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. 2002 Mar 1;539(Pt 2):557–566. doi: 10.1113/jphysiol.2001.012947

Rapid actions of 17β-oestradiol on a subset of lactotrophs in the rat pituitary

H C Christian 1, J F Morris 1
PMCID: PMC2290152  PMID: 11882687

Abstract

Increasingly the role of rapid mechanisms of steroid action in physiological regulation are being recognised. We have investigated rapid effects of 17β-oestradiol (E) on prolactin (PRL) release in vitro. Pituitary segments from male rats were incubated for 5, 10 or 20 min in Earle's balanced salt solution containing 1.2 mm tannic acid (to enable visualisation of exocytosed secretory granules by electron microscopy) either alone (control) or containing 10−10-10−8m E conjugated to bovine serum albumin (E-BSA). PRL and leuteinising hormone (LH) release from pituitary segments were also determined in response to E and E-BSA by radioimmunoassay. Within 10 min E-BSA and E (10−12-10−6m) stimulated a significant (P < 0.05) concentration-dependent release of PRL but not LH. After exposure to experimental media for 5 min, only occasional exocytosis from type I lactotrophs (characterised by large polymorphic secretory granules) was observed in either control or E-BSA treated tissue. In contrast, E-BSA (10−10-10−8m) induced a significant (P < 0.05) increase in the number of exocytotic profiles from type II lactotrophs (characterized by smaller, spherical granules). This effect was not inhibited by removal of extracellular calcium, or by pre-treatment of cells with the RNA synthesis inhibitor actinomycin-D (0.5 μg ml−1), the protein synthesis inhibitor cycloheximide (1 μg ml−1) or the anti-oestrogen ICI 182,780 (1 μm). FACS analysis demonstrated binding of E-BSA-fluorescein isothiocyanate (FITC) (10−10-10−7m) to a subpopulation of anterior pituitary cells. The E-BSA-FITC binding sites assumed a patchy distribution across the cell surface. In conclusion, we report for the first time a rapid, non-genomic effect of E on PRL secretion in normal pituitary tissue.

Many cellular actions of the sex steroid 17β-oestradiol (E) are known to be mediated through the transcriptional regulation of target genes. These effects mainly occur when the steroid binds to intracellular oestrogen receptor proteins (ER) that dimerise and bind to response elements on promoters of target genes and interact with components of the transcriptional machinery to initiate transcription or modify transcription through protein-protein interactions (Halachmi et al. 1994). In addition to these ‘classical’ genomic actions of oestrogens, there is increasing evidence for rapid actions mediated via plasma membrane proteins (for review see Ropero et al. 1999; Schmidt et al. 2000; Israel & Poulain, 2000; Condliffe et al. 2001). Whereas most genomic actions take hours or days to produce their effects, rapid, ‘non-classical’ actions are characterised by their rapid onset, within a few seconds to 10 min, and lack of sensitivity to inhibitors of RNA or protein synthesis. Furthermore, rapid actions are mimicked by E conjugated to macromolecules, such as albumin or peroxidase, that cannot permeate the plasma membrane, and therefore cannot access nuclear receptors (Swenson & Sladek, 1997; Nadal et al. 1998). There is evidence that E can trigger rapid actions within seconds to minutes through a variety of signal transduction events (Kelly & Levin, 2001). These include the stimulation of cAMP synthesis (Aronica et al. 1994), calcium flux (Morley et al. 1992), phospholipase C activation (Le Mellay et al. 1997), protein kinase C activation (Condliffe et al. 2001), MAP kinase cascades (Migliaccio et al. 1996), cGMP-dependant kinase activity (Ropero et al. 1999), phosphatidylinositol-3-OH kinase activation (Simoncini et al. 2000) and nitric oxide (NO) production (Prevot et al. 1999). Rapid actions of E have been attributed to activation of putative membrane oestrogen receptor(s) but, to date, no such protein has been isolated or structurally or functionally characterised.

