Lowering Cyclic Adenosine-3',5'-Monophosphate (cAMP) Levels by Expression of a cAMP-Specific Phosphodiesterase Decreases Intrinsic Pulsatile Gonadotropin-Releasing Hormone Secretion from GT1 Cells
Hiroshi Yoshida,
Luis Beltran-Parrazal,
Paul Butler,
Marco Conti,
Andrew C. Charles and
Richard I. Weiner
Department of Obstetrics Gynecology and Reproductive Sciences (H.Y., P.B., R.I.W.), University of California San Francisco, San Francisco, California 94143; Department of Neurology (L.B.-P., A.C.C.), University of California Los Angeles, Los Angeles, California 90095; and Department of Obstetrics and Gynecology (M.C.), Stanford University, Stanford, California 94350
Address all correspondence and requests for reprints to: Richard Weiner, Ph.D., Department of Obstetrics, Gynecology and Reproductive Sciences, 513 Parnassus Avenue, HSE 1605, Box 0556, University of California San Francisco, San Francisco, California 94143. E-mail: weinerr{at}obgyn.ucsf.edu.
 |
ABSTRACT
|
---|
Pulsatile GnRH secretion is an intrinsic property of GnRH neurons. Since increases in cAMP levels increase excitability and GnRH secretion in the GT11 GnRH cell line, we asked whether cAMP levels play a role in timing excitability and intrinsic pulsatile GnRH secretion. The expression of the cAMP-specific phosphodiesterase (PDE4D1) was used in a genetic approach to lower cAMP levels. Cells were infected with an adenovirus vector (Ad) expressing PDE4D1 (PDE-Ad), or for controls with an empty Ad (Null-Ad) or an Ad expressing green fluorescent protein (GFP-Ad). Infection with the PDE-Ad significantly inhibited forskolin-induced increases in cAMP production, GnRH secretion, and Ca2+ oscillations. Infection of GT11 cells with the PDE-Ad vs. GFP-Ad or Null-Ad controls significantly decreased spontaneous Ca2+ oscillations and inhibited the frequency of GnRH pulses. These data support the hypothesis that the level of cAMP in GT1 neurons is a component of the biological clock timing neuron excitability and pulsatile GnRH secretion. Genetically targeted expression of PDE4D1 represents a powerful approach to study the role of cAMP levels in specific populations of neurons in transgenic animals.
 |
INTRODUCTION
|
---|
REPRODUCTIVE FUNCTION IN male and female mammals is regulated by the hypothalamic/pituitary/gonadal endocrine axis. The pulsatile release of GnRH is the driving force for the secretion of the gonadotropins, LH and FSH, from the anterior pituitary (1). Understanding the molecular mechanisms regulating pulsatile GnRH secretion has been hampered by the low number of GnRH neurons, their scattered localization throughout the preoptic area in rodents, and the complexity of the afferent inputs. The development of the highly differentiated GT1 GnRH neuronal cell lines provided a model to study the signaling mechanisms involved in the complex regulation of GnRH secretion (2). The cells express and process GnRH at high levels (2, 3). The pulsatile release of GnRH is an intrinsic property of cultured GT1 cells (4, 5, 6). Pulsatile GnRH secretion has been observed from endogenous GnRH neurons cultured from the olfactory placode of the monkey (7), rat (8), and sheep (9), although it must be noted that these cultures contain additional neuronal and glial elements in addition to GnRH neurons.
The secretion of GnRH from GT1 neurons was stimulated by elevations in intracellular cAMP (10, 11, 12). Treatment of GT1 cells with dopamine or norepinephrine increased intracellular cAMP levels and stimulated GnRH secretion in a dose-dependent fashion (10, 11). The GnRH-releasing effects of dopamine and norepinephrine are mimicked by pharmacologically increasing the intracellular cAMP levels with 8-bromo-cAMP or forskolin (FSK) treatment (12).
The stimulation of GnRH secretion by elevated cAMP levels may be regulated via cAMP-gated cation channels. We showed that GT1 neurons express the three subunits that comprise the olfactory cyclic nucleotide-gated (CNG) channels (CNG 2, 4.3, and 5) and that the subunits form functional channels with similar electrophysiological properties to those observed in olfactory neurons (13, 14). It is interesting that both the olfactory and GnRH neurons develop from the olfactory placode (15, 16). The same three CNG channel subunits are also expressed in endogenous rat GnRH neurons (17).
CNG channels appear to play an important role in regulating the excitability of GT1 neurons (14). Elevations in cAMP levels increase Ca2+ oscillations in GT1 neurons. Simultaneous on-cell patch clamp and fluorescence imaging studies show that Ca2+ oscillations in GT11 cells were preceded by action potential firing, supporting the idea that Ca2+ oscillations are a reflection of neuron excitability (18). Blockade of CNG channels with L-cis-diltiazem prevents the stimulation of Ca2+ oscillations by increased cAMP levels (14). Since depolarization of GT1 cells results in the opening of voltage-gated Ca2+ channels and exocytosis of GnRH (6, 19), these findings are consistent with the idea that CNG channels were involved in mediating cAMP-induced GnRH release.
