Sequential and opposite regulation of two outward
K+ currents by ET-1 in cultured striatal
astrocytes
R.
Bychkov,
J.
Glowinski, and
C.
Giaume
Institut National de la Santé et de la Recherche
Médicale Unité U114, Collège de France, 75231 Paris, Cedex 05, France
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ABSTRACT |
In the brain,
astrocytes represent a major target for endothelins (ETs), a family of
peptides that can be released by several cell types and that have
potent and multiple effects on astrocytic functions. Four types of
K+ currents (IK) were detected in
various proportions by patch-clamp recordings of cultured striatal
astrocytes, including the A-type IK, the
inwardly rectifying IK IR, the
Ca2+-dependent IK
(IK Ca), and the delayed-rectified
IK (IK DR). Variations
in the shape of current-voltage relationships were related mainly to
differences in the proportion of these currents. ET-1 was found to
regulate with opposite effects the two more frequently recorded outward
K+ currents in striatal astrocytes. Indeed, this peptide
induced an initial activation of IK Ca
(composed of SK and BK channels) and a delayed long-lasting inhibition
of IK DR. In current-clamp recordings, the
activation of IK Ca correlated with a transient hyperpolarization, whereas the inhibition of
IK DR correlated with a sustained
depolarization. These ET-1-induced sequential changes in
membrane potential in astrocytes may be important for the regulation of
voltage gradients in astrocytic networks and the maintenance of
K+ homeostasis in the brain microenvironment.
glial cells; endothelins; calcium-dependent potassium channels
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INTRODUCTION |
ENDOTHELINS (ETs)
constitute a family of peptides that includes at least the following
three isoforms: ET-1, ET-2, and ET-3. Originally described in porcine
endothelial cells as a very potent vasoconstrictor, ET-1 is the most
thoroughly studied member of this family (58-60). ETs
exert multiple effects on vascular and nonvascular tissues and have
been implicated in several physiological functions in the
cardiovascular, endocrine, pulmonary, renal, and nervous system. ET
precursors and receptors are present in situ in the mammalian brain,
including the human brain (53). ET responses are mediated
via at least two ET receptor subtypes, ETA and
ETB, which are coupled to heterotrimeric G proteins and widely distributed in the brain (see Ref. 52). ET-1 and
ET-3 isoforms are present in vivo in brain endothelial cells
(59), some neurons (19), and reactive
astrocytes (27). Primary cultures of astrocytes provide a
suitable model for investigating the ET system, since these cells
naturally contain ET-1 and ET-3 (18, 20), express G
protein-coupled ET receptors (31, 32), and possess
ET-converting enzyme activity (15).
Although less documented than in peripheral tissues, the biological
effects of ETs in the central nervous system have also been
investigated, mainly in astrocytes. In these glial cells, effects of
ETs are widespread, including mobilization of various transduction
pathways, enhancement of glucose uptake (52) and glutamate
efflux (44), increase of c-fos and nerve growth
factor expression, regulation of ionic channel activity
(50), stimulation of mitogenesis (51) and
proliferation (48), inhibition of gap junction-mediated
intercellular communication (4, 20), and triggering of
intercellular Ca2+ waves (54). The
significance of these responses on astrocytic function is still unclear
(17), but ETs are known to be released by reactive
astrocytes and may be involved in various disorders (35, 37,
42). Indeed, ET immunoreactivity and ETB receptor expression are increased significantly in astrocytes after brain injury
(41, 43). In addition, ET levels are also enhanced in
several neurological disorders, including Alzheimer's disease (60), brain inflammation (36), virus
infection (27), subarachnoidal hemorrhage
(32), and ischemia (2, 57).
Interestingly, ET receptor antagonists exert therapeutic effects in
animal models of cerebrovascular diseases (see for reviews Refs.
2 and 37). Undoubtedly, an increased understanding of the
basic effects of ETs on astrocytic properties should clarify how these
peptides intervene in these pathological and experimental situations.
Astrocytes from either primary culture or acute brain slices express a
large spectrum of ionic channels, depending on their brain region of
origin and their reactive state. Although much information is already
available on the regulatory role of ETs on ionic channels in
cardiovascular, endocrine, and muscular tissue, little is known
concerning their influence on the central nervous system. Studies
characterizing membrane potential revealed for the first time that
astrocytes can be a target for ETs (24). However, the
ionic channel subtypes contributing to ET-glial responses have been
poorly characterized so far. Accordingly, this study was undertaken to
determine which K+ channels are regulated by ET-1 in
cultured astrocytes from the rat striatum.
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METHODS |
Cell culture.
Primary cultures were prepared as described previously
(54). Briefly, pregnant OFA rats (IFFA Credo, Lyon,
France) were killed by prolonged exposure to high concentrations of
carbon dioxide. Embryos (18 days old) were removed rapidly from the
uterus and placed in PBS supplemented with glucose (33 mM). Striata
were dissected and mechanically dissociated in PBS-glucose solution. Cells were plated on 12-mm glass coverslips (3 × 105
cells/coverslip) coated with poly-L-ornithine (15 µg/ml).
