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current in bovine adrenocortical
cells: correlation with cortisol secretion
1 Université Claude Bernard Lyon I, Laboratoire de Physiologie des Eléments Excitables, Unité Mixte de Recherche 5123 Centre National de la Recherche Scientifique, 69622 Villeurbanne, France; 2 Institut National de la Santé et de la Recherche Médicale, U 418, Hôpital Debrousse, 69322 Lyon, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ACTH has been shown to depolarize bovine
adrenal zona fasciculata cells by inhibiting a K+ current.
The effects of this hormone on such cells have been reexamined using
perforated and standard patch recording methods. In current clamp
experiments, ACTH (10 nM) induced a membrane depolarization to
36 ± 1 mV (n = 56), which was mimicked by
forskolin (10 µM) or by 8-(4-chlorophenylthio)-cAMP (8 mM).
ACTH-induced membrane depolarizations were associated in the majority
of cells with an increase in membrane conductance. In the other cells, these membrane responses could occur without change or could be correlated with a transient or with a continuous
Cs+-sensitive decrease in membrane conductance. The
depolarizations associated with an increase in membrane conductance
were depressed by Cl
current inhibitors
diphenylamine-2-carboxylic acid (DPC; 1 mM), anthracene-9-carboxylic
acid (9-AC; 1 mM), DIDS (400 µM), verapamil (100 µM), and
glibenclamide (20 µM). In voltage-clamped Cs+-loaded
cells, ACTH activated a time-independent current that displayed an
outward rectification and reversed at 21.5 mV ± 2 (n = 6). This current, observed in the presence of
internal EGTA (5 mM), was depressed in low Cl external
solution and was inhibited by DPC, 9-AC, DIDS,
5-nitro-2-(3-phenylpropylamino)benzoic acid, verapamil, and
glibenclamide. ACTH-stimulated cortisol secretion was blocked by
Cl channel inhibitors DIDS (400 µM) and DPC (1 mM). The
present results reveal that, in addition to inhibiting a K+
current, ACTH activates in bovine zona fasciculata cells a
Ca2+-insensitive, cAMP-dependent Cl current.
This Cl current is involved in the ACTH-induced membrane
depolarization, which seems to be a crucial step in stimulating steroidogenesis.
adrenocorticotropic hormone; calf adrenal zona fasciculata cells; whole cell recording; membrane potential; membrane current; chloride current inhibitors
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ADRENOCORTICOTROPIC
HORMONE (ACTH) is known as a potent activator of the
steroidogenesis in zona fasciculata (ZF) cells. This polypeptide
hormone binds to membrane receptors coupled to a guanine nucleotide
protein (Gs), which stimulates cortisol production through
activation of adenylyl cyclase (16, 26). Although cAMP is
considered as the intracellular messenger of ACTH action (12), molecular mechanisms resulting from the activation
of this metabolic pathway in connection with the steroid biosynthesis remain to be elucidated. Beyond these effects on cellular metabolism, ACTH is involved in the modulation of ionic membrane conductance in
adrenal cells. For example, Chorvatova et al. (4) describe in zona glomerulosa cells isolated from rat adrenal gland a transient Cl current activated in response to ACTH stimulation.
These authors demonstrated that this Cl current was
dependent on a metabolic cascade involving Ras protein. In ZF cells
isolated from bovine adrenal gland, ACTH depolarizes the cell membrane
in a dose-dependent manner by inhibiting a noninactivating ATP-dependent K+ current (IAC) that
sets the resting membrane potential (7, 17). The
inhibition mechanism that requires ATP hydrolysis would be cAMP
dependent but independent of A-kinase activation (9). In
addition, in this cell type, the cortisol secretion would need a
Ca2+ influx via T-type Ca2+ channels activated
by the ACTH-induced membrane depolarization (8).
In the present study, we confirm that ACTH depolarizes the membrane of
ZF cells isolated from calf adrenal gland. However, compared with the
results reported by Mlinar et al. (17), we found that, in
addition to inhibiting a background K+ current, ACTH
activates a Cl current that participates in the membrane
depolarization. Furthermore, we show that the ACTH-stimulated cortisol
secretion is blocked by Cl channel inhibitors.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Cell Preparation
Isolated ZF cells were prepared according to the protocol described by Bilbaut et al. (2). Briefly, fat-free adrenal glands from calves 4-6 mo old were sliced with a Stadie-Riggs microtome. Only the second slice (0.5 mm thick) was used for enzymatic dispersion by sequential trypsination. The dissociation medium contained trypsin (Sigma, St. Louis, MO) at 0.125% in Ham's F-12-DMEM medium (1:1), gentamicin (20 µg/l), penicillin-streptomycin (100 U/ml), L-glutamine (5 mM), and NaHCO3 (14 mM) buffered with HEPES (15 mM) at pH 7.4. Dispersed cells were washed and resuspended in culture medium containing Ham's F-12-DMEM (1:1), L-glutamine (5 mM), penicillin-streptomycin (100 U/ml), NaHCO3 (14 mM), insulin (10 µg/ml), transferrin (10 µg/ml), and vitamin C (104 M) supplemented for 24 h with fetal calf serum (1%). The isolated cells, cultured in a
humidified incubator at 37°C and 5% CO2 in air, were
seeded at low density (4,000-6,000 cells/cm2) in 35-mm
Petri dishes for electrophysiological studies or at high density
(50,000-65,000 cells/cm2) in 12-well dishes for
secretion measurement.
