Corticosteroid binding in plasma of Xenopus laevis. Modifications during metamorphosis and growth

J. steroid Biochem. Vol. 25, No. 3, pp. 343-350, Printed in Great Britain. All rights reserved

1986

0022-4731/86

$3.00 + 0.00

Copyright 0 1986Pergamon Journals Ltd

CORTICOSTEROID BINDING IN PLASMA OF XENOPUS LAEVIS. MODIFICATIONS DURING METAMORPHOSIS AND GROWTH GENEVIEVEJOLIVET-JAUDETand JEANINE LELOUP-H~TZY* Laboratoire de Physiologie C&kale et Cornparke du Musturn National d’Histoire Naturelle and Laboratoire d’Endocrinologie ComparCe associt au CNRS, 7 rue Cuvier, 75231 Paris Ctdex 05, France (Received 9 December 1985) Summary-The binding of corticosteroids has been studied by equilibrium dialysis at 4°C and electrophoresis on polyacrylamide gel. Aldosterone was not bound specifically. Large amounts of corticosterone (B) were bound. A high affinity (K. = 2.8 x lo* M-‘) low capacity (70.1 nM) component was found in tadpoles. Its affinity and its electrophoretic mobility were not significantly modified during metamorphosis and growth until adult. It differs from mammalian CBG with respect to affinity, electrophoretic mobility and specificity. During growth, the capacity of the high affinity component increased significantly (173 nM in adults) and a low affinity (K, = 2.0 x 10’ M-‘) high capacity (18.26 PM) component was detected. This last component has the same electrophoretic mobility as bovine serum albumin. These modifications can be related to the large increase in the level of plasma proteins (especially albumins). The concentrations of bound and free B deduced from our data indicate that in tadpoles the increase in the total concentration in the climax is not a good reflection of modifications of these two fractions since the change of free B is larger than that of bound B. This information is an ‘important consideration in interpreting the physiological role of interrenal secretion during metamorphosis.

INTRODUCndN In the blood of various adult amphibia,

steroids are bound to plasma proteins [l-5]. The presence of high affinity binding sites for corticosterone has been revealed [l, 2? 41. In mammals, it is well established that during both f&al and neonatal life, the amount of corticosteroid bound to the corticosteroid-binding globulin (CBG) undergoes many changes [6,7]. Until now in amphibia,

the binding

of corticosteroids

has

never been studied during the metamorphosis. In Xenopw laevis tadpoles, corticosterone and aldosterone have been identified in interrenal secretions from early developmental stages until the end of metamorphosis, as in adults [8]. In several anuran species, Rana catesbeiuna (9, lo], Bufo bufo [l 11, Xenopus luevis [12,13], the level of these corticosteroids in the plasma (both bound and unbound to plasma proteins) is low during prometamorphosis (beginning of metamorphosis) and a large increase is observed during the climax, shortly before the end of metamorphosis. It has been supposed that only the unbound hormone, named also “free hormone”, represents the active hormone [14]. On the other hand, the specific binding of steroid has been considered as a protection from degradation, or as a steroid storage [15, 161. Further, the binding of hormone in the plasma may be involved in steroid uptake by cells [17]. The variations of the total plasma level cannot be considered reflecting the hormonal supply available towards tissue, the amounts of bound and *Address for correspondence Laboratoire de Physiologic du Museum, 7 rue Cuvier, 75231, Paris Ckdex 05, France. 343

unbound hormone being major physiological parameters. Thus, it was thought worthwhile to determine the extent of the binding of corticosteroids, especially during the climax when the plasma levels are greatly increased before drawing any conclusion concerning the physiological importance of corticosteroids upon metamorphosis. In the present work, the study of the binding of aldosterone and corticosterone in Xenopus laevis tadpoles and adults is presented. EXPERIMENTAL Reagents

Radioactive steroids were purchased from the Radiochemical Center (Amersham, U.K.): [1,2,6,7-‘Hlcorticosterone (sp. act. 94 Ci/mmol) and [1,2,6,7-3H]aldosterone (sp. act. 80 Ci/mmol). The purity of the tritiated steroids was regularly tested by paper chromatography (Whatman No. 1) in the Bush BSsystem [ 181.Aldosterone was given by Ciba-Geigy (Basel, Switzerland), and RU 28 362 (1 l/3,1 7/3-dihydroxy-6-methyl-17a (1-propynyl) androstan- 1,4,6triene-3-one) by Roussel UCLAF (France). Other steroids were obtained from Sigma Chemical Company (St Louis, MO., U.S.A.). All reagents were of analytical grade (Merck, Darmstadt, F.R.G.). Bovine serum albumin was purchased from Miles Laboratories (U.K.). Acrylamide and N,N’-methylenebisacrylamide were obtained from Eastman Kodak Co. Aqua Luma Plus (Lumac) was used for liquid scintillation counting. Regenerated cellulose dialysis membrane was Visking tubing from Union Carbide (Chicago, Ill., U.S.A.).

