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Endocrinology Vol. 148, No. 7 3299-3306
Copyright © 2007 by The Endocrine Society

Frequency of Intrinsic Pulsatile Gonadotropin-Releasing Hormone Secretion Is Regulated by the Expression of Cyclic Nucleotide-Gated Channels in GT1 Cells

B. E. Blackman, H. Yoshida, S. Paruthiyil and R. I. Weiner

Center for Reproductive Sciences, Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco School of Medicine, San Francisco, California 94913

Address all correspondence and requests for reprints to: Richard Weiner, Department of Obstetrics, Gynecology, and Reproductive Sciences, 513 Parnassus Avenue, HSW1475, Box 0556, University of California, San Francisco, San Francisco, California 94143. E-mail: weinerr{at}obgyn.ucsf.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cultures of endogenous GnRH neurons and the GT1 GnRH neuronal cell line release GnRH in pulses (intrinsic pulsatile release) with an interpulse frequency similar to that seen in castrated animals. In both GT1 cells and transgenic rats, lowering cAMP levels by expression of a phosphodiesterase decreased the frequency of intrinsic GnRH pulsatility. We asked whether the cyclic nucleotide-gated cation (CNG) channels expressed in GT1 cells participated in cAMP modulation of intrinsic GnRH pulsatility. Because expression of the CNGA2 subunit is essential for formation of functional CNG channels, we developed an adenovirus (Ad) vector expressing a short interference RNA (siRNA) to the CNGA2 subunit (Ad-CNG-siRNA) or as an infection control, to the coding region of luciferase (Ad-Luc-siRNA). Infection with the Ad-CNG-siRNA of COS cells transfected with a CNGA2 expression vector significantly inhibited CNGA2 protein levels by 74% by Western blot. Infection of GT1-1 cells with Ad-CNG-siRNA resulted in a 68% decrease in the levels of CNGA2 mRNA, a 44% decrease in protein levels, and a clear decrease in immunostaining with an antibody to CNGA2. Infection of GT1-1 cells with Ad-CNG-siRNA decreased spontaneous Ca2+ oscillations compared with Ad-Luc-siRNA-infected or uninfected cells by 71%. Furthermore infection with Ad-CNG-siRNA resulted in a 2-fold increase in the interpulse interval in GnRH secretion (49.4 ± 9.1 min) compared with uninfected cells (25.9 ± 2.5 min) or Ad-Luc-siRNA (29.3 ± 2.8 min)-infected cells. These data provide the first direct evidence that the CNG channel is a downstream signaling molecule in the regulation of the frequency of intrinsic GnRH pulsatility by cAMP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GnRH REGULATES THE synthesis and release of LH and FSH (1). GnRH is released from the median eminence of the hypothalamus into the portal system, in a pulsatile manner. Pulsatile GnRH release is required to maintain gonadotropin secretion (2). The mouse GT1 GnRH neuronal cell line (3, 4, 5) and cultures of endogenous GnRH neurons from several species (6, 7, 8) secrete GnRH in discreet Ca2+-dependent pulses. These findings support the hypothesis that pulsatile release is an intrinsic property of GnRH neurons, i.e. "intrinsic pulsatile release."

The pulsatile release of GnRH is under the control of numerous inhibitory and excitatory inputs (9). Consistent with this observation, the amplitude and frequency of GnRH/LH pulses vary with reproductive states, for example, the frequency increases during the follicular phase and slows during the luteal phase of the human menstrual cycle. Catecholamine-containing neurons play important roles in regulating GnRH secretion (9). GnRH secretion from GT1 cells was increased by both dopamine (DA) and norepinephrine (NE) (10, 11, 12). The DA response is mediated via D1 DA receptors, whereas the NE response is mediated via ß1 adrenergic receptors. Consistent with the fact that both receptors are positively coupled to adenylyl cyclase, treatment with NE or DA resulted in a dose-dependent increase in the intracellular concentrations of cAMP through the activation of adenylyl cyclase (13). Pharmacologically increasing cAMP concentrations by treatment with forskolin or 8-Bromo-cAMP mimicked the stimulation of GnRH release by NE or DA.

GT1 neurons are spontaneously excitable as shown by the appearance of spontaneous Ca2+ oscillation seen in fura 2-loaded cells (14). Because each Ca2+ oscillation was preceded by an action potential, the frequency of Ca2+ oscillations is an index of neuron excitability. Increases in intracellular cAMP levels resulted in an increase in Ca2+ oscillations and neuron excitability (14). Genetically decreasing the intracellular cAMP concentration by the expression of a constitutively active cAMP-dependent phosphodiesterase (PDE4D1) lowered both the frequency of spontaneous Ca2+ oscillations and the frequency of intrinsic pulsatile GnRH release (15). The frequency of pulsatile GnRH release was also decreased in castrated GPR-4 transgenic rats in which PDE4D1 expression was genetically targeted to GnRH neurons (16).

