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Volume 16, Number 11,
Issue of June 1, 1996
pp. 3620-3629
Copyright ©1996 Society for Neuroscience
17 -Estradiol Potentiates Kainate-Induced Currents via
Activation of the cAMP Cascade
Qin Gu and
Robert L. Moss
Department of Physiology, University of Texas Southwestern Medical
Center at Dallas, Dallas, Texas 75235
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Evidence for nongenomic actions of steroids is now coming from a
variety of fields of steroid research. Mechanisms of steroid action are
being studied with regard to the membrane receptors and the activation
of second messengers. The present study investigated the mechanism for
the rapid effect of estrogen on acutely dissociated hippocampal CA1
neurons by using the whole-cell, voltage-clamp recording. Under the
perforated patch configuration, 17 -estradiol potentiated
kainate-induced currents in 38% of tested neurons. The potentiation
was stereospecific, rapid in onset, and reversible after the removal of
the steroid. Dose-response curves show that the potentiation by
17-estradiol was evident at a concentration as low as 10 nM and saturated at 10 µM. 17-Estradiol did not affect the kinetics
(i.e., affinity and cooperativity) and reversal potential of
kainate-induced currents. This suggests that the potentiation did not
result from direct interaction with kainate receptors nor the
activation of ion channels other than kainate receptor-channels. The
potentiation by 17-estradiol was similar to the enhancement of
kainate-induced currents evoked by 8-bromo-cAMP, and was modulated by
an inhibitor of phosphodiesterase (IBMX). The estrogen potentiation was
blocked by a specific blocker of PKA (Rp-cAMPS). Under
standard recording configuration, the effect was significantly affected
by intracellular perfusing with GDP--S or GTP- -S. The data
suggest that the potentiation of kainate-induced currents by
17-estradiol was likely a G-protein(s) coupled, cAMP-dependent
phosphorylation event. By involvement of this nongenomic mechanism,
estrogen may play a role in the modulation of excitatory synaptic
transmission in the hippocampus.
Key words:
steroid hormone;
modulator;
non-NMDA receptor;
inward current;
PKA;
G-protein;
nongenomic
INTRODUCTION
Estrogen acts on a number of different target
organs that contain specific estrogen receptors, including the brain,
to modulate a variety of physiological functions (Stumpf and Sar, 1978 ;
McEwen, 1979, 1991; Maggi and Prerez, 1985). Functionally, the
effects of estrogen range from rapid actions on membrane excitability
and synaptic transmission to long-term actions on sexual
differentiation, development and maturation of the brain, morphological
properties of neurons, endocrine regulation, and initiation of
reproductive behavior (Gould et al., 1990; Woolley and McEwen,
1992).
Two mechanisms have been proposed to characterize the actions of
estrogen (Wehling, 1995). The first is the genomic mechanism of action
that involves the diffusion or transport of estrogen across the
membrane into the neuron, activation of specific intracellular
receptors, and transcriptional regulation of particular genes that
ultimately results in protein synthesis (Pfaff, 1980; Pfaff and
Schwartz-Giblin, 1988; Carson-Jurica et al., 1990; Smith et al., 1993).
In contrast, estrogen has also been found to produce short-term actions
on the electrical properties of neurons and transmitter release. The
rapid onset and time course of these effects suggest that they are
distinct from the genomic mechanism of action and indicate that
estrogen may act on membrane receptors or other cellular components to
alter neuronal events (Nabekura et al., 1986; Smith et al., 1987, 1989;
Minami et al., 1990; Kelly et al., 1992; Majewska, 1992; Paul and
Purdy, 1992). However, the mechanisms to account for the rapid actions
of estrogen have remained elusive.
In hippocampus, 17-estradiol initiated a rapid and reversible
increase in the amplitude of glutamate and kainate-induced or Schaffer
collateral-activated EPSPs in CA1 hippocampal neurons. These responses
were blocked by a non-NMDA antagonist (Wong and Moss, 1991, 1992). In
subsequent experiments using excised patch-clamp recording from CA1
neurons, 17-estradiol did not affect the parameters of single
channel activity induced by kainate, a non-NMDA agonist (Wong and Moss,
1994). Because excised membrane patches lack many important cytoplasmic
enzymes and regulatory cofactors, it is likely that the rapid action
may require that certain intracellular components remain intact.
