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J Am Soc Nephrol 12:1805-1818, 2001
© 2001 American Society of Nephrology

Basolateral Translocation by Vasopressin of the Aldosterone-Induced Pool of Latent Na-K-ATPases Is Accompanied by {alpha}1 Subunit Dephosphorylation: Study in a New Aldosterone-Sensitive Rat Cortical Collecting Duct Cell Line

SABRI DJELIDI, AHMED BEGGAH, NATHALIE COURTOIS-COUTRY, MICHEL FAY, FRANCOISE CLUZEAUD, SAY VIENGCHAREUN, JEAN-PIERRE BONVALET, NICOLETTE FARMAN and MARCEL BLOT-CHABAUD

INSERM U478, Institut Fédératif de Recherches "Cellules Epithéliales," Faculté de Médecine Xavier Bichat, Paris, France.

Correspondence to Marcel Blot-Chabaud, INSERM U478, Faculté de Médecine Xavier Bichat, BP 416, 75870 PARIS, Cedex 18, France. Phone: 331-44856325; Fax: 331-42291644; E-mail: chabaud{at}bichat.inserm.fr .


   Abstract
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 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Abstract. The regulation of plasma membrane Na+-K+-ATPases (NKA) expression by aldosterone and arginin vasopressin (AVP) in the cortical collecting duct (CCD) has been examined in a new rat CCD cell line, designated as RCCD2. This cell line has maintained many characteristics of the CCD—in particular, the expression of the mineralocorticoid receptor. Mineralocorticoid receptor is expressed at the protein level and binds 3H-aldosterone (approximately 15 to 20 fmol/mg protein). Short-circuit current (Isc) experiments showed approximately a twofold increase in Isc associated with a decrease in transepithelial resistance when cells were treated with aldosterone concentrations as low as 10-9 M. This effect on Isc was significant 2 h after aldosterone addition and was still present after 24 h. It was accompanied by an increase in the amount of mRNA encoding for the {alpha} subunit of the epithelial sodium channel (sixfold) and the {alpha}1 subunit of NKA (fourfold) after 24 h of hormone treatment. In addition, mRNA expression of the serum- and glucocorticoid-induced kinase (Sgk) was increased by 10-9 M aldosterone treatment as early as 45 min after hormone addition. As had already been documented in native CCD obtained by microdissection, incubation of RCCD2 cells for 24 h with aldosterone resulted in the constitution of a latent pool of NKA that could be rapidly recruited by AVP (15 min). NKA biotinylation experiments and preparation of membrane fractions show that this latent pool of NKA is present in the intracellular compartment of the cells and is recruited by AVP in the basolateral membrane through a translocation process. This mechanism is accompanied by dephosphorylation of the {alpha}1 catalytic subunit of NKA.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The cortical collecting duct (CCD) is an important site of control of sodium and potassium homeostasis (1,2). It involves ion transporters such as the Na+-K+-ATPase (NKA) located in the basolateral membrane of the cells, the epithelial sodium channel (ENaC) in the apical membrane, and different potassium channels present in both membranes (3). NKA plays a central role because it generates the electrochemical gradients that will be used for sodium absorption and potassium secretion. Its activity is under the control of several hormones—in particular, aldosterone and vasopressin (4,5). It was shown several years ago that aldosterone is required for the constitution of a latent pool of NKA (6,7). These latent pumps are rapidly recruited and/or activated in the basolateral membrane of the cells in response to an increase in sodium entry (6,8), to an increase in cell volume (9), or to arginin vasopressin (AVP) (10), which leads to a synergistic effect of the two hormones. The precise mechanism of this recruitment and/or activation is still unknown. It has been shown (11) that this could involve the activation by AVP of a specific protein phosphatase, namely PP2A. The cellular localization of the latent pool of NKA is also unknown. It may be either intracellular or already present in a inactive form in the membrane. In the first hypothesis, AVP could induce the recruitment of this pool from intracellular stores, as has been already described for Na+ channels or aquaporin 2 (12,13). In the second hypothesis, latent pumps might be activated in situ by a mechanism that remains to be defined.

The goal of this study was to determine the localization of this latent pool of NKA and to give new insights into the mechanism that leads to the rapid increase in NKA activity observed after the addition of AVP. To this end, we used a subclone of the RCCD1 rat CCD cell line (14), referred to as RCCD2. This new cell line has the advantage of expression of the mineralocorticoid receptor (MR; together with the glucocorticoid receptor [GR]) and of increasing its sodium transport after exposure to low doses of aldosterone. Results indicate that in the presence of aldosterone, RCCD2 cells are able to constitute a reserve pool of NKA. This pool of NKA is located in the intracellular compartment and is rapidly recruited to the basolateral membrane of the cells after stimulation with AVP. This phenomenon is accompanied by the dephosphorylation of the {alpha}1 subunit of NKA.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Establishment of the RCCD2 Rat CCD Cell Line
The previously established rat CCD cell line RCCD1 (14) was subcloned after 20 passages of culture in a complete medium that contained Dulbecco's modified Eagle's medium/Ham's F12 1:1 14 mM, NaHCO3, 2 mM glutamine, 5 x 10-8 M dexamethasone, 5 x 10-8 M sodium selenite, 5 mg/ml transferrin, 5 mg/ml insulin, 10 mg/ml epidermal growth factor, 5 x 10-8 M T3, 10 U/ml penicillin-streptomycin, 2% fetal bovine serum (Life Technologies, Cergy Pontoise, France), and 20 mM Hepes (pH 7.4). One of the subclones exhibited a high number of domes, compared with that of RCCD1 cells. Cells from this clone then could be cultured up regularly to passage 40; we referred this cellular clone to as RCCD2. For experiments, cells were seeded on either Transwell or Snapwell filters (Costar Corp., D. Dutscher, Brumath, France) or on Petri dishes previously coated with collagen (Institut J. Boy, Reims, France).

Morphologic Studies
Confluent RCCD2 cells grown on collagen-coated Petri dishes were photographed by use of an inverted microscope equipped with a phase contrast device (Axiovert 10; Zeiss, Esslingen, Germany). For transmission electron microscopy, ultrathin sections were performed on transversally oriented confluent monolayers grown on collagen-treated transwell filters, as described previously (14). Briefly, cells first were rinsed with phosphate-buffered saline (PBS) and then fixed for 1 h with 2.5% glutaraldehyde in PBS at room temperature. Cells then were washed in PBS, postfixed with 1% osmic acid for 15 min, dehydrated in graded series of ethanols, and embedded in Epon. They then were examined with a Philips EM 410 electron microscope (Eindhoven, The Netherlands). Experiments of staining with dolichos biflorus agglutinin (DBA) were performed as described previously (14).