Solutions and Drug Preparation
The control external solution contained (in mM): 135 NaCl, 5 KCl, 2.5 CaCl2, 2 MgCl2, and 10 glucose, buffered with 10 HEPES at pH 7.2 by NaOH. In deficient Cl
solution, 135 mM Cl were exchanged with methanesulfonate
ions. Ca2+-free solution contained 20 mM Ba2+
isosmotically substituted for NaCl. Human synthetic ACTH, fragment 1-24, (Sigma) was prepared from aliquots at 100 µM frozen in
distilled water containing 50 mM acetic acid and 1% bovine serum
albumin. ACTH was used at the final concentration of 10 nM by
successive dilutions in the external solution. Forskolin (FSK),
purchased from Calbiochem (La Jolla, CA), was aliquoted in dimethyl
sulfoxide (DMSO) at a concentration of 10 mM and used at a final
concentration of 10 µM. Cl current inhibitors
diphenylamine-2-carboxylic acid (DPC), anthracene-9-carboxylic acid
(9-AC), 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS),
4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid
(SITS), and niflumic acid were prepared just before use at different concentrations as indicated in the text. The ATP-dependent K+ (KATP) channel modulator glibenclamide and
the Ca2+ channel blocker verapamil were also prepared just
before use at concentrations of 20 and 100 µM, respectively. cAMP
analog 8-(4-chlorophenylthio)-cAMP (8-pcpt-cAMP) was used at 8 mM. All of these drugs were obtained from Sigma and were solubilized in DMSO
(DPC, 9-AC, NPPB, glibenclamide, and niflumic acid), alcohol (8-pcpt-cAMP), or physiological saline (DIDS, SITS, and verapamil). The
membrane properties of isolated cells were not affected by the final
DMSO concentration, which was 
0.1%.
The ionic composition of the pipette filling solution for the perforated patch recording was (in mM): 110 K-aspartate, 20 KCl, 10 NaCl, 2 MgCl2, and 5 EGTA, buffered with 10 mM HEPES at pH 7.2 by NaOH. When needed, K+ conductances were inhibited by substituting Cs+ for K+ in the pipette solution. The cell membrane was perforated using the polyene antibiotic amphotericin B (Sigma). This pore-forming antibiotic, mainly permeable to monovalent cations (24), was used at a concentration of 240 µg/ml and prepared as described by Rae et al. (23): 6 mg of amphotericin B were solubilized in 100 µl of DMSO by sonication for a few seconds, and 20 µl of this solution were then added to 5 ml of internal solution. For the standard patch recording (broken membrane), the ionic composition of the pipette solution was (in mM): 110 K-aspartate, 20 KCl, 10 NaCl, 2 MgCl2, 2ATP Mg, and 5 EGTA, buffered with 10 mM HEPES at pH 7.2 by NaOH.
Electrophysiology
Current- and voltage-clamp recordings were performed in whole cell configuration mainly by use of the perforated patch recording method (23). However, as indicated in the text, some voltage-clamp results were also obtained with the standard recording method (14). Experiments were carried out on isolated cells maintained in primary culture 24-72 h after plating, a period during which bovine ZF cells are known to retain their capacity for synthesizing and secreting steroid hormones in response to ACTH (11). This secretagogue was used at 10 nM, a concentration that maximally stimulates the cortisol secretion of isolated cells (21). For experiments, a Petri dish was transferred from the incubator onto the stage of an inverted microscope, and the culture medium was replaced by the control physiological solution. Further changes of external solution were then performed at a rate of 0.5 ml/min by use of a gravity perfusion system placed close to the cell (~100 µm). Pipettes were pulled from thin-walled borosilicate glass (CG 150T, 1.5 mm OD, Harvard Apparatus, Edenbridge, UK) using a vertical puller (Kopf, Tujunga, CA) and were connected to the headstage of a patch-clamp amplifier RK 400 (Bio-Logic, Claix, France). For perforated patch recordings, the tip of the pipette was dipped into the pipette solution for a few seconds, and then the pipette was backfilled with the solution containing amphotericin B. Patch pipettes had a tip resistance of 2-4 M
in
the control solution. Partition of the cell membrane by amphotericin B
was continuously monitored by applying 20-mV hyperpolarizing pulses
every 30 s from a holding potential of 60 mV. Experiments were
started ~10-15 min after the seal was established when the increase of the transient capacitive current reached a steady-state value indicating a final series resistance of ~8-12 M. The
series resistance was not compensated for, because the voltage error introduced by the maximal activation of membrane currents was estimated
to be <2%. Neither capacitive current nor leak current was subtracted
from membrane current recordings. In experiments where external
Cl was lowered, a 3 M KCl-agar salt bridge was interposed
between the Ag-AgCl reference electrode and the bath solution to
minimize changes in liquid junction potentials. For voltage-clamp
experiments, pulse protocols were generated using the P-Clamp software
(Axon Instruments, Burlingame, CA). In current-clamp experiments,
membrane conductance was monitored by injecting, every 10 s,
constant hyperpolarizing 10-pA current pulses 2 s long. Current
pulses were delivered from a programmable stimulator SMP 300 (Bio-Logic). Membrane conductance was calculated as
I/
V where I was the injected
current and V, the evoked hyperpolarizing potential. All
experiments were performed at room temperature (20-25°C) on
single cells of 15-20 µm in diameter that adhered to the bottom
of the Petri dish.
Membrane signals induced by ACTH stimulation were monitored on both pen (Kipp & Zonen, Delft, the Netherlands) and tape (DTR 1204, Bio-Logic) recorders. Current signals were filtered at 1 kHz, digitized at 4 kHz with an analog-to-digital converter (Labmaster TM 40, Scientific Solutions, Solon, OH), and stored on the hard disk of a computer. For data analysis, Bio-Logic software was used. Results are expressed as means ± SE. When appropriate, data were tested for significance using Student's t-test, where P values of <0.05 were considered to indicate significant differences.
