Rapid Down-regulation of the Type I Inositol 1,4,5-Trisphosphate Receptor and Desensitization of Gonadotropin-releasing Hormone-mediated Ca2+ Responses in alpha T3-1 Gonadotropes*

Gary B. WillarsDagger §, Jean E. RoyallDagger , Stefan R. NahorskiDagger , Faraj El-Gehani, Helen Everest, and Craig A. McArdle

From the Dagger  Department of Cell Physiology and Pharmacology, University of Leicester, Medical Sciences Building, P. O. Box 138, University Road, Leicester LE1 9HN, United Kingdom and the  University Neuroendocrine Unit, Department of Medicine, University of Bristol, Marlborough Street, Bristol BS2 8HW, United Kingdom

Received for publication, September 29, 2000, and in revised form, November 6, 2000



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Despite no evidence for desensitization of phospholipase C-coupled gonadotropin-releasing hormone (GnRH) receptors, we previously reported marked suppression of GnRH-mediated Ca2+ responses in alpha T3-1 cells by pre-exposure to GnRH. This suppression could not be accounted for solely by reduced inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) responses, thereby implicating uncoupling of Ins(1,4,5)P3 production and Ca2+ mobilization (McArdle, C. A., Willars, G. B., Fowkes, R. C., Nahorski, S. R., Davidson, J. S., and Forrest-Owen, W. (1996) J. Biol. Chem. 271, 23711-23717). In the current study we demonstrate that GnRH causes a homologous and heterologous desensitization of Ca2+ signaling in alpha T3-1 cells that is coincident with a rapid (t1/2 < 20 min), marked, and functionally relevant loss of type I Ins(1,4,5)P3 receptor immunoreactivity and binding. Furthermore, using an alpha T3-1 cell line expressing recombinant muscarinic M3 receptors we show that the unique resistance of the GnRH receptor to rapid desensitization contributes to a fast, profound, and sustained loss of Ins(1,4,5)P3 receptor immunoreactivity. These data highlight a potential role for rapid Ins(1,4,5)P3 receptor down-regulation in homologous and heterologous desensitization and in particular suggest that this mechanism may contribute to the suppression of the reproductive system that is exploited in the major clinical applications of GnRH analogues.



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ABSTRACT
INTRODUCTION
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The decapeptide gonadotropin-releasing hormone (GnRH)1 is released from the hypothalamus of mammals in a pulsatile manner to regulate the exocytotic release of luteinizing hormone and follicle-stimulating hormone from pituitary gonadotropes. These hormones are central to the regulation of gonadal steroidogenesis and gamete maturation, and GnRH therefore plays a vital role in the control of vertebrate reproduction. GnRH acts on pituitary gonadotropes through a G-protein-coupled receptor that regulates phospholipase C (PLC) via G-proteins of the Galpha q/11 family (1). GnRH-mediated activation of PLC results in the hydrolysis of phosphatidylinositol 4,5-bisphosphate to generate both inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) and diacylglycerol. These second messengers are able to mobilize Ca2+ from intracellular stores and activate protein kinase C, respectively (2), thereby propagating a signaling cascade that accounts for the biological effects of GnRH.

In contrast to nearly all other known PLC-coupled G-protein-coupled receptors (GPCRs), the GnRH receptor does not undergo rapid (seconds to minutes) desensitization following exposure to agonist (3-8). Recent evidence has suggested that this lack of acute regulation is related to the lack of a C-terminal tail and the absence, therefore, of appropriate regulatory phospho-acceptor sites (8, 9). From a functional perspective, the resistance of the GnRH receptor to rapid desensitization may serve to maintain cellular sensitivity and responsiveness during events such as the pre-ovulatory gonadotropin hormone surge and allow the frequency-encoded pattern of the hypothalamic pulsatile GnRH release (10, 11) to be faithfully maintained at the level of the pituitary gonadotropes.

Despite the lack of acute regulation of the GnRH receptor, sustained exposure to GnRH is able to reduce GnRH-stimulated gonadotropin secretion, and this form of desensitization underlies the suppression of the reproductive system that is exploited in the major clinical applications of GnRH analogues (12). Given the importance of cytosolic Ca2+ elevation in the mediation of GnRH-stimulated gonadotropin secretion (1, 13-15), we have previously explored the potential desensitization of this component of the GnRH receptor-mediated signaling pathway in an immortalized mouse pituitary cell line (alpha T3-1). Despite no evidence for rapid desensitization of the GnRH receptor in these cells, pre-exposure to GnRH can cause a marked suppression of subsequent GnRH-mediated elevations of [Ca2+]i. Both the spike phase of the response (which reflects Ins(1,4,5)P3-dependent mobilization of intracellular Ca2+) and the sustained phase of the response (which is dependent upon Ca2+ entry across the plasma membrane through voltage-operated Ca2+ channels) were attenuated by GnRH pretreatment (4, 5).

