Non-genomic action of estradiol and progesterone on cytosolic calcium concentrations in primary cultures of human granulosa-lutein cells

E.V. Younglai1,3, Y.J. Wu1, T.K. Kwan2 and C.-Y. Kwan2

1 Department of Obstetrics and Gynecology, Reproductive Biology Division and 2 Department of Medicine, McMaster University, Health Sciences Centre, Hamilton, Ontario, Canada L8N 3Z5

3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, McMaster University Medical Centre, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5. Email: younglai{at}mcmaster.ca


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The present study examined whether the sex steroids, estradiol and progesterone, could alter cytoplasmic calcium concentrations ([Ca2+]cyt) in human granulosa-lutein cells. METHODS: Human granulosa cells were obtained at the time of oocyte retrieval for IVF and cultured for 3–7 days. Cells were loaded with Fura-2 AM and changes in [Ca2+]cyt of single cells were studied using a dynamic digital Ca2+ imaging system. RESULTS: Both estradiol and progesterone stimulated elevations of [Ca2+]cyt in Ca2+-containing medium within seconds of exposure of the granulosa-lutein cells to the steroid, but only estradiol caused an increase in [Ca2+]cyt in Ca2+-free medium. Both ICI-182780 and RU 486 stimulated [Ca2+]cyt increases and inhibited the effects of estradiol and progesterone, respectively. Tamoxifen also induced transient increases in [Ca2+]cyt concentrations but inhibited the effects of both estradiol and progesterone. The inhibitory effects of tamoxifen, ICI-182780 and RU 4486 on [Ca2+]cyt responses to estradiol and progesterone could be reversed with higher concentrations of estradiol and progesterone, respectively. The [Ca2+]cyt effects induced with tamoxifen could not be eliminated by prior treatment with RU 486 or ICI-182780. CONCLUSION: These results provide strong evidence that both estradiol and progesterone as well as the steroid antagonists, tamoxifen, RU 486 and ICI-182780, can act on human granulosa-lutein cells through a non-genomic mechanism.

Key words: calcium response/estradiol/granulosa cells/progesterone


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
One of the earliest events of non-genomic action of steroids is an increase in intracellular uptake of calcium (Falkenstein et al., 2000Go). Such rapid actions have been reported for steroids in human ovarian tissue (Machelon et al., 1998Go) where luteinizing granulosa cells were found to respond to androstenedione with a rapid increase in intracellular Ca2+ concentrations ([Ca2+]i) by mobilization of Ca2+ stores from the endoplasmic reticulum and by Ca2+ influx through the voltage-dependent Ca2+ channel. A similar rapid effect on Ca2+ fluxes was found for dichlorodiphenylchloroethylene (DDE, p,p'-DDE) in human granulosa-lutein cells (Younglai et al., 2004Go). These two studies seem to be the only ones to date using human granulosa-lutein cells. This action of DDE was a novel observation for an endocrine-disrupting chemical since DDE had hitherto been known to act as an anti-androgen on the nuclear receptors (Kelce et al., 1995Go). Recent evidence suggest that the isomer of DDE, o,p-DDE binds strongly to the estradiol membrane receptor on breast cancer cells (Thomas et al., 2005Go), lending further support for this additional mechanism of action of endocrine disrupters.

Chicken and porcine granulosa cells can respond to estradiol, but not to progesterone or androgens, with a rapid increase in [Ca2+]i, and the source of Ca2+ for these cells was shown to be exclusively intracellular (Morley et al., 1992Go). Granulosa cells from diethylstilbestrol-treated immature rats failed to respond to estradiol with any increase in Ca2+ (Morley et al., 1992Go). On the other hand, granulosa cells from equine chorionic gonadotrophin-treated, 23-day-old rats or spontaneously immortalized rat granulosa cells do show a rapid response to progesterone (Peluso et al., 2002Go; Peluso 2004Go). In rat granulosa cells, progesterone caused a decrease in [Ca2+]i and inhibited mitosis (Peluso et al., 2002Go). This is in contrast to the studies by Morley et al. (1992)Go who were not able to detect any Ca2+ changes in chicken or porcine granulosa cells when treated with progesterone. In another study, porcine granulosa cells were found to respond with increases in [Ca2+]i when stimulated with estradiol, progesterone and androstenedione (Lieberherr et al., 1999Go). The current study was undertaken to determine whether estradiol and progesterone can induce changes in [Ca2+]i in human granulosa-lutein cells and whether these changes could be influenced by various antagonists.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Unless otherwise stated, all cultures, fluorescent labelling, digital fluorescence calcium ratio imaging and experimental conditions were identical to those described by Younglai et al. (2004)Go.

