Proceedings of the Fourth International Congress on Hormonal Steroids. Mexico City, September 1974

907 Pages·1976·70.993 MB·English

by  V. H. T. James and J. R. Pasqualini (Eds.)

Other titles of interest
ARVY, L.
Histoenzymology of the Endocrine Glands
CONTI, C.
Research on Steroids
DICZFALUSY, E.
Pharmacology of Reproduction
PASQUALINI, J. R.
Recent Advances in Steroid Biochemistry
RASPE, G.
Schering Symposium on Endocrinology
RASPE, G.
Schering Symposium on Biodynamics and Mechanisms of Action of
Steroid Hormones
RASPE, G.
Schering Workshop on Steroid Metabolism
RASPE, G.
Schering Symposium on Mechanisms Involved in Conception
RASPE, G.
Schering Workshop on Pharmacokinetics
RASPE, G.
Schering Symposium on Intrinsic and Extrinsic Factors in Early Mam­
malian Development
RASPE, G.
Schering Workshop on Steroid Hormone "Receptors"
RASPE, G.
Workshop on Mechanisms and Prospects of Genetic Exchange
RASPE, G.
International Workshop on Prostaglandins
RASPE, G.
Schering Symposium on Contraception: The Masculine Gender
RASPE, G.
Workshop on Virus-Cell Interactions
RASPE, G.
Schering Symposium on Immunopathology
RASPE, G.
Hormones and Embryonic Development
RASPE, G.
Workshop on Prognostic Factors in Human Acute Leukemia
RASPE, G.
Schering Workshop on Central Action of Estrogenic Hormones
VOCKAER, R. & DE BOCK, G.
Sexual Endocrinology
VILLEE, C. A.
Control of Ovulation
Proceedings of the Fourth International
Congress on Hormonal Steroids
MEXICO CITY, SEPTEMBER 1974
Edited by
V. H. T. JAMES
St. Mary's Hospital Medical School, London
and
J. R. PASQUALINI
Foundation for Hormone Research, Paris
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First edition 1976
Library of Congress Catalog Card No. 75-24945
Published originally as Volume 6, Numbers 3/4, 5 and 6 of
The Journal of Steroid Biochemistry, 1975, and reprinted here without change of
pagination
Printed in Great Britain by A. Wheaton ά Co., Exeter
ISBN 0 08 019682 9
FOURTH INTERNATIONAL CONGRESS ON
HORMONAL STEROIDS
ORGANIZING COMMITTEE:
E. DICZFALUSY, Sweden (Chairman) V. H. T. JAMES, U.K.
L. MARTINI, Italy (Secretary) E. V. JENSEN, U.S.A.
L. CASTELAZO-AYALA, Mexico K. D. VOIGT, Germany
J. FRIED, U.S.A.
PROGRAMME COMMITTEE:
E. E. BAULIEU, France Μ. G. KOLPAKOV, U.S.S.R.
J. BERTRAND, France G. W. LIDDLE, U.S.A.
R. BORTH, Canada S. LlEBERMAN, U.S.A.
H. BREUER, Germany H. R. LINDNER, Israel
I. E. BUSH, U.S.A. M. B. LIPSETT, U.S.A.
L. CASTELAZO-AYALA, Mexico J. MARTINEZ-MANAUTOU, Mexico
E. DICZFALUSY, Sweden L. MARTINI, Italy
G. DORNER, Germany R. P. MICHAEL, U.K.
L. L. ENGEL, U.S.A. D. R. MISHELL, U.S.A.
J. FERIN, Belgium T. NOMURA, Japan
J. FISHMAN, U.S.A. J. M. ROSNER, Argentina
J. FRIED, U.S.A. A. SEGALOFF, U.S.A.
E. GLAZ, Hungary J. SJOVALL, Sweden
B. HUDSON, Australia S. SOLOMON, Canada
V. H. T. JAMES, U.K. R. VIHKO, Finland
.E. V. JENSEN, U.S.A. K. D. VOIGT, Germany
A. B. KAR, India J. D. WILSON, U.S.A.
N. A. YUDAEV, U.S.S.R.
LOCAL COMMITTEE:
L. CASTELAZO-AYALA (Chairman) A. GALLEGOS
J. L. MATEOS V. GARZA
O. V. DOMINGUEZ A. ZARATE
J. FlNDELMAN
OPENING REMARKS
Mr. President of the Republic, President of the Congress, resume their function when transplanted into another spe­
Ladies and Gentlemen, on behalf of the International cies. Reading this book is a moving experience; one sees
Organizing Committee I would like to express our most the "trial and error" approach of man in operation and
sincere thanks to you for making it possible to organize one recalls Elbert Hubbard saying "truth is an imaginary
the 4th International Congress on Hormonal Steroids here line dividing error into two parts . . .".