The culture medium consisted of a 1:1 mixture of MEM and F-12 nutrient (GIBCO, Gaithersburg, MD) supplemented with glutamine (2 mM), NaHCO3 (13 mM), HEPES (5 mM, pH 7.4), glucose (33 mM),
penicillin-streptomycin (5 IU/ml and 5 mg/ml, respectively), and 10%
Nuserum (Collaborative Research, Bedford, MA). Once plated, cells were
incubated at 37°C in a humidified atmosphere of 95% air and 5%
CO2. The culture medium was changed one time per week. On
day 8, cytosine arabinoside (2 µM) was added for 60 h
to prevent proliferation of fibroblasts and microglia. Cells were
studied between 10 and 20 days of culture. At this time, >95% of the
cells stained positive for glial fibrillary acidic protein, as revealed
by indirect immunofluorescence (INC Biochemicals, Costa Mesa, CA).
Electrophysiology.
Whole cell K+ currents were measured using whole cell,
outside-out, or perforated patch-clamp configurations. The external solution contained (in mM) 140 NaCl, 1.8 CaCl2, 1 MgCl2, 5.4 KCl, and 10 Na-HEPES (pH 7.4), whereas recording
pipettes (resistance 3-8 M
) were filled with a solution
containing (in mM) 80 potassium aspartate, 40 KCl, 20 NaCl, 1 MgCl2, 3 MgATP, 10 EGTA, and 5 K-HEPES (pH 7.4). EGTA was
balanced with Ca2+ to provide 100, 200, or 500 nM free
Ca2+. The cesium pipette solution used to block
K+ channels contained (in mM) 80 cesium aspartate, 40 CsCl,
10 tetraethylammonium (TEA) chloride, 1 MgCl2, 3 MgATP, 10 EGTA, and 5 Cs-HEPES (pH 7.4). When Ca2+-free solution was
used, Na+ substituted for the Ca2+. Solutions
were superfused in the recording chamber (1 ml) by gravity flow with a
renewal time of ~45 s. Drugs were applied using thin tubes connected
to a 2-ml syringe, and superfusion was stopped. In the perforated
patch-clamp experiments, whole cell access was achieved by nystatin
within 5-10 min after seal formation. Nystatin (Sigma Chemicals,
St. Louis, MO) was dissolved in DMSO and was added to the pipette
solution (final concentration, 50-100 µg/ml).
Voltages and currents were recorded at 5-10 kHz using an Axopatch
200A amplifier (Axon Instruments, Foster City, CA). Signals were
filtered at 1 kHz, digitized using a Digidata 1200 interface (Axon
Instruments), and analyzed using pCLAMP software (version 6). Series
resistance and cell capacitance were calculated from the uncompensated
capacitive transients using hyperpolarizing step pulses (10 mV, 10 ms)
and by adjusting the amplifier's series resistance and whole cell
capacitance controls to eliminate current transients. Cell membrane
input resistance was measured using small hyperpolarizing voltage
pulses (10 mV, 10 ms) from a holding potential of
40 mV. All
experiments were performed at room temperature (20-24°C).
Data analysis.
All values are given as means ± SE; n represents the
number of cells tested. Statistical analysis was performed using a
one-way ANOVA test, and a value of P < 0.05 was
considered statistically significant.
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RESULTS |
In agreement with previous publications, we observed that rat
striatal astrocytes in primary cultures exhibit a great heterogeneity of K+ current expression (1, 3, 13, 33, 38, 45,
46). Indeed, among 75 astrocytes initially tested by voltage
steps or ramps, 16% were characterized by a linear current-voltage
(I-V) relationship, whereas the remaining
astrocytes exhibited various patterns of I-V
curves, indicating voltage-dependent properties. Generally, four types
of K+ currents were recorded using the perforated-patch and
the whole cell configuration, including slow-inactivating
delayed-rectifier-type K+ currents
(IK DR), inwardly rectifying K+
currents (IK IR), Ca2+-dependent
K+ currents (IK Ca), and
fast-inactivating A-type K+ currents
(IK A). Variations in the shape of the
I-V relationships were related mainly to
differences in the proportions of these currents.
Voltage-dependent outward K+ currents
in cultured striatal astrocytes.
To determine whether K+ was the major charge carrier of the
net outward current, K+ was replaced by Cs+
(120 mM CsCl) and tetraethylammonium (TEA; 10 mM) in the internal pipette solution. Under these conditions, outward currents were markedly decreases (15 ± 8% of the control value,
n = 7), suggesting that these currents were carried
mainly by K+. The contribution of Cl
to these
outward currents was also tested using Cl
channel
blockers. In the presence of either 5-nitro-2,3-phenylpropylamino benzoic acid (100 µM) or DIDS (100 µM), the outward currents
recorded at +100 mV were reduced only slightly (91 ± 2 and
94 ± 1% of the control value, respectively, n = 5), suggesting only a minor contribution of the Cl
currents under our recording conditions. Further support for K+ as the major charge carrier of the outward currents was
obtained from examining tail currents, which were recorded at various
repolarizing potentials after a prepulse step to +80 mV. The tail
currents indicated an average reversal potential of
78 ± 4 mV
(n = 7), which is close to the calculated Nernst
equation for the K+ reversal potential
(EK =
83 mV).