Electrophysiologic Studies
The measurement of short-circuit current (Isc; µA/cm2), transepithelial voltage (VT; mV), and transepithelial resistance (RT; {Omega} x cm2) was performed on RCCD2 cells grown on collagen-coated Snapwell filters, as described previously (14). Briefly, Snapwell filters were mounted into a voltage clamp system (Costar Corp.), and cells were bathed on each side with 8 ml of medium thermostated at 37°C and circulated by a gaslift (95% O2 and 5% CO2). Isc was measured by clamping VT to 0 mV for 1 s, and RT was calculated from current deflection in response to a -5/+5 mV modification of VT. For studying the effect of hormones on Isc, RCCD2 cells grown on Snapwell filters for several days in the complete medium first were incubated overnight in a minimum medium (MM) containing Dulbecco's modified Eagle's medium/Ham's F12 1:1, 14 mM NaHCO3, 2 mM glutamine, 10 U/ml penicillin-streptomycin, and 20 mM Hepes (pH 7.4). Isc experiments then were performed with the use of this MM as experimental medium.

Northern Blot Experiments
Total RNA (10 to 20 µg) extracted from cells under different experimental conditions were run on a 0.8% denaturing glyoxal agarose gel and blotted onto nylon membranes (Hybond-N; Amersham, Orsay, France). Membranes then were hybridized with random-primed {alpha}32P-dCTP-labeled probes for rat {alpha} ENaC (590 bp; nt 2185 to 2775), NKA {alpha}1 (832 bp; nt 1189 to 2021), NKA ß1 (687 bp; nt 913 to 1600), serum and glucocorticoid-induced kinase (Sgk; 671 bp; nt 314 to 985), and glyceraldehyde phosphate dehydrogenase (851 bp; nt 20 to 871).

Immunoprecipitation and Western Blot Experiments
Immunoprecipitation experiments were performed as described previously (15) on RCCD2 cells previously labeled for 24 h with 37.5 MBq/ml 35S-methionine (37.5 Bq/mmol; Amersham) for MR detection or on unlabeled cells for GR detection. In brief, cells were scraped off the filters and extracted in an ice-cold lysis buffer. Protein extracts were precleared with a Staphylococcus aureus slurry (pansorbin; Calbiochem, Darmstadt, Germany) before incubation overnight at 4°C under end-over-end rotation with antibodies directed against MR (N-17; Santa Cruz Biotechnology Inc., Santa Cruz, CA) or GR (M-20; Santa Cruz Biotechnology Inc.) in the absence or presence of the immunizing peptides (Santa Cruz Biotechnology Inc.). Immunoprecipitates then were incubated with protein A-Sepharose beads (CL-4B; Pharmacia Biotech Inc., Orsay, France) at 4°C for 1 h. Samples of eluted immunoprecipitates were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 7.5%) by use of the Laemmli buffer system before drying and auto-radiography (cells labeled with 35S-methionine) or transfer onto a polyvinylidene difluoride (PVDF) membrane (Amersham) (for unlabeled cells). This membrane then was pretreated overnight in a 5% milk-Tris buffer saline Tween solution, then incubated with GR (1:10,000; 1 h) antibody, followed by incubation with a second goat antibody conjugated to peroxidase (Santa Cruz Biotechnology Inc.; 1:10,000; 1 h). Proteins were visualized by use of enhanced chemiluminescence (ECL) or ECL+ detection kit (Amersham).

For Western blot experiments aimed at detecting the different subunits of ENaC, {alpha}1 NKA, 11ß-hydroxysteroid dehydrogenase of type 2 (11-HSD2), DBA binding sites, and ß-actin, protein extracts were submitted directly to SDS-PAGE, and PVDF membranes were pretreated overnight in 5% milk-Tris buffer saline Tween before incubation with rabbit anti-{alpha}, -ß, or -{gamma} ENaC antibodies (1:10,000; 1 h) (15), anti-{alpha}1 NKA antibody (1:50,000; 30 min), rabbit anti-HSD2 antibody (Neosystem, Strasbourg, France; 1:10,000; 1 h), DBA (Vector; 1:2500; 1 h), and then goat anti-DBA antibody (Vector, Burlingame, CA; 1:10,000; 1 h) or mouse anti-ß actin antibody (Santa Cruz Biotechnology Inc.; 1:10,000; 1 h). The antibody anti-{alpha}1 NKA was generated in the laboratory as described by Girardet et al. (16) and characterized by Western blot and immunolocalization in the rat kidney (data not shown). Membranes then were incubated with peroxidase-conjugated second antibody (1:10,000; 1 h), and proteins were visualized by use of ECL or ECL+ detection kit.

3H-Steroid Binding Experiments
Specific binding of 3H-aldosterone and 3H-dexamethasone was determined in RCCD2 cells grown on 12-mm diameter transwell filters. In a first series of experiments, cells were grown in complete medium before incubation overnight in MM. Then cells were preincubated or not with 10-6 M of the GR antagonist RU486 or 10-6 M of the MR antagonist RU26752 (Roussel Uclaf, Romainville, France) for 1 h before addition of 10-8 M 3H-aldosterone (52 Ci/mmol; Amersham) or 2 x 10-8 M 3H-dexamethasone (42 Ci/mmol; Amersham) for 1 h at 37°C to measure total binding. Nonspecific binding was determined in the same conditions, except that an excess of 1000-fold cold aldosterone or dexamethasone was added. Cells then were rinsed rapidly at 4°C in PBS before steroid extraction with ethanol for 1 h at room temperature. Radioactivity of the ethanol extracts was measured with a liquid scintillation ß counter (Wallac; Pharmacia). Protein content of the cells was assayed by the Bradford method. Specific 3H-steroid binding was determined from the difference between total and nonspecific binding and was expressed as fmol/mg protein. In a second series of experiments aimed at determining the apparent Kd and the Nmax for MR and GR in this cell line, cells were incubated in the conditions described above, except that different concentrations of 3H-aldosterone (0.05 to 1 nM) and 3H-dexamethasone (0.1 to 2.5 nM) ranging over the described Kd were used. Both the total and the nonspecific bindings were determined. The specific 3H-steroid binding was obtained from the difference between total and nonspecific bindings, expressed as fmol/mg protein, and the apparent Kd and Nmax for each receptor was evaluated by Scatchard plots representation and analysis of the regression lines, considering the presence of two sites of binding with high and low affinity for aldosterone and one site of binding for dexamethasone.

