Steroid Hormone Regulation of the Brain: Proceedings of an International Symposium Held at the Wenner-Gren Center, Stockholm, 27-28 October 1980

Sweden

OPENING ADDRESS

K. FUXE,     Department of Histology, Karolinska Institutet, Stockholm, Sweden

Ladies and Gentlemen! On behalf of the organizing committee which consists of Professor Jan-Åke Gustafsson, Professor Lennart Wetterberg and myself, I wish you very welcome to Stockholm and to this Wenner-Gren Symposium on the Steroid Hormone Regulation of the Brain. We are very grateful that so many of you have been able to accept our invitation in spite of other commitments. We are indeed very happy to see so many pioneers in this field here with us this morning. The reason for organizing this symposium has been to bring specialists in the steroid field and specialists in the neuroscience field, especially in the field of chemical neurotransmission, together in order to have a friendly and fruitful discussion on the mechanisms underlying the steroid hormone regulation of the brain. The organization of this symposium also reflects the well accepted view of the importance of a strict interaction between basic and clinical sciences.

Both the steroidal field and the neuroscience field are of course highly recognized. We should mention that Henry Dale and Otto Löwi in 1936 received the Nobel Price for their discoveries relating to chemical transmission of nerve impulses, and in 1970 Ulf von Euler, Katz and Axelrod received the Nobel Price for their discoveries concerning humoral transmitters in nerve terminals and the mechanism for their storage, release and inactivation. Furthermore, in 1950, Kendall, Reich-stein and Hench received the Nobel Price for their discoveries on hormones relating to the adrenal cortex, their structure and biological effects. Finally, in 1966 Charles Brenton Huggins received the Nobel Price for his discoveries concerning hormonal treatment of prostatic cancer. We hope, that by bringing these two fields together in a symposium, we can have a very rewarding scientific debate which may lead to new ideas and also to better understanding of how steroid hormones via target cells in the brain may regulate specific types of synaptic mechanisms in various brain areas and in this way produce their actions of neuroendocrine activity, on instinct behaviours, such as agression and sexual behaviour and on mental activities. We think it is important to emphasize that the heuristic hypothesis in the 1950 of a profound analogy between computer and man is no longer tenable. The new data obtained above all by means of the wet i.e. the neurochemical approach to neurobiology clearly shows that neurons and neuronal networks integrate not only electrical messages but also chemical messages. A basis for this view is the fact that the brain is a target also for blood born signals and among those especially the steroid hormones. The steroid signal and its regulation of neuronal mechanisms is the subject of this symposium. The steroid signal and its possible interaction with other humoral messengers is a matter for a future meeting, since we would like to stress that it is necessary to begin with a study of one factor at each time. However, the aim is obviously a multifactorial analysis of the brain using an heuristic approach like that suggested by Bertalanffy in his system theory in 1950.

With these introductory remarks I have on behalf of the organizing committee the pleasure of opening our symposium and give the word to Professor Luft who is the chairman of the first session of this symposium.

Section I

ACTIONS OF STEROID HORMONES

Outline

Chapter 1: STEROID HORMONES IN THE BRAIN: SEVERAL MECHANISMS?

Chapter 2: THE INTERACTION OF ESTROGENS WITH INTRACELLULAR RECEPTORS AND WITH PUTATIVE NEUROTRANSMITTER RECEPTORS: IMPLICATIONS FOR THE MECHANISM OF ACTIVATION OF SEXUAL BEHAVIOR AND OVULATION

Chapter 3: STUDIES ON THE GLUCOCORTICOID RECEPTOR

Chapter 4: STEROID HORMONE SITES OF ACTION IN THE BRAIN

Chapter 5: DEVELOPMENTAL ASPECTS OF CATECHOLESTROGEN SYNTHESIS

Chapter 6: REGULATION OF HEPATIC STEROID METABOLISM BY THE HYPOTHALAMO-PITUITARY-LIVER AXIS

STEROID HORMONES IN THE BRAIN: SEVERAL MECHANISMS?