As shown in Figs. 1 and
2, the outward K+-currents
were recorded in response to step pulses (±150 mV, 10-mV increments,
300-500 ms) and voltage-ramp (±150 mV, 800 ms) applications,
which were both recorded from a holding potential of
80 mV. In
~30% of the recordings (19 of 75 cells), depolarizing pulses
generated IK A-like currents that inactivated
rapidly (data not shown). In addition, all astrocytes characterized by
voltage-dependent properties exhibited slowly inactivating currents. On
the basis of voltage dependence and pharmacological testing of
K+ currents, two main types of astrocytes were
distinguished. The first type (29 of 124 cells) exhibited an
I-V relationship fitted by a single exponential
function and was characterized by a threshold of activation at
16 ± 3 mV (n = 12; Fig. 1A). The
outward currents elicited by positive step pulses increased
continuously without a saturation plateau. Inward currents resulting
from the activation of IK IR at negative steps
were inhibited by 100 µM Ba2+ (Fig. 1, A and
B). Subsequent applications of apamin (100 nM) slightly
decreased the amplitude of the outward K+ currents over the
entire range of potentials investigated (Fig. 1, A and
B). Furthermore, cumulative applications of iberiotoxin (100 nM) drastically decreased the amplitude of the outward K+
currents, whereas iberiotoxin and apamin together induced saturation of
the resistant K+ outward currents. The second type of
astrocyte (68 of 124 cells) was characterized by a more complex current
profile composed of at least two components. The threshold of
activation for this type of astrocyte was detected at
42 ± 5 mV
(n = 17). In addition, at potentials more positive than
+50 mV, the current amplitude increased continuously after exhibiting a
plateau in the I-V relationship (Fig.
2A). In these cells, IK IR were
inhibited by 100 µM Ba2+, whereas a subsequent
application of iberiotoxin (100 nM) decreased the amplitude of the
outward K+ currents at potentials more positive than +20 mV
(Fig. 2, A and B). Furthermore, cumulative
applications of apamin (100 nM) linearly decreased the amplitude of the
K+ outward currents at all ranges of potential
investigated. The K+ currents resistant to iberiotoxin and
apamin exhibited a saturation plateau face with a slightly pronounced
inactivation of the K+ current at positive potentials.
Finally, 27 cells showed intermediary and more complex
I-V relationships. In addition to step pulses, voltage ramps (800 ms, ±150 mV) were used to establish the
I-V relationship of striatal astrocytes.
Comparison of averaged I-V curves obtained using
the two protocols indicated that they differed slightly in their
amplitude, whereas their shapes were similar (Figs. 1 and 2).

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Fig. 1.
Voltage-dependent K+ currents elicited by step pulses
and voltage ramps in S-G-V astrocytes cultured from the rat striatum.
A: averaged K+ current
(IK) amplitudes obtained from 9 astrocytes and
plotted against corresponding voltages. K+ currents were
recorded in the perforated-patch configuration in response to step
pulses (300-500 ms) applied from 80 mV holding potential with
10-mV step increments. The best fit was performed by a Boltzmann
function from potentials ranging from 80 to +100 mV. Current-voltage
(I-V) relationships were constructed under the
indicated conditions. IbTX, iberiotoxin. B: typical
superimposed K+ current traces used to construct
I-V relationships shown in A. These
recordings were obtained under control conditions and after application
of various channel blockers. C: averaged current amplitudes
obtained from pulse injections and plotted against voltage, as shown in
A for control conditions. The superimposed current trace
represents the average (n = 7) current responses
elicited by a voltage ramp applied from 80 mV (±150 mV, 800 ms).
Inset: slope conductance
(dI/dVm, where
Vm is membrane potential) of the fitting of the
averaged current traces obtained in response to the application of a
voltage ramp from 80 to +100 mV.
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Fig. 2.
Voltage-dependent K+ currents elicited by step pulses
and voltage ramps in N-G-V astrocytes. A: average
K+ current amplitudes obtained from 15 astrocytes and
plotted against the corresponding voltages. K+ currents
were recorded in the perforated-patch configuration in response to step
pulses (300-500 ms) applied from 80 mV holding potential with
10-mV step increments. The best fit was performed by a Boltzmann
function from potentials ranging from 80 to +100 mV.
I-V relationships were constructed under the
indicated conditions. B: typical superimposed K+
current traces used to construct I-V
relationships shown in A. These recordings were obtained
under control conditions and after application of various channel
blockers. C: averaged current amplitudes obtained from pulse
injections and plotted against voltage as shown in A under
control conditions. The superimposed current traces represent the
average (n = 10) current responses elicited by a
voltage ramp applied from 80 mV (±150 mV, 800 ms). Inset:
slope conductance (dI/dVm) of the
fitting of the average current traces obtained in response to the
application of a voltage ramp from 80 to +100 mV.
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Differences in the two main I-V relationships
were analyzed further (20 cells in each case) by computing conductance
(dI/dV) as a function of membrane potential
(Figs. 1C and 2C, insets). The first
category of astrocyte (Fig. 1), characterized by a continuous increase
in current amplitude elicited by step pulses or voltage ramps,
exhibited a single maximum of conductance (i.e., the derivative of the
current to voltage) at +94 ± 6 mV (Fig. 1C,
inset). The second category of astrocyte (Fig. 2),
characterized by a plateau phase around +40 mV in the
I-V relationship and by a continuous increase in
the current amplitude at more positive potentials, exhibited two peaks
of conductance at +47 ± 4 and +100 ± 8 mV (Fig.
2C, inset). From the plots, astrocytes with an
I-V relationship that resulted in a conductance
curve with one maxima were named S-shaped conductance astrocytes
(S-G-V), whereas astrocytes with an I-V
relationship that resulted in a conductance curve with two maxima were
called N-shaped conductance astrocytes (N-G-V).