in Mexico City. We are particularly pleased and delighted, And now the third and fourth phases in the discovery
since there is hardly any other city on earth which had of a steroid hormone: the method of assay, isolation and
such an important historic role in the development of the identification. McGee in the U.S.A. describes effective
steroid field. extraction procedures for the active substance from bull
Since man began to speculate on what forces regulate testes in 1927, and Gallagher and Koch in Chicago develop
the course of life, the idea occurred to him again and again a quantitative method for the testicular hormone in 1930.
that not only external forces dominate him, but that the Now, one can assess the effect of every step in the purifica­
vital processes have their origin in the body itself. Our tion procedure, and already the following year, in 1931,
savage ancestors tried to solve the riddle of life by the Adolf Butenandt in Germany isolates from male urine a
idea of powerful divine beings who arbitrarily pushed them steroid, androsterone, which is believed to be the male hor­
around and whom they must cajole if they wished to pre­ mone. Another error, but a very close guess, and in 1935
serve their health. However, our ancestors soon discovered Professor Ernst Laqueur's group from Holland reports on
the important role of certain internal forces, the role of the isolation of testosterone, the main steroid of the testis.
internal secretions, when they observed the far-reaching With this crystalline material clinical studies are started
metabolic and psychological effects of castration. They all over the world; large quantities of material are needed
noticed major changes in fighting instinct and in persona­ and chemists start to synthesize testosterone and even
lity following the removal of the testes. This is the first more active compounds. These chemical modifications of
step in the history of any biologically active steroid, the the molecule soon lead to the development of the first
observation that the removal of a gland induces a state oral contraceptives by Carl Djerassi and his colleagues in
of deficiency, and Aristotle in his Historia animalium 1952; however, it is impossible to discuss this chapter of
around 350 B.C. describes very well the effects of castration History without considering the role of Mexico, and Pro­
on birds, beasts and men. The next step is always to show fessor Castelazo-Ayala is much more qualified than I am
that the administration of an extract of the gland can re­ to do so. Rather, I would like to mention that I could
store the normal condition; although the Indian Ayurveda show you, Ladies and Gentlemen, the same historic pro­
of Susruta in 1400 B.C. as well as Plinius in ancient Rome cess, with the same cardinal steps, deprivation, substitu­
recommended the administration of testicular tissue for the tion, method of assay, isolation, identification and syn­
cure of impotence and Mesiie, the Elder in the 8th Century thesis in the case of virtually all biologically active steroids.
prescribed such a diet as an aphrodisiac, it is doubtful I could show you how the method of Allen and Doisy
that this therapy had any effect, except perhaps a psycho­ enabled the Doisy group to isolate the female hormone,
logical one. It is first John Hunter in England, who in estradiol, and how the method of Corner and Allen helped
1762 reports on the transplantation of testis in the fowl, Fels and Slotta to isolate progesterone, the hormone of
followed by Professor A. Berthold in Göttingen, who in gestation. I could also tell you that the story of the testicu­
1849 claims to prevent the effects of castration in roosters lar hormone is by no means finished, that ongoing research
by transplanting their testicles into another part of the suggests the existence of another hormone, "inhibin" in
body. Fifty years later, in 1899, the 79 year old Professor the testis, and I could call your attention to the words
Brown-Séquard injects himself with extracts of crushed dog of Shakespeare that "what is past is prologue".
and guinea pig testicles and enthusiastically describes to What is perhaps more important is to realize in a hum­
his astonished and incredulous colleagues in the French ble way that science is a continuously self-correcting pro­
Academy the rejuvenating effect of such therapy, which cess and to remember the words of George Sarton, saying
he calls "la puissance dynamogénique". A few years earlier, that in the final analysis "the acquisition and systematiza-
in 1894, the Russian scientist A. Poehl isolated a crystalline tion of positive information is perhaps the only human
substance, Spermin, which was then believed to be the tes­ activity which is really progressive".
