Effect of mifepristone and antiestrogens on uterine PGF2α and PGE2 concentrations in ovariectomized and pregnant rats

Four antiestrogens (anordiol, tamoxifen, RU 39411, ICI 182780) and the antiprogestin, mifepristone (RU 486), were administered to the following three animal models: (1) ovariectomized rats, (2) mated rats treated post-coitally; and (3) pregnant rats treated post-implantation. The antiestrogens were administered alone or in combination with mifepristone at doses effective in preventing and/or terminating pregnancy in rats. The objective of the study was to determine whether these drugs influenced uterine concentrations of prostaglandins (PGF_2α and PGE₂).Antiestrogens administered alone to ovariectomized rats did not effect uterine PGE₂ or PGF_2α concentrations; whereas the combination of anordiol/mifepristone increased uterine PGF_2α concentration, resulting in an increase in the PGF_2α/PGE₂ ratio.Mated rats were treated post-coitally for three consecutive days with anordiol, tamoxifen, estradiol and mifepristone alone and with the combination of anordiol/mifepristone and tamoxifen/mifepristone. An increase in uterine PGF_2α concentrations and in the PGF_2α/PGE₂ ratio occurred only in anordiol/mifepristone treated group. A decrease in uterine PGE₂ concentrations occurred in animals treated with anordiol, tamoxifen and estradiol, resulting in an increase in the PGF_2α/PGE₂ ratio.Anordiol (5.0 mg/kg/day) and mifepristone (4.0 mg/kg/day) alone and the combination of anordiol/mifepristone (2.5/1.0 mg/kg/day) administered to pregnant rats on days 7, 8 and 9 of pregnancy induced an increase in PGF_2α levels without affecting uterine PGE₂ concentration. The changes in uterine PGF_2α concentrations induced by anordiol and the combination of anordiol/mifepristone resulted in an increase in the PGF_2α/PGE₂ ratio.The antiestrogens tested except for ICI 182780 possessed agonist activity when assayed by measuring their capacity to increase the uterine weights in ovariectomized rats. Also, ICI 182789 was the only antiestrogen that did not influence uterine PG concentrations. It can be concluded that ICI 182780 is the only “pure” antiestrogen among those tested.The present results show that antiestrogens and the combination of mifepristone plus anordiol at doses preventing implantation and terminating pregnancy increase uterine PGF_2α and/or decrease PGE₂ concentrations, resulting in an alteration of PGF_2α/PGE₂ ratio. These findings suggest that there exists a critical balance of PGF_2α to PGE₂ concentrations in the uterus required for the normal passage of fertilized ova through the oviduct, initiating implantation of the blastocysts, development of embryos, and maintenance of pregnancy.     

Cervical mucus concentration of prostaglandins E2 and F2α after pretreatment with mifepristone in the first trimester of pregnancy

Cervical dilatation and softening after pretreatment with mifepristone are well documented. As this effect is similar to that observed after local application of prostaglandin E₂ (PGE₂) it is tempting to speculate that the effect of mifepristone is mediated via an increase of the endogenous secretion of prostaglandins from the cervical mucosa. Eighteen healthy women in the first trimester of pregnancy were treated with oral mifepristone (200 mg) 48 and 24 hours before legal abortion by vacuum aspiration and 18 women in the same age of gestation without any pretreatment served as controls. Cervical mucus was collected for measurement of prostaglandins by radioimmunoassay before administration of the drug and in connection with vacuum aspiration. The cervical dilatation at the time of surgery was significantly increased in women given mifepristone as compared with untreated women (7.6 versus 5.8 mm). The wet weight of collected cervical mucus was significantly increased in mifepristone treated women. The amount of PGE₂ and prostaglandin F_2α per sample was unchanged in mifepristone-treated women, whereas the concentration was lower as an effect of dilution due to an increased yield in cervical secretion observed after mifepristone treatment. The present observation does not give any support to the hypothesis that mifepristone-induced cervical maturation is mediated via an increase in cervical prostaglandin production.     

ER和PRmRNAs在内异症子宫内膜表达的变化

目的 :探讨雌激素受体 (ER)和孕激素受体 (PR)在子宫内膜异位症 (内异症 )子宫内膜的表达。方法 :利用大鼠内异症动物模型 ,采用逆转录聚合酶链反应 (RT PCR)技术 ,检测子宫内膜ER和PRmRNAs的表达情况。结果 :内异症模型组大鼠异位内膜ER、PRmRNAs的表达低于在位内膜和对照组正常子宫内膜 ,与后两者比较差异有显著性意义 (P <0 .0 1) ;而模型组在位内膜ER、PRmRNAs的表达与正常对照组比较差异无显著性意义 (P >0 .0 5 )。内异症模型组异位内膜ER/PRmRNA比值大于在位内膜和正常子宫内膜ER/PRmRNA比值 (P <0 .0 1)。结论 :内异症大鼠异位内膜ERmRNA表达的相对增高在内异症的发生与发展中起着一定的作用。     