Clearly, increasing cAMP levels increases GT1 cell excitability and GnRH secretion, but the effect of decreasing cAMP levels was not known. This question was particularly relevant concerning the role of cAMP levels in timing intrinsic GnRH pulsatility. Intracellular cAMP levels are lowered by hydrolysis by a family of cAMP-specific phosphodiesterases (PDE) (20). We chose a genetic approach to lower cAMP levels by expressing the constitutively active, cAMP-specific PDE4D1 splice variant (21). Stable transfection of MA-10 Leydig cells with PDE4D1 increased PDE activity in the cells and prevented the human chorionic gonadotropin-induced increase in cAMP and steroidogenesis (22). We show that the expression of PDE4D1 in GT1 cells using an adenovirus vector substantially inhibited the FSK-induced stimulation of cAMP levels, Ca2+ oscillations, and GnRH secretion. Supporting the hypothesis that cAMP levels participate in the timing of intrinsic pulsatile GnRH release, we demonstrate that expression of PDE4D1 inhibited spontaneous Ca2+ oscillations and the frequency of spontaneous GnRH pulses.
 |
RESULTS
|
---|
PDE-Ad Infection of GT1 Cells
To decrease cAMP levels, we increased the expression of the constitutively active PDE4D1. Since only a small portion of GT1 cells are transiently transfected by available techniques, we used an Ad vector to direct expression to the majority of GT1 cells. Infection with the green fluorescent protein (GFP)-Ad at 5 plaque-forming units (pfu)/cell resulted in the expression of GFP in 9095% of GT1 cells as indicated by fluorescence microscopy. Infection with up to 30 pfu/cell had no discernable effect on the morphology of the cells. GT11 cells infected for 48 h with 10 pfu/cell of the PDE-Ad showed a 4.2-fold increase in PDE activity compared with the Null-Ad-infected control (Fig. 1A
). The PDE activity increased with the viral load [infection with 10 pfu/cell caused a significantly greater increase in PDE activity than with 5 pfu/cell (P < 0.05)]. The increased PDE activity was inhibited to values below basal levels in the wild-type (WT) cells by addition of Rolipram, a PDE4-specific inhibitor. The observation that endogenous PDE levels were also decreased by Rolipram treatment in WT cells was consistent with the findings that GT11 cells express PDE4B and PDE4D (23). These data showed that the PDE-Ad vector was capable of infecting GT1 cells at low virus concentrations and that the increase in PDE activity by the PDE-Ad was due to the expression of PDE4D1.

View larger version (19K):
[in this window]
[in a new window]
|
Fig. 1. Effect of Infection with Null-Ad or PDE-Ad on PDE Activity (A) or cAMP Levels (B)
A, PDE enzymatic activity (black bars) in uninfected GT11 neurons (WT) or in neurons infected for 48 h with 10 pfu/cell of the Null-Ad vector or 5 or 10 pfu/cell of the PDE-Ad vector. PDE enzymatic activity was also measured in the same groups in the presence of the specific PDE4 inhibitor, Rolipram (50 µM, gray bars). Values are the mean ± SEM, n = 4; *, P < 0.01 relative to the Null-Ad values. B, Release of cAMP into the culture medium (extracellular cAMP) in uninfected GT11 neurons (WT), or neurons infected for 48 h with 20 pfu/cell of the Null-Ad vector or increasing amounts of the PDE-Ad vector (1, 2.5, 5, 10, and 20 pfu/cell). Values are the mean ± SEM; n = 6. *, P < 0.01 relative to the Null-Ad values.
|
|
We also asked whether infection with PDE-Ad decreased basal cAMP levels. GT11 cells were infected with increasing levels of the PDE-Ad (1, 2.5, 5, 10, and 20 pfu/cell) for 48 h. We measured the release of cAMP from the cells for a 30-min period. In many cells, including GT1 cells, increases in intracellular cAMP levels are mimicked by the rapid transport of cAMP into the culture medium (13, 24). There was a dose-dependent inhibition of the release of cAMP from the PDE-Ad-infected cells that reached a maximum of 60% with 20 pfu/cell compared with the Null-Ad-infected cells (P < 0.01) (Fig. 1B
).
Inhibition of FSK-Induced Increases in cAMP and GnRH Secretion
We then determined whether expression of the PDE-Ad would block the FSK-induced increase in cAMP accumulation and GnRH secretion. FSK increases the production of cAMP by stimulating adenylate cyclase activity. We previously reported that increased cAMP levels stimulated GnRH secretion in GT11 neurons, but we had not shown that inhibiting the FSK-induced increase in cAMP would prevent the stimulation of GnRH secretion. Infection with 10 pfu/cell of the PDE-Ad vector inhibited 90% of the FSK-induced increase in GnRH secretion relative to neurons infected with 10 pfu/cell of the Null-Ad vector (Fig. 2A
). FSK-induced stimulation of cAMP release in PDE-Ad-infected neurons was only 48% of that observed in Null-Ad-infected neurons (Fig. 2A
). In a second experiment using infection with GFP-Ad as a control, we obtained similar results (Fig. 2B
). Infection with 10 pfu/cell of the PDE-Ad inhibited 66% of the FSK-induced stimulation of GnRH secretion and 78% of the cAMP release. There was no significant effect of infection with the Null-Ad or GFP-Ad vectors on basal or FSK-induced GnRH or cAMP release relative to uninfected controls.

