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Autoradiographic localization of glucocorticosteroid and progesterone binding sites in the human post-mortem brain
J.sferoid Biochem. Vol. 25,NO. 5B, pp. 717-721, 1986 Pruned in Great Britain. All rights reserved
Copyright
0
OO22-4731/86 $3.OO+0.00 Pergamon Journals Ltd
1986
Proceedings of the XII International Study Group for Steroid Hormones (Rome, 24 December 1985)
AUTORADIOGRAPHIC LOCALIZATION OF GLUCOCORTICOSTEROID AND PROGESTERONE BINDING SITES IN THE HUMAN POST-MORTEM BRAIN ALAIN SARRIEAU*§, MONIQUE DUSSAILLANT*, FRANCE AGID~, DANIEL PHILIBERT~ YVES ACID? and WILLIAM ROSTENE* lINSERM, U55, Centre de Recherches Paris Saint-Antoine, Hopital Saint-Antoine, Paris Saint-Antoine 184, rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, TINSERM U289, Hopital PitiQ-SalpiZtriere, 91, Bd de l’hopital, 75013 Paris and SLaboratoires Roussel Uclaf, 102, route de Noisy, 93230 Romainville, France Summary-The presence of glucocorticosteroid and progesterone binding sites in the human brain has been investigated with an in vitro autoradiographic approach using [3H]RU 38486. The present data revealed that the hippocampus, the entorhinal cortex, the subiculum, the fimbria and the amygdala showed a different pattern of [‘H]RU 38486 labeling using various unlabeled steroids (RU 38486, RU 28362, cortisol, RU 27987 and dexamethasone). RU 38486 is the best competitor in all these structures. The hippocampus seems to possess mainly glucocorticosteroid binding sites whereas the entorhinal cortex and the subiculum reveal the presence of both glucocorticosteroid and progesterone binding sites. Furthermore, the amygdaloid complex and the fimbria show a high density of glucocorticosteroid binding sites.
INTRODUCTION
A large number of investigations have described the presence of corticosteroid binding sites in several human tissues such as the placenta [14], blood lymphoid tissues [9], kidney [lo], cells [5-81, lung[ll, 121 and thymus [13, 141. However, few reports have described the presence of glucocorticosteroid
binding
sites in human
brain
whose
characterization has been carried out on cytosolic preparations [15, 161. The present study reports evidences for the heterogeneity of [3H]RU 38486 binding sites in the post-mortem human brain by means of an in vitro autoradiographic approach on brain sections. EXPERIMENTAL Human tissues
The brain of a 89-year old person with no known neurological disease was used. The brain was removed 15 h after death and kept at -70°C until dissection. Pieces of tissues, taken at the level of hippocampus, were dissected and 32 pm thick frontal sections were cut with a cryostat (Bright) at - 15°C collected on gelatin coated slides and kept at - 70°C until used as previously described for the rat brain [22]. Reagents
[3H]RU 38486 (50.6 Ci/mmol), RU 38486, RU 28362 (a pure glucocorticosteroid), RU 27987 (a synthetic progesterone binding site marker) were §To whom correspondence
should be addressed.
generous gifts from Roussel Uclaf (Romainville, France). Dexamethasone and cortisol were obtained from Sigma. Other chemicals were supplied by Prolabo (Whatman GF/B filter disks), Merck (ammonium molybdate, glycerol), Sigma (gelatin, ATP, dithiotreitol, EGTA and Tris), Baker (scintillation fluid: Aqualyte) and LKB France (Ultrofilm). Incubation and autoradiographic procedures
35 nM [3H]RU 38486 alone or in the presence of 500 PM unlabeled steroid were incubated in TEGMAD buffer (50 mM Tris-HCl pH 7.4, 2mM EGTA, 5% v/v glycerol, 6 mM molybdate, 5 mM ATP and 10 mM dithiotreitol) for 2 h at 4°C. Slides were then washed at 4°C 3 times for 5 min each with 200 ml of 50 mM Tris-HCl (pH 7.4) and sections were wiped off the slides with Whatman GF/B filter disks, placed into plastic vials with 5 ml Aqualyte and treated as previously described in detail [17] for the determination of [3H]RU 38486 radioactivity retained on the slides. For autoradiography, sections were dried after the washings with cold air and placed into Kodak X-ray film holders. Autoradiograms were produced by the apposition of a rigid tritium-sensitive film ([3H]Ultrofilm) on sections and exposed for 26 days at room temperature in a dark room. Films were then developed for 3 min with Kodak D-19 (18, 191. Black and white autoradiograms were arbitrarily converted into pseudo-colour pictures by means of a scanning densitometer (Pericolor 1000, image-assisted Numelec, France) ranging from red dots (where the binding density is the highest) to blue dots (areas which show a low density 717
of binding
sites).