Source of granulosa cells
Patients were treated with a long luteal protocol of GnRH agonist (Lupron; Abbott Laboratories, Montreal, QC, 0.5 mg per day for 10–14 days) and recombinant FSH (12–85 ampoules, 75 IU per ampoule Gonal F; Serono Canada Ltd, Oakville, ON) followed by HCG (Profasi; Serono). After removal of oocyte–cumulus complexes, the remaining follicular aspirates were transported to the research laboratory in polypropylene tubes and the granulosa cells were isolated and cultured. After 3–7 days in culture, areas containing small luteinized cells, characterized by the cytoplasmic–nuclear ratio, were chosen for imaging.

Incubation medium conditions for imaging
Granulosa cells in culture were always exposed to 1–2 mmol Ca2+/l except for the experiments requiring Ca2+-free conditions, where the medium was replaced by the Ca2+-free isotonic physiological medium containing 0.1 mmol EGTA/l immediately prior to the measurement. Although distilled and de-ionized water were used for the preparation of solutions, contaminating Ca2+ from containers and other chemicals may contribute up to 10 µmol Ca2+/l. Therefore, EGTA 0.1 mmol/l was always included in the Ca2+-free medium.

Digital dynamic fluorescence ratio measurements
Changes in Ca2+ concentration were measured using a dynamic digital Ca2+ imaging system (Image-I/FL, Universal Imaging Corporation) with a Zeiss lamp (XBO 100 W/DC) coupled to a Zeiss inverted microscope (Zeiss IM 35) with a 100x oil immersion lens and a numerical aperture of 1.25, as previously described (Low et al., 1997Go). Images were integrated and collected by a Pulnix camera (TM-720, maximal at 3 s/frame) initially at a speed of 15 s/frame. In general, 3–4 probes covering an area of five pixels each were placed on different spatial areas of cells, usually near the plasma membrane and over the nuclear region. Changes in the fluorescence ratio were recorded and the data stored. Since the probes covered small areas of interest within the cell, quantitation of calcium changes was not attempted. Quantitation would vary in the different regions since our previous findings suggested that there are spatial and temporal differences within the cell upon stimulation with agonists (Kwan et al., 2003Go; Younglai et al., 2004Go). Images were saved and, in the event that some areas of interest showed oversaturation of colour during processing, the sequences were rerun with new areas of interest. Cells from at least three different patients had to show a response before the results were accepted as meaningful and quoted. Representative patterns of response are shown in the figures. In some instances, the positions of the probing windows were changed from the original placements to capture the spatial changes in Ca2+ concentrations. Ethical approval was obtained from the institutional research ethics board for this work, and patients signed a consent form agreeing to donate their excess cells for research.

