Influence of phosphatase inhibitors and nucleotides on [3H]dexamethasone binding in cytosol of human placenta

Vol. 21, No. 4, pp. 381-386, Printed in Great Britain. All rights reserved

J. sferoid Biochem.

1984

0022.4731/84

$3.00 + 0.00

Copyright 0 1984 Pergamon Press Ltd

INFLUENCE OF PHOSPHATASE INHIBITORS AND NUCLEOTIDES ON [3H]DEXAMETHASONE BINDING IN CYTOSOL OF HUMAN PLACENTA CLAUDIA HELLER, HECTOR COIRINI and ALWANDRO F. DE NICOLA* Laboratorio de Esteroides, Instituto de Biologia y Medicina Experimental, Obligado 2490, 1428 Buenos Aires, Argentina (Received 20 December 1983) Summary-The purpose of this investigation was to establish the properties of [‘Hldexamethasone binding sites in cytosol of human placenta at term. Cytosol containing 20 mM sodium molybdate (MoO,Na& was incubated for 120 min at 20°C with 40 nM [‘Hldexamethasone. The following properties were observed: (a) a single population of binding sites of high affinity and low capacity was measured by Scatchard analysis; (b) potent glucocorticoids such as dexamethasone and cortisol displaced the tritiated iigand, progesterone showed an intermediate activity, whereas cortisone, testosterone and 17/3-estradiol were ineffective competitors; (c) ultracentrifugation on 1641% glycerol gradients containing 20 mM MoO,Na, yielded sedimentation values of 10.25 k 0.35 S (n = 4 placentas); (d) the binding sites could be differentiated from the enzyme 1lj?-hydroxysteroid dehydrogenase, as the activity of the former, but not that of the latter, was greatly dependent on the presence of MoO,Na, in the incubation medium. Inactivation of binding sites labelled with [3H]dexamethasone by incubation at 20°C was prevented by phosphatase inhibitors such as 20mM MoO,Na, (P < O.Ol), 20mM sodium tungstate (WO,Na,) IP < 0.01) and to a lower extent bv 5 mM ATP and CAMP (P < 0.05). 50 mM NaF. 5 mM GTP or cGMP had no e’ffect. The protection aiforded by MoO,Na, anh WO,N& was correlated with a significant inhibition of the activity of acid phosphatase, but not alkaline phosphatase. Neither ATP nor CAMP modified phosphatase activity. It is suggested that binding sites for [-‘H]dexamethasone in cytosol of human placenta showed properties similar to those described for glucocorticoid receptors in target cells, and that these binding sites are regulated by phosphorylation and dephosphorylation mechanisms.

INTRODUCTION sites for glucocorticoids have been demonstrated in placenta from different species [l-6]. In some of these studies, it was observed that cytosolic binding sites for natural or synthetic hormones resembled glucocorticoid receptors described in other tissues [7], suggesting that placental cells are targets for adrenal hormones. It has been postulated that steroid receptors are phosphorylated proteins, and that the receptor binding capacity depends on its phosphorylation state [g]. Furthermore, phosphatase inhibitors, or phosphorylated compounds such as ATP, prevented receptor degradation [9, lo]. Recent evidences for direct phosphorylation of glucocorticoid, estradiol and progesterone receptors [l I-131 support the role of phosphorylation in receptor binding activity. Cyclic nucleotides also regulate estradiol binding sites, and in endometrial carcinoma cells in culture, CAMP and cGMP have opposite effects 114, 151. In this report, we examined the properties of [‘Hldexamethasone binding sites of human placental cytosol by means of saturation, competition and ultracentrifugation analysis. Second, we studied the effect of phosphatase inhibitors and nucleotides on binding and on the activity of acid and alkaline Binding

‘To whom correspondence should be addressed.

phosphatases, inactivation.