ALAIN SARRIEUA et al
718 RESULTS
Figure 1 describes the competition by several unlabeled steroids for [3H]RU 38486 binding on human brain sections located at the level of the hippocampal formation. [3H]RU 38486 strongly binds to various subfields of the hippocampus mainly following the hippocampal circonvolutions as well as in the fimbria and the subiculum. However, not only the hippocampal formation is labeled with [3H]RU 38486 but also several areas such as the amygdaloid complex and the entorhinal cortex (Fig. la). Addition of unlabeled RU 38486 is able to completely abolish the labeling of [3H]RU 38486 in all the structures studied (Fig. lb). RU 28362, a pure glucocorticosteroid [20], is able to displace [3H]RU 38486 from its binding sites in all these structures but with a lesser extent than unlabeled RU 38486 (Fig. lc). Cortisol, the natural corticosteroid in man, competes with [3H]RU 38486 in the hippocampus, the amygdala and to a lesser extent in the fimbria and the subiculum; in contrast, it has no effect in the entorhinal cortex (Fig. Id). The synthetic progestagen RU 27987 almost completely abolishes the binding of [3H]RU 38486 in the entorhinal cortex and the subiculum but is not able to compete with [3H]RU38486 in the amygdala and weakly in the hippocampus and the fimbria (Fig. le). the Dexamethasone decreases binding of [3H]RU 38486 in the hippocampus and the entorhinal cortex, but slightly in the fimbria, the subiculum and the amygdala (Fig. If).
DISCUSSION
The present paper describes for the first time the binding of [3H]RU 38486 on human brain tissues. Human hippocampus appears a strong target for glucocorticosteroids as it was reported for the rat hippocampus[21]. Both unlabeled antagonist (RU 38486) and agonist (RU 28362) are good competitors for [3H]RU 38486 binding in the human hippocampal formation. In contrast, the natural steroid, cortisol, is much weaker to displace [3H]RU 38486 binding in the hippocampus. This is probably due to the fact that synthetic steroids often have a much higher affinity for binding sites than the natural compounds [22]. RU 27987, a metabolite of the progestin RU 5020, which specifically binds to progesterone receptors [23], is a strong competitor for [3H]RU 38486 binding essentially in the entorhinal cortex which suggests the presence of progesterone binding sites in the cortex in agreement with what was previously reported in the rat brain [24,25]. These data further support previous observations obtained in peripheral tissues suggesting that RU 38486 has both antiglucocorticosteroid and antiprogestagen properties [2630]. Dexamethasone, a synthetic corticosteroid used for the treatment of various human diseases [31], acts at
the level of the central nervous affinity for brain corticosteroid
system showing some binding sites in the
hippocampus and the entorhinal cortex. The use of various synthetic steroids reveals an heterogeneity in the binding capacity of [3H]RU 38486 in different structures of the human brain, as it was reported in the rat brain [32]. The presence of glucocorticosteroid binding sites in human brain is further confirmed by autoradiographic data obtained with the synthetic pure agonist [‘H]RU 28362 which reveal a similar pattern of labeling distribution than what is observed in the present work with the synthetic antagonist [3H]RU 38486 (data not shown). The use of highly potent steroids together with the development of in uitro autoradiographic approaches may give us facilities to detect possible modifications in glucocorticosteroid binding sites in various neurological diseases. authors with to thank Miss Martine Le Hein for typing the manuscript. This work was supported by a grant of Roussel Uclaf and INSERM (no. 84012).
