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Corticosteroid receptors in liver cytosol of the clawed toad, Xenopus laevis: Influence of thyroid and ovarian hormones
GENERAL
AND
COMPARATIVE
73,485-497
ENDOCRINOLOGY
(1989)
Corticosteroid Xenopus
Receptors in Liver Cytosol of the Clawed Toad, laevis: Influence of Thyroid and Ovarian Hormones’
C. B. *Department
of Zoology, and fDepartment
LANGE,*
The University, of Physiology,
W.
HANKE,*,’
AND
W.
K. MORISHIGE~
Kaiserstrasse 12, D-7500 Karlsruhe, John A. Burns School of Medicine, Honolulu, Hawaii 96822
Federal Republic of Germany; University of Hawaii,
Accepted August 26, 1988 The glucocorticoid receptor capacity R, and the dissociation constant Kd were determined in the liver of Xenopus laevis by Scatchard analysis. In S-year-old female toads RO was about three times higher than that in males (153.9,54.3 fmol/mg protein) and Ka was similar in both sexes (4.0, 4.1 n&f). Some of the animals used had abnormal enlarged thyroid glands, atrophic ovaries, or both defects in connection with different levels of R,, but not of Kd, compared to those of normal animals. Females with ovarian atrophy showed significantly lower R, values, in the same range as in normal males, and a high liver weight. In male and female toads with enlarged thyroid glands and in animals with both defects a significantly higher R. occurred compared to that of the corresponding group without this abnormality. To study the influence of thyroid hormones on glucocorticoid receptors, young toads (2-3 years old) received injections of 4-phenyL2thiouraciI T,, or T, on 7 consecutive days. R0 and Kd were determined on the following day. Doses of 50 and 500 ng T, and of 500 and SO00 ng T4 per gram of body weight and day resulted in an increase of R,, up to 250% of the controls. Injections of T, were more efficient in males than in females. The effect of thyroxine was about the same in both sexes. These observations suggest that thyroid and ovarian hormones exert an influence on glucocorticoid receptor capacity and may belong to the factors which regulate glucocorticoid receptors. 0 1989 Academic PWSS, 1~.
The presence of glucocorticoid receptors in the liver of Amphibia is well known. The characteristics of these receptors have been described in a few studies in Rana catesbeiana (Woody and Jaffe, 1982, 1984; Mehdi et al., 1984), in Rana esculenta (Incerpi et al., 1983), and in Xenopus laevis (May and Westley, 1982). In a previous paper, we reported on the binding capacity R, and the dissociation constant Kd in liver cytosol of X. laevis. Diurnal variations of R, were found and correlated with the corticosteroid concentration in the serum of young X. laevis . Seasonal variations showed different patterns ’ This work was supported by Deutsche Forschungsgemeinschaft. ’ To whom requests for reprints should be addressed.
for male and female adult toads with higher receptor value3 always in females.. During the year no correlation occurred between R, and the corticosteroid concentration in serum (Lange and Hanke, 1988). Since glucocorticoid receptors mediate the glucocorticoid effects, the regulation of the receptor protein in vivo is of great interest, although only little information is present (Kalimi and Hubbard, 1983; Rousseau, 1985). A few reports deal with a major area in this line of studies, the hormonal regulation of the receptor. High doncentrations of glucocorticoids dimini,shed the receptor concentration in AtT-20 mouse pituitary tumor cells (Svec, 1985). Injections of dihydrotestosterone reduced the, glucocorticoid receptor levels in thymus a@ bursa of Fabricius tissue in chicken (Coulson et al., 1982). Morishige and co-workers (Mor-
485 0016-6480189 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
486
LANGE,
HANKE,
ishige and Ioun, 1982; Morishige, 1982) showed in a developmental study that the increase of glucocorticoid receptors in rat lung during the first week after birth may be regulated by thyroid hormones and Naito ef al. (1985) found an increase of the receptor capacity in the liver of adult adrenalectomized rats after a single injection of either D- or L-thyroxine. In the adult male and female X. Zuevis used in our experiments, two abnormalities occurred: females with atrophic ovaries and toads of both sexes with enlarged thyroid glands. The finding that these defects were parallel with changed receptor concentrations gave rise to further experiments on the influence of thyroid hormones on glucocorticoid receptors.
AND MOFUSHIGE
[1,2(n)-3H]Dexamethasone (30-50 Wmmol; Amersham Buchler, Braunschweig) was dissolved in double distilled water. The stock solutions of unlabeled dexamethasone (Serva, Heidelberg) were prepared in absolute ethanol. L-3,3’,5-Triiodothyronine (T,), Lthyroxine Cr,), and 4-phenyl-2-thiouracil (PTU) were purchased from Serva (Heidelberg). T,, T4, and PTU were dissolved in 0.1 N NaOH frog Ringer and diluted with pure frog Ringer (6 g NaCl, 0.2 g KCl, 0.2 g CaC!, * 6Hz0, 0.1 g NaHCO, in 1 liter distilled water). The final solution (100 ~1) contained the dose for an animal of 10-g weight.
undeveloped or reduced ovaries, and two female toads showed both defects. Because of the limited amount of collected blood only a few determinations cauld be done with these animals. A significantly higher amount of T4 was found in these preliminary experiments when the thyroid gland was enlarged. Since abnormal thyroid glands occur in only a few cases and can be detected only by dissection, carefully planned experiments were not possible. (b) Experiments with injection of T3, T4, or PTU (2 or 3 years 016). At least 2 months before starting the experiments, male and female toads were kept separately in basins with 10 animals under a daylight regime receiving artificial light from 6:00 to 18:00 hr. They were maintained at 20 + 1” and fed daily with dry food pellets, and the water was changed twice a week. For the experiments groups of five male and five female toads were studied. On 7 consecutive days, between 9:00 and 10:30, each toad was weighed and received an injection of T,, T4, PTU, or vehicle into the dorsal lymphatic vessel. Doses of $50, and 500 ng T,, of 50, 500, and 5000 ng T4, and of 3 pg PTU per gram of body weight were tested. Since 100 ~1 of the prepared solution contained the dose for a 10-g toad, the volume of the injection varied with the weight of the animals. Receptor determination was done the day after the last injection. Experiments were performed in December 1985 (I) and February (II) and June 1986 (III) with toads of about 3 years (I) and 2 years of age (II, III). A total of 65 males and 65 females were sacrificed. To avoid influence of the time of day, the time consuming receptor assay was restricted to 10 animals (2 groups) per day with the following schedule of receptor assays (e.g., Experiment I): Day l-control males and T3 females; Day 2-T, males and T4 females; Day 3--T, males and PTU females; Day 4-PTU males and control females.