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 2. FSK-Induced Stimulation (0.5 µM for 30 min) of GnRH and cAMP Release in Static Culture from
A, Uninfected GT11 neurons (WT) or neurons infected for 48 h with 10 pfu/cell of the Null-Ad vector or 10 pfu/cell of the PDE-Ad vector; and B, uninfected GT11 neurons (WT) or neurons infected for 48 h with 10 pfu/cell of the GFP-Ad vector or 10 pfu/cell of the PDE-Ad vector. Levels of GnRH in the medium (black bars) or medium plus FSK (gray bars). Levels of cAMP in the medium (black bars) or medium plus FSK (gray bars). Values are the mean ± SEM; n = 4. *, P < 0.05 relative to GFP-Ad value; **, P < 0.01 relative to the Null-Ad or GFP-Ad values.
|
|
Inhibition of FSK-Induced Increases in Ca2+ Oscillations
GT11 cells show spontaneous Ca2+ oscillations that are generated by spontaneous depolarizations and bursts of action potentials (18). We had previously shown that FSK-induced increases in cAMP increased the number of Ca2+ oscillations in GT11 cells (14). We asked whether inhibition of the FSK-induced increases in cAMP by the PDE-Ad would inhibit the stimulation of Ca2+ oscillations by FSK. A response to FSK was defined as a Ca2+ oscillation frequency increase of at least 50% compared with baseline. In cells infected with PDE-Ad (5 pfu/cell), the percentage of neurons responding to FSK with an increase in the frequency of Ca2+ oscillations was significantly reduced compared with GFP-Ad-infected neurons (47 ± 12% of PDE-Ad cells vs. 87 ± 7% of GFP-Ad cells, n = 120 cells in four experiments for each condition, P < 0.03) (Fig. 3
, A and B). GFP-Ad cells were used as the primary controls, because we could verify by fluorescence that the cells were, in fact, infected. Results obtained with Null-Ad cells were not significantly different from those obtained with GFP-Ad (not shown). In PDE-Ad cells that did respond to FSK, the response was delayed and the increase in frequency of Ca2+ oscillations was smaller than in control cells. Treatment with Ba2+ (100 µM), which we have previously shown to inhibit K+ channels in GT1 cells (25), evoked either an increase in the frequency of Ca2+ oscillations or a sustained elevation in [Ca2+]i (intracellular Ca2+ concentration) in all PDE-Ad cells that was similar to that observed in control GT1 cells, indicating that PDE-Ad-infected cells were able to respond with an increase in Ca2+ signaling to stimuli not involving cAMP.

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 3. Effect of Infection with (A) GFP-Ad (5 pfu/cell, 48 h) or (B) PDE-Ad (5 pfu/cell, 48 h) on Basal and FSK-Stimulated Ca2+ Signaling in GT11 Cells
Raster plots show changes in [Ca2+]i indicated by change in fura2 fluorescence vs. time for 20 neighboring cells in a microscopic field. Each row in the plot represents an individual cell, with changes in fura2 fluorescence indicated by the gray scale. The line tracing at the top of the raster shows a representative individual cell. The amplitude of F oscillations varied widely from cell to cell. Although GFP caused an increased background fluorescence that affected the F oscillation amplitude, the frequency of Ca2+ oscillations could still be reliably measured in cells expressing GFP. Bath application of FSK (10 µM) evoked a sustained increase in the frequency of Ca2+ oscillations in cells infected with GFP-Ad (A). By contrast, the FSK evoked an increase in Ca2+ oscillation frequency in the minority of PDE-Ad cells (B). In PDE-Ad cells that did respond to FSK, this response occurred after a significant delay. Both PDE-Ad- and GFP-Ad-infected cells showed an increase in Ca2+ oscillation frequency as well as in baseline [Ca2+]i in response to the K+ channel blocker Ba2+.
|
|
Inhibition of Spontaneous Ca2+ Oscillations
We performed additional experiments to quantify the inhibition of spontaneous Ca2+ oscillations by infection with the PDE-Ad over extended time periods. Ca2+ oscillations were defined as transient changes in fura fluorescence lasting at least 3 sec and with an amplitude of at least three times the baseline noise. GT11 neurons expressing PDE4D1 showed a significant reduction in the frequency of spontaneous oscillations in intracellular Ca2+ concentration compared with control cells infected with an GFP-Ad vector (average 0.35 ± 0.02 vs. 0.94 ± 0.11 oscillations/min over 50 min, respectively, n = 120 cells in four experiments for each condition, P < 0.001) (Fig. 4
, A and B). Data from cells infected with the Null-Ad vector were not significantly different from the GFP-Ad cells (not shown). These results demonstrated that lowering cAMP levels decreased spontaneous Ca2+ signaling in GT11 cells, indicating reduced spontaneous excitability.