Chemicals and reagents
Estradiol and progesterone were purchased from Steraloids Inc., Newport, RI. Fluvestrant (ICI 182,780) was obtained from Tocris Cookson Inc., Ellisville, MO. Tamoxifen and mifepristone (RU 486) were purchased from Sigma-Aldrich Chemicals, Oakville, ON. All tissue culture supplies were purchased from Life Technologies, Burlington, ON. Fura-2 acetoxymethyl ester (fura-2AM) was obtained from Molecular Probes, Portland, OR. The steroids were dissolved in dimethylsuphoxide (DMSO) and further diluted with culture medium to achieve a final concentration of ~1 µg/ml which have been shown to be the optimal concentration for estradiol or progesterone to elicit effects in human sperm (Luconi et al., 1998Go; Wennemuth et al., 1998Go). In rat granulosa cells, progesterone at a concentration of 200 ng/ml was effective in altering [Ca2+]i uptake (Peluso et al., 2001Go).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Effects of estradiol and progesterone
Granulosa-lutein cells were treated with steroids at concentrations of up to 1 µg/ml. Figure 1 shows the [Ca2+]cyt response of granulosa cells to estradiol and progesterone. In Figure 1A, four probing windows were placed on three separate cells. Addition of estradiol caused an immediate increase in [Ca2+]cyt above the baseline in all the probed cells, and the changes became oscillatory. The increase in [Ca2+]cyt then declined to baseline and, over the period of observation, started to rise slowly. Addition of EGTA diminished the Ca2+ elevation, thus terminating the experiment. In Figure 1B, three cells were probed. Progesterone caused an immediate elevation in [Ca2+]cyt in all three cells, higher than that elicited with estradiol. EGTA was then added to preserve cell viability when [Ca2+]cyt became excessively elevated.



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Figure 1. Effects of estradiol (A) and progesterone (B), 1 µg/ml each, on [Ca2+]cyt changes in primary cultures of human granulosa-lutein cells. Probes were placed on individual cells and the different colour tracings in the time profile denote changes of the fluorescence ratio at the location of each probe in single human granulosa cells, identified by the dots with corresponding colours in the square frame numbered 1. The ratio image in each frame was taken at the time identified by the numbers corresponding to the image numbers. The ticks along the time profile indicate the time when a chemical is added. Each figure is a representative experiment of cells from three different patients. (A) At 7 min, estradiol was added, followed at 25 min, by 2.5 mmol/l EGTA. Chelation of Ca2+ ions with EGTA caused an immediate drop in the ratio. All three cells showed responses to estradiol. Note the 3-fold increase in Ca2+ with oscillations after addition of estradiol followed by a drop to baseline and a slow increase with oscillations over the next 15 min. (B) At 5 min, progesterone caused an immediate large increase in intracellular Ca2+ and EGTA was quickly added to prevent leaking. All three cells responded.

 
In view of the Ca2+-lowering effect of EGTA seen above, the effects of estradiol and progesterone in Ca2+-free medium were then investigated. Figure 2A shows that estradiol induced an immediate peak in [Ca2+]cyt followed by a return to baseline in Ca2+-free medium. Addition of Ca2+ to the medium to a final concentration of 2.5 mmol/l resulted in a slow oscillatory increase in [Ca2+]cyt. The response to progesterone was different, as shown in Figure 2B. There was no response to progesterone in Ca2+-free medium, and subsequent addition of 2.5 mmol/l Ca2+ also failed to induce an elevation of [Ca2+] as seen for estradiol. However, addition of a further dose of progesterone at 22 min caused a sharp increase in [Ca2+]cyt which was then suppressed with EGTA. This pattern of response for progesterone in Ca2+-free medium was observed in cells from all three different patients.



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Figure 2. Effects of 1 µg/ml each of estradiol (E2) and progesterone (P4) in Ca2+-free medium (containing 0.1 mmol/l EGTA) and following the addition of 2 mmol/l extracellular Ca2+. Each figure is a representative experiment of cells from three different patients. The protocol was identical to that used in Figure 1. (A) Note the rapid increase in [Ca2+]cyt in the absence of extracellular Ca2+, similar to that in Figure 1. Addition of extracellular Ca2+ caused a small increase in [Ca2+]cyt with oscillations. (B) No response was elicited with progesterone in Ca2+-free medium. Addition of Ca2+ to the medium did not elicit an immediate increase in intracellular Ca2+. An additional dose of progesterone to a final concentration of 2 µg/ml then caused an immense increase in [Ca2+]cyt which was quickly terminated with EGTA. The inserts in each figure show the location of the probes within each cell at the start of recordings.

