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Department of Physiology and Biophysics (A.C., L.G., L.B., N.G.-P., M.D.P.) and Service of Endocrinology (L.G., N.G.-P.), Faculty of Medicine, Sherbrooke, Québec, J1H 5N4, Canada
Address all correspondence and requests for reprints to: Dr. Marcel Daniel Payet, Department of Physiology and Biophysics, Faculty of Medicine, University of Sherbrooke, 3001, 12th Avenue North, Sherbrooke, Québec, Canada, J1H 5N4. E-mail: mpayet01{at}courrier.usherb.ca
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sequestrant, demonstrated that Gß subunits transduced the message.
Blockage of the Ras activation using an inhibitor of farnesyl
transferase (BZA-5B) or the monoclonal antibody H-Ras(259) abrogated
the current. Moreover, the addition of Ras-GTPS in the pipette
medium gave rise to the Cl- current. Treatment of the
cells with BZA decreased the aldosterone secretion induced by
10-10 M ACTH but not that induced by
10-8 M ACTH, confirming the involvement of Ras
in steroid secretion. We conclude that ACTH triggers a Cl-
current through the activation of the Ras protein by Gß subunits.
This current, activated at physiological ACTH concentrations (1 to 100
pM) where cAMP production is very low, could play a
significant role in aldosterone production. |
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-GTP and Gß-subunits. G subunits demonstrate effector
specificity and have been classified accordingly. Briefly,
Gs proteins stimulate adenylyl cyclase,
Gi/o proteins mediate inhibition of adenylyl
cyclase and Gq/11 leads to activation of
phospholipase Cß. Gß subunits are also involved in direct
regulation of effector molecules as shown in previous studies (1).
Ionic channels also constitute a class of effectors and can be directly
modulated by interaction with G or Gß-subunits (1, 2, 3). The
receptor tyrosine kinases (RTKs) pathway is the second major mechanism
involved in transmembrane signaling. The Ras/Raf mitogen-activated
protein kinases (MAPKs) cascade (4, 5) plays a pivotal role in
controlling cellular growth, division, and differentiation. Its
activation by peptide growth factors has been studied extensively (6).
There is also recent evidence of cross-talk and feedback actions
between G protein signaling and the Ras/Raf/MAPK cascade, including in
adrenal glomerulosa cells (7, 8). It is now established that G
protein-coupled receptors activate and/or modulate the mitogenic
pathway. This growth factor-like effect could be either PTX-sensitive
or PTX-insensitive and mediated by G or Gß-subunits (for review
see Ref. 9). ACTH is the most potent stimulus of steroid secretion by the adrenal gland. The ACTH receptor belongs to the GPCR family and is coupled to adenylyl cyclase (AC) through a Gs-coupling protein. Recent data indicates that, in bovine zona glomerulosa, ACTH binds two subtypes of receptors, called MC2 and MC5. ACTH binds mainly the MC2 isoform, which is 3.6 fold more abundant in zona glomerulosa than in zona fasciculata. It also binds MC5, which is expressed exclusively in zona glomerulosa (10). These observations could explain the high sensitivity of glomerulosa cells to ACTH. An increase in cAMP production and protein kinase A (PKA)-dependent phosphorylation (11, 12), as well as an increase in intracellular Ca2+ concentration (13) and cytoskeletal reorganization (14), mediate ACTH action in glomerulosa cells. Calcium and potassium channels have been described in glomerulosa and fasciculata cells (15, 16, 17). ACTH increases the amplitude of L-type calcium current but decreases that of the T-type calcium current (18) and of a noninactivating potassium current; these effects are cAMP- dependent. The hormone also blocks a transient outward potassium current in glomerulosa cells (19) and a novel potassium current in fasciculata cells (20). The role of Cl- current in glomerulosa cells has not yet been investigated although it could be involved in steroid secretion. Indeed, a Ca2+-dependent Cl- current, activated by Ang II, has been described in bovine fasciculata cells (21). With regard to these results and to findings describing the role of Cl- ions in LH-induced steroidogenesis of Leydig cells (22, 23), we were interested in determining whether a Cl- current could be involved in the ACTH action in glomerulosa cells and what would be its signaling pathway. Because the ACTH receptor is coupled to cAMP production and also demonstrates trophic effects (24) indicating a putative link to the MAPK cascade, we thus explored these two signaling pathways.
