Extracellular Hypotonicity Increases Na,K-ATPase Cell Surface Expression via Enhanced Na+ Influx in Cultured Renal Collecting Duct Cells
Manlio Vinciguerra*,
Serge Arnaudeau,
David Mordasini*,
Martine Rousselot*,
Marcelle Bens,
Alain Vandewalle,
Pierre-Yves Martin*,
Udo Hasler* and
Eric Feraille*
*Service de Néphrologie, Fondation pour Recherches Médicales, Geneva, Switzerland; Department of Physiology, University of Geneva Medical Center, Geneva, Switzerland; and INSERM U478, Faculté de Médecine Xavier Bichat, Paris, France
Correspondence to Dr. Eric Feraille, Service de Néphrologie, Fondation pour Recherches Médicales, 64 rue de la Roseraie, CH-1211 Geneva 4, Switzerland. Phone: 41-22-382-38-37; Fax: 41-22-347-59-79; E-mail: Eric.Feraille{at}medecine.unige.ch
In the renal collecting duct (CD), the Na,K-ATPase, whichprovides the driving force for Na+ absorption, is under tightmultifactorial control. Because CD cells are physiologicallyexposed to variations of interstitial and tubular fluid osmolarities,the effects of extracellular anisotonicity on Na,K-ATPase cellsurface expression were studied. Results show that hypotonicconditions increased, whereas hypertonic conditions had no effecton Na,K-ATPase cell surface expression in confluent mpkCCDcl4cells. Incubating cells with amphotericin B, which increases[Na+]i, under isotonic or anisotonic conditions, revealed thatNa,K-ATPase recruitment to the cell surface was not directlyrelated to variations of cell volume and osmolarity. The effectsof amphotericin B and extracellular hypotonicity were not additive,and both were prevented by protein kinase A and proteasome inhibitors,suggesting a common mechanism of action. In line with this hypothesis,extracellular hypotonicity induced a sustained stimulation ofthe amiloride-sensitive short-circuit current, indicating increasedNa+ influx through the apical epithelial Na+ channel. Moreover,inhibiting apical Na+ entry by amiloride, a blocker of epithelialNa+ channel, or incubating cells in Na+-free medium preventedthe cell surface recruitment of Na,K-ATPase in response to extracellularhypotonicity. Altogether, these findings strongly suggest thatextracellular hypotonicity stimulates apical Na+ influx leadingto increased [Na+]i, protein kinase A activation, and recruitmentof Na,K-ATPase units to the cell surface of mpkCCDcl4 cells.
In the mammalian kidney, water and solute excretion are tightlycontrolled to maintain body fluid compartment homeostasis. Inthe collecting duct (CD), which is the site of fine tuning ofNa+ balance, principal cells are responsible for Na+ reabsorption,whereas intercalated cells are involved in acid-base secretion.In principal cells, Na+ enters via the luminal epithelial Na+channel (ENaC) and is extruded by basolaterally located Na,K-ATPase,which provides the driving force for vectorial Na+ transport(1). The Na,K-ATPase is under multifactorial control includinghormones, paracrine factors, intracellular Na ([Na]i), and extracellularosmolarity (1–3). Long-term regulation of the enzyme reliesmainly on altered subunit expression, whereas short-term controlis mediated by changes in enzymatic turnover and/or redistributionbetween cell surface and intracellular compartments (1–3).
Mammalian CD cells are physiologically exposed to variationsin both interstitial and luminal osmolalities, which may alterthe Na+ transport by principal cells. Exposure of renal amphibianA6 cells, a model of mammalian principal cells, to extracellularhypotonicity was shown to stimulate Na+ influx through ENaC(4–6). Hypotonic cell swelling is associated with a stimulationof the Na+-pump current in both renal A6 cells (7) and cardiacmyocytes (8). In addition, hypotonic conditions recruit activeNa,K-ATPase units to the cell surface in isolated rabbit corticalCD (CCD) (9). It remains to be determined whether this processrelies on the translocation of an intracellular pool of Na,K-ATPaseunits or alternatively on the activation of silent plasma membraneNa+ pumps.
We investigated the mechanism of control of Na,K-ATPase by extracellulartonicity using cultured mouse principal collecting duct mpkCCDc14cells, a cell line derived from microdissected CCD of an SVPK/Tagtransgenic mouse (10). These transgenic mice carry the SV40large T antigen gene (Tag) under the control of the promoterof the L-type pyruvate kinase (PK) gene and of SV40 early enhancer(SV) (11). The mpkCCDc14 cells retain expression of Na+ andwater transporters that are specific for CD principal cells,such as ENaC and aquaporin-2, as well as transepithelial Na+transport controlled by aldosterone and vasopressin, and representa valuable experimental model (12–14). The results ofthis study demonstrate that Na,K-ATPase cell surface expressionis modulated by Na+ influx independent of cell volume variationin mpkCCDc14 cells.
Cell Culture
The mpkCCDc14 cells (passages 20 to 25) were grown in definedmedium (DM; DMEM:Ham’s F12' 1:1 vol/vol, 60 nM sodiumselenate, 5 µg/ml transferrin, 2 mM glutamine, 50 nM dexamethasone,1 nM triiodothyronine, 10 ng/ml EGF, 5 µg/ml insulin,20 mM D-glucose, 2% [vol/vol] FCS, and 20 mM HEPES, pH 7.4)at 37°C in 5% CO2/95% air atmosphere. Experiments were performedon confluent cells seeded on polycarbonate filters (Transwell,0.4-µm pore size, 1 cm2 growth area; Corning Costar, Cambridge,MA). Cells were kept for 6 to 8 d in DM and then placed in serum-free,hormone-deprived medium 24 h before experiments. For experiments,cells were preincubated for 30 min at 37°C with variousisotonic incubation solutions that contained either 140 mM NaClor 240 mM sucrose, supplemented or not with drugs as describedin Results and figure legends. Afterward, cells were incubatedfor an additional hour at 37°C in the same incubation solutionsto which 1 µg/ml amphotericin B was added or after apicaland basal replacement of the isotonic incubation medium by anequivalent volume of hypotonic (200 mOsm/L) or hypertonic (500mOsm/L) incubation solution. The final composition of the solutionsused is given in Table 1.