Acknowledgements-The
REFERENCES 1. 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 tissues. Am. J. Obstet. Gynec. 145 (1983) 515-523. 2. Heller C.. Coirini H. and De Nicola A. F.: Influence of phosphatase inhibitors and nucleotides on [‘Hldexamethasone binding in cytosol of human placenta. J. steroid Biochem. 21 (1984) 381-386. 3. Lopez Bemal A., Anderson A. B. M. and Turnbull A. C.: The measurement of glucocorticoid receptors in human placental cytosol. Placenta 5 (1984) 105-I 16. 4. Lopez Bemal A. and Turnbull A. C.: High affinity glucocorticoid receptor in human intrauterine tissues. Horm. metab. Res. 17 (1985) 265-266. 5. Tsai B. S., Watt G., Koesnadi K. and Townley R. G.:
Lymphocyte glucocorticoid receptors in asthmatic and control subjects. Clin. Allergy 14 (1984) 363-371. 6. Harmon J. M.. Schmidt T. J. and Thomnson E. B.: Molybdate-sensitive and molybdate-resistant activation-labile glucocorticoid-receptor mutants of the human lymphoid cell line CEM-C7. J. steroid Biochem. 21 (1984) 227-236. 7. Eliard P. H. and Rousseau G. G.: Thermodynamics
of steroid binding to the human alucocorticoid receptor. Biochem. J. 21s (1984) 395404.
8.
Atmanini D.. Strasser T. and Weber P. C.: Parallel determination of glucocorticoid receptors in human mononuclear and Polymorphonuclear-leukocytes after Percoll separation. J. endocr. Inuesf. 8 (1985) 4547.
9. Homo-Delarche
F.: Glucocorticoid receptors and steroid sensitivity in normal and neoplastic human lymphoid tissues: a review. Cancer Res. 44 (1984) 431437. 10. Fuller P. J. and Funder J. W.: Mineralocorticoid and glucocorticoid receptors in human kidney. Kidney Int. 10 (1976) 154-157. 11. lacobelli S., Ranelletti F., Sicca G. and Barile G.: Glucocorticoid receptors in human fetal lung. J. endocr. Invest. 1 (1978) 209-213. 12. Hocchaus G., Rohdewald P., Mullmann H. and Greschuchna D.: Identification of glucocorticoid receptors in normal and neoplastic adult human lung. Res. exp. Med. 182 (1983) 71-78.
Fig. 1. Distribution of [‘H]RU 38486 binding sites in human brain. 35 nM [3H]RU 38486 were incubated alone (la) . , or in the nresence of 500 nM unlabeled RU 38486 (lb).I. RU 28362 (1~). \ II cortisol fld) RU 27987 (le) or dexamethasone (If) as indicated in Experimental. Sections were exposed for 26 days in darkroom at 20°C with [3H]Ultrofilm. Pseudo-colour autoradiograms were generated by an assisted image analyser from black and white autoradiograms [33]. A: amygdala, C: entorhinal cortex, F: fimbria, H: hippocampus, S: subiculum.