The activation of protein kinase A (PKA) is a common downstream signaling event mediating increases in cAMP. However, previous work showed that PKA activation was not necessary for the cAMP-induced GnRH release, nor did inhibition of PKA activity by the specific inhibitor H89 (N-[2-(p-bromo-cinnamylamino)-ethyl]-5-isoquinoline-sulfon-amide 2HCl) block intrinsic pulsatile GnRH secretion (13). Because endogenous GnRH neurons and GT1 cells express all three subunits of the olfactory cyclic nucleotide-gated (CNG) channels (17), we hypothesized that the cAMP-induced secretion of GnRH may be mediated through the regulation of CNG channels. CNG channels are expressed in a wide variety of cell types, including cells that show intrinsic rhythmic activity including heart and pineal gland (18, 19, 20, 21, 22). Patch-clamping studies in GT1 cells demonstrated the presence of functional CNG channels (23). Pharmacological inhibition of CNG channels with L-cis-diltiazem inhibited channel activity and decreased cAMP-induced increases in the frequency of Ca2+ oscillations (23).

The olfactory CNG channel is a tetramer consisting of one CNGA4, one CNGB1b, and two CNGA2 subunits (24, 25). The CNGA2 subunit when expressed alone can form a functional homomeric channel. However, the CNGB1b and CNGA4 subunits when expressed alone do not form a functional channel but, when expressed with the CNGA2 channel, increase the specificity of the channel for cAMP (26). It follows that, by decreasing the expression of CNGA2 channels using short interference RNA (siRNA) technology, the number of functional CNG channels should be decreased. In the present study, the levels of CNGA2 channel protein in GT1-1 cells were decreased by infection with an adenovirus (Ad) expressing an siRNA to the CNGA2 subunit (Ad-CNG-siRNA) vector. We determined the effect of decreasing the levels of the CNGA2 subunit expression on the frequency of spontaneous Ca2+ oscillations and intrinsic pulsatile GnRH release in GT1-1 cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
GT1-1 cells (passages 14–23) were cultured on 10-cm culture plates in DMEM (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (HyClone Laboratories, Logan, UT), 5% horse serum (HyClone Laboratories), 100 U/ml penicillin, and 100 µg/ml streptomycin.

Adenoviral siRNA constructs
Two pairs of oligos (CNGA2#1 and CNGA2#2) were synthesized from sequences corresponding to CNGA2 cDNA sequence (GenBank accession no. NM_007724) (27) (Table 1Go). These oligos were annealed and ligated into linearized pSIREN-Shuttle (Clontech, Palo Alto, CA). The negative control luciferase (Luc) siRNA supplied with the vector was also cloned into pSIREN-Shuttle. Expression of the siRNAs in pSIREN-Shuttle were tested for their ability to knockdown expression of transiently transfected CNGA2 cDNA (Fig. 1Go). Both the CNGA2 siRNA and the Luc siRNA were transferred from the shuttle vector into pAdenoX (Clontech) using the recommended protocol. The recombinant adenoviral DNA was transfected into HEK 293 cells to produce viral particles. Cell lysates were used to reinfect HEK 293 cells for large-scale viral production. The virus was purified on two consecutive cesium chloride gradients, dialyzed with PBS containing 10% glycerol, and titered using the end-point dilution assay. Viral titers were more than 109 pfu/ml.


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  		TABLE 1. Sequences of the siRNA oligonucleotides designed to the mouse CNGA2 mRNA

 

Figure 1
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  		FIG. 1. Western blot analysis of CNGA2 protein levels. Whole-cell extracts from untransfected COS-7 cells or cells transiently transfected with CNGA2 cDNA alone or with the control Luc siRNA, CNGA2-siRNA#1, or CNGA2-siRNA#1, respectively, were immunoblotted with anti-CNGA2 antibody. CNG-siRNA#2 and to a lesser extent CNG-siRNA#1 but not Luc-siRNA inhibited the expression of CNGA2 in transiently transfected COS-7 cells. MW, Molecular weight.

 
Real-time quantitative RT-PCR analysis
GT1-1 cells plated in six-well plates were infected with Ad-CNG-siRNA or Ad-Luc-siRNA, were washed with PBS, and lysed with 1 ml Trizol (Invitrogen). Total RNA was prepared according to the recommended protocol. Real-time quantitative PCR was performed using SYBR Green Supermix (Bio-Rad, Hercules, CA) with an iCycler thermal cycler (Bio-Rad). We used the following primers: CNGA2 forward, 5'-AGGGAAAGGGCACCAAAAAGAAA-3'; CNGA2 reverse, 5'-CCAGCACCAGCCATACCACAAA-3'; ß-actin forward, 5'-GTCCACACCCGCCACCAGTTC-3'; and ß-actin reverse, 5'-GACCCATTCCCACCATCACACC-3'. The data were collected and analyzed using the comparative threshold cycle method using ß-actin expression as the reference gene. Data were collected in duplicate from three separate experiments.

Western blotting
COS-7 cells were plated into six-well plates at a density of 1 x 105 cells per well. Cells were transfected with 0.5 µg of CNGA2 cDNA cloned downstream of the cytomegalovirus promoter in pcDNA-6A (Invitrogen) using Lipofectamine (Invitrogen). After 24 h, the cells were infected with 5 or 10 multiplicity of infection (MOI) (the average number of viral particles per cell) of Ad-CNG-siRNA or Ad-Luc-siRNA for 2 h. Cells were incubated for 48 h and then washed twice in cold PBS, and total cell lysate was collected using radioimmunoprecipitation assay buffer (150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% Na deoxycholate, and 50 mM Tris) containing a complete protease inhibitor cocktail (Roche, Indianapolis, IN). For Western blots on GT1-1 cells, protein was extracted 48 h after infection using the membrane protein extraction kit (Pierce, Rockford, IL) to concentrate the membrane protein. Protein yield was determined by bicinchoninic acid assay (Pierce), and 50 µg of protein in Laemmli sample buffer was resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Bio-Rad). The immunoblots were probed with affinity-purified antirat CNGA2 antibody (Alpha Diagnostic International, San Antonio, TX) at 2.5 mg/ml or the affinity-purified antibody to the CNGA2 channel (1:200; Sigma, St. Louis, MO) in TBS-T [10 mM Tris (pH 7.4), 150 mM NaCl, and 0.05% Tween 20] containing 5% nonfat milk powder and 10% fetal calf serum. Immunoreactive proteins were visualized with the ECL plus Western Blotting Detection System (GE Healthcare Biosciences, Little Chalfont, Buckinghamshire, UK).