Biotinylation of Basolateral NKA Experiments
Biotinylation of basolateral membranes was used to estimate changes in pool size of the plasma membrane sodium pumps. Although this method may not label a subset of sodium pumps located in deep basal infoldings, it is specific for surface pumps and will not label intracellular pumps, so changes in biotinylated pumps likely will reflect changes in the total pool size of the plasma membrane sodium pumps. The protocol was similar to that used by Gottardi et al. (17). Briefly, cells were plated on 24-mm transwell filters. Basolateral membrane proteins were biotinylated by incubation for 1 h with NHS-ss-biotin 1.5 mg/ml (Pierce, Rockford, IL) at 4°C. Then two different protocols were used. In a first series of experiments, cells were rinsed and scraped before addition of packed streptavidin-agarose beads (Pierce). Proteins were eluted from the beads in a sample buffer containing 1% SDS (0.15 M NaCl, 5 mM ethylenediaminetetraacetate, 1% Triton x 100, and 50 mM Tris [pH 7.5]) before 7.5% SDS-PAGE. The gel was transferred onto a PVDF membrane and treated for a Western blot experiment. The membrane was pretreated overnight in a 5% milk-Tris buffer saline Tween solution before incubation with an antiserum raised against the {alpha}1 subunit of NKA (1:50,000 at room temperature for 30 min) and subsequent incubation with peroxidase-conjugated anti-rabbit antibody (1:10,000; 1 h). The signal was visualized with ECL or ECL+ (Amersham) and was quantified by use of an Instant Imager. Results were normalized to the signal obtained by Western blot of ß-actin (anti-ß-actin antibody; 1:10,000; 1 h) in the same cellular samples. In a second series of experiments, after biotinylation of basolateral proteins, the total NKA were immunoprecipitated as described above. After SDS-PAGE and transfer onto a PVDF membrane, total NKA or biotinylated NKA were revealed by use of the anti-{alpha}1 antibody or an anti-biotin antibody (Pierce; 1:10,000; 1 h), respectively. After incubation with a peroxidase-conjugated antibody, proteins were visualized by use of ECL or ECL+ detection.

32P Phosphorylation and Immunoprecipitation of NKA Experiments
RCCD2 cells grown on 24-mm transwell filters were labeled for 24 h at 37°C with 250 µCi/ml [32P]orthophosphate (NEN Life Science Products, Boston, MA) in MM added with 10-8 M aldosterone. Then cells were treated or not for 15 min with 10-8 M AVP before biotinylation of the basolateral membrane, as indicated above. Biotinylated proteins were separated from intracellular proteins by use of streptavidin-agarose beads as described above. Both biotinylated and intracellular proteins were submitted to immunoprecipitation with the NKA anti-{alpha}1 antibody (see above). Then samples were run on SDS-PAGE before drying, autoradiography, and quantification of 32P with an Instant Imager.

Preparation of Membrane Fractions
Experiments were performed according to the "sequential fractionation protocol." In this protocol, different spins were done in series to progressively separate cellular membrane components into progressively smaller or less-dense membrane fragments, including membrane vesicles (microsomes), which pellet with high-speed spins, and plasma membranes, which pellet at lower speeds (18). Briefly, RCCD2 cells were grown on 24-mm transwell filters and treated for 24 h with 10-8 M aldosterone. Then they were treated or not for 15 min with 10-8 M AVP. In each condition, cells from four filters were scraped in 10 ml of an ice-cold solution containing 250 mM sucrose, 10 mM triethanolamine, 0.1 mg/ml phenylmethylsulfonyl fluoride, and 1 µg/ml protease inhibitors (Sigma Chemical Co., St. Louis, MO) and homogenized by use of a tissue homogenizer (Ika Labortechnik, Staufen, Germany), as described previously (19,20,21,22). Then the homogenates were initially spun at low speed (800 x g) for 10 min to pellet incompletely homogenized fragments and nuclei. The supernatants were spun at 17,000 x g for 20 min at 4°C in a Beckmann L8-M ultracentrifuge to obtain a membrane fraction enriched in plasma membrane (low-speed fraction [LS]). The supernatant from this centrifugation was then spun at 200,000 x g for 1 h at 4°C to obtain intracellular membranes (high-speed fraction [HS]).

3H-Ouabain-Binding Experiments
The number of NKA present in the basolateral membrane of RCCD2 cells was determined by use of the 3H-ouabain binding technique. Briefly, RCCD2 cells were grown on collagen-treated Transwell filters and treated by aldosterone and/or AVP, depending on the experiments. The basolateral medium was then replaced twice at 4°C with a sucrose solution containing (in mM) 250 sucrose, 0.8 MgSO4, 5 glucose, 1 MgCl2, 1 CaCl2, 1 alanine, and 20 Hepes/Tris (pH 7.4). Then, cells were incubated at 37°C for 15 min in the sucrose solution to which 5 x 10-5 M 3H-ouabain was added basolaterally (15 to 30 Ci/mmol; Du Pont-New England Nuclear, Boston, MA). This was performed both in the absence and in the presence of a 100-fold excess of unlabeled ouabain, to determine total and nonspecific 3H-ouabain binding, respectively. At the end of the incubation time, cells were rinsed three times at 4°C with the sucrose solution, then incubated for 1 h in this solution at 4°C (to reduce nonspecific binding) before new rinsing. The radioactivity corresponding to each filter was counted with a liquid scintillation beta counter (Wallac; Pharmacia). When 3H counts were used, specific 3H-ouabain binding was calculated and expressed as nmoles/cm2 culture area. To estimate the amount of 3H-ouabain that binds to intracellular NKA because of ouabain diffusion into the cells during the 15-min incubation time at 37°C, RCCD2 cells were treated as described and then submitted to a membrane fractionation (see above). Radioactivity was then counted in the different fractions. About 6% of the total radioactivity was found in the 800 x g fraction, 13% in the 200,000 x g fraction, and 81% in the 17,000 x g fraction. These results indicate that almost all cells are homogenized during the fractionation procedure and that 3H-ouabain binds essentially to NKA present in the plasma membrane of the cells.