ticular hormone, but soon proved to be ineffective. This Mr. President, Ladies and Gentlemen, communication
is the time when commercial exploitation starts, and dur­ is perhaps one of the most complex and most important
ing the decades which follow tons of Spermin and of functions of the human mind. The local Organization
aqueous extracts of bull testes are sold all over the world Committee has provided us with the most excellent facili­
by ambitious men to ambitious men. ties to communicate with each other and to exchange in­
My next note is from 1920; the first World War is over formation during this week and we are very grateful for
and Steinach publishes his famous book on his equally this opportunity. On behalf of the International Organiz­
famous rejuvenating operation, which consists of the liga- ing Committee, I would like to thank once more for all
tion of the Vas Deferens; even this turns out to be of help, generous assistance and effort by our hosts and col­
doubtful, or in the best case of temporary value, just as leagues in Mexico, which will make this Congress a suc­
the next step in the drama. In 1923, Professor S. Voronoff cessful and most memorable one.
publishes his book Greffes testiculaires; he implants mon­
key testes into ageing men, hoping, in vain, that the glands EGON DICZFALUSY
xin
Journal of Steroid Biochemistry, 1975, Vol. 6, pp. 147-159. Pergamon Press. Printed in Great Britain
MECHANISM OF ACTION OF STEROID HORMONES
I. S. EDELMAN
The Cardiovascular Research Institute and the Departments of Medicine, and of Biochemistry and Biophysics
of the University of California, School of Medicine, San Francisco, Ca. 94143, U.S.A.
SUMMARY
Despite the diversity in the target sites and physiological actions of the steroids, an impressive body
of evidence has been assembled in support of a unitary theory of the mechanism of action of these
hormones in vertebrates. The steroids set in motion a train of events, as follows: (a) penetration
into the target cell, (b) stereo specific binding to high affinity receptors, (c) temperature-sensitive acti­
vation of the steroid-receptor complex, (d) attachment of the active complex to chromatin, (e) induction
of RNA and protein synthesis, and (f) physiological expression of the induced protein. Although the
overall sequence is well-defined, our knowledge of the molecular processes involved in each of these
steps is still quite incomplete. Two of the major efforts now underway to elucidate these molecular
processes involve, (a) purification and characterization, in terms of structure-function relations, of the
putative receptors, and (b) studies on the nature of the interaction between steroid-receptor complexes
and the genome. It is now apparent that steroids induce de novo synthesis of both messenger RNA
(mRNA) and ribosomal RNA (rRNA); the role of the former in directing the synthesis of specific
proteins is reasonably clear but that of the latter remains to be elucidated. The mechanism of induction
is also under scrutiny since the observed increases in mRNA synthesis could arise in a variety of
ways, e.g. negative or positive regulation of chromatin template (gene) activity, changes in processing
of heterogeneous RNA to mRNA, effects on RNA polymerase or ribonuclease activities. Although
steroidal regulation of RNA and protein synthesis is a dominant pathway, the possibility of direct
actions on membranes or regulatory enzymes in some circumstances can not be excluded at the present
time.
INTRODUCTION permeability properties of plasma and organelle mem­
branes, or on enzymes (see below). The primary focus
In vertebrates, circulating hormones dominate the
of attention in this report, however, will be on the
morphogenetic and physiological states of almost all
tissues, throughout the life span. In the last 15 years, induction mechanism (Fig. 1).
two unifying theories have emerged that account for
Steroid receptors
most of these diverse cellular responses to hormones ;
the mediating roles of cyclic nucleotides [cyclic The mode of steroid penetration into target cells
adenosine monophosphate and cyclic guanosine has received little attention. In one system, however,
monophosphate] in the actions of peptide and cate- uptake of estrogen by uterine cells appears to involve
cholamine hormones, and induction of protein syn­ facilitated rather than simple diffusion [6].
thesis in the actions of steroid and thyroid hormones. The target cells responsive to a given steroid con­
The interrelationships between these two primary tain high affinity, saturable cytoplasmic binding pro­
mechanisms are also under active study. teins (mol. wt. = 100,000 Dal tons) and the uptake of
A wealth of evidence has led to widespread agree­ the steroid precedes the appearance of the physiologi­
ment on the sequence but not the details of events cal effect. These and other findings, including (1) the
set in motion by the steroids in many well-defined stereospecificity of the high affinity binding proteins,
target tissues [1-5]. An outline of this theory is shown (2) the close correlation between affinity of various
in Fig. 1. The steroid enters the target cells, combines steroids for the binding site and physiological
with a high affinity receptor forming an active com­ potency, and (3) association of the steroid-protein
plex that then binds to selective sites in the chro­ complex with chromatin, lend credence to the infer­
matin. The interaction between the hormone-receptor ence that these high affinity binding proteins are
complex and the genome activates or derepresses receptors that mediate the biological response [1,4].