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 4. Effect of Infection with (A) GFP-Ad (5 pfu/cell, 48 h) or (B) the PDE4D1-Ad (5 pfu/cell, 48 h) on Spontaneous Ca2+ Oscillations in GT11 Cells
In the upper panels, Ca2+ oscillations in 18 neighboring cells in a microscopic field are schematically represented. The lower panels show line tracings of changes in fura2 fluorescence in individual cells (cell no. 2 for A and B). GFP-Ad infected cells showed a significantly higher frequency of spontaneous Ca2+ oscillations compared with PDE-Ad infected cells. Ca2+ oscillation frequency in Null-Ad-infected cells was the same as was observed with GFP-Ad-infected cells (not shown).
|
|
Inhibition of Spontaneous Pulsatile GnRH Release
Finally we determined whether infection with the PDE-Ad would affect spontaneous pulsatile GnRH release observed in perifused GT11 cells (Fig. 5
). In experiment 1, samples were obtained every 4 min from a chamber with a volume of 350 µl, and in experiment two samples were obtained every 2 min from a chamber with a volume of 150 µl. In both experiments infection with 5 pfu/ml of the PDE-Ad vector significantly inhibited the frequency of spontaneous GnRH pulses, interpulse interval, relative to the Null-Ad infected neurons (Table 1
). Infection with the Null-Ad had no significant effect on the frequency of GnRH pulses compared with uninfected GT11 neurons. Therefore, the decrease in spontaneous Ca2+ oscillations after treatment with PDE-Ad correlated closely with a decrease in the frequency of spontaneous GnRH pulses. The mean peak amplitude in PDE-Ad-infected GT11 neurons was unaltered relative to the Null-Ad-infected neurons in both experiments (Table 1
). However, in experiment 2 but not experiment 1 there was a significant decrease (P < 0.01) in the mean peak amplitude in PDE-Ad-infected neurons relative to uninfected neurons. A significant decrease in mean peak amplitude was also observed between the Null-Ad-infected neurons and the uninfected neurons in experiment 2. Therefore, no clear effect of PDE-Ad infection on GnRH pulse amplitude, independent of adenovirus infection, could be observed. It is important to point out that more pulses were detected in experiment 2 than in experiment 1. In experiment 2 more frequent samples were obtained (every 2 vs. 4 min). More frequent sampling can increase the observed frequency of pulses (26). This increase in pulse frequency could also be related to the difference in the volumes of the chambers. In both experiments, GT11 cells were plated at similar densities on a single coverslip. Therefore, the concentration of putative paracrine factors involved in synchronizing pulsatile GnRH release might be different in the two chambers.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5. Spontaneous GnRH Release in Uninfected GT11 Neurons (WT), Neurons Infected for 48 h with 5 pfu/cell of the Null-Ad Vector or 5 pfu/cell of the PDE-Ad
Samples were obtained every 2 min for 180 min from cells perifused in a 125-µl chamber (experiment 2). GnRH levels were measured by RIA in singlicate, and data were analyzed for pulsatile secretion using cluster analysis (*pulse).
|
|
 |
DISCUSSION
|
---|
These findings support the hypothesis that the level of cAMP in GT1 GnRH neurons plays an important role in regulating the frequency of spontaneous Ca2+ oscillations and GnRH pulses. Decreasing cAMP levels by expression of the PDE-Ad significantly decreased the frequency of Ca2+ oscillations and spontaneous GnRH pulses. These findings are in close agreement with studies in the GPR-4 transgenic rat, where targeted expression of PDE4D1 to GnRH neurons resulted in a 2- to 3-fold decrease in the LH pulse frequency in castrated male and female rats (27). In the study with transgenic rats it was hypothesized that expression of PDE4D1 lowered cAMP levels in the GnRH neurons. The findings in the current study strengthen this interpretation. The pacemaker currents underlying spontaneous bursts of Na+-dependent action potentials observed in GT1 neurons are still poorly understood (18). It has been hypothesized that several K+ conductances including inwardly rectified K+ channels may be involved (25). CNG channels have also been identified in GnRH neurons including GT1 cells, and these channels may represent a pathway by which cAMP levels can directly modulate the excitability of GnRH neurons by altering the depolarizing drive that underlies spontaneous oscillations in membrane potentials (14). A mathematical model of the spontaneous excitability of GT1 neurons that includes a cation conductance through cAMP-gated channels reproduces the experimental activity of GT1 neurons (28). The decrease in excitability of GT1 neurons associated with expression of PDE4D1 closely correlated with a decrease in pulsatile GnRH release. The mechanism by which decreased excitability of individual neurons leads to inhibition of coordinated GnRH secretion from a large number of cells (pulse) are still poorly understood. In addition to oscillations in individual cells, increases in [Ca2+]i also occur as intercellular Ca2+ waves that are propagated across hundreds of GT11 neurons (18). Coordination of the wave appears to involve gap junctions in GT1 cells (18). The current data provide a link between decreased neuron excitability and decreased pulsatile GnRH release but do not speak directly to the mechanism involved in the coordination of GnRH pulses. One mechanism that was proposed for the timing of GnRH pulses was the paracrine feedback action of GnRH (29). GnRH receptors were shown to be expressed on GT17 neurons and a switching mechanism between GnRH receptors and Gs and Gi proposed to regulate cyclic changes in activation of adenylate cyclase (30). Low levels of GnRH were reported to increase cAMP levels, whereas high levels inhibited cAMP levels. Although cyclical changes in cAMP are known to function as biological clocks (31), there is no clear evidence that cAMP levels cycle in GnRH neurons or GT1 cells. Global changes in cAMP levels may not be informative in answering this question. Recent data obtained by fluorescence resonance energy transfer analysis in living myocytes showed that ß-adrenergic agonist-induced increases in cAMP levels occurred in discrete microdomains (32).
