Effect of 17 beta-estradiol on thyroliberin responsiveness in GH3/B6 rat prolactin cells

GH3/B6 rat prolactin cells were used to analyse at the cellular level the mechanisms by which 17 beta-estradiol (E2) regulates TRH responsiveness of prolactin cells. Before experiments, cells were grown for up to 7 days in 3 different media: normal medium (N) containing 15% horse serum and 2.5% fetal calf serum, CD medium prepared with charcoal-dextran extracted serum and CDE medium supplemented with 4 x 10(-8) M E2. The binding of 3H-TRH (30 min at 37 degrees C) and the TRH-induced percent increase of prolactin release as a function of TRH doses were compared in the 3 conditions. Preculture in E2 enriched medium increased by 50% the number of TRH high-affinity binding sites without modifying their affinity, increased by up to 3 times the percent of the TRH-induced stimulation of prolactin release and improved by one order of magnitude the ED50 of the TRH effect on prolactin release. The presence of HEPES (10 mM) during TRH challenge masked the effect of E2 on the increase in number of binding sites but respected its potentiating effect on prolactin release.     

Binding of (3H)R5020 to progesterone receptor and serum contaminants in human endometrium.

The binding of [3H]R5020 to cytosol from human endometrium was utilized to evaluate the concentration of progesterone receptor by means of agar gel electrophoresis, using D-norgestrel as cold competitor. It was observed that radioligand binds predominantly to the receptor, and that serum contaminants such as albumin and corticosteroid-binding globulin do not interfere in the assay procedure.     

Progestin receptors in human uterine cytosol

Progestin(norethindrone and norethindrone acetate)-binding protein, exhibiting characteristics similar to uterine progesterone receptor, has been identified in human uterine cytosol. The progestin receptor was characterized by sedimentation coefficient 4.2 S; Stokes radius, 39 Å; frictional ratio 1.29; isoelectric pH 4.6; molecular radius 2.7 nm; and molecualr weight in the range 67 000–74 000. The ammonium-sulfate-precipitated progestin-receptor complex was eluted from a DEAE-cellulose column at 0.18 M KC1. The progestin binding was saturable and stereospecific. The sequential variation in receptor concentration (early proliferative, 3800–4300 sites/cell; late proliferative, 9500–11200 sites/cell; early secretory, 4900–6200 sites/cell; late secretory, 1800–2300 sites/cell) was in conformity for progesterone and the progestins, when concurrently measured. Oral administration of norethindrone significantly reduced the cytoplasmic and nuclear receptor concentration for estradiol and progesterone. A significant observation was that the progestins stabilized the progestin receptor by forming a slowly dissociating complex with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～ 110?130}\text{min}$ as compared with the progesteronereceptor complex dissociating with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{～41}\text{min}$. Thus, the uterine progestin receptor recognizes progestins in general, although with a varying degree of affinity, and the altered rate constants could be of putative importance in determining the biological potency of the progestins.     

Differentiation between steroid hormone receptors CBG and shbg in human target organ extracts by a single-step assay

The steroid complexes of (plasma) corticosteroid-binding globulin can be distinguished from intracellular steroid-receptor complexes by agar electrophoresis at low temperature in neuraminidase-treated tissue extracts. With this method, the presence of progesterone receptor has been demonstrated in heavily plasma-protein-contaminated human uterus “cytosol”, but not in human mammary carcinoma extracts. SHBG and “basic” receptors for estradiol and dihydrotestosterone in human uterus cytosol could also be assayed simultaneously.