344

GENEVIEVEJOLIVET-JAUDET and JEANINE LELOUP-HATEY

Animals

Adults of Xenopus laevis Daudin were obtained from the “Service regional d’elevage d’amphibiens” (S.E.R.E.A., Argenton I’Eglise, France). Spawning and breeding conditions were established as previously described [8]. The normal table of Nieuwkoop and Faber[ 191was used to determine the development stages (St) of larvae.

Sodium phosphate buffer (0.1 M, pH 7.4) was used to dissolve all reagents and to dilute plasma sample. Two hundred ~1 of diluted plasma were put in the first compartment. The other compartment received 200 ~1 of buffer containing tritiated corticosteroid and unlabelled steroid in competitive experiments. All doses of competitive steroids were assayed in duplicate.

Plasma sampling

Data interpretation

Blood was collected by heart puncture, with a heparinized capillary tube, in tadpoles at the beginning of metamorphosis (st 57), during the climax (st 63), in juveniles after the end of metamorphosis (weight: 0.45 g) and in adults (70-130 g). Samples were centrifuged (1800 g, 15 min) and the plasma was stored at -20°C. Mammalian serum was reconstituted from lyophilized horse serum (Seronorm,

When the Scatchard analysis revealed 2 binding sites, calculations of K, and capacities were made using the program WBSIKK 8004 Hypbol[24]. Association constants and capacities are given as also their means f their standard errors. The amounts of bound and free hormone have been calculated from the method described by Labrie[25] as applied to the guinea-pig [26]:

U = KtT - 1 - SaKa - StKt + ,/(SaKa + StKt + 1 - Tkt)’ + 4T(Kt + SaKaKt) 2(Kt + SaKaKt) Nyegard and Co., Oslo, Norway). In order to suppress the possible interaction of large amounts of endogenous steroids during equilibrium dialysis, mammalian serum and plasma of tadpoles that contain usually high levels of steroids have been treated so as to remove endogenous steroids. Mammalian serum was treated by activated charcoal [20]. Tadpole plasma diluted in sodium phosphate buffer 0.1 M, pH 7.4 (1: 10, v/v) was put in dialysis tubing (10 cm length, 0.9 cm dia); the bag was immersed overnight in phosphate buffer (800ml) under stirring at 4°C after which plasma was collected. Protein assay

Proteins were assayed by the method of Bradford[21]. Bovine serum albumin was used as standard. Polyacrylamide gel electrophoresis

Polyacrylamide gel electrophoresis was performed with 7.5% acrylamide gel in Tris 0.05 M-Glycine 0.37 M buffer, pH 8.3 [22]. In order to determine the mobility of the complex steroid-protein, radioactive steroid was added to the gel. The gels were polymerized in 80 x 80 x 2 mm plates. The electrophoresis was run at lO-13°C at 1lOV, 2.5 mA/sample in a Pharmacia gel electrophoresis apparatus GE-4. In order to appreciate the relative mobility of proteins in each plate, plasma proteins were stained by Amido Schwartz (0.001 in 7% acetic acid) in a reference strip. Other strips were sliced transversely for radioactivity counting. The R, was calculated as the ratio of the distance of migration of a component to the distance of migration of the front of the proteins. EquiIibrium dialysis

Equilibrium dialysis was carried out in microdialysis chambers as previously described [23].

La = USaKa Lt = St/(1 + l/UKt) where: Kt = association constant of the high affinity component St = capacity of the high affinity component Ka = association constant of the low affinity component Sa = capacity of the low affinity component T = total concentration of steroid Lt = concentration of steroid bound to the high affinity component La = concentration of steroid bound to the low affinity component U = concentration of unbound steroid Comparisons have been made using the U test of Mann and Whitney for non-parametric values. Slopes of regression lines have been compared by analysis of covariance. RESULTS

Binding of aldosterone

In adult, l&l 7% of [3H]aldosterone was bound to plasma in equilibrium dialysis. The percentage of binding did not vary when aldosterone was added in large excess (7pM, i.e. 10,000 times the concentration of radioactive aldosterone). In tadpoles at the beginning of metamorphosis (st 57), the binding of aldosterone was not detectable. Binding of corticosterone in adults