There is mounting evidence that estrogen may activate intracellular
second messenger systems to modulate neuronal functions. Estrogen has
been shown to change cyclic nucleotide levels in the cell (Gunaga et
al., 1974; Weissman et al., 1975), and act on potassium channels via a
second messenger system involving cAMP to modulate the membrane
potential (Minami et al., 1990). Furthermore, estrogen can directly
stimulate inositol trisphosphates (Favit et al., 1991; Smith, 1991). At
the receptor level, estrogen can rapidly alter ligand-binding
properties of several G-protein-coupled neurotransmitter receptors such
as 5-HT (Bigeon and McEwen, 1982), norepinephrine (Inaba and Kamata,
1979), and D2 dopamine receptors (Levesque and
DiPaolo, 1988; Bazzett and Bender, 1994). Estrogen has also been shown
to induce rapid pharmacodynamic changes of the G-protein-coupled
mechanism of another ligand, decreasing the potency of opioid agonists
in opening inward-rectifying potassium channels (Lagrange et al.,
1994). Moreover, estrogen directly potentiates potassium-stimulated
dopamine release in rat nucleus accumbens (Thompson and Moss,
1994).
In the experiments described here we address the hypothesis that
17-estradiol potentiates the kainate-induced current by the
activation of a cAMP cascade. We show that 17-estradiol potentiates
the kainate-induced current and that this potentiation is mimicked by
cAMP and blocked by Rp-cAMPS. Administration of GDP--S
suppresses, whereas GTP--S prolongs, the potentiation by estrogen.
The findings suggest the nongenomic action of estrogen on
kainate-induced currents involves activation of a cAMP-dependent
phosphorylation process.
MATERIALS AND METHODS
All animal experimentation was conducted in accordance with the
National Institutes of Health Guidelines for the Care and Use of
Laboratory Animals.
Preparation of acutely dissociated neurons. CA1 hippocampal
pyramidal neurons were acutely dissociated using modified procedures of
Kay and Wong (1986). Sprague-Dawley male and female rats from 2 to 4 weeks old were decapitated via a guillotine. Hippocampi were removed
from the brain and quickly blocked and placed in cold PIPES-saline
solution (in mM): NaCl, 120; KCl, 5.0;
CaCl2, 1.0; MgCl2, 1.0;
D-glucose, 25; PIPES, 20; pH 7.4. The blocked
tissue was cut on a vibratome (Oxford) at ~450 µm thick while
bathed in 4°C oxygenated PIPES-saline solution. The slices were
placed on a black surface and punches were made in the CA1 area with a
capillary tube. The punches were incubated at room temperature
(20-22°C) in PIPES-saline solution with 1.5 mg/ml protease (Sigma,
St. Louis, MO). The incubation medium was stirred slowly and smoothly
with 95% O2/5% CO2 blown
at its surface. After 30-45 min of enzymatic digestion, punches were
rinsed three times in oxygenated PIPES-saline and triturated with a
fire-polished Pasteur pipette for mechanical dissociation. The cell
suspension was then plated into the central concave area of a slide
containing the standard extracellular solution (in
mM): NaCl, 140; KCl, 3.0;
CaCl2, 2.0; HEPES, 10; pH 7.3. All chemicals were
obtained from Sigma.
Whole-cell, patch-clamp recordings. Whole-cell recordings
were performed under voltage-clamp mode according to standard
techniques (Hammil et al., 1981). Both conventional as well as
perforated whole-cell, patch-clamp recordings were used in isolated CA1
hippocampal neurons. The dissociated neurons were ~30-40
µM in diameter and were visualized with a Nikon
inverted phase-contrast microscope equipped with Nomarski optics.
Whole-cell recording electrodes were pulled from borosilicated glass
with a Sutter Flaming-Brown electrode puller and fire-polished with a
CPM-2 coating/polishing microforge. The electrode resistance was
typically 2-5 M in bath solution. The standard internal solution
for recording electrode consisted of the following (in
mM): CsCl, 140; NaCl, 4.0; EGTA, 10; HEPES, 10;
CaCl2, 1. The internal solution was adjusted to
pH 7.3 with CsOH. For perforated whole-cell recording, the electrode
was first submerged in standard internal solution for 3-5 sec. A small
amount of solution would reach the tip by capillary action. Then
additional standard internal solution containing 100 mg/ml nystatin was
added to the electrode by backfilling the electrode.
After formation of a gigaohm (>1 G) seal, the membrane was ruptured
by a slight suction in the case of conventional whole-cell recording.
In the case of the perforated whole-cell recording configuration,
however, series resistance and cell capacitance from a capacitive
transient current evoked by voltage pulses ( 10 mV, 20 msec) were
monitored after obtaining a giga-seal. During the development of
nystatin perforation, the amplitude of the capacitive transient
increased and the time constant of the transient decreased. Whole-cell
currents evoked by 20 mV voltage steps (from 80 to +80 mV) gradually
became larger with time, the series resistance decreased to a stable
minimal value (<12 M), indicating the establishment of perforated
patch recording. This usually occurred within 15-25 min. The
subsequent recordings were held for more than 45 min. The holding
potential was 60 mV in both configurations, except in the case of
studying the current-voltage relations where the holding potential was
varied from 100 to + 50 mV. Access resistance was compensated (80%)
electronically and monitored periodically.