Desensitization of voltage-operated Ca2+ channels may account for the desensitization of the plateau phase of the GnRH-mediated response in alpha T3-1 cells (4), but the mechanism underlying attenuated mobilization of Ca2+ from intracellular stores is unclear. Although pre-exposure to GnRH reduces both the number of plasma membrane GnRH receptors and the ability of GnRH to generate Ins(1,4,5)P3, these effects are insufficient to account for the reduced release of intracellular Ca2+ (5). Indeed, this desensitization is heterologous and therefore most probably reflects post-receptor modification(s). Because desensitization of GnRH-stimulated Ca2+ mobilization from intracellular stores cannot be attributed to attenuation of Ins(1,4,5)P3 generation or depletion of hormone-mobilizable intracellular Ca2+ pools, it appears to reflect a reduction in the efficiency with which Ins(1,4,5)P3 mobilizes Ca2+ from intracellular stores (5). There is now accumulating evidence that GPCR-mediated activation of PLC causes down-regulation of Ins(1,4,5)P3 receptors (16-23), most probably through proteolysis that is initiated as a consequence of activation by Ins(1,4,5)P3 (20, 22, 23). However, this down-regulation often requires several hours of agonist stimulation (17, 23), whereas GnRH desensitizes Ca2+ responses in alpha T3-1 cells with pre-stimulation periods of 10-30 min (5). Thus, if Ins(1,4,5)P3 receptor down-regulation underlies desensitization of GnRH-stimulated Ca2+ mobilization, it would have to occur unusually rapidly in alpha T3-1 cells. The current study was therefore undertaken to establish whether GnRH is able to cause Ins(1,4,5)P3 receptor down-regulation and whether any such effect underlies desensitization of Ca2+ mobilization in these cells. alpha T3-1 cells stably transfected with recombinant human M3 muscarinic receptors (7) were used in these experiments to enable comparison of responses to PLC-activating GPCRs that do (M3) and do not (GnRH) show rapid homologous desensitization (7).


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Materials and Cell Culture-- Reagents of analytical grade were obtained from suppliers listed previously (5, 24-26), unless stated, or alternatively from Sigma. Antibodies against PLC isoforms and Galpha q/11 were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), with the exception of PLCdelta 1, which was from Upstate Biotechnology (Lake Placid, NY). The alpha T3-1 gonadotrope cell line was originally a gift from Dr. P. Mellon, University of California, San Diego, CA, and in the current study we used a cell line (alpha T3-1/M3) derived from this, which also expresses the recombinant human muscarinic M3 receptor. Like the endogenously expressed GnRH receptor, this GPCR also couples to the activation of PLC in this cell line (7), and we have demonstrated that muscarinic M3 receptors are subject to rapid but partial desensitization, whereas the endogenously expressed GnRH receptors show no evidence of such regulation (7). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 50 IU/ml penicillin, 50 µg/ml streptomycin, 2 mM glutamine, and 10% (v/v) fetal calf serum. Cultures were maintained at 37 °C in 5% CO2, humidified air and passaged weekly. For experiments, cells were harvested with 10 mM HEPES, 154 mM NaCl, 0.54 mM EDTA (pH 7.4) and re-seeded for use 1-2 days later. Cells were always maintained, and the experimental manipulations were always performed, at 37 °C unless stated otherwise.

Dynamic Video Imaging of Cytosolic Ca2+-- Video imaging of fura-2-loaded cells was performed as described previously (5). Briefly, cells grown on glass coverslips were loaded with the acetoxymethyl ester of fura-2 (2 µM) for 30 min at 37 °C in 1 ml of buffer (pH 7.4, composition (mM): NaCl 127, CaCl2 1.8, KCl 5, MgCl2 2, NaH2PO4 0.5, NaHCO3 5, glucose 10, HEPES 10 with 0.1% bovine serum albumin). Cells were then washed several times and placed within a heated (37 °C) perfusion stage of a Nikon Diaphot inverted microscope. Image capture was performed using MagiCal hardware following alternate excitation at 340 and 380 nm with emission recorded at 510 nm. Values were averaged from 16 or 32 video frames, and background fluorescence was subtracted prior to ratioing. The ratio of fluorescence at 340 and 380 nm was calculated on a pixel-by-pixel basis using maximum and minimum values defined by treatment with 5 µM ionomycin in medium with either 10 mM CaCl2 or 10 mM EGTA and assuming a dissociation constant of 225 nM for fura-2 and Ca2+ at 37 °C, as described (14).

Western Blotting-- Cells were grown to confluence in 6-well multiwell dishes. Medium was removed, and the cells were washed (2 × 1 ml) with medium containing 0.1% bovine serum albumin and incubated in a further 1 ml. GnRH was then added at the appropriate concentration, and the cells were incubated at 37 °C in 5% CO2, humidified air. For immuno-detection of Ins(1,4,5)P3 receptors, medium was aspirated after the required time, the cell monolayer was washed once with ice-cold Krebs/HEPES (7), and 1 ml of ice-cold TE buffer (10 mM Tris, 10 mM EDTA, pH 7.4) was added. Cells were left for 5 min on ice and then scraped from the surface of the plate. Following trituration through a fine gauge needle the resulting suspension was centrifuged (13,000 × g, 4 °C, 15 min). The supernatant was then aspirated, and the pellet was resuspended in 50 µl of TE buffer. An equal volume of sample buffer (100 mM Tris-HCl, 2% SDS, 10% glycerol, 0.1% bromphenol blue, and 200 mM dithiothreitol) was then added, and the samples were boiled for 3 min. Proteins were resolved by 5% SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed using isotype-specific Ins(1,4,5)P3 receptor antibodies. Immunoreactive bands were detected with ECL reagents and exposure to Hyperfilm-ECL (Amersham Pharmacia Biotech). Where required, densitometric analysis of the resulting bands was performed using a Bio-Rad GS-670 imaging densitometer with Molecular Analyst version 1.2 software. For immuno-detection of other proteins (Galpha q/11 and PLC isoforms), Western blotting was carried out as above with the exception that the cell monolayers were solubilized in 200 µl of solubilization buffer (10 mM Tris, 10 mM EDTA, 500 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 100 µg/ml iodoacetamide, 100 µg/ml benzamidine) for 30 min on ice before being processed as described above. Details and use of the polyclonal antibody against the type I Ins(1,4,5)P3 receptor and of the monoclonal antibodies against the type II and type III Ins(1,4,5)P3 receptors have been described previously (26). Other antibodies were at dilutions according to the instructions of the suppliers.