The primary function of pituitary lactotrophs is to produce and secrete prolactin (PRL; Freeman et al. 2000). E controls the expression of PRL in pituitary lactotrophs primarily by a transcription-dependent mechanism (Maurer, 1982). The promoter region of the rat PRL gene contains potential oestrogen response elements (Maurer & Notides, 1987) but recently the ability of oestrogen to induce PRL mRNA and protein has been shown to be dependent on the activity of MAP kinase cascades (Watters et al. 2000). The ERα knockout mouse has provided genetic evidence that ERα plays a critical role in PRL gene transcription and is involved in lactotroph cell growth (Scully et al. 1997). In rodents, two main morphological types of lactotroph can be characterised by electron microscopy (Nogami & Yoshimura, 1982). Type I cells contain large irregular-shaped, electron-dense secretory granules, whereas type II cells contain numerous smaller, spherical, electron-dense granules. In the PRL-secreting cell line GH3/B6, E rapidly stimulates PRL release by acting directly to cause a sustained train in action potentials (Zyzek et al. 1981) and indirectly by reversal of dopamine inhibition (Dufy et al. 1979). More recently, Pappas et al. (1994) have demonstrated membrane oestrogen receptors in GH3/B6 cells in association with rapid oestrogen-induced PRL release. However, these effects have not, to date, been investigated in normal pituitary tissue. Recently, we have demonstrated rapid secretagogue actions (within 5 min of application) of testosterone that affect only the type II lactotrophs in the male rat pituitary (Christian et al. 2000). As part of the investigation we screened a range of steroids and demonstrated preliminary findings of a similar action of E. We now report for the first time the characterisation of selective, rapid actions of E and E-bovine serum albumin (E-BSA) on type II lactotrophs in the rat anterior pituitary gland by use of a combination of tannic acid and electron-microscopic analysis and radioimmunoassay. The present study extends preliminary work published in abstract form (Christian & Morris, 1998).

METHODS

Animals

Adult, normal, male and female Sprague-Dawley rats bred from a colony in the Department of Human Anatomy and Genetics, Oxford, were used. The Principles of Laboratory Animal Care (NIH publication no. 85–23) was followed and the study was in accordance with the UK Animals (Scientific Procedures) Act 1986. The rats were housed, after weaning, in groups of five per cage in a quiet room with 14 h of light and 10 h of darkness and temperature maintained at 20–21 °C; food and water were available ad libitum. Reproductive cycle stage in females was determined by vaginal smears taken daily. All experiments were started between 08.00 h and 09.00 h in order to avoid changes associated with the circadian rhythm. Animals were killed by stunning followed by decapitation, the pituitary gland was removed and the anterior lobe separated from the posterior lobe.

Secretion of PRL in vitro by anterior pituitary segments

Identification of secreting cells

Anterior pituitary glands were cut into four roughly equal segments. The segments were distributed randomly (one segment per well) in the wells of 24-well tissue culture plates (Costar, Cambridge, MA, USA) and incubated at 37 °C for 90 min in 1 ml incubation medium (1 % v/v) aprotonin (Bayer Corp., Saffron Waldon, UK) and 1 % (v/v) penicillin-streptomycin (Sigma, Poole, UK) in oxygenated Earle's balanced salt solution (EBSS; phenol red free; Sigma, pH 7.4) under a humidified atmosphere saturated with 95 % O2-5 % CO2. The segments were then transferred to fresh incubation medium to which had been added 1.2 mm tannic acid (BDH, Poole, UK) without (negative control) or with oestradiol-BSA (E-BSA; 0.1 nm or 10 nm) or 28 mm K+ (positive control) and incubated for an additional 5, 10 or 20 min. To eliminate the possibility of non-specific membrane effects of the steroid or steroid-BSA conjugate on hormone release, a range of other steroids at equal concentrations had previously been tested (Christian et al. 2000). In separate experiments, tissue was equilibrated for 15 min before steroid application with cycloheximide (1 μg ml−1), actinomycin-D (0.5 μg ml−1), H89 (100 μm) or ICI 182,780 (1 μm), which were then included in the medium with E-BSA for the remainder of the experiment. At the end of the final incubation, tissue was fixed by immersion in glutaraldehyde (2.5 % in 0.1 m phosphate buffer, pH 7.2) for 1 h and then processed into Spurr's resin.

Assay of secreted PRL and LH

The experiments in which hormone released into the medium was assayed followed the protocol described above, except that no tannic acid was added to the incubation medium. Medium from the final incubation was collected and stored in aliquots (300 μl, −20 °C) for subsequent measurement of immunoreactive PRL (ir-PRL) and luteinising hormone (ir-LH) by radioimmunoassay. Pituitary segments were weighed on a torsion balance and discarded.