Animals
Preparation of Cytosol
X. Zuevis Daudin used in the experiments were raised from the animal stock in our department. During their lifetime they were kept at about 17” and fed with minced beef heart once a week after metamorphosis. (a) Normal and abnormal adult toads (4-5 years old). Between April 1984 and May 1985 receptor assays were performed in toads which had all passed metamorphosis in June 1980. Each month groups of animals which were last fed 1 week previously were sacrificed between 9:30 and 11:30 hr. In the toads used in these studies two abnormalities occurred in some of the animals. In eight male and five female toads a very enlarged thyroid gland was found, three female toads at the age of more than 4 years had
The liver was rapidly excised, blotted, weighed, and kept on ice. It was cut into small pieces (1 mm) and rinsed twice in chilled buffer (20 mM Tris, pH 7.4 at OX, 250 mM sucrose, 10 mM sodium molybdate, 5 mM dithiothreitol). The tissue was homogenized in 5 vol (w/v) of buffer in a glass-Teflon Potter tissue grinder and centrifuged at 10,OOOgfor 10 min. The lipid layer was discarded and the supernatant centrifuged (100,OOOg for 60 min). The obtained cytosol fraction was used immediately for the binding studies. All subsequent steps were carried out at O-4’. Perfusion of the liver was not possible in groups of these small toads. So, arising from the fact that in toads erythrocytes also contain nuclei which cannot be separated from liver nuclei, a determination of nuclear
MATERIALS
AND METHODS
Materials
CORTCCOSTEROID
RECEPTORS
IN
Xenopus
LIVER
487
ion-like cartilage in male toads. In normal animals of 20-30 g body weight the paired, slightly pigmented, and light-rose gland Binding Studies weighed 5 to 10 mg with both moieties disA Scatchard analysis (Scatchard, 1949) was carried tinguishable from each other. The abnormal out to define Kd (apparent dissociation constant) and thyroid glands were much heavier in the R, (binding capacity, fmohmg protein). After a 15-min range of 50-500 mg. The tissue was dark preincubation in the absence (total binding) or presred and compact. The moieties of the gland ence of 10 pJ4 unlabeled dexamethasone were close together and some of the folli(“nonspecific” binding) aliquots of 0.1 ml cytosol were incubated with varying concentrations of cles were visible with an unaided eye. His[3H]dexamethasone (0.05 ml; final concentrations of tological sections of either a normal or an about 1, 5, 10, 25, 50, and 100 nM) for 24 hr at O-4”. enlarged thyroid gland at the indicated magThe dextran-coated charcoal technique was used to separate free from macromolecular bound labeled ste- nification (Fig. 1) showed differences in structure and size. roid. To each ali,quot 200 p,l of dextran-coated charcoal suspension (20 mM Tris, pH 7.4, 3.75% Norit A, The results in the six occurring groups of 0.375% dextran T70) was added for a 5-min incubation toads, normal toads (N), toads with enwith vortexing. Only the free steroid is adsorbed by larged thyroid gland (T) of both sexes, fethe activated charcoal. In a subsequent centrifugation (ZOOOg, 15 min) the macromolecular bound steroid re- males with ovarian atrophy (0), and femains in the supernatant, which was decanted and males with both defects (OT) are shown in stored in the deep freeze at -20”. Radioactivity was Figs. 2 and 3. measured by liquid scintillation counting (Tri-Carb The dexamethasone binding capacity R, 300; Packard Instrument) in an ahquot of 200 p,l of and the dissociation constant & resulted thawed supematant dissolved in 3 ml counting solution from Scatchard analysis of a saturation (Aqualuma Plus, Lumac, Shaesberg, Netherlands). Quench correction was done by the method of the curve. Significant differences between the spectral index of the external standard. The specific groups were not found for the Kd, with 4.1 binding, the difference between total binding and non+- 0.3 and 4.0 + 0.6 n&fin normal males and specific binding, R,, and Kd were calculated. females, respectively. The R0 values showed statistically significant differences Other Procedures among the six groups. In female toads Ra Blood samples taken from the Truncus arteriosus was about three times higher than that in were centrifuged and the serum was stored in the deep males (Fig. 2). In toads with enlarged thyfreeze at -20” until analyzed. Total corticosterone roid gland a much higher R, occurred comand aldosterone concentration in serum were estipared to that of the corresponding group mated by a RIA (Thurmond et al., 1986) and cytosol without this abnormality. This effect held protein concentration was estimated by the method of Lowry et al. (1951). Statistical evaluation of data was true for male and female normal X. la&is performed by Student’s t test (*: 0.05 > P). Since the and for females with ovarian atrotihy and groups of abnormal toads contain only a few animals was statistically significant in all three (N = 2 to 8) nonparametric statistical tests (Wilcoxcases. In contrast, the presence of abnoron’s U test, Duncan’s new multiple range test) were performed. The design for statistical differences (*) mal ovaries was parallel with a significantly was always the same. lower R, in liver cytosol in comparison to that of normal females. R, was at the same RESULTS low range in these animals as in boreal males. I. Comparison of Kd and R, in Normal To estimate the cytosol receptor capacity and Abnormal Adult Toads in the whole liver, R, was calculated acThe thyroid gland in X. laevis is placed at cording to the formula RL = Ii0 x p x LW/ the ventr+cranial end of the larynx, which 100 Cp = protein concentration in mg/lOO is a flat cartilage in female and a thick cush- mg liver tissue; LW = liver weight in mg). corticosteroid receptors was not performed. Further experiments are planned in this respect.
488
LANGE,
HANKE,
AND MORISHIGE
FIG. 1. Histological section of a normal (a) and an enlarged (b) thyroid gland (Masson-Goldner stain). Bar = 100 p,m.
The same pattern of effects on R, was foun .d for R, with increased values in toads with enlarged thyroid glands and reduced V&l es in toads with ovarian atrophy. The diffe :rences between the groups were statis-
tically more evident for R, than for RL (Fig. 2). The liver weight and the liver body in tdex (liver weight in percentage of the b ody weight) were of the same range in not *mal
CORTICOSTEROJD
RECEPTORS
(a) BINDING CAPACITY
R,
(fmollmg
t
protein)
IN Xenopus
489
LIVER
I
(bl BINOING CAFACITY PER LIVER R,(pmol per Liver)
250
200 150
100
50 1 N T In1
1421
I.91
MALES
N T
N T 0 OT 1321
151
131
121
FEMALES
InI
I621
III
MALES
NTOOT WI
19
13)
I21
FEMALES
FIG. 2. [3H]Dexamethasone binding capacity R, (a) and binding capacity per liver R, (b) in different groups of adult X. Zaevis, in normal toads (N), in toads with enlarged thyroid gland (T), in females with atrophic ovaries (O), or both abnormalities (OT). Lines above the columns depict statistically significant differences between the groups (mean values i- SEM).
toads and toads with enlarged thyroid gland. These parameters were significantly higher in female toads with atrophic ovaries (0 and T, Fig. 3). The corticosterone concentration in serum showed no detectable differences among the groups. The aldosterone concentration in serum was higher in females than in males with the highest concentration in females with both defects (OT). 2. Injluence of Injections of T3, T4, and PTU on I& and R,
The striking results in toads with abnormal thyroid gland prompted us to study the influence of T,, Tq, or PTU injections on the [3H]dexamethasone binding capacity R, and the apparent dissociation constant Kd. In controls, the binding’ capacity of the older animals used in Experiment I was much higher than that in the younger ani-
maIs of Experiments II and III (Figs. 4 and 5). Injections of PTU in the older anim& of Experiment I caused a sign&ant d:ecrease of R, together with a slight increase of the liver weight (males: 768 + 115mg, PTTJ 933 + 260 mg; females: 1182 -+ 200 mg,,,PTU 1480 t 227 mg), Therefore, no change in R, was found. In the younger animals used in Experiment II, PTU had no effect on both R, and RL. Low doses of 5 rig/g T3 and 50 rig/g Tq (I, 11) had no effect on the receptor capacity expressed as either R, or R,. However, after injection of 50 rig/g T3, 500 rig/g T3, %O nglg Tq, or 5000 nglg T,, RD and R, were about two- to threefold significantly, ,higher than those in the control group (II, III). ‘Significant differences were ,not found between the groups of hormone-injected toads in Experiment III (Figs. 4 and 5, Table 1).
490
LANGE,
(a)
HANKE,
LIVER WEIGHT (mg)
AND MORISHIGE
(b)
LIVER BODY INDEX (% of the body weight)
-*
N T In1 ILlI 181 MALES
N T Cl OT 1355151 ml 121 FEMALES
NT In1 IUJ I81 MALES
* *
N T 0 OT I351 (51 131 I21 FEMALES
FIG. 3. The liver weight expressed either in milligrams (a) or in percentage of the body weight (b) in different groups of adult X. Zuevis. Groups and statistics as in Fig. 2.
Table 1 shows the results for male and female toads separately. Sexual differences in R,, but not in R,, were found. They occurred mainly in Experiment I (control, T4, PTU), in which older animals were used, and in controls of Experiment II. R,, and RL values of all three experiments expressed as change versus the corresponding control group are summed up in Fig. 6. The higher. doses of thyroid hormones led to an increase of the receptor capacity in the range of l&251% for R,, and of 184-219% for R, regarding the results of both sexes together. The effect of 500 and 5000 rig/g thyroxine was at about the same level for males as for females (R,, 225-257%; R,, 176-194%). The influence of higher doses of T, was more obvious in male X. laevis. They had increased values of R,, between 229 and 333% and of R, between 272 and 328% in comparison to 158-240% (R,) and 12%182% (RL) in females. Consequently, in male toads T3 was more effective than thyroxine expressed in a higher maximum
change versus control. In females T, was as effective as T4. Injections of thyroid hormones had no effect on the body weight, liver weight, liver body index, and the protein concentration in cytosol. Kd in females was unchanged at lower concentrations and decreased at higher concentrations of thyroid hormones. An increase of the dissociation constant occurred in male toads at the lower concentrations used (5 rig/g T,, 50 and 500 rig/g T4; Table 1). Variations of the concentration of corticosterone and aldosterone in plasma did not occur parallel or inverse parallel to changes in RO. Since the steroid concentration shows a strong variation in amphibia, a statement on the influence of thyroid hormones on these parameters requires a larger number of animals for testing. DISCUSSION 1. Sexual DifSerences and Influence Ovarian Hormones
of
In male and female X. Zaevis older than 4
CORTICOSTEROID
RECEPTORS
IN Xenupus
LIVER
BINDING CAPACITY I fmolfmg protein)
Ro
& P Pe,l$ P E, zms3 OuI-otiii
mP g pz iimm3 8Pflti
; &is; zzl?:aws: ;fLJecti*
EXP I
EXP. II
EXl? III
491
140 120 #O -
80 -
FIG. 4. t3H]Dexamethasone binding capacity R,, in liver cytosol after seven injections of vehicle, T3, Tq, or F’TU at the indicated concentrations on 7 consecutive days. The results of Experiments I-III are shown as mean values + SEM, each column representing a group of 10 toads (5 females, 5 males).
