Glucocorticoid receptor of frog (Rana esculenta) liver

Comp. Biochem. Physiol. Vol. 75B, No. 4, pp. 645-648, 1983 Printed in Great Britain

0305-0491/83 $3.00+0.00 © 1983 Pergamon Press Ltd

GLUCOCORTICOID RECEPTOR OF FROG (RANA ESCULENTA) LIVER SANDRA INCERPI, PAOLO LULY and SERGIO SCAPIN Institute of General Physiology, Faculty of Sciences, I University of Rome, 00185 Rome, Italy (Received 4 January 1983)

The presence of a glucocorticoid soluble receptor is demonstrated in frog liver cytosol. 2. The kinetic characterization of frog liver cytosolic receptor for glucocorticoids is reported and its steroid specificity assessed. 3. Results indicate a gross similarity between frog liver and mammalian glucocorticoid receptor, being a major difference the reduced binding capacity. Abstract--l.

INTRODUCTION

immediately; storage at -4ff'C up to 72hr did not alter appreciably hormonal binding. All operations were performed at 4°C.

Few reports have appeared in recent years which correlate the inducibility of hepatic enzymes in Amphibia with the mechanism of action of glucocorticoid hormones (Chan and Cohen, 1964a,b; Ohisalo and Pispa, 1975). Detailed studies on the presence and subcellular distribution in frog liver of tyrosine aminotransferase (Incerpi et al., 1982; Scapin et al., 1982), an enzyme which is known to be induced by glucocorticoid hormones in higher Vertebrates (Tomkins et al., 1969; Cake and Litwack, 1975), raise the question of the regulatory role of glucocorticoids in Amphibia. On such a basis we started studies on the presence of a glucocorticoid receptor in frog liver employing the synthetic hormone dexamethasone. Dexamethasone was observed to bind specifically to a cytosolic glucocorticoid receptor with features reasonably comparable with those reported for the mammalian receptor (Baxter and Tomkins, 1971; Rousseau et al., 1972) with the only major exception of a reduced binding capacity.

Glucocorticoid binding assay [3H]-Dexamethasone specific binding was tested basically as reported by Rousseau et al. (1972): in a final volume of 0.4 ml, 0. l ml aliquots of the supernatant were incubated for appropriate times at 0°C with labelled dexamethasone and non specific binding was determined in the presence of a 1000-fold excess of the cold hormone. The final protein concentration was 5 mg/ml; preliminary experiments (not reported) indicated that specific binding of labelled dexamethasone was linear over a wide range (1-25 mg/ml) of sample protein concentration. At the end of the incubation period, 50 #1 of a eharcoal~lextran (100-10 mg/ml, in Tricine buffer) suspension were added; the tubes were agitated for 10 sec with a Vortex mixer and then centrifuged for 3 min in a Microfuge; the radioactivity was counted on 0.2 ml aliquots of resulting supernatant using a Packard 2425 scintillation spectrometer. Proteins were estimated by the Lowry et al. (1951) method using bovine serum albumin as a standard.

RESULTS

AND DISCUSSION

MATERIALS AND METHODS

Activated charcoal, cold hormones in the acetate form, dextran and albumin were obtained from Sigma (St. Louis, MO); 1,2,4-[3H]-dexamethasone (40Ci/mmol) was from Amersham International Ltd. (Amersham, Bucks); all other analytical grade reagents were from Merck (Darmstadt,

FRG). Animals Rana esculenta frogs (20-25 g, average body weight), kept an outdoor terrarium, were caught at random during the April-June period. in

Liver cytosol preparation The animals were sacrificed by decapitation; livers, pooled from 15 to 25 frogs, were homogenised with 1 vol of 0.02 M N-Tris-(hydroxymethyl)methyl-glycine (Tricine; Sigma, St. Louis MO), 0.002 M CaC12, 0.001 M MgCI2, pH 7.5, with a motor driven Teflon-glass tissue grinder. The resulting homogenate was centrifuged at 105,000g for l hr; the supernatant was carefully collected in order to minimize lipid contamination and tested for glucocorticoid binding

In Fig. l(A) the specific binding of dexamethasone to frog liver soluble fraction is reported as a function of the free hormone concentration; these data, which are plotted according to Scatchard (1949) analysis in Fig. I(B), suggest the presence of a single class of specific receptor sites. F r o m the results reported in Fig. I(B), the dissociation constant (Kd) for the reaction dexamethasone + r e c e p t o r ~ d e x a m e t h a s o n e receptor complex is 4.17 x 10 9 M (SD 1.6 x 10 9 M, n = 5), and the binding capacity calculated from the X intercept is 51 + 6 x 10 JSmol dexamethasone bound/mg protein (n = 5). The dissociation constant (Kd) derived from equilibrium data is strictly comparable to that reported for mammalian tissues (Baxter and Tomkins, 1971; Rousseau et al., 1972) whereas the binding capacity appears to be about ten times lower. In Fig. 2 the t i m e ~ o u r s e of association to liver soluble receptors is reported; furthermore the addition of a 1000-fold excess of cold hormone induces a 645