Preparation of dispersed pituitary cells

Suspensions of dissociated anterior pituitary cells were prepared according to established protocols (Christian et al. 1997). Briefly, anterior pituitary cells were dissociated with collagenase (0.2 % w/v; Roche Molecular Biochemicals, Lewes, UK) and deoxyribonuclease (DNase, 0.002 % w/v; Sigma) in oxygenated EBSS enriched with BSA (0.4 % w/v, Sigma); the dispersion was aided by gentle trituration (30 s every 10 min). The resulting cell suspension was centrifuged (200 g, 10 min), the pellet was resuspended in 5 ml BSA-enriched EBSS and the suspension was filtered through a 20 μm nylon mesh to remove any large clumps of debris. The filtrate was then centrifuged (300 g, 10 min) and the pellet was resuspended in 5 ml incubation medium. The cells were examined at the light microscope level to verify the effectiveness of the dispersion (> 90 %) and counted using a haemocytometer. Viability of the cells was assessed by the trypan blue exclusion test and was always found to be more than 95 %. Additional samples of the cells were retained for examination with an electron microscope.

Secretion of PRL in vitro by enzymatically dispersed pituitary cells

The cells were diluted in medium (1 % (v/v) aprotonin and 1 % (v/v) penicillin-streptomycin in EBSS, pH 7.4) to a concentration of 2.5 × 105 cells ml−1 per well in 24-well cell culture plates (Costar, Cambridge, MA, USA) and incubated for 90 min at 37 °C in a humidified atmosphere saturated with 95 % O2-5 % CO2. They were then challenged for 10 min with E, E-BSA or 28 mm K+; controls were incubated in an equal volume of incubation medium alone (0.6 ml). After centrifugation (600 g, 4 °C, 10 min) the supernatant fluid was harvested and assayed for ir-PRL. In some experiments, pituitary cells were prepared for electron microscopy in order to assess the quality of cell ultrastructure after incubation.

Quantitative electron microscopy

Anterior pituitary segments and isolated cells were prepared for electron microscopy by standard methods. Briefly, segments were post-fixed in osmium tetroxide (1 % w/v in 0.1 m sodium phosphate buffer) contrasted with uranyl acetate (2 % w/v in distilled water), dehydrated through increasing concentrations of ethanol (70–100 %) and embedded in Spurr's resin (Agar Scientific (UK), Stansted, UK). Ultra-thin sections (50–80 nm) were viewed with a JEM-1010 transmission electron microscope (JEOL USA Inc., Peabody, MA, USA). Sections of pituitary segments were collected as soon as a full block face was presented in order to obtain sections that were relatively superficial. Immunogold labelling for PRL was performed to identify lactotrophs (Nakane, 1975): rabbit anti-rat PRL (National Hormone and Pituitary Program, Gaithersburg, MD, USA) was used at a dilution of 1:5000. For control sections, the primary antibody was omitted and replaced with 0.1 m sodium phosphate buffer containing 1 % w/v egg albumin. Endocrine cells in sections taken systematically from different depths of the embedded tissue were identified on the basis of their secretory granule populations (shape, electron density, size and distribution), and organelle structure, nucleus size and chromatin characteristics (Nogami & Yoshimura, 1982). Cells from individual samples were always identified and counted on four to eight randomised grids according to a systematic random procedure (Weibel, 1973).

Radioimmunoassay of PRL and LH

PRL was determined in duplicate by radioimmunoassay using a primary antibody of defined specificity raised in a rabbit against rat PRL; synthetic PRL was used as a reference preparation and 125I-labelled PRL as tracer (all reagents generously supplied by the National Hormone and Pituitary Program). The assay sensitivity was 0.5 ng ml−1 and the inter-and intra-assay coefficients of variation were 10 % and 4 %, respectively. Dilution curves of test samples were parallel to that of the standard PRL preparation. LH was determined by radioimmunoassay using a primary antibody raised in a rabbit against rat LH; rat LH was used as the reference preparation and rat 125I-labelled LH as tracer (reagents also supplied by the National Hormone and Pituitary program). The assay sensitivity was 1 ng ml−1 and the inter- and intra-assay coefficients of variation were 11 and 8 %, respectively.

