of both series of cpm was evaluated by the t-test. No significant difference could be detected at the 95% probability level. Thus the presence of blank in the assay was negligible, if any.
S
TlOROIDS
The sensitivity of the method, defined as the mass equal to twice the standard deviation of the zero binding, was approximately 25 pg. The accuracy of the assay was assessed by the addition of 5,10, 20 and 40 ng of corticosterone to 2 ml cytosol. After correction for recovery and endogenous corticosterone, the mean values C S.E. (n - 5) were 5.20 + 0.34, 9.30 t- 0.95, 20.16 C 2.15 and 38.37 -t 5.50 ng, respectively. The reproducibility of the method was evaluated in studies of the intra-assay and interassay variations carried out on a homogeneous cytosol pool. The intra-assay coefficient of variation in replicate assays (n = 8) of this pool was 12.0%. The interassay coefficient of variation in assays of the same cytosol pool performed on 5 occasions was 16.6%. Measurement of radioactivity: Radioactivity was measured in a Packard Tri-Carb model 3002 spectrometer. In studies of 3H-dexamethasone binding, aqueous samples (0.5 ml) were mixed with 10 ml Omnifluor in toluene and counted after a pre-equilibration period of at least 2 hrs at 45% efficiency for 3H. In the corticosterone radioinununoassay, aqueous samples (1.1 ml) were counted in 10 ml Aquasol at 25% efficiency for 3H.
&.
Protein assay: Protein was determined by the method of Lowry -et (9) using bovine serum albumin as standard. RESULTS AND DISCUSSION Since the binding of steroid hormones to their intracellular
receptors is a reversible reaction, it should be theoretically
feasible
to detect occupied receptor sites by exchange of the receptor-bound endogenous steroid with labeled hormone. the rate of dissociation
Because in some instances
of stc: i-7 from its receptor is very slow at
O°C, the use of higher temperatures may be necessary for complete equilibration of endogenous and labeled steroid.
Most steroid hormone
receptors, however, have a tendency to be degraded or inactivated upon exposure to higher temperatures or even at O°C for prolonged periods of time.
Thus experimental conditions of temperature and length of
incubation must be carefully standardized to ensure equilibration of endogenous steroid with the labeled hormone in the absence of inactivation of occupied or unoccupied receptor sites. criteria are met, the total number
Once these
(occupied and unoccupied) receptor
S
TDROIDS
sites can be quantitatively measured by incubating aliquots of cytosol with increasing concentrations according to Scatchard
(7).
of labeled steroid and plotting the data
Such quantitative assays have already been
developed for the measurement of receptor sites for estradiol-175 and progesterone
(10)
(11) in the uterus.
To test the stability of
H-dexamethasone-receptor
complexes,
liver cytosol was first incubated for 2 hr at O°C with a saturating concentration
(50 nM) of
at 15OC or 25OC.
3 H-dexamethasone
followed by further incubation
After various time intervals of exposure to 15OC
or 25OC, aliquots of the cytosol were transferred to O°C and assayed after 1 hr for specifically bound The 'H-dexamethasone-receptor
3 H-dexamethasone by the charcoal assay.
complexes appear to undergo rapid degrad-
ation at 25OC and slower but significant degradation at 15OC (Fig.1).
I
30
60
90
Incubation Fig-l.
120 150 180 210 240 Time
(
min.)
3 Stability of H-dexamethasone-receptor complexes at 15Oc or 25OC. Incubation details are given in the text.
Obviously if degradation of the receptor occurs during incubation at 15OC or 25OC, these temperatures cannot be used for a quantitative measurement of the receptor sites. At O°C, the
3 H-dexamethasone-receptor
least 25 hr (Fig.2).
complexes are stable for at
Unoccupied receptor sites are stable at O°C
for 5 hr but undergo significant degradation during longer exposure (Fig.2).
Since non-saturating
concentrations of
3 H-dexamethasone
are
used for the measurement of the number of receptor sites by Scatchard analysis, incubation times longer than 5 hr should not be used in this method.
75
2 ._ > ._
50
a F ._
25
0
‘5 m ._ :
0
0
Steroid - Receptor Free Receptor
5
10
15
Incubation Time Fig.2.
