Desensitization of gonadotropin-releasing hormone action in alphaT3-1 cells due to uncoupling of inositol 1,4,5-trisphosphate generation and Ca2+ mobilization.

Gonadotropin-releasing hormone (GnRH) acts via a G-protein coupled receptor on gonadotropes to increase cytosolic Ca2+ and stimulate gonadotropin secretion. Sustained exposure causes desensitization of these effects, but the GnRH receptor has no C-terminal tail and does not undergo rapid (<5 min) desensitization. Nevertheless, pretreatment of alphaT3-1 cells with GnRH reduced the spike Ca2+ response to GnRH and decreased the GnRH effect on inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) by 30-50%. Ca2+-free medium with or without thapsigargin also decreased GnRH-stimulated Ins(1,4,5)P3 generation, implying that attenuation of the Ca2+ response underlies the Ins(1,4,5)P3 reduction rather than vice versa. Intracellular Ca2+ pool depletion cannot explain desensitization of the Ca2+ response because pool depletion and repletion were faster (half-times, <1 min) than the onset of and recovery from desensitization (half-times 10-20 min and 4-6 h). Moreover, 1-h GnRH pre-treatment attenuated the spike Ca2+ response to GnRH but not that to ionomycin, and brief GnRH exposure in Ca2+-free medium reduced the response to ionomycin more effectively in controls than in desensitized cells. GnRH pretreatment also attenuated the Ca2+ response to PACAP38. This novel form of desensitization does not reflect uncoupling of GnRH receptors from their immediate effector system but rather a reduced efficiency of mobilization by Ins(1,4,5)P3 of Ca2+ from an intact intracellular pool.

Studies over recent years have revealed a general scheme for homologous desensitization of GPCRs 1 involving rapid uncoupling of receptors from their G-protein, subsequent sequestration of receptors from the plasma membrane, and internalization followed by proteolytic degradation (1). Mechanisms underlying rapid desensitization have been documented for a number of GPCRs, which involve rapid agonist-induced recep-tor phosphorylation and consequent uncoupling from the receptors effector system (2). Such phosphorylation may be mediated by protein kinases A or C or by specific G-protein coupled receptor kinases, and in many cases phosphorylation of specific amino acids within the C-terminal tail plays a crucial role (1)(2)(3)(4). For example, specific residues in the C-terminal tail of adenylyl cyclase-activating ␤-adrenergic receptors have been identified that undergo agonist-induced phosphorylation by ␤-adrenergic receptor kinase. This phosphorylation permits association with ␤-arrestin, thereby inhibiting G-protein binding and adenylyl cyclase activation (1,2). The C-terminal tail has also been found to be involved in internalization of a number of receptors of this superfamily (3,4).
GnRH is a hypothalamic decapeptide that acts via GPCRs on gonadotropes to stimulate the exocytotic secretion of luteinizing hormone and follicle-stimulating hormone. Agonist occupancy of GnRH receptors causes a G-protein-mediated activation of PLC that hydrolyzes membrane phosphoinositides yielding diacylglycerol and inositol phosphates, including Ins(1,4,5)P 3 , that respectively activate protein kinase C and mobilize Ca 2ϩ from intracellular stores (5)(6)(7)(8)(9)(10)(11). GnRH also increases Ca 2ϩ entry into gonadotropes, predominantly via voltage-operated Ca 2ϩ channels, and the increase in cytosolic Ca 2ϩ caused by GnRH is primarily responsible for the increase in exocytotic hormone release (7-10, 12, 13). Sustained exposure to GnRH reduces GnRH-stimulated gonadotropin secretion (homologous desensitization), and this underlies the suppression of the reproductive system, which is exploited in the major clinical applications of GnRH analogues (14,15). Sustained treatment with GnRH also reduces the number of cell surface GnRH receptors (down-regulation), and this presumably contributes to desensitization but changes in GnRH receptor number can be uncoupled from those in gonadotrope responsiveness, demonstrating the involvement of additional mechanisms (16 -20).