Secretion Measurement
Freshly isolated ZF cells were seeded in 12-well test plates, each containing 1 ml of culture medium. On the 3rd day, the culture medium was removed and replaced by the following physiological solutions (1 ml): control, control + ACTH (10 nM), control + DIDS (400 µM), control + DPC (1 mM), and control + ACTH (10 nM), in which either DIDS (400 µM) or DPC (1 mM) was added. To test the effects of Cl current inhibitors on the
ACTH-stimulated cortisol secretion, cultured cells were preincubated
for 5 min in the presence of DIDS and DPC before the hormone was added.
After 2 h at 37°C, the cell medium was removed, and cortisol
content was determined by radioimmunoassay using specific antibody
(6, 20). At the end of each experiment, cells were counted
(Coulter, ZBI). The data are presented as means ± SE from
measurements performed in four wells for each condition.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ACTH-Induced Membrane Depolarization
In control physiological solution, the resting membrane potential of isolated ZF cells, determined using the perforated whole cell recording method under current-clamp conditions, was63 ± 2 mV
(n = 48). Usually, this resting potential was more or
less stable. It displayed oscillations that could reach ±5 mV. This was attributed to the strong input membrane resistance of these cells
(3.3 ± 0.3 G, n = 53), presumably related to
stochastic resting ion channel activity. If spontaneous oscillations of
membrane potential were larger than 10 mV, the cells were discarded.
Exposure of ZF cells to ACTH induced a membrane response after a delay
of 58 ± 3 s (n = 56) consisting of a
depolarizing phase followed by a plateau that reached a maximum
value and then decayed, as shown in Fig.
1A. Averaged from 56 cells,
the maximum depolarization induced by ACTH was 36 ± 1 mV. The
duration of the depolarizing phase varied from cell to cell. Measured
at 50 and 90% of the maximum depolarization, this duration was 38 ± 4 and 100 ± 10 s (n = 18), respectively.
Repolarization of the plateau was very slow, and complete recovery of
the membrane potential to its resting value was never achieved, even
when isolated cells were exposed for >30 min to ACTH. In 80% of the
experimented cells (48 of 60), these membrane responses were associated
with an increase in the input membrane conductance from 0.39 ± 0.03 nS (n = 48) in control conditions to 1.8 ± 0.2 nS (n = 48) during the depolarizing plateau. Figure
1B shows the changes both in membrane potential (solid symbols, left axis) and in membrane conductance (open
symbols, right axis) measured every 10 s from the
recording presented in Fig. 1A. In this figure, it can be
seen that, except at the beginning of the response, changes in the
membrane potential paralleled changes in membrane conductance. The
depolarizing phase was accompanied by a progressive increase in
membrane conductance, which reached maximal value during the plateau
and then decayed as the cell membrane repolarized. These records also
revealed that, in the beginning of the depolarizing phase, a large
increase in membrane potential induced by ACTH could be correlated with
very weak or even undetectable changes in membrane conductance. As
subsequently discussed, this could be attributed to the high input
resistance of these cells, where very small ionic currents would be
able to produce large jumps in membrane potential in the absence of detectable change in the membrane conductance.
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Such changes in membrane conductance were not invariably observed.
Thus, in 8 of these 48 cells, this parameter, instead of continuously
increasing at the beginning of the depolarizing phase, as shown in Fig.
1B, displayed an initial transient decrease. Figure
2A illustrates such a pattern.
In this cell, where membrane potential (solid symbols, left
axis) and membrane conductance (open symbols, right axis)
were monitored every 10 s, the beginning of depolarization was
associated with a clear decrease in membrane conductance, which
increased subsequently as the membrane depolarized to reach maximum
during the plateau. Furthermore, in 13% of the cells (8 of 60), the
ACTH-induced membrane response was accompanied by no detectable changes
in membrane conductance. Finally, in 4 of these 60 cells (7%), the
membrane response was associated with a continuous decrease in membrane
conductance (Fig. 2B), which was 0.42 ± 0.06 vs.
0.82 ± 0.15 nS (n = 4) in control conditions. In
these four cells, the maximum value of the membrane depolarization triggered by ACTH was 47 ± 0.8 mV, a potential significantly lower (P = 0.007) than that reported for the responses
associated with an increase in membrane conductance. When
K+ conductances were blocked by substituting K+
for Cs+ in the pipette solution (19 cells), a decrease in
membrane conductance was never detected during the membrane response to
ACTH stimulation. In all of these cells, the ACTH-induced depolarizing
phase was always associated with a continuous increase in membrane
conductance (Fig. 3B), which
decayed subsequently as the membrane repolarized. In these experimental
conditions, the resting membrane potential was 52 ± 4 mV
(n = 19), with values largely scattered and
occasionally more negative than 70 mV (3 of 19 cells), as
illustrated in Fig. 3. This suggests that, in these cells, some
components of background K+ current would be
Cs+ resistant. Exposure of cells to ACTH produced after a
delay of 57 ± 7 s (n = 19), a membrane
response similar to that described in control conditions, which often
began with a fast depolarization resembling a nonovershooting action
potential (Fig. 3A, arrowhead). This type of membrane
activity, previously described after inhibition of the transient
K+ current (2), started when ACTH-induced
membrane depolarization was close to 50 mV. This value corresponds to
the activation potential of voltage-sensitive Ca2+ channels
identified in this preparation (13). With Cs+
in the pipette solution, the maximum value of the depolarizing plateau
potential evoked by ACTH stimulation was 36.5 ± 1.5 mV (n = 19), and membrane conductance, which was 0.38 ± 0.05 nS (n = 19) at rest, rose to 2.9 ± 0.7 nS
(n = 19) during the ACTH-induced membrane response.
This value was significantly higher (P = 0.04) than
that measured in the control solution.
