cAMP Modulates the Excitability of Immortalized Hypothalamic (GT1) Neurons via a Cyclic Nucleotide-Gated Channel

Andrew Charles, Richard Weiner and James Costantin1

Department of Neurology (A.C., J.C.) University of California Los Angeles School of Medicine Los Angeles, California 90095
Department of Obstetrics and Gynecology (R.W.) University of California San Francisco School of Medicine San Francisco, California 94143


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GT1 cells are immortalized hypothalamic neurons that show spontaneous bursts of action potentials and oscillations in intracellular calcium concentration [Ca2+]i, as well as pulsatile release of GnRH. We investigated the role of cyclic nucleotide gated (CNG) channels in the activity of GT1 neurons using patch clamp and calcium imaging techniques. Excised patches from GT1 cells revealed single channels and macroscopic currents that were activated by either cAMP or cGMP. CNG channels from GT1 cells showed rapid transitions from open to closed states typical of heteromeric CNG channels, were selective for cations, and had an estimated single channel conductance of 60 picosiemens (pS). Ca2+ inhibited the conductance of macroscopic currents and caused rectification of currents at increasingly positive and negative potentials. The membrane permeant cAMP analog Sp-cAMP-monophosphorothioate (Sp-cAMPS) increased the frequency of spontaneous Ca2+ oscillations in GT1 cells, whereas the Rp-cAMPS isomer had only a slight stimulatory effect on Ca2+ signaling. Forskolin, norepinephrine, and dopamine, all of which stimulate cAMP production in GT1 cells, each increased the frequency of Ca2+ oscillations. The effects of Sp-cAMPS or NE on Ca2+ signaling did not appear to be mediated by protein kinase A, since treatment with either H9 or Rp-cAMPS did not inhibit the response. The CNG channel inhibitor L-cis-diltiazem inhibited cAMP-activated channels in GT1 cells. Both L-cis-diltiazem and elevated extracellular Ca2+ reversibly inhibited the stimulatory effects of cAMP-generating ligands or Sp-cAMP on Ca2+ oscillations. These results indicate that CNG channels play a primary role in mediating the effects of cAMP on excitability in GT1 cells, and thereby may be important in the modulation of GnRH release.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
cAMP modulates cellular activity by multiple mechanisms, including activation of protein kinases (1, 2), regulation of phosphodiesterases (3), and direct activation of ion channels (4, 5), Cyclic nucleotide gated cation channels (CNG channels) were initially identified in sensory systems, namely in olfactory receptor neurons (6, 7) and retinal photoreceptor cells (8, 9). In these cells, CNG channels mediate the cellular response to sensory stimuli via changes in the levels of cAMP and/or cGMP (10, 11, 12, 13). CNG channels have subsequently been reported in a wide variety of other cell types (14, 15, 16, 17), including cells that show intrinsic rhythmic activity such as heart and pineal cells (14, 18, 19, 20, 21). In cells with spontaneous rhythmic activity, CNG channels have the potential to modulate the excitability of a cell in a manner that results in specific changes in the patterns of spontaneous activity, such as the frequency of firing or the bursting pattern of the cell. In secretory cells, this change in the frequency of spontaneous rhythmic activity could then lead to a change in secretion (22).

GnRH neurons in the mediobasal hypothalamus release GnRH in a pulsatile fashion. This pulsatile release of GnRH is the fundamental "clock" of reproductive cycles (23). The pulsatile release of GnRH has been shown to be an intrinsic property of GnRH neurons that does not require afferent input (23, 24, 25). Primary placodal GnRH neurons (26) and immortalized GnRH neurons (GT1 cells) (27, 28) both show spontaneous pulsatile release of GnRH. Both types of cells also show spontaneous activity characterized by oscillations in [Ca2+]i (26, 29) and in the case of GT1 cells, these oscillations in [Ca2+]i are generated by bursts of action potentials (30). Conditions that modulate spontaneous action potential bursting and Ca2+ signaling in GT1 cells consistently have similar effects on GnRH release in parallel experiments, indicating that this activity is a mechanism that drives secretion (29, 31, 32, 33, 34, 35).

