Rudolf Knuppen

Gonadotropin releasing hormone binding sites in human epithelial ovarian carcinomata

As a first step to investigate whether gonadotropin releasing hormone (GnRH) analogs might be able to modulate directly the proliferation of human epithelial ovarian carcinomata, we checked if binding sites for GnRH are present in these malignancies. Specific binding of [125I][D-Ala6-des Gly10]-GnRH-ethylamide (GnRH agonist = GnRH-A) could be demonstrated in plasma membranes from 32 out of 40 ovarian carcinomata tested. This binding was dependent on temperature, time and plasma membrane concentration. Mathematical analysis of the binding data showed that the interaction of GnRH-A with the binding sites was consistent with a single class of low affinity, high capacity binding sites (Ka = 1.42 +/- 0.14 X 10(5) M-1; range: 0.3-3.8 X 10(5) M-1; R = 209 +/- 69 X 10(-12) M/mg membrane protein; range 16-400 X 10(-12) M/mg MP; means +/- S.E., n = 32). Native GnRH and the GnRH antagonist [D-p-Glu1, D-Phe2, D-Trp3,6]-GnRH had Ka values comparable to those of the GnRH-A used. [125I]GnRH-A binding could not be displaced by oxytocin, thyrotropin releasing hormone and corticotropin releasing factor in concentrations up to 10(-4) M. Somatostatin cross-reacted with binding sites from some carcinomata, while it did not displace GnRH-A binding in membranes from others. Though the functional role of this specific binding site for GnRH in human epithelial ovarian carcinomata is still obscure, it might be part of an autocrine regulatory system and provide a possible point of attack for therapeutic approaches using GnRH analogs in this malignancy.

A simple chemical method for the synthesis of catechol estrogens

Abstract The preparation of 2-hydroxyestrone, 2-hydroxyestradiol-17, 4-hydroxyestrone and 4-hydroxyestradiol-17β by a simple one-step chemical reaction, treatment with potassium nitrosodisulfonate, is described. The structures of the products were established by nmr, ultraviolet, infrared and mass spectra as well as from their chemical and chromatographic properties.

Catecholestrogens are MCF-7 cell estrogen receptor agonists

Catecholestrogens are important metabolites of estradiol and estrone in the human. Considerable interest has focused on the catecholestrogens 2-hydroxy- and 4-hydroxyestradiol since they bind to the estrogen receptor with an affinity in the range of estradiol. Using the MCF-7 cell line, we analysed the capacity of purified catecholestrogens to transform the estrogen receptor into its high affinity nuclear binding form and to affect receptor-dependent processes such as proliferation and expression of the progesterone receptor (PR). Incubations with 2-hydroxy- and 4-hydroxyestradiol at 10(-8) M for 1 h resulted in tight nuclear binding of the estrogen receptor. During treatment of the cells with catecholestrogens we obtained a marked increase in proliferation rate of 36 and 76% for 2-hydroxy- and 4-hydroxyestradiol, respectively, relative to the inductive effect of estradiol (100%). The PR level, was slightly increased by treatment with 2-hydroxyestradiol (10%), whereas treatment with 4-hydroxyestradiol increased the PR level at 28%, compared to estradiol (100%). From these results we conclude that the 2- and 4-hydroxylated derivatives of estradiol are active hormones and are able to initiate estrogen receptor mediated processes in MCF-7 cells.

Formation, metabolism and physiologic importance of catecholestrogens

The metabolism of natural and synthetic estrogens is governed primarily by hydroxylations, leading to polyhydroxylated derivatives of the steroid molecule. In mammals aromatic hydroxylation is most prominent quantitatively. The 2- and 4-hydroxyestrogens (catecholestrogens) formed are secreted not only in high amounts in urine but are also present in significant quantities in different organs, such as the liver, pituitary gland, and hypothalamus. This A ring hydroxylation of primary estrogens is affected by peroxidases, tyrosinases, and unspecific monooxygenases by mechanisms still not completely understood. The activity of the aromatic hydroxylases is regulated not only with respect to the overall extent but also to the relative rate of hydroxylation at C-atoms 2 and 4. The metabolism of catecholestrogens may be divided into reversible and irreversible reactions, of which the reaction with the catechol-O-methyltransferase, and thereby the interaction with catecholamines, the conjugation, and the thioether formation are the most prominent. Low- and high-affinity binding is operative in binding to plasma proteins and receptors. Finally, irreversible binding to cellular macromolecules, such as proteins and deoxyribonucleic acid, and the oncogenic potential of natural and synthetic catecholestrogens are discussed.

A novel radioimmunoassay of allopregnanolone

A radioimmunoassay for determination of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) in serum or plasma has been developed and evaluated. The method employs rabbit antiserum to 3 alpha-hydroxy-5 alpha-pregnane-11,20-dione-11-O-carboxymethyloxime bovine serum-albumin conjugate and tritiated radioligand. The main cross-reactant interfering in the assay, progesterone, is eliminated by permanganate oxidation. Two assay variants were compared, with and without a micro-column chromatography. The simplified variant appeared to be reliable enough for determination of allopregnanolone in normally menstruating women at luteal phase, whereas the column-chromatography step is necessary when analyzing samples of expected low analyte concentration as in women in follicular phase, postmenopausal women, or in men. The levels of allopregnanolone in healthy women correlated excellently with progesterone in agreement with previous findings.