E.-E. BAULIEU,     Inserm U33, Faculté de Médecine de l’Université Paris XI, Lab Hormones, 94270 Biêtre, France

ABSTRACT

Brain cells contain steroid hormone receptors. Aspects of the intracellular steroid receptor system, including the mechanism of action and utilization of estrogens and non-steroidal antiestrogens, are discussed with reference to a recent review (in Central Regulation of the Endocrine System edited by K. Fuxe, T. Hökfelt and R. Luft in 1979, Plenum Press, New York).Since steroids may also act on the plasma membrane of neurons, we report the main characteristics of the only steroid-membrane system of physiological significance which has been studied: progesterone at the surface of maturing Xenopus laevis oocytes. We describe the direct inhibitory effect of progesterone on Adenylate cyclase and discuss steroid-peptide hormone interactions.Finally, we have identified dehydroepiandrosterone sulfate in the rat brain. Still of unknown function, its accumulation (∼ 10 pmol/g tissue) seems independent of the peripheral endocrine system.

KEYWORDS

Steroid hormones

brain

receptors

membrane

Adenylate cyclase

antiestrogens

dehydroepiandrosterone sulfate

estradiol

estriol

insulin

ovalbumin

chick oviduct

breast cancer

endometrium cancer

INTRODUCTION

Radioautographic and biochemical studies of steroid binding in the brain have suggested that, as in other target organs, steroid hormone activities are mediated by intracellular receptors modulating gene expression (Baulieu and colleagues, 1975; Gorski and Gannon, 1976; Jensen and DeSombre, 1972; O’Mailey and Means, 1974). This is the case for sex and adrenal steroids exerting regulatory controls on the availability of pituitary hormones (McEwen and colleagues, 1979; Stumpf and Grant, 1975; Warembourg, 1978). We first summarize some aspects concerning this receptor system, bringing up controversial points related to the mechanism of action of hormones and antihormones. We next review data which were obtained with Xenopus laevis oocytes, establishing that progesterone can act at the cell surface level independently of gene activation, and showing for the first time a direct effect of steroids on Adenylate cyclase activity. We also draw attention to steroid-peptide hormone interactions in this system. Finally, we discuss the recent discovery in the rat brain of rather large quantities of dehydroepiandrosterone sulfate (DS) which, at this stage of our knowledge, appears to accumulate independently of the peripheral endocrine system. The relevance of these observations to steroid influenced functions in the brain has still to be evaluated¹.

INTRACELLULAR RECEPTORS

Sex steroid and glucocorticosteroid receptors have been described in various regions of the brain (see the relevant chapters of this book). Even though there remains still much to do in order to correlate hormone binding and biological activities, (see for instance the still unexplained differential binding of corticosterone and dexamethasone attributed to different receptors (McEwen, 1979)), it has been established that these brain receptors show the same characteristics as the steroid receptors involved in the control of protein synthesis and/or growth of non-neural target cells. For instance the hormonal control of receptor concentration, such as that of the progesterone receptor by estrogen which was originally described in the uterus (Milgrom, Atger, and Baulieu, 1970), also operates in the brain and may be functionally relevant to steroid action (Schwartz, Blanstein, and Wade, 1979; Parsons and colleagues, 1980). We have recently summarized this field (Baulieu, 1978b) and we concentrate here only on three particular aspects of interest for approaching brain functions physiologically and pharmacologically.

Ligand Affinity and Metabolism vs Agonist and Antagonist Activities.

