High resolution screening of plant natural product extracts for estrogen receptor alpha and beta binding activity using an online HPLC-MS biochemical detection system.

A new screening technology that combines biochemical analysis with the resolution power of high-performance liquid chromatography (HPLC), referred to here as high-resolution screening (HRS) technique, is described. The capability of the HRS technology to analyze biologically active compounds in complex mixtures is demonstrated by screening a plant natural product extract library for estrogen receptor (ER) alpha and beta binding activity. The simultaneous structure elucidation of biologically active components in crude extracts was achieved by operating the HRS system in combination with mass spectrometry (MS). In contrast to conventional microtiter-type bioassays, the interactions of the extracts with the ER and the employed label, coumestrol, proceeded at high speed in a closed, continuous-flow reaction detection system, which was coupled directly to the outlet of a HPLC separation column. The reaction products of this homogeneous fluorescence enhancement-type assay were detected online using a flowthrough fluorescence detector. Primary screening of the extract library was performed in the fast-flow injection analysis mode (FlowScreening) wherein the chromatographic separation system was bypassed. The library was screened at high speed, using two assay lines in parallel. A total of 98% of the identified hits were confirmed in a traditional 96-well microplate-based fluorescence polarization assay, indicating the reliability of the FlowScreening process. Active extracts were reassayed in a transcriptional activation assay in order to assess the functional activity of the bioactive extracts. Only functional active extracts were processed in the more time-consuming HRS mode, which was operated in combination with MS. Information on the number of active compounds, their retention times, the molecular masses, and the MS/MS-fingerprints as a function of their biological activity was obtained from 50% of the functional active extracts in real time. This dramatically enhances the speed of biologically active compound characterization in natural product extracts compared to traditional fractionation approaches.

EGF activates highly selective estrogen-responsive reporter plasmids by an ER-independent pathway.

Epidermal growth factor (EGF) mimics the effects of estrogen on some cells, suggesting that it may activate the estrogen receptor (ER). We examined the ability of EGF to increase expression of several different estrogen-responsive luciferase reporters in MCF-7 breast cancer cells. Although EGF increased reporter activity, this effect was not inhibited by estrogen antagonists and was not dependent on estrogen response elements in the reporter plasmid. Similar results were obtained in BG-1 (ovarian) and Ishikawa (uterine) cells. In ER-negative JEG-3 cells, EGF, but not estradiol, increased reporter activity in the absence of transfected ER. The estrogen antagonist ICI 182780 blocked the ability of estradiol, but not EGF, to stimulate proliferation of T47D breast cancer cells, suggesting that the mitogenic effects of EGF are not mediated by ER. EGF does not appear to activate ER-mediated transcription in these experimental systems, although crosstalk between the estrogen and EGF signaling pathways may occur by other mechanisms.

Resveratrol, a polyphenolic compound found in grapes and wine, is an agonist for the estrogen receptor.

The phytochemical resveratrol, which is found in grapes and wine, has been reported to have a variety of anti-inflammatory, anti-platelet, and anti-carcinogenic effects. Based on its structural similarity to diethylstilbestrol, a synthetic estrogen, we examined whether resveratrol might be a phytoestrogen. At concentrations (approximately 3-10 microM) comparable to those required for its other biological effects, resveratrol inhibited the binding of labeled estradiol to the estrogen receptor and it activated transcription of estrogen-responsive reporter genes transfected into human breast cancer cells. This transcriptional activation was estrogen receptor-dependent, required an estrogen response element in the reporter gene, and was inhibited by specific estrogen antagonists. In some cell types (e.g., MCF-7 cells), resveratrol functioned as a superagonist (i.e., produced a greater maximal transcriptional response than estradiol) whereas in others it produced activation equal to or less than that of estradiol. Resveratrol also increased the expression of native estrogen-regulated genes, and it stimulated the proliferation of estrogen-dependent T47D breast cancer cells. We conclude that resveratrol is a phytoestrogen and that it exhibits variable degrees of estrogen receptor agonism in different test systems. The estrogenic actions of resveratrol broaden the spectrum of its biological actions and may be relevant to the reported cardiovascular benefits of drinking wine.