Immunofluorescence
GT1-1 cells (300,000 cells per well) were plated on Matrigel (BD Biosciences Discovery Labware, Bedford, MA) coated 25-mm plastic coverslips in six-well plates. When the cells reached 60–70% confluency, they were infected with 5 MOI of Ad-CNG-siRNA or Ad-Luc-siRNA for 2 h. Cells were washed three times in PBS 48 h after infection and fixed with 100% methanol, for 10 min at –20 C. Cells were washed three times for 5 min each in PBS and then blocked for 20 min in 20 mg/ml BSA in PBS. Cells were incubated at room temperature with anti-CNGA2 (1:200; Sigma). Staining was detected with fluorescein isothiocyanate antirabbit IgG (Jackson ImmunoResearch, West Grove, PA).

Perifusion studies
GT1-1 cells (300,000 cells per well) were plated on Matrigel (BD Biosciences Discovery Labware) coated 25-mm plastic coverslips in six-well plates. When the cells reached 60–70% confluency, they were infected with 5 MOI of Ad-CNG-siRNA or Ad-Luc-siRNA for 2 h. Cells were incubated for 24 h after infection, and the medium was then replaced with Opti-MEM (Invitrogen) and incubated for an additional 24 h before sampling. The coverslips were placed in a modified Sykes-Moore chamber (Bellco Glass, Vineland, NJ), and the cells were perifused with oxygenated Locke’s medium (in mM:154 NaCl, 5.6 KCl, 2.2 CaCl2, 1 MgCl2, 6 NaHCO3, 10 glucose, and 2 HEPES) supplemented with 0.1% BSA and 20 µM bacitracin, at a flow rate of 70–100 µl/min. Chambers were washed for 60 min, and then samples were collected every 2 min for 3 h. Each sample was boiled for 3 min and stored at –80 C for RIA of GnRH. Analysis of the GnRH pulse data were performed using the hormone pulse analysis software Cluster 8 (Michael L. Johnson, University of Virginia, Charlottesville, VA). Cluster analysis was performed on measurements done singly. The coefficient of variation was determined from intraassay controls. Cluster size or nadir was defined by two points that significantly increased or decreased with a t statistic of 4.

GnRH RIA
Levels of GnRH in the media from the perifusion studies were determined by an RIA using rabbit antibody R1245 (obtained from T. Nett, Colorado State University, Fort Collins, CO). This antiserum is specific for intact GnRH (28). Synthetic human GnRH (Sigma) was used for both iodination and as the standard. All samples from an experiment were analyzed in the same assay. The limit of detection of the assay was 0.25–25 pg/tube, and the intraassay coefficient of variation was 10.2%. The limit of detection of the assay was defined as 90% of maximal binding.

Ca2+ assays
Intracellular Ca2+ concentration ([Ca2+]i) was measured using fluorescence ratio imaging with MetaFluor Imaging Software (Universal Imaging Corp., West Chester, PA) as described previously (29). Briefly, GT1-1 cells cultured on Matrigel-coated glass coverslips were loaded with fura 2 (Invitrogen) by incubation in 5 µM fura 2-AM for 45 min at 37 C in oxygenated Locke’s medium supplemented with 0.1% BSA. The cells were then washed in fresh Locke’s medium for 45 min. Coverslips were placed in a temperature-controlled modified Sykes-Moore chamber mounted on a Nikon (Tokyo, Japan) TE2000 inverted fluorescence microscope. Cells were perifused with Locke’s medium or Locke’s medium containing 10 µM forskolin at a flow rate of 50 µl/min. Fura 2 fluorescence at 510 nm was measured at 5 sec intervals for 15 min and an additional 5 min after the addition of forskolin, at excitation settings of 340 and 380 nm. Approximately 40–60 cells were imaged per coverslip, and four coverslips were studied for the control GT1-1 cells as well the Ad-CNG-siRNA- or Ad-Luc-siRNA-infected cells.