enzymes possibly involved in receptor

MATERIALS AND METHODS Materials

[6,7-3H]Dexamethasone (36 Ci/mmol), [ 1,2,6,7-3H]cortisol (93 Ci/mmol) and Omnifluor were obtained from New England Nuclear (Boston, MA). Dextran T-70 was from Pharmacia (Piscataway, NJ) and activated charcoal (Norit A) from Amend Laboratories (New York). Sodium molybdate (MoO,Na,), tungstate (WO,Na,) and fluoride(NaF), ATP,cAMP, guanosine-triphosphate (GTP), 3’,5’-cyclic guanosine-monophosphate (cGMP), methylisobutylxanthine (MIX), p-nitrophenylphosphate and nonradioactive steroids were purchased from Sigma (St Louis, MO). Steroid binding assay

Human term placentas were obtained after vaginal delivery of normal pregnancies. Tissues were transported to the laboratory in ice and fetal membranes discarded after dissection of trophoblastic tissue. Binding assay was similar to that used previously with rat placenta [3]. Thus, placental trophoblast was homogenized at @-4”C in 10 mM Tris buffer pH 7.4, containing 1.5 mM EDTA, 2 mM mercaptoethanol, 10% glycerol (TEMG), by ten up and down strokes

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with a glass homogenizer with a motor-driven pestle. When indicated in the text, TEMG also contained one of the following phosphatase inhibitors: 20 mM Mo04Na2, 20 mM WO,Na,, 50 mM NaF, or one of the following nucleotides at 5 mM:ATP,GTP,cAMP or cGMP. When using nucleotides, the TEMG buffer also contained 0.1 mM MIX to inhibit phosphodiesterase activity. The homogenate was spun at 105,OOOg during 60 min at O+C and the supernatant (cytosol) used for binding assays. 0.3 ml aliquots of cytosol were incubated with 40nM [‘Hldexamethasone during 120 min at 2O”C, and parallel incubations were carried out with labelled substrate in the presence of a 100-fold molar excess of non-radioactive ligand, to determine non-specific binding. After separation of the bound and free steroid fractions with dextran-coated charcoal [ 161, results of specific binding (i.e. total minus nonspecific) were calculated and expressed as fmol of [‘Hldexamethasone bound per mg DNA. DNA was measured in whole tissue according to Burton [ 171. Glycerol gradient centrijiigation

Human placenta1 cytosol was labelled with 40 nM [‘Hldexamethasone in buffer TEMG containing 20 mM MoO,Na, during 34 h at 0°C. The incubate was layered on tubes containing a 1641% linear glycerol gradient in buffer with 20mM MoO,Na,, according to Niu et a1.[18]. Gradient tubes also contained the internal sedimentation marker proteins bovine liver catalase (11.3 S) and human hemoglobin (4.4 S)._Gradients were spun at 200,OOOg for 22 h at 0°C in a swinging bucket rotor (SW-56); at the end of the run, the bottom of the tubes was pierced and 35 fractions each containing 0.12 ml were collected. The position of marker proteins was monitored by recording the O.D. at 410 nm, and the sedimentation coefficient of radioactive peaks determined by the method of Martin and Ames [19].

Measurement of acid and alkaline phosphatase activity

The activity of these enzymes was determined in placental cytosol from incubations with [3H]dexamethasone plus or minus phosphatase inhibitors or nuLleotides as described above. Aliquots of cytosol (2-5 ~1) were used for determination of enzymes according to Sommer[21]. The incubation was carried out during 30 min at 37”C, and contained 2 mM p-nitrophenylphosphate substrate, 1 mM MgCl, and 25 mM sodium bicarbonate buffer pH 10.0 for alkaline phosphatase, or 50 mM sodium acetate buffer pH 5.0 for acid phosphatase. The amount of pmeasured specreleased was nitrophenol trophotometrically by recording O.D. at 405 nm. Results were expressed as nmole product formed per min per mg protein. Statistical analysis

Results were evaluated by analysis of variance followed by the Newman-Keuls test. Equilibrium binding parameters in Scatchard plots were calculated by linear regression analysis using a Hewlett-Packard model 9815A desk computer. RESULTS