Multiple transmitters that modulate GnRH release in vivo, including norepinephrine (NE) and dopamine, activate adenylyl cyclase via G protein-coupled receptors resulting in the formation of cAMP (36, 37, 38, 39). cAMP analogs or ligands that induce the formation of cAMP also consistently increase GnRH release in GT1 cells (40, 41, 42, 43). These and other studies provide strong evidence that cAMP plays a critical role in the modulation of GnRH neuronal signaling and secretion (44, 45). GT1 neurons express a functional CNG channel and express the CNG2, CNG4.3, and CNG5 channel subunits (45). These same subunits are expressed in olfactory neurons, consistent with the common embryological derivation of GnRH and olfactory neurons from the olfactory placode (46). To further investigate the role of cAMP and CNG channels in spontaneous signaling in GT1 cells, we have used the patch clamp technique in conjunction with measurements of [Ca2+]i with fluorescence videomicroscopy. We provide evidence that the activation of CNG channels in GT1 cells represents a primary mechanism by which cAMP modulates their excitability.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Electrophysiological Recording of CNG Channels
Patch-clamp recordings of membrane patches excised from GT1 cells revealed ion channels that were activated by application of cAMP to the intracellular face of the patch (Fig. 1Go). Single channel activity evoked by cAMP was characterized by rapid transitions between the open and closed states typical of CNG channels. In the presence of Ca2+ in the bath and pipette solutions, distinct channel openings to a large conductance state could be clearly discriminated only at large depolarizing potentials. At more negative potentials, the channel showed frequent transitions between the largest conductance and a substate conductance. The channel also displayed substates that were observed infrequently in the absence of cAMP (Fig. 1AGo). The single channel conductance of the largest open state at voltages between 70 and 120 mV was approximately 60 pS (n = 9, Fig. 1Go). Intermittent Ca2+ block of the current may allow the single channel currents to be more clearly distinguished. The visualization of distinct channel openings to a large conductance state only at highly depolarized voltages may be due the relief of Ca2+ blockade of the CNG channel at highly positive and negative potentials (47, 48, 49). No differences in the characteristics of cAMP-activated currents were observed in solutions containing high (144.8 mM) vs. low (5 mM) chloride, indicating that the channels were cationic.



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Figure 1. Single Channel Recording of CNG Channels in GT1–1 Cells

A, Current tracings of consecutive sweeps during voltage steps to 100 mV. In the absence of cAMP, there are no openings to the full conductance state. Occasional openings to a possible substate are infrequent as seen in the bottom trace. Raw tracings were collected without leak subtraction. The first 100 msec of the trace containing the capacitative transient is not plotted. B, Consecutive sweeps at indicated voltages from the same patch shown in panel A after addition of 200 µM cAMP to the cytoplasmic face of the patch. Frequent, rapid transitions from the closed (c-) to open states are seen after the addition of cAMP. The flickering behavior was interrupted occasionally by long closures and null sweeps. C, All-point amplitude histograms from two different patches at six different voltages. The voltage is indicated in parentheses and the unitary current amplitude is shown. The unitary amplitude of the channel was taken from the difference between the peak values of the fit to the two gaussian distributions using the least squares method. Short segments of data (50–3,000 msec) were used to construct the histograms, and thus the relative areas of the distributions do not reflect open probability. D, The current-voltage (I/V) relationship is plotted using data from 14 different patches and 38 amplitude histograms. The single channel conductance between 70 and 120 mV was estimated by linear regression to be 60 pS (r = 0.88).