Assay of cAMP

Anterior pituitary segments were incubated at 37 °C for 90 min in 1 ml incubation medium under a humidified atmosphere saturated with 95 % O2-5 % CO2. The segments were then transferred to fresh incubation medium to which had been added EBSS (negative control), oestradiol-BSA (E-BSA; 1 nm or 100 nm) or vasoactive intestinal peptide (VIP; 10 nm; Peninsula Laboratories, Belmont, CA, USA; positive control) and incubated for an additional 20 min. At the end of this period segments were rapidly put on ice to terminate the reaction. Segments were treated with methanol before sonication and centrifugation at 400 g for 10 min. The supernatant was collected and methanol evaporated. Aliquots were assayed for cAMP using an enzyme immunoassay kit (Amersham Pharmacia Biotech, Little Chalfont, UK).

Detection of cell surface E-binding sites by fluorescence-activated cell sorting(FACS) analysis

Collagenase-dispersed cells were fixed in paraformaldehyde (2 % in PBS, 1 h), washed three times in phosphate-buffered saline (PBS; 3 × 100 g, 10 min) then resuspended in PBS to a concentration of 25 × 106 cells ml−1. Aliquots of cells (50 μl) were incubated with graded concentrations of E-BSA-fluorescein isothiocyanate (FITC) (200 μl in PBS; 1 nm to 1 μm; Sigma) for 20 min at 37 °C in the incubation conditions previously described for release experiments. To determine any non-specific ligand binding, parallel aliquots were incubated with 10−7m BSA-FITC conjugate or co-incubated with an excess of unlabelled E (10−6m). Cells were then washed three times in PBS to remove unbound ligand (3 × 100 g, 10 min), resuspended in PBS (50 μl), smeared onto gelatine-coated slides and mounted in Vectashield (Vecta Laboratories, Peterborough, UK) for confocal microscopy (Leica Microsystems, Heidelberg, Germany).

FACS analysis

Surface fluorescence was quantified by FACS analysis using a FACScan analyser (Becton Dickinson, Oxford, UK) with air-cooled 100 mW argon laser (488 nm) detection and a Consort 32 computer system running Lysis II software. A total of 10 000 cells were counted per sample; particles too large to be single cells as determined by forward light scatter were gated out electronically.

Drugs

The following were used: oestradiol-6-(o-carboxy-methyl) oxime:BSA (20–30 mol steroid per mol BSA), oestradiol-6-(o-carboxymethyl)oxime:BSA-fluorescein isothiocyanate conjugate and BSA-FITC (all Sigma) were initially dissolved in EBSS adjusted to pH 9 with 1 m NaOH and diluted in EBSS pH 7.4. 17α- and 17β-oestradiol (Sigma) and H89 (Calbiochem, Nottingham, UK) were initially dissolved in a small amount of ethanol and subsequently diluted with EBSS; the final concentration of ethanol never exceeded 0.1 % and appropriate controls were included in each experiment. Cycloheximide, actinomycin D (both Sigma) and ICI 182,780 (Tocris Cookson, Bristol, UK) were dissolved and diluted in EBSS immediately prior to use. All solutions were adjusted to pH 7.4 before use.

Statistical analysis

Quantification of exocytosis by electron microscopy was analysed by the Mann-Witney U test. Preliminary analysis of radioimmunoassay (RIA) and FACS data by the Shapiro-Wilks test showed that the data were normally distributed. Subsequent analysis was performed using ANOVA with post hoc comparisons by Duncan's multiple range test. Data are presented as means ± s.e.m. for n independent experiments. Differences were considered significant at P < 0.05. Because the basal rate of ir-PRL and ir-LH release varied between experiments, statistical analyses were made within experiments only. Each of the studies shown was repeated at least three times (for specific details see figure legends) and in all instances a similar data profile was seen.

RESULTS

Effect of E and E-BSA on exocytotic release of PRL from type I and type II lactotrophs in vitro

Stimulation of anterior pituitary segments in vitro with the non-specific depolarizing stimulus of 28 mm K+ (positive control) for 10 min induced a significant (P < 0.01) increase in ir-PRL (Fig. 1A) and ir-LH release (Fig. 1B). Incubation of segments for 10 min with either E-BSA or unconjugated E (10−12-10−6m) significantly (P < 0.05) stimulated ir-PRL release in a concentration-dependent manner (Fig. 1A) but had no detectable effect on the amount of ir-LH released (Fig. 1B). No significant difference between the amount of ir-PRL release in response to E-BSA and to unconjugated E was detectable, but the same concentrations of the stereoisomer 17α-oestradiol were without effect (Fig. 1).