Statistical Analyses
Results are expressed as mean ± SEM. Statistical analysis was performed by use of the t test for unpaired data after ANOVA and correction for multiple comparisons.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Morphologic Characteristics of RCCD2 Cells
When grown on Petri dishes, RCCD2 were organized as closely apposed epithelioid cells and formed numerous domes (Figure 1, A and B). When grown on filters, RCCD2 formed monolayers of cells separated by junctional complexes (Figure 1, D and E). Staining of RCCD2 cells with DBA, which essentially binds to the apical membrane of principal cells in the rat CCD (23), shows that about 50% of the cells are labeled (Figure 1C). This suggests, as in RCCD1 cells (14), the presence of both principal and intercalated cells in cultured RCCD2 cells.



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  		Figure 1. Morphologic features of rat cortical collecting duct (RCCD2) cells. When grown on Petri dishes, RCCD2 cells were organized as layers of epithelioid-shaped cells and formed domes (A, B). When grown on filters, RCCD2 cells grew as a monolayer and were separated by tight junctions (D, E). Dolichos biflorus agglutinin binds approximately 50% of the cells, which suggests that both principal and intercalated cells are present (C). Bars: (A) 250 µm; (B) 50 µm; (C) 10 µm; and (D and E) 1 µm.

 

RCCD2 Cells Express Both MR and GR
The two types of corticosteroid receptors, MR and GR, were detected in RCCD2 cells. Immunoprecipitation experiments revealed MR at about 110 kD. The observed band was displaced when immunoprecipitation was performed in the presence of the immunizing peptide (Figure 2A). GR is also detected in RCCD2 cells at the protein level, with a 90- to 95-kD band totally displaced in the presence of the immunizing peptide (Figure 2B). Figure 3A shows the specific binding of 3H-aldosterone and 3H-dexamethasone observed when RCCD2 cells were preincubated or not with the GR antagonist RU486 or with the MR antagonist RU26752. The specific bindings of 3H-aldosterone and of 3H-dexamethasone were significantly decreased when cells were preincubated with RU486 or RU26752, respectively, a result that is in favor of the presence of both types of receptors in RCCD2 cells. Experiments of specific 3H-aldosterone and 3H-dexamethasone binding at different concentrations, followed by Scatchard plots analysis, allowed us to estimate that RCCD2 cells express about 15 to 20 fmol of MR per mg protein, with an apparent Kd for aldosterone of approximately 1.7 10-10 M and approximately 80 to 90 fmol of GR per mg protein, with an apparent Kd for dexamethasone of approximately 10-9 M (Figure 3, B and C).



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  		Figure 2. Expression of the mineralo- (MR) and glucocorticoid receptors (GR) in RCCD2 cells. (A) Detection of the MR by immunoprecipitation with an anti-MR antibody (MR Ab) after 35S-labeling of the cells. MR is detected at approximately 110 kD. The band was totally displaced when immunoprecipitation was performed in the presence of the immunizing peptide (MR Ab+pept). (B) Detection of the GR by immunoprecipitation with an anti-GR antibody (GR Ab), followed by a blot with the same antibody. GR is detected in RCCD2 cells at the protein level with a 90- to 95-kD band totally displaced when immunoprecipitation is performed in the presence of the immunizing peptide (GR Ab+pept).

 


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  		Figure 3. 3H-steroid binding in RCCD2 cells. (A) The specific binding of 10-8 M 3H-aldosterone and 2 x 10-8 M 3H-dexamethasone was evaluated in RCCD2 cells by difference between the total binding obtained with the 3H-steroid and the nonspecific binding obtained with the 3H-steroid and a 1000-fold excess unlabelled steroid. Experiments were performed in the absence or presence of 10-6 M RU486 and 10-6 M RU26752. **, P < 0.001. (B and C) Dose dependency and Scatchard plots of specific 3H-aldosterone and 3H-dexamethasone bindings in RCCD2 cells grown on porous substrate. Different concentrations of 3H-aldosterone (0.05 to 1 nM) and 3H-dexamethasone (0.1 to 2.5 nM) were used. From these experiments, the apparent Kd and the Nmax were estimated for both types of receptor (see Results section).

 

RCCD2 Cells Exhibit an Aldosterone-Sensitive Isc
When grown on filters in a complete medium (see Materials and Methods section), RCCD2 cells exhibited a high transepithelial resistance and ionic transport, as demonstrated by an associated Isc. In these conditions, the electrophysiological values obtained were Isc = 2.91 ± 0.47 µA cm2; VT = -5.95 ± 0.82 mV lumen negative; and RT = 2100 ± 150 {Omega} x cm2. For examining the effect of aldosterone on ion transport, RCCD2 cells grown on Snapwell filters in complete medium were first incubated overnight in MM. After a control period of 1 h, 10-8 M aldosterone was added or not to medium bathing both the apical and basolateral sides of the cells. Results are shown in Figure 4, A and B. In the absence of hormone, Isc and RT remained constant along the 5 h of the experiment. In contrast, in the experiments in which aldosterone was added, RT decreased as soon as 30 min after addition of the hormone and then remained low. Isc was significantly increased, compared with control cells, 90 min after hormone addition and then remained elevated for at least 24 h. For examining the specificity of the response to 10-8 M aldosterone, Isc experiments were performed in the presence of specific antagonists of the MR (RU26752) or GR (RU486) (Figure 4C). In these experiments, cells first were preincubated for 1 h with the antagonist (10-6 M) before addition of 10-8 M aldosterone and antagonist for 24 h. Incubation of RCCD2 cells for 24 h with 10-8 M aldosterone resulted in a twofold increase in Isc. The presence of 10-6 M RU26752 blocked the aldosterone effect, whereas addition of 10-6 M RU486 did not modify it, which suggests a specific MR-mediated effect at 10-8 M aldosterone. It is surprising that both RU486 and RU26752 added alone modestly but significantly increased Isc, which suggests a partial agonist activity of these compounds when used at relatively high concentration. The dose dependency of aldosterone, RU486, and RU26752 effects on Isc are shown in Figure 4D. Isc was significantly increased from 10-9 to 10-5 M aldosterone, where a fourfold increase in Isc was observed, compared with control cells without aldosterone. In addition, a low but significant agonist activity of RU486 and RU26752 was effectively observed on Isc at 10-6 to 10-5 and 10-5 M, respectively. This partial agonist activity of antagonist-occupied steroid receptors has already been described (24).



