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Department of Obstetrics and Gynaecology, University of Luebeck, Luebeck, Germany
1 Department of Obstetrics and Gynaecology, University of Regensburg, Regensburg, Germany
(Requests for offprints should be addressed to J M Weiss who is now at Department of Obstetrics and Gynaecology, University of Ulm, Prittwitzstr 43, 89075, Ulm, Germany; Email: jmweiss1{at}hotmail.com)
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Abstract |
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T3-1 cells, derived from a murine immortalized gonadotroph cell line, were treated with 100 pM estradiol for 48 h. LH secretion of anterior pituitary cells, additionally stimulated with eight consecutive pulses of 1 nM GnRH for 15 min at an interval of 1 h, was determined by RIA. Gene expression was measured by quantitative RT-PCR and protein expression by immunoblotting. Additionally, quantitative RT-PCR was performed in single rat gonadotrophs to ascribe effects exclusively to intact gonadotrophs. Pulsatile GnRH enhanced the mRNA expression of SNAP-25 and munc-18 in accordance with the LH secretory response with the greatest increase at the third pulse of GnRH. Estradiol treatment further increased GnRH-induced LH secretion at all GnRH pulses. SNAP-25 gene expression was significantly decreased at the fifth GnRH pulse and unaffected at basal after 48 h of estradiol treatment. In contrast, munc-18 mRNA levels were not significantly affected by estradiol at different GnRH-pulses in mixed anterior pituitary cells, whereas munc-18 gene expression was significantly increased at basal. In T3-1 cells and single gonadotrophs, long-term estradiol treatment significantly reduced SNAP-25 protein and gene expression. In contrast, the protein and gene expression of munc-18 was significantly enhanced in both T3-1 cells and single gonadotrophs. In conclusion, munc-18 and SNAP-25 were oppositionally influenced by estradiol. The results suggest that estradiol modulates the expression of exocytotic proteins in gonadotrophs and thus affects LH secretion.
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Introduction |
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Exocytosis, the fusion of secretory vesicles with the plasma membrane, is the final step of signal transduction in neuroendocrine cells such as gonadotrophs to release hormones. Before the secretory vesicles, processed at the trans-Golgi region of the cells, fuse with the plasma membrane, they undergo several modifications such as translocation (transport close to the plasma membrane), docking (attachment to the plasma membrane), and priming (maturation that makes them ready for the final Ca2+-triggered step; Stojilkovic 2005). Functional studies have provided intriguing details into how SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins interact with each other to generate the fusion of lipid bilayers. Regulated exocytosis is controlled and mediated by SNARE proteins. They play a crucial role in the intracellular membrane fusion (Rettig & Neher 2002). SNARE proteins are either residing in the vesicle membrane (v-SNAREs like synaptobrevin=vesicle-associated membrane protein (VAMP) and synaptotag-min; Borisovska et al. 2005) or in the plasma membrane (syntaxin and synaptosomal-associated protein of 25 kDa (SNAP-25); Sorensen et al. 2002). SNAREs bind to each other by forming a very stable four-stranded coiled–coil core complex, constituted by one coil each from syntaxin and VAMP, and two coils from SNAP-25 (Chen & Scheller 2001).
Recently, many proteins have been identified that participate in the exocytosis of the pituitary gland (Thomas et al. 1998). Their exact function, however, remains unclear. SNAP-25, originally identified at neuronal synapses, was found in pituitaries (Aguado 1996, Jacobsson & Meister 1996, Kolk et al. 2004). Since the antisense oligonucleotide of SNAP-25 inhibited TRH-induced prolactin release, SNAP-25 is essential for the release of prolactin in pituitary cell lines (Masumoto et al. 1997). Immunoreactivity of SNAP-25 is enhanced in human prolactinomas as well (Majo et al. 1997). Ca2+-induced LH secretion was significantly decreased after treatment with SNAP-25 antibodies. Furthermore, incubation with GnRH decreased SNAP-25 expression in western blots (Quintanar et al. 2004). Taken together, these results show that SNAP-25 is essential for the secretion of pituitary cells.