Figure 1. Rapid effect of E and E-BSA on PRL secretion but not LH.

Figure 1

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Effects of 17βE (□) and E-BSA (Inline graphic, 10−12-10−6m) for 10 min in vitro on the release of ir-PRL (A) and ir-LH (B) into the medium from anterior pituitary segments from male rats. Values are the mean ± s.e.m. (n = 6). ▪, positive control stimulus, 28 mm K+. The response to 17αE is also shown. The data are typical of those from three replicate experiments. **P < 0.01 vs. corresponding basal control, *P < 0.05 (by ANOVA plus Duncan's multiple range test).

Electron microscopy revealed that in both types of in vitro preparation - pituitary segments and collagenase-dispersed cells - the ultrastructural integrity of the cells was well preserved during the incubations (Fig. 2). Analysis of segments incubated in the presence of tannic acid showed that the positive control stimulus, 28 mm K+, stimulated a significant (P < 0.01) increase in the exocytosis of ir-PRL-containing vesicles from both type I and type II lactotrophs (Fig. 3A and 3B). In contrast, E-BSA (Fig. 3B) induced the exocytosis of PRL-containing vesicles only from the type II lactotrophs. E-BSA (0.1 or 10 nm) for 5, 10 or 20 min in vitro caused a significant (P < 0.01) increase in the mean number of exocytotic events released per type II lactotroph (Fig. 2 and Fig. 3B). The proportion of type II lactotrophs in which exocytosis was visible was also increased (0.1 nm E-BSA, 61 ± 3 %; 10 nm 58 ± 4 % vs. control 22 ± 2 %; P < 0.01). However, E-BSA had no observable effect either on the proportion of type I lactotrophs releasing ir-PRL (E-BSA, 0.1 nm, 24 ± 2 %; 10 nm, 23 ± 1 % vs. control, 23 ± 1 %) or on the number of secretory vesicles exocytosed per cell profile (Fig. 3A). In addition, no significant difference from basal controls was observed with either concentration of steroid on the number of exocytotic profiles occurring from gonadotrophs (data not shown). E-BSA (0.1 or 10 nm) also caused a significant (P < 0.01) stimulation of exocytosis of ir-PRL from anterior pituitary segments from female rats in pro-oestrus and dioestrus (Fig. 4).

Figure 2. Electron micrograph showing exocytosis from a type II lactotroph in response to E-BSA.

Figure 2

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Electron micrograph was taken from an anterior pituitary segment treated with E-BSA (10−8m) for 10 min. Section shows immunogold labelling for PRL. Exocytotic profiles are shown by arrows; N, nucleus. Scale bar, 1 μm.

Figure 3. E-BSA rapidly stimulates exocytosis from type II but not type I lactotrophs.

Figure 3

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Effect of E-BSA (10−10 and 10−8m) for 5, 10 or 20 min in vitro on exocytosis from rat anterior pituitary type I (A) and type II (B) lactotrophs. Values are the mean ± s.e.m. (n = 6). **P < 0.01 vs. corresponding basal control (by Mann-Whitney U test). The data are typical of those from three replicate experiments.

Figure 4. The rapid PRL secretory response to E-BSA is not sex specific.

Figure 4

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Effect of E-BSA (10−10 or 10−8m) for 10 min in vitro on the release of ir-PRL either from male or female anterior pituitary segments taken at pro-oestrus or dioestrus. Values are the mean ± s.e.m. (n = 6). The data are typical of those from three replicate experiments. **P < 0.01 vs. corresponding basal control (by ANOVA plus Duncan's multiple range test).

The E-BSA-stimulated exocytosis of PRL from type II lactotrophs was not influenced by pre-treatment of anterior pituitary segments with either the RNA synthesis inhibitor actinomycin D (0.5 μg ml−1; Fig. 5A) or the protein synthesis inhibitor cycloheximide (1 μg ml−1; Fig. 5B). Furthermore, removal of extracellular Ca2+ (Fig. 5C) had no significant effect on the amount of exocytosis from these cells in response to either concentration of E-BSA tested (10−10m or 10−8m). The pure anti-oestrogen ICI 182,780 (1 μm) was also without effect (Fig. 5D).

Figure 5. Effects of actinomycin D, cycloheximide, ICI 182,780 and removal of extracellular Ca2+ on E-BSA-induced PRL exocytosis.

