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
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ABSTRACT
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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.
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INTRODUCTION
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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.
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RESULTS
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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. 1
).
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. 1A
). The single channel conductance of the largest open
state at voltages between 70 and 120 mV was approximately 60 pS (n
= 9, Fig. 1
). 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 GT11
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 (503,000 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).
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Macroscopic currents activated by cAMP were also measured in a subset
of excised patches from GT11 neurons (Fig. 2
). 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. 3
). 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 GT11 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.
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Effects of cAMP Analogs on Ca2+
Signaling
GT11 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. 26



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. 4
and Table 1
). 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. 5
and Table 1
).

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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,
1256, 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. 2 . 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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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. 6
and Table 1
). The increase in the
frequency of Ca2+ oscillations occurred after a
variable delay of 15 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. 7
and Table 1
) 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.
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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. 8
). 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 510 min in the continued presence of the drug. Subsequent
application of Sp-cAMPS (Fig. 8
) 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 510 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. 5 ).
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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. 9
), 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. 5 . 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.
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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. 10
).
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 (80250
µM) increased the percentage of null sweeps by
approximately 2-fold (range 650% null sweeps in controls to 1398%
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.
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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. 11
).

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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.
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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. 12
), 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.
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DISCUSSION
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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 PKAthe 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 releasestimuli 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.
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MATERIALS AND METHODS
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Cell Culture
GT1-1 cells were maintained in culture in DMEM/F12
media (Mediatech, catalogue no. 10092) 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
6080% confluency and then passaged or transferred onto
poly-D-lysine coated glass coverslips, on which they were
grown for 310 days to a confluence of approximately 6080% before
experimentation.
Electrophysiology
Patch clamp experiments were performed on GT11 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 35 M
. Recordings were made after obtaining
seals greater then 1 G
. 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. 21020, 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. 
Received for publication August 28, 2000.
Revision received February 14, 2001.
Accepted for publication March 8, 2001.
 |
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