 
Macroscopic currents activated by cAMP were also measured in a subset of excised patches from GT1–1 neurons (Fig. 2Go). With symmetric Na+ solutions and no divalent cations, currents activated by cAMP showed linear current/voltage (I/V) curves with a reversal potential of 0 mV. In the presence of Ca2+ in the bath (cytoplasmic face), currents showed reduced conductance with rectification at increasingly positive or negative voltages. In the presence of Ca2+, currents also showed a negative reversal potential. These results are consistent with dual effects of Ca2+ on CNG channels, i.e. 1) Ca2+ blocks monovalent current by a higher affinity binding in the pore and 2) Ca2+ permeates the channel and contributes to a fraction of the overall current under bionic conditions (50, 51). Channel activity was also activated by application of cGMP to the intracellular face of the patch (n = 6 patches, Fig. 3Go). The channels activated by cGMP had a similar conductance and I/V relationship as those activated by cAMP.



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Figure 2. Properties of cAMP-Activated Macrosopic Currents from GT1–1 Cells

A, Currents before the addition of cAMP (Aa) and macroscopic currents activated by the addition of 300 µM cAMP in Ca2+ containing bath solution (Ab). Panel Ac shows currents in the same patch after the removal of Ca2+ from the solution bathing the cytoplasmic face of the patch. Panel Ad shows currents in the same patch after perfusion of a bath solution containing 10 mM Ca2+. Note that the largest currents in the presence of cAMP are in the absence of Ca2+ and symmetric 140 mM Na-MES. B, I/V curves for the background currents and the cAMP-activated macroscopic current. Regions of the I/V curves near the reversal potential are shown on an expanded scale and inset. Addition of cAMP in the presence of 1.3 mM Ca2+ caused the reversal potential to shift to -6.7 mV compared with 0 mV for the background currents, consistent with a higher permeability of Ca2+ vs. Na+ for the cAMP-activated current. C, I/V curves obtained by voltage steps after the removal of Ca2+ from the bath solution and after the addition of Ca2+ (10 mM) in the presence of symmetrical 140 mM Na-MES. The membrane conductance is larger in the absence of Ca2+ and the reversal potential is zero. Note the scale in panel C is larger than in panel B. D, I/V data from the same patch obtained with continuous voltage ramp protocol showing the I/V curve near the reversal potential. In the absence of Ca2+ the reversal potential is 0 mV and in the presence of Ca2+ the reversal potential is -11.1 mV.

 


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Figure 3. Activation of the CNG Channel by cGMP

A, Single channel currents recorded in an excised patch after the addition of 200 µM cGMP. The rapidly flickering channel activity interrupted by long closures is similar to that seen in the presence of cAMP. B, The I/V curve is plotted for voltages between 50 and 120 mV. The best-fit line of the I/V curve between 70 and 120 mV is 63 pS (r = 0.96) similar to that measured in the presence of cAMP.

 
Effects of cAMP Analogs on Ca2+ Signaling
GT1–1 cells show spontaneous oscillations in [Ca2+]i in individual cells that may be limited to single cells or propagated as intercellular waves. The frequency and pattern of these Ca2+ oscillations and intercellular waves vary considerably in individual cells. The raster plots in Figs. 2–6GoGoGoGoGo and 8 show the heterogeneous patterns of Ca2+ signaling of different cells within a microscopic field. Bath application of the cAMP analog Sp-cAMPS (100 µM) resulted in an increase in the frequency of spontaneous Ca2+ oscillations (n > 400 cells in 4 experiments, Fig. 4Go and Table 1Go). In cells that were not showing spontaneous activity, Ca2+ oscillations were often initiated by Sp-cAMPS. The increase in the frequency of Ca2+ oscillations was sustained for 20 min or longer in the continued presence of Sp-cAMPS. The amplitude of Ca2+ oscillations was not significantly changed by Sp-cAMPS. Bath application of the Rp-cAMPS isomer (100 µM) resulted in a slight increase in the frequency of spontaneous Ca2+ oscillations (n > 400 cells in 4 experiments, Fig. 5Go and Table 1Go).