Munc-18 seems to be involved in all eukaryotic vesicle trafficking and fusion (Jahn 2000). It lacks membrane targeting domains and is located in the cytoplasm. Munc-18 binds tightly to syntaxin which blocks its interaction with SNAP-25. The dissociation of munc-18 allows the formation of a complex between syntaxin and SNAP-25. VAMP binds to syntaxin (Sudhof 1995). Plasma membrane and vesicle membrane are now capable of fusing and hormones are released. Munc-18 null mutant mice have no synaptic activity in neurons (Verhage et al. 2000). In the absence of munc-18, Ca2+-triggered exocytosis of neuroendocrine cells is minimized, whereas the overexpression of munc-18 increased exocytosis (Voets et al. 2001). These facts demonstrate the importance of munc-18 in exocytosis. Although many details on munc-18 function in different cell types are known, the function of munc-18 in gonadotrophs and its interaction with estradiol remain unclear.
To shed light on the actions of estradiol, we examined for the first time the effects of estradiol on the exocytotic proteins SNAP-25 and munc-18 in female gonadotrophs. We concentrated on these proteins, because both are critical components for the exocytosis in pituitaries.
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Materials and methods |
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Pituitary cells were obtained from ovarectomized adult (200–250 g) female Sprague Dawley rats (Charles River, Kirchborchen, Germany). Single-cell suspensions were prepared by controlled trypsinization as described previously (Ortmann et al. 1990). Briefly, pituitary glands were collected after decapitation and neural lobe was separated. Anterior pituitaries were cut into small pieces. After digestion with 0.5% trypsin, they were treated with DNase and 0.1% trypsin inhibitor. After gentle pipetting, dispersed cells were separated by centrifugation. Cell viability was determined using the Trypan Blue exclusion method (usually >95%). Cells were incubated in medium 199 (Biochrom KG, Berlin, Germany) with Hank’s salts and L-glutamine, supplemented with 1.4 g/l sodium bicarbonate, 10 µg/ml streptomycin, 100 U/ml penicillin, and 10% horse serum, pretreated with 2% charcoal (Norit A) and 0.2% Dextran T 70 (Pharmacia, Uppsala, Sweden) to eliminate steroids. To allow cell attachment, cultures were incubated for 48 h in humidified incubators at 37 °C in an atmosphere of 5% CO2 and 95% air.
Estradiol and GnRH treatment
After 48 h, cells were treated for another 48 h period with 100 pM estradiol in serum-free media. Estradiol was dissolved in ethanol and the final concentration of ethanol in the culture media was 0.2%. Controls received media containing 0.2% ethanol alone (vehicle). After 48 h treatment with estradiol, mixed anterior pituitary cells were washed and stimulated with eight consecutive GnRH pulses of 1 nM GnRH for 15 min at an interval of 1 h in a static culture system. GnRH pulses were administered by a pipette. After each GnRH pulse, media were removed and replaced by fresh media and kept for 45 min. Media for LH measurements were taken before and after each pulse. Only gonadotrophs react to their physiological agonistic stimulus GnRH. Therefore, we assume that the described effects could be ascribed to gonadotrophs.
LH measurement
Media were collected and analyzed for their hormone content by RIA, using the reference preparation RP-3 from the National Pituitary Agency and Dr Parlow (Harbor-University of California-Los Angeles Medical Center, Torrance, CA, USA). The intra- and inter-assay variations were below 8%.
T3-1 cell culture
Since mixed populations of pituitary cells contain only between 5 and 10% of gonadotrophs we employed T3-1 cells, an immortalized gonadotroph-derived cell line was kindly provided by Dr P L Mellon (San Diego, CA, USA), to save rats and to attribute effects to gonadotrophs. Cells were cultured on six-well dishes (500 000 cells/well) in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Life Technologies, Karlsruhe, Germany) and 100 mg/l gentamycin (Biochrom KG). To allow cell attachment, cells were kept for 36 h in humidified incubators at 37 °C in an atmosphere of 5% CO2 and 95% air. At 70–80% confluency, cells were harvested after detachment with accutase (GiBCO, Wiesbaden, Germany) and treated with 100 pM estradiol in serum-free medium for 48 h.
Immunoblotting
T3-1 cells were lysed in RIPA buffer (containing 1 mM PMSF) for 1 h at 4 °C. Nuclei were removed by centrifugation. Protein concentration was determined according to the method of Bradford (Bio-Rad). Proteins were separated by SDS-polyacrylamide gels (SDS-PAGE), transferred on PVDF membranes and analyzed by immunoblotting as follows: the membranes were blocked in a solution of 5% milkpowder and 3% BSA for 1 h. Primary antibody was diluted in blocking buffer (SNAP-25, 1:3000; munc-18, 1:500) and incubated with the membrane overnight. After washing, peroxidase-conjugated secondary antibody (IgG 1:2000) was added for 1 h. Detection of peroxidase activity was performed with enhanced chemiluminescence hyperfilm and kits (Amersham, Freiburg, Germany).