years, the liver weight, liver body index, the corticosterone concentration in serum, and the Kd of the glucocorticoid receptor were of the same range, However, statistically significant differences existed between both sexes in the receptor capacity RO,with 54.29 fmol/mg protein in males and 153.86 fmollmg protein in females. Expressed in binding sites per gram liver tissue (1.63 X lOI and 4.63 X 1012)these results are in good agreement with values of May and Westley (1982) of 1.4 x lOI* and 2.4 X lOI binding sites per gram liver in the same species. Other studies of amphibian glucocorticoid receptors did not regard sexual differences (Woody and Jaffe, 1982; Mehdi et ai., 1984; Incerpi et al., 1983). In younger toads (2 or 3 years old), which were used in the experiment with injection of thyroid hormones, sexual differences of the receptor capacity were much less expressed than those in ad& toads. Female toads with ovarian atrophy, which occurred incidentally among the animals used in the experiments, showed a
higher liver weight, liver body index, and volume of fat-body. In these animals the capacity of the gonads to store lipids and carbohydrates has been taken over by the liver and fat-body. A high percentage of female X, laevis with ovarian atrophy was found earlier by Alexander and Bellerby (1938) if the toa& were kept i.n an insufficient volume of water. These authors suggest a relation between the ovarian atrophy or retrogression and the seasonal decrease in water volume with overcrowdind of the ponds. Interestingly, females with ovarian atrophy and quite likely low levels of estrogen had low concentrations of glucocarticoid receptors comparable to the results in normal males. Female gonadal steroids thr;refore may be one hormonal factor which affects the glucocorticoid receptor level in the liver. The liver of X. laevis is known as a target organ of estradiol. A direct influ: ence of e&radio1 an toad liver is well known in the synthesis of vitellqgenin, a serum fipophosphoprotein which enters the ovary
492
LANGE, BINDING ( pm01 1
HANKE,
CAPACITY
AND
PER LIVER
MORISHIGE Rt
T
EXf? I EXI? II EXI? III 5. [3H]Dexamethasone binding capacity per liver R, after seven injections of vehicle, T,, T4, or PTU at the indicated concentrations on seven consecutive days. The results of Experiments I-III are shown as mean values f SEM, each column representing a group of 10 toads (5 females, 5 males). FIG.
to be transformed into yolk proteins (Wallace and Jared, 1969; Westley and Knowland, 1979). Experiments with glucocorticoid receptor determination in estradioltreated male toads should give further information if sexual differences and low R, values in ovarian atrophy are due to the different estradiol concentrations. 2. Influence of Thyroid Hormones Glucocorticoid Receptor
on the
Thyroid hormones and glucocorticoids play an important role in lung development in mammals and metamorphosis in amphibia. In mammals both kinds of hormones show comparable effects on different tissues, liver, fibroblasts, pituitary cell cultures, and lung (Shapiro and Sachchidananda, 1982; Smith, 1984; Martial et al., 1979; Nyborg et al., 1984; Gonzales et al., 1986). Results of Koch et al. (1978) in the pituitary of adult rats, of Morishige
(1982) in the lung of neonatal rats, of Naito et al. (1985) in the liver of adult rats, and our experiments in the liver of adult X. luevis demonstrate that thyroid hormones increase the level of glucocorticoid receptors. Parallel effects of both hormones on different tissues in different species therefore may be based on a regulation of glucocorticoid receptor capacity via thyroid hormones . Thyroid hormones and glucocorticoids both support the morphological and biochemical differentiation of fetal lung in lung tissue cultures (Gross et al., 1983; Zimmermann, 1985). Parallel effects of both hormone systems are known too for the carbohydrate metabolism in the liver of mammals. Thyroid hormones and glucocorticoids change the rate of synthesis (Mtiller et al., 1982; Iynedjian and Salavert, 1984; Yaroni and Balinsky, 1984) or the activity of some enzymes in the same sense (Malbon and Campbell, 1984;
CORTICOSTEROID
RECEPTORS
TABLE INFLUENCE Experiment R, (fmoYmg
I pr.)
RL @mol)
ii F M F
Kc+ WO Experiment R,, (fmoumg
II pr.)
Kd (nM)
ii F
Experiment R, (fmoUmg
Kc+ bW
Nore. between
Ill pr.)
79.56 96.93 x 2.54 x 4.82 2.58 2.93
rf 11.18 k 13.72 f 0.41 f 0.77 ? 0.36 ” 0.45
Control M F M
RI. (pmol)
RL f&-d)
Control M
39.74 64.41 x 0.80 n 1.51 4.52 4.59
2 7.63 in 13.02 f 0.16 + 0.11 + 0.64 f 0.49
Control M F M F M F
OF T,,
OF INJECTIONS
45.51 f 11.98 48.51 k 9.72 1.32 k 0.33 1.77 t- 0.39 5.36 2 0.37 8.09 2 1.45
Mean values + SEM of Experiments the sexes (x) and between control
T, 5 n&z 62.23 90.73 3.55 4.51 x 5.91 x 2.87
+ + f -+ f ?
4.48 11.42 0.75 0.38 1.01s 0.43
T3 5 rids 56.62 f 15.89 37.46 f 11.38 0.88 t‘ 0.16 0.71 ” 0.17** x 5.38 t‘ 0.34 x 3.79 k 0.56 T, 50 rig/g 151.36 96.83 4.11 2.29 4.66 5.46
-c -L: tk rt +
21.55** 29.65 0.90* 0.67 0.11 0.62
IN Xenopus
1 TV, AND
PTU
0~
R,, R,,
T4 50. rip/p 89.92 84.90 n 2.49 x 4.90 x 4.17 x 2.51
c f 2 +r t
27.95 12.25 0.45 0.77 0.44* 0.51
-+ 2 2 f + t
9.25** s.27* 0,41** 0.23* 1.10 0.49
PTlJ
t 2 ‘k +i:
21.25* 20.05* 0.61* 0.85 2.27 0.80’
7.9@ 9.88 0.36 0.65 0.67* 0.29
Pm
3 lJ& f 9.40 -c 8.89 f 0.17 + 0.17 zk 1.06 2 0.76
T4 500 nglg 102.31 k 124.39 2 2.46 -t 3.29 + x 8.86 -+ x 4.56 r
I-III for males (M) and females (F) separately. and hormone-injected groups (** **) are indicated.
Stalmans and Laloux, 1979; Malbon and LoPresti, 1981). In amphibia studies on the influence of both hormones on liver metabolism mainly are restricted to the period of metamorphosis. In the liver of R. catesbeiana tadpoles T3 and T4 receptors (Galton and Schaafsma, 1983) and glucocorticoid receptors (Woody and Jaffe, 1984) were demonstrated. Comparable effects of thyroid hormones and corticosteroids on mammalian liver tissue were supposed to be independent from each other in some cases (Miiller et al., 1982; Yaroni and Balinsky, 1984) or synergistic in another (Shapiro and Sachchidananda, 1982). Again the regulation of glucocorticoid receptors via thyroid hormones may be one base of synergistic effects. An increase of the glucocorticoid receptor capacity occurred in the liver of adrenalectomized adult rats 24 hr after an injection of T, (Naito et al., 1985). In amphibia, in the liver of X. laevis, R. was significantly higher in toads with enlarged thyroid gland, whereas &, the liver weight, the liver body
3 Ia43 5 ” t ” C +-
51.63 51.68 1.02 1.33 5.62 4.08
T3 500 ngig 111.93 116.28 3.58 3.24 9.01 3.99
K~
AND
47.75 63.61 x 2.21 x 4.66 x 4.84 x 2.52
T, 50 wk 91.06 101.87 2.62 2.17 x 6.94 x3.99
493
LIVER
26.76 16.22”* 0.72 0.65 0.99*+ 0.19*
Statistically
T4 5000 wk 102.32 121.88 2.55 3.13 4.99 5.46 significant
f -c ” + _” 2
21.04* 29.33* OS5 0.79 0.44 0.61
differences
index, and the concentration of corticosteroids in serum were not different compared to those of normal toads. Our experiments with injections of T, or T4 revealed an increase of R0 up to 250% of the controls. This effect of thyroid hormones, which indicates hyperthyroidism in toads with enlarged thyroid gland, was more obvious in the liver of X. luevis than in the liver of adult rats (Naito et al., 1985; < 160%) and in the lung of neonatal rats (Morishige, 1982; < 170%) * Total concentrations of corticosterone and aldosterone were not different in normal toads and toads with enlarged thyroid gland. After injections of T, or T,,it was not different or slightly higher than that in the control group. Therefore the increase of l?, and RL is possibly not due to a decreased rate of receptor translocation into the nucleus and an enhanced rate of receptor replenishment out of the nucleus, which would be expected together with reduced levels of corticosteroids. Howev,er, changes of the amount of free, available corticosteroids under the influence of T,
494
LANGE,
I
(%
versus
HANKE,
AND
MORISHIGE
control)
Rs
RL
300
0 I
TL (nglgl
T3 (ngfg) 5
5 50
50 500
50 5005000
PTU kg/g)
3 3
I II II III III I II1 III I II FIG. 6. Influence of injections of T,, T,, and PTU on R, and R, in liver cytosol of X. laevis expressed in percentage change versus controls. The columns, arranged according to dose, depict the vaiues for both sexes combined and for male and female toads separately. For experimental conditions see Fig. 4. EXf?