The two classes of astrocytes had different sensitivities to the
K+ channel blockers TEA and 4-aminopyridine (4-AP). Indeed,
in S-G-V astrocytes, TEA (0.1-7 mM) decreased in a
concentration-dependent manner the amplitude of outward K+
currents elicited by ramp voltages mainly at potentials more positive
than +50 mV (Fig. 3A). The
TEA-induced inhibition of the total outward current (measured at +100
mV) was further characterized by plotting the percentage of inhibition
(compared with the control value) against TEA concentration (Fig.
3B). The progressive decline of outward K+
currents in the presence of increasing TEA concentrations was well
fitted by a sigmoid function consistent with a 1:1 binding of TEA to
its binding site, with an IC50 of 475 mM and a slope of
1.59. In contrast, 4-AP used in a concentration ranging from 0.1 to 5 mM had little or no inhibitory effect on S-G-V astrocytes (n = 9, data not shown), whereas N-G-V astrocytes were
sensitive to 4-AP at negative and positive potentials. As shown in Fig. 3C, 4-AP reduced in a concentration-dependent manner outward
currents elicited by voltage ramps in these astrocytes.
Concentration-response curves for the inhibition of outward
K+ currents measured at +20 mV were constructed. The
averaged data were well fitted by a sigmoid function, consistent with a
1:1 binding of 4-AP to its binding site and an IC50 of 1.5 mM. Even at higher concentrations (5-10 mM), 4-AP reduced only
20% of the total outward current (measured at +100 mV). However, the
coapplication of 4-AP (5 mM) and TEA (5 mM) resulted in an 83 ± 9% block of the outward K+ currents in N-G-V astrocytes
(n = 12), indicating the presence of at least two types
of K+ currents in these cells.

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Fig. 3.
Effect of tetraethylammonium (TEA) and 4-aminopyridine (4-AP) on
voltage-dependent outward K+ currents in S-G-V and N-G-V
astrocytes. A: family of current traces elicited by ramp
depolarization before (1) and after (2-7)
TEA application at different concentrations. TEA concentrations were
0.1 (2), 0.25 (3), 0.5 (4), 1 (5), 2 (6), and 5 (7) mM.
B: dose-response relationship showing the effect of TEA on
outward currents. The percentage of current (IK)
inhibition corresponds to the fraction of the total outward current,
which is inhibited by various concentrations of TEA compared with the
control value of this current measured at +100 mV. Data points were
obtained from 5-7 cells and were fit by logistic function.
C: I-V relationships elicited by
voltage ramps before (1) and after (2-5)
application of 4-AP at different concentrations. Concentrations of 4-AP
were 1 (2), 2 (3), 5 (4), and 7 (5) mM. D: dose-response relationship showing the
effect of 4-AP on outward currents measured at +20 mV from voltage
ramps. Current amplitudes are expressed as a percentage of control and
are plotted against 4-AP concentrations. Data points were fitted to
logistic functions. All recordings were performed in the
perforated-patch configuration.
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Action of ET-1 on outward K+
currents.
The effects of ET-1 on the major outward K+ currents were
investigated in S-G-V and N-G-V astrocytes. ET-1 was used at 0.1 µM,
a concentration known to induce maximal responses on the signal transduction pathway in cultured astrocytes (4, 20, 24, 31, 32,
50, 52).
In 14 of 19 S-G-V astrocytes, ET-1 transiently increased the nonlinear
K+ outward currents (Fig.
4A). This ET-1-induced
increase in the amplitude of K+ outward currents reached
189 ± 38% (n = 14) at +50 mV. The ET-1-activated current remained nonlinear and returned to 92 ± 4% of the
control value 19 ± 7 s (n = 14) after the
beginning of the application (Fig. 4A). In this class of
astrocytes, ET-1 elicited a weak voltage-independent outward net
current insensitive to iberotioxin (Fig. 4A). Finally, pretreatment of S-G-V astrocytes with iberotioxin (0.1 µM) prevented the stimulatory effect of ET-1 on the voltage-dependent K+
outward current (n = 5; data not shown).

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Fig. 4.
Effect of endothelin (ET)-1 on voltage-dependent outward
K+ currents recorded from S-G-V and N-G-V astrocytes.
A: perforated-patch recording of outward K+
currents elicited in a type S-G-V astrocyte by ramp depolarization
before (0 s) and after (0.1 µM) ET-1 applied at the indicated times.
ET-1 application induced a large and transient increase in the
amplitude of the outward K+ currents and a shift to the
left of the threshold of activation. When iberiotoxin (100 µM) was
added to the bath solution after a transitory activation of the outward
K+ current was completed, the toxin drastically reduced the
remaining outward K+ currents. B: family of
current traces recorded from a type N-G-V conductance astrocyte before
and after ET-1 application. Note that, 60 s after the peptide
application, the outward K+ currents did not return to the
control value and that the component, which is activated at negative
membrane potentials in the control situation, was lacking (broken
line). Apamin (apm, 0.1 µM) added to the bath solution elicited
further inhibition of the outward K+ currents, whereas
iberiotoxin (0.1 µM) added with apamin (apm + ibtx) blocked the
remaining current.
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In 23 of 45 N-G-V astrocytes, shortly (first 1 min) after the beginning
of its application, ET-1 increased the amplitude of the linear portion
of the outward K+ currents, which often masked the
activation of the iberiotoxin-sensitive component (Fig. 4B).
Thereafter, ET-1 progressively inhibited by 39 ± 7%
(n = 8) the outward K+ current compared
with the control value. After this biphasic effect of ET-1,
I-V curves of the cells were transformed from an
N-G-V to an S-G-V shape computed slope conductance (Fig.