We found that ACTH activate a chloride current. The ACTH-induced
chloride current amplitude was independent of cAMP concentration as
well as its kinetics. It rather appeared dependent on the activation of
the Ras/Raf/MAPK cascade. We effectively demonstrated that Ras was
activated by 10-10 M ACTH.
Pharmacological studies showed that Ras but not Raf-1 or MAPK (ERK1,
ERK2) interacted, in a yet unknown manner, with a chloride channel to
increase the current amplitude. Perfusion of the cell, through the
patch pipette, with Ras-GTPS gave rise to a current with a
current/voltage relationship (I/V curve) similar to that of the
ACTH-induced chloride current. We conclude that the
Cl- channel may be a new effector or constitute
the distal part of a new signaling pathway for Ras protein.
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-subunit and GTPS from Calbiochem (San Diego,
CA); H-Ras (259) azide-free from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA); OP40 Ras antibody from Oncogene Science, Inc. (Scarborough, Ontario, Canada); PD 098059 was a
generous gift from Dr. David T. Dudley (Parke-Davis
Pharmaceuticals Research Division, Warner-Lambett Co., Ann Arbor, MI).
The QEHA peptide was synthesized by the "Service de Séquence de
Peptides de l’Est du Québec" (Le Centre Hospitalier de
l’Université Laval, Québec, Canada). The QEHA peptide was
purified by HPLC (>90%) and its identity verified by mass
spectrometry. Complete protease inhibitor; GST-RBD (Raf-1 Ras binding
domain) from Upstate Biotechnology, Inc. (Lake Placid,
NY); ATP, GTP and GTPS from Roche Molecular Biochemicals (Montréal, Québec, Canada). All
chemicals were of A-grade purity.
Cell isolation and plating
Zona glomerulosa cells were obtained from adrenal glands of
female Long Evans rats weighing 200–250 g. Rats were killed according
to a protocol approved by the Local Ethics Animal Care Committee.
Adrenals glands were isolated according to the method described in
detail elsewhere (25). The successive steps of zona glomerulosa
isolation and cell dissociation were performed in MEM (supplemented
with 100 U/ml penicillin and 100 mg/ml streptomycin). After a 20 min
incubation at 37 C in collagenase (2 mg/ml, 4 capsules/ml) and DNase
(25 µg/ml), the cells were disrupted by gentle aspiration with a
sterile 10 ml pipette, filtered and centrifuged for 10 min at 100
x g. They were then suspended in OPTI-MEM medium
supplemented with 2% FBS, 100 U/ml penicillin and 100 µg/ml
streptomycin and plated in 35-mm tissue culture dishes at a density of
approximately 5 x 104 cells per dish. The
cells were cultured at 37 C in a humidified atmosphere of 95% air-5%
CO2. The cells were used after 1 or 2 days of
culture.
Electrophysiology
The physiological solutions used for the patch clamp experiments
had the following compositions. The basic external solution contained
(mM): NaCl, 100; CaCl2 2;
tetraethylammonium chloride, 35; MgCl2, 1; CsCl,
5.4; HEPES, 5 and glucose 2 g/liter at pH 7.4. The control pipette
solution contained (mM): Cs-aspartate, 120; NaCl, 18;
CaCl2, 1; EGTA, 11; MgCl2,
2; HEPES, 5; ATP, 3, and GTP, 0.4 at pH 7.2. The pipette solution for
experiments in symmetric chloride conditions contained
(mM): CsCl, 126; NaCl, 18; CaCl2, 1;
EGTA, 11; MgCl2, 2; HEPES, 5; ATP, 3, and GTP,
0.4 at pH 7.2. Solutions containing hormones or drugs were freshly
prepared before each experiment.