Maximal binding of [3H]dexamethasone in cytosol of human placenta incubated at 20°C with 20mM MoO,Na, occurred after 60 min and it was maintained for at least 3 h. Low binding levels were observed in the absence of MoO,Na,; therefore specificity, saturation and ultracentrifugation analysis were carried out in the presence of this protective agent. In the specificity studies, cytosol incubated with 40nM [3H]dexamethasone during 120min was exposed to one of three concentrations of competitors: 0.4, 1 and 4pM. Figure 1 shows that the

Determination of 11fi -hydroxysteroid dehydrogenase activity

This enzyme, which catalyzes the conversion of cortisol into cortisone, is present in the soluble fraction of human placenta [20], and it seemed important to differentiate it from the binding sites under study. 5 pCi [3H]Cortisol was incubated in buffer TEMG with or without 20 mM MoO,Na, during 120 min at 20°C. One-half of the incubation was used for binding studies, whereas the other half was additioned of pure standards of cortisol and cortisone (50 pg), extracted twice with dichloromethane and the extract chromatographed on thin layer plates in the system chloroform-methanol-water (188: 12: 1, v/v). The areas with the mobilities of pure cortisol (R, 0.18) and cortisone (R, 0.36) were scraped off the plates, the steroids eluted with methanol and their radioactive content determined [2].

Log Competitor

Concentmtlon

(nM1

Fig. 1. Effect of competitors on [SH]dexamethasone binding in cytosol from human placenta..Cytosol prepared in buffer 10 mM Tris pH 7.4, 1.5 mM EDTA, 2 mM mercaptoethanol, iO% glycerol (TEMG) containing 20 mM sodium molybdate (MoO,NaJ was incubated during 120 min at 20°C with 40 nM [3H]dexamethasone alone or in the presence of a lOO-fold molar excess of non-radioactive hormone, to assess non-specific binding. Competitors were added to cytosol in three different concentrations: 0.4, 1 or 4pM. Bound and free steroid fractions were separated by a dextran-charcoal method [16]. Abbreviations: Dex. dexamethasone; EZ: l7fi-estradiol.

Glucocorticoid

binding in human placenta

sequence of potency to displace the tritiated ligand was: dexamethasone > cortisol > progesterone p cortisone = testosterone = 17P-estradiol, which revealed specificity for active glucocorticoids and for progesterone, a well known partial agonist/ antagonist of glucocorticoids [22]. Figure 2 shows the results of binding parameters at equilibrium with cytosol from fresh human placenta (upper graph) and with placenta kept at -35°C during 2 weeks (lower graph). A single popuiation of high affinity, low capacity sites were visualized after Scatchard plot analysis of binding data from incubations performed with a range of 13H]dexamethasone concentrations (I ,265s nM). In frozen tissue, the affinity of the binding reaction was preserved, together with a decrease in maximal number of sites. The sedimentation coefficient of placental cytosolic sites binding gluc~orticoid was investigated in 20 mM 16-41% glycerol gradients containing MoOpNa2. Figure 3 shows that single, symmetrical peaks were obtained in the gradients, which were bfunted after co-incubation with a lOO-fold excess non-radioactive dexamethasone. In four separate placentas, S values measured 10.25 _+0.35. Binding sites for glucocorticoids in placental cytosol could be differentiated from the enzyme converting cortisol into cortisone by means of their differential reactivity towards MoO,Na,. Human placental cytosol bound [3H]cortisol and the reaction decreased from 212 pM in the presence of 20 rnM MoO,Na, to 55 pM in its absence. However, the conversion of substrate into [3H]cortisone, was lower with (16.2--18.5x) than without MoWa,

383

(26.9-30.8x), clearly differentiating the response of the I I~-hydroxysteroid dehydrogenase to that of the putative glucocorticoid receptor. The effect of phosphatase inhibitors and nucleotides on binding is presented in Fig. 4. The results

Fraction Number Fig. 3. Glycerol gradient centrifugation of f3H]dexamethasone binding sites in human placental cytosol. Cytosol incubated with 40 nM [3H]dexamethasone in buffer TEMG containing 20 mM MOO, Naz was layered on top of l&41% glycerol gradients in buffer containing MoO,Na,, and centrifuged at 200,OOOgfor 22 h. Profile consisting of full circles represent incubation with [3H]steroid only, whereas that with empty circles contained in addition a lOO-fold excess non-radioactive steroid. Sedimentation markers: bovine liver catalase (Ca, S i 1.3) and hemoglobin (Hb, S 4.4). In this placenta, S was 10.5. In four different placentas, mean S values measured 10.25 i: 0.35 (SE).