In diluted plasma (1:20, v/v), the percentage of binding of corticosterone was 70.5 f 2.9 (n = 4). A Scatchard plot for the binding to protein revealed two obvious binding components, a high affinity-low capacity binding one and a low affinity-high capacity

Corticoid binding in Xenopus (tadpoles and adults)

B/F

Ii

. 100

200

300 B nM

Fig. 1. Scatchard plot for the binding of corticosterone to the plasma of adult Xenopus laevis(1: 20, v/v) and tadpoles on st 57 (1:40, v/v). Dialyses were performed at 4°C in a shaking incubator with [‘Hlcorticosterone (0.5 nM) and corticosterone (0 to 72.4 FM). Dialyses were stopped after 20 h incubation. B/F = Bound/Free. B = Bound.

binding one (Fig. 1, Table 1). No sexual difference was observed. More than 91% of corticosterone was bound to the high affinity component, 6.7% to the low affinity component and about 2% remained

unbound [free hormone] (Table 2). Eleven-dehydrocorticosterone, 1l-deoxycorticosterone, cortisol, cortisone and 1 1-deoxycortisol induced a larger inhibition of the binding of [3H]corticosterone than corticosterone itself (Table 3). The inhibition was similar with testosterone. It was considerably smaller with progesterone, estradiol and aldosterone than with corticosterone. Among the synthetic steroids that have been tested, RU 28 362 and dexamethasone provoked a larger inhibition than corticosterone. In return, spironolactone and triamcinolone acetonide were poor inhibitors. After electrophoresis in polyacrylamide gel (10 ~1, 940 pg of proteins), a large peak of radioactivity was observed in fraction 6 of the gel (Rr = 0.43), and a smaller one in fractions 13 and 14 (R, = 1) (Fig. 2). The peak of radioactivity in fraction 6 disappeared after addition of a large excess of unlabelled corticosterone. When Seronorm was studied in parallel (5 ~1, 290 pg of proteins), a prominent peak of competitive binding was observed in fraction 10 (R,= 0.77) and a small one in fraction 13 (Rr= 1). With a very high quantity of bovine serum albumin (1750 pg), two small peaks of radioactivity were detected (R, 0.58 and 1). Binding of corticosterone in tadpoles and juveniles

The percentage of binding in diluted plasma (1:20, v/v) was lower in st 57 and 63 than in adults [38.7 f 2.41 (n = 7). The Scatchard plots for the binding of corticosterone in the plasma of tadpoles at st 57 or 63 and of juveniles 1 month after the end of metamorphosis were linear, revealing only one binding component (Fig. 1). The association constants and maximal binding capacities did not vary

345

significantly during the metamorphosis (Table 1). The K, was not significantly different from the association constant determined in juveniles or in adults as far as the high affinity component was concerned. On the other hand, the maximal binding capacity was significantly lower in tadpoles than in adults (P c 0.01). The increase in total corticosterone in plasma observed between the beginning of metamorphosis and the middle of climax concerned principally the free fraction (15 times) and less the bound fraction [ 1.8 times] (Table 2). After electrophoresis, a single and prominent peak of competitive binding was observed in tadpoles on st 59 and st 62, and in juveniles 8 days after the end of the metamorphosis (Fig. 3). The mobility of this peak was the same in tadpoles, in juveniles and in adults (R, = 0.47 in the 3 cases). Plasma level of proteins

The levels of protein did not vary in tadpoles from st 57 to st 63 (Table 1). A slight increase was observed in juveniles 1 month after the end of metamorphosis. Levels were significantly higher in adults (P c 0.01). DISCUSSION

Our results indicate that in plasma of Xenopus, corticosterone was bound to plasma from early metamorphosis until adult and that the binding system underwent modifications during the development. On the other hand, we observed that aldosterone was not bound in tadpoles and only a small binding was detectable in adults. In man [27] and in numerous mammals [28], aldosterone is also weakly bound to plasma, mainly to albumin. In Xenopus, it can be supposed that albumin-like proteins are responsible for the binding of aldosterone. In adults, two binding components of corticosterone have been characterized. It seems likely that serum albumin is responsible for the low affinity binding. Indeed, the K,, the large capacity and the electrophoretic mobility of the low affinity binding component were similar to that of the bovine serum albumin in the present study, to that of the serum albumin in human [29] and teleosts[3&32]. The characteristics of the high affinity binding component were identical to the values determined by Martin and Ozon[4] in several amphibia. The RI (0.43) of this high affinity component was different from the R, of mammalian CBG (0.77). Since, in various mammalian species (horse, rat, rabbit, bovine), the CBG has a similar electrophoretic mobility [33], our data suggest that the high affinity binding component differs in amphibia and mammals in the electric charge and/or the size of the molecules. Other major differences have been observed between the mammalian CBG and the binding component in Xenopus as indicated by the study of the inhibition of the binding of corticosterone by several compounds. Testosterone and especially synthetic steroids such as