Measurement of Cell Surface Na,K-ATPase
Cell surface Na,K-ATPase was determined on cultured mpkCCDcl4cells as described previously (12,13) using EZ-Link sulfossuccinimidobiotin(Sulfo NHS-S-S-Biotin; Pierce, Rockford, IL) to label cell surfaceproteins. After lysis in homogenizing buffer (HB; 2 mM EDTA,2 mM EGTA, 20 µg/ml leupeptin, 1 µg /ml aprotinin,30 mM NaF, 30 mM Na pyrophosphate, 1 mM PMSF, 1 mM AEBSF, 0.1%[wt/vol] SDS, 1% [vol/vol] Triton X-100, and 20 mM Tris HCl[pH 7.4]), equal amounts of protein were precipitated with streptavidin-agarosebeads (Immunopure immobilized streptavidin; Pierce) dilutedin an antiprotease-supplemented Tris Lysis Buffer (50 mM Tris-HClpH 7.4, 100 mM NaCl, 5 mM EDTA, 20 µg/ml leupeptin, and1 µg /ml aprotinin). After three washes with TLB and onewith 10 mM Tris-HCl (pH 7.4), samples were resuspended in Laemmli’sbuffer (15) and processed for 7% SDS-PAGE, and proteins wereelectrotransferred to polyvinylidene difluoride membranes (Immobilion-P;Millipore, Waters, MA). The Na,K-ATPase -subunit was then detectedwith a polyclonal antibody (dilution 1:10000) raised againstthe rat enzyme (16). The -transferrin receptor was detectedwith an mAb (dilution 1:2000; Zymed, San Francisco, CA), andE-cadherin was detected with a polyclonal antibody (dilution1:5000; Sigma, St. Louis, MO). The protein bands revealed bychemiluminescence (Super Signal Substrate; Pierce) were quantifiedusing a video densitometer and ImageQuant software (MolecularDynamics, Sunnyvale, CA), and the results were expressed aspercentage of control.
Cell Volume Analysis
The mpkCCDc14 cells, grown on polycarbonate filters or glasscoverslips, were rinsed with PBS and incubated for 30 min at37°C in culture medium that contained the fluorescent dyecalcein-AM (5 µM diluted from a 10-mM stock solution inDMSO; Molecular Probes Europe, Leiden, Netherlands). Dye loadingwas terminated by rinsing with PBS; cells were then placed inan open perfusion chamber on a stage of an inverted microscope(Zeiss Axiovert 200M; Carl Zeiss AG, Feldbach, Switzerland),and prewarmed (37°C) incubation solutions were superfusedwith a peristaltic pump (Dynamax, Emeryville, CA). For improvingmeasurements, fluorescence changes were monitored using a spinningdisk confocal microscope, which better preserves living cellsthan a laser scanning confocal microscope and allows normalizationof the fluorescence with respect to the thickness of the opticalslice, improving the accuracy of single-emission dyes. The 488-nmline from a 2.5-W Kr/Ar water-cooled ion laser in multilinemode (Innova 70C Spectrum; Coherent, Santa Clara, CA) was selectedfor calcein excitation by the use of an Acousto-Optical TunableFilter (AOTF; Visitech International, Sunderland, UK). The AOTFoutput was coupled by a single-mode fiber optic (Oz Optics,Ltd., Corp, Ontario, Canada) to a Yokogawa spinning disk confocalscan-head (QLC100; Visitech International) mounted on the invertedmicroscope. Images were collected by a 40 x 0.75 NA Achroplanwater immersion objective (Carl Zeiss AG) and captured witha 12-bit TE/CCD interlined Coolsnap HQ Photometrics camera (RopperScientific, Trenton, NJ). The green emission from calcein wasacquired using a D525/50m emission filter (Chroma TechnologyCorp., Brattleboro, VT). Wavelength selection with the AOTF,camera control, and all of the motorized functions of the microscopewere controlled for image acquisition by Metamorph/Metafluor5.0 software (Universal Imaging, West Chester, PA). The samesoftware was used off-line for image analysis, photo-bleachingcorrection, and curve fitting. Because recorded fluorescencein a given cytoplasmic area is proportional to the intracellularconcentration of calcein, we determined cell volume changesby measurement of variations of calcein fluorescence, as describedpreviously (17). The results were expressed as a percentageof the control period fluorescence intensity.
Electrophysiologic Studies
Confluent mpkCCDcl4 cells that were grown on Snapwell filters(0.4-µm pore size, 12-mm diameter; Corning Costar) weretransferred to a Ussing chamber, and short-circuit current (Isc)was measured under voltage clamp (0 mV) using dual silver-silverchloride electrodes connected to a VCC MC6 voltage-clamp apparatus(Physiologic Instruments, San Diego, CA). Cells were equilibratedat 37°C for 30 min in symmetric isotonic (300 mOsm/L) sucrosebuffer that contained 40 mM Na+ (Table 1) and bubbled with 5%CO2. Isotonic sucrose buffer was then replaced by hypotonicsucrose buffer (200 mOsm/L and 40 mM Na+) using a peristalticpump, and cells were maintained for 60 min under symmetric hypotonicconditions. Then, isotonic conditions were reestablished byexchanging the hypotonic sucrose buffer with isotonic sucrosebuffer. After 30 min of equilibration, 10–6 M amiloridewas added to the apical side of the chamber to measure the amiloride-resistantIsc. The amiloride-sensitive Isc reflecting ENaC-mediated Na+transport was calculated as the total Isc minus the amiloride-resistantIsc. By convention, positive Isc corresponded to a flow of positivecharges from the basal to the apical solution. Results wereexpressed as µA/cm2.
Statistical Analyses
Results are given as means ± SEM from n independent experiments.Each experiment was performed on cultured cells from the samepassage. Statistical analysis of Na,K-ATPase -subunit immunoreactivitywas done using the Mann-Whitney U test or the Kruskal-Wallistest for comparison of two or more than two groups, respectively.Statistical analysis of Isc was done using t test for paireddata. P < 0.05 was considered significant.
Effect of Extracellular Anisotonicity on Na,K-ATPase Cell Surface Expression
We first assessed the influence of extracellular tonicity oncell volume and Na,K-ATPase cell surface expression. Incubationof confluent mpkCCDc14 cells in a hypotonic medium (200 mOsm/L)induced a rapid decrease in fluorescence intensity from calcein-loadedcells corresponding to a 24.5 ± 5.5% increase in cellvolume (Figure 1A). After the hypotonic medium was replacedby an isotonic medium (not shown), fluorescence intensity returnedtoward baseline levels, indicating that the decrease in fluorescencesignal was not due to a dye leakage but rather to intracellularcalcein dilution consecutive to cell swelling. Incubation ofmpkCCDc14 cells for 1 h at 37°C in hypotonic medium increasedNa,K-ATPase cell surface expression by 30% as compared withcells that were incubated under isotonic conditions (Figure 1B).A similar increase of Na,K-ATPase cell surface expressionwas observed when extracellular NaCl was replaced by sucrose(Table 1, Figure 1B, right). Therefore, Na,K-ATPase was recruitedto the cell surface independent of variations of ionic strength.As expected, the fluorescence intensity of calcein-loaded cellsincreased after exposure to a hyperosmotic medium (500 mOsm/L),indicating a 20.1 ± 2.0% decrease in cell volume (Figure 1C).In contrast to the observations made in cells that wereincubated under hypotonic conditions, hypertonic NaCl or sucrosemedium (Table 1) did not significantly alter Na,K-ATPase cellsurface expression (Figure 1D).