0;

100

300

500

(3HI-Dexomethosone

700

900

1100

Bound rtmavrmau)

Fig. 2. Saturation analysis of [3H]dexamethasone binding in of human placenta. Cytosol was prepared in buffer TEMG containing 20 mM MoO,Na, and incubated with a range of [3H]dexamethasone concentrations varying from 1.26 to 55nM, with parallel incubations containing a lOO-fold excess non-radioactive hormone to assess nonspecific binding. The upper graph (A) shows the Scatchard analysis with cytosol from fresh tissue, whereas the lower graph (B) shows the results with placenta kept at -35°C during 2 weeks. Maximal number of binding sites (N,,,) in fmol/mg DNA and dissociation constant (KJ are indicated in the graphs. cytosol

NoF

ATP GTP CAMP cGMP

Fig. 4. Effect of phosphatase inhibitors and nucleotides on binding of [3H]dexamethasone in cytosol of human placenta. Cytosol was incubated in buffer TEMG with 40nM [3H]dexamethasone during 120min at 20°C without further additions (controls, C), with 20mM MoO,Na, (Moo,‘), 20 mM tungstate (WO;), 50 mM sodium fluoride (NaF) or 5 mM concentration of nucleotides: ATP, GTP, CAMP or cGMP. Nucleotide incubations also contained 0.1 mM MIX (methyl-isobutylxanthine) to inhibit phosphodiesterase activity. MIX did not have effects on binding activity by itself. The height of columns represent the mean (+ SE) per cent increase over control values, which were taken as 100%. In controls binding of [3H]dexamethasone was 0.238 + 0.04pmol per mg DNA (n = 10). P values refer to differences with control incubations, Figures inside the columns represent number of placentas studied.

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were expressed as percent of control incubations without additions in order to correct for the variability of binding levels from one placenta to another. Binding of [3H]dexamethasone was increased 6-7fold (P < 0.01) after inclusion of MoO,Na, and WO,Na, in the homogenization and incubation buffer; a smaller, non-statistically significant increment was obtained with NaF (Fig. 4). Of the nucleotides tested, ATP and CAMP at 5 mM increased binding 2-fold, whereas GTP and cGMP were ineffective. Although the increment with ATP and CAMP was of a smaller magnitude than that obtained with the phosphatase inhibitors, it was albeit significant (P < 0.05). It should be mentioned that all nucleotide experiments contained 0.1 mM MIX to inhibit phosphodiesterase; this agent per se had no effect on [3H]dexamethasone binding. Both alkaline and acid phosphatase activities were readily measured in placental cytosol (Fig. 5). Alkaline phosphatase activity was not modified by addition of phosphatase inhibitors or nucleotides. However, MoO,Na, and WO,Na,, which were the best protective agents of the binding reaction, decreased 3-4-fold acid phosphatase activity (P < 0.01). NaF which did not affect [3H]dexamethasone binding significantly, had only a borderline effect on acid phosphatase (P = 0.05). Neither ATP,cAMP, cGMP nor GTP changed acid phosphatase levels.

ACID

PHOSPHATASE

ALKALINE

OL

PHOSPHATASE

I 6

GMP

Fig. 5. Effect of phosphatase inhibitors and nucleotides on the activity of acid phosphatase (upper graph) and alkaline phosphatase (lower graph) in cytosol of human placenta. Concentration of phosphatase inhibitors and nucleotides, and abbreviations as outlined in legend to Fig. 4. Phosphatase activities were determined by measuring spectrophotometrically the release of p-nitrophenol from substrate p-nitrophenylphosphate (2 mM), in buffer containing 1mM‘ MgCl; and 25 m&l sodium bicarbonate buffer pH 10.0 (alkaline nhosohatase) or 50mM acetate buffer (acid phosphatase), according to’[21]. P values refer to differences with control incubations. Figures inside the columns represent number of placentas studied.