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These observations indicate that membrane depolarizations stimulated by ACTH result from complex mechanisms. The modulation of membrane conductance during the response would indicate that ACTH acts by activating one ionic current and inhibiting another. The fact that Cs+ abolishes membrane responses associated with a decrease in membrane conductance suggests strongly that one of the effects of ACTH is to decrease a background K+ conductance. This is in accord with the results reported by Mlinar et al. (17), where the ACTH-induced membrane depolarization of bovine ZF cells was caused by the inhibition of a K+ current. Hence, with the consideration that the mechanisms involved in this process are likely similar to those extensively studied by Enyeart and co-workers (7, 9, 10, 17, 30), depolarization associated with a decrease in membrane conductance was not further characterized in the present work.
FSK- and Permeant cAMP-Induced Membrane Depolarization
As previously emphasized, the binding of ACTH on specific membrane receptors is known to activate the metabolic pathway of adenylyl cyclase via Gs protein. When isolated ZF cells were exposed to 10 µM FSK, a membrane-permeant activator of adenylyl cyclase, a membrane response similar to that triggered by ACTH was observed (Fig. 4A). After a delay of 61 ± 6.5 s (n = 21), the cell membrane began to depolarize and reached maximum value at32 ± 1.5 mV (n = 23) before slowly repolarizing. Compared with
ACTH, the FSK-induced membrane depolarization was significantly higher
(P = 0.03). Membrane conductance changes were similar
to those reported during ACTH stimulation. From a resting value of
0.38 ± 0.05 nS (n = 17), membrane conductance at
first increased up to 1.9 ± 0.45 nS (n = 17) and
then decreased. In one of 24 cells exposed to FSK, a membrane
depolarization to 57 mV from a resting potential of 78 mV was
associated with a continuous decrease in membrane conductance from 0.6 to 0.45 nS.
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Membrane depolarizations were also recorded after isolated cells were
exposed to the membrane-permeant analog 8-pcpt-cAMP (Fig.
4B). After a delay of 93 ± 6.5 s
(n = 3), this metabolite (8 mM) induced a slow
depolarizing phase followed by a steady plateau potential that reached
a maximum value at 27 ± 2.5 mV (n = 3). During
these responses, membrane conductance was drastically increased up to
5 ± 1.5 nS vs. 0.3 ± 0.1 nS (n = 3) in
control conditions. Maximum depolarization and membrane conductance
measured in response to permeant cAMP were significantly different from those measured in response to ACTH stimulation, with P = 0.049 and 0.0006, respectively.
Pharmacology of ACTH-Induced Membrane Depolarization
The aforementioned results indicate that ACTH depolarizes the cell membrane of isolated ZF cells. In most cells, this depolarization is associated with an increase in membrane conductance, suggesting that the hormone stimulates an ionic current whose equilibrium potential would be about30 mV. As a first hypothesis, we consider that ACTH
might evoke an increase of Cl membrane conductance.
Indeed, when DPC (1 mM) or DIDS (400 µM), two nonspecific inhibitors
of Cl currents, were applied during the depolarizing
plateau, the membrane potential returned toward more negative values
(Fig. 5, A and B).
In the presence of DPC, the cell membrane repolarized by 86 ± 4%
(n = 15) and by 50 ± 8% (n = 8)
in the presence of DIDS. As shown in Fig. 5, these effects were
associated with a decrease in membrane conductance. Partial membrane
repolarization was observed for concentrations of DPC ranging from 250 to 500 µM. When FSK was used to trigger the membrane depolarization,
the effects of DPC (1 mM) on the plateau potential were similar to
those observed with ACTH stimulation.
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Other Cl current inhibitors were tested on ACTH- or
FSK-induced membrane depolarizations. Although SITS (400 µM, 3 cells) and niflumic acid (50 µM, 2 cells) were without effect on the depolarizing plateau potential, 9-AC (1 mM) repolarized the cell membrane by 100% (n = 2). Furthermore, verapamil (100 µM) and glibenclamide (20 µM) inhibited these responses by 69 ± 15 (n = 3) and 79 ± 10% (n = 5), respectively (not illustrated).
Effect of Holding Potential Changes on ACTH-Induced Membrane Current
If ACTH increases Cl membrane conductance, as
suggested by the results obtained in current clamp recordings where the
depolarizing plateau potential is decreased by Cl channel
inhibitors, we can expect that under voltage-clamp conditions the
membrane current will be inward for holding potentials more negative
than 30 mV and outward for holding potentials more positive than this
value. To separate the Cl membrane current of an eventual
modulation of K+ membrane conductance by ACTH as observed
in current-clamp experiments, isolated cells were voltage clamped using
the perforated whole cell recording method after Cs+ was
added to the pipette solution. Figure 6
shows that ACTH activated a transient inward current from a holding
potential of 60 mV (Fig. 6A) and a transient outward
current from a holding potential of 10 mV (Fig. 6B). The
increase of the ionic current in the inward or outward direction was
associated with an increase in membrane conductance, which was
determined by applying every 10 s short test pulses 30 mV more
negative than the holding potential of 60 or 10 mV. When the
holding potential was 10 mV, a potential where voltage-dependent
Ca2+ (13) and K+ currents
(2) were both fully inactivated, voltage steps of 75-ms
duration ranging from 100 to +40 mV were delivered by increments of
20 mV to the cell membrane in control conditions and during the maximum
activation of the ACTH-induced current. In the presence of ACTH,
membrane currents recorded during these voltage steps did not display
time dependence and were much larger than those obtained in the control
solution over the entire voltage range studied (Fig.