Puffer electrode and chemical application. A seven-barrel
pipette with a total diameter of 10 µM was used
to puff individual substances on the dendrite of the recorded
dissociated CA1 hippocampal neuron. Ejection of each chemical could be
made separately with a picospritzer unit (General Valve Corp.).
Kainate, 17-estradiol, 17 -estradiol, 8-bromo-cAMP, IBMX,
Rp-cyclic adenosine 3 ,5-monophosphothioate
(Rp-cAMPS), or 8-bromo-cGMP were assigned randomly to one of
the seven barrels. To establish a dose-response curve, each barrel of
the pipette was assigned with one of the different concentrations of
17-estradiol (100 pM, 1 nM, 10 nM, 100 nM, 1 µM, 10 µM) and kainate (100 µM), or with one of the different
concentrations of kainate (1 µM, 10 µM, 100 µM, 1 mM, 10 mM) and
17-estradiol (100 nM). All chemicals were from
Sigma, but Rp-cAMPS was obtained from LC Laboratories.
Kainate currents were induced by pulses of ejection (20 msec, 0.1-1.0
psi) of kainate at the dendrite of CA1 neuron. The application was
repeated once every 30 sec and commenced immediately after the patch
was ruptured. The effects of the different drugs on the kainate-induced
currents were tested by extracellularly perfusing the cell for 3 min.
In some experiments, guanosine 5-O-(2-thiodiphosphate)
(GDP--S), GDP, GTP, or guanosine
5-O-(3-thiotriphosphate) (GTP--S) was added to the
standard internal solution and allowed to diffuse into the cell. These
experiments were conducted under the standard whole-cell recording
configuration, and an ATP regeneration system consisting of 4 mg of
Tris-ATP, 20 mM phosphocreatine, and 50 U/ml
creatine phosphokinase (all from Sigma) was added to the internal
solution to minimize the washout effect on kainate-induced
currents.
Kainate was made as a concentrated stock solution and was diluted in
standard extracellular bath solution before use at a concentration of
100 µM. 17-Estradiol or 17-estradiol was
initially dissolved in DMSO and diluted with standard extracellular
bath solution to one of the following concentrations: 100 pM, 1 nM, 10 nM, 100 nM, 1 µM with the final DMSO concentration not
exceeding 0.01%. 8-Bromo-cAMP and 8-bromo-cGMP were made up in a stock
solution and frozen. Aliquots were diluted with standard extracellular
bath solution immediately before use to concentrations of 500 µM and 1 mM. IBMX was
added directly to the external solution for final concentration of 100 µM. Rp-cAMPS was stored in a sealed
container at 20°C and diluted in external bath solution immediately
before use at 50 µM. GDP--S and GTP--S
were stored in aliquots at 70°C and diluted to 500 µM with standard internal solution.
Data analysis. Whole-cell currents were recorded under
voltage-clamp configuration with Adams/List EPC-9 amplifier and sampled
at 2 kHz and filtered at 2.3 kHz. Data were digitized and stored on an
Atari Mega 4 computer. Analysis of whole-cell current records was
performed with an Atari data analysis program. Peak currents were
normalized as I/I0, where I
represents the amplitude of kainate-induced currents at any testing
time point and I0 is the initial value at
the beginning of the recording. Percentage change in kainate-induced
currents was determined according to the formula
(Idrug/I0 1)100%, where Idrug represents the peak
amplitude of the kainate current in the presence of the test drug.
Dose-response data were fitted with the logistic equation in the form
Idrug I0 = [Imax I0]/[1 + (EC50/D)nH] for
17-estradiol, and I = Imax/[1 + (EC50/D)nH]
for kainate, where Imax is the response to
a saturating concentration of agonist, EC50 is
the concentration that evokes a half-maximal response, and nH
is the estimated Hill coefficient that describes the steepness of the
curve. Fitting was performed with SIGMAPLOT (Jandel Scientific, Corte
Madera, CA). Current-voltage data were obtained by subtraction of leak
currents from currents recorded in the presence of agonists at each
potential. All quantitative data are expressed as mean ± SEM; n
indicates the number of cell tested. Statistical analysis was performed
using the paired or unpaired Student's t test. Results were
considered significant only for p < 0.05.