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Figure 4. Effects of Sp-cAMPS on Ca2+ Signaling in GT1 Cells

The tracing at the top of the figure shows the change in fura2 fluorescence (proportional to change in [Ca2+]i) vs. time in a single representative cell (cell 15). The raster plot shows [Ca2+]i vs. time in 20 different cells in the same microscopic field. Each row in the plot represents a single cell. The gray scale represents the change in fura fluorescence (in gray scale units, 1–256, proportional to change in [Ca2+]i) for each cell. Sp-cAMPS was present in the bath during the time indicated by the bar. Application of Sp-cAMPS resulted in a sustained increase in the frequency of Ca2+ oscillations in most cells in the field, as well as a slight increase in baseline [Ca2+]i.

 


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Figure 5. Effects of Rp-cAMPS on Ca2+ Signaling

The format of the figure is the same as described for Fig. 2Go. The tracing at the top of shows the change in fura fluorescence vs. time for cell 1. The raster plot shows fura fluorescence vs. time for 20 different nearby cells in the microscopic field. Bath application of 100 µM Rp-cAMPS resulted in only a slight increase in the frequency of Ca2+ oscillations, much smaller than that observed with Sp-cAMPS.

 


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Figure 6. Effects of Forskolin on Ca2+ Signaling

The format of the figure is the same as described for Fig. 2Go. Bath application of 1 µM forskolin resulted in an increase in the frequency of Ca2+ oscillations after a short delay. Note the shorter time scale than in Figs. 2Go and 3Go.

 

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Table 1. Average % Change (± SEM) in the Frequency of Ca2+ Oscillations Induced by Different Ligands

 
Bath application of forskolin (1 µM), an activator of adenylyl cyclase, resulted in an increase in the frequency of Ca2+ oscillations that was similar to that observed with Sp-cAMPS (n > 500 cells in 5 experiments, Fig. 6Go and Table 1Go). The increase in the frequency of Ca2+ oscillations occurred after a variable delay of 1–5 min following the application of forskolin.

Both NE and dopamine have been shown to act via receptors that are positively coupled to adenylyl cyclase to stimulate the production of cAMP in GT1 cells. We found that both NE (n > 400 cells in 4 experiments, Fig. 7Go and Table 1Go) and dopamine (n > 300 cells in 3 experiments, not shown) resulted in a significant increase in the frequency of Ca2+ oscillations, as well as an increase in the baseline [Ca2+]i.



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Figure 7. Effects of NE on Ca2+ Signaling

Bath application of 2 µM NE resulted in an increase in the frequency of Ca2+ oscillations, as well as an increase in baseline [Ca2+]i in many cells.

 
Inhibition of Protein Kinase A (PKA) Does Not Inhibit the Effects of Sp-cAMPS or NE
To determine if the stimulation of GT1 cells by Sp-cAMPS and NE was mediated by the activation of PKA by cAMP, we investigated whether the PKA inhibitor H9 inhibited the effects of Sp-cAMPS and NE. Previous studies showed that a similar PKA inhibitor H89 effectively inhibited phosphorylation in the same concentration range (45). We found that H9 (10 µM) induced an immediate increase in the frequency of Ca2+ oscillations, as well as an increase in baseline [Ca2+]i (Fig. 8Go). This initial stimulatory effect is consistent with the previously reported acute increase in cAMP and GnRH release in response to H89 (45). The frequency of Ca2+ oscillations returned to control levels after 5–10 min in the continued presence of the drug. Subsequent application of Sp-cAMPS (Fig. 8Go) or NE (not shown) resulted in an increase in the frequency of Ca2+ oscillations that was similar to that observed in cells that had not been exposed to H9 (n > 300 cells in 3 experiments for each condition).



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Figure 8. The PKA Inhibitor H9 Does Not Inhibit the Effects of Sp-cAMPS

Bath application of 10 µM H9 resulted in a transient increase in the frequency of Ca2+ oscillations and baseline [Ca2+]i. The frequency of Ca2+ oscillations recovered to control levels after 5–10 min in the continued presence of H9. Application of Sp-cAMPS after 12 min of continuous exposure to H9 resulted in an increase in the frequency of Ca2+ oscillations similar to that observed in cells not exposed to H9 (Fig. 5Go).

