Antioestrogen inhibition of oestradiol-induced alterations in hypothalamic noradrenaline turnover.

Long-term (4-6 weeks) ovariectomized rats were injected with either oestradiol benzoate (OB; 20 micrograms s.c.) or monohydroxytamoxifen (MTAM; 0.2 mg i.p.) plus OB. Oestradiol benzoate was administered at 12.00 h on day 0 and MTAM was given immediately before OB, followed by further injections twice daily to maintain sufficiently high antioestrogen levels. When given alone, OB reduced the serum levels of LH during the morning (08.00-09.00 h) and afternoon (17.30-18.30 h) hours of day 3 after priming. The feedback actions of OB on LH release were accompanied by time-dependent alterations of noradrenaline turnover in the preoptic-anterior hypothalamic brain area (POAH). On day 3 after priming the noradrenaline turnover rate was reduced in the morning and increased in the afternoon. The increase correlated with an enhanced sensitivity of the LH secretory system to progesterone. The antioestrogen MTAM blocked the OB-induced sensitization of LH release to the stimulatory action of progesterone and interfered with the stimulatory long-term effect of oestradiol on hypothalamic noradrenaline turnover. The data strongly support the view that the oestrogen-induced afternoon increase of noradrenaline turnover in the POAH represents a prerequisite for the induction of LH surges. The stimulatory effect of oestradiol on hypothalamic noradrenaline turnover seems to be mediated by a classical oestrogen receptor mechanism.

Effects of oestradiol benzoate and progesterone on luteinizing hormone release and catecholamine turnover in the preoptic-hypothalamic brain area of ovariectomized rats.

At noon, long-term (4-6 weeks) ovariectomized rats were exposed for 6-78 h to a single subcutaneous injection of oestradiol benzoate (20 micrograms) which significantly reduced the serum levels of LH over the whole time-interval investigated. The negative feedback action of oestradiol was accompanied by reduced turnover of both noradrenaline and dopamine in the preoptic-anterior hypothalamic brain area (POAH), but not in the mediobasal hypothalamus, 6, 68 and 72 h after administration of the hormone. Between 72 and 78 h after oestradiol-priming an afternoon increase of noradrenaline turnover was observed in the POAH. In rats primed with oestradiol benzoate for 72 h, short-term exposure (6 h) to progesterone (2.5 mg) induced a marked surge of serum LH and FSH in the late afternoon. In the POAH of these rats progesterone did not interfere with the afternoon increase of noradrenaline turnover induced by oestradiol-priming. However, it markedly increased the dopamine turnover rate of primed rats, thus reversing the inhibitory action of oestradiol benzoate on the dopaminergic system of the POAH. It is concluded that both the noradrenergic and the dopaminergic neurones of the POAH are involved in the negative and positive feedback actions of oestradiol and progesterone on LH and FSH release. The paper discusses whether the oestradiol-induced afternoon increase in noradrenaline turnover represents a prerequisite for the induction of LH surges by progesterone.

Action of oestrogens and antioestrogens on oestrogen-inducible cytoplasmic and androgen-inducible microsomal activity of 3 alpha-hydroxysteroid dehydrogenase in male rat kidney.

The influence of steroidal and non-steroidal antioestrogenic compounds on the effect of systemically administered oestradiol (OE2) and diethylstilboestrol (DES) was investigated in adult male rats with intact gonads. In this animal model, oestrogens induced the NADP-dependent cytoplasmic activity and prevented the inductive action of androgens on NADP-dependent microsomal activity of renal 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDH). Simultaneous administration of tamoxifen (0.5 mg/day) with OE2 (5 microgram/day) or DES (10 microgram/day) for 10 days completely blocked the inductive effect of OE2 on cytoplasmic 3 alpha-HSDH, whereas, in the case of the microsomal enzyme, the repressive effects of OE2 and DES were antagonized only to 28 and 16% respectively. Simultaneous administration of 5 alpha-dihydrotestosterone (DHT; 0.5 mg/day) for 10 days antagonized the inductive effect of OE2 on the cytoplasmic enzyme activity to 86% and completely by-passed the repressive effects of OE2 and DES on the microsomal enzyme activity. It is concluded that oestrogenic induction of renal cytoplasmic 3 alpha-HSDH involves an oestrogen receptor mechanism which, in this animal model, can be antagonized by tamoxifen. In contrast, oestrogenic repression of renal microsomal 3 alpha-HSDH is obviously the consequence of the strong antigonadotrophic activity of oestrogens leading to subsequent repression of testicular androgen secretion by mechanisms which can be only weakly antagonized by tamoxifen. Exogenous DHT, even in the presence of OE2 or DES, completely compensates for this centrally mediated deficit of peripheral androgen.