Determination of Ins(1,4,5)P3 Receptor Density by Binding of [3H]Ins(1,4,5)P3-- Ins(1,4,5)P3 receptor binding was determined in membranes of alpha T3-1 cells as described (16). Incubations were for 45 min at 4 °C in 200 µl of incubation buffer with 2-10 µg of membrane protein, 10 nCi of [3H]Ins(1,4,5)P3, and 0 or 10-9-10-5 M unlabeled Ins(1,4,5)P3. The incubations were terminated by centrifugation and removal of supernatants by aspiration. Pellets were then solubilized in NaOH and transferred to scintillant for beta -counting.

Ins(1,4,5)P3-mediated Release of 45Ca2+ from Intracellular Stores of Permeabilized Cells-- 45Ca2+ release assays were performed in cytosol-like buffer (composition (mM): KCl 120, KH2PO4 2, (CH2COONa)2 5, MgCl2 2.4, HEPES 20, ATP 2, pH 7.2) using a previously described method (27). The [Ca2+] of the cytosol-like buffer was determined using fura-2 (28) and buffered to 120-190 nM with EGTA. Cells (2 ml containing 4-6 mg of protein) were permeabilized by the addition of beta -escin (25 µg/ml). The suspension was then centrifuged (500 × g, 2 min) and resuspended in 6 ml of cytosol-like buffer, and 45Ca2+ was added (equivalent to 0.4 µl/ml 45Ca2+ at 1.98 mCi/ml). After gentle vortexing, the cells were left for 15-20 min at room temperature. To initiate release of loaded 45Ca2+, 50 µl of cells were added to 50 µl of Ins(1,4,5)P3. After 60 s, 500 µl of silicon oil was added, and the cells were centrifuged at 16,000 × g for 2 min. The aqueous phase and most of the silicon oil phase were aspirated, the tubes were inverted, and the remaining oil was allowed to drain. The pellets were solubilized in scintillant, and the unreleased 45Ca2+ was determined. Release was calculated as a percentage of the total 45Ca2+ loaded. The size of the rapidly releasable pool was also determined using 10 µM ionomycin to indicate the amount of 45Ca2+ loaded into the intracellular stores. This was ~80% of the total 45Ca2+.


    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Pretreatment of alpha T3-1/M3 cells for 1 h with maximal concentrations (7) of either GnRH (1 µM) or methacholine (1 mM) resulted in both homologous and heterologous desensitization of agonist-mediated Ca2+ signaling (Fig. 1, a and b). The homologous and heterologous loss of Ca2+ signaling as a result of GnRH or methacholine pretreatment were maximal following 30-60 min of pretreatment and were sustained at this level for at least 24 h of pretreatment (Fig. 1c).



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Fig. 1.   Homologous and heterologous desensitization of Ca2+ signaling in alpha T3-1/M3 cells. Cells were cultured, loaded with fura-2, and prepared for imaging as described under "Experimental Procedures." Before imaging, they were pretreated for 60 min (a and b) or for the times indicated (c) in buffer containing 100 nM GnRH, 1 mM methacholine, or no addition (control). The cells were then washed extensively and mounted on the microscope stage. During imaging the cells were stimulated as indicated with either 100 nM GnRH (a and c) or 1 mM methacholine (b). In panel c, showing the time course of homologous and heterologous desensitization of GnRH-mediated Ca2+ signaling, the GnRH-stimulated increase in [Ca2+]i (calculated by subtraction of pre-stimulation values from maximal post-stimulation values) is shown as a function of pretreatment time. Control responses to GnRH were determined at each time point but were not time-dependent and have, therefore, been pooled and plotted at the 0 time point for clarity. Each trace shows the mean ± S.E. derived from 3 separate experiments (a and b), 3-7 separate experiments (c), or >16 experiments (c, 0 h) with 20-50 cells imaged in each experiment.

Expression of type I, II, and III Ins(1,4,5)P3 receptors was examined by Western blotting of solubilized alpha T3-1/M3 cells using isoform-specific antibodies (26). Expression of all three isoforms was detected, although bands representing type II and III Ins(1,4,5)P3 receptors were faint and then only apparent after exposure of the Western blot to film for longer periods of time (data not shown). Although we are unable to quantitate precisely the relative levels of the different types of Ins(1,4,5)P3 receptors, the predominant expression of type I is consistent with that in the pituitary, which expresses type I, II, and III Ins(1,4,5)P3 receptors at 73, 24, and 3% of the total receptor population, respectively (19).