 
In addition to its variable effects on CNG channels, the cAMP analog Rp-cAMPS inhibits activation of PKA. We found that 100 µM Rp-cAMPS did not inhibit the stimulatory effect of forskolin on Ca2+ signaling (n > 500 cells in 5 experiments, Fig. 9Go), nor did it inhibit the stimulatory effects of NE or dopamine (n = 3 experiments each, not shown).



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Figure 9. Rp-cAMPS Does Not Inhibit the Effects of Forskolin

Bath application of the Rp-cAMPS, which inhibits activation of PKA, resulted in minimal effect on spontaneous Ca2+ signaling as shown in Fig. 5Go. Subsequent application of forskolin in the continued presence of Rp-cAMPS resulted in an increase in frequency of Ca2+ oscillations that was similar to that observed in control experiments in the absence of Rp-cAMPS.

 
The Effects of L-cis-Diltiazem (LCD)
Application of the CNG channel antagonist LCD inhibited cAMP-activated channels in excised membrane patches from GT1 cells (n = 5 patches, Fig. 10Go). Estimation of closed time of the channel by calculating the percentage of "null" sweeps (sweeps in which no channel activity was observed) before and after application of LCD showed that LCD significantly increased the closed time in all patches. In 4 patches, LCD (80–250 µM) increased the percentage of null sweeps by approximately 2-fold (range 6–50% null sweeps in controls to 13–98% null sweeps after LCD), whereas in 1 patch, there was a nearly 40-fold increase in the percentage of null sweeps (1% to 39%).



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Figure 10. LCD Inhibits cAMP-Activated Channels in GT1 Cells

Tracings show consecutive sweeps of channel activity (after subtraction of capacitative artifact) in the presence of 200 µM cAMP before and immediately after the addition of 80 µM LCD. The voltage of the pipette was stepped from 0 mV to -110 mV in all sweeps. Two traces are omitted during LCD addition where indicated by the arrow. In this experiment, LCD immediately inhibited channel activity. Channel activity was observed in 69 of 150 sweeps before LCD, but only 3 of 297 sweeps after LCD. Channel activity was restored after washout of LCD (not shown). LCD had a similar inhibitory effect on channel activity in all patches, although in other patches the inhibition was less pronounced.

 
We then used LCD to investigate the role of CNG channels in the stimulation of GT1 cells by Sp-cAMPS and NE. In some experiments, application of 40 µM LCD resulted in an immediate increase in the frequency of Ca2+ oscillations, which returned to basal levels after 10 min. Subsequent addition of NE or Sp-cAMPS in the presence of LCD resulted in no activation of Ca2+ signaling. After washout of LCD, the stimulatory effect of NE and Sp-cAMPS on Ca2+ signaling was restored. (n > 400 cells in 4 experiments for each condition, Fig. 11Go).



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Figure 11. LCD Inhibits the Stimulatory Effect of Sp-cAMPS on Ca2+ Signaling

Application of 40 µM LCD resulted in an increase in the frequency of spontaneous Ca2+ oscillations. In the presence of LCD, NE did not change the frequency of Ca2+ oscillations. After washout of LCD, the typical stimulatory effect of NE on Ca2+ oscillations was restored.

 
Elevated Extracellular Ca2+ Inhibits cAMP-Stimulated Ca2+ Signaling
Since CNG channels are inhibited by divalent cations, we investigated the effects of elevated extracellular Ca2+ before and after stimulation with forskolin, NE, or Sp-cAMPS. Increasing extracellular Ca2+ to 4 mM or higher resulted in a slight increase in baseline [Ca2+]i, followed by a slight decrease in the frequency of spontaneous Ca2+ oscillations. However, in the presence of elevated extracellular Ca2+, there was a marked inhibition of the stimulatory effects of forskolin (n > 500 cells in 5 experiments, Fig. 12Go), NE, or Sp-cAMPS (n > 300 cells in 3 experiments each, not shown). The inhibition of forskolin-stimulated Ca2+ signaling by elevated extracellular Ca2+ was rapidly reversible (Fig. 12).