4B). In 8 of these 45 cells, the transitory stimulatory
effect of ET-1 did not occur, with only the reduction of the
voltage-gated K+ current being observed. Furthermore,
pretreatment of N-G-V astrocytes with apamin (0.1 µM) prevented the
initial transient linear current evoked by ET-1 (n = 6). When toxin was applied after the ET-1-induced transient changes of
K+ currents, a further decrease (38 ± 8% of the
control value) in the amplitude of the outward K+ currents
was observed. Finally, a larger inhibitory response (19 ± 6% of
the control value, n = 8) occurred under the
coapplication of apamin (0.1 µM) and iberotioxin (0.1 µM) after
ET-1 treatment (Fig. 4B).
Because ET-1 triggers complex intracellular Ca2+
concentration ([Ca2+]i) elevations in
striatal astrocytes (4, 55, 56), whole cell recordings
were also performed in N-G-V cells with 10 mM EGTA in the recording
pipette solution to prevent [Ca2+]i
transients. Outward K+ currents were generated by either
voltage ramps (±100 mV) or voltage steps (10 mV) applied from
80 to
+100 mV, starting from a holding potential of
80 mV (Fig.
5, A and B). Under
these conditions, ET-1 application (0.1 µM) inhibited an outward
current having typical kinetics of IK DR (Fig.
5, A, inset, and B). This ET-1-induced
inhibition of IK DR was concentration dependent (Fig. 5C) and was observed at peak and steady-state levels.
IC50 and curve slope, estimated from the best fit of the
logistic function, were 112 nM and 1.72, respectively.

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Fig. 5.
Inhibitory effect of ET-1 on voltage-dependent outward
K+ currents. A: I-V
relationship elicited by voltage ramp before (control) and 2 min after
ET-1 (0.1 µM) treatment. Inset: isolation of the current
inhibited by ET-1 was carried out by subtraction of the current
recorded in the presence of ET-1 from the control current
(ICon IET-1).
B: superimposed current traces elicited by step pulses to
+50 mV, applied from a 80-mV holding potential, before (control) and
2 min after ET-1 application. The current inhibited by the peptide
(ICon IET-1) is
shown in current trace on right. C: dose-response
curve showing the inhibitory effect of ET-1 on outward K+
currents in response to +50-mV voltage steps. Current plot corresponds
to the normalized current expressed as the percentage of the control
value. Averaged data (means ± SE from 4-8 cells) were fitted
with logistic functions with an EC50 value and slope of
1.11 µM and 1.7, respectively. D: inactivation of outward
K+ currents in the presence and absence of ET-1. Plots of
the relationship between peak outward K+ currents recorded
at a test potential of +50 mV and membrane potential (preconditioning
potential) before ( ) and after ( ) ET-1
application (n = 5). The membrane potential was held at
80 mV, stepped to a preconditioning voltage (90 s) ranging from 80
to 0 mV (10-mV increments), and then depolarized to +40 mV (500 ms).
Data obtained before and after application of ET-1 were fitted by
curves describing the Boltzmann function, with midpoints of
inactivation (V0.5) of 30.6 ± 1.6 and
21.5 ± 2.5 mV, and steepness factor (k) was
11.2 ± 0.2 and 9.7 ± 0.6 mV, respectively. E:
typical K+ currents elicited by test pulse to +50 mV under
control conditions and after application of ET-1. All recordings were
obtained in the whole cell configuration.
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The voltage dependence of steady-state inactivation of
IK DR was studied using a double-pulse
protocol. The extent of channel inactivation was assessed by measuring
the peak outward K+ current (+50 mV, 500 ms) after holding
the membrane potential for 20 s at values ranging from
80 to +20
mV with 10-mV increments (Fig. 5, D and E).
Typically, as indicated by plotting
I/Imax vs. the membrane potential,
the initial depolarization increased the inactivation of the outward
K+ current (Fig. 5D). The inactivation curve of
the outward K+ current was well fitted by the Boltzmann
equation as follows: I/Imax = (Imax
Imin)/[1 + exp(V
Vh)/k] + Imin, where I (pA) is the amplitude
of the outward current elicited by the test pulse V (mV),
preceded by the conditioning prepulse; Imax (pA)
is the amplitude of the current elicited by a test pulse from a
conditioning prepulse of
80 mV; Imin (pA) is
the noninactivating current component; Vh (mV)
is the value of the conditioning potential leading to 50% inactivation
of the K+ current; and k is the steepness factor
characterizing the voltage sensitivity of the channels.
Effects of ET-1 on single-channel activities.
Single- channel recordings were performed in outside-out configuration
with internal and external solution containing 150 and 5.4 mM
K+, respectively, and Ca2+ concentration in the
pipette solution was fixed at 200 nM. Under these conditions, two main
types of noninactivating K+ channel activities were
distinguished by comparing currents recorded at potentials ranging from
40 to +90 mV. On the basis of their distinct amplitudes, two channel populations were observed in either
the same (Fig. 6A) or in different excised patches of
membrane (Fig. 7). The amplitude of these
two types of unitary currents were analyzed and plotted against
membrane potential to determine their unitary conductance. Plots of
large unitary currents were well fitted by linear regression (Fig. 6C),
and an averaged unitary conductance of 189 ± 7 pS
(n = 7), which corresponds to characteristic BK
channels, could be determined. The conductance of K+
channels exhibiting low-amplitude currents was estimated using a linear
fit from 0 to +40 mV (Fig. 6D). The unitary
I-V relationship constructed from averaged data
gave an average unitary conductance of 21 ± 3 pS
(n = 6), corresponding to the small conductance of SK
channels (Fig. 6D).