Challenge of alpha T3-1/M3 cells with 1 µM GnRH resulted in a rapid and marked loss of type 1 Ins(1,4,5)P3 receptor immunoreactivity (Fig. 2, a and c), which had a half-time of ~20 min, was maximal by 60 min (<20% of immunoreactivity remaining), and sustained for at least 24 h in the continued presence of agonist (Fig. 2, a and c). Comparable data were obtained using 100 nM GnRH (Fig. 2c). Challenge of alpha T3-1/M3 cells with 1 mM methacholine also resulted in a marked loss of type I Ins(1,4,5)P3 receptor immunoreactivity, albeit with a rate and magnitude that was less than that observed with GnRH (Fig. 2, b and c). In contrast to treatment with 1 µM GnRH, there was some recovery of type I Ins(1,4,5)P3 receptor immunoreactivity during 24-h treatment with methacholine (Fig. 2, b and c). The GnRH-mediated loss of type I Ins(1,4,5)P3 receptor immunoreactivity was concentration-dependent, with an EC50 of -9.91 ± 0.61 (log10, M; n = 4; 0.12 nM) (all data with errors are mean ± S.E.) at 60 min of treatment (data not shown).



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Fig. 2.   a-c, agonist-mediated down-regulation of type I Ins(1,4,5)P3 receptor immunoreactivity in alpha T3-1/M3 cells. Cells were challenged with 1 µM GnRH, 100 nM GnRH, or 1 mM methacholine for the times indicated, and Western blots for the type I Ins(1,4,5)P3 receptor were performed. Western blots, representative of three experiments, show the type I Ins(1,4,5)P3 receptor in the absence of agonist treatment or following treatment with either 1 µM GnRH (a) or 1 mM methacholine (b) for the indicated times. The density of the band representing the type I Ins(1,4,5)P3 receptor was quantified and expressed as a percentage of that in the absence of agonist (c). Data are mean ± S.E.; n = 3. , 1 µM GnRH; open circle , 100 nM GnRH; black-square, 1 mM methacholine. d and e, time course of the recovery of type I Ins(1,4,5)P3 receptor immunoreactivity following down-regulation with GnRH. Following treatment with GnRH (1 µM) for 1 h, cells were washed, and the incubation was continued in the presence of an antagonist of the GnRH receptor (antide, 1 µM). Cells were then either solubilized immediately or allowed the indicated recovery time before solubilization. Western blotting for the type I Ins(1,4,5)P3 receptor was then performed. A representative Western blot is shown (d); below the blot are the mean densitometric data (e). The density of the band representing the type I Ins(1,4,5)P3 receptor was quantified and expressed as a percentage of that under basal (no agonist treatment) conditions. The data are the mean ± S.E.; n = 3.

In experiments designed to examine the rate of recovery of type I Ins(1,4,5)P3 receptor immunoreactivity, cells were first treated with 1 µM GnRH for 1 h to induce down-regulation. Agonist activation of GnRH receptors was then stopped by washing the cell monolayer and continuing the incubation in the presence of the GnRH receptor antagonist, antide (1 µM). Type I Ins(1,4,5)P3 receptor immunoreactivity was then examined over the subsequent 24 h. The 1-h pretreatment with GnRH resulted in a marked loss of type I Ins(1,4,5)P3 receptor immunoreactivity that was further reduced after 1 h of "recovery" time (Fig. 2, d and e). Levels of receptor immunoreactivity then increased back to basal levels by 24 h (Fig. 2, d and e). Similar results were obtained when cells were treated with 1 µM GnRH and washed, but antagonist was not added (data not shown).

In binding experiments, pretreatment of intact cells for 60 min with 1 µM GnRH reduced the binding of [3H]Ins(1,4,5)P3 to alpha T3-1 membranes to 51 ± 7% (n = 4) of control without measurably altering the Kd (Fig. 3).



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Fig. 3.   Loss of [3H]Ins(1,4,5)P3 binding to membranes prepared from alpha T3-1/M3 cells pretreated with GnRH. Cells were pretreated for 60 min in medium with 0 () or 1 µM GnRH (open circle ) as indicated, and competition binding was then used to determine the number and affinity of Ins(1,4,5)P3 receptors for [3H]Ins(1,4,5)P3 as described under "Experimental Procedures." The data shown are the mean ± S.E. (n = 3) from a single representative experiment. Pooling data from four such experiments revealed that GnRH pretreatment reduced the Bmax to 51 ± 7% of control without measurably altering the Kd (4.9 ± 2.2 nM).

Permeabilization of alpha T3-1/M3 cells with beta -escin allowed intracellular Ca2+ stores to be loaded with tracer 45Ca2+, which could then be released by the addition of Ins(1,4,5)P3. This exogenous Ins(1,4,5)P3 was able to release a maximum of ~60% of the 45Ca2+ that had been loaded over a 15-min period, with an EC50 of -6.8 ± 0.2 (log10, M; n = 4; 0.16 µM) (Fig. 4). Treatment of cells for 1 h with 1 µM GnRH significantly reduced the magnitude of Ins(1,4,5)P3-mediated release of 45Ca2+ from the intracellular stores (p < 0.001, two way analysis of variance) but had no significant effect on the EC50 for Ins(1,4,5)P3, which was -6.5 ± 0.3 (log10, M; n = 4; 0.32 µM) (Fig. 4).