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  		Figure 4. Effects of aldosterone on short-circuit current (Isc) and transepithelial resistance in RCCD2 cells. The effect of 10-8 M aldosterone was tested on both Isc (A) and transepithelial resistance (RT) (B) and compared with cells in control condition. Whereas no modification was observed on Isc or RT in control condition, the addition of aldosterone (arrow) resulted in a rapid decrease in RT and an associated increase in Isc. RT was significantly decreased as soon as 30 min after the addition of aldosterone, whereas Isc was significantly increased after 2 h. Each point is the mean ± SEM from five experiments. *, P < 0.05; #, P < 0.001, 10-8 M aldosterone versus control. (C) Mineralocorticoid specificity of the response to 10-8 M aldosterone. The effect of 24 h of treatment with 10-8 M aldosterone was tested on Isc after incubation of the cells in the presence of 10-6 M RU26752 and of 10-6 M RU486 (each bar gives the mean ± SEM from six experiments). Cells were preincubated with the antagonist for 1 h before treatment or not with aldosterone. *, P < 0.05; §, P < 0.01; #, P < 0.001. (D) Dose dependency of aldosterone, RU486, and RU26752 effects on Isc. The dose dependency was evaluated after 24 h of treatment with the compound. Isc was significantly increased from 10-9 to 10-5 M aldosterone. Isc was weakly but significantly increased with 10-6 and 10-5 M RU486 and 10-5 M RU26752. Results are given as mean ± SEM from three to five experiments. *, P < 0.05; §, P < 0.01; #, P < 0.001 experimental versus control without the compound.

 

RCCD2 Cells Exhibit Amiloride-Sensitive Sodium Transport and Respond to Vasopressin
Figure 5A shows that RCCD2 cells treated for 24 h with 10-8 M aldosterone present an increased Isc, compared with untreated cells. RCCD2 cells transport sodium through an amiloride-sensitive apical ENaC and a basolateral NKA. Whereas basolateral addition of 10-5 M amiloride did not modify Isc (data not shown), apical addition of the drug resulted in a significant decrease in Isc. Likewise, 5 x 10-5 M ouabain added in the basolateral side significantly decreased Isc. Finally, basolateral addition of 10-8 M vasopressin for 15 min resulted in approximately a 50% increase in Isc, in addition to the effect of aldosterone. The dose-response curve of AVP effect on Isc in RCCD2 cells pretreated for 24 h with 10-8 M aldosterone is shown in Figure 5B. A significant effect of the hormone is observed with AVP concentrations as low as 10-10 M.



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  		Figure 5. Effect of amiloride, ouabain, and vasopressin (AVP) treatment on Isc of aldosterone-treated RCCD2 cells. (A) Confluent RCCD2 cells grown on Snapwell filters were used to test the effect of administration for 15 min of 10-5 M amiloride (apical), 5 x 10-5 M ouabain (basolateral), and 10-8 M AVP (basolateral) on the Isc of RCCD2 cells treated for 24 h with 10-8 M aldosterone (A). Mean ± SEM from 6 to 12 filters are given. **, P < 0.01; ***, P < 0.001. (B) The dose dependency of treatment for 15 min with AVP is shown. In these experiments, RCCD2 cells were pretreated for 24 h with 10-8 M aldosterone. Mean ± SEM from three to six filters are given. *, P < 0.05; **, P < 0.01, AVP versus control.

 

Effect of Aldosterone on the Expression of mRNA Encoding for ENaC and NKA
Different studies have reported that aldosterone is able to increase the amount of mRNA encoding for the {alpha} subunit of ENaC and for the {alpha} subunit of NKA (reviewed in reference 25). Thus, we checked whether these effects were observed in RCCD2 cells. Figure 6A shows that RCCD2 cells express the three subunits of ENaC as visualized by Western blot. Figure 6B shows that treatment of RCCD2 cells for 24 h with 10-9 M aldosterone largely increases (approximately sixfold) the amount of mRNA encoding for {alpha} ENaC, as detected by Northern blot. Likewise, the amount of mRNA encoding for {alpha}1 NKA was increased (approximately fourfold; Figure 6C). The amount of mRNA encoding for ß1 NKA also was increased but to a lower extent (approximately 50%; Figure 6D).



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  		Figure 6. Regulation of epithelial sodium channel (ENaC) and Na+-K+-ATPases (NKA) mRNA expression by aldosterone in RCCD2 cells. The three subunits {alpha}, ß, and {gamma} of ENaC are present in RCCD2 cells as demonstrated by Western blot (A). A single band was observed with the immune serum for each subunit. (B, C, and D) A representative experiment of Northern blot with {alpha} ENaC and {alpha}1 and ß1 NKA on control cells (C) or on cells treated for 24 h with 10-9 M aldosterone. Glyceraldehyde phosphate dehydrogenase was used as an internal control. Approximately a sixfold increase in the amount of mRNA encoding for the {alpha} ENaC subunit (B) was observed after aldosterone treatment. Approximately a fourfold increase in {alpha}1 mRNA also was observed (C). Finally, a small effect of aldosterone (50% increase) was observed on ß1 mRNA (D).

 

Effect of Aldosterone on the Expression of mRNA Encoding for Sgk
It was shown recently (26,27,28) that Sgk is induced rapidly by aldosterone in the CCD and in the colon. We checked whether this effect could be observed at the mRNA level in RCCD2 cells. Figure 7 shows that 10-9 M aldosterone significantly increases Sgk mRNA expression, as detected by Northern blot, as soon as 45 min after the beginning of the treatment (approximately a 50% increase). In addition, the effect is dose dependent, given that increasing the concentration of aldosterone results in an increased expression of Sgk mRNA.



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  		Figure 7. Regulation of serum and glucocorticoid-induced kinase (Sgk) mRNA expression by aldosterone in RCCD2 cells. Northern blot detection of the mRNA encoding for Sgk in RCCD2 cells treated or not with 10-9 M aldosterone for different times is shown. Sgk mRNA expression is significantly increased as soon as 45 min after the beginning of the treatment. The effect was dependent on the aldosterone concentration (10-9 to 10-7 M). Each point is the mean ± SEM from four experiments, and data are corrected for the expression of glyceraldehyde phosphate dehydrogenase, which is used as an internal control. *, P < 0.05; **, P < 0.01 experimental versus control.