and T4 cannot be excluded because inconsistent results have been found in mammals (Westphal, 1986; Henning et al., 1986). Further experiments with determination of nuclear receptor will shed further light on this question. In R. catesbeiana tadpoles the glucocorticoid receptor capacity decreased during metamorphosis. It was not changed by the addition of T, to the tap water (Woody and Jaffe, 1984). These results certainly are impaired by the increasing levels of corticosteroids (Jaffe, 1981) during spontaneous and induced metamorphosis. The effective concentrations of T3 (50 ng/ g) and T4 (500 rig/g) found in our experi-
ments were high in comparison to maximal physiological concentrations of about 15 pg T, and 1000 pg T, per milliliter of plasma in other species of amphibia (Kuhn et al., 1985; Tasaki et al., 1986). However, they correspond with results of McNabb (1969), who found significant alterations of the nitrogen excretion of R. pipiens after injections of 1000 ng but not of 100 ng T4 per gram and day. A clear dose-response relationship might not be found because of the treatment for as long as 7 days. Experiments with shorter treatments may show a gradual increase in receptor levels with increasing T, and T, doses.
CORTICOSTEROID
RECEPTORS
T3 injections were more potent in male toads than in females to increase &,, whereas T4 showed about the same effect in both sexes. In her study on T3 and T, receptors Galton (1986) concluded that liver nuclei of R. catesbeiana contain one set of high affinity, low capacity thyroid hormone binding sites, with similar capacity and two- to threefold higher affinity for T3 than for T4. With this assumption the sexual differences in effectiveness of T, and T4 found are not due to a different R,. They may be due to sexual differences in the & of the thyroid hormone receptor, in transport rate, or metabolism of T, and T,. A decrease of R, after injections of PTU is not perceptible in our experiments. Accordingly, the glucocorticoid receptor capacity was not reduced after thyroidectomy in adult rats (Naito et al., 1985; Koch et al., 1978). The limited amount of collected blood did not allow us to measure the steroid and the T, and T, concentrations in parallel. Therefore, the known hypothyroid effect of PTU was not proved. The high values of R, after treatment with T3 and T4 may be based (1) on a higher rate of synthesis, (2) on a lower rate of degradation of the glucocorticoid receptor, or (3) on the activation of inactive receptor molecules. Rousseau (1984) suggested a model of an intracellular receptor cycle. The receptor is recycled in the cytoplasm in an oxidized, nonbinding form, which is then converted to the form that binds steroids by a NADPH-dependent, thioredoxin-mediated reducing system. Naito et al. (1985) propose that the increase of R. after injections of thyroid hormones may be correlated with its conversion to the steroid binding form; thyroid hormones induce NADPdependent enzymes and in this way enhance NADPH production and shift the equilibrium toward the form that binds steroids. Similar effects of thyroid hormones and corticosteroids in liver metabolism, e.g., on NADPH-dependent enzymes in
IN Xenopus
495
LIVER
adult amphibia (Weindel and Hanke, 1988; Kasprzyk and Obuchowicz, 1980), are in contradiction to this explication. Like thyroid hormones, corticosteroids should increase the level of the steroid binding receptor form, but only the contrary is known (Svec, 1985). REFERENCES Alexander, S. S., and Bellerby, C. W. (1938). Experimental studies on the sexual cycle of the South African clawed toad (Xenopus laevis). I. J. Exp. Biol.
15, B-81.
Coulson, P. B., Thornwaite, J. T., S&afar, D. F., and Seaver, S. S. (1982). Modulation of glncocorticoid hormone receptor levels in chicken lymphoid tissue following treatment with androgens in viva. J. Steroid Biochem. 17, l-9. Galton, V. A. (1986). Throxine and 3,5’,3’-triiodothyronine bind to the same putative receptor in hepatic nuclei of Rana catesbeinana tadpoles. Endocrinology 118, 11144118. Galton, V. A., and Schaafsma, J. (1983). Binding of thyroid hormones to isolated hepatic nuclei from Rana catesbeiana tadpoles. Endocrinology 11, 1999-2006. Gonzales, L. W., Ballard, P. L., Ertsey, R., and Williams, M. C. (1986). Glucocorticoids and thyroid hormones stimulate biochemical and morphological differentiation of human fetal lung in organ culture. J. Clin. Endocrinol. Metab. 62, 678-691. Gross, I., Ballard, P. L., Ballard, R. A., Jones, C. T., and Wilson, C. M. (1983). Corticosteroid snmulation of phosphatidylcholine synthesis in cultured fetal rabbit lung: Evidence for de nova protein synthesis mediated by glucocorticoid receptors. Endocrinology
112,829-837.
Henning, S. J., Leeper, L. L., and Dieu, D. N. (1986). Circulating corticosterone in the infant rat: The mechanism of age and thyroxine effects. Pediarr. Res. 20, 87-92.
Incerpi, S., Luly, P., and Scapin, S. (1983). Glucocorticoid receptor of frog (Rana escuienttc) liver. Comp.
Biochem.
Physiol.
B. 75, 645-648,
lynedjian, P. B. and Salavert, A. (19@).. Effects of glucagon, dexamethasone and triiodothyronine on phosphoenolpyruvate carboxykinase (GTP) synthesis and mRNA level in rat liver cells. Ear. J. Biochem.
145,483-1497.
Jaffe, R. C. (1981). Plasma concentration of. corticosterone during Rana catesbeiamz tadpole metamorphosis. Gen. Camp. Endocrinot. 44,316.328. Kalimi, M., and Hubbard, J. R. (1983). Glucocorticoid receptors. In “Principles of Receyterology”
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HANKE,
(M. K. Agarwal, Ed.), pp. l-67. de Gruyter, Berlin/New York. Kasprzyk, A., and Obuchowicz, L. (1980). The effect of thyroxine and triiodothyronine on glucose6-phosphate and 6-phosphogluconate dehydrogenase activity in liver and fat body of the frog Rana esculenta. Gen. Comp. Endocrinol. 42, 384-388. Koch, B., Lutz-Bucher, B., Briaud, B., and Mialhe, C. (1978). Inverse effects of corticosterone and thyroxine on glucocorticoid binding sites in the anterior pituitary gland. Acta Endocrinol.-88, 2937. Kuhn, E. R., Darras, V. M., and Verlinden, T. M. (1985). Annual variations of thyroid reactivity following thyrotropin stimulation and circulating levels of thyroid hormones in the frog Rana ridibunda. Gen. Comp. Endocrinol. 57, 2&273. Lange, C. B., and Hanke, W. (1988). Corticosteroid receptors in liver cytosol of the clawed toad, Xenopus laevis: Daily and seasonal variations. Gen. Comp. Endocrinol. 71, 141-152. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. Z. Biol. Chem. 193,265275. Malbon, C. C., and Campbell, R. (1984). Thyroid hormones regulate hepatic glycogen synthase. Endocrinology 115, 681686. Malbon, C. C., and LoPresti, J. J. (1981). Hyperthyroidism impairs the activation of glycogen phosphorylase by epinephrine in rat hepatocytes. J. Biol. Chem. 256, 12199-12205. Martial, J. A., Seeburg, P. H., Matulich, D. T., Goodman, H. M., and Baxter, J. D. (1979). Regulation of growth hormone messenger RNA. Zn “Glucocorticoid Hormone Action” (J. D. Baxter and G. G. Rousseau, Eds.), pp. 279-289. SpringerVerlag, Berlin/Heidelberg/New York. May, F. E. B., and Westley, B. R. (1982). Glucocorticoid receptor of Xenopus laevis: Possible effect of phosphorylation on hormone binding. Mol. Cell. Endocrinol. 26, 103-1’17. McNabb, R. A. (1969). The effects of thyroxine on glycogen stores and oxygen consumption in the leopard frog, Rana pipiens. Gen. Comp. Endocrinol. 12, 276-281. Mehdi, A. Z., DiBattista, J. A., and Sandor, T. (1984). Glucocorticoid receptors of the American bullfrog (Rana catesbeiana). Gen. Comp. Endocrinol. 53, 475. Morishige, W. K. (1982). Thyroid hormone influences glucocorticoid receptor levels in the neonatal rat lung. Endocrinology 11, 1017-1019. Morishige, W. K, and Joun, N. S. (1982). Influence of glucocorticoids on postnatal lung development in the rat: Possible modulation by thyroid hormone. Endocrinology 111, 1587-1594.