Experiments were performed at room temperature and if needed, in the dark. The Petri dish (1 ml volume solution) was mounted on the stage of an inverted microscope (Nikon) and cells were observed at magnification of 100x. Ionic currents were recorded using the whole-cell configuration of the patch-clamp method (26). Patch electrodes with resistance of 3 to 5 megohms were pulled from Pyrex Glass capillaries (Corning 7740, Corning, Inc., Corning, NY). Ionic currents were recorded with an axopatch 1B (Axon Instruments, Burlingame, CA), whereas pulse stimulation and data acquisition were performed with an A/D interface DAS 16F (Metrabyte Taunton, MA) and an IBM-compatible computer under the control of a custom built program. Ionic currents were filtered at 1 kHz and sampled at 2 kHz. Voltage ramps applied from +55mV to -90 mV at a rate of 30 mV/s were filtered at 50 Hz and sampled at 100 Hz. All reported voltages were corrected where appropriate for the 10 mV junction potential between the low-Cl- pipette solution and the high-Cl- normal bath solution. Analyses were performed using custom-made software.
The Ras peptide (10 µM) was added in 360 µl with 200
µM GTPS overnight at 4 C and then dialyzed against 200
ml of control pipette solution for 10 h at 4 C; the dialysis was
repeated 3 times.
cAMP determination
Intracellular cAMP production was determined by measuring the
conversion of [3H]-ATP into
[3H]-cyclic AMP, as previously described (27).
Briefly, isolated cells were incubated 2h at 37 C in OPTI-MEM culture
medium containing 25 µCi/ml [3H]-adenine.
After washing, centrifugation and incubation in cold HBSS (NaCl,
130 mM; KCl, 3.5 mM;
CaCl2, 1.8 mM;
MgCl2, 0.5 mM;
NaHCO3, 2.5 mM; HEPES, 5
mM, supplemented with 1 g/liter glucose and 0.5% BSA) with
1 mM isobutyl methylxanthine (IBMX) for 15 min, ACTH (1
nM) was added for another 15 min incubation. ATP and cAMP
were separated on Dowex 1 x 8 and alumine columns.
p21 Ras activity determination
The assay was performed as previously described (28, 29).
Briefly, glomerulosa cells were cultured for 24 h in 60-mm Petri
dishes (1.0 x 106 cells/dish). After
hormonal stimulation, cells were harvested in the lysis buffer A (50
mM Tris-HCl, pH 7.5, 15 mM NaCl, 20
mM MgCl2, 5 mM EGTA,
Complete protease inhibitor, 1% Triton X-100, 1% N-octyl-glucoside)
for 15 min at 4 C. Insoluble material was removed by centrifugation at
12,000 x g for 2 min at 4 C. Proteins from lysates (1
mg) were incubated with 30 µg of GST-RBD fusion protein—where RDB is
amino acids 81–131 of Raf-1 and is the minimal domain required for
binding of Ras-GTP (30)—preadsorbed to glutathione-Sepharose beads for
2 h at 4 C. Precipitates were washed three times with buffer A.
The presence of p21ras was detected by
resuspending the final pellet in 20 µl of 2 x Laemmli buffer,
followed by protein separation on 12% polyacrylamide gels, and Western
blotting with antisera OP40 (1:100) recognizing
p21ras.
Statistical analysis
The data are presented as the mean ± SEM.
Statistical analyses were performed using a Student’s t
test, and P values were obtained from Dunett’s table, with
n indicating the number of experiments.
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) and in an
inward direction at -60 mV (Fig. 1B). Once activated, the current
decreased slowly, despite the presence of ACTH in the bath, with a time
constant of 108 ± 15 sec (n = 13). To determine the ionic
nature of the activated current, we first tested the contribution of
chloride ions by experiments where the pipette
Cl- concentration was changed so as to work in
symmetrical and asymmetrical Cl- conditions. The
contribution of potassium ions was excluded because of the presence of
cesium ions in the external and internal media. The holding membrane
potential (HP) was maintained at -40 mV and voltage pulses were
applied every 5 sec to -80, 0, +20 or +60 mV for 1500 ms. An example
of ACTH action (10-10 M), observed
in asymmetric (filled symbols) and symmetric (open
symbols) chloride conditions in two different cells is shown in
Fig. 2A. For statistical purposes, the
current measured from the original recording at each potential was
divided by the value of the cell input capacitance (mean value:
29.7 ± 0.9 pF, n = 17). Thus, the obtained normalized
current values were comparable among cells of various sizes. The I/V
curves recorded in control conditions (squares,
Ic) and at maximal ACTH action (inverted
triangles, Imax) are illustrated in Fig. 2B.