 

Effect of Aldosterone and Vasopressin on Specific 3H-Ouabain Binding
The amount of active NKA present in the basolateral membrane of RCCD2 cells was examined as a function of the hormonal status by specific 3H-ouabain binding technique. Results are shown in Figure 8. Figure 8A shows the time course of specific 3H-ouabain binding in RCCD2 cells grown on porous substrate and incubated for different elapses of time at 37°C with 5 x 10-5 M 3H-ouabain. Experiments show that the rate of 3H-ouabain binding is linear from 0 to 20 min incubation time. In view of these results, further studies were performed at 15 min incubation time. Figure 8B shows that, compared with the control condition in the absence of hormone, addition of AVP for 15 min resulted in a small but nonsignificant increase in specific 3H-ouabain binding. In contrast, treatment of RCCD2 cells for 24 h with 10-8 M aldosterone resulted in a significant increase. Finally, when 10-8 M AVP was added for 15 min on aldosterone-treated cells, a further significant increase in specific 3H-ouabain binding was observed, compared with cells treated only with aldosterone.



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  		Figure 8. Effects of aldosterone and AVP on specific 3H-ouabain binding. (A) The time course of specific 3H-ouabain binding was measured in RCCD2 cells grown on porous substrate and incubated at 37°C for 0 to 20 min with 5 x 10-5 M 3H-ouabain. During this period the binding was linear. (B) Specific 3H-ouabain binding was measured on confluent RCCD2 cells grown on Transwell filters and incubated in minimum medium (MM) for 24 h (C), incubated for 24 h in MM and then for 15 min with 10-8 M AVP (C+AVP), treated for 24 h with 10-8 M aldosterone (A), or first incubated 24 h with 10-8 M aldosterone and then with 10-8 M AVP for 15 min (A+AVP). Each bar is the mean ± SEM from five experiments. **, P < 0.01; ***, P < 0.001.

 

Effect of Aldosterone and AVP on the Total Amount of NKA and on the Amount of NKA Accessible to Biotinylation
The total amount of NKA present in the RCCD2 cells was determined by Western blot (Figure 9A) in different hormonal conditions (control cells treated or not for 15 min with 10-8 M AVP and cells pretreated for 24 h with 10-8 M aldosterone and then treated or not for 15 min with AVP). Treatment of the cells with 10-8 M aldosterone for 24 h resulted in a two- to threefold increase in the amount of total NKA present in the cells without any effect of the treatment for 15 min with 10-8 M AVP. The amount of NKA present in the basolateral membrane of the cells was determined after biotinylation of membrane proteins in the same hormonal conditions. Results are given in Figure 9, B and D. In Figure 9B, cells submitted to biotinylation of basolateral proteins first were submitted to immunoprecipitation of the whole {alpha}1 NKA before detection by Western blot of total {alpha}1 NKA or biotinylated {alpha}1 NKA. Total {alpha}1 NKA were increased in both aldosterone and aldosterone + AVP-treated cells, whereas biotinylated {alpha}1 NKA were increased only in aldosterone + AVP-treated cells. In Figure 9D, cells first were submitted to biotinylation before biotinylated proteins were recovered with streptavidin-agarose beads and before detection of biotinylated NKA. The total amount of ß-actin present in the cells in each experimental condition also is shown. The average of the results obtained in seven different experiments are given. Whereas the amount of ß-actin is not affected by the hormonal treatment, differences in the amounts of biotinylated NKA were observed. Compared with the control condition, neither 15 min of treatment with 10-8 M AVP nor 24 h of treatment with 10-8 M aldosterone modified the amount of biotinylated NKA. In contrast, in cells treated for 24 h with aldosterone and further treated for 15 min with AVP, a large increase in the number of NKA accessible to biotinylation was observed. As a control, Figure 9C shows that ß-actin is not detected in the fraction corresponding to the biotinylated proteins, whereas it is detected in the nonbiotinylated one, which attests to a biotinylation of plasma membrane proteins only.



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  		Figure 9. Effects of aldosterone and AVP on the total amount of NKA and on the amount of NKA accessible to biotinylation. (A) The total amount of NKA was measured by Western blot on confluent RCCD2 cells grown on Transwell filters and incubated in MM for 24 h (lane 1), incubated for 24 h in MM and then for 15 min with 10-8 M AVP (lane 2), treated for 24 h with 10-8 M aldosterone (lane 3), or first incubated 24 h with 10-8 M aldosterone and then with 10-8 M AVP for 15 min (lane 4). The assay was standardized for the same amount of proteins in the four conditions. (B) The total amount of NKA and the amount of NKA accessible to biotinylation were determined on RCCD2 cells grown on porous substrate, incubated in the four conditions described for A, and submitted to biotinylation of basolateral proteins before immunoprecipitation of the whole NKA. Then the total amount of NKA was revealed with an anti-{alpha}1 antibody (Total {alpha}1 NKA), and the biotinylated NKA was revealed with an anti-biotin antibody (Biotinylated {alpha}1 NKA) (see Materials and Methods section). (C) The amount of ß-actin was evaluated in the two fractions obtained after biotinylation of the basolateral proteins, i.e., the biotinylated proteins recovered with streptavidin-agarose beads (Biot) and the remaining proteins (Non biot) (see Materials and Methods section). ß-actin is detected only in the second fraction. (D) The amount of NKA localized in the basolateral membrane of RCCD2 cells was assessed after biotinylation of the whole proteins present in this membrane, recovering of the whole biotinylated proteins, and then detection of the NKA (see Materials and Methods section). Cells were treated as in A. An example of an experiment is shown, and the average from seven experiments is given. ***, P < 0.001.

 

Influence of AVP on the Localization of the {alpha}1 Catalytic Subunit of NKA
The influence of 15 min of treatment with 10-8 M AVP was examined on the localization of the aldosterone-dependent pool of NKA. To this end, preparations of membrane fractions corresponding to the intracellular compartment and to the plasma membrane were performed, and the {alpha}1 subunit of NKA was detected in each fraction by Western blot (Figure 10). Two fractions were obtained: an LS fraction enriched in plasma membrane and an HS fraction enriched in intracellular membranes. Figure 10A shows that the microsomal enzyme 11-HSD2 is detected effectively at approximately 38 kD in the HS fraction. In contrast, Figure 10B shows that DBA binding sites, which are located essentially in the apical membrane of RCCD2 cells (Figure 1C and confocal microscopy data not shown), are detected in the LS fraction. The DBA-binding glycoproteins exhibit molecular weights ranging from <30 kD to >200 kD, as already described in LLC-PK1 cells (29). In addition, after biotinylation of the basolateral membrane proteins (see Materials and Methods section), biotin is detected exclusively in the LS fraction (Figure 10 C). Results presented in Figure 10D show that AVP treatment of RCCD2 cells pretreated for 24 h with 10-8 M aldosterone results in a decreased number of NKA present in the HS fraction, i.e., the intracellular compartment, and a parallel increase in the amount of NKA present in the LS fraction, i.e., the plasma membrane. This result is in favor of a translocation of pumps from the intracellular compartment to the basolateral membrane under the influence of AVP.