Time-dependent effects of oestradiol and progesterone on hypothalamic catecholamine turnover in ovariectomized rats.

Long-term ovariectomized rats received a single injection of 20 micrograms oestradiol benzoate (OB) which reduced the serum levels of LH for at least 3 days. The inhibitory effects were accompanied by time-dependent alterations of noradrenaline and dopamine turnover rates in the mediobasal hypothalamus (MBH) and the preoptic-anterior hypothalamic brain area (POAH). Oestradiol markedly interfered with the time-dependent variations of noradrenaline and dopamine turnover seen in the MBH of untreated ovariectomized animals during daylight hours. In the POAH the turnover rate of noradrenaline decreased 2 days after priming with OB and then increased in the afternoon of day 3. The increase of noradrenaline turnover in the POAH was accompanied by a low afternoon turnover rate of dopamine in the MBH and by an increased sensitivity of the LH secretory system to progesterone. Dopamine and noradrenaline turnover involve a time element. While the negative feedback actions of oestradiol do not seem to be associated with changes in dopamine or noradrenaline turnover, the results support the view that the induction of LH afternoon surges depends upon an increase of stimulatory noradrenergic inputs to the POAH and a decrease of inhibitory dopaminergic inputs in the MBH.

Enzyme activity in kidney, adrenal and gonadal tissue of rats treated neonatally with androgen or oestrogen.

A single injection of 300 mug oestradiol benzoate (OEB) or 1.25 mg testosterone propionate (TP) on day 1 of life led to significant changes in the activity of enzymes involved in steroid hormone metabolism in kidney, adrenal and gonadal tissues of adult rats. In the kidney, the enzyme activities of male rats reacted to OEB, but not TP, by the development of normal female levels. With one exception the enzyme activities of the kidney of female rats did not respond to either steroid. In the adrenal of both sexes 5alpha-reductase reacted to OEB, but not TP treatment, by a fourfold increase in activity. In the ovary all the enzymes investigated responded both to OEB and TP treatment by a fall in activity; 20alpha-hydroxysteroid dehydrogenase activity fell to undetectable levels. In the testis, OEB and TP treatment led to contrasting effects. With the exception of 5alpha-reductase all the enzymes tested in this organ responded to OEB by a rise in activity. Where TP had any effect, it produced a slight decrease in activity.

Endocrine regulation of sex-dependent hydroxysteroid dehydrogenase activities in rat kidney: NADP-dependent microsomal 3alpha- and 20beta-hydroxysteroid dehydrogenase.

The NADP-dependent microsomal kidney enzymes, 3alpha- and 20beta-hydroxysteroid dehydrogenase (HSDH), which exhibit considerable sex differences in their activities (male:female activity ratios, 16:1 and 30:1 respectively), were investigated after interference with the pituitary-gonad and pituitary-adrenal systems. Prepubertal gonadectomy as well as hypophysectomy of mature male rats led to a decline in HSDH activity to almost that found in the normal female rat, whereas activities in female rats were unaffected. Testosterone induced typical male 3alpha-HSDH activity in both gonadectomized and hypophysectomized rats of either sex. Administration of 5alpha-dihydrotestosterone (5alpha-DHT) or 5alpha-androstane-3alpha, 17beta-diol to hypophysectomized male rats was equally effective in restoring full 3alpha- and 20beta-HSDH activities whereas 5alpha-androstane-3beta, 17beta-diol was less effective and dehydroepiandrosterone was ineffective. Simultaneous administration of cyproterone acetate did not block the inductive action of 5alpha-DHT. Administration of chorionic gonadotrophin, pregnant mare serum gonadotrophin or a combination of luteinizing hormone and follicle-stimulating hormone to hypophysectomized male rats all led to parallel increases in the weight of the seminal vesicles and in both renal enzyme activities; administration of growth hormone, prolactin or thyroid-stimulating hormone was ineffective. Adrenalectomy of gonadectomized, but not of hypophysectomized male rats, caused a further drop in activity to the normal female level. Adrenalectomy of otherwise intact rats did not affect either enzyme activity. The hypophysis was involved in the regulation of the two NADP-dependent renal HSDH activities through its gonadotrophic function in male rats; adrenal secretions were of little physiological significance.