They rectified in an outward direction with a reversal potential that,
when compared among different cells, reached a mean value of
-35.83 ± 1.99 mV (n = 6) in asymmetric and +0.16 ±
0.78 mV (n = 5) in symmetric conditions. In symmetric chloride
conditions, the reversal potential was shifted positively by
approximately 36 mV compared with the value obtained in asymmetric
conditions, demonstrating the chloride nature of the investigated
current. Pharmacological evidence of the nature of the current was
further gained by using Cl- current inhibitors.
When applied to the bath medium, SITS (100 µM),
a nonspecific inhibitor of chloride currents (31), induced a rapid
decrease of the ACTH-provoked current (10-10
M) toward control values, as demonstrated in Fig. 2C. The time constant of the phase of decrease of the current was
21 ± 2 sec (n = 3). A second Cl-
channel blocker, DPC (31) was also tested. As shown in Fig. 3C, application of DPC (50
µM) induced a decrease of the current with a
time constant of 46 sec (mean value: 47 ± 1; n = 3).
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, cAMP production was measured as
the percentage of transformation of ATP to cAMP in control conditions
(C) and 15 min after application of ACTH (10-8
and 10-10 M). Under similar
experimental conditions, in single cells, the ACTH-induced increase of
the absolute value of maximal current amplitude, measured at a holding
potential of -50 mV, was not significantly different for
10-10 M and
10-8 M (Fig. 3B). Because of the
lack of correlation between the maximal amplitude of the ACTH-induced
current and cAMP production, two different approaches were used to
investigate a possible role of cAMP in the activation of the chloride
current by ACTH. First, the permeant cAMP analog 8Br-cAMP (1
mM) was added to the bath medium (symmetrical chloride
condition) but no current was activated (Fig. 3C). Later application of
ACTH (10-8 M) triggered a current
which was blocked by DPC (50 µM). Secondly, as shown in
Fig. 3D, forskolin (FSK), applied to the cell in the external medium at
a concentration of 10-6 M, had no
effect (n = 3). As demonstrated in Fig. 3D, when ACTH
(10-8 M) was then applied on the
FSK-treated cell, a current with the same characteristics as in the
absence of the drug was activated. In addition, pretreatment of the
cells with IBMX increased cytoplasmic cAMP concentration induced by
10-8 M ACTH 2.5 fold (data not
shown), but the amplitude of the current was similar in the absence or
in the presence of IBMX (mean amplitude, -3.38 ± 1.05 pA/pF,
n = 3 for 500 µM IBMX).
Phosphorylation is a widely used mechanism for control of protein
activation or inactivation including ionic channels (32, 33). To
determine if phosphorylation could play a role in the ACTH-induced
chloride current, the patch pipette was filled with a solution
containing nonspecific alkaline phosphatase (ALPase, 10 U/ml) (32).
Application of ACTH (10-8 M) in
these conditions gave rise to a current (Fig. 3Eb; mean current
-8.2 ± 1.6 pA/pF; n = 3) that was significantly greater
than the ACTH-control current (Fig. 3Ea). The kinetics of the current
were also affected; specifically, the phase of decrease was slowed down
(Fig. 3Eb). Phosphorylation by the cAMP-dependent protein kinase A
(PKA) was excluded based on the results that the amplitude of the
ACTH-induced current was not affected by the PKA inhibitor H-89 (10
µM, 30 min; mean current: -3.3 ± 1.5 pA/pF; n
= 5).
The ACTH-induced chloride current is activated by the Ras
protein
As recently described, receptors coupled to
Gs protein can activate the Ras/MAPK cascade
through Gß-subunits released from the
Gs-activated protein (34, 35). To see if it could
be the case for the ACTH receptor, we measured
p21ras activity. Using the Ras-binding domain of
Raf (RBD) as a specific trap to selectively precipitate
p21ras only in its GTP bound state, we
demonstrated that application of 10-10
M of ACTH induced a rapid increase in Ras-GTP (Fig. 4). The effect culminated at 5 min, with
a stimulation of 2.1-fold increase over basal level, then decreased at
10 min, but remained elevated even after 30-min incubation.