RESULTS
Kainate-induced currents
Under the conventional whole-cell recording configuration,
application of kainate (100 µM) to the
dendrites of CA1 hippocampal neurons initially evoked an inward
current. The initial amplitude of kainate-induced current was 560 ± 214 pA (n = 12). The amplitude declined by 50% within 20 min (Fig. 1A,B). This washout effect was
apparently attributable to intracellular dialysis (Korn et al., 1991)
and was similar to that previously reported for kainate and NMDA
currents (MacDonald et al., 1989; Wang et al., 1991). The application
of 17-estradiol (100 nM) significantly
retarded the decline in kainate-induced current amplitude observed
during washout (Fig. 1A,C). The effect of 17-estradiol
was observed in 3 out of 10 neurons tested, occurred within 5 min of
application, and reversed within 3-5 min. To circumvent the washout of
the kainate-induced currents, the perforated patch technique was used
to record whole-cell currents. In this configuration, whole-cell
recording can be done without diluting important substances from the
cytoplasm, and physiologically relevant mechanisms remain operational.
Repeated application of kainate (100 µM) onto a
cell yielded responses with a peak value of 691 ± 382 pA (n = 82). The amplitude rarely varied (within ±10%) and was stable for a
period of more than 45 min (Fig. 1A,D). All subsequent
results were obtained under this perforated configuration except in the
case where GTP--S or GDP--S was included in the recording
pipette.
Fig. 1.
Comparison of kainate-induced currents using
conventional and perforated whole-cell patch recording techniques on
acutely dissociated CA1 hippocampal neurons. A, Two cells
were recorded under the conventional whole-cell recording
configuration. A 20 msec pressure application of kainate (100 µM) to the dendrites of these CA1 neurons
evoked an inward current, the amplitude of which declined to 50% of
its initial value within 20 min (open triangles). One cell
(open circles) was tested with 17-estradiol (100 nM). The application of 17-estradiol
(solid circles) significantly retarded the decline in the
amplitude of kainate-induced currents. Also in A, the
amplitude of kainate-induced currents, obtained using the perforated
patch recording, was stable for a period of up to 25 min (open
squares). Individual current traces recorded under the
conventional whole-cell patch configuration in the absence
(B) and presence (C) of 17-estradiol, and
under perforated whole-cell patch configuration (D), are
shown to the right of the graph.
[View Larger Version of this Image (17K GIF file)]
Potentiation of kainate-induced currents by 17-estradiol
The amplitude of kainate-induced currents was increased in the
presence of 17-estradiol (100 nM, 3 min) by
45.86 ± 4.1% of its original value (31 of 82 cells tested, 38%). The
potentiation was rapid in onset (within 3 min of application) and
reversible. However, in the cells (n = 16) responsible to
17-estradiol, the application of 17-estradiol (100 nM, 3 min), an inactive isomer of
17-estradiol, induced no obvious change in the kainate current
(104.76 ± 5.81% compared with control as 100%) (Fig.
2). It suggested that the potentiation was a
stereospecific effect.
Fig. 2.
17-Estradiol potentiated the kainate-induced
current. The illustrated example shows the effects of 17- and
17-estradiol on kainate-induced currents recorded from an individual
hippocampal neuron. The amplitude of kainate-induced currents was
increased in the presence of 17-estradiol (100 nM) but not 17-estradiol (100 nM). The potentiation was rapid in onset (2-3
min from its application) and reversible after removal of the chemical.
Actual current traces selected at specific time points (filled
circles) are displayed to the right of the graph.
[View Larger Version of this Image (17K GIF file)]
The potentiation by 17-estradiol was evident at a concentration as
low as 10 nM and appeared to be saturated at 10 µM (n = 5). The dose-response curve
gave an EC50 16.370 ± 2.763 nM and a nH 0.8878 ± 0.0918 (Fig.
3A). In the absence of kainate,
17-estradiol alone had no obvious effect on membrane current (data
not shown). Dose-response curves for various concentrations of kainate
before 17-estradiol (100 nM) application show
an EC50 114.20 ± 0.52 µM
and a nH 1.33 ± 0.11. The application of 17-estradiol significantly
(p < 0.02) increased the amplitude of kainate-induced
currents (56 ± 5%; n = 5) at the tested kainate
concentrations of 100 µM, 1 mM, and 10 µM.
17-Estradiol had no evident effect on the values of
EC50 (102.96 ± 0.92 µM)
and nH (1.15 ± 0.16) (Fig. 3B).
Fig. 3.
Potentiation of kainate-induced currents by
17-estradiol was dose-dependent. A, The effect of
17-estradiol was not evident at concentrations below 10 nM and appears saturated at 10 µM (n = 5). B,
Dose-response curves for various concentrations of kainate before
( ) and after ( ) 17-estradiol (100 nM)
application show that 17-estradiol increased the amplitude of
kainate-induced currents with no effect on the values of
EC50 and nH (n = 5).
[View Larger Version of this Image (15K GIF file)]
Current-voltage curves show a nearly identical reversal potential
(0.350 ± 0.3561 mV compared with control 0.7625 ± 0.1322 mV;
n = 6) and linearity. However, the slopes were significantly
different (2.220 ± 1.090 nS compared with control 1.6333 ± 0.1291 nS;
p < 0.01; n = 6). This result is consistent with
the involvement of a nonspecific cationic conductance of
kainate-induced currents. In the illustrated recording (Fig.