Corticosterone selectively increases follicle-stimulating hormone beta-subunit messenger ribonucleic acid in primary anterior pituitary cell culture without affecting its half-life.

We demonstrated previously that glucocorticoids differentially affect the levels of the two pituitary gonadotropins, LH and FSH, both in vivo and in vitro. In vivo, the effect of glucocorticoids is GnRH independent, indicating a direct action on the gonadotrope, and it leads to selective up-regulation of the pituitary content of FSH and FSH beta-subunit messenger RNA (mRNA). The objective of the present study was to confirm the direct action of corticosterone (B) on FSH beta-subunit mRNA in primary anterior pituitary cell culture and to assess whether the selective B-induced rise in FSH beta mRNA is mediated through altered stability of the FSH beta transcript. Anterior pituitary glands collected from randomly cycling female rats were dissociated with trypsin. Cells were incubated at 37 C for 48 h and subsequently exposed to vehicle or B (1.7 microM) for an additional 42 h. At the end of the incubation, media were sampled for FSH and LH, cells were lysed, and total RNA was isolated for Northern blot analysis. Exposure to B for 42 h caused direct and selective upregulation of FSH release, FSH content, and FSH beta mRNA; decreased alpha-subunit mRNA; and had no significant effect on LH release, LH content, or LH beta mRNA. To evaluate the mRNA stability of the three subunits, cells were exposed to the transcription blocker actinomycin D (act D; 5 micrograms/ml) for an additional 6 h. The combined 6-h treatment with B and act D slightly, but significantly, suppressed alpha-subunit mRNA and did not change LH beta mRNA, confirming a long half-life of the two gonadotropin subunit mRNAs. In contrast, FSH beta mRNA was significantly suppressed by act D to the same level in vehicle- and B-treated cells. The posttranscriptional decay rate was examined by sampling at 0, 1, 2, 3, and 6 h during the 6-h act D treatment period. Decay curves for FSH beta mRNA were parallel in vehicle- and B-treated cells, indicating that B did not alter FSH beta mRNA stability. We conclude that the selective B-induced rise in FSH beta mRNA is mediated at the level of transcription rather than mRNA stabilization.

Effects of corticosterone and testosterone on pituitary gonadotropin content, secretion, bioactivity and messenger RNA levels in the presence or absence of GnRH in male rats.

The effects of corticosterone (B) and testosterone (T) on pituitary and serum bioactive and immunoreactive gonadotropins and on gonadotropin hormone subunit messenger RNA levels were compared in the absence of GnRH. Male rats were implanted with pellets of either cholesterol, B or T. At implantation, 2 and 4 days later half of each group received GnRH antagonist and animals were killed 5 days after implantation. As expected, GnRH antagonist lowered bioactive and immunoreactive serum FSH and LH, pituitary FSH, LHß and FSHß mRNA. B treatment alone lowered bioactive and immunoreactive serum FSH and immunoreactive serum LH. B reversed the antagonist effect on bioactive and immunoreactive pituitary FSH and FSHß mRNA. T alone lowered bioactive and immunoreactive serum FSH and LH levels. T reversed the antagonist effect on bioactive and immunoreactive pituitary FSH. T lowered bioactive and immunoreactive pituitary LH and LHß mRNA and partially reversed the antagonist effect on FSHß mRNA. The data suggest that either B or T enhance FSH synthesis by acting directly at the gonadotrope, but that B does not affect LH variables to the same extent as T. The results suggest that in stressed animals, when T levels are reduced, B can substitute for T in sustaining FSH synthesis.

Age-related changes in the secretion of LH in vivo and in vitro in infantile and prepubertal Holstein bull calves.

