Subcellular Distribution of 3α-Hydroxysteroid Dehydrogenase and Antiestrogen Action on Androgen-Metabolizing Enzymes in Rat Pituitary Gland

Abstract: Gonadectomy of male rats led to a threefold increase of 3α-hydroxysteroid dehydrogenase (3α-HSDH) activity in pituitary homogenates that could be completely reversed by chronic administration of estradiol or 5α-dihydrotestosterone (DHT). 3α-HSDH was found to be distributed mainly between the 10,000 g and 100,000 g sediments from whole homogenates. The microsomal enzyme activity showed a substantial specificity for NADH whereas the cytosolic enzyme (100,000 g supernatant) demonstrated a slight preference for NADPH. The changes in V _max found in homogenates following gonadectomy and gonadal steroid administration reflected changes in NADH- linked activity of the microsomal, but not the cytosolic enzyme. Estradiol-induced suppression of NADH-linked 3α-HSDH activity in pituitary homogenates from gonadectomized rats of either sex was accompanied by a similar suppression of NADPH-linked 5α-reductase activity and a marked decrease of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release. In the ovariectomized rat chronic administration of nonsteroidal antiestrogens had strong estrogenic effects on 3α-HSDH activity and LH release, but not on 5α-reductase activity and FSH release. In the gonadectomized male rat, which was much less sensitive to intrinsic estrogenicity of the antiestrogens tested, nafoxidine completely blocked estradiol-induced suppression of 5α-reductase activity and FSH release and partially antagonized suppression of LH release. The trans -isomeric, substituted triphenylethylenes, tamoxifen, and enclomiphene, as well as nitromifene (mixture of trans and cis isomers) were able partially to counteract estradiol-induced suppression of 5α-reductase, but not 3α-HSDH activity. It is concluded that estradiol action on pituitary 5α-reductase, but not 3α-HSDH activity, involves an estrogen receptor mechanism.     

Nongenomic effects of an anti-idiotypic antibody as an estrogen mimetic in female human and rat osteoblasts

We investigated the early effects of the anti-idiotypic antibody (clone 1D₅), which recognized the estrogen receptor (ER), on cytosolic free calcium concentration ([Ca²⁺]i) and its long term effects on creatine kinase (CK) specific activity in female human and rat osteoblasts. These actions were compared to the known membrane and genomic effects of 17β estradiol (E₂). Like E₂, clone 1D₅ increased within 5 s [Ca²⁺]i in both cell types by two mechanisms: 1) Ca²⁺ influx through voltage-gated Ca²⁺ channels as shown by using EGTA, a chelator of extracellular Ca²⁺, and nifedipine, a Ca²⁺ channel blocker; 2) Ca^2+; mobilization from the endoplasmic reticulum as shown by using phospholipase C inhibitors, such as neomycin and U-73122, which involved a Pertussis toxin-sensitive G-protein. Clone 1D₅ and E₂ stimulated CK specific activity in human and rat osteoblasts with ten fold higher concentrations than those needed for the membrane effects (0.1 μg/ml and 10 pM, respectively). Both effects were gender-specific since testosterone and 5α-dihydotesterone were uneffective. Tamoxifen and Raloxifene, two estrogen nuclear antagonists, inhibited CK response to 1D₅ and E₂ and Ca²⁺ response to 1D₅, but not CA²⁺ response to E₂. By contrast, (Fab′)₂ dimer, a proteolytic fragment of 1D₅ with antagonist properties, inhibited both membrane and genomic effects of 1D₅ and E₂. In conclusion, these results imply that clone 1D₅ has an estrogen like activity both at the membrane and nuclear levels in female human and rat osteoblasts. 1D₅ must therefore interact with membrane binding sites, penetrate the cells, and reach the nuclear receptors by an as yet uncharacterized mechanism. J. Cell. Biochem. 65:53–66. © 1997 Wiley-Liss, Inc.     

Synthesis and structure–activity relationships of analogs of EM-652 (acolbifene), a pure selective estrogen receptor modulator. Study of nitrogen substitution

EM-652 (acolbifene) analogs have been synthesized as selective estrogen receptor modulators. Substitution on the nitrogen atom of these 2H-1-benzopyran derivatives has been studied for its influence on antiestrogenic activity. Binding to the rat estrogen receptor, inhibition of estradiol-stimulated proliferation of T-47D breast cancer cells, as well as antiuterotrophic and uterotrophic activities in ovariectomized mice have been evaluated. 2H-1-Benzopyran 1b (EM-343, racemic form of EM-652), which contains a piperidine ring, shows the best pharmacological profile; RBA=380, IC₅₀value=0.110?nM (in T-47D cells), as well as 63% and 84% antiuterotrophic inhibitions at the 7.5 and 75?nmol doses, respectively.     

Induction of riboflavin-carrier protein in the immature male rat by estrogen: Kinetic and hormonal specificity

The kinetics of estrogen-induced accumulation of riboflavin-carrier protein in the plasma was investigated in immature male rats using a specific and sensitive homologous radio-immunoassay procedure developed for this purpose. Following a single injection of the steroid hormone, plasma riboflavin-carrier protein levels increased markedly after an initial lag period of approximately 24 h, reaching peak levels around 96 h and declining thereafter. A 1.5 fold amplification of the inductive response was evident on secondary stimulation with the hormone. The magnitude of the response was dependent on hormonal dose, whereas the initial lag phase and the time of peak riboflavin-carrier protein induction were unaltered within the range of the steroid doses (0.1–10 mg/ kg body wt.) tested. Simultaneous administration of progesterone did not affect either the kinetics or the maximum level of the protein induced. The hormonal specificity of this induction was further adduced by the effect of administration of antiestrogens viz., En and Zu chlomiphene citrates, which effectively curtailed hormonal induction of the protein. That the induction involvedde novo-protein synthesis was evident from the complete inhibition obtained upon administration of cycloheximide. Passive immunoneutralization of endogenous riboflavin-carrier protein with antiserum to the homologous protein terminated pregnancy in rats confirming the earlier results with antiserum to chicken riboflavin-carrier protein.     