Figure 1. Effect of extracellular hypo- or hypertonicity on cell volume and Na,K-ATPase cell surface expression. (A and C) Confluent mpkCCDc14 cells that were grown on glass coverslips were loaded with calcein-AM and mounted in an open perfusion chamber placed on a stage of an inverted microscope. After a 30-min equilibration period in isotonic incubation solution (300 mOsm/L; iso), cells were superfused with either hypotonic (200 mOsm/L; hypo; A) or hypertonic (500 mOsm/L; hyper; C) incubation solutions and fluorescence intensity was recorded. The graph illustrates the changes in calcein fluorescence (line) and the maximal variation of cell volume (bars) expressed as a percentage of baseline. Results are means ± SEM from three separate experiments. (B and D) Confluent mpkCCDcl4 cells that were grown on polycarbonate filters were first preincubated in the presence of isotonic (300 mOsm/L) NaCl- or sucrose-containing incubation solution (Table 1) for 1 h at 37°C and then incubated under isotonic, hypotonic (B), or hypertonic conditions (D) for 1 h at 37°C. The Na,K-ATPase -subunit was detected by Western blotting performed after biotinylation and streptavidin precipitation of cell surface proteins. (Top) Representative immunoblots showing Na,K-ATPase cell surface expression. (Bottom) The densitometric values of labeled Na,K-ATPase -subunit bands were expressed as a percentage of the optical density value of cells that were incubated in isotonic medium (100%). Results are means ± SEM from six separate experiments. *P < 0.05 versus 300 mOsm/L values.
The question arises whether cell swelling controls Na,K-ATPaseexpression to the cell surface. Using mpkCCDcl4 cells, we showedthat amphotericin B, which forms artificial pores that enhanceNa+ and K+ plasma membrane permeabilities (18), increases Na,K-ATPasecell surface expression in the presence of physiologic concentrationsof extracellular Na+ (19). Because amphotericin B induces cellswelling in MDCK cells (17), we tested whether variations ofcell volume may account for the Na+ ionophore-induced Na,K-ATPaserecruitment. For this purpose, mpkCCDc14 cells were preincubatedin isotonic NaCl medium (Table 1) for 1 h at 37°C and thenfor an additional hour in isotonic or hypotonic NaCl medium(200 mOsm/L) with or without 1 µg/ml amphotericin B. Figure 2A and 3A show that under isotonic conditions, amphotericinB did not alter calcein fluorescence intensity, indicating theabsence of a detectable variation of cell volume. In the absenceof amphotericin B, incubation of cells with hypotonic medium(200 mOsm/L) decreased calcein fluorescence, reflecting cellswelling (Figure 1A), whereas calcein fluorescence slightlyincreased in cells that were challenged with both amphotericinB and hypotonic medium (Figure 2B). The latter increase in calceinfluorescence most likely reflected a moderate cell shrinkage(9.9 ± 1.5%) consecutive to the diffusion of ions andaccompanying water through amphotericin B pores from the cytosolto the less concentrated extracellular medium (18). We nextanalyzed the combined effects of extracellular hypotonicityand amphotericin B permeabilization on Na,K-ATPase cell surfaceexpression to determine whether they potentially share a commonmechanism of action despite dissimilar effects on cell volume.Amphotericin B and extracellular hypotonicity increased Na,K-ATPasecell surface expression to a similar extent and in a nonadditivemanner (Figure 2C). These results suggest that both stimuliactivate similar or convergent signaling pathways.
Figure 2. Effect of amphotericin B and extracellular hypotonicity on cell volume and Na,K-ATPase cell surface expression. (A and B) Confluent mpkCCDc14 cells that were grown on glass coverslips were loaded with calcein-AM as described in the legend of Figure 1. After equilibration with isotonic incubation solution (300 mOsm/L; iso), cells were superfused with 1 µg/ml amphotericin B diluted in isotonic (Ampho B; A) or hypotonic (200 mOsm/L; hypo; B) solutions and fluorescence intensity was recorded. The graph illustrates the changes in calcein fluorescence (line) and the maximal variation of cell volume (bars) expressed as a percentage of baseline. Results are means ± SEM from three separate experiments. (C) Confluent mpkCCDcl4 cells that were grown on polycarbonate filters were first preincubated in the presence of isotonic (300 mOsm/L) NaCl-containing incubation solution (Table 1) for 1 h at 37°C. Cells were then permeabilized or not with 1 µg/ml amphotericin B and then incubated with isotonic (300 mOsm) or hypotonic (200 mOsm) solution for 1 h at 37°C. The Na,K-ATPase -subunit was then detected by Western blotting performed after biotinylation and streptavidin precipitation of cell surface proteins. (Top) Representative immunoblots showing Na,K-ATPase cell surface expression. (Bottom) The densitometric values of labeled Na,K-ATPase -subunit bands were expressed as a percentage of variations of the optical density value of cells that were incubated in isotonic medium in the absence of amphotericin B (100%). Results are means ± SEM from eight separate experiments. *P < 0.05 versus 300 mOsm/L without amphotericin B values.
Figure 3. Effect of amphotericin B and extracellular hypertonicity on cell volume and Na,K-ATPase cell surface expression. (A and B) Confluent mpkCCDc14 cells that were grown on glass coverslips were loaded with calcein-AM, equilibrated with the isotonic incubation solution (Iso), and superfused with 1 µg/ml amphotericin B as described in the legend of Figure 2. Amphotericin B was diluted in either isotonic (Ampho B; A) or hypertonic (500 mOsm/L; hyper; B) incubation solution and fluorescence intensity was recorded. The graph illustrates the changes in calcein fluorescence (line) and the maximal variation of cell volume (bars) expressed as a percentage of baseline. Results are means ± SEM from three separate experiments. (C) Confluent mpkCCDcl4 cells that were grown on polycarbonate filters were first preincubated in the presence of isotonic (300 mOsm/L) NaCl incubation solution (Table 1) for 1 h at 37°C. Cells were then permeabilized or not with 1 µg/ml amphotericin B and then incubated under either isotonic (300 mOsm/L) or hypertonic (500 mOsm/L) solution for 1 h at 37°C. The Na,K-ATPase -subunit was then detected by Western blotting performed after biotinylation and streptavidin precipitation of cell surface proteins. (Top) Representative immunoblots showing Na,K-ATPase cell surface expression. (Bottom) The densitometric values of labeled Na,K-ATPase -subunit bands were expressed as a percentage of variations of the optical density value of cells that were incubated in isotonic medium in the absence of amphotericin B (100%). Results are means ± SEM from eight separate experiments. *P < 0.05 versus 300 mOsm/L without amphotericin B values.