As with adenylyl cyclase coupled receptors, many PLC-coupled receptors clearly undergo rapid desensitization (21,22), and evidence exists that similar regulatory mechanisms operates for receptors coupled to both effector system (21)(22)(23). However, GnRH receptors are unique amongst the currently known G-protein coupled receptors in that they lack the C-terminal cytoplasmic tail, which has proven to be so important for desensitization and/or internalization of other GPCRs (5-7), and recent work on the kinetics of GnRH-stimulated PLC activation in ␣T3-1 cells (a gonadotrope-derived cell line) indicates that the GnRH receptor does not undergo rapid (Ͻ10 min) homologous desensitization (24). We have shown, however, that pretreatment for 60 min with GnRH causes a pronounced reduction the GnRH-induced increase in cytosolic Ca 2ϩ concen-tration ([Ca 2ϩ ] i ) in these cells (25). Both the spike phase of the response (which reflects mobilization of intracellular Ca 2ϩ ) and sustained phase of the response (which is dependent upon Ca 2ϩ entry across the plasma membrane) were attenuated by GnRH pretreatment (25). Here we have addressed the relationship between Ins(1,4,5)P 3 generation and Ca 2ϩ mobilization in order to define more closely the cellular loci involved in desensitization of the spike response.
Dynamic Video Imaging of Cytosolic Ca 2ϩ -Video imaging of fura 2-loaded ␣T3-1 cells was performed as described (27,28). Cells were washed and pretreated for 60 min at 37°C in 0.9 ml of PSS (127 mM NaCl, 1.8 mM CaCl 2 , 5 mM KCl, 2 mM MgCl 2 , 0.5 mM NaH 2 PO 4 , 5 mM NaHCO 3 , 10 mM glucose, 0.1% bovine serum albumin, and 10 mM HEPES, pH 7.4) containing 0 (control) or 0.1 M GnRH with fura 2 (100 l of 20 M fura 2/AM) added 30 min before the end of this incubation. They were then washed several times in PSS, and the coverslips were loaded into a holder that was fitted into a heating chamber at 37°C. Image capture was typically performed within 10 -25 min of loading in approximately 500 l of PSS or in PSS with 100 M EGTA and no CaCl 2 (Ca 2ϩ -free medium) using MagiCal hardware, Tardis software, and a Nikon Diaphot microscope. The cells were excited alternately at 340 and 380 nm, and emitted light was collected at 510 nm averaging the data from 8 or 16 video frames and subtracting background values before ratioing. The ratio of fluorescence at 340 and 380 nm was calculated on a pixel-by-pixel basis and used to determine the Ca 2ϩ concentration assuming a dissociation constant of 225 nM for fura 2 and Ca 2ϩ at 37°C. Calibration was performed as described (27,28).
Quantification of Intracellular D-Ins(1,4,5)P 3 -D-Ins (1,4,5)P 3 mass was determined using a modification of a radioreceptor assay previously validated for stereospecificity and positional specificity (29,30). Briefly, ␣T3-1 cells were grown to approximately 80% confluence and then washed and incubated for 30 min in 1 ml of Krebs/HEPES at 37°C. The medium was aspirated and replaced with 150 l of buffer with the indicated concentration of GnRH. Incubations were performed in triplicate and were terminated at 5-300 s by addition of 150 l of ice-cold 1 M trichloroacetic acid. For zero time points, trichloroacetic acid was added before the GnRH. The D-Ins(1,4,5)P 3 was then extracted as described (30) using duplicate aliquots for each of the triplicate samples and standards (0.1 nM to 3 M). Protein content of the wells was determined in 0.1 M NaOH digests of control cells using bovine serum albumin as standard.