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Fig. 6.
Unitary currents recorded from nonactivating K+
channels. A: unitary K+ channel currents
recorded in outside-out configuration at steady-state potentials of 0 mV. Histogram of the current amplitude was fitted by the sum of a
Gaussian distribution with the peaks centered at 0.94, 2.01, and 4.9 pA. B: unitary currents of K+ channels recorded
in perforated-patch configuration after holding the cell at 0 mV for 10 min. For these current traces elicited by step pulses at the indicated
voltages, background current was subtracted without additional
filtering of the recorded signal. C: averaged (from 4-8
cells) amplitudes of unitary currents were recorded in outside-out
( ) and perforated-patch ( )
configurations after inactivation of the delayed-rectifier
K+ current (IK DR) by prolonged
holding of the cell to 0 mV. The amplitudes were fitted with a linear
function. The unitary slope conductance was 189 ± 7 pS for
voltages ranging from 0 to +80 mV. D: averaged (from 4-9
cells) amplitudes of unitary currents recorded from outside-out
( ) and perforated-patch ( )
configurations after inactivation of IK DR.
The amplitudes were fitted with a linear function (solid line) and with
the Goldman-Hodgkin-Katz function (broken line). The unitary slope
conductance was 21 ± 3 pS.
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Fig. 7.
Activation of SK and BK channels by ET-1. Single channel
activity recorded before (A and C) and after
(B and D) ET-1 application (0.1 µM). Histograms
were fitted by the sum of Gaussian distributions with peak centers at
0.08 and 0.95 pA in A; 0.11, 0.99, 1.87, and 2.75 pA in
B; 0.08 and 5.6 pA in C; and 0.09, 5.7, 10.4, and
15.1 pA in D. All points were constructed during 3-s
recording periods. Single-channel recordings were performed at 0 mV
using the perforated-patch configuration. Broken lines indicate the 0 current level in each trace. Current traces 7 (in
B and D) correspond to the inhibition of
single-channel activity by apamin (100 nM) and iberiotoxin (100 nM),
respectively. (Arrows indicate cooperative opening of BK channels.)
|
|
Because ET-1 (0.1 µM) did not affect the behavior of the single
channels recorded in the outside-out configuration (data not shown), the perforated-patch configuration was used to study
single-channel activity induced by this peptide (Fig. 6B).
An interesting observation was made from perforated-patch recordings
performed with N-G-V astrocytes. Indeed, a resting whole cell current
reduced to 20-40 pA could be recorded after inactivation of the
dominating IK DR obtained by holding the
membrane potential at
10 or 0 mV for 10-15 min. Under these
recording conditions, single-channel activity superimposed on the whole
cell current could be recorded in 13 from 34 N-G-V astrocytes. The
current traces in Fig. 6B show these macroscopic currents
with superimposed unitary K+ channel activity. The
amplitude of these unitary currents was calculated and plotted vs.
voltage in a similar way as the measurements under outside-out
conditions (Fig. 6, C and D). It is noteworthy that the measured conductances obtained under these recording conditions were very similar to those obtained from outside-out patches. The perforated-patch configuration, which preserved
intracellular Ca2+ gradients, was used to correlate local
Ca2+ changes with the modulation of single channel
activity. We observed that unitary current recorded from BK channels
had a different pattern of activity when recorded from either the
perforated-patch or outside-out configuration. Indeed, bursts of BK
channel activity were recorded when the integrity of the cytoplasmic
environment was preserved, suggesting that simultaneous opening of
these channels may occur.
For further analyses, the background resting current was subtracted and
digitally filtered at 400 Hz while ET-1 was applied in
Ca2+-free solution. ET-1 (0.1 µM) application increased
the open-state probability of SK and BK channels from 0.24 ± 0.03 to 0.81 ± 0.08 (n = 7) and 0.15 ± 0.07 to
0.64 ± 0.017 (n = 5), respectively. In addition,
maximal time opening of single SK and BK channels was significantly
enhanced from 81 ± 12 to 394 ± 41 ms (n = 7) and 31 ± 4 to 58 ± 9 ms (n = 5),
respectively (Fig. 7, A-D). Apamin (0.1 µM, Fig.
7B) but not iberiotoxin (0.1 µM, data not shown) inhibited
the ET-1-induced activity of SK channels. In contrast, BK channels
activated by ET-1 were highly sensitive to iberiotoxin (0.1 µM, Fig.
7D). BK channels were also found to be activated differently
than SK channels in response to the rise in
[Ca2+]i. Indeed, because of the simultaneous
opening of several BK channels (from 4 to 8 channels), which resulted
in current transients, BK channels exhibited a highly cooperative
behavior under ET-1 application (Fig. 7D). This cooperative
behavior was not observed for SK channels.
Changes in membrane potential elicited by ET-1 in astrocytes.
Finally, the effect of ET-1 on membrane potential of cultured striatal
astrocytes was investigated in current-clamp mode. As previously
reported (16, 33, 49), cultured astrocytes exhibited a
wide heterogeneity of resting membrane potentials, ranging from
78 to
29 mV, with an average value of
48 ± 18 mV (n = 35).