Inhibition of synaptosomal high-affinity uptake of dopamine and serotonin by estrogen agonists and antagonists.

High-affinity uptake of dopamine and serotonin into a synaptosomal preparation from rat cerebral cortex was inhibited by a number of estrogen agonists and antagonists in vitro in a stereoselective and competitive manner. The most potent estrogenic inhibitors in the dopaminergic and serotonergic system were ethinylestradiol (KI = 558 nM) and 2-hydroxyethinylestradiol (KI = 226 nM), respectively. Structure-activity relationships are discussed and compared with the effects of estrogens on noradrenaline uptake. However, as all physiologically occurring estrogens inhibited amine uptake only in the micromolar concentration range it seems unlikely that this direct interaction of estrogen with the amine carrier is responsible for the changes in dopamine and serotonin uptake observed during the estrous cycle or after in vivo administration of estrogens and/or progesterone.

Re-establishment of stimulatory estradiol effects on luteinizing hormone secretion in long term ovariectomized rats.

The ability of a single injection of estradiol benzoate (EB, 20 micrograms) to induce proestrus-like surges of luteinizing hormone (LH) on the day following EB administration was tested in long term ovariectomized rats which had been pretreated with EB (20 micrograms) and/or progesterone (P, 2.5 mg). LH afternoon surges could be evoked only in those animals which had previously received both ovarian hormones, EB and P. Pretreatment with a single hormone was ineffective. Moreover, in rats preinjected only with P the LH surge was shifted from afternoon to morning hours.

Effects of short-term exposure to catecholestrogens on catecholamine turnover in the preoptic-hypothalamic brain of ovariectomized rats.

The effects of a series of catecholestrogens (2-hydroxyestrogens and 4-hydroxyestrogens) were compared to those of a primary estrogen, ethynylestradiol (EE2), on the catecholaminergic system in the preoptic-hypothalamic rat brain. Adult ovariectomized rats received single injections (100 microgram s.c.) of EE2, 2-hydroxyestradiol (2-OHE2), 2-hydroxyethynylestradiol (2-OHEE2), 4-hydroxyestradiol (4-OHE2) or 4-hydroxyethynylestradiol (4-OHEE2). Eight hours after estrogen administration the animals were killed, and the concentrations of dopamine (DA), noradrenaline (NA) and adrenaline (A) were determined radioenzymatically in the preoptic area (POA) and the mediobasal hypothalamus (MBH). EE2 and all the catecholestrogens tested uniformly suppressed (P less than 0.01) serum LH levels. This correlated well with the decreased turnover rate of A in the POA. In both the POA and the MBH of EE2-treated animals the turnover rate of NA was markedly decreased whereas the concentrations of catecholamines remained unaffected. The catecholethynylestrogens, potent inhibitors of catechol O-methyltransferase, caused two-fold increases of NA concentrations in the POA (2-OHEE2 and 4-OHEE2) and MBH (2-OHEE2) without affecting the turnover rate. Thus, although EE2 and all the catecholestrogens tested uniformly suppressed LH release they induced highly different effects on the noradrenergic system In the preoptic-hypothalamic brain.

Estrogen induction of progestin receptors in pituitary, hypothalamic and uterine cytosol of androgenized female rats.

Female rats treated neonatally with a single dose (1.25 mg/animal) of testosterone propionate and ovariectomized when adult did not respond to a priming dose (20 micrograms/animal) of estradiol-17 beta 3-benzoate and subsequent application of progesterone (2.5 mg/animal) 72 h later with an afternoon surge of luteinizing hormone, which could be induced by the same hormonal regimen in neonatally oil-treated long-term ovariectomized female rats. However, both treatment groups responded equally well to the estrogen stimulus with an increase in cytosolic progestin receptors in hypothalamic and pituitary, as well as uterine tissue. It therefore seems unlikely that the observed loss of sensitivity of the gonadotropin release mechanism in neonatally androgenized, estrogen-primed female rats to a progesterone stimulus can be explained by a loss of progestin receptor induction capacity of estrogen/progestin target tissues involved in gonadotropin secretion.

Preferential uptake of norepinephrine into dopaminergic terminals of a synaptosomal preparation from rat cerebral cortex.