Quantification of Ca2+ concentration and Ca2+ oscillations
To distinguish living cells from dead cells, only cells that showed a 20% increase in Ca2+ concentration after treatment with forskolin for 5 min were used for analysis. The [Ca2+]i was estimated from the ratio of the emissions and comparison with fura 2 standards (30). These values were plotted in raster plots using Transform (Fortner Software, Sterling, VA). The mean Ca2+ concentration before and after forskolin treatment of individual cells was measured and normalized for the length of the sampling period, giving a mean value of the [Ca2+]i. The data were analyzed with two-way ANOVA using Prism (GraphPad Software, San Diego, CA). Ca2+ oscillations were quantified using PeakFit, (SeaSolve Software, San Jose, CA) using the Autoscan Residuals function. A linear two-point baseline subtraction was performed, and the threshold amplitude was set at 50%. The number of oscillations was measured for each cell over the sampling period and calculated as a percentage change relative to the uninfected cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Development of CNGA2 siRNA
We designed two siRNAs specific to the mouse CNGA2 cDNA (CNGA2#1 and CNGA2#2) (Table 1Go). Before cloning these oligonucleotides into the Ad expression vector, we tested their ability to knockdown expression of a CNGA2 cDNA. We performed these experiments in COS cells transiently expressing the CNGA2 cDNA. The oligonucleotides were cloned into pSIREN-Shuttle and expressed in COS cells transiently cotransfected with a CNGA2 expression construct. Western blot analysis clearly showed that CNGA2#2 was more effective than CNGA2#1, decreasing the expression of the CNGA2 cDNA (Fig. 1Go).

Because only a small portion of GT1 cells are transiently transfected by available techniques, for subsequent experiments in GT1 cells, we developed an Ad vector to direct expression of the CNGA#2 siRNA. The CNGA2#2 as well as the control Luc siRNA were cloned into an Ad vector for all additional studies. These constructs are referred to as Ad-CNG-siRNA and Ad-Luc-siRNA, respectively.

Inhibition CNGA2 protein levels by Ad-CNG-siRNA in COS cells
To show that infection with the Ad-CNG-siRNA lowered the protein levels of the CNGA2 subunit, we performed experiments in COS cells transiently transfected with an expression vector expressing the mouse CNGA2 cDNA. The effect of Ad-CNG-siRNA infection on COS-7 cells was evaluated by Western blot analysis (Fig. 2Go). A highly significant 74% decrease in CNGA2 protein levels was observed after infection with Ad-CNG-siRNA at 5 MOI compared with a 7% nonsignificant increase in expression seen with Ad-Luc-siRNA infection. At 10 MOI of Ad-CNG-siRNA, a similar 69% decrease was observed in CNGA2 protein levels, but a nonspecific decrease in CNGA2 protein levels was observed with a 10 MOI of Ad-Luc-siRNA.


Figure 2
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  		FIG. 2. Infection with Ad-CNG-siRNA but not Ad-Luc-siRNA inhibited the expression of CNGA2 in transiently transfected COS cells. A, Immunoblot of CNGA2 in COS cells: lane 1, untransfected COS cells; lanes 2–6, COS cells transiently transfected with CNGA2 cDNA; lane 2, uninfected; lane 3, infected with Ad-CNG-siRNA 5 MOI; lane 4, infected with Ad-CNG-siRNA 10 MOI; lane 5, infected with Ad-Luc-siRNA 5 MOI; and lane 6, infected with Ad-Luc-siRNA 10 MOI. B, Data from two separate experiments was quantitated and represented as a percentage of the control COS cells transiently transfected with CNGA2 cDNA (lane 2 above).

 
Inhibition expression of CNGA2 mRNA and protein by Ad-CNG-siRNA in GT1-1 cells
We investigated the mRNA levels of CNGA2 in GT1-1 cells 48 h after infection by real-time quantitative RT-PCR. At 5 MOI, Ad-CNG-siRNA caused a 68% average decrease in mRNA levels of CNGA2 (Fig. 3Go). No effect was seen with 5 MOI of Ad-Luc-siRNA. At 10 MOI of Ad-CNG-siRNA, a 90% decrease in CNGA2 mRNA levels were observed, but a nonspecific effect on CNGA2 mRNA was observed with Ad-Luc-siRNA. Presumably, this effect was attributable to the high viral load in the cells and was consistent with previous findings (15).


Figure 3
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  		FIG. 3. Ad-CNG-siRNA decreased CNGA2 mRNA in GT1-1 cells. GT1-1 cells were infected with Ad-CNG-siRNA (5 and 10 MOI) or Ad-Luc-siRNA (5 and 10 MOI), and RNA was isolated 48 h after infection. The data were analyzed using the comparative threshold cycle method with ß-actin as the references gene. The data, expressed as a percentage of the control CNGA2 mRNA in uninfected cells, represent the mean ± SEM from three separate experiments. Ad-CNG-siRNA (5 MOI) but not Ad-Luc-siRNA (5 MOI) inhibited the expression of CNGA2 mRNA in GT1-1 cells as shown by quantitative RT-PCR.

 
The siRNA appeared specific because no affect was observed of infection with the Ad-CNG-siRNA on the mRNA levels of ß-actin (data not shown). Because infection with 10 MOI of the Ad-Luc-siRNA had a nonspecific effect on both the levels of CNGA2 mRNA and protein, all subsequent experiments were done with 5 MOI of the siRNA vectors. This viral load was sufficient to infect the majority of GT1 cells, because infection with 5 MOI of the same Ad vector expressing green fluorescent protein (GFP) resulted in GFP expression in 90–95% of GT1 cells (data not shown).

A specific band corresponding to the CNGA2 protein was observed in immunoblots of membrane protein extracted from GT1-1 cells (Fig. 4AGo). The blots were quantified and band intensity was normalized for loading using an antibody to glyceraldehyde-3-phosphate dehydrogenase. In agreement with findings in COS cells, a 44% decrease in the intensity of the CNGA2 band relative to the uninfected GT1-1 cells was observed after infection with 5 MOI of the Ad-CNG-siRNA (Fig. 4BGo). Only a small 6% decrease in the CNGA2 band intensity was observed after infection with 5 MOI of the Ad-Luc-siRNA. Additionally, a specific decrease in CNGA2 immunostaining was also observed in GT1-1 cells infected with 5 MOI of the Ad-CNG-siRNA relative to uninfected or with cells infected with 5 MOI of the Ad-Luc-siRNA (Fig. 4CGo).