transcription or post-transcriptional regulation of The physical and biological characteristics of all
RNA synthesis. The products, mRNA and rRNA, dic­ of the steroid receptors, including that of 1,25 dihyd-
tate the synthesis of specific proteins whose properties rocholecalciferol (Vitamin D derivative) are remark­
determine the morphogenetic and physiological re­ ably homologous [1,4]. The main features of these
sponses to the hormones. Although this receptor- homologies will be illustrated for the most part by
effector system mediates many of the actions of ster­ reference to studies with aldosterone, since this ster­
oid hormones, other basic mechanisms may also play oid has been the focus of my work for more than
a role, especially at very high concentrations of the a decade [7].
hormones. This possibility will be alluded to briefly The association of steroids with target cell nuclei
in relation to possible direct effects of steroids on the was first revealed by autoradiography [7,8]. 3H-
147
148 I. S. EDELMAN
CAPILLARY
TARGET CELL
Translation
Steroid Steroid
Induced Physiological
Proteins Effects
Receptor
Fig. 1. General model of the receptor-induction mechanism of steroid hormone action. Modified from
Feldman et al.[2].
Aldosterone was localized to the nuclear and peri- independently proposed by Gorski et al.[10] and by
nuclear areas of toad bladder epithelium. In contrast, Jensen et α/.[11]. An important feature of this
3H-progesterone, an inactive steroid at low con­ mechanism is the requirement for temperature acti­
centrations compared to aldosterone, was randomly vation of the complex for binding to the nucleus.
distributed. Bogoroch (cited in Edelman [9] ) explored The cytoplasmic-nuclear transfer process and other
the specificity of the observed binding in competition features of the steroid-receptor system have been in­
studies (Table 1). Excess estradiol-17ß, which is inac­ corporated into the model shown in Fig. 2. The recep­
tive with respect to Na+ transport in the toad blad­ tor is assumed to exist in an inactive and an active
der, had no effect on the distribution of 3H-aldoster- conformation in equilibrium, as formulated by Rubin
one but 9a-fluorocortisol, an active mineralocorticoid, and Changeux[12] for allosteric enzymes. This allos-
significantly diminished both nuclear and cytoplasmic teric equilibrium model was applied to steroid recep­
localization. These findings imply the existence of tors by Samuels and Tomkins[13] in order to account
cytoplasmic and nuclear receptors, in vivo. for the behaviour of steroids witl* mixed agonist and
The receptors appear to reside in the cytoplasm antagonist properties [suboptimal inducers—in their
pending availability of their respective steroids and nomenclature]. The phenomenon of partial agonist-
then bind to chromatin sites after formation of the antagonist behaviour is illustrated in Table 2. In the
cytoplasmic complex. This "two-step" mechanism was isolated toad bladder system, 11-deoxycortisol elicited
Table 1. Quantitative distribution of 3H-aldosterone between cytoplasm and nucleus
of toad bladder epithelium by autoradiography
Competitive steroid (100:1) Grains/nucleus Grains/cytoplasm
None 2-66 0-79
17/?-estradiol 2-75 0-96
9a-fluorocortisol 0-84 0-45
Toad bladders were exposed to 3H-aldosterone (5-3 x 10" 8M) for 30min with
or without added steroid (5-3 x 10" 6M). Average of 200 cells counted per section.
[Bogoroch, R. & Edelman, I. S., cited in [9]].
Table 2. Agonist/antagonist activity of 11-deoxycortisol on Na+ transport in the isolated toad bladder
Agonist/antagonist
No. of Increase in SCC (6 h)* activity
pairs Steroid (%) (%)
Aldosterone (7 x 10" 8 M) 219 + 28
11-Deoxycortisol (5 x 10"6M) 66 ± 16 + 30
Aldosterone (7 x 10" 8 M) 88 ± 21
Aldosterone (7 x 10" 8 M) -71
26+ 10
11-Deoxycortisol (5 x 10"6M)
* SCC denotes short-circuit current. Mean + S.E.M. Edelman, I. S.: Unpublished results.