719
Steroid binding sites in human brain 13. Ranelletti F. O., Carmignani M., Iacobelli S. and Tonali P.: Glucocorticoid-binding components in human thymus hyperplasia. Cuncer Res. 38 (1978) 516-520. 14. Weatherill P. J. and Bell P. A.: Comparison of the physical characteristics of the molybdate-stabilized glucocorticoid receptor from rat, pig and human tissues. J. steroid Biochem. 21 (1984) 287-291. 15. Tsuboi S., Kawashima, R., Tomioka O., Nakata M., Sakamoto N. and Fujita T.: Glucocorticoid binding proteins of human brain cytosol. Brain Res. 179 (1979) 181-185. 16. Yu Z-Y., Wrange O., Boethius J., Gustafsson J. A. and Granholm L.: A qualitative comparison of the glucocorticoid receptor in cytosol from-human brain and rat brain. Brain Res. 223 (1981) 325-333. 17. Sarrieau A., Vial M., ‘Philibert D., Moguilewsky M., Dussaillant M., McEwen B. S. and Rostene W.: In vitro binding of tritiated glucocorticoids directly on unfixed rat brain sections. J. steroid Biochem. 20 (1984) 1233-1238. 18. Sarrieau A., Vial M., Philibert D. and Rostene W.: In vitro autoradiographic localization of ‘H corticosterone binding sites in rat hippocampus. Eur. J. Pharmac. 98 (1984) 151-152. 19. Palacios J. M., Niehoff D. L. and Kuhar M. J.: Receptor autoradiography with tritium-sensitive film: potential for computerized densitometry. Neurosci. Let& 25 (1981) 101-105. 20. Philibert D. and Moguilewsky M.: RU 28362, a useful tool for the characterization of glucocorticoid and mineralocorticoid receptors. 65th Ann. Mt. Endocr. Sot. Abstr. no. 1018 (1983) 335. 21. McEwen B. S., Gerlach J. L. and Micco D. J.: Putative glucocorticoid receptors in hippocampus and other regions of the rat brain. In The Hippocampus.: A Comprehensive Treatise (Edited by E. Isaacson and K. Pribram). Plenum Press, New York (1975) pp. 285-322. 22. Raynaud J. P. and Ojasoo T.: The relevance of structure-affinity relationships in the study of steroid hormone action. In Steroid Hormone Receptors: Structure and Function (Edited by H. Eriksson and J-A. Gustafsson). Elsevier Science Publishers B. V., Amsterdam (1983) pp. 141-170.
721
23. Raynaud J. P., Ojasoo T., Jouquey A., Moguilewsky M. and Teutsch G.: Probes for steroid receptors. In Endocrinology (Edited by F. Labrie and L. Proulx). Elsevier Science Publishers B. V., Amsterdam (1984) 533-536. 24. McEwen B. S., Davis P. G., Gerlach J., Krey L. C., MacLusky N. J., McGinnis M. Y., Parsons B. and Rainbow T. C.: Progestin receptors in the brain and pituitary gland. In Progesterone and Progesrin (Edited by C. W. Bardin, P. Mauvais-Jarvis and E. Milgrom). Raven Press, New York (1983) 59-76. 25. Moudgil V. K.: Progesterone receptors. In Principles of Receprerology (Edited by M. K. Agarwal). Walter de Gruyter, Berlin (1983) 273-379. 26. Healy D. L., Baulieu E. E. and Hodgen G. D.: Induction of menstruation by an antiprogesterone steroid (RU486) in nrimates: site of action. dose-resnonse relationship, and hormonal effects. Fer;. Ster. 40 (1983) 253-257. 27. Philibert D.: RU 38486: An original multifaceted antihormone in vivo. In Adrenocorticoid Antagonist (Edited by M. K. Agarwal). Walter de Gruyter, Berlin (1984) pp. 77-101. 28. Neef G., Beier S., Elger W., Henderson D. and Wiechert R.: New steroids with antiprogestational and antiglucocorticoid activities. Sreroidr 44 (1984) 349-372. 29. Gravanis A., Schaison G., George M., De Brux J., Satyaswaroop P. G., Baulieu E. E. and Robe1 P.: Endometrial and pituitary responses to the steroidal antiprogestin RU 486 in postmenopausal women. J. c/in. Endocr. Metab. 60 (1985) 156163. 30. Gagne D., Pons M. and Philibert D.: RU 38486. A potent antiglucocorticoid in vitro and in vivo. J. steroid Biochem. 23 (1985) 247-251.