To investigate further the relationship between extracellulartonicity, Na+ permeability, and Na,K-ATPase cell surface expression,we examined the effect of extracellular hypertonicity on theamphotericin B–induced increase in Na,K-ATPase cell surfaceexpression. For this purpose, mpkCCDc14 cells were preincubatedin NaCl isotonic medium (Table 1) for 1 h at 37°C and incubatedfor an additional hour in isotonic or hypertonic NaCl medium(500 mOsm/L) with or without 1 µg/ml amphotericin B. Inthe absence of amphotericin B, extracellular hypertonicity inducedcell shrinkage (Figure 1C), whereas the addition of amphoterinB to the hypertonic solution induced a slight decrease in calceinfluorescence, reflecting a moderate cell swelling (12.2 ±1.2%; Figure 3B). This latter effect most likely relies on extracellularion entry and accompanying water through the membrane poresformed by amphotericin B (18). Adding 200 mOsm/L NaCl or sucrose(corresponding to the 500 mOsm/L hypertonic solution) did notsignificantly modify Na,K-ATPase cell surface expression (Figure 3C).However, addition of amphotericin B to both hypertonicand isotonic solution increased Na,K-ATPase cell surface expression(Figure 3C). These results indicate that hypertonicity doesnot modulate the effect of increased Na+ influx caused by amphotericinB on Na,K-ATPase recruitment to the cell surface. Altogether,our results demonstrate that the recruitment of Na,K-ATPaseto the cell surface of mpkCCDcl4 cells is not directly correlatedto changes in cell volume (Table 2).
Table 2. Correlation between cell volume variation, Na+ entry, and increase in Na,K-ATPase cell surface expression
Extracellular Hypotonicity Increases Na,K-ATPase Cell Surface Expression via a Protein Kinase A– and Proteasome-Dependent Pathway
The lack of additive effects of amphotericin B and extracellularhypotonicity on Na,K-ATPase membrane recruitment suggests thatboth stimuli activate convergent signaling pathways. In CD principalcells, Na,K-ATPase cell surface recruitment induced by amphotericinB–dependent Na+ influx is mediated by cAMP-independentPKA activation that requires proteasomal activity (19). To determinewhether a similar pathway is involved in the observed hypotonicity-inducedincrease in Na,K-ATPase cell surface expression, we preincubatedmpkCCDcl4 cells in the absence or presence of 10–5 M H89,a protein kinase A (PKA) inhibitor, for 1 h at 37°C beforeexposure or not to a hypotonic (200 mOsm/L) NaCl or sucrosemedium with or without H89 for an additional hour. Inhibitionof PKA by H89 fully prevented the increase in Na,K-ATPase cellsurface expression induced by NaCl or sucrose hypotonic solution(Figure 4). We next investigated whether the hypotonicity-inducedincrease in Na,K-ATPase cell surface expression also requiresproteasomal activity. For this purpose, we preincubated mpkCCDc14cells for 2 h at 37°C in the absence or presence of 10–6M of the proteasomal inhibitor lactacystin before exposure ornot to hypotonic (200 mOsm/L) NaCl medium with or without lactacystinfor an additional hour. Figure 5 shows that lactacystin preventedthe hypotonicity-induced Na,K-ATPase recruitment. Similar resultswere achieved using 10–5 M MG132, a proteasomal inhibitorthat is structurally unrelated to lactacystin (data not shown).Taken together, these results indicate that amphotericin B andextracellular hypotonicity both increased Na,K-ATPase cell surfaceexpression via the same mechanism.
Figure 4. The hypotonicity-induced increase in Na,K-ATPase cell surface expression is dependent on protein kinase A (PKA). Confluent mpkCCDcl4 cells that were grown on polycarbonate filters were first incubated in the presence of isotonic (300 mOsm/L) NaCl (A) or sucrose (B) incubation solution (Table 1) and with (–) or without (+) 10–6 M H89, a PKA inhibitor, for 1 h at 37°C. Cells were then incubated under either isotonic or hypotonic (200 mOsm/L) solution supplemented or not with H89 for 1 h at 37°C. Na,K-ATPase -subunit was then detected by Western blotting performed after biotinylation and streptavidin precipitation of cell surface proteins. (Top) Representative immunoblots showing Na,K-ATPase cell surface expression. (Bottom) The densitometric values of labeled Na,K-ATPase -subunit bands were expressed as a percentage of the optical density value of cells that were incubated in isotonic medium in the absence of H89 (100%). Results are means ± SEM from five separate experiments. *P < 0.05 versus 300 mOsm/L without H89 values.
Figure 5. The hypotonicity-induced increase in Na,K-ATPase cell surface expression is dependent on proteasomal activity. Confluent mpkCCDcl4 cells that were grown on polycarbonate filters were first preincubated in the presence of isotonic (300 mOsm/L) NaCl incubation solution with (–) or without (+) 10–6 M lactacystin, a proteasomal inhibitor, for 1 h at 37°C. Cells were then incubated under either isotonic or hypotonic (200 mOsm/L) conditions and in the presence or absence of lactacystin for 1 h at 37°C. Na,K-ATPase -subunit was then detected by Western blotting performed after biotinylation and streptavidin precipitation of cell surface proteins. (Top) Representative immunoblots showing Na,K-ATPase cell surface expression. (Bottom) The densitometric values of labeled Na,K-ATPase -subunit bands were expressed as a percentage of the optical density value of cells that were incubated in isotonic medium and in the absence of lactacystin (100%). Results are means ± SEM from five separate experiments. *P < 0.05 versus 300 mOsm/L without lactacystin values.
Hypotonicity Stimulates the Amiloride-Sensitive ISC in mpkCCDcl4 Cells
In renal amphibian A6 cells, a model of CD principal cells,extracellular hypotonicity was shown to stimulate transepithelialNa+ transport by increasing both the number of ENaC (4–6)present in the apical membrane and Na,K-ATPase activity (7).We therefore determined whether a hypotonic challenge stimulatesthe amiloride-sensitive ISC in mpkCCDc14 cells. Cells were firstequilibrated in isotonic sucrose solution (300 mOsm/L) thatcontained 40 mM Na+ (Table 1). Under these conditions, ISC wasstable for at least 120 min. After exposure of mpkCCDc14 cellsto a hyposmotic sucrose solution (200 mOsm/L) in the continuouspresence of 40 mM extracellular Na+, the amiloride-sensitiveIsc gradually increased from 4.8 ± 0.4 to 8.7 ±0.6 µA/cm2 (P < 0.01) during the first 20 min of incubation.and this stimulation was sustained for at least 40 min (Figure 6A).The amiloride-sensitive ISC promptly returned to a nearlybasal levels (5.3 ± 0.3 µA/cm2) after reestablishmentof isotonic conditions (Figure 6). However, the amiloride-insensitiveISC was similar under isotonic and hypotonic conditions (0.4± 0.1 µA/cm2). These findings indicate that extracellularhypotonic challenge enhances the apical Na+ influx through ENaCand therefore may increase [Na+]i in mpkCCDcl4 cells.