4), the conductance to kainate was increased from 1.8 nS
to 2.5 nS by 17-estradiol (100 nM).
Fig. 4.
Current-voltage curves obtained in the absence
(open circles) and presence (filled
circles) of 17-estradiol (100 nM).
Both relationships are linear with a nearly identical reversal
potential; however, the slopes were significantly different.
17-Estradiol (100 nM) increased the
conductance to kainate (100 µM) from 1.8 to 2.5 nS, as determined from the slope of the current-voltage relation.
Individual kainate-induced currents used to construct the plot are
shown to the right of the graph.
[View Larger Version of this Image (20K GIF file)]
Mediation of cyclic nucleotide
To investigate the mechanism underlying the potentiation of
kainate-induced currents by 17-estradiol, possible modulatory agents
including cyclic nucleotides (cAMP, cGMP) and IBMX were tested on the
neurons responsive to 17-estradiol. 8-Bromo-cAMP (500 µM), a membrane-permeant and
phosphodiesterase-resistant analog of cAMP, increased the amplitude of
kainate-induced currents (39.60 ± 3.88%; n = 10), whereas
8-bromo-cGMP (500 µM) had no effect on
kainate-induced currents (105.64 ± 8.52% compared with control as
100%; n = 10). This finding suggests the possibility of
specific effect of cAMP on kainate-induced currents. The enhancement
evoked by 8-bromo-cAMP was similar to that of 17-estradiol in the
latency and magnitude of current increase (Fig. 5).
IBMX, a membrane-permeant phosphodiesterase inhibitor, was tested to
identify whether 17-estradiol was exerting its effect via the cAMP
cascade. IBMX (100 µM) enhanced the effects of
a subsaturating concentration (100 nM) of
17-estradiol (28.97 ± 6.03%; p < 0.05; n = 4) (Fig. 6A). IBMX applied alone had no
obvious effect on membrane current or kainate-induced currents
(n = 4; data not shown). Furthermore, the presence of
17-estradiol at its saturating concentration (10 µM) occluded the enhancement induced by
8-bromo-cAMP (500 µM) (Fig. 6B),
even though both agents could potentiate the kainate-induced current
when applied alone (n = 3).
Fig. 5.
The enhancement of kainate-induced currents by
cAMP, 8-bromo-cAMP (500 µM) increased the
amplitude of kainate-induced currents, whereas 8-bromo-cGMP (500 µM) had no obvious effect on kainate-induced
current. The enhancement evoked by 8-bromo-cAMP was similar to that of
17-estradiol in the latency and magnitude. Sample current traces
before and after 8-bromo-cAMP and 8-bromo-cGMP application are shown to
the right of the graph.
[View Larger Version of this Image (17K GIF file)]
Fig. 6.
Potentiation of kainate-induced currents by
17-estradiol was mediated by cAMP. A, Application of IBMX
(100 µM) enhanced the effect of 17-estradiol
(100 nM). B, The presence of
17-estradiol at its saturating concentration (10 µM) almost occluded the enhancement induced by
8-bromo-cAMP (500 µM), even though both agents
could potentiate the kainate-induced currents when applied alone (data
not shown). Sample current traces selected at specific time points
(filled circles) are shown to the right of the
graphs.
[View Larger Version of this Image (22K GIF file)]
Involvement of cAMP-dependent protein kinase and
GTP-binding protein(s)
In hippocampal CA1 neurons, the principle cellular target site of
cAMP is the regulatory subunit of cAMP-dependent protein kinase (PKA).
Because the results from the present study indicate that the effects of
17-estradiol could be mediated by cAMP, inhibiting PKA should block
the potentiation of 17-estradiol on kainate currents. Experiments
treating neurons with an inhibitor of PKA were consistent with this
hypothesis. The administration of Rp-cAMPS (50 µM; n = 5), a particularly potent
competitive inhibitor of PKA (Dostmann et al., 1990), completely
eliminated the potentiation normally observed with the application of
17-estradiol. An example of the effect of Rp-cAMPS on
17-estradiol potentiation of kainate-induced currents is illustrated
in Figure 7.
Fig. 7.
Involvement of cAMP-dependent protein kinase.
A, The administration of Rp-cAMPS (50 µM) completely eliminated the potentiation
normally observed with the application of 17-estradiol (100 nM). Sample current traces selected at specific
time points (filled circles) are shown to the right of
the graph.