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  		Figure 10. Influence of AVP on the localization of the {alpha}1 catalytic subunit of NKA in aldosterone-treated cells. (A, B, and C) The two membrane fractions obtained by differential centrifugation, i.e., the high-speed fraction (HS) corresponding to intracellular membranes and the low-speed fraction (LS) corresponding to plasma membranes were characterized by specific detection of the 11-ß hydroxysteroid dehydrogenase (11-HSD2), the DBA binding sites, and the biotinylated proteins recovered after biotinylation of the basolateral membrane proteins in the two fractions. DBA binding sites and biotinylated proteins are detected in the LS fraction, whereas 11-HSD2 is detected in the HS fraction. (D) The {alpha}1 subunit of NKA was detected in HS and LS fractions of membranes obtained from RCCD2 cells treated with 10-8 M aldosterone for 24 h and treated (A + AVP) or not (A) with 10-8 M AVP for 15 min. AVP treatment (A + AVP) results in a decreased number of {alpha}1 NKA in the HS fraction and a parallel increase in the amount of {alpha}1 NKA present in the LS fraction, compared with cells treated only with aldosterone (A). Results are given as mean ± SEM of four experiments. *, P < 0.05; **, P < 0.01.

 

Influence of AVP on the Phosphorylation State of the {alpha}1 Catalytic Subunit of NKA
The phosphorylation state (phosphorylated versus dephosphorylated) of the {alpha}1 subunit of NKA was examined both in the intracellular compartment and in the basolateral membrane as a function of the hormonal treatment. To this end, experiments of whole protein phosphorylation, followed by a biotinylation step and immunoprecipitation with anti-{alpha}1 NKA antibody (see Materials and Methods section), were performed. Results (Figure 11) show that NKA pumps that reside in the intracellular compartment after 24 h of treatment with 10-8 M aldosterone are present under a phosphorylated form. Treatment of these cells with 10-8 M AVP for 15 min results in a decreased number of phosphorylated proteins. NKA that reside in the basolateral membrane (accessible to biotinylation) are always present under a dephosphorylated state. These results suggest that a dephosphorylation of NKA occurs in parallel with the NKA translocation to the basolateral membrane after AVP treatment.



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  		Figure 11. Influence of vasopressin on the phosphorylation state of the {alpha}1 catalytic subunit of NKA in aldosterone-treated cells. The phosphorylation state of the {alpha}1 subunit of NKA was examined in RCCD2 cells treated for 24 h with 10-8 M aldosterone and treated (A + AVP) or not (A) for 15 min with 10-8 M AVP. To this end, experiments of whole-protein phosphorylation, followed by a biotinylation step and immunoprecipitation with the NKA anti-{alpha}1 antibody, were performed (see Materials and Methods section). Both the biotinylated proteins, corresponding to proteins present in the basolateral membrane, and the proteins that were not accessible to biotinylation, corresponding to intracellular proteins, were submitted to immunoprecipitation. After treatment for 24 h with aldosterone, numerous NKA were present in the intracellular compartment under a phosphorylated form. AVP treatment for 15 min decreased the number of intracellular NKA present under this form. The NKA present in the basolateral membrane were always dephosphorylated. Results are expressed taking into account ß-actin in each condition. Results are given as mean ± SEM of four experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Use of RCCD2 Cells to Study Physiologic Effects of Aldosterone in the CCD
The CCD is involved in the fine adjustments of sodium reabsorption and potassium secretion, two processes under the hormonal control of aldosterone and vasopressin (1,4,5). Amphibian cellular models derived from the toad urinary bladder or from the distal nephron of Xenopus Laevis (A6 cells) (30) have been widely used to study the mechanisms that operate in the CCD. Several mammalian CCD cells lines have been produced but do not retain the response to aldosterone (14,31,32). Recently, a subclone of MDCK cells (MDCK-C7) was shown to exhibit aldosterone sensitivity (33). Likewise, a mouse CCD principal cell line has been described (34). In these studies (33,34), high doses of aldosterone were used to induce physiologic effects, which suggests that both the MR and GR were involved in the observed effects. In a previous study, we characterized a rat CCD cell line, the RCCD1 cells (14). They have retained many properties of the CCD, such as high transepithelial resistance, the presence of both principal and intercalated cells, and the response to polypeptidic hormones (e.g., AVP) or to glucocorticoids. However, RCCD1 cells have lost the response to aldosterone. The RCCD2 subclone described in the present study exhibits properties similar to those of the parental line and, in addition, has maintained sensitivity to aldosterone. The MR is present in RCCD2 cells (Figure 2). The protein is detected at approximately 110 kD when a specific anti-MR antibody is used. Using this antibody, Silvestre et al. (35) also detected a single band corresponding to the rat MR in the heart and in the kidney. In the CCD, two classes of corticosteroid receptors are present, the MR and GR. GR also are present in RCCD2 cells (Figure 2), as visualized by a single band at approximately 90 kD. Tritiated ligand binding experiments and Scatchard plots analysis allowed us to estimate that, in RCCD2 cells, GR are five to six times more abundant than MR. Isc experiments showed that aldosterone was able to induce a significant increase in Isc with hormone concentrations as low as 10-9 to 10-8 M. At these concentrations, the aldosterone-dependent increase in Isc is essentially mediated by the MR. At higher concentrations of aldosterone (10-7 to 10-5 M), one can speculate that the occupancy of both MR and GR by aldosterone results in more important effects on Isc, as previously reported (36). The time course of effects on Isc and on the RT observed with low doses of aldosterone fits well with the previously described aldosterone effects in A6 cells (37). After a 2-h period during which a high decrease in RT was observed, the response involved an increase in the transepithelial sodium reabsorption through the amiloride-sensitive ENaC and the basolateral NKA. The different subunits that constitute these transporters are expressed in RCCD2 cells. As previously described in different models (34,38,39,40), 24 h of treatment with aldosterone 10-9 M increases the amount of mRNA encoding for the {alpha} subunit of ENaC and for the {alpha}1 subunit of the NKA (Figure 6). It is interesting that the serum and glucocorticoid serine/threonine kinase Sgk, which was shown recently to be increased rapidly by aldosterone (26,27,28), also is present in RCCD2 cells. Treatment with 10-9 M aldosterone significantly increases the amount of mRNA encoding for Sgk very rapidly (as soon as 45 min after hormone addition). On the whole, this RCCD2 subclone seems to constitute a good cellular model to study the physiologic effects of aldosterone in the rat CCD.