Figure 6. Hypotonicity stimulates the amiloride-sensitive short-circuit current (ISC) in mpkCCDcl4. Confluent mpkCCDcl4 cells that were grown on polycarbonate filters (Snapwell) were transferred to a Ussing chamber, and Isc was measured under voltage clamp (0 mV) as described in Materials and Methods. Cells were first equilibrated for 30 min at 37°C in the presence of isotonic (300 mOsm/L) sucrose incubation solution and then exposed to hypotonic (200 mOsm/L) sucrose incubation solution for 60 min before reestablishing isotonic conditions. All incubation solutions contained 40 mM Na+. (A) Representative recording showing ISC as a function of time and extracellular osmolarity. (B) Amiloride-sensitive ISC under isosmotic (300 mOsm/L), hyposmotic (200 mOsm/L), and reestablishment of isosmotic condition. Results from five separate experiments are shown.
Increase in Na,K-ATPase Cell Surface Expression in Response to Extracellular Hypotonicity Depends on Apical Na+ Entry
Our results showed that extracellular hypotonicity stimulatedthe amiloride-sensitive Isc in mpkCCDcl4 cells, suggesting thatincreased Na,K-ATPase cell surface expression in response toextracellular hypotonic challenge relies on enhanced Na+ influxthrough ENaC. To test this hypothesis, we preincubated mpkCCDcl4cells that were grown on filters under isotonic conditions for30 min at 37°C with or without 10–6 M amiloride, aninhibitor of ENaC, before exposure to a hypotonic (200 mOsm/L)extracellular solution with or without amiloride for an additionalhour. Amiloride fully prevented the increase in Na,K-ATPasecell surface expression induced by extracellular hypotonicity(Figure 7). Experiments were then performed using a nominallyNa+-free medium (Table 1) to investigate further the Na+ dependenceof the hypotonicity-induced recruitment of Na,K-ATPase to thecell surface. Figure 8, A and B, shows that exposure of calcein-loadedcells to hypotonic (200 mOsm/L) sucrose medium induced a similarincrease in cell volume in the presence of a residual 40 mMNa+ (23.0 ± 2.2%) or in the nominal absence of Na+ (21.9± 2.6%). A significant increase in cell surface Na,K-ATPasewas still observed when using a hypotonic (200 mOsm/L) solutionin which NaCl was substituted by sucrose but still containeda residual 40 mM Na+ as compared with that measured using anisotonic (300 mOsm/L) sucrose solution that contained the sameconcentration of Na+ (Figure 8C, left). In contrast, extracellularhypotonicity did not increased Na,K-ATPase cell surface expressionwhen mpkCCDcl4 cells were incubated in a nominally Na+-freesolution in which NaCl was substituted by sucrose and in whichthe remaining 40 mM Na+ was substituted by K+ (Table 1, Figure 8C,right). The hypotonicity-induced recruitment of Na,K-ATPasewas blunted in the absence of Na+ despite a similar increasein cell volume and therefore cell surface area, suggesting thatNa,K-ATPase is specifically targeted to the cell surface inthe presence of extracellular Na+. Figure 8D shows that, inagreement with the functional link between Na,K-ATPase and E-cadherinobserved in epithelial cells, exposure of mpkCCDcl4 cells toNaCl hypotonic medium induced a parallel increase of the cellsurface expression of both proteins (20). In contrast, the cellsurface expression of the ubiquitously expressed and quicklyrecycling -transferrin receptor (21) remained unchanged. Theseresults demonstrate that extracellular hypotonicity specificallyinduces the recruitment of Na,K-ATPase and functionally linkedE-cadherin to the cell surface. Moreover, this effect criticallydepends on apical Na+ influx mediated by ENaC.
Figure 7. The hypotonicity-induced increase in Na,K-ATPase cell surface expression is dependent on epithelial NaCl channel activity. Confluent mpkCCDcl4 cells that were grown on polycarbonate filters were first incubated in the presence of isotonic (300 mOsm) NaCl (A) or sucrose (B) incubation solution with (–) or without (+) 10–6 M amiloride for 30 min at 37°C. Cells were then incubated in either an isotonic or a hypotonic (200 mOsm/L) solution and in the presence or absence of amiloride for 1 h at 37°C. The Na,K-ATPase -subunit was detected by Western blotting performed after biotinylation and streptavidin precipitation of cell surface proteins. (Top) Representative immunoblot showing Na,K-ATPase cell surface expression. (Bottom) The densitometric values of labeled Na,K-ATPase -subunit bands were expressed as a percentage of the optical density value of cells that were incubated in isotonic medium and in the absence of amiloride (100%). Results are means ± SEM from four separate experiments. *P < 0.05 versus 300 mOsm/L without amiloride values.
Figure 8. The hypotonicity-induced increase of Na,K-ATPase cell surface expression is dependent on the presence of extracellular sodium. (A and B) Confluent mpkCCDc14 cells that were grown on glass coverslips were loaded with calcein-AM and mounted in an open perfusion chamber placed on a stage of an inverted microscope. After a 30-min equilibration period in isotonic sucrose incubation solution (300 mOsm/L; iso), cells were superfused with hypotonic sucrose (200 mOsm/L; hypo) incubation solutions that contained (A) or not (B) 40 mM Na+ and fluorescence intensity was recorded. The graph illustrates the changes in calcein fluorescence (line) and the maximal variation of cell volume (bars) expressed as a percentage of baseline. Results are means ± SEM from two separate experiments. (C) Confluent mpkCCDcl4 cells that were grown on polycarbonate filters were first preincubated in the presence of isotonic (300 mOsm/L) sucrose incubation solution that contained (40 mM) or not (0 mM) Na+ (Table 1) for 1 h at 37°C. Cells were then incubated in either an isotonic or a hypotonic (200 mOsm/L) solution and in the presence or absence of Na+ for 1 h at 37°C. The Na,K-ATPase -subunit was detected by Western blotting performed after biotinylation and streptavidin precipitation of cell surface proteins. (Top) Representative immunoblot showing Na,K-ATPase cell surface expression. (Bottom) The densitometric values of labeled Na,K-ATPase -subunit bands were expressed as a percentage of the optical density value of cells that were incubated in isotonic medium (100%). Results are means ± SEM from four separate experiments. *P < 0.05 versus control 300 mOsm/L values. (D) Confluent mpkCCDcl4 cells that were preincubated in the presence of isotonic (300 mOsm/L) NaCl incubation solution were incubated in either an isotonic or a hypotonic (200 mOsm/L) NaCl solution for 1 h at 37°C. The -transferrin receptor (top), E-cadherin (middle), and Na,K-ATPase -subunit (bottom) were detected by Western blotting performed on the same membrane after biotinylation and streptavidin precipitation of cell surface proteins.