A model for the pulsatile behavior of GnRH neurons, which predicts that a network of interconnected cells with periodic alterations in cell excitability would show pulsatile behavior, has been described (33). This model does not require pacemaker cells. cAMP levels could constitute a timing mechanism for pulsatile GnRH release by periodically altering cell excitability. Alterations in cell excitability would make it more or less likely that a sufficient number of GnRH neurons concurrently depolarize to generate a pulse of GnRH.
The findings with the PDE-Ad in GT1 cells demonstrate the promise of this approach in overcoming problems associated with the transient expression of cDNAs in these cells. Even with low levels of virus, the vast majority of cells expressed the targeted protein. Increased levels of PDE activity were obtained by infection with relatively low levels (510 pfu/cell) of Ad vectors that did not appear to have significant nonspecific effects on the cells. These data also demonstrate the effectiveness of the use of expression of PDE4D1 to decrease cAMP levels. This genetic approach should have wide application both in studies with cultured cells and in vivo in transgenic animals. One potential problem that must be kept in mind when using this approach is that at least 2448 h post infection are necessary to obtain sufficient levels of PDE. During this time the prolonged decrease in cAMP could increase or decrease the level of expression of numerous genes.
The physiological significance of findings in GT1 cells must always be confirmed in animal experiments. In the case of the role of cAMP regulating the secretion of GnRH from endogenous neurons, the concurrence of the findings is impressive. The FSK-induced increase in cAMP levels in fragments of the rat median eminence stimulated GnRH release (34) as it does in GT1 cells. We have shown by in situ hybridization and double immunostaining that endogenous GnRH neurons express the same three CNG channel subunits expressed in GT1 cells (17). Some mechanism must exist for GnRH neurons to coordinate secretion from the subset of neurons constituting a pulse. The median eminence is the only region in which large numbers of GnRH neurons can directly communicate through physical approximation or via paracrine mechanisms involving GnRH. Since the CNG channel subunit proteins are observed in GnRH nerve terminals in the median eminence, CNG channels could participate in the coordination of pulsatile secretion. As discussed earlier, the observation that expression of PDE4D1 in GT11 cells decreased the GnRH pulse frequency is in perfect agreement with the decrease in pulsatile LH secretion in GPR-4 transgenic rats (27).
 |
MATERIALS AND METHODS
|
---|
Production of PDE-Ad, GFP-Ad, or Null-Ad Vectors
We used the Adeno-X Expression System (CLONTECH, Palo Alto, CA) to construct the Ad expression vectors. The PDE4D1 (obtained from Marco Conti) or enhanced GFP (CLONTECH) cDNAs were inserted into the pShuttle vector. The expression cassette from the pShuttle was excised and inserted into the Adeno-X viral DNA. For the Null-Ad the expression cassette containing no insert was ligated into the Adeno-X viral DNA. Recombinant Adeno-X viral DNA was purified and analyzed by restriction mapping to confirm that it contained the PDE4D1 and enhanced GFP cDNA. The recombinant adenoviral DNA was digested with PacI and transfected into HEK 293 cells by Lipofectamine 2000 (Life Technologies, Inc., Carlsbad, CA). Lysates were used to reinfect HEK293 cells for large-scale production. The virus was purified on two consecutive cesium chloride gradients, dialyzed, and titered (35). The titer of the purified recombinant virus was determined in HEK 293 cells by a cell lysis assay in 96-well microtiter plates. The titer of viral preparations used in the studies ranged from 109 to 1010 pfu/ml.
Static Culture Studies
GT11 cells (passages 1923) were cultured on 10-cm culture plates in DMEM (Life Technologies, Inc.) supplemented with 5% fetal bovine serum (HyClone Laboratories, Inc., Logan, UT), 5% horse serum (HyClone Laboratories, Inc.), 100 U/ml penicillin, and 100 µg/ml streptomycin. For static culture experiments, GT11 cells (100,000 cells per well) were plated on 24-well plates. At 3040% confluency, cells were infected with 510 pfu/cell of PDE-Ad, GFP-Ad, or Null-Ad for 2 h. Forty-eight hours later, cells were preincubated for 30 min in Lockes medium (154 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl2, 1 mM MgCl2, 6 mM NaHCO3, 10 mM glucose, 2 mM HEPES) supplemented with 0.1% BSA and 20 µM bacitracin. Drugs were added for 30 min in Lockes medium. Conditioned media were collected, centrifuged, boiled for 3 min, and stored at -20 C for RIA of GnRH and cAMP.