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-subunits are known to be involved in Ras activation in the
CGRP-coupled receptors (9), we used a peptide scavenger, the QEHA
peptide, which binds to and thus specifically blocks Gß action (3, 36). The QEHA peptide was added to the patch pipette medium at a
concentration of 250 µM to dialyze the cell interior (3).
A 5-min period was allowed after breaking through the patch membrane.
In these conditions, the effect of ACTH (10-10
M) was abrogated. In five separate experiments, ACTH
induced a current of very small amplitude (-0.35 ± 0.11 pA/pF
n = 5; P < 0.05, compared with the control
current) (Fig. 5). To confirm the
involvement of Gß subunit in the signalization to the
Cl- channel, the Gß-subunit (100
nM) was added to the pipette medium. Figure 6A shows the current that was activated.
The I/V curves obtained after breaking in the membrane and at the time
of the maximal effect are illustrated in Fig. 5. The mean amplitude of
the current measured at -50 mV was -1.56 ± 0.68 pA/pF (n =
4). We then looked at the next step which is the activation of the Ras
protein. The Ras protein is synthesized as a cytosolic precursor which
must be anchored at the inner surface of the plasma membrane by a
farnesyl group to be functional (37). Glomerulosa cells were
preincubated with an inhibitor of farnesyl transferase, benzodiazepine
peptidomimetic (BZA) (38), BZA-5B (200 µM),
45 h before experiments. As shown in Fig. 5, the ACTH-induced
current was blunted by BZA-5B treatment. Its increase of -0.81 ±
0.20 pA/pF (n = 7) was lower (P < 0.05) than in
normal conditions. BZA-5B treatment has the drawback that farnesylation
of several proteins could have been impaired. A more specific
inhibition was obtained by using the monoclonal antibody H-Ras (259),
which has been shown to suppress the functional activities of
p21ras (39). Figure 5 shows that the ACTH-induced
current was abolished by addition of the H-Ras (259) (85.5 µg/ml) in
the pipette medium. When heat-inactivated H-Ras (259) (90 C, 10 min)
was added in the pipette instead of the active antibody, ACTH did
activate the Cl- current (n = 3). However,
the amplitude of the ACTH-activated current was less than in control
conditions. This could be due to an incomplete inactivation of the
H-Ras or a residual activity of the inactivated protein.
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S (200 nM of Ras-GTPS). In such conditions, we
observed a transient activation of a current with characteristics
similar to that provoked by ACTH (Fig. 6C). The current activated by
the Ras protein had a mean amplitude of -1.02 ± 0.36 pA/pF
(n = 7) and lasted longer (time constant of decrease 885 ±
197 sec; n = 5) than that provoked by ACTH, but still remained
transient. The I/V curves of the current observed before dialysis of
the cell with Ras (dots) and at maximum of Ras action
(dashes), are represented at Fig. 6D. The observation that
Gß-, Ras-, and ACTH-induced current reversed at similar voltages
suggested that we were dealing with the same Cl-
current. The possibility that the current may depend on Raf-1
activation or downstream kinases was tested. This does not seem to be
the case because ACTH-induced current was not affected when cells were
treated with geldanamycin (mean current: -3.59 ± 0.8 pA/pF;
n = 3), a drug shown to induce a specific decrease in Raf-1
protein levels without any effect on Ras (40). Specific inhibition of
the MAPKK by PD 098059 (41) confirmed that the
Cl- current was not activated by the MAPK as
postulated for the K+ current induced by PACAP 38
(42) (mean current: -3.4 ± 0.98 pA/pF; n = 5). Finally,
because the effect of ACTH on the chloride channel was mediated by Ras,
we tested the effect of BZA on aldosterone secretion. Indeed, a 18
h preincubation with BZA (50 µM) did decrease
aldosterone secretion induced by 10-10
M ACTH but has little effect on that induced by
10-8 M ACTH (Fig. 7).
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subunits transduced the signal. Of
importance, the Cl- current was activated for
low concentrations of ACTH where aldosterone production is not
dependent of cAMP.
In zona glomerulosa cells, ACTH (10-10 and
10-8 M) induced, in
K+-free solutions, a transient increase of a
current with a mean amplitude of approximately -3.5 pA/pF at -50 mV.