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Fig. 4.   Reduced ability of exogenous Ins(1,4,5)P3 to release 45Ca2+ from the intracellular stores of alpha T3-1/M3 cells following pretreatment with GnRH. Cells were incubated in the presence or absence of 1 µM GnRH for 1 h and permeabilized with beta -escin, and intracellular Ca2+ stores were loaded with 45Ca2+. Exogenous Ins(1,4,5)P3 was then added, and the amount of 45Ca2+ release was determined by measuring the radioactivity remaining associated with the cell pellet. Results are expressed as the percentage of 45Ca2+ released by Ins(1,4,5)P3 compared with the total amount loaded. The data are the mean ± S.E.; n = 3.

Thimerosal has been reported to increase Ins(1,4,5)P3 receptor sensitivity (29). In naive alpha T3-1/M3 cells, 100 µM thimerosal increased the spike [Ca2+]i response to a sub-maximal (5 nM) (Fig. 5a) but not maximal (1 µM) (Fig. 5c) concentration of GnRH when cells were challenged in the absence of extracellular Ca2+ (to assess the effects on Ca2+ release only). This suggests that the Ins(1,4,5)P3 receptor does not limit the magnitude of the Ins(1,4,5)P3-mediated Ca2+ release in naive cells stimulated with a maximal concentration of GnRH but can limit the response to submaximal agonist concentrations. When cells were pretreated for 1 h with 100 nM GnRH (in the presence of extracellular Ca2+), thimerosal had little effect on the subsequent response (again in the absence of extracellular Ca2+) to a submaximal concentration of GnRH but markedly potentiated the response to a maximal concentration (Fig. 5, b and d), suggesting that, in GnRH-pretreated cells, Ins(1,4,5)P3 receptor activation is rate-limiting for GnRH-stimulated Ca2+ mobilization.



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Fig. 5.   The effect of thimerosal on mobilization of Ca2+ by GnRH in control and GnRH-pretreated alpha T3-1/M3 cells. Cells were pretreated with 0 (a and c; Control) or 1 µM GnRH (b and d; GnRH pretreated) for 60 min and then prepared for Ca2+ imaging as described in the legend to Fig. 1. During imaging, the cells were transferred to Ca2+-free medium (first vertical arrow) and then to Ca2+-free medium containing 0 or 100 µM thimerosal (second vertical arrow) as indicated. Finally, the cells were stimulated with 5 nM (a and b) or 1 µM (c and d) GnRH (still in Ca2+-free medium with 0 or 100 µM thimerosal) as shown by the horizontal arrows. Each trace shows the mean ± S.E. derived from three separate experiments, with 20-50 cells imaged in each experiment. After control pretreatments, thimerosal increased the response to 5 nM GnRH but not that to 1 µM GnRH, whereas in GnRH-pretreated (desensitized) cells, thimerosal increased the response to 1 µM GnRH but not that to 5 nM GnRH.

Although challenge of cells with 1 µM GnRH resulted in a dramatic reduction in type I Ins(1,4,5)P3 receptor immunoreactivity (see above), exclusion of Ca2+ from the extracellular buffer prevented the GnRH-mediated loss of Ins(1,4,5)P3 receptors but had no significant effect on the basal (nonagonist-stimulated) levels over a 1-h period (Fig. 6a). Furthermore, the absence of extracellular Ca2+ partially prevented the homologous desensitization of GnRH-mediated spike and plateau Ca2+ signaling (Fig. 6, b and c). Incubation of cells for 4 h with the cysteine protease (and proteasome) inhibitor N-acetyl-Leu-Leu-norleucinal (ALLN, 100 µg/ml) (20, 22, 23) prior to and during a 1-h incubation with 1 µM GnRH also markedly attenuated the agonist-induced loss of type I Ins(1,4,5)P3 receptor immunoreactivity (Fig. 7a). The effects of ALLN on the desensitization of the agonist-mediated Ca2+ response were, however, difficult to interpret. Thus, even in the absence of GnRH pretreatment, ALLN markedly inhibited the spike and plateau [Ca2+]i responses to GnRH (data not shown), and we therefore used the more specific proteasome inhibitor lactacystin (10 µM, 4 h) (22). Lactacystin also markedly protected type I Ins(1,4,5)P3 receptor immunoreactivity against GnRH-mediated down-regulation (control (untreated), 100%; 1 h, 1 µM GnRH, 44.6 ± 4.5%; lactacystin, 80.6 ± 23.0%; 1 h, 1 µM GnRH + lactacystin, 102.5 ± 34.1%). Furthermore, lactacystin attenuated, but did not completely prevent, GnRH-mediated desensitization of [Ca2+]i mobilization (Fig. 7, b and c). There was also some inhibition of GnRH-mediated spike [Ca2+]i signaling in the presence of lactacystin (Fig. 7b), although the plateau was unaffected (data not shown).