Statistical Analysis and Data Presentation-The figures show data from a single experiment (representative of four experiments) or the mean Ϯ S.E. of data pooled from n independent experiments (raw data or data normalized as described in the figure legends). Data are reported in the text as mean Ϯ S.E., and statistical analysis was by Student's t test, accepting p Ͻ 0.05 as statistically significant. EC 50 values were estimated by nonlinear regression using Graphpad Prism (San Diego, CA). For Ca 2ϩ measurements, image analysis was used to quantify the mean ionized Ca 2ϩ in all of the cells in each field of view (which typically contained 10 -50 cells) as well as in individual cells. The figures show the mean (with or without S.E.) of data pooled from the indicated number of fields of view. Where spike and plateau Ca 2ϩ values are reported these were defined arbitrarily as the maximum response within 10 s of stimulation and the response after 1 min, respectively.

RESULTS
In the first series of experiments Ca 2ϩ imaging was performed with ␣T3-1 cells pre-treated with 0 or 0.1 M GnRH for 60 min and then after extensive washing stimulated with GnRH during image capture. As shown (Fig. 1, main panel) 0.1 M caused the expected biphasic [Ca 2ϩ ] i increase in control cells from a basal value of 52 Ϯ 9 nM to a spike of 556 Ϯ 76 nM followed by a plateau of 165 Ϯ 13 nM (mean Ϯ S.E., n ϭ 7). In cells pre-treated for 60 min with GnRH basal [Ca 2ϩ ] i was unaffected (52 Ϯ 7 nM, n ϭ 7), but the spike and plateau responses to GnRH were attenuated (to 127 Ϯ 16 and 124 Ϯ 12 nM, respectively, p Ͻ 0.05). Further studies characterizing the time course of desensitization of the spike phase indicated a significant (p Ͻ 0.05) reduction after only 10 min of pretreatment with GnRH (from 605 Ϯ 80 to 308 Ϯ 73 nM) and that the spike response to GnRH was maximally attenuated following a 20 min pretreatment ( Fig. 1, inset).
Although the effect of GnRH pretreatment on GnRH-stimulated Ins(1,4,5)P 3 production may contribute to desensitization of the Ca 2ϩ response, PLCs are Ca 2ϩ -dependent (31), so it is equally possible that desensitization of the Ca 2ϩ response underlies the attenuated Ins(1,4,5)P 3 response. To address this, we assessed the effects of manipulations that alter the effect of GnRH on [Ca 2ϩ ] i In normal Ca 2ϩ -containing medium GnRH caused the expected rapid and sustained increase in Ins(1,4,5)P 3 , from 16 Ϯ 4 at 0 s to 325 Ϯ 79 pmol/mg protein at 5 s (Fig. 4). This response was not measurably altered by 2 M thapsigargin but was decreased in Ca 2ϩ -free medium and in Ca 2ϩ -free medium with thapsigargin (to 38 Ϯ 11 and 33 Ϯ 9% of control respectively, at 300 s). The effect of Ca 2ϩ -free medium was most pronounced at the later time points (1 and 5 min), and only the combined treatment with thapsigargin in Ca 2ϩ -free medium reduced the initial response (to 42 Ϯ 10% of control at 5 s).