Perifusion Studies
Approximately 250,000 GT11 cells (passages 1825) were plated on Matrigel (Collaborative Biomedical Products, Bedford, MA) or laminin (Sigma, St. Louis, MO)-coated 25-mm plastic coverslips (Nulge Nunc International, Naperville, IL). At 50% confluency, cells were infected by 5 or 10 pfu/cell of PDE-Ad or Null-Ad for 48 h as above. Medium was replaced with Opti-MEM 24 h before sampling. One cell-coated coverslip was perifused in modified Sykes-Moore chambers (Bellco Glass, Inc., Vineland, NJ). The coverslip formed one side of the chamber, separated from the top of the chamber by an o-ring. The volume of the chamber was either 125 or 350 µl depending on the thickness of the o-ring used. Cells were perifused at flow rates from 100150 µl/min with oxygenated Lockes medium. Chambers were washed for 60 min, and then samples were obtained every 4 min for 2 h with the 350-µl chamber (experiment 1), and every 2 min for 3 h with the 125-µl chamber (experiment 2). Each sample was boiled for 3 min and stored at -20 C for RIA. Analysis of the GnRH pulse data was performed using the hormone pulse analysis software Cluster 7. In experiments with samples obtained every 2 min from the 125-µl chamber, cluster analysis was performed on measurements done in singlicate (36). The coefficient of variation was determined from intraassay controls. In experiments with samples obtained from the 350-µl chamber every 4 min, cluster analysis was performed on measurements done in duplicate. Cluster size or nadir was defined by three points that significantly increase or decrease with a t statistic of 3.
Imaging Ca2+ Oscillations
[Ca2+]i was measured using a fluorescence video imaging system that was previously described in detail (37). GT11 cells cultured on poly-D-lysine-coated glass coverslips were loaded with fura2 by incubation in 5 mM fura2-AM for 40 min. Coverslips were excited with a mercury lamp through 340- and 380-nm bandpass filters, and fluorescence at 510 nm was recorded through a x10 or x20 objective with a silicon-intensified target camera or charge-coupled device camera to VHS videotape or to an optical memory disc recorder. Images were digitized, background subtracted, and shading corrected, after which [Ca2+]i was calculated on a pixel-by-pixel basis by a Data Translation frame grabber and image analysis board (Data Translation, Marlboro, MA). Alternatively, fluorescence data recorded on the VHS videotape were digitized and analyzed with an Axon Image Lightning board and Axon Image Workbench software (Axon Instruments, Inc., Foster City, CA). Mini Analysis Program (Synaptosoft) and a peak detection program written with Labview (National Instruments, Austin, TX) were used to quantify the frequency and duration of fura2 fluorescence changes. The software program Transform (Research Systems Inc., Boulder, CO) was used to generate raster plots and line graphs of Ca2+ data. Experiments were carried out in Hanks balanced salt solution with 10 mM HEPES buffer (pH 7.4), at 22 C. Hanks balanced salt solution consisted of in mM concentration: 136.9 NaCl, 0.34 Na2PO4, 5.6 KCl, 0.44 KH2PO4, 0.81 MgSO4, 1.26 CaCl2, and 5.5 D-glucose (Mediatech, Herndon, VA). FSK was applied by perifusion of the coverslips with at least 2 ml of buffer containing the agent.
PDE Assay
To measure PDE activity, cells were harvested in hypotonic homogenizing buffer [10 mM Tris HCl (pH 7.4), 2 mM EDTA, 2 mM EGTA, 5 mM sodium pyrophosphate] supplemented with a protease inhibitor cocktail (Roche Diagnostic Co., Indianapolis, IN). Supernatants were added to the reaction buffer [40 mM Tris HCl (pH 8.0), 10 mM MgCl2, 5 mM 2-mercaptoethanol, 1 µM cAMP, 0.1% BSA, 12.5 nM [H3]cAMP (NEN Life Science Products, Boston, MA)] (38). The reaction was stopped by boiling, and 50 µg of snake venom (Opiophagus hannah) was added to convert [3H]5'-AMP to [3H]adenosine. [3H]Adenosine was separated from other labeled molecules using Bio-Rad anion exchange resin (AG1-x2, 200400 mesh, Bio-Rad Laboratories, Hercules, CA) and counted by liquid scintillation. Activity was expressed as nanomoles of cAMP hydrolyzed per min/mg of cellular protein. To identify the cAMP-specific phosphodiesterase activity, a specific inhibitor of the cAMP-specific PDE4, Rolipram (Sigma, St. Louis, MO), was added to the incubation mixture at a final concentration of 50 µM.
GnRH RIA
Levels of GnRH in media from static culture and perifusion studies were determined by a RIA using rabbit polyclonal antibody R1245 (obtained from T. Nett). This antiserum is specific for intact GnRH (39). Synthetic human GnRH (Sigma) was used both for iodination and as the standard. All samples from an experiment were analyzed in the same assay. The limit of detection of the assay was 0.2525 pg/tube, and the intraassay coefficient of variation was 7.0%. The limit of detection of the assay was defined as 90% of maximal binding.
cAMP RIA
Levels of cAMP in the media, perifusates, and cell extracts were determined in duplicate with a RIA using a rabbit anti-cAMP polyclonal antibody (Calbiochem, San Diego, CA). Samples were acetylated with triethylamine-acetic anhydride (2:1). All samples from an experiment were analyzed in the same RIA in duplicate. The range of detection of the assay was 0.5250 fmol/tube, and the intraassay coefficient of variation was 7.8%. The limit of detection of the assay was defined as 90% of maximal binding.