The objectives of this study were (i) to determine whether age-related changes in the secretion of LH are associated with alterations in secretory activity or numbers of gonadotrophs, and (ii) to determine whether gonadotrophs obtained from the pars distalis and pars tuberalis undergo similar age-related changes in function. Blood samples were collected from Holstein bull calves every 15 min for 12 h at < 1, 12, and 42 weeks of age (n = 5 per age group) to characterize the secretion of LH. Calves were killed 3-5 days later. The pars distalis and pars tuberalis were enzymatically dispersed into suspensions of single cells. Cells from the pars distalis were (i) extracted with 0.01 mol NaHCO3 l-1, (ii) fixed for immunocytochemical analysis, and (iii) cultured in six-well plastic plates at a density of 500,000 cells per well in media containing 2.5% homologous calf serum for 18 and 72 h. Cells from the pars tuberalis were cultured as for pars distalis cells. As expected, LH pulse frequency increased (P < 0.01) between one and 12 weeks of age and then declined. The percentage of cells from the pars distalis containing immunoreactive LH averaged 8.4%, and did not change with age. The mass of the pars distalis and the total number of cells recovered increased with age (P < 0.05); consequently, the number of gonadotrophs recovered also increased. The initial content of LH of pars distalis cells changed with age and was greatest at 12 weeks.(ABSTRACT TRUNCATED AT 250 WORDS)

Corticosterone in vivo increases pituitary follicle-stimulating hormone (FSH)-beta messenger ribonucleic acid content and serum FSH bioactivity selectively in female rats.

Experimental objectives were to determine: 1) if the native glucocorticoid, corticosterone (B), can selectively increase pituitary FSH and FSH beta messenger RNA (mRNA) in the presence or absence of a GnRH signal; and 2) if B affects the biological activity of the gonadotropins. Metestrous female rats were implanted with cholesterol or B. Each implant group received 100 micrograms GnRH antagonist or control injections every 48 h beginning at the time of implantation, and were killed 5 days later. B significantly increased bioactive serum FSH, with or without GnRH antagonist. GnRH antagonist decreased bioactive serum FSH. Immunoreactive serum FSH was not affected by any treatment. B did not affect bioactive serum LH, but GnRH antagonist significantly suppressed bioactive serum LH. Immunoreactive serum LH was significantly lowered by either B or GnRH antagonist. Neither bioactive nor immunoreactive pituitary FSH or LH content were affected by B, GnRH antagonist, or combined treatments, and no treatment affected alpha or LH beta mRNA. B significantly increased FSH beta mRNA specifically, in the presence or absence of GnRH antagonist. These results demonstrate that corticosterone can increase biological activity of secreted FSH and increase FSH beta mRNA in the absence of a GnRH signal, suggesting a direct effect on the anterior pituitary gland.

Age-related changes in the secretion of growth hormone in vivo and in vitro in infantile and prepubertal Holstein bull calves.

The average concentration of GH in blood is high at birth and declines during the period of sexual maturation in bulls. The objectives of these studies were (1) to define age-related changes in vivo in the pulsatile secretion of GH from birth to puberty, (2) to determine whether pituitary cell content of GH and characteristics of the secretion of GH in vitro reflect age-related changes in vivo, and (3) to examine whether responsiveness to GH-releasing hormone (GHRH) and somatostatin (SRIF) in vitro changed with age in Holstein bull calves. In experiment 1, calves were bled every 15 min for 12 h at < 1, 12 and 42 weeks of age (n = 5/group), these being representative of infantile, juvenile and pubertal stages of development. Calves were killed 3 to 5 days later and the pars distalis of the anterior pituitary gland was enzymatically dispersed into a suspension of single cells. Aliquots of cells were extracted with 0.01 mol NaHCO3/l to determine the content of GH and cultured for 18 and 72 h. As expected, the average concentration of GH in plasma decreased with age (P < 0.001). The initial decrease in GH was caused by a reduction in the baseline concentration between birth and 12 weeks of age. There was a marked decrease in GH pulse amplitude between 12 and 42 weeks of age and a further reduction in the baseline concentration. In contrast, the pulse frequency of GH increased (P < 0.05) from < 1 week to 12-weeks of age and remained constant thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)

Cortisol in vivo increases FSH beta mRNA selectively in pituitaries of male rats.

Cortisol treatment for 6 or 12 days had no effect on serum FSH in intact males and animals castrated for 1 or 7 days, but pituitary FSH was increased by the steroid in both intact and castrate groups. In contrast, cortisol inhibited serum LH in both intact and castrated animals while only increasing pituitary levels of LH in 7 day castrates. Cortisol also increased the mRNA for FSH beta without affecting alpha or LH beta mRNAs. These data suggest that the selective increase in pituitary content of FSH may be due to a selective increase in FSH beta mRNA following exposure to cortisol.