31. Raynaud J. P., Lando D. and Moguilewsky M.: Les Steroides. Bases moleculaires de l’emploi des sttro’ides en nsvchiatrie. Acta Psvchiat. bek 80 (1980) 639-657. 32. MC&en B. S., De Kloet E. R. and R&t&e W.: Adrenal Steroid receptors and actions in the nervous system. Physiol. Rev. (1986) In press. 33. Rostene W., Quirion R., Beaudet A. et Maziere B.: Images des recepteurs des messagers chimiques et des medicaments dans le cerveau. Mid-Sci. 1 (1985) 419424.
Copyright
0
OO22-4731/86 $3.OO+0.00 Pergamon Journals Ltd
1986
Proceedings of the XII International Study Group for Steroid Hormones (Rome, 24 December 1985)
AUTORADIOGRAPHIC LOCALIZATION OF GLUCOCORTICOSTEROID AND PROGESTERONE BINDING SITES IN THE HUMAN POST-MORTEM BRAIN ALAIN SARRIEAU*§, MONIQUE DUSSAILLANT*, FRANCE AGID~, DANIEL PHILIBERT~ YVES ACID? and WILLIAM ROSTENE* lINSERM, U55, Centre de Recherches Paris Saint-Antoine, Hopital Saint-Antoine, Paris Saint-Antoine 184, rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, TINSERM U289, Hopital PitiQ-SalpiZtriere, 91, Bd de l’hopital, 75013 Paris and SLaboratoires Roussel Uclaf, 102, route de Noisy, 93230 Romainville, France Summary-The presence of glucocorticosteroid and progesterone binding sites in the human brain has been investigated with an in vitro autoradiographic approach using [3H]RU 38486. The present data revealed that the hippocampus, the entorhinal cortex, the subiculum, the fimbria and the amygdala showed a different pattern of [‘H]RU 38486 labeling using various unlabeled steroids (RU 38486, RU 28362, cortisol, RU 27987 and dexamethasone). RU 38486 is the best competitor in all these structures. The hippocampus seems to possess mainly glucocorticosteroid binding sites whereas the entorhinal cortex and the subiculum reveal the presence of both glucocorticosteroid and progesterone binding sites. Furthermore, the amygdaloid complex and the fimbria show a high density of glucocorticosteroid binding sites.
INTRODUCTION
A large number of investigations have described the presence of corticosteroid binding sites in several human tissues such as the placenta [14], blood lymphoid tissues [9], kidney [lo], cells [5-81, lung[ll, 121 and thymus [13, 141. However, few reports have described the presence of glucocorticosteroid
binding
sites in human
brain
whose
characterization has been carried out on cytosolic preparations [15, 161. The present study reports evidences for the heterogeneity of [3H]RU 38486 binding sites in the post-mortem human brain by means of an in vitro autoradiographic approach on brain sections. EXPERIMENTAL Human tissues
The brain of a 89-year old person with no known neurological disease was used. The brain was removed 15 h after death and kept at -70°C until dissection. Pieces of tissues, taken at the level of hippocampus, were dissected and 32 pm thick frontal sections were cut with a cryostat (Bright) at - 15°C collected on gelatin coated slides and kept at - 70°C until used as previously described for the rat brain [22]. Reagents
[3H]RU 38486 (50.6 Ci/mmol), RU 38486, RU 28362 (a pure glucocorticosteroid), RU 27987 (a synthetic progesterone binding site marker) were §To whom correspondence
should be addressed.