We next assessed whether depletion of the GnRH-mobilizable Ca 2ϩ pool underlies desensitization of the Ca 2ϩ response. We have shown that treatment with GnRH in Ca 2ϩ -free or Ca 2ϩ -containing medium can deplete the hormone-mobilizable Ca 2ϩ pool (33), but the imaging experiments described herein were not performed until at least 10 min after the GnRH pretreatment (the time required to wash the cells and prepare for imaging), and we suspected that pool refilling may well have occurred. To assess the time required for pool refilling, ␣T3-1 cells were stimulated first for 1 min with 0.1 M GnRH in Ca 2ϩ -free medium (in order to deplete the GnRH-mobilizable pool). They were then washed extensively (Ͼ5 min) in Ca 2ϩfree medium and then either stimulated with 0.1 M GnRH in Ca 2ϩ -free medium or exposed briefly (5-60 s) to Ca 2ϩ -containing medium before being returned to Ca 2ϩ -free medium and stimulated again with 0.1 M GnRH. In cells incubated throughout in Ca 2ϩ -free medium, the second exposure failed to increase [Ca 2ϩ ] i (Fig. 5, upper trace). However, brief exposure (5-60 s) to Ca 2ϩ -containing medium both increased the [Ca 2ϩ ] i and enabled GnRH to cause a spike of [Ca 2ϩ ] i elevation during the subsequent (Fig. 5, middle and lower traces), indicating that pool refilling had occurred. In cells exposed to Ca 2ϩ -containing medium for 1 min, the response to the second GnRH challenge was comparable with that seen in the first, and the half-time for pool refilling was estimated to be 5-20 s. In parallel studies the influence of such manipulations on GnRHstimulated Ins(1,4,5)P 3 accumulation was also addressed. As shown (Fig. 6), GnRH caused a rapid increase in Ins(1,4,5)P 3 levels in control cells (pool intact) stimulated in Ca 2ϩ -free medium. This response was attenuated in cells that had been pre-treated for 1 min with 0.1 M GnRH in Ca 2ϩ -free medium in order to deplete the intracellular Ca 2ϩ pool (pool emptied) and was returned to control values by 1 min of exposure to normal Ca 2ϩ -containing medium in order to allow pool refilling after the GnRH pretreatment (pool emptied and refilled).
Although the experiments described above indicated that rapid refilling of the GnRH-mobilizable Ca 2ϩ pool had occurred, recovery from desensitization was much slower (Fig. 7). Indeed, ␣T3-1 cells pre-treated for 60 min with 0.1 M GnRH did not recover from such desensitization until at least 4 h after pretreatment. In order to test the possible involvement of pool depletion more directly, we compared the effects of GnRH and the Ca 2ϩ ionophore ionomycin on [Ca 2ϩ ] i in control cells (Fig. 8,  left panel) and in cells pre-treated for 60 min with 0.1 M GnRH (Fig. 8, right panel). In control cells stimulated in Ca 2ϩ -free  , left panel). These data indicate that the two stimuli mobilize Ca 2ϩ from a shared intracellular pool and that the ionomycin response can therefore be used as a measure of the filling state of the GnRH-mobilizable intracellular Ca 2ϩ pool (see also Ref. 32). In cells pre-treated for 60 min with 0.1 M GnRH, ionomycin alone caused an increase in [Ca 2ϩ ] i , comparable with that seen in control cells (compare lower traces in left and right panels), indicating the retention of this intracellular pool. Stimulation with 1 nM GnRH in Ca 2ϩ -free medium did not increase [Ca 2ϩ ] i or reduce the subsequent response to ionomycin. Stimulation with 0.1 M GnRH caused only a modest increase in [Ca 2ϩ ] i and reduction of the subsequent ionomycin response. The data shown in Fig. 8 are from a series of experiments from which concentration response curves for the increase in [Ca 2ϩ ] i caused by GnRH and the reduction in ionomycin response due to GnRH were constructed (Fig. 9). As shown, GnRH caused a dose-dependent increase in [Ca 2ϩ ] i in Ca 2ϩ -free medium with an EC 50 of 6 nM and also attenuated the ionomycin response to approximately 15% of control, with an IC 50 of 33 pM. In GnRH pre-treated cells, the EC 50 for the increase in Ca 2ϩ could not be calculated but is apparently greater than 1 M. In these cells GnRH decreased the ionomycin response to approximately 50% of control with an IC 50 of 4.2 nM.