Recordings from 21 astrocytes indicated that ET-1 (0.1 µM) evoked
several patterns of changes in membrane potential. Indeed, addition of
peptide triggered either a transient membrane hyperpolarization of
22 ± 4 mV (n = 6; Fig.
8A), a sustained
depolarization of 28 ± 3 mV (n = 7; Fig. 8B), or
a biphasic change in membrane potential characterized by a transient
hyperpolarization followed by a sustained depolarization (n = 4; Fig. 8C). However, no modification in membrane
potential could be observed in 4 of the 21 cells exposed to ET-1.

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|
Fig. 8.
Three different effects (A-C) of ET-1 on
membrane potential. All experiments were performed in the
perforated-patch configuration. Broken line indicates the resting
membrane potential before ET-1 application (0.1 µM).
|
|
Additional experiments were made to determine the relationship between
the ET-1-induced activation of IK Ca and
inhibition of IK DR and the various effects of this
peptide on membrane potential. Several observations suggested that
transient hyperpolarization resulted from the activation of
IK Ca linked to
[Ca2+]i elevation. First, ionomycin
application (1 µM) in the absence of external Ca2+
was always followed by hyperpolarization (30 ± 5 mV,
n = 5). Second, although no change in membrane
potential was observed when iberiotoxin (0.1 µM) was added to the
bath solution (n = 8), ET-1 application showed no
effect (n = 3) or induced only a 22 ± 2-mV
depolarization (n = 3). Third, when cells were treated with apamin (0.1 µM), which alone induced a slight depolarization of
11 ± 3 mV (n = 7), ET-1 (0.1 µM) evoked an
additional depolarization of 18 ± 4 mV (3 of 4 cells). Finally,
experiments performed with the IK Dr blocker
4-AP (1 mM) indicated that the sustained depolarization observed in 7 of 21 cells was the result of inhibition of this current by ET-1.
Indeed, this IK DR blocker, which alone
depolarized the cells by 31 ± 2 mV (n = 5),
prevented the ET-1 (0.1 µM)-induced depolarization (n = 4).
 |
DISCUSSION |
The present study demonstrates that ET-1 exerts sequential and
opposite effects on the outward K+ currents of cultured
astrocytes from the rat striatum. Indeed, this peptide first activates
two subtypes of Ca2+-dependent channels contributing to
IK Ca, BK and SK channels, according to their
pharmacological and biophysical properties. Second, ET-1 reduces total
outward K+ currents through a prolonged inhibition of
IK DR channels. These effects were observed on
the same cells or separately on distinct cells. Astrocyte heterogeneity
is likely responsible for this variety of responses. Because
IK Ca and IK DR are
involved in several cell functions, the regulatory effects of ET-1
could account for some of the central effects of ETs.
As shown, astrocytes express at least four types of K+
currents (8, 30, 46). Three currents, which are
voltage-dependent and Ca2+-independent, were identified as
IK A, IK DR, and IK IR by their pharmacological and kinetic
properties (1, 3, 7, 34, 40, 46). These three
K+ currents have been recorded extensively in either
primary cultures or acute brain slices (9, 13, 14, 38,
39). Finally, an IK Ca has also been
described in astrocytes (30, 50). This latter current
results from the contribution of BK channels, characterized by a large
unitary conductance (>80 pS), sensitivity to voltage, and blockade by
charybdotoxin and iberiotoxin (11, 12, 25), and from SK
channels, characterized by a smaller unitary conductance (<30 pS), a
weak sensitivity to voltage, and blockade by apamin (6, 16,
23).
These four K+ currents have already been described in
several studies on astrocytes. Depending on the preparation (primary cultures or brain slices) and the brain region investigated (1, 3, 40, 46), the contribution of these four K+
currents was estimated previously and indicated the existence of
astrocyte heterogeneity. In cultured striatal astrocytes, variation in
the shape of the I-V relationship was related to
differences in the proportions of the channel subtype. In fact, the
following two distinct forms of I-V relationships
were distinguished: 1) S-G-V astrocytes, characterized by
high sensitivity to TEA and iberotioxin, and 2) N-G-V
astrocytes, in which IK DR was the prevalent
current under resting conditions. Moreover, no morphological differences between the astrocytes were correlated with the variations in the I-V relationship.
Generally, SK channels are distinguished from BK channels by their
higher sensitivity to intracellular Ca2+ and their slower
and lower responses to changes in potential (6, 34). In
striatal astrocytes, transient increases in
[Ca2+]i induced by ET-1 enhanced the activity
of BK and SK channels with different profiles. The open time of SK
channels was increased much more than that of BK channels. Several BK
channels, but not SK channels, were opened simultaneously within a
short duration (cooperative opening). Because Ca2+
signaling plays a pivotal role in astrocyte function (21, 54, 56), the occurrence of SK and BK channels in these cells may be
engaged in physiological processes. Indeed, in astrocytes, SK channels
at negative potentials (from
40 to 0 mV) and at moderate [Ca2+]i increases might maintain long-lasting
potential-independent hyperpolarizations. Also, SK channels are
generally involved in the same process, which follows repetitive action
potential firing in neurons (6, 26), a phenomenon that
occurs in astrocytes in pathological conditions (8, 47).