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Fig. 1. (A) Specific binding of [3H]-dexamethasone to frog liver cytosol. Tubes containing various concentrations of [3H]-dexamethasone were incubated for 2 hr at 0°C in the absence and presence of a 1000-fold excess of cold hormone. Free steroid was calculated subtracting bound steroid from total steroid present in the incubation medium (Baxter and Tomkins, 1971). Each point is the average of five duplicate experiments carried out on different cytosol preparations. For other information, see the text. (B) Scatchard plot (r > 0.99, as from regression analysis) o f specific binding data reported in (A).

marked dissociation of bound labelled steroid which reaches about 90~ within 5 hr. Association to soluble glucocorticoid receptor shows second order kinetics in mammalian cells (Baxter and Tomkins, 1971): the linearity of a plot of time vs log (free steroid concentration/free receptor site concentration) suggests (Figs 3A,B,C) that this applies also in the case of frog liver (Baxter and Tomkins, 1971; Maron and Prutton, 1958). From

7

data reported in Figs. 3(A), (B) and (C) the average association rate constant obtained at three steroid concentrations is 1.41 × 109 M - ' per min. Conversely, the linearity of a plot of log (bound steroid concentration) vs time (Fig. 3D) indicates that also frog hepatic receptor for glucocorticoids obeys first order kinetics of dissociation as previously reported for mammalian cells (Baxter and Tomkins, 1971; Maron and Prutton, 1958; Stevens, 1961); the dissociation

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time ( h ) Fig. 2. Association and dissociation kinetics of [3H]-dexamethasone to/from liver cytosol receptors. [3H]-dexamethasone was added at various concentrations (5 x 10 e M, • • ; 1 × 10 -g M, O - - O ; 1 × 10 -9 M, • i ) in the absence and presence of a 1000-fold excess of cold hormone to samples of frog liver cytosol and incubated at 0°C for the time indicated. For dissociation experiments ( O - - O ) an excess of cold hormone was added to an aliquot at the indicated time in order to have a final steroid concentration of 1 x 10 5 M. Each point is the average of five experiments, as reported for Fig. I(A).

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Fig. 3. (A)-(C) Kinetic data analysis. Time vs log (bound steroid/unbound receptor site concentration) derived from association data of Fig. 2 is reported being [3H]-dexamethasone concentration: (A) l x 10-9M; (B) l x 10-SM; (C) 5 x 10-SM. Unbound steroid as well as unbound receptor site concentration were estimated as reported by Baxter and Tomkins (1971). (D) Dissociation data from Fig. 2 are plotted as log (bound steroid) vs time. The correlation coefficientwas r > 0.99 in all cases, as from regression analysis. For further details, see text. rate constant determined from the half-dissociation time (84 min) of the averaged experiments reported in Fig. 3(D) was 8.25 x 10-3/min. From an inspection of reported kinetic data in comparison with similar experiments on mammalian soluble receptors (Baxter and Tomkins, 1971) it appears that frog liver glucocorticoid receptors bind the hormonal ligand at a higher rate with respect to the mammalian counterpart as indicated by the association rate constant; the same observation holds when a comparison of dissociation data is made. The equilibrium (dissociation) constant determined from the dissociation and association rate constants was 2.25 × 10- ' I M , which is about two order of

magnitude lower than the Ka determined from equilibrium data reported above. The competition for 1 x 10 SM [3H]-dexamethasone binding at equilibrium was assayed over a wide range of concentrations of the cold analog as well as of other steroid hormones (Fig. 4). The results obtained show the following sequence of binding affinities: dexamethasone > corticosterone = hydrocortisone > aldosterone = estradiol; thus receptor specificity appears to be high for the synthetic hormone dexamethasone, intermediate for other glucocorticoids tested and low for two additional steroids. When the experimental work reported so far was

648

SANDRA INCERPI et al. REFERENCES

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Fig. 4. Steroid binding specificity to frog liver cytosol. Samples were incubated 2 h r at 0 C with l x 10 S M [3H]-dexamethasone in the presence of various concentrations of cold steroids. Dexamethasone (C)), corticosterone (×), hydrocortisone (Q), aldosterone (A), estradiol (/%).

already completed, a paper appeared on the partial characterization of hepatic glucocorticoid receptor in the American bullfrog Rana catesbeiana (Woody and Jaffe, 1982). Our results are at variance with those reported for the bullfrog, essentially for the binding capacity of the soluble receptor and for the shorter dissociation time observed in the present study; these discrepancies might be ascribed, at least partially, to species differences as well as to adaptive mechanisms being serum levels of glucocorticoids in the frog higher in winter with respect to summer period (Jungreis et al., 1970; Leboulenger et al., 1982). Acknowledgement--This investigation was supported by a financial aid of the Italian Ministry of Education.

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