Radioimmunoassay of 2-hydroxyestrone

Under the protection of ascorbic acid a 2-hydroxyestrone bovine serum albumin conjugate was prepared containing intact 2-hydroxyestrone as determined by gas chromatographymass spectometry. Using this antigen highely specific antibodies were raised in rabbits. Cross-reactivity for 2-hydroxyestradiol and 2-hydroxyestriol was 26 and 4.5%, respectively. An assay procedure of 2-hydroxyestrone in human plasma is described. Using special precautions the assay allows the determination of 2-hydroxyestrone in plasma samples of women (50-95 pg/ml), pregnant women (105-220 pg/ml), men (45-65 pg/ml) and children(20-40 pg/ml).

Weak estrogenic activity of phenol red in the pituitary gonadotroph: re-evaluation of estrogen and antiestrogen effects.

Phenol Red (Phr) which is widely used as a pH indicator in cell culture media has recently been described to possess estrogenic activity in different cell types. In the present study we investigated if the dye shows such activity on LH secretion of cultivated rat pituitary cells and controlled the established effects of estradiol (E2) and keoxifene (K) in this model in the absence of Phenol Red. 24 h treatment of pituitary cell cultures with Phr led to enhancement of GnRH-stimulated LH secretion whereas 4 h treatment reduced LH secretion. When the cells received E2 instead of Phr for the indicated incubation periods we observed nearly identical results i.e. a short-term inhibitory and a long-term stimulatory effect on LH secretion. 24 h treatment of pituitary cell cultures with increasing concentrations of Phr led to a stimulatory effect on GnRH-stimulated LH secretion an effect that occurred at 10 microM got maximal at 100 microM and was lost at higher concentrations resulting in a bell-shaped dose-response curve. The inhibitory action of Phr was present at concentrations greater than or equal to 10 microM. Both effects could be blocked by the antiestrogen K indicating their specificity. K has recently been described to induce an antigonadotrophic effect in this model. Although high concentrations of the antiestrogen were still able to inhibit LH secretion this effect was not present at lower concentrations when Phr-free culture medium was used in the experiments. Thus Phr showed weak estrogenic activity in the gonadotroph. The established actions of E2 and K on LH secretion were qualitatively reproducible when Phr was excluded from the culture medium.

Specific low affinity binding sites for gonadotropin-releasing hormone in human endometrial carcinomata

Specific low-affinity high-capacity binding sites for gonadotropin-releasing hormone (GnRH) have recently been discovered in human breast and ovarian carcinomata. We checked whether similar binding sites are present in human endometrial cancer. Plasma membrane preparations were incubated with [125I,D-Ala6-desGly10]-GnRH-ethylamide in the presence or absence of unlabelled GnRH agonists or other peptides. GnRH-binding could be demonstrated in all 12 tumor samples tested. The mathematical analysis of the binding data was consistent with a single class of low affinity (Ka = (0.8-1.4) x 10(5) M-1) and high-capacity (Bmax = (134-142) x 10(-12) M/mg membrane protein) binding sites. Native GnRH had a similar affinity to the binding sites as the GnRH agonist used. Other peptides such as oxytocin, somatostatin and thyrotropin-releasing hormone did not crossreact with the binding sites. A photolabelled derivative of [D-Lys6]-GnRH was prepared with the bifunctional photolabile reagent (4-azidobenzyl)-N-hydroxysuccinimide. Photoaffinity labelling of endometrial carcinoma membranes and subsequent sodium dodecyl sulfate electrophoresis in 10% polyacrylamide gel revealed the presence of a single molecular mass component of 62 +/- 1.9 kDa. The appearance of this photolabelled binding site could be largely suppressed by the addition of unlabelled GnRH-agonist (10(-4) M) and thus represents the specific binding site for GnRH in endometrial cancer.

Inhibitory effects of the antiprogestin, RU 486, on progesterone actions and luteinizing hormone secretion in pituitary gonadotrophs

The effects of RU 486 on the modulation of LH release by progesterone were investigated in cultured anterior pituitary cells from ovariectomized adult female rats. The inhibitory effect of progesterone on LH secretion was demonstrable in estrogen-treated pituitary cells, in which addition of 10(-6) M progesterone to cells cultured in the presence of 10(-9) M estradiol for 52 h reduced the LH response to GnRH (10(-11) to 10(-7) M). When RU 486 was superimposed upon such combined treatment with estradiol and progesterone, the suppressive effect of progesterone on GnRH-induced LH release was completely abolished. The converse (facilitatory) effect of progesterone on LH secretion was observed in pituitary cells pretreated with 10(-9) M estradiol for 48 h and then with 10(-6) M progesterone for 4 h. When RU 486 was added together with progesterone during the 4 h treatment period, the facilitatory effect of progesterone was blocked and LH release fell to below the corresponding control value. The direct effect of RU 486 on LH secretion in the absence of exogenous progesterone was evaluated in cells cultured in the absence or presence of 10(-9) M estradiol and then treated for 4 to 24 h with increasing concentrations of RU 486 (10(-12) to 10(-5) M) and stimulated with GnRH (10(-9) M) during the last 3 h of incubation. In estrogen-deficient cultures, 4 h exposure to RU 486 concentrations of 10(-6) M and above decreased the LH response to GnRH by up to 50%. In cultures pretreated with 10(-9) M estradiol, GnRH-stimulated LH responses was inhibited by much lower RU 486 concentrations, of 10(-9) M and above. After 24 h of incubation the effects of RU 486 were similar in control and estradiol-pretreated pituitary cell cultures. Thus, RU 486 alone has a significant inhibitory effect on LH secretion that is enhanced in the presence of estrogen. The antiprogestin is also a potent antagonist of both the inhibitory and the facilitatory actions of progesterone upon pituitary gonadotropin release in vitro.

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.