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Figure 12. Extracellular Ca2+ Inhibits Forskolin-Stimulated Ca2+ Signaling

Increasing the [Ca2+] in the extracellular medium from 1.3 mM to 5 mM resulted in a slight decrease in the frequency of spontaneous Ca2+ oscillations, although the baseline [Ca2+]i and the amplitude of Ca2+ oscillations was increased in some cells. Application of forskolin in the presence of elevated extracellular Ca2+ resulted in no increase in the frequency of Ca2+ oscillations. Return to normal (1.3 mM) [Ca2+]e in the continued presence of forskolin evoked an immediate increase in the frequency of Ca2+ oscillations, which was abolished by subsequent addition of increased [Ca2+] medium and restored by returning again to normal [Ca2+]e.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The electrophysiological characteristics of the channels activated by cAMP in GT1 neurons are similar to those that have been described for CNG channels in other systems. Channel activity was evoked by both cAMP and cGMP and had a flickering behavior that is typical of heteromeric CNG channels (52, 53, 54). The activation of channels by both cAMP and cGMP is similar to CNG channels recorded in olfactory cells and is consistent with a channel comprised of the CNG2, CNG4.3, and CNG5 subunits that we have previously reported to be expressed in GT1 cells (45). The value that we estimated for the channel conductance (60 pS) is in the range of those reported for other cloned and native CNG channels (54, 55, 56). Distinct single channel currents were most clearly observed in the presence of Ca2+ at highly depolarized potentials. This may be because the intermittent blockade of the channel by Ca2+ results in more distinct channel openings, and because partial relief of Ca2+ blockade of the channel occurs at highly positive and negative potentials (47, 48, 49). The higher selectivity for Ca2+ vs. Na+, the increased Ca2+ permeability with increased membrane potential, and the inhibition of the cAMP current by Ca2+ are all consistent with the reports of other investigators (47, 48, 49, 50). The inhibition of the channel by LCD is also characteristic of CNG channels described in other cell systems (53, 57, 58, 59).

Ca2+ oscillations and intercellular Ca2+ waves in GT1 cells result from spontaneous bursts of Na+-dependent action potentials that cause influx of Ca2+ through L-type voltage gated channels (30). The increase in the frequency of Ca2+ oscillations observed in response to cAMP analogs is consistent with an increase in excitability due to depolarization of the membrane potential. A similar increase in the frequency of Ca2+ oscillations is observed in response to depolarizing stimuli, such as increased extracellular K+ or Ba2+ (29). Our results suggest that activation of CNG channels by cAMP modulates the excitability of GT1 cells by altering the depolarizing drive that underlies spontaneous oscillations in membrane potential. Although CNG channels are generally permeable to cations, at resting membrane potential the opening of CNG channels would be expected to result in an influx of Na+ and Ca2+. Influx of either Na+ or Ca2+ would result in depolarization that would bring baseline membrane potential closer to the threshold for firing of Na+ dependent action potentials, thereby increasing the frequency of action potential bursts and the resulting Ca2+ oscillations. In this manner, CNG channels could regulate the frequency of the spontaneous oscillatory activity of GT1 cells.

cAMP could also be modulating Ca2+ signaling through PKA-mediated phosphorylation of other ion channels such as voltage-gated Ca2+ channels, a mechanism that has been shown in a number of other cellular systems. However, the kinase inhibitor H9 did not inhibit the Ca2+ signaling response to Sp-cAMP or NE, indicating that phosphorylation mediated by activation of PKA is not required for the signaling response. The specific mechanism of the initial stimulatory effect of H9 on Ca2+ signaling is unclear. Previous studies have shown that H89 resulted in an acute increase in cAMP levels and GnRH secretion, suggesting an inhibition of an inhibitory feedback effect of PKA on cAMP production (45).