generous gifts from Roussel Uclaf (Romainville, France). Dexamethasone and cortisol were obtained from Sigma. Other chemicals were supplied by Prolabo (Whatman GF/B filter disks), Merck (ammonium molybdate, glycerol), Sigma (gelatin, ATP, dithiotreitol, EGTA and Tris), Baker (scintillation fluid: Aqualyte) and LKB France (Ultrofilm). Incubation and autoradiographic procedures
35 nM [3H]RU 38486 alone or in the presence of 500 PM unlabeled steroid were incubated in TEGMAD buffer (50 mM Tris-HCl pH 7.4, 2mM EGTA, 5% v/v glycerol, 6 mM molybdate, 5 mM ATP and 10 mM dithiotreitol) for 2 h at 4°C. Slides were then washed at 4°C 3 times for 5 min each with 200 ml of 50 mM Tris-HCl (pH 7.4) and sections were wiped off the slides with Whatman GF/B filter disks, placed into plastic vials with 5 ml Aqualyte and treated as previously described in detail [17] for the determination of [3H]RU 38486 radioactivity retained on the slides. For autoradiography, sections were dried after the washings with cold air and placed into Kodak X-ray film holders. Autoradiograms were produced by the apposition of a rigid tritium-sensitive film ([3H]Ultrofilm) on sections and exposed for 26 days at room temperature in a dark room. Films were then developed for 3 min with Kodak D-19 (18, 191. Black and white autoradiograms were arbitrarily converted into pseudo-colour pictures by means of a scanning densitometer (Pericolor 1000, image-assisted Numelec, France) ranging from red dots (where the binding density is the highest) to blue dots (areas which show a low density 717
of binding
sites).
ALAIN SARRIEUA et al
718 RESULTS
Figure 1 describes the competition by several unlabeled steroids for [3H]RU 38486 binding on human brain sections located at the level of the hippocampal formation. [3H]RU 38486 strongly binds to various subfields of the hippocampus mainly following the hippocampal circonvolutions as well as in the fimbria and the subiculum. However, not only the hippocampal formation is labeled with [3H]RU 38486 but also several areas such as the amygdaloid complex and the entorhinal cortex (Fig. la). Addition of unlabeled RU 38486 is able to completely abolish the labeling of [3H]RU 38486 in all the structures studied (Fig. lb). RU 28362, a pure glucocorticosteroid [20], is able to displace [3H]RU 38486 from its binding sites in all these structures but with a lesser extent than unlabeled RU 38486 (Fig. lc). Cortisol, the natural corticosteroid in man, competes with [3H]RU 38486 in the hippocampus, the amygdala and to a lesser extent in the fimbria and the subiculum; in contrast, it has no effect in the entorhinal cortex (Fig. Id). The synthetic progestagen RU 27987 almost completely abolishes the binding of [3H]RU 38486 in the entorhinal cortex and the subiculum but is not able to compete with [3H]RU38486 in the amygdala and weakly in the hippocampus and the fimbria (Fig. le). the Dexamethasone decreases binding of [3H]RU 38486 in the hippocampus and the entorhinal cortex, but slightly in the fimbria, the subiculum and the amygdala (Fig. If).
DISCUSSION
The present paper describes for the first time the binding of [3H]RU 38486 on human brain tissues. Human hippocampus appears a strong target for glucocorticosteroids as it was reported for the rat hippocampus[21]. Both unlabeled antagonist (RU 38486) and agonist (RU 28362) are good competitors for [3H]RU 38486 binding in the human hippocampal formation. In contrast, the natural steroid, cortisol, is much weaker to displace [3H]RU 38486 binding in the hippocampus. This is probably due to the fact that synthetic steroids often have a much higher affinity for binding sites than the natural compounds [22]. RU 27987, a metabolite of the progestin RU 5020, which specifically binds to progesterone receptors [23], is a strong competitor for [3H]RU 38486 binding essentially in the entorhinal cortex which suggests the presence of progesterone binding sites in the cortex in agreement with what was previously reported in the rat brain [24,25]. These data further support previous observations obtained in peripheral tissues suggesting that RU 38486 has both antiglucocorticosteroid and antiprogestagen properties [2630]. Dexamethasone, a synthetic corticosteroid used for the treatment of various human diseases [31], acts at
the level of the central nervous affinity for brain corticosteroid
system showing some binding sites in the
hippocampus and the entorhinal cortex. The use of various synthetic steroids reveals an heterogeneity in the binding capacity of [3H]RU 38486 in different structures of the human brain, as it was reported in the rat brain [32]. The presence of glucocorticosteroid binding sites in human brain is further confirmed by autoradiographic data obtained with the synthetic pure agonist [‘H]RU 28362 which reveal a similar pattern of labeling distribution than what is observed in the present work with the synthetic antagonist [3H]RU 38486 (data not shown). The use of highly potent steroids together with the development of in uitro autoradiographic approaches may give us facilities to detect possible modifications in glucocorticosteroid binding sites in various neurological diseases. authors with to thank Miss Martine Le Hein for typing the manuscript. This work was supported by a grant of Roussel Uclaf and INSERM (no. 84012).