Figure 4
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  		FIG. 4. Infection with Ad-CNG-siRNA but not Ad-Luc-siRNA inhibited the expression of CNGA2 protein in GT1-1 cells. A, Immunoblot of membrane protein with antibodies to CNGA2 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in GT1-1 cells. Lane 1, Uninfected GT1-1 cells; lane 2, GT1-1 cells infected with Ad-Luc-siRNA 5 MOI; and lane 3, GT1-1 cells infected with Ad-CNG-siRNA 5 MOI. B, Immunoblot data were quantitated from two separate experiments and represented as a percentage of the uninfected GT1-1 cells (lane 1 above). C, Immunofluorescence with anti-CNGA2 in uninfected GT1-1 cells (1 ), Ad-Luc-siRNA 5 MOI (2 ), and Ad-CNG-siRNA 5 MOI (3 ) infected cells.

 
Effect of infection Ad-CNG-siRNA on frequency of Ca2+ oscillations
We then asked whether lowering the levels of CNGA2 expression in GT1-1 cells would affect the occurrence of spontaneous Ca2+ oscillations. Calcium was measured using fura 2 imaging in living GT1-1 cells with fluorescence microscopy. In untreated, fura 2-labeled cells, spontaneous Ca2+ oscillations were observed with a similar frequency to those reported previously (14). A 71% decrease (from 5.3 to 1.5 oscillations per 15 min experiment) in spontaneous Ca2+ oscillations (P < 0.0001) was observed in GT1-1 cells infected with the Ad-CNG-siRNA (Fig. 5BGo) relative to the uninfected cells (Fig. 5CGo). No difference was observed in the Ad-Luc-siRNA-infected cells (Fig. 5AGo).


Figure 5
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  		FIG. 5. Effect of Ad-CNG-siRNA on [Ca2+]i. Fura 2 Ca2+ imaging experiments in GT1-1 neurons infected with 5 MOI of Ad-Luc-siRNA (A), Ad-CNG-siRNA (B), and uninfected (C) cells. The tracings at the top of each section (A–C) show the changes in [Ca2+]i vs. time in a representative cell. The raster plots show [Ca2+]i vs. time for 10 representative cells from a single field. The grayscale represents the [Ca2+]i from 0 to 600 nM. Forskolin (10 µM; Fsk) treatment is indicated by the bar. Given the flow rate and the volume of the chamber, approximately 2 min are required before forskolin reaches the cells.

 
After recording spontaneous Ca2+ oscillations, cells were treated with forskolin to identify responsive cells. In this way, any nonresponsive cells were eliminated from analysis. Forskolin treatment caused a rapid, large, pharmacological increase in cAMP levels that resulted in a dramatic increase in Ca2+ oscillations in GT1 cells (23). Only cells showing a 20% increase in [Ca2+]i after forskolin treatment were used for analysis of spontaneous Ca2+ oscillations. Although forskolin treatment stimulated Ca2+ oscillation in the Ad-CNG-siRNA-treated cells, the average increase in [Ca2+]i was significantly decreased relative to controls (P < 0.001) (Fig. 6Go). This finding is consistent with the conclusion that Ad-CNG-siRNA-treated cells expressed fewer CNG channels.


Figure 6
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  		FIG. 6. Infection with Ad-CNG-siRNA but not Ad-Luc-siRNA causes a decrease in the mean [Ca2+]i in response to forskolin (Fsk). The mean [Ca2+]i before and after forskolin (10 µM) stimulation from 30 cells from a single microscopic field. These cells were selected based on the criteria that they all showed a greater than 20% increase in [Ca2+]i after forskolin stimulation. Data are the mean ± SD. *, P < 0.001 relative to uninfected and Ad-Luc-siRNA-infected cells

 
Inhibition of spontaneous pulsatile GnRH release
Finally, we tested the effect of lowering CNGA2 subunit levels on intrinsic pulsatile GnRH release from GT1-1 cells. Uninfected GT1-1 cells or cells infected with the Ad-CNG-siRNA or Ad-Luc-siRNA were perifused, and samples were obtained every 2 min for 3 h for measurement of GnRH by RIA. A dramatic decrease in the number of intrinsic GnRH pulses per experiment was observed after infection with Ad-CNG-siRNA, whereas no difference was seen between uninfected cells and the Ad-Luc-siRNA-infected cells (2.6 ± 0.4, n = 8 compared with 5.1 ± 0.9, n = 8 and 4.8 ± 0.5, n = 8 pulses per experiment, respectively; P < 0.05) (Fig. 7Go, Table 2Go). Similarly the interpulse interval was significantly increased (P < 0.01) by infection with the Ad-CNG-siRNA (49.4 ± 9.10 min) vs. uninfected (25.9 ± 2.5 min) or Ad-CNG-siRNA-infected (29.3 ± 2.8 min) cells. Interestingly, no significant change in the pulse amplitude was observed (Table 2Go).