Finally, we compared the effects of GnRH pretreatment on Ca 2ϩ response with GnRH and to PACAP38, another peptide that acts via GPCRs to activate PLC in ␣T3-1 cells (36,37). As expected, pretreatment for 60 min with 0.1 M GnRH abolished the spike response to GnRH seen in Ca 2ϩ -free medium (Fig. 10,  upper panel). PACAP38 (0.1 M) caused a comparable spiketype increase in Ca 2ϩ to GnRH, and this effect was also greatly diminished by GnRH pretreatment (Fig. 10, lower panel). DISCUSSION Gonadotropin-releasing hormone is released from the hypothalamus in a pulsatile manner and elicits pulsatile gonadotropin secretion. This stimulation pattern maintains responsiveness to GnRH, whereas continuous stimulation causes desensitization and attenuates gonadotropin secretion (16,38). Recent studies (24) have revealed, however, that GnRH-stimulated [ 3 H]IP total accumulation (an indicator of PLC activity measured in cells labeled with [ 3 H]inositol and stimulated in the presence of LiCl) remains linear for 10 min after stimulation. These data argue against rapid agonist-induced uncoupling of the GnRH receptor from its effector system (G-proteins activating PLC), possibly because it lacks the C-terminal tail, which has been implicated in homologous desensitization of other GPCRs and rhodopsin (39,40). Desensitization of GnRHinduced Ca 2ϩ responses can, however, be demonstrated within an intermediate time frame. Pretreatment for 60 min with GnRH causes a dose-dependent reduction in the ability of GnRH to increase Ca 2ϩ in ␣T3-1 cells (25) and comparable effects are seen in primary cultures of rat pituitary cells 2 and with GnRH receptors transfected into HEK-293 cells (34). This pretreatment caused desensitization of both spike and plateau phases of the Ca 2ϩ response to GnRH in ␣T3-1 cells and also decreased the effect of KCl on [Ca 2ϩ ] i (25), supporting the suggestion that desensitization occurs at the level of voltage operated Ca 2ϩ channels in gonadotropes and ␣T3-1 cells (25,41) but leaving the mechanism underlying desensitization of the spike Ca 2ϩ response unresolved.
In addition to attenuation of the effect of GnRH on [Ca 2ϩ ] i , pretreatment with GnRH has been shown to decrease GnRHstimulated [ 3 H]IP total accumulation by approximately 25% (25), but in these experiments total [ 3 H]IP total accumulation was measured in cells labeled with [ 3 H]inositol. This has the possible disadvantages that PLC activity directed against phospholipids other than PIP 2 may be quantified (21) and that the GnRH pretreatment may have altered the specific activity of the [ 3 H]inositol-labeled phospholipid substrate pool. Accordingly, we have now examined the relationship between GnRH effects on Ins(1,4,5)P 3 mass levels and on [Ca 2ϩ ] i in control and GnRH pre-treated cells. We show here that pretreatment for 60 min with 0.1 M GnRH causes desensitization of both spike and plateau phases of the Ca 2ϩ response to GnRH (Fig. 1, see also Ref. 25) and that desensitization of the spike response is relatively rapid in onset (half-time 10 -20 min) but is only slowly reversed (half-time 4 -6 h). Desensitization of the Ca 2ϩ response was associated with a reduction in the effect of GnRH on  6. Influence of depletion and refilling of the GnRH-mobilizable intracellular Ca 2؉ pool Ins(1,4,5)P 3 responses to GnRH in ␣T3-1 cells. Cells were pre-treated for 1 min in Ca 2ϩ -free medium with 0 (pool intact) or 0.1 M (pool emptied) GnRH and then washed and maintained in Ca 2ϩ -free medium for a period of 5 min during which one group of pool-depleted cells were exposed to Ca 2ϩ -containing medium for 1 min before being returned to Ca 2ϩ -free medium (pool emptied and refilled). The cells were then stimulated as indicated in Ca 2ϩ -free medium with 0.1 M GnRH before processing for Ins(1,4,5)P 3 measurement. The data are from three separate experiments normalized as a percentage of internal control responses (the maximum responses seen in pool intact cells) of 48.2 Ϯ 8.9 pmol/well. Ins(1,4,5)P 3 levels (to between 50 and 80% of control) without any measurable change in EC 50 .