7A, inset). From
current-voltage relationships illustrated in Fig. 7, A and
B, membrane currents induced during ACTH stimulation exhibited an outward rectification and reversed at 21.5 ± 2 mV (n = 6). When FSK was used, similar observations were
made on the membrane current activated from these two different holding potentials. Current-voltage relationships established from a holding potential of 10 mV indicated that the FSK-induced outward current reversed at 20 ± 2.5 mV (n = 3) (not
illustrated).
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Ionic and Pharmacological Characterization of the ACTH-Activated Membrane Current
The Cl component of the ACTH-induced membrane
current was tested by lowering the external Cl
concentration to 14 mM instead of 140 mM to shift
ECl
toward a more positive potential. In
such conditions, the membrane current recorded from a holding potential
of 10 mV was more inward by 5-6 pA when isolated cells were
exposed to Cl-deficient solutions (not illustrated). On
the other hand, ACTH-induced outward current was strongly depressed in
a reversible manner when the ZF cell was briefly exposed to low
Cl solution. Figure 8
illustrates the effects of solution changes on the membrane current
generated by ACTH stimulation. In 14 mM Cl, the
ACTH-induced outward current was not only diminished
quasi-instantaneously but was inward, signifying that, under these
circumstances, its reversal potential was more positive than 10 mV.
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The pharmacological properties of the outward ionic current activated
by ACTH stimulation were studied from a holding potential of 10 mV
using different Cl channel blockers. A large inhibition
of the outward current was obtained by exposing isolated cells to 1 mM
DPC (4 cells) or 9-AC (2 cells). At maximum inhibition, the membrane
current was more inward than the control current, suggesting that these
two substances could block a resting Cl component (Fig.
9A). These effects were fully
reversible. DIDS used to the concentration of 400 or 250 µM (7 cells)
was also a potent inhibitor of the membrane current activated by ACTH
(Fig. 9A) or by FSK (not illustrated). For lower
concentrations, DIDS (100 µM) partially inhibited this ionic current
(Fig. 9B), suggesting a dose-dependent effect on the
membrane response. The ACTH-induced outward current was also suppressed
in six cells by 100 µM NPPB (Fig. 9B) and was sensitive to
100 µM verapamil (2 cells) and 20 µM glibenclamide (5 cells) (not
illustrated).
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Ca2+-Independence of the ACTH-Induced Membrane Current
As discussed later, these results indicate that the ionic current induced by ACTH stimulation is dominated by a Cl current.
ACTH is known to increase internal Ca2+ concentration, and
the presence of a Ca2+-dependent Cl current
activated in response to angiotensin II has been reported in isolated
ZF cells by Chorvatova et al. (5). To verify whether the
Cl current induced by ACTH could be Ca2+
dependent, experiments were performed using the conventional patch
recording method in the presence of 5 mM EGTA in the pipette solution.
In three cells, from a holding potential of 60 mV, ACTH activated an
inward current that was fully inhibited by DIDS (400 µM) (Fig.
10A). This indicates that
the ACTH-induced membrane current is partly or entirely carried by a
Ca2+-independent Cl current. An eventual
involvement of extracellular Ca2+ in the ACTH-induced
membrane current was also examined by replacing 2.5 mM Ca2+
with 20 mM Ba2+. From a holding potential of 90 mV, a
value 40 mV more negative than the activation potential of
voltage-sensitive Ca2+ currents (13), ACTH
induced a large inward current that was inhibited by DIDS (400 µM)
(Fig. 10B).
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Secretion
ACTH is known to activate cortisol secretion in cultured isolated ZF cells (11). The present study reveals that ACTH induces a membrane depolarization that can be blocked by various Cl current inhibitors. To know whether correlations could
exist between membrane depolarization and cell secretion,
ACTH-stimulated cortisol production was measured in control conditions
and in the presence of two Cl current inhibitors, DIDS
(400 µM) and DPC (1 mM). The results shown in Fig.
11 are representative of three
experiments performed on cell populations submitted to similar
protocols. From these results, basal cortisol secretion was detected in
control physiological solution. Similarly, basal cortisol secretion
also was measured after incubation of isolated ZF cells for 2 h in
the presence of DIDS or DPC. This suggests that these two
Cl channel inhibitors do not exert toxic effects on cell
metabolism. Compared with measurements obtained in control conditions,
ACTH stimulated cortisol secretion by a factor of >100. On the other hand, when cells were preincubated with DIDS or DPC for 5 min before
exposure for 2 h in a solution containing ACTH and
Cl channel inhibitors, cortisol secretion was diminished
by 95.5 and 99.3%, respectively.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ACTH-Induced Cl Current
current
in ZF cells isolated from bovine adrenal glands. This membrane current was characterized from results obtained in current- and voltage-clamp recordings. In current-clamp, the membrane depolarization, associated in the majority of the studied cells with a strong increase in membrane
conductance, was sensitive to different inhibitors of Cl
channels. In voltage-clamp, the ACTH-activated membrane current was
identified as a Cl current. It was depressed when the
external Cl concentration was lowered, and it was
sensitive to a large range of Cl channel inhibitors,
including DPC, 9-AC, DIDS, and NPPB and also to verapamil and
glibenclamide. Its reversal potential was about 20 mV, a potential
that is near the expected equilibrium potential for Cl.
Moreover, the ACTH-induced Cl current was a
cAMP-dependent current, because membrane depolarizations similar to
those induced by ACTH were evoked by direct exposure of cells to FSK or
to membrane-permeant analog 8-pcpt-cAMP. Although the properties of the
membrane current activated by 8-pcpt-cAMP were not studied, the
biophysical and pharmacological characteristics of ionic current
induced by FSK known to increase cytosolic cAMP via the direct
activation of adenylyl cyclase were comparable to those observed in the
presence of ACTH.