Mechanism of action 149
Fig. 2. Allosteric-equilibrium model of the steroid-receptor system. Modified from Feldman e tal.[2].
an increase in active Na+ transport, measured by the amino-transferase by glucocorticoids and occupied
short-circuit current (SCC) technique, that was 30% of cytoplasmic glucocorticoid receptor sites but failed to
that caused by maximum doses of aldosterone and in generate intra-nuclear complexes. Kaiser et al. [15, 16]
combination with aldosterone inhibited the response reported that in rat thymocytes, the anti-glucocorti-
by 71%. Thus total activity accounts for 100% occu­ coid, cortexolone bound to the receptor but failed
pancy of the putative receptors and can be ration­ to transfer to nuclear binding sites. In our studies,
alized as indicating that the affinity of 11-deoxy- the anti-mineralocorticoid, spirolactone-mineralocor-
cortisol for the two conformations is in the ratio of ticoid-receptor complexes (SC-26304) did not bind to
30:70, active vs inactive. Primary agonists (e.g., aldos­ nuclear acceptor sites in vivo (see Table 3), nor in
terone) would presumably have a high affinity for the reconstitution experiments with isolated nuclear or
active conformation and negligible affinity for the in­ chromatin fractions [17]. These results are consistent
active conformation. The converse should hold for with the allosteric-equilibrium model. Selective
primary antagonists. Support for this model was affinity for the inactive form of the receptor, however,
obtained in studies with steroid antagonists. Baxter need not be the only mechanism of action of an anta­
et a/. [14] found that in hepatoma cells in tissue cul­ gonist. Clark et al. [18] reported that the anti-estrogen
ture, progesterone inhibited induction of tyrosine nafoxidine-HCl, acted by causing prolonged nuclear
retention of the estrogen receptor, and depletion of
cytoplasmic receptor content, without inducing com­
Table 3. Cytoplasmic and nuclear binding of 3H-aldoster- mensurate uterine stimulation.
one and 3H-SC-26304, in vivo
Numerous investigators have explored the physical
3H-Aldosterone 3H-SC-26304 characteristics of the cytoplasmic and nuclear forms
of the steroid receptors. In high salt density gradients,
2 min 10 min 2 min 10 min the cytoplasmic steroid-receptor complexes migrate
Cytoplasm 31 10 6-4 1-2
at 4 to 5S and in low salt at 7 to 9S (Table 4). Some
Tris-soluble 11-3 19-7 -2-2 -0-8
KCl-extract 5-9 9-6 -1-4 0 antagonist-receptor complexes do not aggregate in
low salt solutions; 3H-SC-26304-mineralocorticoid
Serum 10-6 5-9 54-6 23-4 receptor complexes and 3H-cortexolone-glucocorti-
coid-receptor complexes migrated at 3S to 4S in both
After ligation of the portal vein and hepatic artery,
adrenalectomized rats were injected i.v. with either (1) low and high salt density gradients [15, 17]. These
0-7 x 10~9mol 3H-aldosterone + 10 x dexamethasone, findings are consistent with the inference that binding
or (2) 10-4 x 10~9mol 3H-SC-26304 + 10 x dexametha­ of the agonist (but not these antagonists) activates
sone and the kidneys were removed at 2 or 10 min after
the receptor by inducing a change in receptor confor­
injection. All fractions were extracted with dichlorometh-
mation or sub-unit interactions (Fig. 2). Sherman and
ane and corrected for non-specific labeling based on injec­
tions in paired rats with 100 x d-aldosterone. The tissue co-workers [25,26] recently proposed a subunit
results are expressed in units of x 10_14mol/mg protein. model for the progesterone receptor consisting of a
The negative values indicate that the non-specific quanti­ globular subunit that binds the steroid and an asso­
ties exceeded the total bound in the absence of excess cold
ciated asymmetric subunit(s) that may control speci­
steroid. The serum concentrations (xl0"9M) of the
dichloromethane extractable 3H-steroid are given below fic binding of the complex to chromatin. The steroid-
the line. Mean of two experiments. From Marver et α/.[17]. binding subunit was obtained by treatment of par-
150 I. S. EDELMAN
Table 4. Sedimentation of cytoplasmic steroid receptor-complex in density gradients
Authors Steroid Tissue Low salt High salt
Marver et α/.[19] Aldosterone Kidney 8-5S/4S 4-5S
Kaiser et α/.[15] Triamcinoloneacetonide Thymus 7S/3-5S 4S
Baxter and Tomkins[20] Dexamethasone Hepatoma 8S 4S
Baulieu et α/.[21] Dihydrotestosterone Ventral 8S/4-5S 4^5S
Mainwaring and Mangan[22] prostate
Jensen et al.[23] Estradiol Uterus 8S/4S 4-5S
Sherman et α/.[24] Progesterone Oviduct 8S/5S 3-7S
Marver et α/.[17] Spirolactone Kidney 3S 4S
Kaiser et α/.[15] Cortexolone Thymus 3-5S 3-5S
tially purified receptors with Ca2+ or other divalent to purify and characterize the receptors with the aid
cations and is a low molecular weight, compact, basic of photo-affinity labels are also in progress [31,32].