The present study shows that extracellular hypotonicity andamphotericin B induce the recruitment of Na,K-ATPase units tothe cell surface of renal CD principal cells independent ofcell volume variation via increased Na+ influx, leading to theproteasomal-dependent activation of PKA. Renal CD cells areexposed to physiologic variations in luminal fluid and/or interstitialosmolality, which influence cell volume (22) and trigger adaptiveprocesses aimed at restoring initial cell volume (23,24). Indeed,renal epithelial cells undergo cell swelling or cell shrinkagein response to extracellular hypotonicity or hypertonicity,respectively (25–27). That Na,K-ATPase provides the drivingforce for vectorial ion transport and plays a key role in themaintenance of a constant composition of the intracellular milieuand cell volume (28) led us to analyze the existing relationshipbetween cell volume variation and Na,K-ATPase cell surface expression,a major determinant of Na,K-ATPase activity in CD principalcells (3). In agreement with a previous study on isolated mouseCCD (9), the present findings demonstrate that hypotonicity,which induced cell swelling, is associated with Na,K-ATPaserecruitment to the plasma membrane but that hypertonicity, whichinduced cell shrinkage, does not alter Na,K-ATPase cell surfaceexpression. Modification of the intracellular concentrationor activity of solutes may also lead to variations of cell volume(26,27,29), particularly in secreting or absorptive epithelialcells. For example, apical Na+-alanine and Na+-glucose co-transporteractivation induced a sustained increase in cell volume in isolatedrabbit proximal tubules (26). Likewise, inhibition of activeNa+ extrusion by ouabain, which specifically inhibits the Na,K-ATPase,increases cell volume in CCD (29). It is interesting that theNa+ ionophore amphotericin B induces a similar increase in Na,K-ATPasecell surface expression under isotonic conditions, i.e., inthe absence of cell volume variation, as well as under hypotonicor hypertonic conditions associated with cell shrinkage or cellswelling, respectively. These results strongly suggest thatthe expression levels of cell surface Na,K-ATPase are not directlycontrolled by cell volume variation in renal CD principal cells(Table 2).
Previous studies performed in renal A6 cells have shown thata short hypotonic challenge stimulates both Na,K-ATPase activity(7) and apical Na+ entry via an increase in the number of conductingENaC (4–6). Results of the present study confirm thatextracellular hypotonicity stimulates apical Na+ influx thoughENaC (Figure 6) and show that elimination of the apical Na+entry through ENaC either by addition of 10–6 M amilorideto the apical side of mpkCCDcl4 cells grown on filters or byincubation of cells in a nominally Na+-free solution fully preventedthe increase in Na,K-ATPase cell surface expression inducedby extracellular hypoosmolality (Figures 7 and 8). Moreover,exposure of mpkCCDcl4 cells to hypotonic sucrose medium thatcontained or not 40 mM Na+ induced a similar increase in cellvolume. These observations therefore suggest that the increasein [Na]i consecutive to the stimulation of ENaC-dependent Na+entry and not cell swelling triggers the increase in Na,K-ATPasecell surface expression in response to a short-term hypotonicchallenge in mpkCCDcl4 cells. However, our results do not ruleout the possibility that cell swelling triggers ENaC activationin response to extracellular hypotonicity. This finding is inagreement with the previously described intracellular Na+ concentration-dependentrecruitment of Na,K-ATPase in mpkCCDcl4 cells (19). Altogether,our observations suggest that CD principal cells exhibit anintracellular Na+ sensing mechanism that triggers a signalingcascade that leads to Na,K-ATPase recruitment from an inactiveintracellular pool to the basolateral plasma membrane, as describedpreviously in response to cAMP (12), aldosterone (13), activationof G protein–coupled receptors (30), or increased [Na+]i(19,31).
We have previously shown that a rise in [Na+]i induced by Na+ionophores leads to proteasomal-dependent PKA activation andsubsequent increased expression of cell surface Na,K-ATPasein mpkCCDcl4 cells (19). Here we show that both hypotonicityand the Na+ ionophore amphotericin B stimulate to the same extentand in a nonadditive manner the recruitment of cell surfaceNa,K-ATPase (Figure 2). Furthermore, Na,K-ATPase cell surfacerecruitment could be prevented after pharmacologic inhibitionof either PKA or proteasomal activity. Altogether, these resultsstrongly suggest that extracellular hypotonicity and amphotericinB share the same signaling pathway. The Na+-induced activationof PKA does not require cAMP (19), thus excluding the classicalpathway involving dissociation of the PKA holoenzyme inducedby cAMP binding to regulatory subunits, which consequently alleviatesautoinhibitory contacts and releases the active PKA catalyticsubunit (PKAc) (32,33). Ferraris et al. (34) recently showedthat, in response to extracellular hypertonicity, PKA can beactivated independent of cAMP after the release of free activePKAc as a result of the dissociation of a protein complex thatcomprises a discrete pool of PKAc associated with the tonicity-responsiveenhancer binding protein. However, such a mechanism of PKA activationcaused by increased cellular Na+ influx seems to be very unlikelybecause extracellular hypertonicity, a hallmark of tonicity-responsiveenhancer binding protein activation (34), does not alter Na,K-ATPasecell surface expression (Figure 1). Recently, Zhong et al. (35)demonstrated that the transcriptional activity of NF-B is regulatedby the IB-associated PKAc subunit activation through a cAMP-independentpathway. Inducers such as LPS and IL-1 initiate a cytoplasmicsignaling cascade that ultimately leads to the proteasomal degradationof IB, nuclear translocation of NF-B, and transcriptional activationof proinflammatory response genes. It is interesting that amongthe cascade of events that control this complex process, theproteasomal degradation of IB was shown to result in cAMP-independentactivation of PKAc, which forms a multimeric PKAc/IB/NF-B p65complex under inactive state (35,36). The proteasomal dependenceof both amphotericin B–induced (19) and extracellularhypotonicity-induced cell surface recruitment of Na,K-ATPase(Figure 5) is compatible with the mechanism reported above andrequires further investigation. It remains to be determinedwhether PKA directly induces Na,K-ATPase redistribution, e.g.,through phosphorylation of the Na,K-ATPase -subunit (37–39),or requires further downstream signaling intermediate(s).
In conclusion, our results strongly suggest that Na+ ionophoresand extracellular hypotonicity induce recruitment of Na,K-ATPaseunits to the plasma membrane after similar cAMP-independentactivation of PKA, most likely through the release of activePKAc from a protein complex after proteasomal degradation ofa regulatory protein in response to increased [Na+]i. This processis not directly dependent of cell volume variation and mostlikely relies on the activation of an intracellular Na+-sensingpathway.
Acknowledgments
This work was supported in part by Swiss National FoundationGrant 31-67878.02 and a Carlos and Elsie deReuter Foundationgrant to E.F.
