Dalton Cardiovascular Research Center, Department of Veterinary Biomedical Sciences, University of Missouri, Columbia, Missouri 65211
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ABSTRACT |
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Li, Zhicheng and
Meredith Hay.
17--Estradiol Modulation of Area Postrema Potassium Currents.
J. Neurophysiol. 84: 1385-1391, 2000.
The purpose of this study was to determine the effects of
17-
-estradiol on area postrema neuronal activity in vivo and on area
postrema potassium currents (IK) in vitro. In anesthetized rats,
intravenous injection of 17-
-estradiol (10 ng/kg bw) -inhibited area
postrema neuronal activity in 8/8 neurons tested. The averaged firing
rate decreased from 2.9 ± 1.1 to 1.1 ± 0.3 Hz. The
inhibitory effects of 17-
-estradiol on area postrema neuronal
activity were rapid in onset (within 1 min) and long-lasting (>8 min).
To study the cellular mechanisms involved in this response, the effects of 17-
-estradiol were examined in dissociated area postrema neurons. In these cells, 17-
-estradiol (0.5 nM) increased the averaged peak
IK 27 ± 8%. The time course for the potentiation was observed within ~0.5-1 min after the application of 17-
-estradiol. Full recovery from the potentiation usually occurred within ~3-4 min after the washout of 17-
-estradiol. The biologically inactive 17-
-estradiol had no effect on area postrema IK and the
17-
-estradiol antagonist, ICI 182,780 blocked the effects of
17-
-estradiol on area postrema IK. Finally, big conductance
calcium-activated potassium current (MaxiK+) was identified
in area postrema neurons (n = 12/12). Blockade of
MaxiK+ with 100 nM iberiotoxin blocked the effects of
17-
-estradiol on IK. These results suggested 17-
-estradiol might
modulate area postrema neuronal activity by increasing
MaxiK+ current.
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INTRODUCTION |
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Estrogen, especially
17--estradiol, has long been used as a replacement hormone in
postmenopausal women to achieve a wide range of health benefits,
including among others, cardiovascular protection against hypertension
and stroke (Stampfer et al. 1991
). However, little is
known about the estrogen's effects on the CNS or nuclei within the CNS
involved in cardiovascular regulation.
Circumventricular organs are unique central structures that allow for
the communication of information between circulating hormones and
peptides and the CNS. The area postrema is a circumventricular organ in
the hindbrain which is known to be important in many physiological
functions including the central regulation of cardiovascular function.
The area postrema is known to send dense projections to the nearby
dorsomedial and dorsal lateral nucleus tractus solitarius (Morest 1967; Shapiro and Miselis
1985
; van der Kooy and Koda 1983
),
lateral parabrachial nucleus, and the dorsal motor nucleus of the vagus
(Shapiro and Miselis 1985
), all of which are
known to be important for the autonomic neuronal activity control. The central projections from the area postrema to these nuclei have been
suggested to be important for the cardiovascular regulatory effects of
area postrema activation.
Estrogen receptors have been identified in the rat area postrema
(Laflamme et al. 1998) and have been suggested to be
involved in the regulation of area postrema neuronal activity. The
purpose of present study was to 1) determine the role of
estrogen on area postrema neuronal activity, and 2) to begin
to evaluate some of the cellular mechanisms underlying
17-
-estradiol's modulation of area postrema neurons.
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METHODS |
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In vivo electrophysiology
Intact female Sprague-Dawley rats (150-300 g) were anesthetized
with urethane (0.6 g/kg). Animals were randomized as to their stage in
the estrous cycle. The responses of area postrema neurons to an acute
dose of 17--estradiol were similar in all animals tested, regardless
of the stage in the estrous cycle. Femoral arterial and venous
catheters (PE-50 Intramedic) were inserted to monitor blood pressure
and to deliver drugs, respectively. Animals were placed in a
stereotaxic head holder (Kopf). The area postrema was exposed by
removing the atlantooccipital membrane between the occipital bone and
first vertebrae. Animals were allowed to breathe spontaneously. An
automatic heating pad was used to maintain rat body temperature at
37 ± 1.0°C.