The current/voltage relationship presented a rectification in the
outward direction with a reversal potential at -36 mV, characteristic
of value found for Cl- permeable channels. The
ionic nature of the current was further assessed by changing the
Cl- concentration of the internal medium. It
turned out that the value of the reversal potential shifted toward
positive voltage (0.2 mV) according to a Cl-
permeable channel. However, the experimental value in asymmetrical
Cl- concentrations was different from that
calculated with the Nernst equation (-48.2 mV). This could be due to a
poor selectivity of the channel for Cl- ions
(43). Cl- channels can be activated by
extracellular ligands, cAMP, intracellular Ca2+,
voltage, mechanical stretch and swelling, and coupling G proteins (44).
Because the experiments were performed with a medium pipette containing
EGTA, it could be concluded that the Cl- channel
was not activated by cytosolic Ca2+. Moreover, we
have previously shown that ACTH increased cytoplasmic
Ca2+ concentration (13) with slow kinetics not
compatible with that of the ACTH-induced current. Considering that cAMP
is the main second messenger of the ACTH signaling pathway, we
investigated whether it could potentially activate the ACTH-induced
current. The ACTH receptor is coupled to AC through a
Gs coupling protein, and cAMP is thought to be
the main second messenger involved in ACTH-induced secretion, although
a discrepancy between the ACTH thresholds for aldosterone secretion and
cAMP production suggests that other signaling pathways may be involved
(27). In our experiments, we used two concentrations of ACTH,
10-10 M and
10-8 M, corresponding to the
EC50 for aldosterone and cAMP production,
respectively (27). As cAMP-dependent Cl-
channels have been described in numerous cell types, we performed
experiments to determine if this was also the case for the
ACTH-dependent channel. The first observation against a cAMP-dependence
of the current was the lack of correlation between its amplitude and
cAMP production for the two concentrations of ACTH used. As seen in
Fig. 3, A and B, although cAMP production induced by
10-8 M ACTH was six times greater
than that for 10-10 M ACTH, both
current amplitudes were similar. The time-course of the current and
cAMP production were also different: whereas the current amplitude
decreased after 160 sec, cAMP concentration remained at its maximal
level for at least 15 min (data not shown), which is not the
hallmark of a cAMP-dependent Cl- current, as in
the CFTR-induced current, whose amplitude rapidly follows the changes
of cAMP concentration (45). More straightforward arguments were
produced using FSK, a direct activator of adenylyl cyclase or 8Br-cAMP,
a direct activator of PKA. In glomerulosa cells, application of
10-6 M of FSK gave rise to an
increase in cAMP similar to that obtained with
10-9 M ACTH (14). Figure 3, C and D,
shows that no current was triggered by FSK or 8Br-cAMP but a later
application of ACTH (10-10 M)
activated a current. Confirming the results obtained in the presence of
IBMX, the amplitude of the current obtained in these conditions was
comparable to that obtained with ACTH alone. Furthermore, preincubation
of the glomerulosa cells with the PKA inhibitor H-89 (13, 46) was
ineffective on the amplitude of the ACTH-induced current, which is at
odds with the observation with the PKA-regulated CFTR-channel (47).
Moreover, the activity of the CFTR channel was blocked by the
nonspecific ALPase (48), whereas we found that ALPase increased the
amplitude of the ACTH-induced current. Modulation of channel activity
by protein kinase-dependent phosphorylation has been widely reported
(33), but the effects of this modification can vary, depending on cell
and channel types. Our findings support the hypothesis that
phosphorylation results, in part, in the inhibition of the ACTH-induced
current, whereas its rise stems from the activation of a different
pathway that does not involve cAMP.