Regardless of the precise mechanism of the stimulatory effect of H9, however, this effect does not detract from the key conclusion drawn here, that inhibition of PKA did not inhibit the response to Sp-cAMP or NE. Consistent with these finding, H89 did not inhibit the increase in GnRH release induced by cAMP analogs or dopamine (45). Rp-cAMPS has also been shown to inhibit activation of PKA—the observation that Rp-cAMPS did not inhibit cAMP-stimulated Ca2+ signaling in GT1 cells provides additional evidence that PKA is not required for the stimulatory effect of cAMP.

Rp-cGMPS and Rp-cAMPS have variable effects on CNG channels (60, 61). Our observation that Rp-cAMPS resulted in a minimal increase in the frequency of Ca2+ oscillations compared with Sp-cAMPS is consistent with studies that have shown that the different phosphorothioate isomers have different effects on olfactory CNG channels (60), and the finding that Rp-cGMPS had a stimulatory effect on rat olfactory channels (61). The observation that the effects forskolin and NE were not inhibited by Rp-cAMPS indicates that the effects of 100 µM Rp-cAMPS on CNG channels could be overcome by increasing concentrations of cAMP.

LCD has been shown to block CNG channels at concentrations similar to those used here (53). Although LCD has been shown to inhibit CNG channels more effectively from the cytoplasmic side, a qualitatively similar inhibition of CNG channels has been reported from the extracellular surface (58). The reversible inhibition of the Ca2+ signaling response to NE and Sp-cAMPS by LCD in the extracellular medium therefore provides additional evidence that the Ca2+ signaling response to cAMP is mediated by the CNG channel. However, the mechanism of the initial stimulatory effect of LCD is unknown, and this stimulatory effect could play a role in the inhibitory effect of LCD on cAMP-stimulated Ca2+ signaling.

Since Ca2+ is another inhibitor of CNG channels, the inhibitory effect of extracellular Ca2+ provides additional evidence for CNG channels as mediators of the effects of cAMP. While extracellular Ca2+ had a slight inhibitory effect on spontaneous Ca2+ signaling, it had a marked inhibitory effect on the Ca2+ oscillations stimulated by forskolin, Sp cAMP, and NE. This inhibition of cAMP-stimulated Ca2+ oscillations by extracellular Ca2+ supports the results obtained with LCD and is consistent with a role for CNG channels in the stimulatory effects of cAMP on Ca2+ signaling.

There is strong evidence that the cAMP pathway plays a primary role in the modulation of GnRH release by neurotransmitters. Both dopamine and NE stimulate GnRH secretion in GT1 cells through receptors that are positively coupled to adenylyl cyclase (41, 42). The results described in this study suggest that CNG channels directly mediate the stimulatory effect of dopamine and NE on GnRH secretion. The frequency of Ca2+ oscillations in GT1 cells is highly correlated with GnRH release—stimuli that increase the frequency of Ca2+ oscillations (including high K+, Ba2+, GABA, and cAMP analogs) all stimulate GnRH release, whereas conditions that inhibit Ca2+ oscillations [tetrodotoxin (TTX), Ca2+ channel blockers] inhibit GnRH release (29, 33, 34, 35, 62). The activation of CNG channels by ligands that induce the formation of cAMP provides a mechanism by which these ligands can increase the frequency of Ca2+ oscillations and thereby increase GnRH release.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Culture
GT1-1 cells were maintained in culture in DMEM/F12 media (Mediatech, catalogue no. 10–092) supplemented with 5% FBS, 5% horse serum, 100 IU/ml penicillin and 100 µg/ml streptomycin in 252-mm flasks. Cells were grown to approximately 60–80% confluency and then passaged or transferred onto poly-D-lysine coated glass coverslips, on which they were grown for 3–10 days to a confluence of approximately 60–80% before experimentation.