 
Controls
The effects of various other steroids on [Ca2+]cyt were investigated in order to demonstrate that the [Ca2+]cyt changes were not non-specific effects. Figure 3 shows the results with 1 µg/ml each of androstenedione, testosterone, dehydroepiandrosterone (DHEA) and estrone. Estradiol and progesterone were the positive controls. Figure 3A is a representative of cells from four of eighr patients which showed a [Ca2+]cyt response to androstenedione, and two doses (10 µg/ml each) of progesterone were needed to elicit a [Ca2+]cyt response. Cells from four of six patients responded to testosterone with increases in [Ca2+]cyt, and Figure 3B is a representative of one of these. There was a delay in the [Ca2+]cyt response to progesterone. The solvent used for dissolving the steroids, DMSO, as well as DHEA and estrone, had no effects on [Ca2+]cyt changes (Figure 3C). Pregnenolone (four patients), dihydrotestosterone (three patients) and estriol (three patients) at concentrations of 1 and 10 µg/ml also had no effects. In other experiments, a single addition of a control steroid was followed by either progesterone or estradiol alone 6–10 min later, and these control steroids also had no effect on [Ca2+]cyt. The effects of Ca2+-free medium with these steroids were not investigated.



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Figure 3. Effects of addition of other steroids (1 µg/ml) on Ca2+ changes in granulosa-lutein cells. Ad=androstenedione; P4=progesterone; T=testosterone; E2=estradiol; Dh=dehydroepiandrosterone; E1=estrone; DMSO=dimethylsulphoxide. Inserts show the location of probes at the start of recordings. Each figure is a representative experiment of at least three separate patient samples. Each control steroid was also tested against progesterone or estradiol alone in separate experiments.

 
Effects of antagonists
Steroid antagonists are known to act at the nuclear level and therefore were not expected to affect [Ca2+]cyt changes induced by exogenous steroids. Figure 4A shows that RU 486 at 1 µg/ml, by itself induced a slight Ca2+ transient in two of four cells probed, but inhibited the subsequent response to progesterone up to a final concentration of 2 µg/ml. Pre-incubation of cells with RU 486 for 1 h prior to labelling with fura-2AM and exposure to progesterone also inhibited the Ca2+ response to progesterone but not to estradiol (Figure 4B). In one cell with two probes (green and purple), an apparent secondary release of [Ca2+]cyt was observed in the presence of estradiol.



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Figure 4. Effects of steroid antagonists on intracellular Ca2+ changes in granulosa cells. Inserts are similar to those in Figure 2. (A) Changes in [Ca2+]cyt induced by RU 486 (1 µg/ml per application), followed by two doses of 1 µg/ml progesterone. This figure is representative of four different patients. (B) Cells were pre-treated with 2 µg of RU 486/ml for at least 1 h prior to stimulation with 1 µg of progesterone/ml followed 3 min later by 1 µg of estradiol/ml. This figure is representative of three patients with different time intervals between progesterone and estradiol. (C) ICI-182780 (two doses of 1 µg/ml each) was added to the medium at the times indicated, followed ~7 min apart by 2 µg of esradiol/ml and 2 µg of progesterone/m;. (D) Three doses of 1 µg of estradiol/ml for a final concentration of 3 µg of estradiol/ml reversed the inhibitory effect of ICI-182780. (E) Tamoxifen was used at a concentration of 5 µg/ml which inhibited the effects of both estradiol and progesterone. (F) The two doses of tamoxifen (0.5 µg/ml) resulted in a final concentration of 1 µg/ml, the second dose of estradiol led to a final concentration of 2.3 µg/ml and progesterone was added to a final concentration of 1.3 µg/ml. E2=estradiol; P4=progesterone; RU=RU 486; ICI=ICI-182780; TAM=tamoxifen; Ion=ionomycin. Inserts show the location of probes at the start of recordings.