Antibodies used
SNAP-25a (1:3000), BabCO, Berkley, CA, USA; munc-18-2 (1:500), Becton Dickinson, Heidelberg, Germany; ß-actin (1:7500), Sigma; IgG (1:2000), Amersham, Braunschweig, Germany.
Quantitative RT-PCR
RNA was isolated from rat pituitary cells and T3-1 cells using TRI reagent (Sigma) according to the manufacturer’s instructions. Total RNA (0.5–2 µg) was reverse transcribed using SuperScript II reverse transcriptase (Invitrogen Life Technologies, Inc.) and 10 pM poly-(deoxythymidine) primer in a 10 µl total volume reaction. The first cDNA synthesis reaction was heated to 55 °C for 5 min to inactivate the reverse transcriptase and then diluted with 10 mM Tris–HCl (pH 7.5) to a final volume of 100–300 µl. An aliquot (4 µl) of cDNA was used for quantification of gene expression. We used DNase digestion after RNeasy silica membrane-based RNA purification to remove any trace of DNA. Specific primers for LH, SNAP-25, and munc-18 were used (Tables 1
and 2). All primers were designed over introns based on the exon/intron gene structures. MgCl2 concentrations were optimized. A typical reaction involved an initial denaturation at 95 °C for 2 min, followed by 40 cycles of 50 °C for 1 min, and elongation at 60 °C for 30 s. A standard melting curve cycle was used to check the quality of amplification and to confirm the absence of primer dimer formation. To ensure accuracy, each reaction was repeated 2–5 times per sample. The mean value of the repeat was used for each gene per sample to calculate the ratio between the mean value of the target gene and the internal control gene. The relative level of each gene was expressed by the ratio of the number of transcripts of the gene to an internal control gene which was ß-actin. The data were analyzed using the comparative threshold cycle method, because the efficiency of the target amplification was approximately equal to that of the ß-actin amplification.
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We have established a quantitative reverse transcriptase-PCR (RT-PCR) approach for the analysis of RNA transcript levels in single rat pituitary cells. This approach allowed us to trace back the effects exclusively to intact rat gonadotrophs. Primary pituitary cell cultures were dispersed in 60 mm tissue culture plates at a density of 1x104 cells and incubated as described before. Cells were detached with accutase (PAA-Laboratories GmbH Catalogue no. L11-007, Vienna, Austria). The pellet was washed twice in cold (4 °C) Dulbecco’s PBS (without Mg2+ and Ca2+) and diluted in 1 ml 10% polyvinylpyrrolidone (PVP; Sigma P-5288) to ease cell collection in viscous medium.
A drop of the cell mixture was observed under an inverted microscope. Single cells were isolated with a micromanipulator fitted with a pulled microcapillary. A single cell was expelled into a PCR tube filled with 10 µl ice-cold 1xPCR buffer and frozen immediately in –80 °C overnight. First-strand cDNA synthesis was performed using standard protocol with SuperScript II Reverse Transcriptase (Invitrogen). The PCR program consisted of the following cycles: one cycle (2 min at 50 °C and 2 min at 95 °C) followed by 50 cycles (95 °C for 15 s, 59 °C for 15 s, and 72 °C for 20 s.).
All primer pairs span at least one intron so that the PCR product could be distinguished by size from contaminating genomic DNA. All primer pairs were designed to anneal at 59 °C optimally. Primers are listed in Table 3. Real-time PCR was performed on a DNA Engine Opticon 2 System (MJ Research, Waltham, USA) in a volume of 25 µl including 12.5 µl 2xPlatinum SYBR Green qPCR SuperMix (Invitrogen), 0.4 µM primer (final concentration) and 2 µl template cDNA. All components without cDNA, but DEPC water instead served as negative control. To ensure ‘hot start conditions’, the Platinum Taq DNA polymerase was complexed with a proprietary antibody that blocks polymerase activity at ambient temperatures. Activity is restored after a denaturation step in PCR cycling, providing an automatic hot start in PCR for increased sensitivity, specificity, and yield. PCRs were optimized for magnesium concentration, primer concentration, and annealing temperature. PCR products were verified with both melting curve analysis and DNA gel electrophoresis. Gonadotrophs were identified by simultaneous detection of LH-ß, which is unique for gonadotrophs.