Figure 5

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Effect of E-BSA (10−10 and 10−8m) for 10 min on exocytosis from type II lactotrophs in the presence and absence of actinomycin D, 0.5 μg ml−1(A), cycloheximide, 1 μg ml−1 (B), no extracellular Ca2+ (C), or ICI 182,780, 1 μm (D). □, control,Inline graphic, test treatment. Values represent the mean ± s.e.m. (n = 6). The data are typical of those from three replicate experiments. NS, not significant to control.

To investigate the intracellular mechanism(s) underlying the rapid response to E we determined whether the adenylate cyclase-protein kinase A cascade was activated. The positive control stimulus vasoactive intestinal peptide (VIP; 10−8m) induced the expected significant (P < 0.01) increase in cAMP content of pituitary segments, but E-BSA (10−10-10−7m caused no change in pituitary cAMP (Fig. 6A). Consistent with this, the protein kinase A inhibitor H89 (100 μm) significantly (P < 0.01) blocked the increase in ir-PRL release in response to VIP (10−8m), but did not influence the amount of ir-PRL released in response to E-BSA (10−10-10−8m; Fig. 6B).

Figure 6. The mechanism for E-BSA-induced PRL release does not involve the cAMP-protein kinase A pathway.

Figure 6

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Effect of E-BSA (10−10-10−7m) on intracellular cAMP concentration (A) and on ir-PRL release from anterior pituitary segments (B) in the presence and absence of a protein kinase A inhibitor, H89 (100 μm). □, control; Inline graphic, H89 treated. VIP (10−8m) positive control is also shown. The data are typical of those from three replicate experiments. **P < 0.01 vs. corresponding basal control, †† P < 0.01 significantly different from VIP only. Values represent the mean ± s.e.m. (n = 6).

E-BSA-FITC binding to the surface of collagenase-dispersed anterior pituitary cells

Figure 7A shows that the effect of E-BSA and un-conjugated E on the isolated anterior pituitary cell preparation was similar to that on pituitary segments. Both E-BSA and E (10−12-10−6m) caused significant concentration-dependent increases in the release of ir-PRL within 10 min. Incubation of isolated anterior pituitary cells with E-BSA-FITC (10−8m) in the same conditions resulted in fluorescent labelling of 10 % of the dispersed cells (Fig. 7B). In the majority of labelled cells, E-BSA-FITC labelling was not localised to any particular area of the plasma membrane, but surface labelling was punctate (Fig. 7B). Figure 7C shows that binding of E-BSA-FITC (10−10-10−7m) to the cells was concentration dependent and abolished by co-incubation of cells with unlabelled E in molar excess; incubation of cells with 10−7m BSA-FITC alone resulted in very low amounts of non-specific fluorescence.

Figure 7. Binding of E-BSA-FITC to isolated anterior pituitary cells.

Figure 7

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A, effects of E and E-BSA (10−12-10−6m) for 10 min on the release of ir-PRL into the medium from collagenase-dispersed male rat anterior pituitary cells. Values represent the mean ± s.e.m. (n = 6). The data are typical of those from three replicate experiments. **P < 0.01 vs. corresponding basal control, *P < 0.05 (by ANOVA plus Duncan's multiple range test). B, distribution of E-BSA-FITC binding sites on the surface of collagenase-dispersed rat anterior pituitary cells (arrowed) visualised by confocal microscopy. Scale bar, 50 μm. No binding was detected in cells incubated with BSA-FITC (100 nm) (data not shown). C, binding of E-BSA-FITC to collagenase-dispersed anterior pituitary cells. Concentration-dependent binding of E-BSA-FITC (10−10-10−7m) (expressed as median fluorescence intensity per cell) is shown, which is abolished in the presence of 1 μm free E. Each point represents the mean ± s.e.m. (n = 3). **P < 0.01 vs. corresponding control (by ANOVA and Duncan's multiple range test). The data are typical of those from three replicate experiments.