DISCUSSION

The present results demonstrate that human placental cytosol contains binding molecules for [3H]dexamethasone which resembled glucocorticoid receptors, confirming previous findings in humans and other species[l-61. This assertion receives support from the following data: (a) a single population of sites of high affinity and low capacity was measured by Scatchard analysis (b) there was good specificity for potent glucocorticoids such as dexamethasone and cortisol, whereas cortisone, androgen and estrogen were ineffective competitors. Progesterone, which is a partial agonist/antagonist for glucocorticoid receptors and action [22], also displaced the bound ligand (c) Glycerol gradient ultracentrifugation yielded sedimentation coefficients (av. 10.25 S) coincident with those shown by others as representing molybdate-stabilized holoreceptors [l&23-25] (d) The binding site was not that of the enzyme 11fi -hydroxysteroid dehydrogenase, judged by the sensitivity of the former and the insensitivity of the latter, to the stabilizing action of MoO,Na,. The properties described above for the human placenta are similar to those shown in the rat placenta [2,3]. In the latter reports, exchange between radioactive [3H]dexamethasone and bound hormone took place after incubation for 120 min at 20°C in the presence of MoO,Na,, suggesting that the binding capacity found in our experiments (1.l pmol/mg DNA) represented total and not only available binding sites. This value is close to that reported by Lageson et a[.[61 for the glucocorticoid receptor in third trimester placenta: I .9 pmol/mg DNA. Assay at 20°C instead than at 0°C would have the advantage of increasing the affinity and steroid specificity of the binding site, as has been shown with the glucocorticoid receptor in splenic lymphocytes [26]. Further investigation of the properties of [-‘HIdexamethasone binding sites in placental cytosol, demonstrated that inactivation of the occupied sites was prevented by MoO,Na> and WO,Na,, while NaF had a slight effect, suggesting that phosphatase inhibition may stabilize the sites. This is in agreement with Pratt and coworkers [9, 11,27, 281 which established that these compounds inhibit several phosphatases. In our studies, with cytosol exposed to MoO,Na, and WO,Na,, alkaline phosphatase remained unchanged, whereas acid phosphatase was greatly diminished, which suggests that the latter enzyme may be involved in binding site inactivation. However, stabilization of receptors by MoO,Na, appears to involve additional mechanisms, such as inhibition of proteolytic activity [29] and protection of an SH group necessary for ligand binding[9]. Stabilization of binding sites in human placental cytosol was also obtained with ATP and CAMP, although to a lower extent than with MoO,Nar and WO,Na,. GTP and cGMP were ineffective. It is possible that stabilization by nucleotides was not due to inhibition of dephosphorylation but to increasing

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phosphorylation, considering that the nucleotides had no effect on acid or alkaline phosphatase activity. This view receives support by early work with thymus cells and fibroblasts, showing that ATP and phosphorylated sugar derivatives protected the receptor [g, IO] and by the direct demonstration of in vitro phosphorylation of steroid receptors [ 1l-l 31. Nevertheless, it is possible that the actions of nucleotides are more complex and may involve a direct allosteric interaction of ATP, ADP, other nucleotides and even PPi with a nucleotide binding site on the receptor protein [30,31]. The nucleotide specificity for receptor stabilization may also depend on the target tissue and the ligand used, as Fleming et a1.[14, 151 have shown that ATP, GTP and cGMP increased estradiol binding in human endometrial cancer cells, whereas CAMP had the opposite effect. Finally, it is tempting to suggest that binding sites for glucocorticoids present in placental cytosol [l-6], and this communication or vesicles [32] mediate glucocorticoid effects on the placenta [33-351. It is therefore important to understand which factors regulate the phosphorylation and dephosphorylation of binding sites, as potential controlling mechanisms of the function of the human placenta. Acknowledgements-This

work was supported in part by Research Council and the Atomic Energy Agency of Argentina. We deeply appreciate the collaboration of the Department of Obstetrics and Gynecology, Centro de Education Medica e Investigation Clinica (CEMIC) for providing human placentas for this study. the National