AND
MORISHIGE
Miiller, M. J., Thomsen, A., Sibrowski, W., and Seitz, H. J. (1982). 3,5,3’-Triiodothyronineinduced synthesis of rat liver phosphoenolpyruvate carboxykinase. Endocrinology 111, 14691475.
Naito, K., Isohashi, F., Tsukanaka, K., Horiuchi, M., Okamoto, K., Matsunaga, T., and Sakamoto, Y. (1985). Effects of D- and t-thyroxine on the glucocorticoid binding capacity of adult rat liver. Biochem. Biophys. Res. Commun. 129, 447-452. Nyborg, J. K., Nguyen, A. P., and Spindler, S. R. (1984). Relationship between thyroid and glucocorticoid hormone receptor occupancy, growth hormone gene transcription, and mRNA accumulation. J. Biol. Chem. 259(20), 12377-12381. Rousseau, G. G. (1984). Structure and regulation of the glucocorticoid hormone receptor. Mol. Cell. Endocrinol. 38, l-l 1. Scatchard, G. (1949). The attractions of proteins for small molecules and ions. Ann. N.Y. Acnd. Sci. 51, -72.
Shapiro, L. E., and Sachchidananda, J. (1982). Regulation of proteins by thyroid hormone and glucocorticoid: The responses of hepatic a,,-globulin and pituitary growth hormone differ in adult male hypothyroid rats. Endocrinology 111, 653-660. Smith, T. J. (1984). Dexamethasone regulation of glycosaminoglycan synthesis in cultured human skin fibroblasts. J. Clin. Invest. 74, 2157-2163. Stalmans, W., and Laloux, M. (1979). Glucocorticoids and hepatic glycogen metabolism. In “Glucocorticoid Hormone Action” (J. D. Baxter and G. G. Rousseau, Eds.), pp. 517-533. Springer-Verlag, Berlin/Heidelberg/New York. Svec, F. (1985). Glucocorticoid receptor regulation. Life Sci. 36, 23.59-2366. Tasaki, Y., Inoue, M., and Ishii, S. (1986). Annual cycle of plasma thyroid hormone levels in the toad, Bufo japonicus. Gen. Comp. Endocrinol. 62,404-410. Thurmond, W., Kloas, W., and Hanke, W. (1986). Circadian rhythm of interrenal activity in Xenopus laevis. Gen. Comp. Endocrinol. 61, 260-271. Wallace, R. A., and Jared, D. W. (1969). Studies on amphibian yolk. III. The estrogen-induced hepatic synthesis of a serum lipophosphoprotein and its selective uptake by the ovary and transformation into yolk platelet proteins in Xenopus Zaevis. Dev. Biol. 19, 498-526. Weindel, K., and Hanke, W. (1988). The effect of corticosteroids on the lipogenesis of the liver of the clawed toad Xenopus laevis. Acta Endocrinol. 117 Suppl. 287, 223-224. Westley, B., and Knowland, J. (1979). Estrogen causes a rapid, large and prolonged rise in the level of nuclear estrogen receptor in Xenopus lae-
CORTICOSTEROID
RECEPTORS
vis liver. Biochem. Biophys. Res. Commun. 88, 1167-I 172. Westphal, U. (1986). “Steroid-Protein Interactions II.” Springer-Verlag, Berlin/New York. Woody, C. J., and Jaffe, R. C. (1982). Partial characterization of frog (Rana catesbeiana) hepatic glucocorticoid receptor. Gen. Comp. Endocrinol. 47, 28-35. Woody, C. J., and Jaffe, R. C. (1984). Binding of dexamethasone by hepatic, intestine, and tailfin
IN
Xenopus LIVER
497
cytosol in Rana catesbeiana tadpoles during spontaneous and triiodothyronine-induced metamorphosis. Gen. Camp. Endocrinol. 54, 194-202. Yaroni, S., and Balinsky, J. B. (1984). Hormonal effects on liver glutamate dehydrogenase in adrenalectomized. and thyroidectoniized rats. Endocrinology 114, 2337-2343. Zimmermann, B. (1985). Influence of corticosteroids and thyroxine on lung development in vitro. Acta Endocrinol. 108, Suppl. 267, 35-36.
AND
COMPARATIVE
73,485-497
ENDOCRINOLOGY
(1989)
Corticosteroid Xenopus
Receptors in Liver Cytosol of the Clawed Toad, laevis: Influence of Thyroid and Ovarian Hormones’
C. B. *Department
of Zoology, and fDepartment
LANGE,*
The University, of Physiology,
W.
HANKE,*,’
AND
W.
K. MORISHIGE~
Kaiserstrasse 12, D-7500 Karlsruhe, John A. Burns School of Medicine, Honolulu, Hawaii 96822
Federal Republic of Germany; University of Hawaii,
Accepted August 26, 1988 The glucocorticoid receptor capacity R, and the dissociation constant Kd were determined in the liver of Xenopus laevis by Scatchard analysis. In S-year-old female toads RO was about three times higher than that in males (153.9,54.3 fmol/mg protein) and Ka was similar in both sexes (4.0, 4.1 n&f). Some of the animals used had abnormal enlarged thyroid glands, atrophic ovaries, or both defects in connection with different levels of R,, but not of Kd, compared to those of normal animals. Females with ovarian atrophy showed significantly lower R, values, in the same range as in normal males, and a high liver weight. In male and female toads with enlarged thyroid glands and in animals with both defects a significantly higher R. occurred compared to that of the corresponding group without this abnormality. To study the influence of thyroid hormones on glucocorticoid receptors, young toads (2-3 years old) received injections of 4-phenyL2thiouraciI T,, or T, on 7 consecutive days. R0 and Kd were determined on the following day. Doses of 50 and 500 ng T, and of 500 and SO00 ng T4 per gram of body weight and day resulted in an increase of R,, up to 250% of the controls. Injections of T, were more efficient in males than in females. The effect of thyroxine was about the same in both sexes. These observations suggest that thyroid and ovarian hormones exert an influence on glucocorticoid receptor capacity and may belong to the factors which regulate glucocorticoid receptors. 0 1989 Academic PWSS, 1~.
The presence of glucocorticoid receptors in the liver of Amphibia is well known. The characteristics of these receptors have been described in a few studies in Rana catesbeiana (Woody and Jaffe, 1982, 1984; Mehdi et al., 1984), in Rana esculenta (Incerpi et al., 1983), and in Xenopus laevis (May and Westley, 1982). In a previous paper, we reported on the binding capacity R, and the dissociation constant Kd in liver cytosol of X. laevis. Diurnal variations of R, were found and correlated with the corticosteroid concentration in the serum of young X. laevis . Seasonal variations showed different patterns ’ This work was supported by Deutsche Forschungsgemeinschaft. ’ To whom requests for reprints should be addressed.
for male and female adult toads with higher receptor value3 always in females.. During the year no correlation occurred between R, and the corticosteroid concentration in serum (Lange and Hanke, 1988). Since glucocorticoid receptors mediate the glucocorticoid effects, the regulation of the receptor protein in vivo is of great interest, although only little information is present (Kalimi and Hubbard, 1983; Rousseau, 1985). A few reports deal with a major area in this line of studies, the hormonal regulation of the receptor. High doncentrations of glucocorticoids dimini,shed the receptor concentration in AtT-20 mouse pituitary tumor cells (Svec, 1985). Injections of dihydrotestosterone reduced the, glucocorticoid receptor levels in thymus a@ bursa of Fabricius tissue in chicken (Coulson et al., 1982). Morishige and co-workers (Mor-
485 0016-6480189 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
486
LANGE,
HANKE,
ishige and Ioun, 1982; Morishige, 1982) showed in a developmental study that the increase of glucocorticoid receptors in rat lung during the first week after birth may be regulated by thyroid hormones and Naito ef al. (1985) found an increase of the receptor capacity in the liver of adult adrenalectomized rats after a single injection of either D- or L-thyroxine. In the adult male and female X. Zuevis used in our experiments, two abnormalities occurred: females with atrophic ovaries and toads of both sexes with enlarged thyroid glands. The finding that these defects were parallel with changed receptor concentrations gave rise to further experiments on the influence of thyroid hormones on glucocorticoid receptors.