 
ICI-182780 at 2 µg/mLl had effects on [Ca2+]cyt changes similar to those shown for RU 486 in Figure 4A (four patients). However, when an equivalent amount of estradiol or progesterone (2 µg/ml) was added to cells exposed to 2 µg of ICI-182780/ml, [Ca2+]cyt increased within seconds of exposure to the steroid (Figure 4C). Increasing the concentration of estradiol in the medium can over-ride the effects of ICI-182780 (Figure 4D). Tamoxifen, another anti-estrogen, markedly increased [Ca2+]cyt (Figure 4E). In addition, it completely suppressed the [Ca2+]cyt response to estradiol and progesterone. The Ca2+ ionophore, ionomycin, immediately elevated [Ca2+]cyt. As with RU 486 and ICI-182780, increasing the concentration of estradiol and progesterone can overcome the inhibitory effects of the antisteroids (Figure 4F). The effects of tamoxifen were not prevented by prior treatment with RU 486 or ICI-182780 (Figure 5).



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Figure 5. Effect of steroid antagonists, 1 µg/ml, on the calcium response to tamoxifen. RU=RU 486; ICI=ICI-182780; Tam=tamoxifen. Inserts show the location of probes at the start of recordings.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The non-genomic actions of estradiol and progesterone on reproductive tissues are well known (Revelli et al., 1998Go; Gerdes et al., 2000Go; Levin, 2001Go, 2002Go; Bramley, 2003Go; Sak and Everaus, 2004Go). Although this non-genomic action has been demonstrated in gonadal cells from several animal species such as chicken, rat and pig granulosa cells (Morley et al., 1992Go; Revelli et al., 1998Go; Lieberherr et al., 1999Go; Peluso et al., 2001Go), to our knowledge the present communication is the first report on a similar action on human granulosa-lutein cells. The non-genomic action of estradiol has been demonstrated in chicken and porcine granulosa cells (Morley et al., 1992Go) and that for progesterone in rat granulosa cells (Peluso et al., 2002Go). Androstenedione has been shown to have a rapid effect on Ca2+ changes in human granulosa-lutein cells (Machelon et al., 1998Go) and to have an inhibitory effect on the non-genomic response of oocytes to estradiol (Tesarik and Mendoza, 1997Go). Thus it would appear that human granulosa-lutein cells can have non-genomic responses to a variety of steroids. Porcine granulosa cells also respond to estradiol, progesterone and androstenedione with a rapid increase in [Ca2+]i (Lieberherr et al., 1999Go). Both estradiol and progesterone receptors have been detected on human sperm membranes (Luconi et al., 1998Go, 1999Go), and there appears to be an interference of estradiol receptors with progesterone effects in sperm. Progesterone and estradiol can increase [Ca2+]i changes in sperm, and this is mediated through the membrane receptors. It is possible that the steroid effects on granulosa-lutein cells are also mediated by membrane receptors.

The probes for detection of [Ca2+]cyt changes in individual cells each covered an area of five pixels. Up to nine probes have been used in the past to follow the progression of Ca2+ ion changes through the cell membrane to the nucleus (Kwan et al., 2003Go). Thus changes can be detected adjacent to the cell membrane, over intracellular organelles or in the nucleus, and the magnitude of responses in individual cells would depend on the location of the probes. Usually two probes were used per cell—one near the membrane and the other near the nucleus. The differences in responses could also be due to patient variation as well as cell type. Small and large cells derived from the mural, cumulus or theca layers in the corpus luteum have different abilities to produce progesterone (Lemoin and Mauleon, 1982Go).

Non-specific effects of supraphysiological doses of steroids have been described (Losel et al., 2003Go). The concentrations of estradiol and progesterone used are consistent with levels commonly found in mature human antral follicles (Kreiner et al., 1987Go; Yie et al., 1995Go) and used for demonstrating non-genomic effects in human spermatozoa (Wennemuth et al., 1998Go; Luconi et al., 2001Go). We have also used 10-fold concentrations of non-active steroids such as estriol, which has a similar structure to estradiol, to show the absence of non-specific effects.