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Unknown amounts of molecules were determined from gene-specific standard curves using the Opticon Monitor Analysis Software (MJ Research). The cDNA standards were prepared from purified PCR products (NucleoSpin Extract II, Machery-Nagel, Düren, Germany). The purified PCR products were analyzed and quantified photometrically at 260 nm. Quantified DNA was diluted from 107 to 102 molecules (standard curve).
Statistical analysis
Data obtained from 3–4 experiments run in triplicate were pooled, and all the results are expressed as percentage (%) of control (vehicle-treated), set to 100%. P values <0.05 were considered as statistically significant. Statistical differences between each experimental and the control group were analyzed using two-way ANOVA repeated measures and Bonferroni post-test, one-way ANOVA with Dunnett’s post-test or unpaired t-test respectively.
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Results |
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LH secretion was highest and significantly higher than control (from 4 up to 18 ng/ml, P<0.01) at the fourth GnRH pulse. Estradiol further increased the LH response of mixed anterior pituitary cells to GnRH pulses with the highest LH secretion of 22 ng/ml (P<0.01) at the third GnRH pulse (Fig. 1A).
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After 48 h of estradiol treatment, the gene expression of SNAP-25 was diminished, whereas munc-18 mRNA levels were significantly enhanced (P=0.012; basal in Fig. 2B).
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Presence of SNAP-25 and munc-18 in T3-1 cells
Since it was not known whether SNAP-25 and munc-18 are present in T3-1 cells, we performed immunoblots to identify these proteins in our cell line. We could demonstrate the presence of SNAP-25 and munc-18 in our T3-1 cells (Fig. 3).
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T3-1 cells
Figure 4A shows a significant decrease (–22%) in SNAP-25 gene expression by treatment with 100 pM estradiol for 48 h (P<0.01). On the contrary, the gene expression of munc-18 was significantly increased (+ 33%; P<0.05) by 100 pM estradiol for 48 h (Fig. 4B). Incubation with 100 pM estradiol for only 24 h had no effect on the gene expression of neither SNAP-25 nor munc-18 (data not shown).
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T3-1 cells
The data on the protein expression of the exocytotic proteins are in keeping with the results on the gene expression. Again, long-term treatment with 100 pM estradiol significantly decreased SNAP-25 protein expression by 34% (P<0.0001, Fig. 5A), whereas the expression of munc-18 was significantly enhanced by 46% (P<0.01, Fig. 5B). The 24 h incubation of the cells with 100 pM estradiol had no effect on SNAP-25 and munc-18 protein expression (data not shown).
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To confirm our previous results, we performed experiments in single cells of rat pituitary gonadotrophs. Mixed populations of anterior pituitary cells contain around 10% gonadotrophs in intact rats and up to 30% in ovariectomized rats. Therefore, it is not possible to trace back effects exclusively to gonadotrophs in mixed anterior pituitary cell cultures. This was the reason why we here used immortalized murine-derived T3-1 tumor cells consisting only of gonadotrophs. The normal secretion of the complete LH in this cell line is disturbed, however. We cannot exclude the possibility that this disturbed secretion is due to a defect in the exocytotic machinery. To solve that dilemma, we used single rat pituitary gonadotrophs. To identify gonadotrophs, we performed single-cell quantitative RT-PCR simultaneously always for LH-ß and either SNAP-25 ormunc-18.
Our results in single rat gonadotrophs were congruent to the results gained in T3-1 cells. SNAP-25 gene expression (Fig. 6A) was significantly decreased by 87% (P=0.014) and munc-18 gene expression (Fig. 6B) was significantly increased (+181%) in single gonadotrophs after long-term (48 h) estradiol treatment with 100 pM (P<0.01). Figure 6C shows a representative gel with the PCR products for LH-ß, munc-18, SNAP-25, and ß-actin.