DISCUSSION

This study demonstrates for the first time that physiological concentrations of E or E bound to BSA can very rapidly stimulate significant release of PRL selectively from type II lactotrophs by a mechanism that appears not to involve classical nuclear receptors. The effects were detected in male and female pituitary tissue and in both in vitro anterior pituitary preparations tested - anterior pituitary segments in which the intimate cell-cell contacts and three-dimensional tissue arrangements are intact, and isolated anterior pituitary cells where these arrangements are lost but diffusion of test ligands to target sites might be expected to be more efficient. These data suggest it is likely that E acts directly on type II lactotrophs and not on adjacent cells to regulate the release of a local intrapituitary regulator of PRL secretion. Neither type I lactotrophs nor gonadotrophs were found to respond to E and this could not be attributed to a lack of effect of tannic acid on these cells because K+-stimulated release of hormone from these cells was readily detectable. The rapid effects of E on PRL release from type II lactotrophs appear to be specific because only this population of cells was affected by exposure to the steroid and the inactive stereoisomer of E, 17α-oestradiol, was without effect. In gonadotrophs, the steroid 3α-hydroxy-4-pregnen-20-one has been shown to rapidly (within minutes) suppress gonadotrophin-releasing hormone (GnRH)-stimulated follicle-stimulating hormone (FSH) release (Wiebe, 1997). We were unable to detect any change in basal LH release from anterior pituitary segments exposed to E or E-BSA for 10 min although other investigators have demonstrated slight stimulatory effects of E on basal and GnRH-induced LH release in isolated anterior pituitary cells when treated with E for longer periods (e.g. 28 h; Liu & Jackson, 1988, 1990). The treatment time tested here would not be expected to be sufficiently long to reveal a similar stimulation of basal release that may be activated by nuclear oestrogen receptor (ER) pathways and any rapid effect on LH release might well be an inhibition or stimulation of GnRH-stimulated release that we did not test.

While the classical ER is thought to be located mostly in the nucleus, oestrogen-binding proteins have also been described within the plasma membrane (Pietras & Szego, 1977; Ramirez et al. 1996; Prevot et al. 1999). We have identified such binding sites on the surface of isolated anterior pituitary cells by use of FACS analysis and the fluorescent ligand E-BSA-FITC to reveal, by confocal microscopy, a patchy distribution of binding sites on the surface of pituitary cells. The E binding is high affinity, concentration dependent and abolished by excess unlabelled E. A single class of E-specific binding sites on pituitary membranes from female rats has previously been reported (Bression et al. 1986) but these binding sites have not been isolated nor structurally or functionally characterised. It is not known whether this binding is restricted to type II lactotrophs, and this possibility is currently under investigation by us. Little is known about the structure of membrane ER. It is not clear whether membrane ERs are derived from the nuclear ‘classical’ oestrogen receptor types or represent a novel member of the oestrogen receptor family. Three intracellular ER isoforms are expressed in the anterior pituitary: ERα, the ERα truncated isoform TERP, and ERβ (Mitchner et al. 1998). It is not known which are expressed by type II lactotrophs but we are currently testing whether some difference in expression profile to type I lactotrophs might explain the selectivity of the rapid E response and/or selective expression of a distinct putative membrane ER. Studies in Chinese hamster ovary cells have shown that expression of a single cDNA encoding either ERα or ERβ gives rise both to membrane and nuclear receptors (Razandi et al. 1999). Furthermore, others who have made use of antibodies raised against a variety of domains in the nuclear ERα can identify the membrane ER in several cell types including GH3/B6 cells (Watson et al. 1999). GH3/B6 cells are a pituitary somatolactotroph tumour cell line which release PRL rapidly in response to E and are widely used as an in vitro model for lactotrophs (Zyzek et al. 1981; Pappas et al. 1994). These data suggest that some membrane ER and nuclear ERα must have a similar amino acid composition and a strong structural similarity. However, in primary pituitary tissue we found the rapid PRL response to E to be insensitive to the pure ER antagonist ICI 182,780 indicating that the effect is not mediated by receptors related to intracellular ER.

Steroids conjugated to BSA have been shown to be impermeant to the plasma membrane and therefore cannot access cytoplasmic receptors. They have been used increasingly as tools to investigate non-genomic, cell surface effects of steroids (Swenson & Sladek, 1997). The PRL-secretagogue effect of E-BSA strongly suggests an effect on membrane E-binding sites. The effects of E conjugated to BSA were indistinguishable from those of non-conjugated E in both in vitro anterior pituitary preparations tested so the possibility that the BSA part of the conjugate is responsible for mediating the effect is unlikely. The effects of both E and E-BSA occurred rapidly, within 5 min, and were independent of DNA transcription or RNA translation and are therefore very unlikely to represent an action via a nuclear receptor. However, others have argued that it is not possible to conclude that any proteins E-BSA interacts with are truly in the plasma membrane. Stevis et al. (1999) demonstrated that commercially available E-BSA has free E associated with it and if associated free E is removed by dialysis E-BSA does not itself bind to ER. Despite this, several laboratories have shown that E-BSA does not activate nuclear ER (Watters et al. 1997; Razandi et al. 2000) and that it is limited to action at the plasma membrane.