Identification of the functional domain of glucocorticoid receptor involved in RU486 antagonism

Mifepristone, also known as RU486, is a potent glucocorticoid receptor (GR) antagonist that inhibits GR-mediated transactivation. As an alternative to existing antidepressants, RU486 has been shown to rapidly reverse psychotic depression, most likely by blocking GR. Although a number of studies have demonstrated RU486-induced GR antagonism, the precise mechanism of action still remains unclear. To identify the GR domain involved in RU486-induced suppression, GR transactivation and nuclear translocation were examined using cells transfected with human GR (hGR), Guyanese squirrel monkey GR (gsmGR), and GR chimeras into COS-1 cells. RU486 showed a much more potent suppressive effect in gsmGR-expressing cells versus hGR-expressing cells, without significant cortisol- or RU486-induced changes in nuclear translocation. A GR chimera containing the gsmGR AF1 domain (amino acids 132–428) showed a marked decrease in luciferase activity, suggesting that this domain plays an important role in RU486-induced GR antagonism. Furthermore, fluorescence recovery after photobleaching (FRAP) analysis indicated that, in the presence of RU486, gsmGR AF1 domain contributes to GR mobility in living COS-1 cells. Taken together, these results demonstrate, for the first time, that the antagonistic effects of RU486 on GR transactivation involve a specific GR domain.     

Agonist and antagonist-induced qualitative and quantitative alterations of progesterone receptor from breast cancer cells

T47D cells, cultured in medium containing serum stripped of endogenous steroids, proliferate in response to treatment with the progesterone receptor (PR) agonist, R5020 or the PR agonist/antagonist, RU486, whereas the full PR antagonist, ZK98299 has no proliferative effects. Under estrogenized conditions, all of the PR ligands tested inhibit cell growth [23]. In order to determine whether the levels or phosphorylation state of PR are reflected in the growth patterns of T47D cells, we monitored the effects of these PR ligands on the immunoblotted PR band intensities, the relative intensities, of PR-A and PR-B, and their phosphorylation states that are reflected in their altered mobility during SDS-PAGE. Under conditions where the PR ligands inhibit cell proliferation, each ligand had distinctively different qualitative and quantitative effects on PR. Short term treatment of the cells with R5020 or RU486 induced a characteristic phosphorylation-dependent upshift of both PR-A and PR-B. The phosphorylated PR was stable for up to 4 days after treatment of the cells with RU486, but was down regulated between 6-24 h after treatment with R5020. No replenishment of PR in cells treated with R5020 was detected. ZK98299, at concentrations tested, had no qualitative or quantitative effects on PR. Culturing cells for 8 days in medium containing steroid-depleted serum caused a significant reduction in the PR band intensity without causing a change in the ratio of PR-A and PR-B or their phosphorylation states. This decrease in the PR band intensity was reversed by maintaining the cells in 1 nM estrogen, but was potentiated by RU486 or ZK98299. These observations support the view that decreased PR levels may play a role in the stimulatory effects of R5020 and RU486 when cells are cultured under non-estrogenized conditions.     

米非司酮(RU486)对吗啡成瘾后新颖环境探究活动量的影响

精神成瘾性药物急性戒断后行为活动量增加是一个突出表现,本实验以米非司酮(RU486)为干涉药物,抑制糖皮质激素与受体结合,间接抑制戒断期间DA递质升高所引起活动量的增加。结果表明,米非司酮可以明显降低成瘾后急性戒断期间的活动量,但是对腹腔注射生理盐水动物活动量影响不显著。结论:进一步验证糖皮质激素在吗啡成瘾戒断期间的易化作用,同时也表明RU486可以在一定程度上缓解戒断后药物敏感化行为     

睾丸酮对离体培养人蜕膜细胞形态的影响

实验选用早孕人工流产蜕膜组织进行体外培养,观察睾丸酮对蜕膜细胞形态的影响并与RU 486加以比较。研究结果提示:(1)睾丸酮(6.9×10~(-5)mol/L)能抑制离体培养人蜕膜细胞的生长发育,但这种抑制作用是暂时和可恢复的且与用药剂量及持续时间有关。(2)睾丸酮对蜕膜细胞形态的影响与RU 486(4.7×10~(-4)mol/L)的作用效果相似。     

Mapping of the fate of cell lineages generated from cells that express the Wnt4 gene by time-lapse during kidney development