Area postrema single-unit activities were recorded using
glass-recording electrodes (~5-10 M, filled with 3.0 M sodium
chloride). Extracellular single-unit recordings were obtained to a
maximal depth of 400 µm around the center of area postrema and to 200 µm around the edge of area postrema. Electrical signals were
amplified on a Grass P5 amplifier, discriminated via WPI window
discriminator, and then digitized using MacLab (Chart software). A
minimum 2-min spontaneous single-unit activity was recorded before the
administration of 17-
-estradiol (10 ng/kg). This single dose of
17-
-estradiol was selected to mimic the maximal total circulating
estradiol a female rat might reach during proestrus (Smith et
al. 1975
). The effect of 17-
-estradiol administration on the
spontaneous activity of an area postrema single-unit activity was then
evaluated. An excitatory or inhibitory response is defined by a
25%
increase or decrease in neuronal firing frequency, respectively.
Histological locations of the recorded neurons were obtained by
ejecting 2.5% Chicago blue through a injection pipette attached to the
recording electrode.
At the end of experiment, the animal was euthanized and perfused intracardially with phosphate-buffered saline followed by 4% formaline. The hindbrain was stored in 4% Formalin plus 30% sucrose solution overnight. The locations of recording electrode tracts in the AP were verified histologically.
Area postrema neurons dissolation
Ten-day-old Sprague-Dawley rats of mixed gender were used in all the described studies. The hindbrain and cerebellum were rapidly removed and placed in 4°C physiological buffer containing (mM) 124 NaCl, 5 KCl, 2.0 CaCl2, 1.0 MgCl2, 26.0 NaHCO3, 10.0 glucose, pH 7.35. A 500-µM-thick, horizontal medullary slice, which included the area postrema, was obtained using a vibratome. Under a dissecting microscope, the area postrema was easily visualized and cut away from the surrounding tissue. The tissue was incubated at 37°C for 30 min in an Earl's balanced salt solution containing 5 mg/ml papain (Sigma), 1.5 mg/ml bovine serum albumin. The tissue was triturated in a papain-free solution with serially smaller pipettes until most of the tissue was dissociated. Dissociated cells were rinsed in minimum essential media (MEM) and plated on poly(lysine)-coated coverslips. Cells were maintained in MEM with 8 ng/ml nerve growth factor (Gibco).
Patch-clamp recordings
All experiments were performed on area postrema neurons
following 2-3 days in culture before the growth of extended neurites. Patch-clamp electrodes were constructed from No. 8161-type capillary glass (WPI Glass) pulled on a Flaming/Brown micropipette puller and
polished on a Narishige microforge. Final electrode resistance was
between 1-3 M. Standard whole-cell patch-clamp techniques used were
similar to those described by other investigators (Hamill et al.
1981
). Patch pipettes filled with the appropriate solutions were attached to the head-stage amplifier, which was mounted to a
hydraulic micromanipulator (Narishige). The reference electrode consisted of an Ag-AgCl plug immersed in a 150 mM KCl agar bridge, which was placed in the bath. Recordings were made using an Axopatch 1-D patch clamp amplifier and filtered at 3 kHz using a four-pole Bessel filter. Currents were digitized on-line at 10 kHz using Axodata
software (Axon Instruments) and stored on a Macintosh computer for
analysis. Current-voltage relationships were corrected for linear
leakage current measured from hyperpolarizing command pulses from
90
to
120 mV. Junction potentials were electronically compensated and
balanced to zero with the pipette immersed in the bath solution. Data
are reported as mean ± standard error. Results were analyzed
using t-test or ANOVA test.
Pharmacological application
Experiments were performed on cells which had been plated onto 9-mm-square coverslips. The coverslips were placed in a recording chamber filled with 260 µM bath solution. The flow of fluid (2 ml/min) through the changer was controlled by a multiport solenoid valve system that governed the gravity-fed flow onto the cell.
Solutions
Potassium currents (IK). (All solution components were obtained
from Sigma.) Ion substitution and pharmacological agents were used to
isolate the K+ current. The bath solution
consisted of (in mM) 137.0 N-methyl-D-glucamine, 5.4 KCl, 0.2 CaCl2 1.0 MgCl2, 10.0 glucose, 10.0 HEPES, pH ~7.35-7.40. The pipette solution consisted
of (in mM) 145.0 KAsp, 1.0 MgCl2, 10.0 HEPES, 0.2 CaCl2, 10.0 EGTA, and pH 7.20. The final calcium concentration in the pipette solution is 1.0 µM (Fabiato
1988).