The Aldosterone-Dependent Pool of NKA Is Localized in the Intracellular Compartment before Translocation to the Basolateral Membrane by AVP
We and others have shown that, in the mammalian CCD, aldosterone is responsible for the constitution of a latent pool of NKA (6,7). This pool of latent pumps can be rapidly recruited and/or activated in an active form by different stimuli, including an increase in intracellular sodium concentration or in cellular volume, or by treatment with vasopressin (8,9,10). Thus, in addition to a synergistic effect at the apical membrane (10), aldosterone and AVP act synergistically at the basolateral membrane to increase sodium reabsorption and thus maintain the sodium homeostasis of the organism. However, the precise mechanism involved is unknown. In particular, the exact localization of the latent pool of pumps, either intracellular or within the membrane but in an inactive form, is not known. In RCCD2 cells, the experiments showed that treatment for 24 h with 10-8 M aldosterone increased the total amount of NKA present in the cells, which suggests, as in the native CCD, that aldosterone is responsible for the constitution of a pool of pumps. The 3H-ouabain binding experiments revealed that RCCD2 cells also reproduce the phenomenon observed in the native CCD isolated by microdissection, i.e., a synergistic effect of aldosterone and AVP on the specific 3H-ouabain binding. However, because ouabain binds to active NKA, one cannot discriminate between intracellular pumps or latent pumps in the plasma membrane. Experiments of subcellular localization by confocal or electronic microscopy did not allow us to answer the question clearly because of the close vicinity of the basolateral membrane and its basolateral infoldings with intracellular membranes. Thus, we decided to perform experiments of biotinylation of basolateral proteins and preparation of membrane fractions. Control experiments were performed to ensure the validity of these methods. In particular, the microsomal enzyme 11-HSD2, which is a key enzyme in the mechanism of mineralocorticoid selectivity and which is present in most of the aldosterone-sensitive cells (41), is detected in RCCD2 cells, exclusively in the HS fraction that corresponds to intracellular membranes. Both types of experiments showed that, after 24 h of pretreatment with 10-8 M aldosterone, treatment with 10-8 M AVP for 15 min resulted in a decreased number of intracellular NKA that was associated with a parallel rise in the number of NKA present in the basolateral membrane of the cells. Thus, as has already been described for ENaC or aquaporin 2 (12,13), AVP permits the translocation of transport proteins that initially are localized in the intracellular compartment.

Dephosphorylation of the {alpha}1 Catalytic Subunit Occurs in Parallel with the Recruitment of NKA to the Basolateral Membrane
We observed in this study that treatment of RCCD2 cells for 24 h with aldosterone leads to the constitution of a pool of NKA. This pool is intracellular. When the measures of NKA amounts (Figure 10) and measures of {alpha}1 NKA phosphorylation (Figure 11) are compared in the two different pools of NKA, one can observe that treatment with aldosterone alone leads to an increase in intracellular pumps that present a high degree of phosphorylation. Treatment of these cells for 15 min with AVP leads to a parallel decrease in the number of intracellular pumps and in the level of phosphorylation of the intracellular {alpha}1 subunit, which suggests that pumps that initially are phosphorylated leave the intracellular compartment. In addition, the number of pumps present in the basolateral membrane of the cells increases. When localized in this membrane, NKA are totally dephosphorylated, which suggests that dephosphorylated pumps reach the basolateral membrane. Thus, dephosphorylation of the {alpha}1 subunit might be associated with the recruitment of NKA in RCCD2 cells. Along the same line, we showed previously in native mouse CCD that inhibition of a specific protein phosphatase, namely PP2A, blocked pump recruitment, which suggests that a dephosphorylation process was involved (11). Here, we show that the {alpha}1 subunit is dephosphorylated, a finding that does not exclude that other proteins also could be dephosphorylated. The question that arises is whether dephosphorylation is required either for NKA activation or for NKA exocytosis or both. Likewise, we cannot totally exclude that both phenomena occur in parallel without any cause—effect relationship. It has been shown that the {alpha} subunit of NKA can serve as a substrate for protein kinase A or protein kinase C (42,43). In addition, several studies demonstrated that NKA is active under its dephosphorylated form and inactive under its phosphorylated form (44,45). Likewise, it has been shown that exocytic/endocytic events often are coupled with dephosphorylation/phosphorylation processes (46,47,48). In the CCD, it has been shown that aquaporin2 phosphorylation by PKA stimulation is required for its exocytosis in the apical membrane (49,50,51). Concerning NKA, Chibalin et al. (52,53) showed in the renal proximal tubule that the dopamine-induced endocytosis of NKA is initiated by phosphorylation of serine 18 in the rat {alpha} subunit and is responsible for the decreased activity. In these studies, the authors suggested that it is the removal of the {alpha} subunit from the plasma membrane rather than its phosphorylation that results in decreased cell NKA activity. In our study, we showed that in CCD cells, aldosterone and AVP act synergistically to recruit NKA from intracellular stores and that this translocation is accompanied by a dephosphorylation of the NKA {alpha}1 subunit. Further studies will be necessary to determine the putative role of dephosphorylation in the exocytic process and/or the NKA activation in these cells.


   Acknowledgments
 
This work was supported by the INSERM. We thank Drs. M. E. Rafestin-Oblin, M. Lombès, M. C. Zennaro, and J. Fagart for helpful discussion concerning the mineralo- and glucocorticoid receptors. We thank Roussel Uclaf for the generous gift of RU486 and RU26752. We thank P. Disdier and S. Roger for photographic mounting.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication September 18, 2000. Accepted for publication March 3, 2001.




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