AND MOFUSHIGE
[1,2(n)-3H]Dexamethasone (30-50 Wmmol; Amersham Buchler, Braunschweig) was dissolved in double distilled water. The stock solutions of unlabeled dexamethasone (Serva, Heidelberg) were prepared in absolute ethanol. L-3,3’,5-Triiodothyronine (T,), Lthyroxine Cr,), and 4-phenyl-2-thiouracil (PTU) were purchased from Serva (Heidelberg). T,, T4, and PTU were dissolved in 0.1 N NaOH frog Ringer and diluted with pure frog Ringer (6 g NaCl, 0.2 g KCl, 0.2 g CaC!, * 6Hz0, 0.1 g NaHCO, in 1 liter distilled water). The final solution (100 ~1) contained the dose for an animal of 10-g weight.
undeveloped or reduced ovaries, and two female toads showed both defects. Because of the limited amount of collected blood only a few determinations cauld be done with these animals. A significantly higher amount of T4 was found in these preliminary experiments when the thyroid gland was enlarged. Since abnormal thyroid glands occur in only a few cases and can be detected only by dissection, carefully planned experiments were not possible. (b) Experiments with injection of T3, T4, or PTU (2 or 3 years 016). At least 2 months before starting the experiments, male and female toads were kept separately in basins with 10 animals under a daylight regime receiving artificial light from 6:00 to 18:00 hr. They were maintained at 20 + 1” and fed daily with dry food pellets, and the water was changed twice a week. For the experiments groups of five male and five female toads were studied. On 7 consecutive days, between 9:00 and 10:30, each toad was weighed and received an injection of T,, T4, PTU, or vehicle into the dorsal lymphatic vessel. Doses of $50, and 500 ng T,, of 50, 500, and 5000 ng T4, and of 3 pg PTU per gram of body weight were tested. Since 100 ~1 of the prepared solution contained the dose for a 10-g toad, the volume of the injection varied with the weight of the animals. Receptor determination was done the day after the last injection. Experiments were performed in December 1985 (I) and February (II) and June 1986 (III) with toads of about 3 years (I) and 2 years of age (II, III). A total of 65 males and 65 females were sacrificed. To avoid influence of the time of day, the time consuming receptor assay was restricted to 10 animals (2 groups) per day with the following schedule of receptor assays (e.g., Experiment I): Day l-control males and T3 females; Day 2-T, males and T4 females; Day 3--T, males and PTU females; Day 4-PTU males and control females.
Animals
Preparation of Cytosol
X. Zuevis Daudin used in the experiments were raised from the animal stock in our department. During their lifetime they were kept at about 17” and fed with minced beef heart once a week after metamorphosis. (a) Normal and abnormal adult toads (4-5 years old). Between April 1984 and May 1985 receptor assays were performed in toads which had all passed metamorphosis in June 1980. Each month groups of animals which were last fed 1 week previously were sacrificed between 9:30 and 11:30 hr. In the toads used in these studies two abnormalities occurred in some of the animals. In eight male and five female toads a very enlarged thyroid gland was found, three female toads at the age of more than 4 years had
The liver was rapidly excised, blotted, weighed, and kept on ice. It was cut into small pieces (1 mm) and rinsed twice in chilled buffer (20 mM Tris, pH 7.4 at OX, 250 mM sucrose, 10 mM sodium molybdate, 5 mM dithiothreitol). The tissue was homogenized in 5 vol (w/v) of buffer in a glass-Teflon Potter tissue grinder and centrifuged at 10,OOOgfor 10 min. The lipid layer was discarded and the supernatant centrifuged (100,OOOg for 60 min). The obtained cytosol fraction was used immediately for the binding studies. All subsequent steps were carried out at O-4’. Perfusion of the liver was not possible in groups of these small toads. So, arising from the fact that in toads erythrocytes also contain nuclei which cannot be separated from liver nuclei, a determination of nuclear
MATERIALS
AND METHODS
Materials
CORTCCOSTEROID
RECEPTORS
IN
Xenopus
LIVER
487
ion-like cartilage in male toads. In normal animals of 20-30 g body weight the paired, slightly pigmented, and light-rose gland Binding Studies weighed 5 to 10 mg with both moieties disA Scatchard analysis (Scatchard, 1949) was carried tinguishable from each other. The abnormal out to define Kd (apparent dissociation constant) and thyroid glands were much heavier in the R, (binding capacity, fmohmg protein). After a 15-min range of 50-500 mg. The tissue was dark preincubation in the absence (total binding) or presred and compact. The moieties of the gland ence of 10 pJ4 unlabeled dexamethasone were close together and some of the folli(“nonspecific” binding) aliquots of 0.1 ml cytosol were incubated with varying concentrations of cles were visible with an unaided eye. His[3H]dexamethasone (0.05 ml; final concentrations of tological sections of either a normal or an about 1, 5, 10, 25, 50, and 100 nM) for 24 hr at O-4”. enlarged thyroid gland at the indicated magThe dextran-coated charcoal technique was used to separate free from macromolecular bound labeled ste- nification (Fig. 1) showed differences in structure and size. roid. To each ali,quot 200 p,l of dextran-coated charcoal suspension (20 mM Tris, pH 7.4, 3.75% Norit A, The results in the six occurring groups of 0.375% dextran T70) was added for a 5-min incubation toads, normal toads (N), toads with enwith vortexing. Only the free steroid is adsorbed by larged thyroid gland (T) of both sexes, fethe activated charcoal. In a subsequent centrifugation (ZOOOg, 15 min) the macromolecular bound steroid re- males with ovarian atrophy (0), and femains in the supernatant, which was decanted and males with both defects (OT) are shown in stored in the deep freeze at -20”. Radioactivity was Figs. 2 and 3. measured by liquid scintillation counting (Tri-Carb The dexamethasone binding capacity R, 300; Packard Instrument) in an ahquot of 200 p,l of and the dissociation constant & resulted thawed supematant dissolved in 3 ml counting solution from Scatchard analysis of a saturation (Aqualuma Plus, Lumac, Shaesberg, Netherlands). Quench correction was done by the method of the curve. Significant differences between the spectral index of the external standard. The specific groups were not found for the Kd, with 4.1 binding, the difference between total binding and non+- 0.3 and 4.0 + 0.6 n&fin normal males and specific binding, R,, and Kd were calculated. females, respectively. The R0 values showed statistically significant differences Other Procedures among the six groups. In female toads Ra Blood samples taken from the Truncus arteriosus was about three times higher than that in were centrifuged and the serum was stored in the deep males (Fig. 2). In toads with enlarged thyfreeze at -20” until analyzed. Total corticosterone roid gland a much higher R, occurred comand aldosterone concentration in serum were estipared to that of the corresponding group mated by a RIA (Thurmond et al., 1986) and cytosol without this abnormality. This effect held protein concentration was estimated by the method of Lowry et al. (1951). Statistical evaluation of data was true for male and female normal X. la&is performed by Student’s t test (*: 0.05 > P). Since the and for females with ovarian atrotihy and groups of abnormal toads contain only a few animals was statistically significant in all three (N = 2 to 8) nonparametric statistical tests (Wilcoxcases. In contrast, the presence of abnoron’s U test, Duncan’s new multiple range test) were performed. The design for statistical differences (*) mal ovaries was parallel with a significantly was always the same. lower R, in liver cytosol in comparison to that of normal females. R, was at the same RESULTS low range in these animals as in boreal males. I. Comparison of Kd and R, in Normal To estimate the cytosol receptor capacity and Abnormal Adult Toads in the whole liver, R, was calculated acThe thyroid gland in X. laevis is placed at cording to the formula RL = Ii0 x p x LW/ the ventr+cranial end of the larynx, which 100 Cp = protein concentration in mg/lOO is a flat cartilage in female and a thick cush- mg liver tissue; LW = liver weight in mg). corticosteroid receptors was not performed. Further experiments are planned in this respect.
488
LANGE,
HANKE,
AND MORISHIGE
FIG. 1. Histological section of a normal (a) and an enlarged (b) thyroid gland (Masson-Goldner stain). Bar = 100 p,m.