Figure 7
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  		FIG. 7. Spontaneous GnRH release in uninfected GT1-1 neurons [wild type (WT)] and neurons infected for 48 h with 5 MOI of Ad-Luc-siRNA or 5 MOI of Ad-CNG-siRNA. Samples were obtained every 2 min for 180 min from perifused cells. Data are shown for four of eight experiments for each treatment. GnRH levels were measured singly by RIA, and data were analyzed for pulsatile secretion using cluster analysis (arrowhead denotes a pulse).

 

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  		TABLE 2. Summary of GnRH pulse parameters determined by cluster analysis

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Substantial data exist that the level of cAMP in GT1 cells regulated spontaneous cell excitability and the frequency of intrinsic GnRH pulses (15, 23). GT1 neurons show spontaneous activity characterized by oscillations in membrane potential, bursts of action potentials, and oscillatory increases in [Ca2+]i that are generated by the influx of Ca2+ through voltage-gated Ca2+ channels (14). Previously, it was demonstrated that lowering cAMP levels in GT1 cells by expression of PDE4D1, a cAMP-specific PDE, decreased spontaneous Ca2+ oscillations and the frequency of intrinsic GnRH pulses (15). Activation of PKA did not appear essential for the effects of increasing cAMP because pharmacological blockade of PKA had no effect on intrinsic pulsatile GnRH release (13). Although GT1 cells were shown to express CNG channels subunits and functional CNG channels, the importance of this downstream signaling pathway in regulating alterations in neuron excitability and intrinsic GnRH pulse frequency were unclear (17). The current findings support the hypothesis that CNG channels play an important role as a downstream signaling event in regulation of neuron excitability by cAMP levels.

The lowering of the levels of CNGA2 subunit mRNA by 68% and protein by 44% by infection with the Ad-CNG-siRNA decreased spontaneous Ca2+ oscillations by 71% and doubled the interpulse frequency of GnRH pulses. The doubling in the GnRH interpulse frequency was almost identical to that seen by lowering cAMP levels by expression of PDE4D1 in GT1 cells (15). Also similar to the results with PDE4D1 expression, there was no effect of lowering CNG channel expression on the GnRH pulse amplitude.

We hypothesize that increased levels of cAMP result in increased conductance through the CNG channels of presumably Na+ and/or Ca2+ (Fig. 8Go). Although CNG channels are permeable to cations in general, there would be a depolarizing influx of Na+ or Ca2+ into the cell at physiologic membrane potentials, i.e. the resting potential is near the equilibrium potential for K+ and far from the equilibrium potential for Na+ or Ca2+. Binding of cAMP to the CNG channel proteins opens the channels, allowing cations to enter the neurons that increase the depolarization drive (23). The magnitude of the cAMP-regulated conductance through the CNG channels is not sufficient to trigger an action potential. Instead, the increase in the resting potential presumably leads to the activation of inwardly rectified K+ channels that are responsible for triggering the action potentials in GT1 cells (31, 32). By decreasing the number of CNG channels by infection with the Ad-CNG-siRNA, the cAMP levels in the neuron would take longer to increase the resting potential sufficiently to activate the inwardly rectified K+ channels. This would result in fewer action potentials and Ca2+ oscillations. Generation of an action potential is dependent on the opening of fast, voltage-gated Na+ channels (33, 34). The firing of the action potential in turn results in the opening of L-type voltage-gated channels, an intracellular Ca2+ oscillation, and the exocytosis of GnRH (5, 14).


Figure 8
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  		FIG. 8. The cAMP signaling pathway regulating the increased excitability of GT1 cells. Increased production of cAMP by adenylyl cyclase (AC) increases the occupancy of cAMP binding sites on the CNG channel subunits and cation conductance through the channel. The opening of the CNG channel increases Ca2+ and Na+ influx, increasing the resting potential and increasing the probability of the generation of an action potential. It has been hypothesized that inwardly rectified K+ channels play a key role in the triggering of an action potential (31 32 ). The opening of voltage-regulated fast Na+ channels and the depolarization of the cells leads to the opening of the L-type voltage-gated Ca2+ channels (observed as Ca2+ oscillations in fura 2-loaded cells). The increase in [Ca2+]i is essential for coupling the exocytosis of GnRH from intracellular stores.

 
Performing these experiments depended on the ability of expressing specific siRNAs that would sufficiently lower CNG channel expression to alter cell function. Several lines of experiments have shown that expression of the CNGA2 subunit is essential for the formation of functional CNG channels. The CNGA2 subunit constitutes two of the four subunits composing a cAMP-sensitive CNG channel (24, 25). We reasoned, therefore, that lowering CNGA2 levels would decrease the number of functional channels. We demonstrated that the siRNA used in the experiments dramatically lowered the level of CNGA2 protein level in transfected COS cells and in GT1 cells. No nonspecific effects were seen on nonrelated gene expression, e.g. ß-actin. An observation consistent with the conclusion that infection with the Ad-CNG-siRNA lowered the level of CNG channel expression was that the increase in [Ca2+]i after forskolin treatment in Ad-CNG-siRNA-infected GT1 cells was significantly lower than in Ad-Luc-siRNA-infected cells. This observation is consistent with the conclusion that there are fewer CNG channels in the cells available to permit Ca2+ to enter the cell to regulate the resting potential. CNG channels have long been recognized as Ca2+ channels (35). The level of CNG channel expression has been shown to affect increases in [Ca2+]i after increases in cAMP levels (36).