Complex
20
25
(Hours)
3 Stability of H-dexamethasone-recepgor complexes and unoccupied receptor sites (free receptor) at 0 C. The sta$ility of free receptors was tested by incubating cytosol at 0 C for the time periods indicated and then assaying for specific binding after a further 2-hr incubation at O°C with 50 nM 3H-dexamethasone.
The effect of endogenous corticosterone on the quantitative detection of the total dexamethasone preincubating
liver cytosol from adrenalectomized
O°C with nonlabeled corticosterone 0 to 300 nM.
receptor sites was evaluated by rats for 1 hr at
at concentrations ranging from
Following the incubation, the specific binding of
3 H-dexamethasone
was measured either directly or after brief treat-
ment with charcoal to remove excess
(unbound) corticosterone.
In
control experiments using buffer or heated cytosol to inactivate the receptor, it was established that a charcoal concentration of 3-4 mg/ml was sufficient to absorb corticosterone at concentrations up to 1 MM almost quantitatively.
This amount of charcoal also absorbed
3-5% of cytosol protein in a non-specific manner so that the levels of receptor detectable after charcoal treatment remained unaltered. Pre-treatment
of cytosol with higher amounts of charcoal resulted in
detection of somewhat reduced concentrations of the receptor.
A
linear relationship between receptor and protein concentration was found at least over the range of 5-30 mg of protein per ml of cytosol. In all assays the cytosol was adjusted to a protein concentration of 12-15 mg/ml. The number of dexamethasone
receptor sites in cytosols treated
as described above was measured by two methods.
In the first method,
aliquots of the cytosol were incubated for 5 hr at O°C with increasing concentrations
of
3 H-dexamethasone
amount of nonlabeled dexamethasone
with or without a lOO-fold
and the concentration of the
receptors was determined by plotting the data according to Scatchard (7) (Scatchard analsis).
In the second procedure, the cytosol was
incubated at O°C with a saturating concentration
(50 nM) of 3H-dexa-
metfiasone
in the presence
or absence of 5
j_iM af
nonlabeLed dexameth-
asone and aliquots wexe assayed at various time intervals until maximum specific binding was obtained
(single point analysis).
Preincubation of cytosol with increasing amounts of corticosterone resulted in a gradual decrease of the number of detectable dexamethasone
3.5
3.0
_____.
- Charcoal + Charcoal Control 0.05 pM Corticosterone 0.10 p M Cor ticosterone 0.30 pM Corticosterone
2.5
Fig. 3.
Effects of cytosoi preincubation with corticostesone and of remwval of unbound carticosterone by brief treatment with charcoal on the estimation of the number of specific 3Hdexamethasone binding sites by Scatchard analysis. Experimental details are given in the text.
receptor
sites (Rn) and an increase of the apparent dissociation constant
(Kd) of the binding reaction as determined by Scatchard analysis (Figs.3 and 4).
The effect of corticosterone on both Rn and Kd was diminished
30
25
0
-
Charcoal
0
+
Charcoal
T
20
5 - 15 y” 10
5
50 100 [ Fig.4.
Corticosterone
300
1(nM)
Effects of cytosol preincubation with corticosterone and of removal of unbound corticosterone by brief treatment with charcoal on the apparent dissociation constant (Kd) of 3Hdexamethasone binding determined by Scatchard analysis. Experimental details are given in the text.
S
TIIROIDS
but not completely eliminated by brief treatment of the cytosol with charcoal to remove excess hormone prior to the assay.
Both binding parameters
3 H-dexamethasone binding
(En and Kd), however, were not signif-
icantly affected by corticosterone
concentrations up to 50 nM (Figs. 3
and 4). The effect of corticosterone on the detection of Rn by single point analysis is shown in Fig.5.
In control samples, not preincubated with
corticosterone, maximum specific binding of
3 H-dexamethasone was ob-
tained by 5 hr of incubation and remained unchanged up to 30 hr.
0.6
.---___. A
a
5
Fig.5.
10 Incubation :i”,e
Control 0.05 pM 0.10 pM 0.30 pM
(l&s)
- Charcoal + Charcoal Corticosterone Corticosterone Corticosterone
25
3o
Effects of cytosol preincubation with corticosterone and of removal of unbound corticosterone by brief treatment with charcoal on the estimation of the number of specific 3Hdexamethasone binding sites by single point analysis. Conditions of incubation are given in the text.