The same pattern of effects on R, was foun .d for R, with increased values in toads with enlarged thyroid glands and reduced V&l es in toads with ovarian atrophy. The diffe :rences between the groups were statis-
tically more evident for R, than for RL (Fig. 2). The liver weight and the liver body in tdex (liver weight in percentage of the b ody weight) were of the same range in not *mal
CORTICOSTEROJD
RECEPTORS
(a) BINDING CAPACITY
R,
(fmollmg
t
protein)
IN Xenopus
489
LIVER
I
(bl BINOING CAFACITY PER LIVER R,(pmol per Liver)
250
200 150
100
50 1 N T In1
1421
I.91
MALES
N T
N T 0 OT 1321
151
131
121
FEMALES
InI
I621
III
MALES
NTOOT WI
19
13)
I21
FEMALES
FIG. 2. [3H]Dexamethasone binding capacity R, (a) and binding capacity per liver R, (b) in different groups of adult X. Zaevis, in normal toads (N), in toads with enlarged thyroid gland (T), in females with atrophic ovaries (O), or both abnormalities (OT). Lines above the columns depict statistically significant differences between the groups (mean values i- SEM).
toads and toads with enlarged thyroid gland. These parameters were significantly higher in female toads with atrophic ovaries (0 and T, Fig. 3). The corticosterone concentration in serum showed no detectable differences among the groups. The aldosterone concentration in serum was higher in females than in males with the highest concentration in females with both defects (OT). 2. Injluence of Injections of T3, T4, and PTU on I& and R,
The striking results in toads with abnormal thyroid gland prompted us to study the influence of T,, Tq, or PTU injections on the [3H]dexamethasone binding capacity R, and the apparent dissociation constant Kd. In controls, the binding’ capacity of the older animals used in Experiment I was much higher than that in the younger ani-
maIs of Experiments II and III (Figs. 4 and 5). Injections of PTU in the older anim& of Experiment I caused a sign&ant d:ecrease of R, together with a slight increase of the liver weight (males: 768 + 115mg, PTTJ 933 + 260 mg; females: 1182 -+ 200 mg,,,PTU 1480 t 227 mg), Therefore, no change in R, was found. In the younger animals used in Experiment II, PTU had no effect on both R, and RL. Low doses of 5 rig/g T3 and 50 rig/g Tq (I, 11) had no effect on the receptor capacity expressed as either R, or R,. However, after injection of 50 rig/g T3, 500 rig/g T3, %O nglg Tq, or 5000 nglg T,, RD and R, were about two- to threefold significantly, ,higher than those in the control group (II, III). ‘Significant differences were ,not found between the groups of hormone-injected toads in Experiment III (Figs. 4 and 5, Table 1).
490
LANGE,
(a)
HANKE,
LIVER WEIGHT (mg)
AND MORISHIGE
(b)
LIVER BODY INDEX (% of the body weight)
-*
N T In1 ILlI 181 MALES
N T Cl OT 1355151 ml 121 FEMALES
NT In1 IUJ I81 MALES
* *
N T 0 OT I351 (51 131 I21 FEMALES
FIG. 3. The liver weight expressed either in milligrams (a) or in percentage of the body weight (b) in different groups of adult X. Zuevis. Groups and statistics as in Fig. 2.
Table 1 shows the results for male and female toads separately. Sexual differences in R,, but not in R,, were found. They occurred mainly in Experiment I (control, T4, PTU), in which older animals were used, and in controls of Experiment II. R,, and RL values of all three experiments expressed as change versus the corresponding control group are summed up in Fig. 6. The higher. doses of thyroid hormones led to an increase of the receptor capacity in the range of l&251% for R,, and of 184-219% for R, regarding the results of both sexes together. The effect of 500 and 5000 rig/g thyroxine was at about the same level for males as for females (R,, 225-257%; R,, 176-194%). The influence of higher doses of T, was more obvious in male X. laevis. They had increased values of R,, between 229 and 333% and of R, between 272 and 328% in comparison to 158-240% (R,) and 12%182% (RL) in females. Consequently, in male toads T3 was more effective than thyroxine expressed in a higher maximum
change versus control. In females T, was as effective as T4. Injections of thyroid hormones had no effect on the body weight, liver weight, liver body index, and the protein concentration in cytosol. Kd in females was unchanged at lower concentrations and decreased at higher concentrations of thyroid hormones. An increase of the dissociation constant occurred in male toads at the lower concentrations used (5 rig/g T,, 50 and 500 rig/g T4; Table 1). Variations of the concentration of corticosterone and aldosterone in plasma did not occur parallel or inverse parallel to changes in RO. Since the steroid concentration shows a strong variation in amphibia, a statement on the influence of thyroid hormones on these parameters requires a larger number of animals for testing. DISCUSSION 1. Sexual DifSerences and Influence Ovarian Hormones
of
In male and female X. Zaevis older than 4
CORTICOSTEROID
RECEPTORS
IN Xenupus
LIVER
BINDING CAPACITY I fmolfmg protein)
Ro
& P Pe,l$ P E, zms3 OuI-otiii
mP g pz iimm3 8Pflti
; &is; zzl?:aws: ;fLJecti*
EXP I
EXP. II
EXl? III
491
140 120 #O -
80 -
FIG. 4. t3H]Dexamethasone binding capacity R,, in liver cytosol after seven injections of vehicle, T3, Tq, or F’TU at the indicated concentrations on 7 consecutive days. The results of Experiments I-III are shown as mean values + SEM, each column representing a group of 10 toads (5 females, 5 males).
years, the liver weight, liver body index, the corticosterone concentration in serum, and the Kd of the glucocorticoid receptor were of the same range, However, statistically significant differences existed between both sexes in the receptor capacity RO,with 54.29 fmol/mg protein in males and 153.86 fmollmg protein in females. Expressed in binding sites per gram liver tissue (1.63 X lOI and 4.63 X 1012)these results are in good agreement with values of May and Westley (1982) of 1.4 x lOI* and 2.4 X lOI binding sites per gram liver in the same species. Other studies of amphibian glucocorticoid receptors did not regard sexual differences (Woody and Jaffe, 1982; Mehdi et ai., 1984; Incerpi et al., 1983). In younger toads (2 or 3 years old), which were used in the experiment with injection of thyroid hormones, sexual differences of the receptor capacity were much less expressed than those in ad& toads. Female toads with ovarian atrophy, which occurred incidentally among the animals used in the experiments, showed a
higher liver weight, liver body index, and volume of fat-body. In these animals the capacity of the gonads to store lipids and carbohydrates has been taken over by the liver and fat-body. A high percentage of female X, laevis with ovarian atrophy was found earlier by Alexander and Bellerby (1938) if the toa& were kept i.n an insufficient volume of water. These authors suggest a relation between the ovarian atrophy or retrogression and the seasonal decrease in water volume with overcrowdind of the ponds. Interestingly, females with ovarian atrophy and quite likely low levels of estrogen had low concentrations of glucocarticoid receptors comparable to the results in normal males. Female gonadal steroids thr;refore may be one hormonal factor which affects the glucocorticoid receptor level in the liver. The liver of X. laevis is known as a target organ of estradiol. A direct influ: ence of e&radio1 an toad liver is well known in the synthesis of vitellqgenin, a serum fipophosphoprotein which enters the ovary
492
LANGE, BINDING ( pm01 1
HANKE,
CAPACITY
AND
PER LIVER
MORISHIGE Rt
T
EXf? I EXI? II EXI? III 5. [3H]Dexamethasone binding capacity per liver R, after seven injections of vehicle, T,, T4, or PTU at the indicated concentrations on seven consecutive days. The results of Experiments I-III are shown as mean values f SEM, each column representing a group of 10 toads (5 females, 5 males). FIG.
to be transformed into yolk proteins (Wallace and Jared, 1969; Westley and Knowland, 1979). Experiments with glucocorticoid receptor determination in estradioltreated male toads should give further information if sexual differences and low R, values in ovarian atrophy are due to the different estradiol concentrations. 2. Influence of Thyroid Hormones Glucocorticoid Receptor
on the
Thyroid hormones and glucocorticoids play an important role in lung development in mammals and metamorphosis in amphibia. In mammals both kinds of hormones show comparable effects on different tissues, liver, fibroblasts, pituitary cell cultures, and lung (Shapiro and Sachchidananda, 1982; Smith, 1984; Martial et al., 1979; Nyborg et al., 1984; Gonzales et al., 1986). Results of Koch et al. (1978) in the pituitary of adult rats, of Morishige
(1982) in the lung of neonatal rats, of Naito et al. (1985) in the liver of adult rats, and our experiments in the liver of adult X. luevis demonstrate that thyroid hormones increase the level of glucocorticoid receptors. Parallel effects of both hormones on different tissues in different species therefore may be based on a regulation of glucocorticoid receptor capacity via thyroid hormones . Thyroid hormones and glucocorticoids both support the morphological and biochemical differentiation of fetal lung in lung tissue cultures (Gross et al., 1983; Zimmermann, 1985). Parallel effects of both hormone systems are known too for the carbohydrate metabolism in the liver of mammals. Thyroid hormones and glucocorticoids change the rate of synthesis (Mtiller et al., 1982; Iynedjian and Salavert, 1984; Yaroni and Balinsky, 1984) or the activity of some enzymes in the same sense (Malbon and Campbell, 1984;
CORTICOSTEROID
RECEPTORS
TABLE INFLUENCE Experiment R, (fmoYmg
I pr.)