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Discussion |
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Only a few other investigators have tested the effects of estradiol on exocytotic proteins and the results have been conflicting. Jacobsson et al.(1998) demonstrated that estradiol in vivo suppressed SNAP-25 gene expression in anterior lobes of pituitaries. Rats were treated for 21 days with estradiol capsules. They, however, found no influence of estradiol on other exocytotic proteins like VAMP-2, Hrs-2 and munc-18. In keeping with that line, a lower expression of SNAP-25 protein in estrogen-induced tumors of the pituitary gland in Fisher 344 rats was detected (Majo et al. 1999). In contrast, others concluded that the expression of synaptic proteins including SNAP-25 and munc-18 in ewes is not changed in the anterior pituitary at the time when LH secretion is maximal, i.e. after estradiol treatment (Thomas et al. 1998). Estradiol treatment of GH4C1 rat pituitary cells even increased SNAP-25 levels about 1.5-fold (Lee et al. 2000).
These results are in part contradictory to our findings. The differences might be due to different species and different cell lines used. These data suggest that SNAP-25 regulation is not the same in GH4C1 cells, a prolactin-secreting cell line, and in ewes as it is in the normal rodent pituitary gland. Since no investigator examined the effects of estradiol particularly on gonadotrophs but on the entire anterior lobe of the pituitary, the differences in estradiol regulation might be due to the dominance of other compartments as well. The pituitary gland consists of several hormone-secreting cell types with only a small fraction of gonadotrophs (5–10%). The literature to date deals either with the anterior pituitary on the whole, prolactin secreting cells, or tumors. Reviewing these articles concerning the effects of estradiol on SNAP-25, one may speculate that estradiol treatment decreased SNAP-25 in gonadotrophs, whereas in prolactin-secreting cells, SNAP-25 is increased after estradiol treatment. It fits well that human prolactinomas express more SNAP-25 than normal glands (Majo et al. 1997). That points at a different regulation of exocytosis in gonadotrophs and lactotrophs. Further studies on the different regulation of SNAP-25 mediated exocytosis in lacotrophs and gonadotrophs are warranted.
We aimed to delineate, how our findings fit into the current concepts of exocytosis and its regulation. Treatment of permeabilized male rat gonadotrophs with SNAP-25 antibodies diminished calcium-induced LH secretion, but did not influence basal LH secretion. Treatment with 100 nM GnRH, either in vitro for 4 or 72 h or in vivo for 5 days twice a day, decreased SNAP-25 protein expression (Quintanar et al. 2004). This is in striking contrast to our results. We found a profound increase in both SNAP-25 and munc-18 gene expression after treatment with GnRH pulses. These differences might well be due to a very different experimental setting: we used female, not male rats and we measured gene, not protein expression in vitro. Furthermore, we applied 1 nM GnRH for 15 min at 1 h intervals. This procedure is well established to mimic the physiological situation and has been used by other groups (Turgeon & Waring 1999). Furthermore, we could demonstrate a significant decrease of SNAP-25 in GnRH-stimulated gonadotrophs by estradiol. On the other hand, the expression of munc-18 was significantly increased in non-agonist stimulated gonadotrophs treated with estradiol. The effects of estradiol on GnRH-stimulated cells were not that distinctive, since GnRH pulses alone led to a higher increase in munc-18 than in SNAP-25 expression. This vigorous rise might mask the additional effects of estradiol.
In our experiments, the LH secretory pattern in response to the agonistic stimulus GnRH is congruent to the response of SNAP-25 and munc-18 to GnRH with the maximal increase at the third pulse of GnRH. It is therefore conceivable that GnRH-induced LH secretion is associated with corresponding changes in the exocytotic proteins SNAP-25 and munc-18. As yet another contrast, here we gained evidence that the estradiol-induced increase in basal LH release leads to a reduced SNAP-25 expression. This indicates that basal LH release is not independent of SNAP-25. This may be explained by the fact that the enhanced LH secretion precipitates a depletion of SNAP-25. The function of SNAP-25 in the modulation of LH secretion by estradiol remains not fully elucidated. It is not clear what happens to SNAP-25 after membrane merger. Walch-Solimena et al.(1995) found evidence that SNAP-25 is at least partially recycled at the synaptic cleft of neurons. Pituitary cells are able to recycle components of the exocytotic machinery (Ferraro et al. 2005). Massive stimulation of prolactin release in vitro leads to a depletion of the cellular levels of prolactin up to 80%. Prolactin, for example, remains connected with the cell membrane after exocytosis and is ready for recycling and reuse. Others components like PAM-1, a membrane enzyme participating in the final steps of the biosynthesis of hormones, can be functionally recycled. There are no reports that clarify the fate of SNAP-25 after exocytosis in the pituitary gland occurred.