Acknowledgements-The
REFERENCES 1. 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 tissues. Am. J. Obstet. Gynec. 145 (1983) 515-523. 2. Heller C.. Coirini H. and De Nicola A. F.: Influence of phosphatase inhibitors and nucleotides on [‘Hldexamethasone binding in cytosol of human placenta. J. steroid Biochem. 21 (1984) 381-386. 3. Lopez Bemal A., Anderson A. B. M. and Turnbull A. C.: The measurement of glucocorticoid receptors in human placental cytosol. Placenta 5 (1984) 105-I 16. 4. Lopez Bemal A. and Turnbull A. C.: High affinity glucocorticoid receptor in human intrauterine tissues. Horm. metab. Res. 17 (1985) 265-266. 5. Tsai B. S., Watt G., Koesnadi K. and Townley R. G.:
Lymphocyte glucocorticoid receptors in asthmatic and control subjects. Clin. Allergy 14 (1984) 363-371. 6. Harmon J. M.. Schmidt T. J. and Thomnson E. B.: Molybdate-sensitive and molybdate-resistant activation-labile glucocorticoid-receptor mutants of the human lymphoid cell line CEM-C7. J. steroid Biochem. 21 (1984) 227-236. 7. Eliard P. H. and Rousseau G. G.: Thermodynamics
of steroid binding to the human alucocorticoid receptor. Biochem. J. 21s (1984) 395404.
8.
Atmanini D.. Strasser T. and Weber P. C.: Parallel determination of glucocorticoid receptors in human mononuclear and Polymorphonuclear-leukocytes after Percoll separation. J. endocr. Inuesf. 8 (1985) 4547.
9. Homo-Delarche
F.: Glucocorticoid receptors and steroid sensitivity in normal and neoplastic human lymphoid tissues: a review. Cancer Res. 44 (1984) 431437. 10. Fuller P. J. and Funder J. W.: Mineralocorticoid and glucocorticoid receptors in human kidney. Kidney Int. 10 (1976) 154-157. 11. lacobelli S., Ranelletti F., Sicca G. and Barile G.: Glucocorticoid receptors in human fetal lung. J. endocr. Invest. 1 (1978) 209-213. 12. Hocchaus G., Rohdewald P., Mullmann H. and Greschuchna D.: Identification of glucocorticoid receptors in normal and neoplastic adult human lung. Res. exp. Med. 182 (1983) 71-78.
Fig. 1. Distribution of [‘H]RU 38486 binding sites in human brain. 35 nM [3H]RU 38486 were incubated alone (la) . , or in the nresence of 500 nM unlabeled RU 38486 (lb).I. RU 28362 (1~). \ II cortisol fld) RU 27987 (le) or dexamethasone (If) as indicated in Experimental. Sections were exposed for 26 days in darkroom at 20°C with [3H]Ultrofilm. Pseudo-colour autoradiograms were generated by an assisted image analyser from black and white autoradiograms [33]. A: amygdala, C: entorhinal cortex, F: fimbria, H: hippocampus, S: subiculum.