Steroid hormone receptors have very high affinity for their natural hormones (corresponding to Kd,eg ∼ 1-0.1 nM). Usually weak agonists have lower affinity and strong agonists show higher affinity, related to faster or slower dissociation rates, respectively.in vivo however, effects are also very much dependent on ligand availability at the receptor level, and therefore on its overall metabolism measured by the Metabolic Clearance Rate, MCR (Tait and Burstein, 1964). An interesting (and historical) example is the case of estriol, a low affinity natural estrogen showing a fast dissociation from the receptor and a high MCR. After a single injection, its very brief availability at the receptor level explains its weak effect as compared that of estradiol on rat uterus growth. Regardless of the dose injected, estriol is prevented from giving a full estradiol effect (impeded estrogen: Huggins and Jensen, 1955). However, repeated injection of the steroid allows the maximal estrogenic response to be obtained (Lan and Katzenellenbogen, 1976). The beginning of estradiol action includes an early water imbibition of the uterine tissue which is followed secondarily by generalized protein synthesis (Astwood, 1938). The response to the single injection of estriol is mostly confined to the early imbibition, and consequently a qualitative difference between the two estrogens has been suggested. In fact, we believe that this so-called qualitative difference is due primarily to the short presence of estriol in the target cell, which makes it incapable of inducing the protein synthesis response. Repeated injection of estriol can compensate for its rapid clearance. This point is of importance, since it may be argued that complexes between different agonist ligands and the estrogen receptor may interact with different effector sites in the gene machinery. That there may not be such molecular differences is supported by unpublished experiments performed by P. Pennequin with the chick oviduct system: early effects of estradiol and estriol on the accumulation of the mRNAs of egg white proteins (as measured by the relative rate of synthesis according to Palmiter, Oka, and Schimke, 1971), are exactly the same. Therefore we believe that different steroids inducing the same receptor conformation which is able to interact identically with the same components of the gene machinery may provoke different qualitative responses, through differences in binding affinity and/or general metabolism governing their availability at the target level. In this context, a detailed study of the positive and negative effects of estrogens on LH release would be interesting, as would that of the mechanism of action of a weak estrogen-antiestrogen such as Clomiphen in inducing ovulation, and also of sex steroids and antiestrogens on sexual behavior (Etgen, 1979). The use of impeded estrogen for obtaining an antiestrogenic effect could also be discussed along these lines.

If the proper hormonal response to an agonist can occur regardless of its affinity for the binding site, conversely other ligands interacting with the same receptor site may be antagonists, again whatever their affinity. Tamoxifen and 4-hydroxytamoxifen are non-steroidal antiestrogens which do not display any estrogenic activity in the chick oviduct system (Baulieu and colleagues, 1980). However, the former has less affinity and the latter more affinity than estradiol itself for the estrogen receptor. Incidently, it is interesting to note that tamoxifen is approximately as effective as hydroxytamoxifenin vivo because of its very low MCR. The simple global explanation postulates a 2 state model of receptor conformation one dealing with hormone activity and favored by the binding of agonists, and the other dealing with non-active receptor complexes and favored by the binding of antagonists. Whatever the mechanism of the allosteric transition between the two forms, the steric differences of ligands provoking one or the other receptor conformation, are not reflected by hormone binding affinities. If we consider the extreme situations, we measure either the binding of an agonist ligand to the active conformation of the receptor, or the binding of an antagonist ligand to the inactive conformation of the receptor. The simplest logic suggests that in each series the ligands with highest affinity provoke the most important effects, irrespective of whether they are agonists or antagonists. The difficult problem of the mixed agonist-antagonist compounds has not been solved. Ligands may modulate the equilibrium between the two forms of the receptor differently.

Naturally occurring steroids may be studied in the light of this discussion. Dihydrotestosterone probably favors the agonist conformation of the androgen receptor more than testosterone (Anderson and Liao, 1968; Bruchovsky and Wilson, 1968; Baulieu, Lasnitzki, and Robel, 1968), and estradiol binding to the androgen receptor may provoke an antagonist conformation (Feyel-Cabanes and colleagues, 1978). Conversely, the binding of androgens to the estrogen receptor seems to stabilize its estrogenic form (Rochefort, Lignon, and Capony, 1972).