Preincubation of cytosol with 50 nM corticosterone had no significant effect on the detection of E&Iand on the time required to obtain maximum binding, indicating that bound corticosterone exchanges with 'H-dexamethasone within 5 hr.
That such an exchange is taking place at O°C is
further supported by the observation that maximum binding of 'H-dexamethasone in the presence of higher concentrations (100-300 nM) of corticosterone was not observed until after about 20 hr.
In the presence
of 100 or 300 nM corticosterone, however, the maximum detectable P.nwere below control levels and were not affected by further incubation suggesting that a fraction of the receptor sites are occupied with corticosterone which either does not exchange with exchanges at an extremely slow rate.
3 H-dexamethasone or it
This results in an underestimation
of in at corticosterone concentrations higher than 50 nM. As in the case of Scatchard analysis (Fig.3), pretreatment with charcoal results in a modest increase of detectable Rn but it does not abolish the corticosterone effect (Fig.5). Fig.6 summarizes the effects of cytosol preincubation with corticosterone and of removal of excess hormone by subsequent charcoal treatment on the Pn detectable by Scatchard and single point analyses. Both methods detect virtually 100% of the total Rn in the presence of corticosterone concentrations up to 50 nM.
Charcoal pretreatment is
not necessary at these levels of corticosterone. The presence of higher concentrations of corticosterone results in underestimation of Rn by both methods. Charcoal pretreatment in the latter cases improves the assays but does not eliminate the corticosterone effect. Thus in the presence of 100 XI!+ corticosterone, 62% of Pn are detected by Scatchard analysis and 82% by single point analysis. After charcoal
treatment, the corresponding values are 76 and 86%. of 300 nM corticosterone,
0 0
Fig-G.
In the presence
Scatchard analysis detects 35% and single
Scatchard analysis Single point analysis - Charcoal + Charcoal
Percentage of the receptor sites for 'H-dexamethasone detectable by Scatchard or single point analysis following preincubation of liver cytosol from adrenalectomized rats with various amounts of corticosterone with or without charcoal pretreatment. Values were estimated from data shown in Figs. 3 and 5.
point analysis detects 62% of R.n. The latter values increase to 52 and 70%,
respectively,
after charcoal treatment.
It therefore appears that
when the cytosol contains corticosterone at concentrations
higher than
50 nM charcoal treatment followed by single point analysis gives the best quantitative
estimation of the total Rn.
To assess the effect of endogenous corticosterone on the quantitative detection of Rn, the levels of corticosterone from fetal, immature and adult rats were measured. the endogenous corticosterone
concentration
in liver cytosols
As shown in Table 1,
in liver cytosols ranged
between 0.8 nM in immature rats and 27 nM in fetal rats.
These values
TABLE 1. Levels of endogenous corticosterone from developing rats
in liver cytosols
Corticosterone ng/ml
Age of rats
Fetal (19.5 - 21.5 days) Fetal (22 days) Immature (6-day-old) Adult
9.48 4.12 0.28 1.40
Concentration nM
_+ 1.45a(10)b 3~ 0.52 (10) + 0.06 ( 7) ? 0.28 ( 6)
27.40 11.91 0.81 4.05
k + + +
4.19 1.50 0.17 0.81
aMean j: S.E. b
Number in parentheses
indicates number of determinations
are below the minimum corticosterone
concentrations
(50 nM) required
to produce any significant effect on the quantitative measurement of Pn by Scatchard or single point analysis
(Fig.6).
is supported by the data shown in Table 2.
This conclusion
Similar concentrations
of
receptor were detected in liver cytosols from fetal or adult rats by both methods.
Pretreatment
of the cytosols with charcoal did not
increase the amount of detectable Rn indicating a minimal effect, if any, of endogenous corticosterone on the detection of receptor sites. CONCLUSION Due to instability of the receptor at higher temperatures and the slow rate of dissociation costerone-receptor
of at least a fraction of the corti-
complexes at OOC, we were not successful in devel-
- Charcoal 0.195 f 0,021(5) 0.315 t 0.018(8) 0.483 t 0.016(10)
+ Charcoal 0.187 I!I 0.018(6) 0.280 f 0.006(8) 0.512 f 0.015(16)
0.180 k 0.015C(6)d 0.300 k O.Ol.3(8) 0.534 r 0.012(16)
'Mean k S.E. d Number in parentheses indicates number of determinations
a
0.195 + 0.025(5) 0.308 i- 0.03.9(8) 0.475 r 0.020(10)
+ Charcoal
Single p oint analysis
- Charcoal
Scatchard analysis
tpmol/mg protein)
Untreated (- charcoal) or charcoal-treated (t charcoal) cytosol was incubated for 22 hr at Q°C with a saturating concentration (50 r&I) of 3H-dexamethasone and assayed for specific binding by the charcoal assay. b 20 days of gestation
a
Fetalb (Intact) Adult (Intact) Adult (Adrenaletomized)
Age of rats
Receptor concentration
Concentrations of glucocorticoid receptors in liver cytosols from fetal and adult rats estimated by Scatchard or single point analysis with or without charcoal pretreatment
TABLE 2.