A second key requirement for success of using siRNA expression in GT1 cells was the ability to direct expression to the majority of GT1 cells in the cultures. We had previously used Ad vectors to direct expression of PDE4D1 to GT1 cells (15). In these studies as well as in the current studies, as long as the viral load was not raised above 5 MOI, little if any nonspecific effect of viral infection was observed. As an additional control for these experiments, the same viral vector expressing Luc was used to infect cells at 5 MOI. In this way, we controlled for both viral infection and effects of expressing large amounts of a protein. No effects on the frequency of Ca2+ oscillations or GnRH pulses were observed after infection with 5 MOI of Ad-Luc-siRNA. Infection of GT1 cells with 5 MOI of an Ad vector expressing GFP resulted in observable green fluorescence in better than 90% of the cells.

The physiological significance of observations in GT1 GnRH neurons relative to endogenous GnRH can always be questioned. Experiments to date strongly support the parallel role of cAMP signaling in GT1 neurons and endogenous GnRH neurons. Both GT1 cells and rat GnRH neurons express all three of the CNG channel subunits (17). The lowering of the GnRH pulse frequency by expression of PDE4D1 in GT1 cells was almost identical to the lowering of the LH pulse frequency in castrated GPR-4 transgenic rats (16). The expression of PDE4D1 was demonstrated to be specifically targeted to GnRH neurons in the GPR-4 rats using the GnRH promoter/enhancer sequences.

One remaining question is whether cAMP levels are maintained at a relatively constant level to maintain GnRH neuron excitability (permissive role) or whether the levels of cAMP cycle provide a mechanism for timing intrinsic pulsatile GnRH release (clock). Cycles in cAMP levels have been shown to constitute a biological clock in Dictyostelium (37). Measuring the fluctuations of cAMP concentrations in cells has been limited to the availability of sensitive molecular tools. Both PKA (38) and more recently the exchange protein activated by cAMP (39, 40) have been used to monitor cytosolic cAMP levels using fluorescence resonance energy transfer. Future studies will use these techniques to directly address this question in GT1 cells.


   Footnotes
 
This work was supported by National Institutes of Health Grant HD041996.

Present address for H.Y.: Department of Obstetrics and Gynecology, Yokohama City University, Graduate School of Medical Research, 3-9 Fukuura, Kanazwa-ku, Yokohama, Kanagawa 236-0004, Japan.

Disclosure Statement: B.E.B., H.Y., S.P., and R.I.W. have nothing to declare.

First Published Online March 29, 2007

Abbreviations: Ad, Adenovirus; [Ca2+]i, intracellular Ca2+ concentration; CNG, cyclic nucleotide-gated; DA, dopamine; GFP, green fluorescent protein; Luc, luciferase; MOI, multiplicity of infection; NE, norepinephrine; PDE, phosphodiesterase; PKA, protein kinase A; siRNA, short inference RNA.

Received October 24, 2006.