Cl current activation in response to ACTH exposure was
reported in zona glomerulosa cells isolated from the rat adrenal gland by Chorvatova et al. (4). This membrane current displayed
an outward rectification and was sensitive to the Cl
channel blockers DPC and SITS. Contrary to the Cl current
described in this study on bovine ZF cells, the ACTH-induced Cl current in rat glomerulosa cells was activated neither
by FSK nor by cAMP analogs. This indicates that the metabolic pathway of adenylyl cyclase was not involved in the activation of this cAMP-independent Cl current, which, for Chorvatova et al.
(4), was dependent on the activation of Ras protein by
G
subunits.
Up to the present time, the activation of a Cl membrane
current by ACTH has not been described in adrenal ZF cells. From the results previously reported by Mlinar et al. (17), the
membrane depolarization induced by ACTH in bovine ZF cells was
attributed to the inhibition of a background K+ current.
These discordant effects of ACTH on this cell type could be related to
differences in experimental appraoches. Our experiments were usually
performed using the perforated patch recording method on primary
culture of adrenal ZF cells (from 24 to 72 h after plating)
obtained from calves 4-6 mo old. The experiments of Enyeart et al.
(7) and Mlinar et al. (17) were carried out
using the standard recording method on freshly isolated adrenal ZF
cells (usually 12 h after plating) obtained from steers 1-2 yr
old. However, the presence of the Cl current identified
in this study in response to ACTH cannot be explained either by the
recording method used or by the age of animals. In the present work,
when the standard whole cell recording was used (Fig. 10A),
ACTH-induced Cl current was observed. To verify whether
age could be a determining factor, we performed experiments on adrenal
ZF cells isolated from 3-yr-old steers. The results obtained were
similar to those described on calves; ACTH induced a depolarization
associated with an increase in the Cl membrane
conductance thus excluding a difference in the cell maturation of the
adrenal glands. Consequently, although no experiment was performed on
freshly isolated cells, the hypothesis according to which the
expression of the ACTH-induced Cl current in adrenal ZF
cells would be dependent on time in culture cannot be excluded.
Development of ionic currents in relation to time in culture have been
reported in bovine ZF cells (13) as in other cell types
(25, 31).
Identification of the Cl Current
channels have been described in
different cells that belong to cAMP-regulated,
Ca2+-dependent, voltage-dependent, and swelling-dependent
or volume-regulated Cl channels (28). The
identification of these different Cl channels on the
basis of their electrophysiological and pharmacological properties is
often problematic, because similarities in biophysical characteristics
may exist among these channels and because no specific pharmacological
tools are available at the present time.
The possibility that the ACTH-induced Cl current
identified in isolated ZF cells corresponds to CFTR cAMP-regulated
Cl current must be discarded, even though these two
Cl currents are sensitive to sulfonylurea glibenclamide.
Indeed, unlike the cyctic fibrosis transmembrane conductance regulator (CFTR) Cl current (1), the ACTH-induced
Cl current in isolated ZF cells displayed an outward
rectification and was DIDS sensitive. In addition, sulfonylureas are
known to inhibit other types of Cl channels than CFTR
Cl channels (22, 27), and mRNA coding for
this channel protein was never detected in adrenal tissue.
Another experimental result suggests that the ACTH-induced
Cl current is not a Ca2+-dependent
Cl current. The activation of this current was not
prevented either after the internal cell compartment was loaded with
EGTA or after external Ca2+ was replaced by
Ba2+. However, Chorvatova et al. (5) described
in isolated ZF cells from bovine adrenal gland exposed to angiotensin
II a small component of Ca2+-dependent Cl
current coactivated with a large apamin sensitive
Ca2+-dependent K+ current. In the absence of
selective inhibitors of Ca2+ dependent Cl
current and considering that ACTH induced oscillating Ca2+
rise in bovine adrenal ZF cells (15), we therefore cannot
exclude that such a component was activated together with a
Ca2+-independent current component which would dominate the
total Cl current induced by ACTH. The ACTH-dependent
increase of cytosolic Ca2+ concentration measured by Kimoto
et al. (15) raises the question why, in the present study,
the activation of a Ca2+-dependent K+ current
was never detected during the exposure of ZF cells to this hormone. We
suppose that the increase of cytosolic Ca2+ would be
insufficient to activate Ca2+-dependent K+
channels or that ACTH would activate a metabolic pathways that would
inhibit these K+ channels.
On the basis of the present results, we cannot decide whether the
ACTH-induced Ca2+-independent Cl current
belongs to the family of voltage-activated or volume-regulated Cl currents. Indeed, these two types of Cl
currents can display outward rectification and show a sensitivity to
various pharmacological agents including verapamil (18,
29).
Physiological Role of the Cl Current
Membrane depolarization.
This study confirms previous observations of Mlinar et al.
(17), who reported in bovine isolated ZF cells a membrane
depolarization induced by ACTH. These authors attributed this membrane
response to the inhibition of a background Cs+-sensitive
K+ current termed IAC. This ionic
current, observed using the standard recording method, was
characterized by a continuous growth during the course of experiment
(17) and by its sensitivity to intracellular ATP supplied
by the patch pipette (7). In the present study, where the
perforated patch recording method was used and the intracellular ATP
level could not be controlled, we showed that, in addition to
inhibiting a background Cs+-sensitive membrane conductance
that likely corresponds to IAC, ACTH also
activates a Cl current. These different observations
indicate that, in our hands, ACTH has a dual effect on the modulation
of the membrane conductance of isolated cells. This peptide would
stimulate two opposite mechanisms, which would act synergistically to
depolarize ZF cell membranes. The respective participation of
these two mechanisms in the membrane depolarization was not quantified
in this work. However, some experimental evidence suggests that the
activation of the Cl current exerts a significant role in
ACTH-stimulated membrane depolarization. We have observed that
Cl channel blockers applied during the ACTH-induced
plateau could repolarize the cell membrane to the control value. This
signifies that, in these cells, the ACTH-induced membrane
depolarization was caused only by the activation of the
Cl current. Furthermore, in the majority of cells (80%),
the ACTH depolarizing plateau was correlated with an increase in
membrane conductance. This indicates that, during this depolarizing
phase, the cell membrane conductance was dominated by the activation of
the Cl conductance.
channels would be sufficient
to produce a large membrane depolarization. In addition, examination of
membrane responses obtained in Cs+-loaded cells shows that,
during the depolarizing phase, the increase in Cl
membrane conductance is a slow process. Consequently, we can conceive
that, during the experiments performed in control conditions where the
two opposite mechanisms involved in the membrane depolarization are
stimulated by ACTH, discrete changes in membrane conductance could be
correlated with large jumps in membrane potential.