6

JnVeItih?@

11.4 > 9.6

1.4 1 10.2 8.8 f 0.6 8.6

46.9 rt 3.9t

proteins (mg.ml-‘)

Levelof

76.6 73.6 132.7

2.2

78 88.6 33.6 t 70. I * 9.5 **II

Maximal binding capacity (nW

2.4 1.8

2.8 & 0.47 NSll

Association constant (K,) (lOsM_‘)

Means are given with their standard errors. *N: number of animals constituting each plasma sample. tMean of values found for 8 adult plasmas with its standard error. SND: Non detectable. ~iDi&rences have been tested between tadpoles and adults (U Test of Mann and Whitney). ~Juveniks have been selected 1 month after the end of metamorphosis. **Significant for P < 0.01. NS: non significant.

50 50

Climax (st 63)

N’

19 19 I$ 1s 29 and 18

Developmental stage

Adults

-

Animals - ._~

bindine oroteins in

First component

Table I. Association constant (K-1and maximal bindine caoacitv of corticosterone plasma

ND ND ND ND

ND ND ND

Maximal binding capacity (PM)

ND ND

ND ND

NDf

Association constant (K,) (IO’M-‘)

Second component

from Xenoous Levis at 4°C

Corticoid binding in Xenopus (tadpoles and adults)

347

Table 2. Concentrationof total corticosterone, bound fraction and unbound fraction in the plasma of

Xenopu~ heuis

tadpoles and adults (at 4°C)

Boundcorticosteronet Total corticosterone* ng.ml-’

c

ng.ml-’ *

,

Unbound corticosteronet ng.ml-’

Lt

La

4.6 + 1.47 (4)

4.2

0.3

Beginning of metamorphosis st 54 to 58

14.2 + 1.44 (11)

12.6

Beginning of climax st 59 to 61

21.3 + 1.80 (11)

17.45

-

3.6

Middle of climax st 62 to 63

45.8 + 3.96 (5)

22.5

-

23.1

End of climax st 64 and 65

17.1 +3.11 (41

14.8

-

Adults

0.1 1.5

2.25

Meansare givenwith their standarderrors,in brackets, number of determinations. *Concentrations evaluated in the present study (adults) or previously [13] (tadpoles) by radioimmunoassay chloride extracts. tCalculatcd in according to [25] and [26]; Lt: corticosterone bound to the high affinity component; La: corticosterone bound to the low affinity component.

dexamethasone or RU 28 362 were effective in inhibiting the binding of [3H]corticosterone to Xenopus plasma. That is quite unexpected, since in mammals, testosterone binding to CBG is not very strong whatever the considered species (4 to 100 times less strongly than corticosterone) [28] and synthetic steroids are not good competitors for the binding of corticosteroids [34]. Some comments have to be added about the specificity of the binding of corticosterone. The fact that cortisol or cortisone are effective competitors is of minor physiological importance since these steroids are synthesized in Xeno pus neither in adult [35] nor in tadpoles [8]. In return, it is worthwhile to point out the large inhibition induced by 1 1-dehydrocorticosterone because this steroid is synthesized in large amounts by the interrenal cells in vitro in Xenopus laevis during metamorphosis [8] and not in adults [35]. However, the physiological consequences of this competitive effect cannot be estimated because it has never been

on methylene

determined until now whether high levels of this steroid exist in the plasma. In the plasma of tadpoles, only one binding component was characterized. No significant difference could be detected in the K. and in the electrophoretic mobility in tadpoles, juveniles and adults concerning this high affinity binding; it could be supposed that qualitatively, the same high affinity component was present in plasma. Two major quantitative modifications have been observed after the end of metamorphosis, an increase of the binding capacity of the high affinity component and the appearance of a low affinity component. The capacity of the high affinity component increased simultaneously with the concentration of the proteins in the plasma (Table 1) that was previously observed in other amphibians [36-391. The appearance of the second binding component could be referred to the increase of the concentration of albumin. In the present study, no attempt was made to demonstrate the rise of the concentration of

Table 3. Specificity of the binding of corticosterone in the plasma of adult Xenopus /muis Inhibition Corticosterone 1I-Dchydrocorticosterone I I-Deoxycorticosterone Aldosterone

100 123 147 4

Cortisol Cortisone 1I -Deoxycortisol

123 153 130

Sexual steroids

Progesterone Testosterone Estradiol

48 102 56

Synthetic steroids

RU 28 362 Dexamethasone Triamcinolone acctonide Spironolactone

152 112 63 24

Natural corticosteroids in Xenopus laevis

(no 17 K-OH) Non-synthesized corticosteroids in Xenopus laeuis

(presence of 17 a-OH)

Equilibrium dialysis were performed at 4°C with [‘IQxvticosterone (0.32 nM) and unlabelled competitors (72 nM). The inhibition induced by corticosterone was arbitrarily fixed at 100.