We thank Fabio Carrozzino and Michelangelo Foti (Departmentof Morphology, University of Geneva, Geneva, Switzerland) forhelpful discussion and technical skills.
Feraille E, Doucet A: Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: Hormonal control. Physiol Rev 81: 345–418, 2001[Abstract/Free Full Text]
Therien AG, Blostein R: Mechanisms of sodium pump regulation Am J Physiol 279: C541–C566, 2000
Feraille E, Mordasini D, Gonin S, Deschenes G, Vinciguerra M, Doucet A, Vandewalle A, Summa V, Verrey F, Martin PY: Mechanism of control of Na,K-ATPase in principal cells of the mammalian collecting duct. Ann N Y Acad Sci 986: 570–578, 2003[Medline]
Niisato N, Marunaka Y: Hyposmolality-induced enhancement of ADH action on amiloride-sensitive Isc in renal epithelial A6 cells. Jpn J Physiol 47: 131–137, 1997[CrossRef][Medline]
Niisato N, Van Driessche W, Liu M, Marunaka Y: Involvement of protein tyrosine kinase in osmoregulation of Na+ transport and membrane capacitance in renal A6 cells. J Membrane Biol 175: 63–77, 2000[CrossRef][Medline]
Niisato N, Marunaka Y: Activation of the Na+-K+ pump by hyposmolality through tyrosine kinase-dependent Cl– conductance in Xenopus renal epithelial A6 cells. J Physiol 518: 417–432, 1999[Abstract/Free Full Text]
Bewick NL, Fernandes C, Pitt AD, Rasmussen HH, Whalley DW: Mechanisms of Na+-K+ pump regulation in cardiac myocytes during hyposmolar swelling. Am J Physiol 276: C1091–C1099, 1999
Coutry N, Farman N, Bonvalet JP, Blot-Chabaud M: Role of cell volume variations in Na+-K+-ATPase recruitment and/or activation in cortical collecting duct. Am J Physiol 266: C1342–C1349, 1994
Bens M, Vallet V, Cluzeaud F, Pascual-Letallec L, Kahn A, Rafestin-Oblin ME, Rossier BC, Vandewalle A: Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line. J Am Soc Nephrol 10: 923–934, 1999[Abstract/Free Full Text]
Miquerol L, Cluzeaud F, Porteu A, Alexandre Y, Vandewalle A, Kahn A: Tissue specificity of L-pyruvate kinase transgenes results from combinatorial effects of proximal promoter and distal activator regions. Gene Expression 5: 315–330, 1996[Medline]
Gonin S, Deschenes G, Roger F, Bens M, Martin PY, Carpentier JL, Vandewalle A, Doucet A, Feraille E: Cyclic AMP increases cell surface expression of functional Na,K-ATPase units in mammalian cortical collecting duct principal cells. Mol Biol Cell 12: 255–264, 2001[Abstract/Free Full Text]
Summa V, Mordasini D, Roger F, Bens M, Martin PY, Vandewalle A, Verrey F, Feraille E: Short term effect of aldosterone on Na,K-ATPase cell surface expression in kidney collecting duct cells. J Biol Chem 276: 47087–47093, 2001[Abstract/Free Full Text]
Hasler U, Mordasini D, Bens M, Bianchi M, Cluzeaud F, Rousselot M, Vandewalle A, Feraille E, Martin PY: Long term regulation of aquaporin-2 expression in vasopressin-responsive renal collecting duct principal cells. J Biol Chem 277: 10379–10386, 2002[Abstract/Free Full Text]
Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685, 1970[CrossRef][Medline]
Carranza ML, Féraille E, Favre H: Protein kinase C-dependent phosphorylation of Na+,K+-ATPase -subunit in rat kidney cortical tubules to the plasma membrane of rat proximal convoluted tubule cells. Am J Physiol Cell Physiol 271: C136–C143, 1996[Abstract/Free Full Text]
Zelenina M, Brismar H: Osmotic water permeability measurements using confocal laser scanning microscopy. Eur Biophys J 29: 165–171, 2000[CrossRef][Medline]
Fournier I, Barwicz J, Tancrede P: The structuring effects of amphotericin B on pure and ergosterol- or cholesterol-containing dipalmitoylphosphatidylcholine bilayers: A differential scanning calorimetry study. Biochim Biophys Acta 1373: 76–86, 1998[Medline]
Vinciguerra M, Deschênes G, Hasler U, Mordasini D, Rousselot M, Doucet A, Vandewalle A, Martin PY, Féraille E: Intracellular Na+ controls cell surface expression of Na,K-ATPase via a cAMP-independent PKA pathway in mammalian kidney collecting duct cells. Mol Biol Cell 14: 2677–2688, 2003[Abstract/Free Full Text]
Rajasekaran SA, Palmer LG, Moon SY, Peralta Soler A, Apodaca GL, Harper JF, Zheng Y, Rajasekaran AK: Na,K-ATPase activity is required for formation of tight junctions, desmosomes, and induction of polarity in epithelial cells. Mol Biol Cell 12: 3717–3732, 2001[Abstract/Free Full Text]
Presley JF, Mayor S, McGraw TE, Dunn KW, Maxfield FR: Bafilomycin A1 treatment retards transferring receptor recycling more than bulk membrane recycling. J Biol Chem 272: 13929–13936, 1997[Abstract/Free Full Text]
Montrose-Rafizadeh C, Guggino WB: Cell volume regulation in the nephron. Annu Rev Physiol 52: 761–772, 1990[CrossRef][Medline]
Lang F, Busch GL, Ritter M, Volk H, Waldegger S, Gulbins E, Haussinger D: Functional significance of cell volume regulatory mechanisms. Physiol Rev 78: 247–306, 1998[Abstract/Free Full Text]
Jakab M, Furst J, Gschwentner M, Botta G, Garavaglia ML, Bazzini C, Rodighiero S, Meyer G, Eichmueller S, Woll E, Chwatal S, Ritter M, Paulmichl M: Mechanisms sensing and modulating signals arising from cell swelling. Cell Physiol Biochem 12: 235–258, 2002[CrossRef][Medline]
Sun AM, Saltzberg SN, Kikeri D, Hebert SC: Mechanisms of cell volume regulation by the mouse medullary thick ascending limb of Henle. Kidney Int 38: 1019–1029, 1990[Medline]
Kirk KL, Schafer JA, Di Bona DR: Cell volume regulation in rabbit proximal straight tubule perfused in vitro. Am J Physiol 252: F922–F932, 1987
Beck JS, Breton S, Laprade R, Giebisch G: Volume regulation and intracellular calcium in the rabbit proximal convoluted tubule. Am J Physiol 260: F861–F867, 1991
Skou JC: Nobel Lecture. The identification of the sodium pump. Biosci Rep 18: 155–169, 1998[CrossRef][Medline]
Strange K: Volume regulation following Na+ pump inhibition in CCT principal cells: apical K+ loss. Am J Physiol 258: F732–F740, 1990
Bertorello AM, Komarova Y, Smith K, Leibiger IB, Efendiev R, Pedemonte CH, Borisy G, Sznajder JI: Analysis of Na+,K+-ATPase motion and incorporation into the plasma membrane in response to G protein-coupled receptor signals in living cells. Mol Biol Cell 14: 1149–1157, 2003[Abstract/Free Full Text]