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Fig. 6.   The influence of extracellular Ca2+ on GnRH-mediated down-regulation of type I Ins(1,4,5)P3 receptor immunoreactivity and GnRH-mediated [Ca2+]i signaling in alpha T3-1/M3 cells. For determination of Ins(1,4,5)P3 receptor immunoreactivity (a), cells were challenged with GnRH (1 µM, 1 h) in Krebs/HEPES buffer with either 1.3 mM Ca2+ or no added Ca2+. Western blotting for the type I Ins(1,4,5)P3 receptor was then performed. The density of the bands representing the type I Ins(1,4,5)P3 receptor was quantified and expressed as a percentage of that in the presence of extracellular Ca2+ but in the absence of GnRH. The data are the mean ± S.E.; n = 4. For determination of [Ca2+]i signaling (b and c), cells were pretreated with 0 (b) or 1 µM GnRH (c) for 60 min in buffer (filled symbols) or Ca2+-free buffer (open symbols), then washed in normal buffer, and prepared for Ca2+ imaging as described in the legend to Fig. 1. During imaging, the cells were transferred to Ca2+-free buffer (vertical arrow) and then stimulated with 100 nM GnRH (still in Ca2+-free buffer) as indicated by the horizontal arrows. Each trace shows the mean ± S.E. derived from three separate experiments, with 20-50 cells imaged in each experiment.



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Fig. 7.   The influence of protease inhibition on GnRH-mediated down-regulation of type I Ins(1,4,5)P3 immunoreactivity and homologous desensitization of [Ca2+]i signaling in alpha T3-1/M3 cells. For determination of Ins(1,4,5)P3 receptor immunoreactivity (a), cells were incubated with (+) or without (-) the cysteine protease inhibitor ALLN (100 µg/ml) for 4 h. Agonist (1 µM GnRH or 1 mM methacholine) was then added, and the incubation was continued for an additional 1 h. Western blotting for the type I Ins(1,4,5)P3 receptor was then performed. The densities of the bands representing the type I Ins(1,4,5)P3 receptor were quantified and expressed as a percentage of that under basal conditions (no ALLN or agonist). The data shown are the mean ± S.E.; n = 4. For determination of [Ca2+]i signaling (b and c), cells were pretreated for 3 h in buffer with 0 (control, filled symbols) or 10 µM (open symbols) lactacystin with 0 (b) or 100 nM GnRH (c) added for the final 60 min of the pre-incubation and fura-2-acetoxymethyl ester present for the final 30 min. The cells were then washed in normal buffer and prepared for Ca2+ imaging as described in the legend to Fig. 1. During imaging, the cells were transferred to Ca2+-free buffer and then stimulated with 100 nM GnRH. Each trace shows the mean ± S.E. derived from three separate experiments, with 20-50 cells imaged in each experiment.

Using commercially available antibodies against the PLC isoforms beta 1-4, gamma 1-2, and delta 1-2, the expression of PLCbeta 1, beta 3, gamma 1, and gamma 2 was demonstrated in alpha T3-1/M3 cells (Fig. 8). The immunoreactivity of those antibodies that did not detect proteins in alpha T3-1/M3 cells was confirmed using extracts from either SH-SY5Y neuroblastoma cells or rat brain (data not shown). Given that GnRH receptor-mediated responses are via Galpha q/11 and most likely, therefore, via PLCbeta isoforms, we examined the influence of GnRH or methacholine treatment on the expression of Galpha q/11, PLCbeta 1, and beta 3. Exposure of alpha T3-1/M3 cells for up to 1 h with maximal concentrations of either GnRH (1 µM) or methacholine (1 mM) had no consistent effects on the levels of Galpha q/11 or the PLC isoforms beta 1 and beta 3 (Fig. 8).



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Fig. 8.   Lack of effect of acute agonist treatment (<= 1 h) on the expression of Galpha q/11, PLCbeta 1, and PLCbeta 3 in alpha T3-1/M3 cells. Western blotting for the PLC isoforms beta 1-4, gamma 1-2, and delta 1-2 demonstrated the expression of PLCbeta 1, beta 3, gamma 1, and gamma 2 in alpha T3-1/M3 cells. Agonist treatment for up to 1 h had no effect on the expression levels of Galpha q/11, PLCbeta 1, and PLCbeta 3. This was also reflected in the densitometric scan data from three separate experiments (data not shown).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has been known for over two decades that sustained stimulation of gonadotropes with GnRH causes desensitization of GnRH-stimulated gonadotropin secretion (30), an effect that can be either exploited or avoided in clinical applications of GnRH analogues (12). The recent discovery that mammalian GnRH receptors do not show rapid homologous desensitization (3-8) reveals that desensitization of GnRH-stimulated gonadotropin secretion must reflect changes distal to the receptor, as implied by earlier work showing that GnRH receptor regulation does not explain desensitization of gonadotropin secretion (5). Our work has revealed that pretreatment of alpha T3-1 cells with GnRH causes a pronounced desensitization of GnRH-stimulated mobilization of Ca2+ from intracellular stores (4, 5), which is of particular interest in the light of the established importance of Ca2+ mobilization in mediation of GnRH-stimulated gonadotropin secretion. Because this desensitization is heterologous (cross-desensitization is seen with responses to other PLC-activating stimuli), it most likely reflects changes in the amount or activity of effector proteins distal to the GnRH receptor.