REFERENCES 1. Wong

M. D. and Burton A. F.: Studies on corticosterone-receptor complexes from mouse placenta. Can. J. Biochem. 52 (1974) 190-195. 2. Heller C., Coirini H. and De Nicola A. F.: Binding of (3H)-dexamethasone by rat placenta. Endocrinology 108 (1981) 1697-1702. 3. Heller C., Coirini H. and De Nicola A. F.: The placenta as a glucocorticoid target tissue: exchange assay of (‘H)-dexamethasone binding and effect of steroids on receptor content. Horm. Metab. Res. 15 (1983) 41-45. 4. Giannopoulos G., Hanzzan A. and Solomon S.: Glucocorticoid receptors in fetal and rabbit tissues. J. biol. Chem. 249 (1974) 24242427.

5. Speeg K. V. and Harrison R. W.: The ontogeny of the human placenta glucocorticoid receptor and inducibility of heat-stable alkaline phosphatase. Endocrinology 104 (1979) 1364-1368. 6. Lageson J. M., Spelsberg T. C. and Coulam C. B.: Glucocorticoid receptor in human placenta: Studies of concentration and functional differences of preterm and term tissue. Am. J. Obstet. Gynec. 145 (1983) 515-523. 7. Cake M. H. and Litwack G.: The glucocorticoid receptor. In Biochemical Actions of Hormones (Edited by G. Litwack). Academic Press, New York, Vol. III (1975)

pp. 3 17-390. 8. Munck A., Wira C., Young D. A., Mosher K. M., Hallahan C. and Bell P. A.: Glucocorticoid-recentor complexes and the earliest steps in the action of glucocorticoids on thymus cells. J. steroid Biochem. 3 (1972) 567-578.

9. Housley P. R., Dahmer M. K. and Pratt W. B.: Inactivation of glucocorticoid binding capacity by proS.B *I,&--c

10.

11. 12.

13.

14.

15.

385

tein phosphatases in the presence of molybdate and complete reactivation by dithiothreitol. J. biol. Chem. 257 (1982) 86158618. Ishii D. N. and Aronow L.: In oitro degradation and stabilization of the glucocorticoid binding component from mouse fibroblasts. J. steroid Biochem. 4 (1973) 593-603. Housley P. R. and Pratt W. B.: Direct demonstration of glucocorticoid receptors phosphorylation by intact L-cells. J. biol. Chem. 258 (1983) 463&4635. Migliaccio A., Lastoria S., Moncharmont B., Rotondi A. and Auricchio F.: Phosphorylation of calf uterus 17/3-estradiol receptor by endogenous Cal+-stimulated kinase activating the hormone binding of the receptor. Biochem. biophys. Res. Commun. 109 (1982) 1002-I 110. Dougherty J. J., Puri R. K. and Toft D. 0.: Phosphorylation in vitro of chicken oviduct progesterone receptor. J. biol. Chem. 257 (1982) 1422614230. Fleming H., Blumenthal R. and Gurpide E.: Effects of cyclic nucleotides on estradiol binding in human endometrium. Endocrinology 111 (1982) 1671-1677. Fleming H., Blumenthal R. and Gurpide E.: Rapid changes estrogen binding elicited by cGMP or - in specific _ CAMP in cytosol from human endometrial cells. Proc. natn. Acad. Sci., U.S.A. 80 (1983) 24862490.

16. Baxter J. D. and Tomkins G. M.: Specific cytoplasmic glucocorticoid receptors in hepatoma tissue culture cells. Proc. natn. Acad. Sci. U.S.A. 68 (1971) 932-937. 17. Burton K.: A study of conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62 (1956) 315-323.