The Wnt4 gene encodes a secreted signaling molecule controlling the development of several organs, such as the kidney, adrenal gland, ovary, mammary gland and pituitary gland. It is thought to act in the embryonic kidney as an auto-inducer of nephrogenesis controlling mesenchyme-to-epithelium transition, and Wnt4-deficient mice die soon after birth, probably of kidney failure. Given the requirement for Wnt4 signaling in the control of organogenesis, the targeting of Cre recombinase under the control of the Wnt4 promoter would provide a valuable tool for fate mapping and functional genomics. We report here on the generation and characterization of a Wnt4^EGFPCre knock-in allele where the EGFPCre fusion cDNA and Neo selection cassette were targeted into the Wnt4 locus. EGFP-derived fluorescence was observed in the pretubular aggregates of the E14.5 embryonic kidney that normally express Wnt4 mRNA. Characterization of the pattern of recombination of the floxed Rosa26^LacZ reporter with the Wnt4^EGFPCre allele revealed that in addition to the embryonic kidney, reporter-derived staining was observed in the embryonic gonad, spinal cord, lung and adrenal gland, i.e. the sites of Wnt4 gene expression. Time-lapse fate mapping of the Wnt4^EGFPCre-activated yellow fluorescent protein (YFP) from the Rosa26 locus in organ culture revealed that the cells that had expressed the Wnt4 gene contributed to the nephrons, some of the cells around the stalk of the developing ureter and also certain presumptive medullary stromal cells. Moreover, the time-lapse movies suggested that the first few pretubular cell aggregates may not mature into nephrons but instead appear to disintegrate. In association with this, Rosa26^YFP-positive stromal cells emerge around these disintegrating structures. Such cells may be transient, since their derivatives are neither detected later in the more mature kidney nor is there an overlap of the Wnt4^EGFPCre; Rosa26^LacZ-marked cells with those of the endothelial cells, the smooth muscle cells or the macrophages. The Wnt4^EGFPCre allele provides a useful new tool for conditional mutagenesis and provides the first time-lapse-based map of the fate of nephron precursor cells.     

The Effect of Mifepristone (RU 486) on Plasma Cortisol in Alzheimer’s Disease

The glucocorticoid receptor (GR) antagonist mifepristone (RU-486) has been reported to increase early morning plasma ACTH/cortisol in diverse non-demented populations. This pilot study examined the cortisol response to RU 486 in patients with Alzheimer’s disease (AD), a condition associated with abnormalities in various aspects of the hypothalamic-pituitary-adrenal (HPA) axis. Nine AD subjects were randomized in a placebo-controlled parallel study: 4 in the placebo group and 5 in the RU 486 group. Subjects received oral doses of RU 486 (200 mg) or placebo daily for 6-weeks. Morning plasma cortisol was determined at baseline, at 12 h following the first study drug dose, and weekly thereafter. RU 486 resulted in a significant increase in cortisol levels [F(1,6)=65.32; P<0.001]. The magnitude of this increase grew over the course of the study [F(1,6)=63.17; P<0.001], was not related to cortisol suppression after dexamethasone and appeared greater than that reported in the literature in younger populations in response to the same drug regimen. However, further studies with age-matched controls should be done to determine possible AD related changes in this response.     

RU486对兔输卵管平滑肌的收缩效应

应用肌肉机械－电换能器和Ｇｉｌｓｏｎ生理记录仪,观察ＲＵ４８６对假孕４ｄ兔离体输卵管平滑肌的收缩效应。结果显示：（１）ＲＵ４８６可直接作用输卵管平滑肌,使其收缩频率增加,而未明显改变收缩张力及振幅,与在体肌内注射ＲＵ４８６观察到的结果相似;（２）ＲＵ４８６部分抑制ｃａ~（２＋）诱发的平滑肌收缩活动,它还与Ｖｅｒａｐａｍｉｌ诱发的抑制效应有协同作用,与ＮＥ诱发的收缩张力有拮抗作用,而对Ｆｏｒｓｋｏｌｉｎ诱发的效应未产生任何影响。以上结果表明,ＲＵ４８６对输卵管平滑肌的作用似乎是改变细胞内游离Ｃａ（２＋）的结果,可能干扰Ｃａ（２＋）的流入、或／和内质网Ｃａ（２＋）释放以及Ｃａ（２＋）－Ｉｐ３信息传递机制。     

中枢神经系统特异性表达Cre重组酶的转基因小鼠

利用从129sv小鼠基因组文库克隆得到的1．8kb的胶质细胞原纤维酸性蛋白(GFAP)基因的5′端调控序列,构建了含有2个β—珠蛋白绝缘子、GFAP5′端调控区、Cre基因和人生长激素基因(hGH)polyA的转基因载体pGFAP—Cre—hGH。以显微注射的方法将7．6kb的转基因片段pGFAP—Cre—hGH引入191枚小鼠基因组受精卵,其中176枚分别移植至8只假孕母鼠的输卵管中使其发育,共获得子代小鼠25只。经PCR和Southern杂交鉴定其中7只小鼠基因组上整合有Cre基因,整合率为28％。用整合有Cre基因的转基因小鼠与基因组上整合有LoxP位点和LacZ表达框的ROSA26鼠杂交,以检测Cre酶的活性、组织特异性及其介导的两个LoxP位点间的重组。LacZ染色结果表明,GFAP—Cre转基因小鼠只在中枢神经系统中表达Cre重组酶并能在体内成功介导LoxP位点间的重组。