[View Larger Version of this Image (17K GIF file)]
To verify whether the pathway mediating the actions of 17-estradiol
on kainate currents involves a G-protein(s), isolated hippocampal
neurons (n = 38) were recorded under the conventional
whole-cell recording configuration and intracellularly perfused with
GDP--S (500 µM) or GTP--S (500 µM). Ten of 27 cells tested (37%) were
responsive to 17-estradiol (100 nM). The
potentiation was significantly (p < 0.001) suppressed by
the application of GDP--S (46 ± 11%; n = 3; Fig.
8A). Whereas intracellular administration of
GDP(500 µM) alone, as well as GTP (500 µM; data not shown), had no effect on the
kainate-induced currents (n = 3; Fig. 8B). The
potentiation induced by 17-estradiol was significantly (p < 0.001) prolonged in the presence of GTP--S compared with control
(n = 4; Fig. 8C). Intracellular diffusion of
GDP--S or GTP--S alone caused no evident effect on membrane
current or kainate-induced currents (n = 11; data not
shown).
Fig. 8.
Effect of G-protein(s) on the 17-estradiol
potentiation of kainate-induced currents. Isolated hippocampal neurons
were recorded under the conventional whole-cell configuration and
dialyzed with GDP--S (500 µM), GDP alone, or
GTP--S (500 µM). As illustrated in
A, the
potentiation induced by 17-estradiol shortly after
membrane rupture was suppressed as GDP--S diffused into the cell (46 ± 11%; n = 3), whereas in B, intracellular
perfusion with GDP alone had no effect on the kainate-induced currents
(n = 3). C, The intracellular administration of
GTP--S prolonged the potentiation by 17-estradiol (n = 4).
[View Larger Version of this Image (10K GIF file)]
DISCUSSION
The results indicate that 17-estradiol potentiated
kainate-induced currents in a subpopulation (38 and 37% from
perforated recording and standard recording, respectively) of acutely
dissociated hippocampal CA1 neurons. The effects of 17-estradiol
were stereospecific, reversible, rapid in onset, and persisted for the
duration of its application. Furthermore, the effects of
17-estradiol on kainate-induced currents were mimicked by
8-bromo-cAMP and modulated by IBMX. The estrogen-induced potentiation
was blocked by a specific blocker of PKA (Rp-cAMPS),
suppressed by GDP--S, and prolonged by GTP--S. The data suggest
that the potentiation of kainate-induced currents by 17-estradiol
appears to be mediated by a G-protein(s) coupled, cAMP-dependent
phosphorylation of kainate receptors.
Despite previous electrophysiological evidence for modulation of
glutamate receptors by estrogen, Wong and Moss reported that a
single-channel analysis of both NMDA and non-NMDA receptors in the
excised membrane patch failed to find evidence of direct glutamate
receptor-channel modulation by estrogen, even though pregnenolone
sulfate was shown to potentiate NMDA receptor-channel activity in the
experiments (Wong and Moss, 1994). Their studies argued for an indirect
interaction between estrogen and glutamate receptor ligands. However,
there is more evidence from both non-neural and neural tissues that
estrogen may directly activate the production of the cyclic nucleotide
second messengers (Szego and Davis, 1967; Weissman et al., 1975; Mugge
et al., 1993). Biochemical studies suggest that estrogen can directly
stimulate adenylate cyclase activity in uterine cells (Bergamini et
al., 1985). Estrogen can acutely elevate the level of cAMP in
hypothalamic neurons (Gunaga et al., 1974), and estrogen-induced
depolarization associated with decreased potassium conductance is
enhanced by a specific adenylate cyclase activator and a
phosphodiesterase inhibitor (Minami et al., 1990). In the cerebellum,
the estrogen-induced potentiation of excitatory responses to glutamate
may be mediated by the inositol triphosphate second messenger system,
as acute estrogen treatment increases quisqualate-stimulated
phosphatidyl inositol in cerebellar neurons (Smith, 1991).
Several studies have suggested that brain second messenger systems,
well known for altering nerve cell activity by modifying
characteristics of voltage-gated channels (Kaczmarek and Levitan,
1987), also play a role in the modulation of ligand-gated ionotropic
glutamate receptors (Wang et al., 1991, 1994; Blackstone et al., 1994;
Lieberman and Mody, 1994). In cultured hippocampal neurons, the
whole-cell current response to glutamate and kainate was enhanced by
forskolin, an activator of adenylate cyclase, and the opening frequency
and the mean open time of the non-NMDA-type glutamate receptor channel
was increased by adenosine 3,5-monophosphate-dependent protein kinase
(PKA) (Greengard et al., 1991). Based on the sequences of cloned
subunits, most of the ligand-gated ionotropic glutamate receptors
contain potential phosphorylation sites for PKC and
Ca2+/calmodulin-dependent protein kinase II
(McGlade-McCulloh et al., 1993). The GluR6 subunit contains a consensus
sequence for phosphorylation by PKA. Phosphorylation of kainate
receptor complexes that contain the GluR6 subunit increases the
glutamate- or kainate-induced responses (Raymond et al., 1993; Wang et
al., 1993). The phenomenon of washout in the conventional whole-cell
recording of kainate induced-currents resulted from the decreased
ability of the neuron to maintain such physiologically relevant
mechanisms, especially intracellular phosphorylation. Intracellular
application of the catalytic subunit of PKA and cAMP prevented the
rundown of kainate-induced currents (Wang et al., 1991). The
retardatory effect of 17-estradiol occurred with a short latency and
appeared similar to that of PKA in preventing the washout of
kainate-induced currents. This result suggested that the effect of
17-estradiol likely involved the maintenance or activation of
intracellular cAMP-dependent phosphorylation rather than a genomic
mechanism.