Statistical Analysis
All values are presented as the mean ± SEM unless stated otherwise. All statistical analyses were performed by one-way ANOVA and unpaired t test. In one instance the percentage of cells showing an increase in Ca2+ oscillations in response to FSK was statistically analyzed using the Mann-Whitney U test.
 |
ACKNOWLEDGMENTS
|
---|
We would like to recognize the participation of Jean Louis Vigne, Amy Choi, and Sreenivasan Paruthiyil in technical aspects of this work.
 |
FOOTNOTES
|
---|
This work was supported by NIH Grants HD08924 and HD41996 (R.W.) and National Science Foundation Grant IBN-9982585 (A.C.).
Abbreviations: Ad, Adenovirus vector; [Ca2+]i, intracellular calcium concentration; CNG, cyclic nucleotide gated; FSK, forskolin; GFP, green fluorescent protein; PDE, phosphodiesterase; pfu, plaque-forming units.
Received for publication February 21, 2003.
Accepted for publication June 19, 2003.
 |
REFERENCES
|
---|
- Knobil E 1980 The neuroendocrine control of the menstrual cycle. Recent Prog Horm Res 36:5388[Medline]
- Mellon PL, Windle JJ, Goldsmith PC, Padula CA, Roberts JL, Weiner RI 1990 Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5:110[Medline]
- Wetsel WC, Mellon PL, Weiner RI, Negro-Vilar A 1991 Metabolism of pro-luteinizing hormone-releasing hormone in immortalized hypothalamic neurons. Endocrinology 129:15841595[Abstract]
- Martinez de la Escalera G, Choi AL, Weiner RI 1992 Generation and synchronization of gonadotropin-releasing hormone (GnRH) pulses: intrinsic properties of the GT11 GnRH neuronal cell line. Proc Natl Acad Sci USA 89:18521855[Abstract]
- Wetsel WC, Valenca MM, Merchenthaler I, Liposits Z, Lopez FJ, Weiner RI, Mellon PL, Negro-Vilar A 1992 Intrinsic pulsatile secretory activity of immortalized luteinizing hormone-releasing hormone-secreting neurons. Proc Natl Acad Sci USA 89:41494153[Abstract]
- Krsmanovic LZ, Stojilkovic SS, Merelli F, Dufour SM, Virmani MA, Catt KJ 1992 Calcium signaling and episodic secretion of gonadotropin-releasing hormone in hypothalamic neurons. Proc Natl Acad Sci USA 89:84628466[Abstract]
- Terasawa E, Keen KL, Mogi K, Claude P 1999 Pulsatile release of luteinizing hormone-releasing hormone (LHRH) in cultured LHRH neurons derived from the embryonic olfactory placode of the rhesus monkey. Endocrinology 140:14321441[Abstract/Free Full Text]
- Funabashi T, Daikoku S, Shinohara K, Kimura F 2000 Pulsatile gonadotropin-releasing hormone (GnRH) secretion is an inherent function of GnRH neurons, as revealed by the culture of medial olfactory placode obtained from embryonic rats. Neuroendocrinology 71:138144[CrossRef][Medline]
- Duittoz AH, Batailler M 2000 Pulsatile GnRH secretion from primary cultures of sheep olfactory placode explants. J Reprod Fertil 120:391396[Abstract/Free Full Text]
- Martinez de la Escalera G, Gallo F, Choi AL, Weiner RI 1992 Dopaminergic regulation of the GT1 gonadotropin-releasing hormone (GnRH) neuronal cell lines: stimulation of GnRH release via D1-receptors positively coupled to adenylate cyclase. Endocrinology 131:29652971[Abstract]
- Martinez de la Escalera G, Choi AL, Weiner RI 1992 ß1-Adrenergic regulation of the GT1 gonadotropin-releasing hormone (GnRH) neuronal cell lines: stimulation of GnRH release via receptors positively coupled to adenylate cyclase. Endocrinology 131:13971402[Abstract]
- Martinez de la Escalera G, Choi AL, Weiner RI 1995 Signaling pathways involved in GnRH secretion in GT1 cells. Neuroendocrinology 61:310317[Medline]
- Vitalis B, Costantin JL, Tsai P-S, Sakakibara H, Paruthiyil S, Iiro T, Martini J-F, Taga M, Choi ALH, Charles AC, Weiner RI 2000 Role of the cAMP signaling pathway in the regulation of GnRH secretion in GT1 cells. Proc Natl Acad Sci USA 97:18611866[Abstract/Free Full Text]
- Charles A, Weiner R, Costantin J 2001 cAMP modulates the excitability of immortalized hypothalamic (GT1) neurons via a cyclic nucleotide gated channel. Mol Endocrinol 15:9971009[Abstract/Free Full Text]
- Schwanzel-Fukuda M, Pfaff DW 1989 Origin of luteinizing hormone-releasing hormone neurons. Nature 338:161164[CrossRef][Medline]
- Wray S, Grant P, Gainer H 1989 Evidence that cells expressing luteinizing hormone-releasing hormone mRNA in the mouse are derived from progenitor cells in the olfactory placode. Proc Natl Acad Sci USA 86:81328136[Abstract]