719
Steroid binding sites in human brain 13. Ranelletti F. O., Carmignani M., Iacobelli S. and Tonali P.: Glucocorticoid-binding components in human thymus hyperplasia. Cuncer Res. 38 (1978) 516-520. 14. Weatherill P. J. and Bell P. A.: Comparison of the physical characteristics of the molybdate-stabilized glucocorticoid receptor from rat, pig and human tissues. J. steroid Biochem. 21 (1984) 287-291. 15. Tsuboi S., Kawashima, R., Tomioka O., Nakata M., Sakamoto N. and Fujita T.: Glucocorticoid binding proteins of human brain cytosol. Brain Res. 179 (1979) 181-185. 16. Yu Z-Y., Wrange O., Boethius J., Gustafsson J. A. and Granholm L.: A qualitative comparison of the glucocorticoid receptor in cytosol from-human brain and rat brain. Brain Res. 223 (1981) 325-333. 17. Sarrieau A., Vial M., ‘Philibert D., Moguilewsky M., Dussaillant M., McEwen B. S. and Rostene W.: In vitro binding of tritiated glucocorticoids directly on unfixed rat brain sections. J. steroid Biochem. 20 (1984) 1233-1238. 18. Sarrieau A., Vial M., Philibert D. and Rostene W.: In vitro autoradiographic localization of ‘H corticosterone binding sites in rat hippocampus. Eur. J. Pharmac. 98 (1984) 151-152. 19. Palacios J. M., Niehoff D. L. and Kuhar M. J.: Receptor autoradiography with tritium-sensitive film: potential for computerized densitometry. Neurosci. Let& 25 (1981) 101-105. 20. Philibert D. and Moguilewsky M.: RU 28362, a useful tool for the characterization of glucocorticoid and mineralocorticoid receptors. 65th Ann. Mt. Endocr. Sot. Abstr. no. 1018 (1983) 335. 21. McEwen B. S., Gerlach J. L. and Micco D. J.: Putative glucocorticoid receptors in hippocampus and other regions of the rat brain. In The Hippocampus.: A Comprehensive Treatise (Edited by E. Isaacson and K. Pribram). Plenum Press, New York (1975) pp. 285-322. 22. Raynaud J. P. and Ojasoo T.: The relevance of structure-affinity relationships in the study of steroid hormone action. In Steroid Hormone Receptors: Structure and Function (Edited by H. Eriksson and J-A. Gustafsson). Elsevier Science Publishers B. V., Amsterdam (1983) pp. 141-170.
721
23. Raynaud J. P., Ojasoo T., Jouquey A., Moguilewsky M. and Teutsch G.: Probes for steroid receptors. In Endocrinology (Edited by F. Labrie and L. Proulx). Elsevier Science Publishers B. V., Amsterdam (1984) 533-536. 24. McEwen B. S., Davis P. G., Gerlach J., Krey L. C., MacLusky N. J., McGinnis M. Y., Parsons B. and Rainbow T. C.: Progestin receptors in the brain and pituitary gland. In Progesterone and Progesrin (Edited by C. W. Bardin, P. Mauvais-Jarvis and E. Milgrom). Raven Press, New York (1983) 59-76. 25. Moudgil V. K.: Progesterone receptors. In Principles of Receprerology (Edited by M. K. Agarwal). Walter de Gruyter, Berlin (1983) 273-379. 26. Healy D. L., Baulieu E. E. and Hodgen G. D.: Induction of menstruation by an antiprogesterone steroid (RU486) in nrimates: site of action. dose-resnonse relationship, and hormonal effects. Fer;. Ster. 40 (1983) 253-257. 27. Philibert D.: RU 38486: An original multifaceted antihormone in vivo. In Adrenocorticoid Antagonist (Edited by M. K. Agarwal). Walter de Gruyter, Berlin (1984) pp. 77-101. 28. Neef G., Beier S., Elger W., Henderson D. and Wiechert R.: New steroids with antiprogestational and antiglucocorticoid activities. Sreroidr 44 (1984) 349-372. 29. Gravanis A., Schaison G., George M., De Brux J., Satyaswaroop P. G., Baulieu E. E. and Robe1 P.: Endometrial and pituitary responses to the steroidal antiprogestin RU 486 in postmenopausal women. J. c/in. Endocr. Metab. 60 (1985) 156163. 30. Gagne D., Pons M. and Philibert D.: RU 38486. A potent antiglucocorticoid in vitro and in vivo. J. steroid Biochem. 23 (1985) 247-251.
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