An interesting feature of potentiation by 17-estradiol on the
kainate-induced current is the concentration of the steroid. In
vivo, the gonads are the most likely source of estrogen and
progestin. After release into the circulation, steroids can access most
of the brain uniformly. At present there is no evidence for de
novo synthesis of estrogens in the brain, however, the brain
(glial cells and synaptic terminals) does contain a necessary enzyme
(aromatase) for the conversion of circulating testosterone to
17-estradiol. Pregnenolone and dehydroepiandrosterone are derived
from cholesterol and are found in the brain at concentrations superior
to those found in the blood on the order of 10 nM
and 100 nM (Baulieu and Robel, 1991). In the
present study, the effect of 17-estradiol was dose-dependent;
concentrations as low as 10 nM potentiated the
kainate-induced current. As 17-estradiol was applied to the
extracellular solution, the real concentration affecting the cell was
lower than 10 nM and probably within the
effective concentration range of other neurosteroids (Wong and Moss,
1992, 1994; ffrench-Mullen et al., 1994, 1995).
Dose-response curves show that the concentration of kainate required
to evoke a half-maximal response was the same before and during the
application of 17-estradiol as were the calculated Hill slopes. This
finding suggests that no substantial changes in either the apparent
affinity or cooperativity of the response occurred during the
application of 17-estradiol. Analysis of current-voltage
relationships before and after the introduction of 17-estradiol
showed that 17-estradiol caused no obvious changes in the reversal
potential of the kainate-induced current. This result suggests that the
potentiation by 17-estradiol did not result from activation of
additional ionic conductance but was caused by increasing the
conductance of the kainate receptor-channels. As the potentiation by
17-estradiol was similar to the enhancement of kainate currents by
8-bromo-cAMP both in the time course and degree, 17-estradiol may
have activated an intrinsic second messenger system rather than acted
directly on the kainate receptors.
Evidence that 17-estradiol acted through elevation of cAMP levels
was obtained by application of IBMX, an agent that prolongs the
lifetime of cAMP by inhibiting phosphodiesterase. IBMX enhanced the
effect of 17-estradiol. The ability of a saturating concentration of
17-estradiol to occlude the modulation produced by 8-bromo-cAMP also
argues for a shared pathway. The fact that a specific PKA inhibitor,
Rp-cAMPS, was able to block the effect of 17-estradiol
makes a more convincing case for the involvement of cAMP-dependent
phosphorylation in the estrogenic modulation of kainate-induced
currents.
Involvement of a G-protein(s) in the 17-estradiol potentiation of
kainate-induced currents was addressed by including the nonhydrolyzable
GTP analogs, GDP--S and GTP--S, in the recording electrode.
GDP--S and GTP--S compete with intracellular GTP for the binding
site of G-protein at a subunit. GDP--S locks the GTPase cycle in an
inactive status, and GTP--S activates G-proteins irreversibly
(Gilman, 1987; Hepler and Gilman, 1992). The estrogenic potentiation on
kainate-induced currents was affected by intracellular perfusion of
GDP--S or GTP--S, which suggests the action of 17-estradiol
was likely to be G-protein(s) coupled. The identity of the G-protein(s)
and the specific subclass cannot be determined from the present
data.
Recent reports indicate that some neurosteroids modulate voltage-gated
calcium currents in hippocampal CA1 neurons via a pertussis
toxin-sensitive G-protein-coupled mechanism and the data suggests that
the steroids can access their binding site from the extracellular
surface (ffrench-Mullen et al., 1994, 1995). At the present time and to
our knowledge, no evidence exists to support the notion of specific
membrane binding sites for estrogen in hippocampus. Although high
affinity membrane estrogen-binding sites have been identified from
several specific brain regions, such as hypothalamus, cerebellum, and
olfactory bulb (Zheng and Ramirez, 1994). In our recent preliminary
experiments, 17-estradiol conjugated with BSA had no observable
effect on kainate-induced currents when applied to the outside of the
cell (data not shown). Estrogen-BSA has been demonstrated to be
biologically active (Lieberherr et al., 1993; Tesarik and Mendoza,
1995). Because estrogen is lipophilic and readily crosses the membrane
and the conjugate (BSA) is membrane impermeable, this finding suggests
that 17-estradiol might have to cross the membrane to affect
kainate-induced currents. a subpopulation of cytoplasmic steroid
receptors that are not translocated to the nucleus has been described
(Welshons and Judy, 1995). The estrogen could bind to these receptors
and, theoretically, participate in rapid, nongenomic estrogen
events.