The genomic estrogen receptors have been located in human granulosa cells (Suzuki et al., 1994Go; Saunders et al., 2000Go; Jakimiuk et al., 2002Go), but not in corpus luteum which contained progesterone receptors (Suzuki et al., 1994Go). Genomic progesterone receptors have also been detected in human corpus luteum cells by immunohistochemistry (Maybin and Duncan, 2004Go). Although there have been suggestions that the genomic and membrane receptors may have some similarities (Saner et al., 2003Go; Sak and Everaus, 2004Go) and may arise from a single transcript (Razandi et al., 1999Go), there is no evidence to date on the nature of the human granulosa-lutein membrane receptor for estradiol or progesterone. In addition, there may be multiple classes of proteins which can function as non-genomic steroid receptors (Watson and Gametchu, 2003Go), and recent evidence suggests that these membrane receptors may be G protein-like-coupled receptors (Edwards, 2005Go).

The different patterns of [Ca2+]cyt responses observed with estradiol and progesterone suggest that there are distinct and separate mechanisms by which these agonists act on the granulosa-lutein cell. The [Ca2+]cyt response to estradiol in Ca2+-free medium suggests that Ca2+ is mobilized from intracellular stores such as the smooth endoplasmic reticulum, and Ca2+ influx could also occur. The Ca2+ response to progesterone, however, suggests that the mechanism by which progesterone acts is different from that of estradiol, in that intracellular sources are not utilized. The failure of the cells in the presence of progesterone to respond on addition of Ca2+ to the medium was unexpected. This suggests that the membrane factors through which progesterone acts are more sensitive to the destabilizing effect of the lack of calcium (Webb and Bohr, 1978Go) and that calcium is required for restoration of membrane stability (Ou et al., 1997Go). In porcine granulosa cells, on the other hand, progesterone triggers rapid transmembrane Ca2+ influx and/or calcium mobilization from endoplasmic reticulum (Machelon et al., 1996Go). The concentration of Ca2+ within the cell is controlled by two general mechanisms: (i) entry from extracellular fluid by voltage-operated channels; or (ii) release from endoplasmic reticulum by capacitative calcium entry which activates the store-operated channel permitting influx from the extracellular fluid (Brini and Carafoli, 2000Go). Calcium oscillations have been shown to be required for cell division (Swann et al., 2004Go) and it is possible that these oscillations trigger mitosis in the cultured granulosa cells. Calcium oscillations also represent a physiological mechanism to prevent a rapid rise of cytosolic Ca2+ concentration to toxic levels (Miyazaki, 1995Go; Bootman et al., 2001Go). Further studies with the use of inhibitors are required to eludicate the different intracellular mechanisms involved in estradiol- and progesterone-induced Ca2+ fluxes in human granulosa-lutein cells.

Although a previous communication revealed that human luteinizing granulosa cells did not show a [Ca2+]cyt response to testosterone as they did to androstenedione (Machelon et al., 1998Go), in four of six patients we found that testosterone could induce [Ca2+]cyt uptake in granulosa-lutein cells. In addition, cells from only four of eight patients responded to androstenedione. This may reflect patient variability or possible high levels of androgens or estrogens produced during stimulation protocols, attenuating the effects of these steroids. A similar attenuation has been found in estrogen-treated microvessels (Kakucs et al., 2001Go). Moreover, testosterone at a concentration of 10–5 mol/l can induce [Ca2+]cyt influx in chicken granulosa cells (Morley et al., 1992Go) as well as activated T cells (Benten et al., 1997Go). This observation with testosterone is validated further by the absence of [Ca2+]cyt responses to the structurally similar steroids, DHEA and dihydrotestosterone.

Neither DMSO, estrone, estriol, pregnenolone, DHEA or dihydrotestosterone stimulated Ca2+ uptake in the human granulosa-lutein cells. The use of these control steroids, while not eliciting increases in [Ca2+]cyt concentrations, raised the possibility that these treatments could enhance or inhibit the responses to estradiol and progesterone. Such a possibility is reflected in Figure 3A and B where the rapid response to progesterone is apparently delayed. This inhibition of a steroid effect by another steroid is being examined in more detail. The media used in our experiments do not exceed 0.5% DMSO. A concentration of 0.2–1% DMSO in the culture medium can induce a 2- to 6-fold increase in Ca2+ uptake in chicken granulosa cells (Morley and Whitfield, 1993Go), pointing to species variation in sensitivity of membrane receptors.