Receptor Activation and Distribution vs Agonist and Antagonist Activities

Non-steroidal antiestrogens which bind to the estrogen receptor translocate it from the cytoplasm to the nucleus (Clark, Anderson, and Peck, 1973; Rochefort and Garcia, 1976), even if the antihormone is not agonist, as in the case of tamoxifen applied to the chick oviduct system (Sutherland, Mester, and Baulieu, 1977). Therefore, the presence of receptor in the nucleus is not necessarily associated with hormone action, and the translocation of cytoplasmic receptor to the nucleus does not always lead to estrogen activity, the nature of the ligand being decisive. This concept should be kept in mind in the interpretation of receptor measurements and of cytological visualization of receptor.

Estradiol can trigger an estrogen response in tamoxifen treated cells when most receptor is nuclear. Conversely tamoxifen can suppress estradiol activity when injected at the same time or after estradiol administration, provided that the respective affinities and concentrations of the hormone and the antiestrogen allow the appropriate replacement of one by the other. Therefore, contrary to previous suggestions (Clark, Anderson, and Peck, 1973), the antiestrogenic effects can take place whether the estradiol receptor is present in the cytoplasm or in the nucleus at the time of antihormone administration. The estrogenic effect can also take place even if the estradiol receptor is predominantly located in the nucleus and eventually complexed with antagonist when estrogen is administered.

Transformation of the receptor has been described in terms of change of sedimentation coefficient of the receptor observedin vitro upon binding estradiol, and implicitly related to the hormone dependent translocation of the cytoplasmic receptor (8S) to the nucleus (5S) (Jensen and DeSombre, 1972). Activation was originally described as a hormone dependent increase of affinity of the receptor for polyanions (acidophilic activation (Higgins, 1973; Milgrom, Atger, and Baulieu, 1973)), including nuclei and has been correlated to the translocation of this receptor from the cytoplasm to the nucleus. More recently, the rate of dissociation of estradiol from activated estrogen receptor was found to be slower than that from non-activated receptor (Weichman and Notides, 1977). Using molybdate (Nielsen, Vogel, and Pratt, 1977) as a tool for monitoring transformation of the chick oviduct receptor, we found a correlation between its change of sedimentation coefficient, acidophilic activation and the decrease of the rate of dissociation of the hormone (Wolfson and colleagues, 1980). Since high affinity, non-agonist 4-hydroxytamoxifen provokes the transfer of the receptor to the nucleus in the chick oviduct, J. Mester and C. Geynet have recently undertaken experiments with the chick estradiol receptor in order to see whether the antiestrogen does provoke actual activation. Preliminary (Mester and colleagues, 1980) and unpublished experiments of C. Geynet indicate that under heat activation conditions, in the absence but not in the presence of molybdate, 4-hydroxytamoxifen complexes show a change in sedimentation coefficient and a slowing down of the dissociation rate. Thesein vitro results confirm the contrast between the lack of estrogenic activity of 4-hydroxytamoxifen and the transfer of the drug-receptor complex into the nucleus which we observed in vivo. A non-active ligand can therefore provoke receptor activation and binding to a nuclear receptor which is its probable consequence, suggesting that these steps are not the only critical determinants for promoting hormone action. Recent experiments of M.C. Lebeau and N. Massol at the chromatin level (Massol, Lebeau, and Baulieu, 1978) suggest binding differences for the estrogen- and antiestrogen-receptor complexes.

Antiestrogen and Multihormonal Effects

Tamoxifen does not display the same balance between estrogenic and antiestrogenic activities in different animal species. Purely antiestrogen in the chick, it is mostly antiestrogenic and weakly estrogenic in the human and other primates (this was the basis of its early use in breast cancer) (Walpole, 1968). In the rat uterus, although the antiestrogenic action is evident, tamoxifen is also an estrogen and in the mouse uterus the estrogenic response is largely predominant. It could be that the estrogen receptor differs in the various species studied so far, in such a way that the balance between the active and inactive conformations upon binding of antiestrogen will be different and thus explain these results. Other factors may be involved however, and in mouse L cells in culture, tamoxifen is a very potent antiestrogen (Jung-Testas and Baulieu, 1979). Moreover, the possible role of specific antiestrogen binding sites has yet to be considered (Faye, Lasserre, and Bayard, 1980; Sutherland and Foo, 1979). We wish to emphasize here another aspect of the complexity which we have to deal with, in revealing that the hormonal milieu may dramatically change the activity of the antiestrogen drug.