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RESULTS |
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17--estradiol inhibits area postrema neuronal activity
Figure 1A is a
photomicrograph illustrating the recorded area postrema neurons stained
with 2.5% Chicago blue. Figure 1B is an example of area
postrema single-unit recording. In the present study, a total of eight
area postrema neurons were recorded and tested with 17--estradiol
injection. The frequency of area postrema spontaneous neuronal activity
ranged from a minimum 0.2-9.9 Hz. The average rate of discharge of the
neurons was 2.9 ± 1 Hz. Intravenous injection of 17-
-estradiol
inhibited area postrema neuronal activity in all eight neurons (Fig.
2B), an example of which was
shown in Fig. 2A. As summarized in Fig. 2B,
17-
-estradiol decreased the average firing rate by 62%, from
2.9 ± 1.1 to 1.1 ± 0.4 Hz (P < 0.05).
These effects on area postrema neuronal activity were rapid in onset
(within 1 min, Fig. 2A) and long-lasting (>8 min).
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17--estradiol facilitates area postrema IK
The modulation of voltage-gated IKs has been shown to be important
in the regulation of neuronal excitability. 17--estradiol has been
shown to modulate certain types of IKs (Joels and Karst 1995
). To determine the specific effects of 17-
-estradiol on area postrema neuronal activity, we studied the voltage and
pharmacological properties of IKs in area postrema neurons in the
presence of 17-
-estradiol using whole-cell patch-clamp methodology.
Figure 3A is a 3-day-cultured
area postrema neuron. Figure 3B illustrates a typical total
IK isolated from an area postrema neuron evoked by 10-mV step
depolarizations from 80 mV holding potential to +30 mV. Figure
3C demonstrates the voltage-current relationship of this
area postrema IK.
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Figure 4 illustrates the effect of
17--estradiol on evoked area postrema IK. Application of
17-
-estradiol (50 nM) potentiated IK. Five minutes after washout of
17-
-estradiol and replacement of the bath solution, peak IK
recovered toward the control level. In all the neurons tested
(n = 13/13), application of 17-
-estradiol (50 nM)
potentiated IK at every voltage level (Fig. 4D).
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The effects of 17--estradiol on area postrema IK were dose related.
An example is shown in Fig.
5A. For the generation of accumulative concentration-response curve, 17-
-estradiol was applied
to an area postrema neuron consecutively from low to high concentration
with 10-fold increases. In other experiments, some cells were lost
during increasing 17-
-estradiol concentrations, thus not all
concentrations were tested in a single cell. The responses to
17-
-estradiol were normalized within each cell to the control peak
IK level and averaged across cells. Although variability analysis
suggests that the effects at different concentration levels were not
statistically different, there was an obvious trend toward an increase
of effects with increasing concentrations.
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Selectivity of 17--estradiol facilitation of area postrema IK
17--estradiol has the same molecular formula as
17-
-estradiol but it does not have any known physiological function.
ICI 182,780 is an estrogen antagonist that does not have a partial agonist effect on estrogen receptors. The unique
pharmacological properties of ICI 182,780 were used to determine
whether the effects of 17-
-estradiol on area postrema IK were
specific for 17-
-estradiol activation of an estrogen receptor. As
shown in Fig. 6A,
17-
-estradiol had no effect on area postrema IK-evoked
depolarization from
80 mV holding potential to 20 mV. The averaged
data are shown in Fig. 6B (n = 4). To test
whether 17-
-estradiol's effects on area postrema IK were due to the
binding of the agent to area postrema estrogen receptors, 1 µM ICI
182,780, together with 17-
-estradiol (50 nM), was applied to the
bath solution. As demonstrated in Fig. 6C, in the presence
of ICI 182,780, 17-
-estradiol did not have any effects on area
postrema IK. The averaged results were shown in Fig. 6D
(n = 4).
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17--estradiol modulation of area postrema big conductance
calcium-activated IK (MaxiK+)
Using a specific MaxiK+ channel blocker,
iberiotoxin, we were able to isolate MaxiK+ in
area postrema neurons. As shown in Fig.