In conclusion, the present results demonstrate that physiological
levels of 17-estradiol reversibly potentiate the amplitude of
kainate-induced currents. This modulation is associated with a
G-protein(s) coupled, cAMP dependent phosphorylation process. This
potentiation of kainate-induced currents may participate in the
regulation of glutamatergic transmission in hippocampus, where the
glutamate receptors have been implicated in memory, epilepsy,
excitotoxicity, and some neurodegenerative diseases (Collingridge and
Lester, 1989; Meldrum and Garthwaite, 1990). Future studies are
required to identify the binding sites on which 17-estradiol exerts
this nongenomic effect and to clarify the physiological significance of
the enhancement of kainate-induced currents by 17-estradiol in
hippocampus.
FOOTNOTES
Received Jan. 5, 1996; revised Feb. 27, 1996; accepted March 8, 1996.
This work was supported by Research Grant 1 RO1-MH 47418 from the
National Institute of Mental Health, National Institutes of Health,
awarded to R.L.M. We thank Carol Dudley for her comments and criticisms
on this manuscript and Dr. Tina Thompson for her technical assistance.
We also thank Drs. Susan Mumby and Al Gilman for their generous gift of
purified GDP--S and GTP--S.
Correspondence should be addressed to Dr. Robert L. Moss, Department of
Physiology, University of Texas Southwestern Medical Center at Dallas,
5323 Harry Hines Boulevard, Dallas, TX
75235-9040.
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C. E. Navarro, S. A. Saeed, C. Murdock, A. J. Martinez-Fuentes, K. K. Arora, L. Z. Krsmanovic, and K. J. Catt
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J. Qiu, M. A. Bosch, S. C. Tobias, D. K. Grandy, T. S. Scanlan, O. K. Ronnekleiv, and M. J. Kelly
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O. A. Sukocheva, L. Wang, N. Albanese, S. M. Pitson, M. A. Vadas, and P. Xia
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Mol. Endocrinol.,
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C. E. Navarro, S. Abdul Saeed, C. Murdock, A. J. Martinez-Fuentes, K. K. Arora, L. Z. Krsmanovic, and K. J. Catt
Regulation of Cyclic Adenosine 3',5'-Monophosphate Signaling and Pulsatile Neurosecretion by Gi-coupled Plasma Membrane Estrogen Receptors in Immortalized Gonadotropin-Releasing Hormone Neurons
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N. Foldvary-Schaefer and T. Falcone
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I. M. Abraham, S.-K. Han, M. G. Todman, K. S. Korach, and A. E. Herbison
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R. M. LOSEL, E. FALKENSTEIN, M. FEURING, A. SCHULTZ, H.-C. TILLMANN, K. ROSSOL-HASEROTH, and M. WEHLING
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S. L. Hardy, G. M. Anderson, M. Valent, J. M. Connors, and R. L. Goodman
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C. N. Rudick and C. S. Woolley
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A. Kumar and T. C. Foster
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H C Christian and J F Morris
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L. Curtis, B. Buisson, S. Bertrand, and D. Bertrand
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E. Falkenstein, H.-C. Tillmann, M. Christ, M. Feuring, and M. Wehling
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M. R. Zamani, N. L Desmond, and W. B Levy
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E. J. Oh, L. P. Thompson, and D. Weinreich
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A. B Ropero, E. Fuentes, J. M Rovira, C. Ripoll, B. Soria, and A. Nadal
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D. B. Dubal, P. J. Shughrue, M. E. Wilson, I. Merchenthaler, and P. M. Wise
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P. M. Wise, M. J. Smith, D. B. Dubal, M. E. Wilson, K. M. Krajnak, and K. L. Rosewell
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B. S. McEwen, P. Tanapat, and N. G. Weiland
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M. R. Foy, J. Xu, X. Xie, R. D. Brinton, R. F. Thompson, and T. W. Berger
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A. E. Herbison
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Q. Gu and R. L Moss
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K. Obrietan and A. N. van den Pol
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A. H. Lagrange, O. K. Rønnekleiv, and M. J. Kelly
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H C Christian and J F Morris
Rapid actions of 17{beta}-oestradiol on a subset of lactotrophs in the rat pituitary
J. Physiol.,
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539(2):
557 - 566.
[Abstract]
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[PDF]
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