protein. In contrast, the intact receptor is a high Recently, Gorell et al.[33] reported purification of an
molecular weight, asymmetric, acidic protein, imply­ estradiol-receptor complex extracted from calf uterine
ing an association of dissimilar subunits. nuclei and obtained sufficient yields for determination
Impressive evidence that the steroid receptors are of molecular weight (^72,000 Daltons) and amino
bi-functional (or multi-functional) has been obtained acid composition. More detailed information on these
in studies of genetic variants of lymphoma cells in and related studies may answer many of the funda­
tissue culture [27]. Cell selection was accomplished mental questions on structure-function determinants
by resistance to the killing action of dexamethasone in the expression of receptor activity.
in the medium. As shown in Table 5, three variants
have been isolated and characterized; r~ lacks de­
Nuclear binding of steroid-receptor complexes
monstrable high affinity glucocorticoid binding sites,
nt~ is deficient in nuclear transfer of the complex to Nuclear steroid-receptor complexes appear to be
chromatin and d~ fails to respond despite formation derived from the cytoplasmic pool. In vivo, formation
of the chromatin-bound complex. The existence of of the cytoplasmic complex precedes appearance of
the nt~ variant indicates that the steroid-binding site the nuclear bound form and cytoplasmic receptor
is distinct from the determinant for binding of the content is depleted as nuclear-binding proceeds
complex to chromatin and is consistent with the Sher­ [10,11]. In the excised uterus, generation
man model of dissimilar "subunits" and her specula­ of the 5S nuclear 3H-estradiol complex and
tion that steroid-binding is the province of one sub- depletion of the cytoplasmic 8S complex is tempera­
unit and binding to chromatin the province of the ture-dependent and these shifts are in phase. Similar
other. findings have been obtained in reconstitution exper­
Characterization of the primary and higher order iments with steroid-labeled cytosol fractions and un-
structure of the cytoplasmic and nuclear forms of the labeled nuclear or chromatin fractions from many tis­
steroid receptors will be possible when sufficient sues [1]. One such experiment is shown in Fig. 3.
amounts of pure material are available for analysis. Renal cytoplasmic fractions from adrenalectomized
At the present time, a number of serious efforts to rats were labeled with 3H-aldosterone and incubated
purify these receptors are underway. Significant pro­ with washed renal nuclei at 25°C. On a quantitative
gress has been reported in the purification of cyto­ basis nuclear uptake of 3H-aldosterone complexes (re­
plasmic estrogen receptors by affinity chromat­ covered by 01 M Tris-HCl and 0-4 M KCl elution)
ography [28, 29] and to some extent in the purifica­ accounted for 60% of the receptor content lost from
tion of cytoplasmic progesterone receptors by DEAE- the cytoplasmic fraction during incubation [19].
cellulose chromatography [30]. Promising attempts '' Moreover, dissociation of the cytoplasmic complex by
Table 5. Genetic variants in glucocorticoid responsive lymphoma cells in tissue culture
Genetic Cytoplasmic Cytoplasmic Nuclear Physiological
type reactants product product response
Receptor + Steroid —► Complex --* Chromatin-bound —> I Transport and growth
4 Wild complex death
_*
r~~
nt~ + + -
d~ + + +
* The r cells may either lack receptors entirely or the receptor may be present but defective in the binding reaction.
From Sibley et α/.[27].