In a synaptosomal preparation from male rat cerebral cortex only 34% of total norepinephrine (NE) uptake could be inhibited by nanomolar concentrations of desmethylimipramine (DMI) with an apparent IC50 value of 0.37 nM. The residual uptake was efficiently inhibited by micromolar concentrations of DMI (IC50 = 4.0 microM). In synaptosomes from the hypothalamus, 74% of total NE uptake could be blocked by DMI with an IC50 of 0.1 nM whereas in synaptosomes from the striatum the IC50 for DMI inhibition was 3.8 microM. It is concluded that in synaptosomes from rat cerebral cortex only 34%, and in synaptosomes from the hypothalamus 74% of total NE are taken up by noradrenergic nerve terminals whereas the residual NE uptake occurs in dopaminergic nerve endings.

Competition by monophenolic estrogens and catecholestrogens for high-affinity uptake of [3H](-)-norepinephrine into synaptosomes from rat cerebral cortex and hypothalamus.

High affinity uptake of [3H](-)-norepinephrine (NE) was investigated in synaptosomes from rat cerebral cortex (Km = 360 +/- 30 nM) and hypothalamus (Km = 307 +/- 90 nM). Estrogens but not androgens, glucocorticoids or progestin interfered competitively with NE uptake. Ethinylestradiol was the most effective competitor tested, its Ki value being 200 nM in the cortex and 144 nM in the hypothalamus. Stereospecificity of the inhibitory effect of estradiol-17 beta with a preference for the 17 beta-hydroxy group was indicated by the ineffectiveness of estradiol-17 alpha and estrone as competitors. A-ring substitution of estradiol-17 beta or ethinylestradiol by hydroxyl groups in positions 2 and 4 (yielding catecholestrogens) or methyl substitution in positions 2 and 4 (yielding methylestrogens) significantly reduced the inhibitory potency of the estrogen. Methoxylation in positions 2, 4 or 11 beta completely abolished the competitive action of estradiol-17 beta or ethinylestradiol on NE uptake.

Neonatal defeminization of the luteinizing hormone release mechanism by catecholestrogens: different potencies of 2- and 4-hydroxyestradiol.

Female rats were neonatally treated with estradiol-17 beta-benzoate or the long-acting dibenzoate esters of the isomeric catecholestrogens, 2-hydroxyestradiol-17 beta and 4-hydroxyestradiol-17 beta. Estrogen benzoates were administered subcutaneously from day 1 to 5 of life at doses of 0.05, 0.10, 0.50 and 1.00 micrograms/day. All rats were ovariectomized as adults and, 4 weeks later, the luteinizing hormone (LH) response to progesterone (2.5 mg) was tested after priming with estradiol-17 beta-benzoate (20 micrograms). At a dose of 0.5 micrograms/day, estradiol-17 beta-benzoate and 4-hydroxyestradiol-17 beta-dibenzoate were equally effective in neonatally defeminizing the LH surge mechanism. In contrast, up to a dose of 1.00 micrograms/day, 2-hydroxyestradiol-17 beta-dibenzoate did not interfere with the LH response in adult life. In the pituitary gland and uterus of the neonatally defeminized rats estrogen responsiveness of cytosolic progestin receptor induction was unimpaired. Moreover, in the uterus of these rats nuclear translocation of cytosolic progestin receptors was intact.

Estrogen agonistic and antagonistic action of 8 non-steroidal antiestrogens on progestin receptor induction in rat pituitary gland and uterus.

All 8 non-steroidal antiestrogens tested considerably increased progestin receptor concentration in the uterus and, to a lesser extent, in the pituitary of ovariectomized rats. However, the pituitary was more sensitive than the uterus to the estrogen antagonistic action of these compounds, in that monohydroxytamoxifen, LY 117,018, enclomiphene, nitromifen, nafoxidine and trans-tamoxifen completely blocked progestin receptor induction by estradiol benzoate. In these tissues the order of the in vitro binding affinity of antiestrogens to cytoplasmic estrogen receptors was not correlated with either their in vivo estrogen agonistic or antagonistic potency.

Interactions of methylestrogens with cytoplasmic and nuclear estrogen receptors in rat pituitary gland, hypothalamus and uterus.