Oestrogens have previously been shown to induce rapid increases in intracellular Ca2+ in rat and human oocytes (Morley et al. 1992; Tesarik & Mendoza, 1995), rat and human osteoblasts (Somjen et al. 1997) and rat enterocytes (Picotto et al. 1996). Our experiments using Ca2+-free EBSS have demonstrated extracellular Ca2+ is not required for the rapid PRL response to E and this was also the case for the rapid testosterone-induced effects on type II lactotrophs (Christian et al. 2000). Similarly, we have previously shown that the effect of E on the exocytosis of oxytocin and vasopressin from the dendrites of magnocellular neurosecretory neurones is not affected by the absence of Ca2+ in the bathing medium (Wang et al. 1995). Tse & Tse (2000) have recently demonstrated stimulation of Ca2+-independent exocytosis of LH via a G protein acting on a pool of vesicles or granules distinct from the Ca2+-dependent pool. We are currently investigating the possibility that similar pools may exist in type II lactotrophs that are regulated in a similar manner.

Increases in cAMP, induced via rapid activation of membrane-bound adenylate cyclase in response to oestrogens, are well known (Aronica et al. 1994; Gu & Moss, 1996). cAMP stimulates PRL secretion and it is by this pathway that the peptide VIP acts. Therefore it appeared possible that E-BSA induced PRL release via mobilisation of cAMP. However, no change in intrapituitary cAMP was detected in response to E-BSA and prior exposure to the protein kinase A inhibitor H89 was without effect on E-BSA-induced PRL release, so activation of the cAMP-protein kinase A signalling cascade does not appear to be involved.

These selective, rapid effects of E are very similar to those that we recently reported for testosterone (T) on PRL release from male rat pituitary glands (Christian et al. 2000). We demonstrated the effects of unconjugated testosterone were not significantly different from those of testosterone conjugated to BSA. Therefore it would appear unlikely that the rapid effects of T-BSA are via conversion to E by aromatase in lactotrophs as aromatase activity is largely intracellular. However, there is evidence to indicate that aromatase is expressed at the cell surface in some cell lines (Amarneh & Simpson, 1996) and the possibility of conversion by membrane-bound aromatase in pituitary cells cannot be excluded. Local metabolism of steroids could produce acute changes in steroid milieu in vivo that would not be expected to occur systemically.

E-BSA induced a comparable fold induction of PRL release in anterior pituitary segments from dioestrus and pro-oestrus females as males. Similarly, in osteoblasts and osteocytes rapid E and T effects of similar potency in both male and female cells have been reported (Kousteni et al. 2001). However, in rat distal colonic epithelium E but not T acutely inhibits Cl− secretion in female but not male cells - showing sex-specific actions do exist in some cell types (Condliffe et al. 2001). Physiologically, the rapid effect of E on type II lactotrophs may contribute to the regulation of the reproductive axis. Testosterone and E, by rapidly facilitating PRL release, may contribute to the negative feedback effects of these steroids on gonadotrophin release as stimulation of lactotrophs is known to inhibit gonadotrophins (Lewis et al. 1986; Makino et al. 1987).

In conclusion, we report for the first time rapid effects of physiological concentrations of E to induce PRL secretion from a defined subpopulation of normal pituitary lactotrophs. Previous studies demonstrating rapid effects of E in the pituitary have made use of pituitary-derived GH3 tumour cell lines alone and we have confirmed for the first time that these effects are also characteristic of primary pituitary cells. The effect is not mediated via a membrane adenylate cyclase pathway and is not sex specific. Further studies are now underway to characterise the putative pituitary membrane ER and to determine the signalling cascades activated by E and T in lactotrophs.

Acknowledgments

Research in the authors' laboratory is supported by the Wellcome Trust. We thank the National Hormone and Pituitary Programme (Gaithersberg, MD, USA) for the generous supply of reagents. We thank Lynne Scott, Sarah Rodgers, Derek Hardiman and Bob Wickens for expert technical help.

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