7A, 800-ms, 10-mV step
depolarizations from 80 mV holding potential evoked a series of area
postrema IK. In Fig. 7B, 100 nM iberiotoxin was applied to
the bath solution to block MaxiK+. The before-
and after-iberiotoxin area postrema potassium peak IKs are plotted
against their corresponding depolarization voltages in Fig.
7D. Figure 7E illustrates digitally subtracted
MaxiK+ current. The MaxiK+
activated near
30 mV and increased its peak amplitude linearly with
more positive depolarization voltages. MaxiK+ was
found in all area postrema neurons tested (n = 12/12).
The MaxiK+ constitutes from 39 to 68% of total
area postrema IK, with an average contribution of 52 ± 5%. These
results suggested that MaxiK+ might play an
important role in area postrema neuronal activity.
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To test whether 17--estradiol can selectively potentiate area
postrema MaxiK+, the following protocol was
followed: 1) A neuron was first tested with 17-
-estradiol
and allowed to recover. 2) The same neuron was then treated
with iberiotoxin to block the MaxiK+ current.
3) A second application of 17-
-estradiol together with iberiotoxin was applied to the neuron to determine whether the facilitory effects of 17-
-estradiol could still be observed during blockade of MaxiK+. As shown in Fig.
8A, area postrema IK evoked by
an 800-ms depolarization to 20 mV was potentiated by 17-
-estradiol
by 30%. Application of 100 nM iberiotoxin revealed a
MaxiK+ of 0.63 nA, which was 60% of the total
IK. The second application of 17-
-estradiol in the presence
of iberiotoxin did not increase the remaining IK. Averaged data are
illustrated in Fig. 8B. These results suggest that
17-
-estradiol inhibits area postrema neuronal activity by increasing
MaxiK+ current.
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DISCUSSION |
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The results from this study are the first to demonstrate that a
sex hormone can modulate area postrema neuronal activity and area
postrema IK channel function. Furthermore, these results suggest that
17--estradiol may inhibit area postrema activity via nongenomic mechanisms.
17--estradiol's modulation of area postrema neuronal activity is
most likely to involve excitation or inhibition of its IKs, a major
determinant of neuronal excitability. Previous studies have shown that
area postrema neurons possess at least two types of IKs 1) a
rapid-activating and rapid-inactivating (IA) current and 2)
a delayed rectifier current which is slowly activating and
noninactivating (Hay and Lindsley 1995
). In the present
study, we have shown area postrema neurons also express an
iberiotoxin-sensitive Ca2+-activated potassium
channel, the MaxiK+ channel. The
MaxiK+ is a category of big conductance (15-250
pS) potassium channels that is sensitive to both changes in
intracellular calcium concentration and membrane potential. In
neurons, MaxiK+ contributes to the
afterhyperpolarization that modulates repetitive firing and overall
neuronal excitability. The large presence of MaxiK+ in area postrema neurons might underlie
the exhibited low level of spontaneous activity observed in these cells.
In reports from other laboratories, the effects of 17--estradiol on
IKs have been varied and appear to depend on the cell type that has
been tested. For example, 17-
-estradiol can stimulate IA-type
transient outward currents (Rusko et al. 1995
) in rabbit aortic endothelial cells but inhibit the IA-type transient currents in
rat myometrial cells (Erulkar et al. 1994
). However, it
does not affect the IA-type current in rat hippocampal neurons
(Joel and Karst 1995
) nor delays an inward rectifier
current expressed on Xenopus oocytes (Waldegger et
al. 1996
). In the present study, area postrema neurons show an
increase in their total IK to 17-
-estradiol application. Our results
are in agreement with a number of studies which show 17-
-estradiol
increases IK and results in hyperpolarization (Kelly et al.