The in vitro affinities to cytoplasmic estrogen receptors of the methylestrogens 2-methylestradiol-17 beta, 4-methylestradiol-17 beta and 4-hydroxy-2-methylestradiol-17 beta, which are useful probes to test the biological importance of 2- or 4-hydroxylation of estradiol-17 beta (catecholestrogen formation), have been determined in hypothalamic, pituitary and uterine tissue of the ovariectomized rat. Moreover, the in vivo capacity of these compounds to translocate estrogen receptors into the cell nucleus of pituitary and uterine tissue has been studied. Methylestrogens exhibited estrogen receptor affinities which were not significantly different from the binding affinity of estradiol-17 beta. When given at a high dose (100 micrograms/animal) their nuclear translocation capacity was equal (4-methylestradiol-17 beta) or even higher (2-methylestradiol-17 beta) than that of estradiol-17 beta. However, at a low dose (5 micrograms/animal) 4-methylestradiol was completely ineffective in both the pituitary gland and the uterus, and 2-methylestradiol-17 beta was less potent than estradiol-17 beta in the pituitary gland.

Estrogen responsiveness of progestin receptor induction in the pituitary, preoptic-hypothalamic brain and uterus of neonatally estrogenized female rats.

Female rats were defeminized by neonatal treatment with estradiol-17beta benzoate, moxestrol (RU 2858), monohydroxytamoxifen ICI 79,280) or the dibenzoate esters of the catecholestrogens, 2-hydroxyestradiol-17beta and 4-hydroxyestradiol-17beta. When ovariectomized as adults and primed with estradiol-17beta benzoate all these rats demonstrated a deficient luteinizing hormone response to progesterone administration. However, estrogen responsiveness of progestin receptor induction was unimpaired in both the pituitary gland, the preoptic-hypothalamic brain and the uterus.

Actions of sex hormones on the brain.

1. The brain is a target for sex steroid hormones. As a result of sex hormone actions on the brain various behavioral changes are observed in animal and man. This paper gives a brief overview over the multiple central nervous functions that are under modulatory control of sexual hormones and describes the complex sex steroid actions on the brain by giving an example for "activating" and "organizing" effects of estrogens on noradrenergic neurons in the brain of rats. 2. Estradiol-17 beta induced sex specific alterations in the turnover of noradrenaline in the preoptic area and mediobasal hypothalamus showing "female" or "male" responses. 3. Neonatal manipulations of female rat pups by testosterone, estradiol-17 beta or 4-hydroxyestradiol-17 beta defeminized the "female" response of the noradrenaline turnover in the preoptic area. 4. Defeminization was not observed when neonatal females received the non aromatizable sex steroid dihydrotestosterone. 5. Activating and organizing effects of sex steroids on animal brain as shown here for noradrenergic neurons are discussed in relation to the regulation of behavior in man. Special regard is given to psychic disorders that might be associated with abnormalities in the production or metabolism of or the response to gonadal hormones.

Distribution and properties of thiol S-methyltransferase in rat brain.

Microsomal thiol S-methyltransferase (TMT) of rat brain catalysed the methylation of dithiothreitol (Km = 84 microM) and other thiol compounds using S-adenosylmethionine as methyl donor (Km = 3.7 microM). With increasing polarity of thiol substrates there was a decrease in the maximal velocities of reaction and an increase in the apparent Km values. TMT was found to be unevenly distributed amongst various brain regions, with highest activities in the medulla oblongata and the hippocampus.

Interaction of non-steroidal antiestrogens with dopamine receptor binding.

The ability of various estrogen antagonists and agonists to compete with [3H]spiroperidol, [3H]domperidone, [3H]dihydroalprenolol, [3H]dihydroergocryptine, [3H]dopamine or [3H]5-hydroxytryptamine for binding to membrane preparations from rat brain tissue was tested. The non-steroidal triphenylethylene-type antiestrogens with an amine side chain--enclomiphene, nitromifene, tamoxifen and zuclomiphene--were found to be competitive inhibitors of [3H]spiroperidol (Kd = 0.12 nM; Bmax = 101 fmol/mg protein) and [3H]domperidone (Kd = 0.62 nM; Bmax = 86 fmol/mg protein) binding to striatal membranes. The Ki values ranged from 4-12 microM. Estradiol-17 beta (Ki = 480 microM) or diethylstilbestrol (Ki = 63 microM) were much less effective inhibitors exhibiting noncompetitive interaction with the in vitro binding of [3H]spiroperidol. The pharmacological relevance of the antiestrogen interactions with dopamine receptor binding is discussed with respect to adverse effects of the in vivo administered compounds such as nausea and vomiting.