Although pretreatment with GnRH clearly reduced the effect of GnRH on Ins(1,4,5)P 3 levels, this effect alone cannot explain the desensitization of the Ca 2ϩ response because the Ins(1,4,5)P 3 responses to 0.1 M GnRH in control cells and to 1 M GnRH in GnRH pre-treated cells were indistinguishable, yet 1 M GnRH caused very little increase in [Ca 2ϩ ] i in GnRH pre-treated cells, whereas the [Ca 2ϩ ] i response to 0.1 M GnRH is maximal in control cells (Figs. 3 and 9). An alternative possibility is that the attenuated [Ca 2ϩ ] i increase in the desensitized cells is the cause of the decrease in Ins(1,4,5)P 3 because in other systems Ca 2ϩ mobilization supports agonist stimulated Ins(1,4,5)P 3 generation, and depletion of intracellular Ca 2ϩ pools can underlie desensitization of Ins(1,4,5)P 3 responses (42,43). The demonstration that the GnRH effect on Ins(1,4,5)P 3 levels is attenuated in Ca 2ϩ -free medium supports this interpretation. Interestingly, the immediate (5 s) effect of GnRH on Ins(1,4,5)P 3 was only reduced by thapsigargin in Ca 2ϩ -free medium (Fig. 4). Thapsigargin specifically blocks the ATPase, which sequesters Ca 2ϩ into rapidly releasable pools in many cells (32), and as a consequence of continuous leak of Ca 2ϩ from these pools causes their depletion. Thapsigargin, thereby prevents mobilization of intracellular Ca 2ϩ by many ligands acting via PLC-linked receptors, including GnRH in ␣T3-1 cells (33)(34)(35). Inhibition of the immediate effect of GnRH on Ins(1,4,5)P 3 levels by thapsigargin in Ca 2ϩ -free medium implies that the maximal Ins(1,4,5)P 3 response requires Ca 2ϩ elevation and the lack of effect of thapsigargin in Ca 2ϩ -containing medium or of Ca 2ϩ -free medium alone suggests that the Ca 2ϩ requirement can be met either by mobilization or entry. Similarly, depletion of the hormone mobilizable Ca 2ϩ pool by brief pretreatment with GnRH reduced the immediate effect of GnRH on Ins(1,4,5)P 3 in Ca 2ϩ -free medium, and this inhibition was reversed by brief exposure to normal medium in order to replenish the Ca 2ϩ pool. Together these observations demonstrate that Ca 2ϩ mobilized from intracellular stores during the spike phase of the response to GnRH exerts a positive feedback effect on Ins(1,4,5)P 3 production, probably reflecting the Ca 2ϩ dependence of PLC (31). Accordingly, the data suggest that desensitization of the Ca 2ϩ response contributes to the observed reduction in Ins(1,4,5)P 3 rather than vice versa. Confirmation of the fact that this uncoupling does not occur at the level of the GnRH receptor is provided by the demonstration that GnRH pretreatment causes comparable inhibition of the effects of GnRH and of PACAP on [Ca 2ϩ ] i in these cells (Fig. 10).
The observations above raise the question of why Ins-(1,4,5)P 3 failed to increase [Ca 2ϩ ] i in GnRH pre-treated cells, and we have addressed the possibility that this simply reflects depletion of the hormone mobilizable Ca 2ϩ pool as seen in other systems (44). In Ca 2ϩ -free medium GnRH caused only a transient increase in [Ca 2ϩ ] i presumably because the hormonemobilizable Ca 2ϩ pool became rapidly depleted. This is verified by the fact that after extensive washing, a second stimulation with GnRH does not increase [Ca 2ϩ ] i unless the cells are first exposed to normal Ca 2ϩ -containing medium to enable pool refilling (Fig. 5, see also Ref. 32). Using this protocol, and assuming the maximal increase in [Ca 2ϩ ] i caused by GnRH to be directly proportional to the filling state of the GnRH mobilizable Ca 2ϩ pool, we estimate that pool refilling occurs with a half-time of 5-20 s (in Ca 2ϩ -containing medium without GnRH), which is in stark contrast to the slow recovery from desensitization of the spike effect of GnRH on [Ca 2ϩ ] i (halftime 4 -6 h). In all of the Ca 2ϩ imaging experiments described here, cells were removed from the pretreatment solution and then washed extensively in Ca 2ϩ -containing medium before transfer to the microscope stage for imaging. Imaging was therefore not started until at least 10 min after termination of the pretreatment, and it is most unlikely that pool depletion had been maintained for this time. Comparison of the rates of onset of desensitization and of pool depletion also argue against a causal relationship, because desensitization of the spike phase of the Ca 2ϩ response to GnRH occurred with a half-time of 10 -20 min (Fig. 1), whereas depletion of the GnRH-mobilizable Ca 2ϩ pool was complete within 1 min (Fig. 5).