BK channels with fast voltage-dependent kinetics operate at potentials
more positive than 0 mV and provide a negative feedback to rapidly
hyperpolarize the cell. Moreover, the cooperative opening of BK
channels usually results in the appearance of spontaneous transient
outward currents (STOCs; see Refs. 5 and 11), but these
are not observed in striatal astrocytes. As shown for cardiomyocytes
and skeletal and smooth muscle cells, besides the expression of BK
channels, STOCs generally require the close association of other
defined cellular elements, including voltage-gated Ca2+
channels, sarcoplasmic reticulum ryanodine receptor channels, and
Na+/Ca2+ exchangers (5, 10). The
absence of voltage-gated Ca2+ channels and the weak
ryanodine response in striatal astrocytes (21) may
contribute to the lack of STOCs in these cells. Alternatively, the
activation of these two types of Ca2+-dependent
K+ channels could occur during the propagation of
intercellular Ca2+ waves (22). Thus SK and BK
channels could constitute a target for these waves. These channels
could be activated not only in cells directly stimulated by ET-1 but
also in neighboring astrocytes to which the Ca2+ waves propagate.
Our results indicate that ET-1 transiently activates
IK Ca in cultured striatal astrocytes. A
similar observation was made after ionomycin application in the absence
of external Ca2+ (data not shown), indicating that
Ca2+ released from internal stores activates SK and BK
channels in these cells. Interestingly, in response to ET-1 (0.1 µM)
application, the activation of IK Ca (the
present study) and the inositol trisphosphate-evoked increase in
[Ca2+]i (4, 21, 54) have the
same duration (30 s). From these observations, it is expected that
other peptides, neurotransmitters, or hormones that induced similar
increase in [Ca2+]i (see Ref.
22) may also activate SK and BK channels in astrocytes. ET-1 also reduced IK DR, but this effect was
long lasting and occurred with a delay. The differing time courses for
the ET-1 effects on K+ currents occurred not only when both
responses were observed on the same cell but also when the responses
were recorded separately on distinct cells. This suggests that these
two ET-1-mediated regulatory processes are independent. Consequences on
resting membrane potential of the sequential regulations of
Ca2+-dependent K+ channels and
IK DR by ET-1 were determined in experiments carried out using current-clamp mode. Because membrane potentials were
monitored in the perforated-patch configuration with imposed ion
gradients, the measured values may differ from the physiological situation measured by intracellular microelectrodes (24).
However, the relative time course changes in membrane potential induced by ET-1 and their sensibility to the various blockers used allowed us
to dissect the participation of identified K+ channels. The
following three modalities of potential changes were observed: a
biphasic effect resulting from a transient hyperpolarization followed
by a sustained depolarization, a single transient hyperpolarization, or
a prolonged depolarization. The similarity in the time courses of the
ET-1 responses suggested that the hyperpolarization results from the
activation of IK Ca, while the subsequent
depolarization corresponds to the inhibition of
IK DR. Further supporting this statement, the
ET-1-induced hyperpolarization and depolarization were prevented by the
K+- and Ca2+-dependent channel blockers and
4-AP (IK DR blocker), respectively.
All of these observations were obtained from primary cultures. In the
future, they should be complemented by using a more integrated
preparation, such as acute brain slices. However, these results can be
discussed in the light of previous studies indicating that ETs can
affect several astrocytic functions. Indeed, ETs exert several effects
in astrocytes, including an increase in glucose uptake
(52), mitogenic and proliferative responses (28, 29,
41, 48), and an initiation followed by an inhibition of the
propagation of intercellular Ca2+ waves (4,
54). Some of these responses could be attributed to the
ET-1-induced regulation of K+ outward currents, since
several of these currents were found to contribute to astrocytic
functions. For instance, IK DR seems to be
critical for astrocytic proliferation (17). However, astrocytic proliferation induced by ETs should not be related directly
to IK DR regulation. In fact, this current is upregulated in reactive gliosis, whereas proliferation is inhibited and
scar repair is delayed by the IK DR blocker
4-AP. On the other hand, the ET-1-induced inhibition of
IK DR could affect K+ homeostasis
in the following two ways: either by modulating K+
buffering or by affecting the other voltage-sensitive channels through
changes in membrane potential. Indeed, both
IK IR and IK DR are
thought to be involved in K+ redistribution and
elimination, a process referred to as "spatial buffering" (see Ref.
46). Alternatively, ET-1 could modify the shape of
"glial spikes," since in astrocytes, according to Bordey and
Sontheimer (8) and Sontheimer and Ritchie
(47), IK DR contributes to the
slow afterhyperpolarization that follows the depolarization evoked by
current injections. Finally, because ET-1 is presumably produced
preponderantly by endothelial cells, it is likely that perivascular
astrocytes contributing to the brain-blood barrier constitute a
preferential target of this peptide. The sequential regulation of
K+ channels reported here could also be involved in the
regulation of K+ exchange between the circulating blood and
the extracellular space in the central nervous system, which could
affect the activity of local neuronal networks.
 |
ACKNOWLEDGEMENTS |
We thank K. D. Peusner for comments on the manuscript.
 |
FOOTNOTES |
This work was supported by the European Community Grant
QLK6-1999-02203, and R. Bychkov was supported by a fellowship
from the Institut National de la Santé et de la Recherche
Médicale (Poste Vert).
Address for reprint requests and other correspondence: C. Giaume, INSERM U114, Collège de France, 11, Place Marcelin
Berthelot, 75231 Paris, Cedex 05, France (E-mail:
christian.giaume{at}college-de-france.fr).
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 18 July 2000; accepted in final form 15 May 2001.
 |
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