Once more, the chick oviduct system serves as a model. Progesterone increases, as does estradiol, the acccumulation of egg white proteins such as ovalbumin and conalbumin. Tamoxifen does not, as indicated before, and we took advantage of this situation to examine whether progesterone action was dependent on oestradiol. If that were the case, the administration of tamoxifen together with progesterone would decrease the effects of the latter. We did not observe such a result, thus, confirming the likelihood that progesterone directly increases protein synthesis via its own receptor. However, we observed an increase of progesterone effects, and the conalbumin synthesis was much potentiated as were other parameters of oviduct cell function also (M.G. Catelli and N. Binart, in preparation). Even more curiously, the same sort of potentiation effects of tamoxifen were observed with androgen and glucocorticosteroid stimulated syntheses of specific proteins in the chick oviduct (Y. Mainguy and Y. Lebouc, to be published). The mechanisms involved in these unexpected results are beyond our present understanding. However, in addition to their theoretical interest, such side-effects must be considered when using an antiestrogen clinically, and one should not reason only in terms of a simple antiestrogen activity. For instance it has been observed that tamoxifen can increase the concentration of progesterone receptors in the estrogen receptor containing breast and endometrium cancers in women. This obviously indicates that tamoxifen is not a pure antagonist, but it may be of practical interest for the prediction of hormone responsiveness and for the monitoring of therapy (Namer, Lalanne, and Baulieu, 1980). The application of these principles to the central nervous system may help to explain the complex multihormonal activities observed there.

STEROID HORMONE ACTION AT THE MEMBRANE LEVEL

The very structure of steroid hormones has led several investigators to postulate early on that their interaction with cell membranes may be biologically important. Even if interesting experiments with artificial lipid systems have been performed several years ago (Bangham, Standish, and Weissman, 1965; Willmer, 1961), only the reinitiation of meiosis in Xenopus laevis oocytes by progesterone has provided convincing data demonstrating that the interaction of a steroid hormone with the cell surface can be of physiological significance (Baulieu and colleagues, 1978).

The Progesterone-Oocyte Model System for Steroid Membrane Interaction

In studies conducted with S. Schorderet-Slatkine, we have studied meiosis, which is quite different from any process occurring in the brain. However, being only concerned with the initial events which occur when steroid hormones interact with membranes, we may find in the primitive germ cells biochemical mechanisms which are of significance even for the most differentiated brain cells.

The progesterone effect on oocytes does not exhibit the same narrow specificity as the binding of steroid hormones to intracellular receptors in somatic target cells. One can obtain the meiotic response by exposing oocytes to testosterone, cortisol or various steroid metabolites which are not involved in vivo. Specificity can however be demonstrated since minor chemical changes of active steroids, which do not affect their polarity, may abolish their activity. In addition, we could observe antagonism of progesterone action, apparently of competitive nature, by using inactive or very weakly active steroids such as 17α-ethynyl-estradiol. These results led to the suggestion that there are a limited number of specific membrane sites for steroids at the surface of oocytes (review in Baulieu and colleagues, 1978). Indeed, we cannot decide at the present time whether they belong to a receptor protein or to specific lipidic areas, or to a combination of both. Involvement of Ca²+ movements (Review in Baulieu and colleagues, 1978), inositol phospholipid metabolism (Schorderet-Slatkine, Schorderet, and Baulieu, 1977) and methylation Processes² have been observed which may help to further understand the transmission of the biological signal at the membrane level (Hirata and Axelrod, 1980).