Electrophysiology
Patch clamp experiments were performed on GT1–1 cells using the on-cell and excised patch configurations (63). Pipettes were constructed from 7740 borosilicate tubing (catalog no. TW150F, WPI, Corning, Inc., Sarasota, FL) and fire polished to a tip resistance of between 3–5 M{Omega}. Recordings were made after obtaining seals greater then 1 G{Omega}. For single-channel experiments, symmetrical solutions were used consisting of HBSS, 140 mM Na-methanesulfonate (MES) or 140 K-MES. HBSS consisted of (in mM) 136.9 NaCl, 0.34 Na2HPO4, 4.17 NaHCO3, 5.36 KCl, 0.44 KH2PO4, 0.81 MgSO4, 1.26 CaCl2, and 5.5 mM D-glucose (catalog no. 21–020, Mediatech, Herndon VA). Na-MES (Na-CH3SO3) and K-MES were used as low Cl- solutions. All solutions contained 10 mM HEPES titrated to pH 7.2 with NaOH or KOH. For macroscopic current experiments, symmetric HBSS or Na-MES solutions were used. To investigate effects of Ca2+, Na-MES was used in the pipette and HBSS or Na-MES with or without added CaCl2 was used in the bath. When 5 mM Na-MES or K-MES was used in the pipette solutions, 5 mM NaCl or KCl was added to keep the Ag/AgCl pipette wire stable. The bath ground consisted of an Ag/AgCl wire connected to the bath via a 100 mM KCl agar bridge.

Electrophysiological recordings were performed using pClamp 7 software, an Axopatch 200 amplifier, and a Digidata 1200 A/D board (Axon Instruments, Foster City, CA). Single channel and macroscopic membrane currents were measured under voltage clamp. For voltage ramp experiments, currents were activated with continuous voltage ramps from -100 to +100 mV over 1 sec. Before the ramp, the voltage was held at -100 mV for 250 msec to achieve a steady current after time-dependent changes due to voltage-dependent relief of channel block by divalent cations (50).

Measurement of [Ca2+]
[Ca2+]i was measured using a fluorescence imaging system that has previously been described in detail (64). Briefly, cells on poly-D-lysine-coated glass coverslips were loaded with fura2 by incubation in 5 µM fura2-AM for 40 min. Cells were then washed and maintained in normal medium for 30 min before experimentation. Coverslips were excited with a mercury lamp through 340 and 380 nm bandpass filters, and fluorescence at 510 nm was recorded through a 10x or 20x objective with a silicon-intensified target (SIT) camera or charge coupled device (CCD) camera to VHS videotape or to an optical memory disc recorder. Images were then digitized and subjected to background subtraction and shading correction, after which [Ca2+]i was calculated on a pixel-by-pixel basis, as previously described, by a frame grabber and image analysis board (Data Translation, Marlboro, MA). Alternatively, fluorescence data recorded on VHS videotape were digitized and analyzed with an Axon Image Lightning board and Axon Imaging Workbench software.

Mini Analysis Program (Synaptosoft) was used to quantify characteristics of calcium signals, including the frequency, amplitude, and decay time of calcium oscillations. The software program Transform (Research Systems, Inc., Boulder, CO) was used to generate raster plots and line graphs of calcium data.

Experiments were carried out in HBBS solution with 10 mM HEPES buffer, pH 7.4 (HBSS/HEPES) at 22 C. Ligands were applied by perfusion of coverslips with at least 2 ml of HBSS/HEPES containing the particular agent. Fura2 was obtained from Calbiochem (La Jolla, CA). All other reagents were obtained from Sigma (St. Louis, MO).


    FOOTNOTES
 
Address requests for reprints to: Andrew Charles, Department of Neurology, University of California Los Angeles, 710 Westwood Plaza, Los Angeles, California 90095. E-mail: acharles{at}ucla.edu

This work was supported by the following grants: NIH Grant P01 NS-02808 and National Science Foundation Grant IBN-9982585 to A.C.

1 Present address: Department of Molecular and Cellular Biology, University of California, Berkeley, Berkeley, California 94720. Back

Received for publication August 28, 2000. Revision received February 14, 2001. Accepted for publication March 8, 2001.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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