RL @mol)
ii F M F
Kc+ WO Experiment R,, (fmoumg
II pr.)
Kd (nM)
ii F
Experiment R, (fmoUmg
Kc+ bW
Nore. between
Ill pr.)
79.56 96.93 x 2.54 x 4.82 2.58 2.93
rf 11.18 k 13.72 f 0.41 f 0.77 ? 0.36 ” 0.45
Control M F M
RI. (pmol)
RL f&-d)
Control M
39.74 64.41 x 0.80 n 1.51 4.52 4.59
2 7.63 in 13.02 f 0.16 + 0.11 + 0.64 f 0.49
Control M F M F M F
OF T,,
OF INJECTIONS
45.51 f 11.98 48.51 k 9.72 1.32 k 0.33 1.77 t- 0.39 5.36 2 0.37 8.09 2 1.45
Mean values + SEM of Experiments the sexes (x) and between control
T, 5 n&z 62.23 90.73 3.55 4.51 x 5.91 x 2.87
+ + f -+ f ?
4.48 11.42 0.75 0.38 1.01s 0.43
T3 5 rids 56.62 f 15.89 37.46 f 11.38 0.88 t‘ 0.16 0.71 ” 0.17** x 5.38 t‘ 0.34 x 3.79 k 0.56 T, 50 rig/g 151.36 96.83 4.11 2.29 4.66 5.46
-c -L: tk rt +
21.55** 29.65 0.90* 0.67 0.11 0.62
IN Xenopus
1 TV, AND
PTU
0~
R,, R,,
T4 50. rip/p 89.92 84.90 n 2.49 x 4.90 x 4.17 x 2.51
c f 2 +r t
27.95 12.25 0.45 0.77 0.44* 0.51
-+ 2 2 f + t
9.25** s.27* 0,41** 0.23* 1.10 0.49
PTlJ
t 2 ‘k +i:
21.25* 20.05* 0.61* 0.85 2.27 0.80’
7.9@ 9.88 0.36 0.65 0.67* 0.29
Pm
3 lJ& f 9.40 -c 8.89 f 0.17 + 0.17 zk 1.06 2 0.76
T4 500 nglg 102.31 k 124.39 2 2.46 -t 3.29 + x 8.86 -+ x 4.56 r
I-III for males (M) and females (F) separately. and hormone-injected groups (** **) are indicated.
Stalmans and Laloux, 1979; Malbon and LoPresti, 1981). In amphibia studies on the influence of both hormones on liver metabolism mainly are restricted to the period of metamorphosis. In the liver of R. catesbeiana tadpoles T3 and T4 receptors (Galton and Schaafsma, 1983) and glucocorticoid receptors (Woody and Jaffe, 1984) were demonstrated. Comparable effects of thyroid hormones and corticosteroids on mammalian liver tissue were supposed to be independent from each other in some cases (Miiller et al., 1982; Yaroni and Balinsky, 1984) or synergistic in another (Shapiro and Sachchidananda, 1982). Again the regulation of glucocorticoid receptors via thyroid hormones may be one base of synergistic effects. An increase of the glucocorticoid receptor capacity occurred in the liver of adrenalectomized adult rats 24 hr after an injection of T, (Naito et al., 1985). In amphibia, in the liver of X. laevis, R. was significantly higher in toads with enlarged thyroid gland, whereas &, the liver weight, the liver body
3 Ia43 5 ” t ” C +-
51.63 51.68 1.02 1.33 5.62 4.08
T3 500 ngig 111.93 116.28 3.58 3.24 9.01 3.99
K~
AND
47.75 63.61 x 2.21 x 4.66 x 4.84 x 2.52
T, 50 wk 91.06 101.87 2.62 2.17 x 6.94 x3.99
493
LIVER
26.76 16.22”* 0.72 0.65 0.99*+ 0.19*
Statistically
T4 5000 wk 102.32 121.88 2.55 3.13 4.99 5.46 significant
f -c ” + _” 2
21.04* 29.33* OS5 0.79 0.44 0.61
differences
index, and the concentration of corticosteroids in serum were not different compared to those of normal toads. Our experiments with injections of T, or T4 revealed an increase of R0 up to 250% of the controls. This effect of thyroid hormones, which indicates hyperthyroidism in toads with enlarged thyroid gland, was more obvious in the liver of X. luevis than in the liver of adult rats (Naito et al., 1985; < 160%) and in the lung of neonatal rats (Morishige, 1982; < 170%) * Total concentrations of corticosterone and aldosterone were not different in normal toads and toads with enlarged thyroid gland. After injections of T, or T,,it was not different or slightly higher than that in the control group. Therefore the increase of l?, and RL is possibly not due to a decreased rate of receptor translocation into the nucleus and an enhanced rate of receptor replenishment out of the nucleus, which would be expected together with reduced levels of corticosteroids. Howev,er, changes of the amount of free, available corticosteroids under the influence of T,
494
LANGE,
I
(%
versus
HANKE,
AND
MORISHIGE
control)
Rs
RL
300
0 I
TL (nglgl
T3 (ngfg) 5
5 50
50 500
50 5005000
PTU kg/g)
3 3
I II II III III I II1 III I II FIG. 6. Influence of injections of T,, T,, and PTU on R, and R, in liver cytosol of X. laevis expressed in percentage change versus controls. The columns, arranged according to dose, depict the vaiues for both sexes combined and for male and female toads separately. For experimental conditions see Fig. 4. EXf?
and T4 cannot be excluded because inconsistent results have been found in mammals (Westphal, 1986; Henning et al., 1986). Further experiments with determination of nuclear receptor will shed further light on this question. In R. catesbeiana tadpoles the glucocorticoid receptor capacity decreased during metamorphosis. It was not changed by the addition of T, to the tap water (Woody and Jaffe, 1984). These results certainly are impaired by the increasing levels of corticosteroids (Jaffe, 1981) during spontaneous and induced metamorphosis. The effective concentrations of T3 (50 ng/ g) and T4 (500 rig/g) found in our experi-
ments were high in comparison to maximal physiological concentrations of about 15 pg T, and 1000 pg T, per milliliter of plasma in other species of amphibia (Kuhn et al., 1985; Tasaki et al., 1986). However, they correspond with results of McNabb (1969), who found significant alterations of the nitrogen excretion of R. pipiens after injections of 1000 ng but not of 100 ng T4 per gram and day. A clear dose-response relationship might not be found because of the treatment for as long as 7 days. Experiments with shorter treatments may show a gradual increase in receptor levels with increasing T, and T, doses.
CORTICOSTEROID
RECEPTORS
T3 injections were more potent in male toads than in females to increase &,, whereas T4 showed about the same effect in both sexes. In her study on T3 and T, receptors Galton (1986) concluded that liver nuclei of R. catesbeiana contain one set of high affinity, low capacity thyroid hormone binding sites, with similar capacity and two- to threefold higher affinity for T3 than for T4. With this assumption the sexual differences in effectiveness of T, and T4 found are not due to a different R,. They may be due to sexual differences in the & of the thyroid hormone receptor, in transport rate, or metabolism of T, and T,. A decrease of R, after injections of PTU is not perceptible in our experiments. Accordingly, the glucocorticoid receptor capacity was not reduced after thyroidectomy in adult rats (Naito et al., 1985; Koch et al., 1978). The limited amount of collected blood did not allow us to measure the steroid and the T, and T, concentrations in parallel. Therefore, the known hypothyroid effect of PTU was not proved. The high values of R, after treatment with T3 and T4 may be based (1) on a higher rate of synthesis, (2) on a lower rate of degradation of the glucocorticoid receptor, or (3) on the activation of inactive receptor molecules. Rousseau (1984) suggested a model of an intracellular receptor cycle. The receptor is recycled in the cytoplasm in an oxidized, nonbinding form, which is then converted to the form that binds steroids by a NADPH-dependent, thioredoxin-mediated reducing system. Naito et al. (1985) propose that the increase of R. after injections of thyroid hormones may be correlated with its conversion to the steroid binding form; thyroid hormones induce NADPdependent enzymes and in this way enhance NADPH production and shift the equilibrium toward the form that binds steroids. Similar effects of thyroid hormones and corticosteroids in liver metabolism, e.g., on NADPH-dependent enzymes in
IN Xenopus
495
LIVER
adult amphibia (Weindel and Hanke, 1988; Kasprzyk and Obuchowicz, 1980), are in contradiction to this explication. Like thyroid hormones, corticosteroids should increase the level of the steroid binding receptor form, but only the contrary is known (Svec, 1985). REFERENCES Alexander, S. S., and Bellerby, C. W. (1938). Experimental studies on the sexual cycle of the South African clawed toad (Xenopus laevis). I. J. Exp. Biol.
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