1980
; Nabekura et al. 1986
). However, in area
postrema neurons, 17-
-estradiol does not change IA or the delayed
rectifier current. Thus, MaxiK+ is the principal
component responsible for the area postrema total IK increase observed
with 17-
-estradiol. Our results are similar to observations in
cardiac myocytes and coronary artery smooth muscle cells where
17-
-estradiol activates MaxiK+ (Node et
al. 1997a
,b
; Wellman et al. 1996
). Since
MaxiK+ activation requires an elevation of
[Ca2+]i, one explanation
for this enhanced MaxiK+ is the intracellular
calcium surge resulting from activation of calcium channel by
17-
-estradiol (Joels and Karst 1995
). However, it has
yet to be tested whether 17-
-estradiol can affect area postrema
voltage-gated Ca2+ channels.
In the present study, both the in vivo and in vitro neuronal response
to 17--estradiol were rapid. The increase of area postrema IK was
observed 45 s after 17-
-estradiol and maximal responses appeared within ~1-2 min. Similar rapid responses to
17-
-estradiol have been observed in other studies on medial amygdala
neurons (Nabekura et al. 1986
). In the present study,
ICI 182,780, an estrogen receptor antagonist without partial estrogen
bioactivity, can totally block the effects of 17-
-estradiol on area
postrema IK. These results suggest that estrogen receptors participate in mediating the action of 17-
-estradiol in area postrema neurons. However, the exact signal transduction pathway of 17-
-estradiol's effects is yet to be determined.
Since the genomic effects of estrogen require a complex cascade of
events including the hormone-receptor binding, targeted gene
expression, and protein synthesis, it may take hours for the hormone
signal to be translated into membrane excitability changes. The time
course of the response in the present study suggests that genomic
effects are unlikely and nongenomic effects are the reasonable
alternative mechanism underlying 17--estradiol's action in these
studies. In contrast to genomic mechanism, the nongenomic effects are
rapid in onset and do not require nuclear estrogen receptors or gene
expression. However, it does need membrane receptors and/or other
cellular second-messenger systems to change neuronal activity
(Node et al. 1997a
).
MaxiK+ channels comprise two subunits: the
subunit, which forms the pore of the channels (Adelman et al.
1992
; Butler et al. 1993
; Tseng-Crank et
al. 1994
), and the
subunit, which forms the regulatory site
sensitive to intracellular Ca2+ (McManus
et al. 1995
; Meera et al. 1996
). Recently,
17-
-estradiol has been shown to directly bind to the
subunit of
MaxiK+ expressed on Xenopus oocytes
and can acutely activate this channel (Valverde et al.
1999
). These studies in the Xenopus oocyte may suggest a total new mechanism by which 17-
-estradiol may modulate MaxiK+ channel activity.
The area postrema, one of seven circumventricular organs, has the
highest estrogen receptor density among cardiovascular-related brain
nuclei (Laflamme et al.1998). Functionally, the area
postrema is known to be involved in regulation of body fluid balance,
feeding behaviors, emesis, and cardiovascular regulation
(Shapiro and Miselis 1985
). It sends dense projections
to the nearby dorsomedial and dorsal lateral nucleus tractus
solitarius, lateral parabrachial nucleus, and the dorsal motor nucleus
of the vagus (Morest 1967
; Shapiro and Miselis
1985
; van der Kooy and Koda 1983
), all of which
are known to be important for the autonomic control of blood pressure.
Several lines of evidences have suggested that circulating 17-
-estradiol may modulate autonomic and cardiovascular function in
particular via CNS modulation of sympathetic outflow (Akaishi et
al. 1996a
,b
; Chu and Beilin 1997
;
Colucci et al. 1982
; Condon et al. 1989
;
Crofton and Share 1997
; He et al.
1998
). Because of the area postrema's dense population of
estrogen receptors, its known involvement in the modulation of
sympathetic activity, and its inhibition by circulating estradiol, it
is reasonable to suggest that the area postrema may be one central site
whereby circulating estradiol may act to modulate sympathetic outflow and potentially cardiovascular function. Future studies will be designed to determine the role of the area postrema in estradiol modulation of cardiovascular regulatory function.
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ACKNOWLEDGMENTS |
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The authors thank K. Lindsley for expert technical support and Dr. Jayabala Pamidimukkala for area postrema neuron image processing.
This project was supported by the American Health Association and National Heart, Lung, and Blood Institute Grant HL-62261.
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FOOTNOTES |
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Address for reprint requests: M. Hay (E-mail: haym{at}missouri.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 29 November 1999; accepted in final form 31 May 2000.
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REFERENCES |
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