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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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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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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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 (10Solutions 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 ClThe 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 MMembrane 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 ![]() |
RESULTS |
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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, wasExposure 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 at
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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 about
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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
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Ionic and Pharmacological Characterization of the ACTH-Activated Membrane Current
The Cl
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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
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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
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DISCUSSION |
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ACTH-Induced Cl Current
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
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.
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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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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