oping a quantitative assay for the measurement of glucocorticoid receptor sites in liver cytosols containing high amounts of endogenous corticosterone, similar to the exchange assays previously described for estradiol-17B (10) and progesterone (11) receptors. However, the assays described in this report measure quantitatively receptor sites in liver cytosols as long as the concentration of endogenous corticosterone does not exceed 50 nM.
Since this value is higher
than the corticosterone levels found in liver cytosols from rats at various developmental stages, the present assays can be used for the quantitative measurement of cytoplasmic receptor sites in rat liver under most physiological conditions. When the levels of corticosterone in cytosol are higher than 50 nM, the assays are only semiquantitative. It is therefore recommended that the assay of glucocorticoid receptor sites in liver cytosol should include measurement of the endogenous corticosterone concentrations in cytosol preparations. The present results indicate that the postnatal increase in the levels of cytoplasmic glucocorticoid receptors in rat liver described earlier (1) is not due to methodological limitations resulting from variations in the concentration of endogenous corticosterone in cytosol preparations. It is, however, possible that the concentration of the receptor in liver cells does not change significantly during development but the observed variations in the levels of receptor sites in the cytosol reflects changes in the distribution of the receptor between cytoplasmic and nuclear compartments. This latter possibility will be examined in a subsequent communication.
66
S
fJ?~EOfDB
This research was supported by Grant MT-4474 from the Medical Research Council of Canada. The author is a Scholar of the Medical Research Council of Canada. PEFERENCES Giannopoulos, G., J. Biol. Chem. 250, 5847(1975). The following trivial names are used: dexamethasone r 118, 17% 21-trihydroxy-9)(-fluoro-~~-methyl-l,4-pregnadiene-3,20-dione; cortisol z corticosterone t 118, 21-dihydroxy-4-pregnene-3,20-dione; Il-deoxycortisol ll$, lP, 2f-trihydroxy-4-pregnene-3,20-dione; 17=, 2f-dihydroxy-4-pregnene-3,20-dione;cortisone = 174, 21dihydroxy-4-pregnene-3,11,20+rione; dexoycorticosterone= Zlhydroxy-4-pregnene-3,20-dione;aldosterone = 118, 21-dihydroxy3,20-dioxo-4-pregnen-18-al;progesterone = 4-pregnene-3,20-dione; androstenedione = 4-androstene-3,20-dione;testosterone = 17Bhydroxy-4-androsten-3-one;estradiol-176 = estra-1,3,5(10)-triene3, 178-dial. Chem. 248, 3876(1973). 3. Giannopoulos, G., J. Sol. 4. Dupouy, J.P., Coffigny, H. and Magre, S., J. Endocr. 65, 347(1975) 5. Malinowska, K.W., Hardy, R.N., and Nathaniel%, P.W., Experientia 28, 1366(1972). 6. Giannopoulos, G., J. Biol. Chem. 250, 2896(1975). 7. Scatchard, G., Ann. N.Y. Acad. Sci. 51, 660(1949). 8. Brenner, P.F., Guerrero, R.# Cekan, Z., and Diczfalusy, E., Steroids 22, 775(1973). 9. Lowry, O-H., Rosebrough, N.J., Farr, A.L., and Randall, R-J., J. Biol. Chem. 193, 265(1951). 10. Katzenellenbogen, J.A., Johnson, H-J., Jr., and Carson, K.E., Biochemistry 12, 4092(1973). 11. Milgrom, E., Perrot, M., Atger, M., and Baulieu, E.-E., Endocrinology 90, 1064(1972). 1.
2.