Our results are the first to demonstrate that Ras is activated by ACTH,
in primary cultures of glomerulosa cells. This could ultimately leads
to the activation of MAPK pathway as demonstrated for ACTH in Y1
adrenal cells (49) or for several receptors coupled to AC through
Gs (9). However, recent studies from our group
have demonstrated that ACTH did not activate ERK1/ERK2 (50). Moreover,
aldosterone secretion induced by low concentrations of ACTH is
sensitive to Ras inhibition, but not to MEK1 inhibition (50). These
observations corroborate present results showing that the ACTH-induced
current was not sensitive to geldanamycin and PD98059. In contrast, we
found that ACTH-induced Cl- current was
abolished when the QEHA peptide, a Gß subunit sequestrant (3, 36),
was added to the pipette medium. Our observations indicate that the
Gß subunit released from the G protein coupled to the ACTH
receptor is involved in the signaling pathway from the ACTH receptor to
ACTH-induced Cl- current. This was further
strengthened by the activation of the current by Gß added in the
pipette medium. However, the Cl- current could
be directly triggered by Gß subunits (1) or by a downstream
effector such as Ras. Two results demonstrated that Ras, activated by
ACTH, was directly involved in the ACTH-induced current. First, the
ACTH-induced current was abolished when Ras activation was blocked in
glomerulosa cells pretreated with the permeant benzodiazepine
peptidomimetic (BZA-5B), an inhibitor of farnesyl transferase (38) or
with the H-Ras (259) antibody added in the pipette medium (51).
Secondly, the addition of the Ras-GTPS form in the pipette medium
resulted in the activation of a current that displayed characteristics
similar to the ACTH-induced current. Taken altogether, our results
point to the role of Ras in the activation of the ACTH-induced
Cl- current and open the possibility of a new
effector for Ras.
It has been shown that the Raf-1 protein kinase is a direct downstream effector of Ras. However, other effectors of Ras have been discovered including the phosphatidylinositol 3-kinase, RalGDS, a GEF for Ral, a RalGDS-related protein and Rin1, a protein of unknown function (see Ref. 52). Recently, a role for Ras signaling has been observed in synaptic transmission and long-term memory (53) as well as in regulation of rat neuronal voltage-dependent Ca2+ channels (51). It is thus conceivable to postulate that Ras may directly or indirectly interact with the Cl- channel. Protein(s) such as the Rho/Rac p21 GTPases involved in morphogenesis and cytoskeletal organization (see (54)) could be considered as putative intermediates between Ras and the Cl- channel. For example, microinjection of Rho into Swiss 3T3 fibroblasts induced the rapid formation (1–2 min) of stress fibers and focal adhesion formation (55), and activation of a Cl- current, distinct from the Cl- current activated by swelling, CA2+ or cAMP. This activation paralleled the Rho-mediated cytoskeletal changes also described in this cell type (56). Recent work by our group (14) has shown that in glomerulosa cells, ACTH induced a rapid (within 1 min) redistribution of actin fibers at the membrane level, which decreased to control levels after 15 min. The mechanism of this effect, necessary for ACTH-induced secretion of steroid (14), is not yet understood, but the cascade Ras-Rho-actin could be involved, as well as the ACTH-induced current. Chloride channels play a pivotal role in cell volume regulation, stabilizing membrane potential and transepithelial transport (57). The ACTH-induced Cl- current could be involved at several levels. Firstly, glomerulosa cells are particularly sensitive to membrane depolarization, which allows the opening of voltage-dependent Ca2+ channels and an increase in cytostolic Ca2+ concentration (13). Activation of a Cl- current, depending on the value of the equilibrium potential of the chloride system, leads to a change in the membrane potential. In zona glomerulosa, the depolarization of the membrane observed upon ACTH application (ACTH, 10-10 M) (58), where the outward K+ current is not blocked (19), can be due to the ACTH-induced Cl- current. Indeed, Cl--mediated depolarization has been observed in fibroblasts (56) and smooth muscle cells (59). Secondly, the efflux of Cl- ions from the cell upon activation of the ACTH-induced current could have a stimulating effect on the Steroidogenic Acute Regulatory protein [StAR; (60)] as demonstrated on LH-induced testosterone production in Leydig cells (23, 61).
In conclusion, we have demonstrated the presence of a Ras-dependent Cl- current in glomerulosa cells. This current is activated by low concentrations of ACTH below the threshold for cAMP production but above that for aldosterone production. It may play a role in the previously described cAMP-independent aldosterone production (27, 50).
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Acknowledgments |
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Footnotes |
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Received May 13, 1999.
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References |
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), induced by ACTH 10-10 M
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