348

GENEVIEVE JOLIVET-JAUDETand

Adult Xenopus

dm

Seronorm

I

BSA

40 000

30 000

I

20 000

10

000

5

10

15

Gel slice Fig. 2. Polyacrylamide gel electrophoresis of plasma of X&pus la&is (IO ~1, 94Opg of proteins), Seronorm (5 ~1, 290 ,_ ue of oroteins) and bovine serum albumin (BSA) (1750 . pg). Tritiated corticosterone (0.78 nM) with (b---o) or without (e-@) corticosterone (0.78 PM) was incorporated in gel. Duration of electrophoresis: 3 h. The arrows indicate the front of migration of proteins. Four millimeter gel slices were incubated 4 h in scintillation liquid before counting. albumin in the plasma during metamorphosis and growth, but it was shown previously by several authors [3&39]. Nevertheless, it is not possible to conclude that a low affinity binding is totally absent in tadpoles, since our methods do not allow to observe any binding when only small amounts of low affinity component are present in the sample. In various mammals, quantitative variations of plasma CBG have been described in perinatal life [26]. The ontogeny of CBG is regulated primarily by increasing concentrations of thyroxine [7,26]. It is well known that in amphibia, the circulating level of thyroid hormones increases largely during the climax of metamorphosis. In Xenopus Zaeuis tadpoles, the

JEANINE LELOUP-HKTEY

highest levels of triiodothyronine have been observed from st 59 until st 63 [40,41]. In Ambprornu mexicanurn, thyroid hormones can determine an increase of the concentration of serum proteins, especially in cr-globulins [42]. This could be of interest since the mammalian CBG has been identified as an x-globulin [33]. It could be supposed that a relationship exists between the high plasma levels of thyroid hormones, the increase in the level of proteins (including x-globulins) and the modification of the binding capacity of the high affinity component. However, in Xenopus. the binding capacity of the high affinity component increased only in juveniles, several weeks after the peak of triiodothyronine. The aim of this study was to investigate the relationship between the modifications of the billding of corticosteroids and the changes of the plasma levels of aldosterone and corticosterone previously observed (131. Since it was shown that aldosterone was not bound to a high affinity component in plasma, the variation of the plasma levels can be considered as a good index of the changes of the supply of available hormone towards tissue. The increase in the plasma level of this hormone during climax indicates an actual elevation of this supply. It is not the same for corticosterone. During metamorphosis, both fractions are largely modified. but to a different degree (Table 2). In tadpoles from the beginning of metamorphosis to the middle of the climax, the concentration of unbound corticosterone increases 15 times whereas the bound corticosterone increases about twice and the total hormone about 3 times. Thus, the increase in the total concentration during the climax is largely amplified in the free hormone level. Despite the controversial hypothesis regarding the physiological activity of the bound and the unbound fraction [15-l 71, our data indicate obviously that it is necessary to consider the relative amounts of bound and free hormone in the plasma before making any interpretation of the physiological importance of corticosteroids towards the control of metamorphosis.

dprn.103

40

Acknowledgements-This study has been supported in part by a grant from the Comite de l’action concertee de la DGRST: Biologic de la Reproduction et du Developpement (Contrat BRD-71399). We wish to express our gratitude to Professor S. Ishii for the communication of the calculation program for 2 binding sites written by Doctor K. Kubokawa and we address our thanks to Dr C. Salmon

I

st 62

J

t 5 10 15 Gel slice

5

for the adaptation of this program to an Apple 2E computer. We should like to thank br Desaulle and Dr Scheibli (Ciba Geigy, Base], Switzerland) for the gift of aldosterone and Dr Deraedt (Roussel UCLAF, France) for the gift of RU 28 362.

10 15

Fig. 3. Polyacrylamide gel electrophoresis of plasma of tadpoles at the beginning of climax (st 59), at the middle of climax (st 62) in juveniles 8 days after the end of the metamorphosis (J) and in adult. Volume of each plasma sample = 20 ~1. Duration of electrophoresis: 4 h. Other conditions were as in Fig. 2.

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