Secretion.
This study establishes the existence of correlations between
ACTH-induced membrane depolarization and ACTH-induced cortisol secretion. Our results discard a toxic effect of DIDS or DPC on intracellular pathways that stimulate cortisol secretion. In these conditions, membrane depolarization appears as a crucial step to
trigger cell secretory activities in response to ACTH. From studies
performed by Yanagibashi et al. (32) and confirmed
subsequently by Enyeart et al. (8), ACTH-induced cortisol
secretion in bovine ZF cells was inhibited in the presence of
Ca2+ channel blockers. In agreement with the conclusions of
Enyeart et al., we proposed that the membrane depolarization induced by ACTH would be a sufficient condition to activate voltage-dependent Ca2+ channels identified in this cell type
(13), even though this depolarization failed to trigger
action potentials in control solution. Indeed, the activation threshold
of T- and L-type Ca2+ currents described by Guyot et al.
(13) in bovine adrenal ZF cells was about 50 mV in
control physiological saline (2.5 mM Ca2+), a potential
15-20 mV more negative than that reached during the ACTH-induced
membrane depolarization. In adrenal cells, an increase of cytosolic
Ca2+ concentration seems to be requisite to triggering
steroidogenesis (3). Recently Kimoto et al.
(15) have reported the existence of ACTH-induced cytosolic
Ca2+ oscillations in bovine adrenal ZF cells, whereas
Nishikawa et al. (19) have shown, in the same preparation,
that the expression of the steroidogenic acute regulatory protein,
involved in the mitochondrial transport of cholesterol, could be
regulated by a Ca2+/calmodulin-dependent protein kinase
whose activity required an increase of cytosolic Ca2+.
Consequently, the ACTH-induced membrane depolarization would correspond
to the primary cell signal required in the triggering of the cortisol secretion.
| |
ACKNOWLEDGEMENTS |
|---|
We are grateful to J. Diez, J.-L. Andrieu (Unité Mixte de Recherche 5123, Centre National de la Recherche Scientifique), and M. C. Berthelon (Institut National de la Santé et de la Recherche Médicale U 418) for help in preparing isolated cells.
| |
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: A. Bilbaut, Université Claude Bernard Lyon I, Laboratoire de Physiologie des Eléments Excitables, UMR CNRS 5123, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.
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.
10.1152/ajpendo.00218.2001
Received 18 May 2001; accepted in final form 17 September 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
|
|---|
1.
Begenisich, T,
and
Melvin JE.
Regulation of chloride channels in secretory epithelia.
J Membr Biol
163:
77-85,
1998[ISI][Medline].
2.
Bilbaut, A,
Chorvatova A,
Ojeda C,
and
Rougier O.
The transient outward current of isolated bovine adrenal zona fasciculata cells: comparison between standard and perforated patch recording methods.
J Membr Biol
149:
233-247,
1996[ISI][Medline].
3.
Capponi, AM,
Rossier MF,
Davies E,
and
Vallotton MB.
Calcium stimulates steroidogenesis in permeabilized bovine adrenal cortical cells.
J Biol Chem
263:
16113-16117,
1988
4.
Chorvatova, A,
Gendron L,
Bilodeau L,
Gallo-Payet N,
and
Payet D.
A Ras-dependent chloride current activated by adrenocorticotropin in rat adrenal zona glomerulosa cells.
Endocrinology
141:
684-692,
2000
5.
Chorvatova, A,
Guyot A,
Ojeda C,
Rougier O,
and
Bilbaut A.
Activation by angiotensin II of Ca2+-dependent K+ and Cl currents in zona fasciculata cells of bovine adrenal gland.
J Membr Biol
162:
39-50,
1998[ISI][Medline].
6.
Darbeida, H,
and
Durand P.
Glucocorticoid enhancement of glucocorticoid production by cultured ovine adrenocortical cells.
Biochim Biophys Acta
972:
200-208,
1988[Medline].
7.
Enyeart, JJ,
Gomora JC,
Xu L,
and
Enyeart JA.
Adenosine triphosphate activates a noninactivating K+ current in adrenal cortical cells through nonhydrolytic binding.
J Gen Physiol
110:
679-692,
1997
8.
Enyeart, JJ,
Mlinar B,
and
Enyeart JA.
T-type Ca2+ channels are required for adrenocorticotropin-stimulated cortisol production by bovine adrenal zona fasciculata cells.
J Mol Endocrinol
7:
1031-1040,
1993.
9.
Enyeart, JJ,
Mlinar B,
and
Enyeart JA.
Adrenocorticotropic hormone and cAMP inhibit nonactivating K+ current in adrenocortical cells by an A-kinase-independent mechanism requiring ATP hydrolysis.
J Gen Physiol
108:
251-264,
1996
10.
Gomora, JC,
and
Enyeart JJ.
Dual pharmacological properties of a cyclic AMP-sensitive potassium channel.
J Pharmacol Exp Ther
290:
266-275,
1999
11.
