ACTH-induced Clminus current in bovine adrenocortical cells: correlation with cortisol secretion

Sylvie Dupré-Aucouturier1, Armelle Penhoat2, Oger Rougier1, and André Bilbaut1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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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 (10-4 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 MOmega 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 MOmega . 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/Delta V where I was the injected current and Delta 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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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, was -63 ± 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 GOmega , 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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Fig. 1.   ACTH-induced membrane depolarization associated with increase in membrane conductance. A: original recording showing the effect of ACTH on the membrane potential of an isolated cell. Brief hyperpolarizing deflections of the membrane potential superimposed on the voltage trace were evoked in this and the other recordings by current pulses of 10 pA injected for 2 s every 10 s. C, control solution. B: this graph drawn from the recording illustrated in A established quantitative correlations between membrane potential (left axis, ) and membrane conductance (right axis, open circle ) 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.

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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Fig. 2.   ACTH-induced membrane depolarization associated with decrease in membrane conductance. A: initial depolarizing phase (left axis, ) induced by ACTH is clearly correlated with a transient decrease of the membrane conductance (right axis, open circle ); compare with Fig. 1B. B: ACTH-induced membrane depolarization (left axis, ) correlated with a continuous decrease in membrane conductance (right axis, open circle ).



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Fig. 3.   ACTH-induced membrane depolarization in Cs+-loaded cells. A: original recording showing the beginning of an ACTH-induced membrane depolarization obtained after dialysis of the inner cell compartment by Cs+. An initial spike that peaks at about -20 mV (arrowhead) characterizes this response. For more clarity, the first hyperpolarizing potentials evoked by the injection of current pulses have been truncated in this recording. B: this graph drawn from the recording shown in A illustrates the change of both the membrane potential (left axis, ) and the membrane conductance (right axis, open circle ) during the exposure of the Cs+-loaded cell to ACTH.

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 -32 ± 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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Fig. 4.   cAMP-dependent membrane depolarization. A: forskolin (FSK; 10 µM), an activator of adenylyl cyclase, induces a membrane depolarization associated with membrane conductance changes comparable to those observed in the presence of ACTH (see Fig. 1A). B: membrane depolarization induced by 8-(chlorophenylthio)-cAMP (8-pcpt-cAMP; 8 mM), a membrane-permeant form of cAMP. Note the slow time course of the depolarizing phase (compare with Figs. 1A and 4A) and the membrane conductance decrease that accompanies the membrane depolarization.

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 -30 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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Fig. 5.   Effect of Cl- current inhibitors on the depolarizing plateau. A: slow membrane repolarization produced by 1 mM diphenylamine-2-carboxylic acid (DPC) applied during the ACTH-induced depolarizing plateau potential. This reversible effect was correlated with a progressive decrease in membrane conductance. B: effect of DIDS (400 µM) on the depolarizing plateau potential. At this concentration, DIDS incompletely repolarized the cell membrane.

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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Fig. 6.   Effect of the holding potential on the ACTH-induced membrane current. Whole cell membrane current activated by application of ACTH is inward from a holding potential of -60 mV (A) and outward from a holding potential of -10 mV (B).



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Fig. 7.   Current-voltage relationships of the ACTH-induced membrane current. A: current-voltage relationships in control conditions () and at the maximum of ACTH-activated membrane current (open circle ) recorded from a holding potential of -10 mV. Inset: original traces of the total membrane current obtained in control conditions (top recordings) and during application of ACTH (bottom recordings). The cell membrane was stepped from -100 to +40 mV during 75 ms by 20-mV increments. B: current-voltage relationships of the ACTH-induced membrane current obtained by subtracting procedures from the current-voltage curves illustrated in A. This curve displays outward rectification and intercepts the voltage axis at -21.5 ± 2 mV (n = 6).

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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Fig. 8.   Effect of low external Cl- solution on the ACTH-induced outward current. The membrane current activated by ACTH from a holding potential of -10 mV is outward in control solution (140 mM Cl-) and inward when the cell was briefly exposed to deficient Cl- solution (14 mM).

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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Fig. 9.   Effects of different Cl- channel inhibitors of the ACTH-induced outward current. A: blocking effects of the ACTH-induced outward current (holding potential, -10 mV) by DPC (1 mM), anthracene-9-carboxylic acid (9-AC; 1 mM), and DIDS (400 µM). Control holding current, +8 pA. B: blocking effects of the ACTH-induced outward current obtained in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 100 µM) and DIDS (100 µM). Control holding current: +10 pA.

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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Fig. 10.   Ca2+-independence of the ACTH-induced membrane current. A: DIDS-sensitive inward current activated by ACTH from a holding potential of -60 mV in the presence of 5 mM EGTA in the pipette solution; standard patch recording. Control holding current, -12 pA. B: DIDS-sensitive inward current activated by ACTH from a holding potential of -90 mV in the absence of external Ca2+ replaced by 20 mM Ba2+.

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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Fig. 11.   Inhibition of the ACTH-induced cortisol secretion by Cl- current blockers DIDS and DPC. This graph is representative of 3 series of experiments performed from 3 different cell dissociations. Error bars correspond to the mean of measurements effected in 4 wells for each experiment. Results are expressed for 106 cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACTH-Induced Cl- Current

This study reveals that ACTH activates a Cl- 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 Gbeta gamma 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

Various types of Cl- 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.

On the other hand, during the ACTH-induced depolarizing phase that precedes the plateau phase, large membrane potential changes could be associated with very weak changes in membrane conductance. Because the resting membrane conductance of these cells is low (~0.3 nS), a quasi-undetectable increase in membrane conductance caused by ACTH-activation of a few Cl- 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.


    REFERENCES
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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[ISI][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[Abstract/Free Full Text].

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[Abstract].

10.   Gomora, JC, and Enyeart JJ. Dual pharmacological properties of a cyclic AMP-sensitive potassium channel. J Pharmacol Exp Ther 290: 266-275, 1999[Abstract/Free Full Text].

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[ISI][Medline].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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[ISI][Medline].

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[Abstract/Free Full Text].

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[ISI][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[Abstract].

28.   Valverde, MA, Hardy SP, and Sepulveda FV. Chloride channels: a state of flux. FASEB J 9: 509-515, 1995[Abstract/Free Full Text].

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[Abstract/Free Full Text].

30.   Xu, L, and Enyeart JJ. Properties of ATP-dependent K+ channels in adrenocortical cells. Am J Physiol Cell Physiol 280: C199-C215, 2001[Abstract/Free Full Text].

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[ISI][Medline].

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].


Am J Physiol Endocrinol Metab 282(2):E355-E365
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society




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