It is interesting that tamoxifen by itself had a membrane effect in stimulating an increase in [Ca2+]cyt. While this was unexpected, it has been reported that tamoxifen has a non-genomic effect in stimulating membrane-bound guanylate cyclase in porcine proximal tubular LLC-PK1 cells (Chen et al., 2003Go) and can stimulate an increase in [Ca2+] uptake in MCF-7 breast cancer cells (Chang et al., 2002Go). Tamoxifen has also been found to stimulate Ca2+ influx in human sperm and to inhibit the progesterone-induced Ca2+ influx (Luconi et al., 2001Go). In chicken granulosa cells, tamoxifen had no effect (Morley et al., 1992Go). Tamoxifen, which is a selective estrogen receptor modulator, can have antagonist activity in breast cancer cells but is an agonist for endometrial growth (Hermenegildo and Cano, 2000Go). In our study, tamoxifen completely inhibited the effects of both estradiol and progesterone. With chicken granulosa cells, tamoxifen prolonged the carbachol-triggered [Ca2+]cyt surges (Morley and Whitfield, 1994Go).

ICI-182780 prevented the [Ca2+]cyt response to estradiol, but further addition of estradiol could reverse this inhibition. The relative binding of ICI-182780 is 0.89, compared with that of estradiol 1.0 (Wakeling et al., 1991Go), and therefore excess estradiol would be expected to reverse the non-genomic effect of ICI-182780. The inhibitory effect of ICI-182780 on the non-genomic action of estradiol has also been observed in rat astrocytes (Chaban et al., 2004Go). This estrogen receptor antagonist can activate large conductance, calcium-activated potassium channel (BKCa) activity in smooth muscle (Dick, 2002Go) just like estradiol and tamoxifen (Valverde et al., 1999Go; Dick et al., 2001Go), but in cultured endothelial cells of human coronary artery it is inhibitory for BKCa channel activity (Liu et al., 2003Go). Thus these two estrogen antagonists, tamoxifen and ICI-182780, act by different mechanisms at the cell membrane. However, more recent evidence suggests that tamoxifen and ICI-182780 have high affinities for the membrane receptor for estradiol in the SKBR3 breast cancer cell line (Thomas et al., 2005Go), confirming our observations on the non-genomic effects of these compounds. Competition between estradiol and the antagonists can explain the reversal of effects when excess steroid is added.

RU 486 has a Kd of ~10–9 mol/l (Cadepond et al., 1997Go) and has both anti-progestational and anti-glucocorticoid activities. At a concentration of 1 µg/ml used in these experiments, RU 486 showed a slight increase in [Ca2+]cyt uptake but inhibited the [Ca2+]cyt response to progesterone. Similar to the effects of ICI-182780, excess progesterone can over-ride the inhibitory effect of RU 486. Concentrations of RU 486 used in the past have ranged from 640 nmol/l to inhibit the effects of a similar concentration of progesterone (Peluso et al., 2001Go) to 100 µmol/l to show an increase in apoptosis in luteinizing granulosa cells (Svensson et al., 2001Go). The inability of RU 486 and ICI-182780 to inhibit the [Ca2+]cyt responses to tamoxifen suggests that tamoxifen is acting at a different site compared with the other two antagonists. Taken together, these data suggest that the sex steroids, estradiol and progesterone, as well as the antagonists, tamoxifen, ICI-182780 and RU 486, can act as genomic and non-genomic agents.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Michael Neal and the physicians of the Centre for Reproductive Care, Hamilton Health Sciences, for providing the granulosa cells. This work was supported by the Canadian Institutes of Health Research. Y.J.W. is a Visiting Scholar from the Department of Obstetrics and Gynecology, The Second Hospital of Hebei Medical University, China.


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 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on October 27, 2004; resubmitted on March 18, 2005; accepted on April 14, 2005.