18. Niu E. M., Neal R. M., Pierce V. K. and Sherman M. R.: Structural similarity of molybdate-stabilized steroid receptors in human breast tumors, uteri and leukocytes. J. steroid Biochem. 15 (1981) l-10. 19. Martin R. G. and Ames B. N.: A method for determining the sedimentation behaviour of enzymes: application to protein mixtures. J. biol. Chem. 236 (1961) 1372-1379. 20. Lopez Bernal A., Anderson A. B. M., Demers L. M. and Turnbull A. C.: Glucocorticoid binding by human fetal membranes at term, J. clin. Endocr. Metab. 55 (1982) 862-865.

21. Sommer A. J.: The determination of acid and alkaline phosphatase using p-nitrophenylphosphate as substrate. Am. J. Med. Tech. 20 (1954) 244. 22. Rousseau G. G.: Interaction of steroids with hepatoma cells: molecular mechanisms of glucocorticoid hormone action. J. steroid. Biochem. 6 (1975) 75-89. 23. Hutchens T. W., Markland F. S. and Hawkins E. F.: Physicochemical analysis of reversible molybdate effects on different molecular forms of glucocorticoid receptor. Biochem. biophys. Res. Commun. 103 (1981) 6c67. 24. Sherman M. R. and Stevens J.: Structure of mammalian steroid receptors: evolving concepts and methodological developments. A. Rev. Physiol. In press. 25. Coirini H., Marusic E. T., De Nicola A. F., Rainbow T. C. and McEwen B. S.: Identification of mineralocorticoid binding sites in rat brain by competition studies and density gradient centrifugation. Neuroendocrinology 37 (I 983) 354-360. 26. MacDonald R. G. and Cidlowski J. A.: Alterations in specificity of glucocorticoid receptors with temperature in rat splenic lymphocytes. J. steroid Biochem. 10 (I 979) 21-29.

27. Leach K. L., Dahmer K. M., Hammond N. D., Sando J. J. and Pratt W. B.: Molvbdate inhibition of alucocorticoid receptor inactivat;on and transformation. J. biol. Chem. 254 (1979) 1188411890. 28. Nielsen C. L., Sando J. S., Vogel W. M. and Pratt W. B.: Glucocorticoid receptor inactivation under cell-free conditions. J. biol. Chem. 252 (1977) 7568-7578.

386

CLAUDIAHELLERet al.

29. Sherman M. R., Moran M. C., Tuazon F. B. and Stevens Y. W.: Structure, dissociation and proteolysis of ~arnn~alian steroid receptors: muttiplicity of glucocorticoid receptor forms and proteolytic enzymes in rat liver and kidney cytosols. J. biol. Chem. 258 (1983) 10366-10473. 30. Barnett C. A., Palmour R. M., Litwack G. and See-

gniiler J. E.: In V&O stabilization of the unoccupied glucocorticoid receptor by adenosine-5’-diphosphate. Endocrinology 112 (1983) 2059-2068. 31. Holbrook N. J., Bodwell J. E. and Munck A.: Effects of ATP and pyrophosphate on properties of glucoco~icoid-r~ptor complexes from rat thymus ceils. J. biol. Chem. 258 (1983) 14885-14894.

32. Fant M. E., Harbison

R. D. and Harrison R. W.: Gluc~orti~id uptake into human placental membrane vesicles. J. bioi. Chem. 254 (1979) 621%6221. 33. Liggins G. C., Fairclough R. J., Grieves S. A., Kendall J. K. and Knox B. S.: The mechanism of initiation of parturition in the ewe. Recent Prog. Horm. Res. 29 (1973) 11l-150. 34. Heller C. and De Nicola A. F.: Inhibition

of progesterone synthesis in placenta by administration of dexamethasone to pregnant rats. J. steroid Biochem. 19 (1983) 1339-1343. 35. Mano T. and Chou J. Y.: Regulation of human chorionit gonadotropin synthesis in cultured human placental cells by glucocorticoids. Endocrinology 109 (1981) 888-892.