Korteweg et al.(2005) tested hormone levels in the anterior pituitaries of munc-18 null mutant mice. They found very low levels of circulating hormones and enhanced intracellular hormones. This underlines the importance of munc-18 for the secretion of hormones from anterior pituitary cells. They suggested that munc-18 is important for the docking of vesicles, because large dense-core vesicles assembled close to, but not directly at the terminal membrane (Korteweg et al. 2005). This is in contrast to the findings of Verhage et al.(2000) that synaptic vesicles are distributed normally in synapses of munc-18 null mutant mice. This shows that the transfer of results derived from experiments in neuronal synapses to gonadotrophs is restricted.
Since it has been suggested that munc-18 hampers the ability of syntaxin to form SNARE complexes, munc-18 might be a negative regulator of exocytosis and an overexpression of munc-18 should reduce hormone release. This could be true if the concentration of munc-18 and syntaxin is equal, because they interact with a 1:1 stoichiometry (Misura et al. 2000). Schütz et al.(2005) found that syntaxin 1 expression is 20 times higher than munc-18 expression. In accordance with that, Toonen et al.(2005) argued against the possibility that munc-18 acts as a simple chaperone for syntaxin. Munc-18 has recently been identified as a positive regulator of exocytosis in PC12 cells (Schütz et al. 2005). They proposed a dual role for munc-18 switching between syntaxin binding and mint1 binding. Overexpression of mint1 inhibits exocytosis in PC12 cells. Binding of munc-18 to mint1 prevents mint1’s negative effect on exocytosis. The first target of munc-18 is syntaxin, because of the high-affinity of their binding. Higher cytosolic munc-18 however leads to a higher binding to mint1 and consecutively to an increase in hormone release. An overexpression of the cytoplasmic region of syntaxin thus leads to an inhibition of secretion from PC12 cells (Dulubova et al. 1999). Furthermore, munc-18 is required for the stability of syntaxin. Munc-18 null mutant mice expressed lower levels of syntaxin 1, but other proteins of the exocytotic machinery remained unaffected. Even in the absence of munc-18, syntaxin 1 can be targeted properly (Toonen et al. 2005). The lack of munc-18 may lead to an uncontrolled binding of syntaxin 1 to other, non-cognate, exocytotic proteins, which in turn could hamper cell viability. Toonen et al.(2005) did not find evidence for detrimental effects of the promiscuous syntaxin 1 binding on cell viability. The SNARE complexes formed might not be accessible for normal exocytosis, since munc-18 null-mutant mice have no transmitter release (Verhage et al. 2000). Munc-18 may have a crucial and autonomous function in conjunction with syntaxin 1 in exocytosis (Toonen et al. 2005).
The concepts of Schütz et al.(2005) and Toonen et al.(2005) are in keeping with our results. Estradiol treatment leads to an up-regulation of munc-18 and consecutively to increased LH secretion. Therefore, munc-18 is a crucial component of the modulation of LH secretion by estradiol.
Other concepts on how estradiol could positively regulate LH secretion are well established. Estradiol modulates GnRH receptor number (Gregg et al. 1990) and its gene expression (Bauer-Dantoin et al. 1993). Moreover, estradiol could affect components of the GnRH signal transduction such as calcium signaling and inositol phosphates (Ortmann et al. 1992, 1995).
However, those concepts could not fully explain the positive modulatory effect of estradiol on basal and agonist induced LH secretion. Therefore, our findings add novel evidence to the hypothesis that estradiol affects even distant mechanisms of signal transduction like exocytosis.
In conclusion, long-term estradiol treatment negatively regulates the exocytotic protein SNAP-25 and positively affects the regulation of munc-18 in female gonadotrophs. The up-regulation of munc-18 is another mechanism by which estradiol might enhance LH secretion from female gonadotrophs.
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Acknowledgements |
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T3-1 cells.
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Funding |
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This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG Or 52/8-1). There is no conflict of interest that would prejudice our impartiality.
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References |
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Received in final form 3 November 2006
Accepted 30 November 2006
Made available online as an Accepted Preprint 13 December 2006
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P<0.05 compared with control (unpaired t-test).