To more directly test the involvement of Ca 2ϩ pool depletion, we exploited the fact that GnRH and ionomycin mobilize Ca 2ϩ from shared intracellular Ca 2ϩ pools so that responses to ionomycin in Ca 2ϩ -free medium can be used to measure the filling state of the hormone-mobilizable pool (33). In control cells GnRH and ionomycin caused comparable transient increase of [Ca 2ϩ ] i in Ca 2ϩ -free medium, and 0.1 M GnRH dramatically diminished the [Ca 2ϩ ] i increase on subsequent treatment with ionomycin. In cells pre-treated for 60 min with 0.1 M GnRH, ionomycin caused comparable increases in [Ca 2ϩ ] i to that seen in control cells indicating that the intracellular Ca 2ϩ pool was intact at the time of ionomycin addition. In contrast, in GnRHdesensitized cells, 0.1 M GnRH caused only a minor increase in Ca 2ϩ and very little reduction of the subsequent response to ionomycin (Fig. 8), suggesting that in these cells, GnRH is unable to increase Ca 2ϩ because it fails to mobilize this extant intracellular Ca 2ϩ pool. Comparison of concentration response curves from such experiments support this interpretation (Fig.  9). In control cells the brief exposure to GnRH in Ca 2ϩ -free medium reduced the subsequent effect of ionomycin to Ͻ15%, indicating that the vast majority of the ionomycin mobilizable pool is accessible to GnRH and did so with an IC 50 of 33 pM. In contrast, in the GnRH-desensitized cells the brief GnRH treatment only reduced the ionomycin response to 50%, with an IC 50 of 4200 pM. Comparison of EC 50 values for GnRH-stimulated Ins(1,4,5)P 3 accumulation (Fig. 3) and inhibition of the ionomycin effect on [Ca 2ϩ ] i provides an indication of the efficiency with which Ins(1,4,5)P 3 mobilizes Ca 2ϩ . In control cells GnRH is at least 2000 times more potent at mobilization of Ca 2ϩ than at elevation of Ins(1,4,5)P 3 (EC 50 values of 33 pM and 83 nM, respectively), whereas for the GnRH pre-treated cells the difference is less than 40-fold (EC 50 values of 4.2 and 134 nM, respectively). Thus desensitization of the [Ca 2ϩ ] i response to GnRH reflects both a reduction in the proportion of the intracellular ionomycin mobilizable Ca 2ϩ pool, which can be released by GnRH, and a reduction in the efficiency of such mobilization.
In summary, we have found no evidence for uncoupling of GnRH receptors from their immediate effector system (PLC activated by G q/11 ) even after a relatively protracted period of stimulation with GnRH. Instead, GnRH pretreatment apparently impairs the efficiency with which Ins(1,4,5)P 3 mobilizes Ca 2ϩ from intracellular stores in ␣T3-1 cells. Although the reason for this reduction is unknown, we are currently investigating the possible involvement of Ins(1,4,5)P 3 receptor regulation and cellular compartmentalization of the effector system. We suggest that this effect, together with desensitization of voltage-operated Ca 2ϩ channels, may underlie desensitization of GnRH-stimulated gonadotropin secretion and that ␣T3-1 cells provide a valuable model system for investigation of the post-receptor regulation of cellular responsiveness to PLC activating ligands.