Accepted for publication March 15, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Schally AV, Arimura A, Baba Y, Nair RM, Matsuo H, Redding TW, Debeljuk L 1971 Isolation and properties of the FSH and LH-releasing hormone. Biochem Biophys Res Commun 43:393–399[CrossRef][Medline]
  2. Knobil E 1980 The neuroendocrine control of the menstrual cycle. Recent Prog Horm Res 36:53–88[Medline]
  3. Martinez de la Escalera G, Choi AL, Weiner RI 1992 Generation and synchronization of gonadotropin-releasing hormone (GnRH) pulses: intrinsic properties of the GT1-1 GnRH neuronal cell line. Proc Natl Acad Sci USA 89:1852–1855[Abstract/Free Full Text]
  4. 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 89:4149–4153[Abstract/Free Full Text]
  5. 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 89:8462–8466[Abstract/Free Full Text]
  6. Duittoz AH, Batailler M 2000 Pulsatile GnRH secretion from primary cultures of sheep olfactory placode explants. J Reprod Fertil 120:391–396[Abstract]
  7. 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 71:138–144[CrossRef][Medline]
  8. 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 140:1432–1441[Abstract/Free Full Text]
  9. Kordon C, Drouva S, Martinez de la Escalera G, Weiner R 1994 Role of classic and peptide neuromediators in the neuroendocrine regulation of LH and prolactin. In: Knobil EN, ed. The physiology of reproduction. New York: Raven; 1621–1681
  10. 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 131:1397–1402[Abstract]
  11. 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 131:2965–2971[Abstract]
  12. Uemura T, Nishimura J, Yamaguchi H, Hiruma H, Kimura F, Minaguchi H 1997 Effects of noradrenaline on GnRH-secreting immortalized hypothalamic (GT1–7) neurons. Endocr J 44:73–78[Medline]
  13. 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 97:1861–1866[Abstract/Free Full Text]
  14. Charles AC, Hales TG 1995 Mechanisms of spontaneous calcium oscillations and action potentials in immortalized hypothalamic (GT1–7) neurons. J Neurophysiol 73:56–64[Abstract/Free Full Text]
  15. Yoshida H, Beltran-Parrazal L, Butler P, Conti M, Charles AC, Weiner RI 2003 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. Mol Endocrinol 17:1982–1990[Abstract/Free Full Text]
  16. 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 99:17191–17196[Abstract/Free Full Text]
  17. El Majdoubi M, Weiner RI 2002 Localization of olfactory cyclic nucleotide-gated channels in rat gonadotropin-releasing hormone neurons. Endocrinology 143:2441–2444[Abstract/Free Full Text]
  18. Biel M, Zong X, Distler M, Bosse E, Klugbauer N, Murakami M, Flockerzi V, Hofmann F 1994 Another member of the cyclic nucleotide-gated channel family, expressed in testis, kidney, and heart. Proc Natl Acad Sci USA 91:3505–3509[Abstract/Free Full Text]
  19. Darvish N, Russell JT 1998 Neurotransmitter-induced novel modulation of a nonselective cation channel by a cAMP-dependent mechanism in rat pineal cells. J Neurophysiol 79:2546–2556[Abstract/Free Full Text]
  20. Ding C, Potter ED, Qiu W, Coon SL, Levine MA, Guggino SE 1997 Cloning and widespread distribution of the rat rod-type cyclic nucleotide-gated cation channel. Am J Physiol 272:C1335–C1344
  21. Hundal SP, DiFrancesco D, Mangoni M, Brammar WJ, Conley EC 1993 An isoform of the cGMP-gated retinal photoreceptor channel gene expressed in the sinoatrial node (pacemaker) region of rabbit heart. Biochem Soc Trans 21:119S
  22. Sautter A, Biel M, Hofmann F 1997 Molecular cloning of cyclic nucleotide-gated cation channel subunits from rat pineal gland. Brain Res Mol Brain Res 48:171–175[Medline]
  23. Charles A, Weiner R, Costantin J 2001 cAMP modulates the excitability of immortalized hypothalamic (GT1) neurons via a cyclic nucleotide gated channel. Mol Endocrinol 15:997–1009[Abstract/Free Full Text]
  24. Shapiro MS, Zagotta WN 1998 Stoichiometry and arrangement of heteromeric olfactory cyclic nucleotide-gated ion channels. Proc Natl Acad Sci USA 95:14546–14551[Abstract/Free Full Text]
  25. Zheng J, Zagotta WN 2004 Stoichiometry and assembly of olfactory cyclic nucleotide-gated channels. Neuron 42:411–421[CrossRef][Medline]
  26. Bradley J, Bonigk W, Yau KW, Frings S 2004 Calmodulin permanently associates with rat olfactory CNG channels under native conditions. Nat Neurosci 7:705–710[CrossRef][Medline]
  27. Ruiz ML, London B, Nadal-Ginard B 1996 Cloning and characterization of an olfactory cyclic nucleotide-gated channel expressed in mouse heart. J Mol Cell Cardiol 28:1453–1461[CrossRef][Medline]
  28. Nett TM, Akbar AM, Niswender GD, Hedlund MT, White WF 1973 A radioimmunoassay for gonadotropin-releasing hormone in serum. J Clin Endocrinol Metab 36:880–885[Medline]
  29. Yoshida H, Paruthiyil S, Butler P, Weiner RI 2004 Role of cAMP signaling in the mediation of dopamine-induced stimulation of GnRH secretion via D1 dopamine receptors in GT1–7 cells. Neuroendocrinology 80:2–10[CrossRef][Medline]
  30. Grynkiewicz G, Poenie M, Tsien RY 1985 A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450[Abstract/Free Full Text]
  31. Bosma MM 1993 Ion channel properties and episodic activity in isolated immortalized gonadotropin-releasing hormone (GnRH) neurons. J Membr Biol 136:85–96[Medline]
  32. Costantin JL, Charles AC 2001 Modulation of Ca2+ signaling by K+ channels in a hypothalamic neuronal cell line (GT1-1). J Neurophysiol 85:295–304[Abstract/Free Full Text]
  33. Mellon PL, Windle JJ, Goldsmith PC, Padula CA, Roberts JL, Weiner RI 1990 Immortalization of hypothalamic GnRH neurons by genetically targeted tumorigenesis. Neuron 5:1–10[CrossRef][Medline]
  34. Hales TG, Sanderson MJ, Charles AC 1994 GABA has excitatory actions on GnRH-secreting immortalized hypothalamic (GT1–7) neurons. Neuroendocrinology 59:297–308[CrossRef][Medline]
  35. Frings S, Seifert R, Godde M, Kaupp UB 1995 Profoundly different calcium permeation and blockage determine the specific function of distinct cyclic nucleotide-gated channels. Neuron 15:169–179[CrossRef][Medline]
  36. Fagan KA, Schaack J, Zweifach A, Cooper DM 2001 Adenovirus encoded cyclic nucleotide-gated channels: a new methodology for monitoring cAMP in living cells. FEBS Lett 500:85–90[CrossRef][Medline]
  37. Firtel RA 1996 Interacting signaling pathways controlling multicellular development in Dictyostelium. Curr Opin Genet Dev 6:545–554[CrossRef][Medline]
  38. Adams SR, Harootunian AT, Buechler YJ, Taylor SS, Tsien RY 1991 Fluorescence ratio imaging of cyclic AMP in single cells. Nature 349:694–697[CrossRef][Medline]
  39. Nikolaev VO, Bunemann M, Hein L, Hannawacker A, Lohse MJ 2004 Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem 279:37215–37218[Abstract/Free Full Text]
  40. Nikolaev VO, Gambaryan S, Engelhardt S, Walter U, Lohse MJ 2005 Real-time monitoring of the PDE2 activity of live cells: hormone-stimulated cAMP hydrolysis is faster than hormone-stimulated cAMP synthesis. J Biol Chem 280:1716–1719[Abstract/Free Full Text]




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