Activation-dependent down-regulation of effector proteins is emerging as an important mechanism for post-receptor adaptive responses, and such responses have already been observed in response to GnRH. In alpha T3-1 cells, GnRH causes a loss of Galpha q (31) and of regulatory and catalytic subunits of protein kinase A (32). It also causes the apparent proteolysis of protein kinase C delta  and epsilon  (33). Here we have focused on the possible effects of GnRH on effector proteins involved in Ca2+ metabolism and found that a 60-min pretreatment with GnRH causes pronounced desensitization of GnRH-stimulated Ca2+ mobilization without measurably altering cellular levels of PLCbeta 1, PLCbeta 3, or Galpha q. These data are consistent with earlier studies demonstrating that GnRH reduces Galpha q levels extremely slowly (half-time > 6 h) (31) and that reduced Ins(1,4,5)P3 generation alone does not account for desensitization of Ca2+ mobilization in these cells (5). In contrast, we show that GnRH pretreatment causes a pronounced down-regulation of Ins(1,4,5)P3 receptors (as demonstrated by radioligand binding and immunological quantification of type I Ins(1,4,5)P3 receptors), that this effect is functionally significant (as demonstrated by a reduction in Ins(1,4,5)P3-stimulated 45Ca2+ mobilization in permeabilized cells), and that the onset of this effect, and recovery from the effect, have similar kinetics to the onset of, and recovery from, desensitization of Ca2+ mobilization (4, 5).

In several systems, activation of PLC-coupled GPCRs has been shown to down-regulate Ins(1,4,5)P3 receptors, an effect attributed to Ca2+-dependent proteolysis of active Ins(1,4,5)P3 receptors (17, 20, 22, 23). Activation of these receptors has been shown to cause their ubiquitination while still in the endoplasmic reticulum membrane (22). This is thought to target Ins(1,4,5)P3 receptors for proteasomal degradation, as demonstrated by the fact that proteasome inhibitors can prevent GPCR-mediated down-regulation (20, 22, 23). Our data are in accord with this model, because we have found that GnRH-mediated down-regulation of type I Ins(1,4,5)P3 receptor immunoreactivity is prevented in Ca2+-free medium and by the two protease inhibitors ALLN and lactacystin. Interestingly, we have found that the down-regulation of type I Ins(1,4,5)P3 receptors caused by GnRH is more rapid, more pronounced, and more slowly reversed than that caused by methacholine (muscarinic M3 receptor activation). This is despite the fact that both stimuli cause comparable increases in [Ca2+]i in these cells and occur even when the concentrations of GnRH and methacholine are matched to give comparable maximal increases in Ins(1,4,5)P3 levels in these cells (100 nM GnRH, 1 mM methacholine) (7). However, the muscarinic M3 receptor undergoes a partial rapid homologous desensitization and therefore causes a transient increase in Ins(1,4,5)P3 mass, reducing to a sustained plateau after a peak at 10 s, whereas the GnRH receptor does not rapidly desensitize and therefore causes a sustained increase in Ins(1,4,5)P3 mass, which reaches maximal levels within 20-30 s (7). This clearly implies that the Ins(1,4,5)P3 receptor down-regulation is sensitive not just to the magnitude of the Ins(1,4,5)P3 response but also to its duration, precisely as expected if it is the Ins(1,4,5)P3 occupied (active) receptor conformation that is sensitive to proteolysis (22). Thus, the lack of GnRH receptor desensitization may contribute to the unusual rapidity of Ins(1,4,5)P3 receptor down-regulation in these cells. Typically, Ins(1,4,5)P3 receptor down-regulation occurs with a half-time of 4-24 h (17, 23), as compared with <20 min in GnRH-stimulated alpha T3-1/M3 cells (Fig. 2). Presumably with other PLC-activating GPCRs, receptor desensitization attenuates Ins(1,4,5)P3 responses and thereby reduces the rapidity and/or magnitude of Ins(1,4,5)P3 receptor down-regulation. It should be noted, however, that bombesin and cholecystokinin reduce Ins(1,4,5)P3 receptor levels with a half-time of <30 min in AR4-2J cells (19) and that methacholine caused Ins(1,4,5)P3 receptor down-regulation with a half-time of <60 min in alpha T3-1/M3 cells, demonstrating that relatively rapid down-regulation can occur, even with receptors that do desensitize.