- El Majdoubi M, Weiner RI 2002 Localization of olfactory cyclic nucleotide-gated channels in rat gonadotropin-releasing hormone neurons. Endocrinology 143:24412444[Abstract/Free Full Text]
- Costantin JL, Charles AC 1999 Spontaneous action potentials initiate rhythmic intercellular calcium waves in immortalized hypothalamic (GT11) neurons. J Neurophysiol 82:429435[Abstract/Free Full Text]
- Charles AC, Hales TG 1995 Mechanisms of spontaneous calcium oscillations and action potentials in immortalized hypothalamic (GT17) neurons. J Neurophysiol 73:5664[Abstract/Free Full Text]
- Conti M, Nemoz G, Sette C, Vicini E 1995 Recent progress in understanding the hormonal regulation of phosphodiesterases. Endocr Rev 16:370389[Medline]
- Sette C, Vicini E, Conti M 1994 The ratPDE3/IVd phosphodiesterase gene codes for multiple proteins differentially activated by cAMP-dependent protein kinase. J Biol Chem 269:1827118274[Abstract/Free Full Text]
- Swinnen JV, DSouza B, Conti M, Ascoli M 1991 Attenuation of cAMP-mediated responses in MA-10 Leydig tumor cells by genetic manipulation of a cAMP-phosphodiesterase. J Biol Chem 266:1438314389[Abstract/Free Full Text]
- Sakakibara H, Conti M, Weiner RI 1998 Role of phosphodiesterases in the regulation of gonadotropin-releasing hormone secretion in GT1 cells. Neuroendocrinology 68:365373[CrossRef][Medline]
- Strewler GJ 1984 Release of cAMP from a renal epithelial cell line. Am J Physiol 246:C224C230
- Costantin JL, Charles AC 2001 Modulation of Ca(2+) signaling by K(+) channels in a hypothalamic neuronal cell line (GT11). J Neurophysiol 85:295304[Abstract/Free Full Text]
- Urban RJ, Johnson ML, Veldhuis JD 1989 Biophysical modeling of sensitivity and positive accuracy of detecting episodic endocrine signals. Am J Physiol 257:E88E94
- Paruthiyil S, El Majdoubi M, Conti M, Weiner RI 2002 Phosphodiesterase expression targeted to gonadotropin-releasing hormone neurons inhibits luteinizing hormone pulses in transgenic rats. Proc Natl Acad Sci USA 99:1719117196[Abstract/Free Full Text]
- LeBeau AP, Van Goor F, Stojilkovic SS, Sherman A 2000 Modeling of membrane excitability in gonadotropin-releasing hormone-secreting hypothalamic neurons regulated by Ca2+-mobilizing and adenylyl cyclase-coupled receptors. J Neurosci 20:92909297[Abstract/Free Full Text]
- Krsmanovic LZ, Martinez-Fuentes AJ, Arora KK, Mores N, Navarro CE, Chen HC, Stojilkovic SS, Catt KJ 1999 Autocrine regulation of gonadotropin-releasing hormone secretion in cultured hypothalamic neurons. Endocrinology 140:14231431[Abstract/Free Full Text]
- Krsmanovic LZ, Mores N, Navarro CE, Arora KK, Catt KJ 2003 An agonist-induced switch in G protein coupling of the gonadotropin-releasing hormone receptor regulates pulsatile neuropeptide secretion. Proc Natl Acad Sci USA 100:29692974[Abstract/Free Full Text]
- Tomchik KJ, Devreotes PN 1981 Adenosine 3',5'-monophosphate waves in Dictyostelium discoideum: a demonstration by isotope dilution-fluorography. Science 212:443446[Medline]
- Zaccolo M, Pozzan T 2002 Discrete microdomains with high concentration of cAMP in stimulated rat neonatal cardiac myocytes. Science 295:17111715[Abstract/Free Full Text]
- Gordan JD, Attardi BJ, Pfaff DW 1998 Mathematical exploration of pulsatility in cultured gonadotropin-releasing hormone neurons. Neuroendocrinology 67:217[CrossRef][Medline]
- Ojeda SR, Urbanski HF, Katz KH, Costa ME 1988 Prostaglandin E2 releases luteinizing hormone-releasing hormone from the female juvenile hypothalamus through a Ca2+-dependent, calmodulin-independent mechanism. Brain Res 441:339351[CrossRef][Medline]
- Kanegae Y, Lee G, Sato Y, Tanaka M, Nakai M, Sakaki T, Sugano S, Saito I 1995 Efficient gene activation in mammalian cells by using recombinant adenovirus expressing site-specific Cre recombinase. Nucleic Acids Res 23:381638121[Abstract]
- Veldhuis JD, Johnson ML 1986 Cluster analysis: a simple, versatile, and robust algorithm for endocrine pulse detection. Am J Physiol 250:E486E493
- Charles AC, Merrill JE, Dirksen ER, Sanderson MJ 1991 Intercellular signaling in glial cells: calcium waves and oscillations in response to mechanical stimulation and glutamate. Neuron 6:983992[Medline]
- Thompson WJ, Appleman MM 1971 Multiple cyclic nucleotide phosphodiesterase activities from rat brain. Biochemistry 10:311316[Medline]
- Nett TM, Akbar AM, Niswender GD, Hedlund MT, White WF 1973 A radioimmunoassay for gonadotropin-releasing hormone in serum. J Clin Endocrinol Metab 36:880885[Medline]