Hormonal modulation of reproduction-specific thiamin carrier protein in the rat

The hormonal modulation of thiamin carrier protein in the plasma and uterine luminal secretion during the normal reproductive phases of the animal (estrous cycle and pregnancy) as well as during experimental estrogenisation was investigated in the rat using a specific and sensitive homologous radioimmunoassay procedure developed for this purpose. Following a single injection of estrogen to immature male rats, thiamin carrier protein rapidly accumulated in plasma attaining peak concentration at 48 h and declining thereafter. A 1.5-fold amplification of the inductive response was observed on secondary stimulation with the hormone. The magnitude of the response exhibited a clear dependency on the dose of the steroid hormone, whereas the time at which peak levels of thiamin carrier protein production was remained unaltered in the concentration range of the steroid tested. The inductive effect of estrogen was severely curtailed by the antiestrogens,viz., En- and Zu-clomiphene citrates, while progesterone was incapable of either modulating the estrogen-induced response or eliciting an induction by itself. Cycloheximide drastically blocked the response to estrogen. Evidence for the ability of uterus to serve as yet another independent site of thiamin carrier protein synthesis was obtained byin vitro incorporation of radioactive amino acids into immunoprecipitable thiamin carrier protein in the tissue explants of estrogenised female rats. The levels of thiamin carrier protein in uterine luminal fluid measured during estrous cycle, pregnancy and experimental estrogenisation exhibited remarkable similarity to the plasma thiamin carrier protein profiles.     

Effect of antiestrogen regimen on prostacyclin and thromboxane A2 in postmenopausal patients with breast cancer: Evidence of significance of hypertension, smoking or previous use of estrogen therapy

To explore the mechanism(s) by which antiestrogens may protect against the development of cardiovascular disorders, we measured the production of vasodilatory, antiaggregatory prostacyclin (PGI₂ and that of vasoconstrictive, proaggregatory thromboxane A₂ (TxA₂) before and after 6 months' use of antiestrogens in postmenopausal patients after operation for stage II breast cancer (n = 38). Urine samples were assayed by high performance liquid chromatography and radioimmunoassays for 2,3-dinor-6-ketoprostaglandin F1α (=metabolite of PGI₂, dinor-6-keto) and for 2,3-dinor-thromboxane B₂ (=metabolite of TxA₂, dinor-TxB₂). In addition, in 35 of these 38 patients we assayed the capacity of platelets to produce thromboxane A2 during standardized blood clotting. The 4 patients using low-dose aspirin had low thromboxane production, and were excluded from further analysis of the data. An antiestrogen regimen consisting either of tamoxifen (n = 15) or of toremifene (n = 19) caused no changes in production of PGI₂ or TxA₂, or in their ratio, and in this regard, these antiestrogens behaved similarly. Hypertensive patients (n = 7) using different antihypertensive agents were characterized by reduced urinary out-put of dinor-6-keto (18.5 ± 6.1 vs 35.5 ± 18.5 ng/mmol, mean ± SD, p < 0.05) and reduced platelet capacity to produce TxA₂ (62.6 ± 67.8 vs 134.6 ± 75.6 ng/mL, p < 0.05). The patients (n = 15) who had used estrogen replacement therapy (ERT) up until diagnosis of breast cancer showed reduced dinor-TxB₂ excretion (15.5 ± 12.7 vs 29.9 ± 20.9 ng/mmol, p < 0.05) before initiation of antiestrogens, and elevated dinor-6-keto output during the antiestrogen regimen (32.4 ± 21.2 vs 22.7 ± 8.7 ng/mmol, p = 0.07). Smokers (n = 6) had elevated dinor-TxB2 output before and during antiestrogen use. Thus we conclude that the cardiovascular protection provided by an antiestrogen regimen is unlikely to be mediated through vaso- and platelet active PGI₂ and TxA₂.     

Volume-activated chloride channels in neuroblastoma cells are blocked by the antiestrogen toremifene

Summary The presence of volume-activated chloride channels has been examined in neuroblastoma C1300 cells using the whole-cell configuration of the patch-clamp technique. Chloride channels could not be detected under isotonic conditions. However, hypotonic challenge induced slowly developed inward and outward anionic currents that exhibited outward rectification and inactivation at the most depolarizing potentials, features that were similar to the currents described in other cell preparations where volume-activated Cl^– channels have been associated with the expression of P-glycoprotein. This hypotonicity-activated Cl^– currents could be reversibly blocked by extracellular exposure to toremifene, a novel synthetic antioestrogen. The fact that toremifene and its analog tamoxifen, have been shown to block P-glycoprotein-associated chloride channels and to reverse P-glycoprotein associated multidrug resistance in a number of cell lines suggest that P-glycoprotein could be involved in the generation of hypotomic-induced chloride conductance in neuroblastoma cells.