The major question raised by our data is whether down-regulation of Ins(1,4,5)P3 receptors contributes to or underlies desensitization of Ca2+ mobilization. Our investigations of response kinetics are entirely compatible with this possibility because we have found a) that the time-course of Ins(1,4,5)P3 receptor down-regulation in response to GnRH is comparable with that for the onset of desensitization (Figs. 2 and 1c, respectively) and b) that both effects are maintained as GnRH pretreatment is extended to 24 h. Further support for the possible causal relationship is provided by the demonstrations a) that the GnRH-mediated Ins(1,4,5)P3 receptor loss and desensitization of Ca2+ signaling are associated with reduced Ins(1,4,5)P3-stimulated mobilization of 45Ca2+ from permeabilized cells (directly establishing the functional significance of Ins(1,4,5)P3 receptor regulation in this system), b) that Ca2+-free medium prevents and attenuates GnRH-mediated Ins(1,4,5)P3 receptor down-regulation and desensitization of Ca2+ mobilization, respectively, c) that lactacystin prevents and attenuates GnRH-mediated Ins(1,4,5)P3 receptor down-regulation and desensitization of Ca2+ mobilization, respectively, and d) that thimerosal partially reverses desensitization of Ca2+ mobilization. Because thimerosal increases the affinity of Ins(1,4,5)P3 receptors for Ins(1,4,5)P3 (29), it would only be expected to influence responses to Ins(1,4,5)P3 under conditions where Ins(1,4,5)P3 receptor activation is limiting. Thus, the ability of thimerosal to amplify Ca2+ mobilization by 5 nM GnRH, but not by 1 µM GnRH, implies that Ins(1,4,5)P3 receptor activation is limiting for the response to the low concentration of GnRH but not to the high concentration. In desensitized cells, however, thimerosal increased the response to the high concentration of GnRH, demonstrating that in these cells Ins(1,4,5)P3 receptor activation has become limiting. This is precisely what would be expected if Ins(1,4,5)P3 receptor loss leaves the desensitized cells with insufficient Ins(1,4,5)P3 receptors for efficient mobilization of Ca2+ even in the face of sufficient GnRH-stimulated Ins(1,4,5)P3 levels.

Although our data are largely consistent with the possibility that GnRH-mediated Ins(1,4,5)P3 receptor down-regulation underlies desensitization of Ca2+ mobilization, several lines of evidence might argue against this interpretation. Thus, stimulation of muscarinic receptors results in a time course of heterologous desensitization of GnRH-mediated [Ca2+]i elevation similar to the homologous desensitization caused by GnRH pretreatment. This is despite the finding that GnRH causes a more rapid and greater loss of Ins(1,4,5)P3 receptor immunoreactivity than muscarinic receptor stimulation. Furthermore, the retention of ~50% of Ins(1,4,5)P3 receptors (Fig. 3) and the fact that maximal Ins(1,4,5)P3-stimulated 45Ca2+ mobilization is only reduced by ~42% (Fig. 4) stand in contrast to the almost complete loss of the spike phase [Ca2+]i response to GnRH in desensitized cells (Fig. 1). Similarly, complete inhibition of type I Ins(1,4,5)P3 receptor down-regulation by pretreatment in Ca2+-free medium, or in the presence of ALLN or lactacystin, contrasts to only partial inhibition, or no measurable inhibition, of desensitization. These apparent inconsistencies could reflect contributions from other as yet unidentified mechanisms of desensitization or may reflect differences in the relative contributions of Ins(1,4,5)P3 receptor subtypes, or of receptors in different cellular locations, to the end points quantified. Thus, type II Ins(1,4,5)P3 receptors, which are relatively resistant to down-regulation in other systems (19), may contribute disproportionally to the 45Ca2+ mobilization response, and local down-regulation of Ins(1,4,5)P3 receptors in the immediate vicinity of GnRH receptors may be more extreme than that revealed by global measurements of all Ins(1,4,5)P3 receptors. Alternatively, it is possible that the 50% loss of Ins(1,4,5)P3 receptors and the consequent increase in mean distance between functional Ins(1,4,5)P3 receptors are sufficient to prevent propagation of Ca2+ mobilization by calcium-induced calcium release (34) and therefore have a disproportionately large effect on Ca2+ responses in intact cells (as compared with permeabilized cells or membrane preparations). It is equally possible, however, that other modifications of Ins(1,4,5)P3 receptors (e.g. phosphorylation, ATP binding, ubiquitination) inhibit Ins(1,4,5)P3 receptor signaling in the desensitized cells without altering immunoreactivity or radioligand binding in membrane preparations. Some of these modifications, particularly ubiquitination, appear to be involved in the targeting of Ins(1,4,5)P3 receptors for degradation (22). That such targeting occurs is demonstrated by our finding that Ins(1,4,5)P3 receptor immunoreactivity continues to decline following removal of GPCR activation (Fig. 2).

Whereas a number of studies have demonstrated the principle of agonist-induced Ins(1,4,5)P3 receptor down-regulation (16-23), the current study provides evidence of a setting in which such regulation may be functionally relevant. Thus, loss of Ins(1,4,5)P3 receptors following either pre-ovulatory surges in GnRH or, in particular, the clinical use of GnRH agonists may play a part in the suppression of gonadotrope function. It should be noted that such a mechanism would also result in a compromised function of other Ins(1,4,5)P3-dependent, Ca2+-mobilizing receptors expressed on pituitary cells (e.g. pituitary adenylyl cyclase-activating polypeptide receptors). Such heterologous loss of function by this mechanism may be less apparent in other systems in which GPCR desensitization may serve to limit the down-regulation of signaling components shared with other receptors.


    FOOTNOTES

* This work was supported by Grants 16895/1.5 and 054949 from the Wellcome Trust (to S. R. N. and C. A. M., respectively).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 0116-2523094; Fax: 0116-2525045; E-mail: gbw2@leicester.ac.uk.

Published, JBC Papers in Press, November 7, 2000, DOI 10.1074/jbc.M008916200


    ABBREVIATIONS

The abbreviations used are: GnRH, gonadotropin-releasing hormone; PLC, phospholipase C; Ins(1, 4,5)P3, inositol 1,4,5-trisphosphate; GPCR, G-protein-coupled receptor; ALLN, N-acetyl-Leu-Leu-norleucinal.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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