Goodyer, CG,
Torday JS,
Smith B,
and
Giroud CJP
Preliminary observations of bovine adrenal fasciculata-reticularis cells in monolayer culture: steroidognesis, effect of ACTH and cyclic AMP.
Acta Endocrinol
83:
373-385,
1976.
12.
Grahame-Smith, DG,
Butcher RW,
Ney RL,
and
Sutherland EW.
Adenosine 3',5'-monophosphate as the intracellular mediator of the action of adrenocorticotropic hormone on the adrenal cortex.
J Biol Chem
242:
5535-5541,
1967
13.
Guyot, A,
Dupré-Aucouturier S,
Ojeda C,
Rougier O,
and
Bilbaut A.
Two types of pharmacologically distinct Ca2+ currents with voltage-dependent similarities in zona fasciculata cells isolated from bovine adrenal gland.
J Membr Biol
173:
149-163,
2000[ISI][Medline].
14.
Hamill, OP,
Marty A,
Neher E,
Sakmann B,
and
Sigworth FJ.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch
391:
85-100,
1981[ISI][Medline].
15.
Kimoto, T,
Ohta Y,
and
Kawato S.
Adrenocorticotropin induces calcium oscillations in adrenal fasciculata cells: single cell imaging.
Biochem Biophys Res Commun
221:
25-30,
1996[ISI][Medline].
16.
Lefkowitz, RJ,
Roth J,
and
Pastan I.
ACTH-receptor interaction in the adrenal: a model for the initial step in the action of hormones that stimulate adenyl cyclase.
Ann NY Acad Sci
185:
195-209,
1971[ISI][Medline].
17.
Mlinar, B,
Biagi BA,
and
Enyeart JJ.
A novel K+ current inhibited by adrenocorticotropic hormone and angiotensin II in adrenal cortical cells.
J Biol Chem
268:
8640-8644,
1993
18.
Monaghan, AS,
Mintenig GM,
and
Sepulveda FV.
Outwardly rectifiying Cl channels in guinea pig small intestinal villus enterocytes: effect of inhibitors.
Am J Physiol Gastrointest Liver Physiol
273:
G1141-G1152,
1997
19.
Nishikawa, T,
Omura M,
and
Suematsu S.
Possible involvement of calcium/calmodulin-dependent protein kinase in ACTH-induced expression of the steroidogenic acute regulatory (StAR) protein in bovine adrenal fasciculata cells.
Endocr J
44:
895-898,
1997[ISI][Medline].
20.
Penhoat, A,
Jaillard C,
and
Saez JM.
Synergistic effects of corticotropin and insulin-like growth factor I on corticotropin receptors and corticotropin responsiveness in cultured bovine adrenocortical cells.
Biochem Biophys Res Commun
165:
355-359,
1989[ISI][Medline].
21.
Peytremann, A,
Nicholson WE,
Brown RD,
Liddle GW,
and
Hardman JG.
Comparative effects of angiotensin and ACTH on cyclic AMP and steroidogenesis in isolated bovine adrenal cells.
J Clin Invest
52:
835-842,
1973.
22.
Rabe, A,
Disser J,
and
Frömter E.
Cl channel inhibition by glibenclamide is not specific for the CFTR-type Cl channel.
Pflügers Arch
429:
659-662,
1995[ISI][Medline].
23.
Rae, J,
Cooper K,
Gates P,
and
Watsky M.
Low access resistance perforated patch recordings using amphotericin B.
J Neurosci Methods
37:
15-26,
1991[ISI][Medline].
24.
Rae, J,
and
Fernandez J.
Perforated patch recordings in physiology.
News Physiol Sci
6:
273-277,
1991
25.
Richard, S,
Neveu D,
Carnac G,
Bodin P,
Travo P,
and
Nargeot J.
Differential expression of voltage-gated Ca2+ currents in cultivated aortic myocytes.
Biochim Biophys Acta
1160:
95-104,
1992[Medline].
26.
Saez, JM,
Morera AM,
and
Dazord A.
Mediators of the effects of ACTH on adrenal cells.
In: Advances in Cyclic Nucleotides Research, , edited by Dumont JE,
Greengard P,
and Robinson AG.. New York: Raven, 1981, vol. 14, p. 563-579.
27.
Sakaguchi, M,
Matsuura H,
and
Ehara T.
Swelling-induced Cl-current in guinea pig atrial myocytes: inhibition by glibenclamide.
J Physiol (Lond)
505:
41-52,
1997[ISI][Medline].
28.
Valverde, MA,
Hardy SP,
and
Sepulveda FV.
Chloride channels: a state of flux.
FASEB J
9:
509-515,
1995[Abstract].
29.
Von Weikersthal, SF,
Barrand MA,
and
Hladky SB.
Functional and molecular characterization of a volume-sensitive chloride current in rat brain endothelial cells.
J Physiol (Lond)
516:
75-84,
1999
30.
Xu, L,
and
Enyeart JJ.
Properties of ATP-dependent K+ channels in adrenocortical cells.
Am J Physiol Cell Physiol
280:
C199-C215,
2001
31.
Yaari, Y,
Hamon B,
and
Lux HD.
Development of two types of calcium channels in cultured mammalian hippocampal neurons.
Science
235:
680-682,
1987
32.
Yanagibashi, K,
Kawamura M,
and
Hall PF.
Voltage-dependent Ca2+ channels are involved in regulation of steroid synthesis by bovine but not rat fasciculata cells.
Endocrinology
127:
311-318,
1990[Abstract].
| ||||||||||||||||||||||||||||||||
) and membrane
conductance (right axis, 
) during the
exposure of the cell to ACTH. In this and the other graphs, membrane
potential and membrane conductance were measured every 10 s and
plotted against time.