Mechanism of action 151
Table 6. Intranuclear distribution of 3H-aldosterone
TRIS-SOLUB LENUCLEAR
-O
Fraction , Total Bound
Tris-CaCl2 Extract 27-6 ± 0-9 63*
Chromatin 55-1 ± 2-1 76t
Residual 17-3 + 0-3
Mean + S.E.M. Bound refers to the proportion of the total
0 6 3H-Aldosterone in the particular fraction that was bound
INCUBATION TIME (min) to a macromolecule.
* Determined by precipitation with 50% saturated
Fig. 3. Formation of nuclear 3H-aldosterone-receptor com­
(NH ) S0 and G-50 Sephadex gel filtration.
plexes in reconstituted mixtures of cytosol and nuclear 4 2 4
t The chromatin was sheared and the resultant soluble-
fractions of kidneys from adrenalectomized rats. Cytosol
nucleohistone was passed through G-50 Sephadex
was pre-labeled with 3H-aldosterone (1-3 x 10~8 M) + 9a-
columns. From Swaneck et α/.[34].
fluorocortisol (1-3 x 10-6M) and incubated with washed
nuclei at 25°C. From Marver et α/.[19].
or no binding to chromatin, inhibit steroid action
heating to 37°C eliminated nuclear uptake; in con­ [15,17], and nuclear transfer negative (nt~) lym-
trast pre-heating the nuclear fraction did not impair phoma variants are resistant to cytolysis by glucocor-
uptake. ticoids [27]. Recently, Gehring and Tomkins[36]
Direct association of the steroid-receptor com­ found that temperature-activated dexamethasone-
plexes derived from the cytoplasm with chromatin receptor complexes from glucocorticoid-sensitive lym-
accounts for much of the nuclear uptake process. phoma cells bound to isolated nuclei from both sensi­
Thus, in nuclei prepared from adrenalectomized rats, tive and resistant cells, and to homologous and heter-
after injection of the steroid in vivo, binding of ologous DNA. The cytoplasmic complex from resis­
3H-aldosterone-receptor complexes to chromatin tant cells, however, was deficient in binding to either
accounted for at least 55% of total nuclear accumu­ nuclear or DNA fractions.
lation (Table 6) [34]. Moreover, chromatin and intact The molecular reactions involved in the association
nuclei manifest very similar acceptor activity in recon- of steroid-receptor complexes with chromatin and the
stitution experiments. For example, Mainwaring and mechanism of modulation of gene expression by this
Peterken[35] showed that undenatured cytoplasmic event are, for the most part, still undefined. Chro­
receptor proteins (from rat prostate) were needed to matin acceptor activity apparently has some degree
transfer 3H-5a-dihydrotestosterone to isolated chro­ of specificity in recognizing steroid-receptor com­
matin and that extraction of the labeled chromatin plexes, at least in some systems. Lippman and
with KCl yielded a 4-5S complex indistinguishable Thompson[37] found that HTC cell nuclei bound
from that found in intact prostate. glucocorticoid-receptor complexes from L cells after
The relevance of chromatin binding of these com­ saturation with HTC cell complexes and vice versa.
plexes to physiological action is indicated by In many systems, however, tissue specificity with re­
numerous findings: (1) In well-characterized target tis­ spect to nuclear or chromatin acceptor activity is not
sues, the ability of various steroids to block formation demonstrable. The significance of binding of steroid-
of the chromatin-bound specific steroid-receptor com­ receptor complexes to chromatin in reconstitution ex­
plexes correlates closely with their potencies as periments, however, has been called into question by
agonist or antagonists [1-4]. This phenomenon is Chamness et al. [38] who found no evidence of satu­
illustrated in Table 7 with competition experiments ration of nuclear binding of estrogen-receptor com­
on the binding of 3H-aldosterone complexes to renal plexes even when nuclear uptake was several-fold
chromatin [34]. In addition, as noted above, steroids greater than that obtained with maximal physiologi­
that form cytoplasmic receptor complexes with little cal doses in vivo. These results raise the possibility
Table 7. Steroidal specificity for 3H-aldosterone-binding sites in renal
chromatin
Relative specific activity
No. Competing steroid*
24 None 100
9 Progesterone (100:1) 06+ 18
6 17^-Estradiol (100:1) 89 + 12
10 Cortisol (100:1) 51 + 10
6 9a-Fluoro-cortisol ( 100:1 ) 11 + 0-9
8 Spirolactone (10,000:1) 21 + 3
Mean + S.E.M. No. denotes the number of experiments.
* Molar ratio of competing steroid to 3H-aldosterone is given in parentheses.
From Swaneck et α/.[34].