A concentration of progesterone of 1 μM is necessary to obtain 50 % of the effect, suggesting that the interaction with the oocyte membrane is of low affinity. However, this value corresponds to the naturally occurring concentration of the hormone delivered directly by follicle cells to the oocyte. The anaesthetic action of large pharmacological doses of progesterone, deoxycorticosterone and steroids of the 1l-oxo-5α-pregnane series observed in the rat and in the human may be related to a molecular mechanism similar to that of progesterone action on the oocytes. Physiologically, the biosynthesis of estradiol (Naftolin and colleagues, 1975) and of catechol-estrogens (Fishman and colleagues, 1976) may create zones of high steroid concentration which could operate on the membranes of neighbouring cells. Alternatively, one should also consider the possibility that neurons have a typical high affinity membrane receptor similar to that described for uterine cells (Pietras and Szego, 1977), and several experiments (Dufy and colleagues, 1979; Kelly and colleagues, 1977) have demonstrated high affinity, stereo-specific, short latency electrophysiological responses of neuro-cells to steroids, suggesting membrane effects rather than a genomic response (Baulieu, 1978a). With B. Carette and J. Barry, we have used acidic steroids which do not bind to intracellular receptors and do not enter target cells and we observed specificity (Carette and colleagues, 1979). However, these iontophoretic experiments did not establish any correlation between the electrical effects and the biological function of the tested cells. Recent experiments of D. Poulain (this volume) have even indicated that the effects of acidic derivatives of steroids and of their corresponding natural hormones introduced by micro-pressure method are not correlated. It is clear that the present approaches in this field are only very preliminary. Finally, we must stress that effects obtained in the oocyte system with drugs active on neurons, such as propranolol and other β-adrenergic inhibitors, local anaesthetics, neuroleptics, antidepressants, … are probably not directly relevant to the studies of neuromechanisms since no correlation between their pharmacological properties and their effect on oocytes has been found (Schorderet-Slatkine and Schorderet, 1976).

Steroid Effect on Adenylate Cyclase

A clearcut effect of progesterone on membrane function has recently been revealed in studies carried out by J. Labbé-Lepicard and J. Hanoune on Xenopus laevis oocyte Adenylate cyclase (in preparation). Previous findings of S. Schorderet-Slatkine and M. Schorderet (in preparation) had established a rapid and sustained decrease of cAMP provoked by progesterone in oocytes. The germ cells were homogenized and after removal of 1000×15 gxmin pellet, the 10,000×20 gxmin sedimenting fraction was studied. It contains ∼ 30 % of the total Adenylate cyclase activity of the homogenate, and 5–8 % total protein. The specific activity of Adenylate cyclase is the highest in this subcellular fraction (80 pmol/g protein/30 min) and its Km ∼ 0.7 mM ATP. An examination of the 10,000 g fraction by electron microscopy showed membrane vesicle-like structures. The Adenylate cyclase activity of this fraction is stimulated several fold by previous exposure of oocytes to 10 pM cholera toxin for 6 h, while the soluble enzyme, obtained in the 100,000×30 gxmin supernatant and representing 50-60 % of oocyte total Adenylate cyclase activity, was unchanged. Progesterone 10 μM applied for 1.5 min to untreated or cholera toxin treated oocytes profoundly decreased Adenylate cyclase activity of the 10,000 g fraction. In vitro, the enzymatic activity of the same fraction was stimulated by NaF 20 mM and by Gpp(NH)p 10 μM and progesterone decreased both basal or stimulated Adenylate cyclase activity in a dose-dependent manner. None of these changes were observed when the soluble enzyme was tested. Steroid specificty was studied in the 10,000 g fraction directly by comparing the effects of 10 μM progesterone and other steroids. Testosterone was very active (as it is in intact cells), and also cortisone and to a lesser degree cortisol, but 17α-ethynyl-estradiol was not, again reminiscent of effects on the intact oocytes. Simultaneous incubation of the 10,000 g fraction with 17α-ethynyl-estradiol 10 μM showed a displacement of the dose-response curve of progesterone by 1 order of magnitude, compatible with a competitive antagonist effect, again as observed in the entire