Barlet-Bas C, Khadouri C, Marsy S, Doucet A: Enhanced intracellular sodium concentration in kidney cells recruits a latent pool of Na-K-ATPase whose size is modulated by corticosteroids. J Biol Chem 265: 7799–7803, 1990[Abstract/Free Full Text]
Francis SH, Corbin JD: Structure and function of cyclic nucleotide-dependent protein kinases. Annu Rev Physiol 56: 237–272, 1994[CrossRef][Medline]
Taylor SS, Buechler JA, Yonemoto W: cAMP-dependent protein kinase: Framework for a diverse family of regulatory enzymes. Annu Rev Biochem 59: 971–1005, 1990[CrossRef][Medline]
Ferraris JD, Persaud P, Williams CK, Chen Y, Burg M: Activity of the TonEBP/OREBP transactivation domain varies directly with extracellular NaCl concentration. Proc Natl Acad Sci U S A 99: 16800–16805, 2002[Abstract/Free Full Text]
Zhong H, SuYang H, Erdjument-Bromaage H, Tempst P, Ghosh Z: The transcriptional activity of NF-B is regulated by the IB-associated PKAc subunit through a cyclic AMP-independent mechanism. Cell 89: 413–424, 1997[CrossRef][Medline]
Dulin NO, Niu J, Browning DD, Ye RD, Voyno-Yasenetskaya T: Cyclic AMP-independent activation of protein kinase A by vasoactive peptides: J Biol Chem 24: 20827–20830, 2001
Beguin P, Beggah AT, Chibalin AV, Burgener-Kairuz P, Jaisser F, Mathews PM, Rossier BC, Cotecchia S, Geering K: Phosphorylation of the Na,K-ATPase alpha-subunit by protein kinase A and C in vitro and in intact cells. Identification of a novel motif for PKC-mediated phosphorylation. J Biol Chem 269: 24437–24445, 1994[Abstract/Free Full Text]
Fisone G, Chieng SXJ, Nairn AC, Czernic AJ, Hemmings HC, Höäg JO, Bertorello AM, Kaiser R, Bergman T, Jörnvall H, Aperia A, Greengard P: Identification of the phosphorylation site for cAMP-dependent protein kinase on Na+,K+-ATPase and effects of site-directed mutagenesis. J Biol Chem 269: 9368–9373, 1994[Abstract/Free Full Text]
Feschenko MS, Sweadner KJ: Structural basis for species-specific differences in the phosphorylation of Na,K-ATPase by protein kinase C. J Biol Chem 269: 30436–30444, 1994[Abstract/Free Full Text]
Received for publication April 29, 2004.
Accepted for publication July 7, 2004.
This article has been cited by other articles:
C. E Hills, P. E Squires, and R. Bland Serum and glucocorticoid regulated kinase and disturbed renal sodium transport in diabetes
J. Endocrinol.,
December 1, 2008;
199(3):
343 - 349.
[Abstract][Full Text][PDF]
A. R. Cinelli, R. Efendiev, and C. H. Pedemonte Trafficking of Na-K-ATPase and dopamine receptor molecules induced by changes in intracellular sodium concentration of renal epithelial cells
Am J Physiol Renal Physiol,
October 1, 2008;
295(4):
F1117 - F1125.
[Abstract][Full Text][PDF]
D. Mordasini, M. Bustamante, M. Rousselot, P.-Y. Martin, U. Hasler, and E. Feraille Stimulation of Na+ transport by AVP is independent of PKA phosphorylation of the Na-K-ATPase in collecting duct principal cells
Am J Physiol Renal Physiol,
November 1, 2005;
289(5):
F1031 - F1039.
[Abstract][Full Text][PDF]
U. Hasler, M. Vinciguerra, A. Vandewalle, P.-Y. Martin, and E. Feraille Dual Effects of Hypertonicity on Aquaporin-2 Expression in Cultured Renal Collecting Duct Principal Cells
J. Am. Soc. Nephrol.,
June 1, 2005;
16(6):
1571 - 1582.
[Abstract][Full Text][PDF]
Top
Abstract
Introduction
-subunit was then detected with a polyclonal antibody (dilution 1:10000) raised against the rat enzyme (16). The 
37°C in hypotonic medium increased Na,K-ATPase cell surface expression by 
show that under isotonic conditions, amphotericin B did not alter calcein fluorescence intensity, indicating the absence of a detectable variation of cell volume. In the absence of amphotericin B, incubation of cells with hypotonic medium (200 mOsm/L) decreased calcein fluorescence, reflecting cell swelling (Figure 1A), whereas calcein fluorescence slightly increased in cells that were challenged with both amphotericin B and hypotonic medium (Figure 2B). The latter increase in calcein fluorescence most likely reflected a moderate cell shrinkage (9.9 ± 1.5%) consecutive to the diffusion of ions and accompanying water through amphotericin B pores from the cytosol to the less concentrated extracellular medium (18). We next analyzed the combined effects of extracellular hypotonicity and amphotericin B permeabilization on Na,K-ATPase cell surface expression to determine whether they potentially share a common mechanism of action despite dissimilar effects on cell volume. Amphotericin B and extracellular hypotonicity increased Na,K-ATPase cell surface expression to a similar extent and in a nonadditive manner (Figure 2C). These results suggest that both stimuli activate similar or convergent signaling pathways. 
). Moreover, exposure of mpkCCDcl4 cells to hypotonic sucrose medium that contained or not 40 mM Na+ induced a similar increase in cell volume. These observations therefore suggest that the increase in [Na]i consecutive to the stimulation of ENaC-dependent Na+ entry and not cell swelling triggers the increase in Na,K-ATPase cell surface expression in response to a short-term hypotonic challenge in mpkCCDcl4 cells. However, our results do not rule out the possibility that cell swelling triggers ENaC activation in response to extracellular hypotonicity. This finding is in agreement with the previously described intracellular Na+ concentration-dependent recruitment of Na,K-ATPase in mpkCCDcl4 cells (19). Altogether, our observations suggest that CD principal cells exhibit an intracellular Na+ sensing mechanism that triggers a signaling cascade that leads to Na,K-ATPase recruitment from an inactive intracellular pool to the basolateral plasma membrane, as described previously in response to cAMP (12), aldosterone (13), activation of G protein–coupled receptors (30), or increased [Na+]i (19,31). 
B is regulated by the I
