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Vol. 14, Issue 7, 2677-2688, July 2003
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*Division de Néphrologie, Fondation pour
Recherches Médicales, CH-1211 Genève 4, Switzerland;
Service de Néphrologie
Pédiatrique, Hôpital A. Trousseau, F-75571 Paris cedex 12,
France; Laboratoire de Physiologie et
Génomique des Cellules Rénales, FRE 2468, Institut des
Cordeliers, IFR 58, 75270 Paris cedex 6, France; and
INSERM U478, Faculté de Médecine
Xavier Bichat, BP416, F-75870 Paris Cedex 18, France
Submitted November 11, 2002;
Revised February 13, 2003;
Accepted March 13, 2003
Monitoring Editor: Guido Guidotti
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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), is tightly
regulated (Therien and Blostein,
2000; Féraille and
Doucet, 2001). Long-term regulation of Na,K-ATPase relies mainly
on alteration of the expression of its subunits, whereas short-term control is
mediated by changes in enzymatic turnover and/or redistribution between cell
surface and intracellular compartments.
In the mammalian cortical collecting duct (CCD), a rise in intracellular
Na+ concentration ([Na+]i) rapidly increases the
activity of Na,K-ATPase and the number of specific ouabain binding sites
(Barlet-Bas et al.,
1990b; Blot-Chabaud et
al., 1990). It has been shown that
[Na+]i-dependent increase of Na,K-ATPase activity does not require
transcriptional regulation and/or de novo protein synthesis
(Barlet-Bas et al.,
1990b). These findings raise the possibility that silent Na-pumps
already located at the cell membrane are activated or alternatively, that
preexisting intracellular Na,K-ATPase units are shuttled to the cell surface.
The latter hypothesis was supported by recent experimental evidence showing
that an intracellular pool of Na,K-ATPase units can be rapidly recruited to
the cell surface in response to cAMP
(Gonin et al., 2001)
or to aldosterone (Summa et al.,
2001).
The aim of the present study was to investigate the effect of an increase
in [Na+]i on Na,K-ATPase cell surface expression in CCD cells and
to determine the intracellular signaling pathway mediating this effect. To
answer these questions, we used both microdissected intact rat CCDs and
confluent cultured mouse principal collecting duct mpkCCDc14 cells
(Bens et al., 1999), a
cell line characterized by retained-expression of transporters specific for
CCD principal cells including ENaC and aquaporin-2 as well as by controlled
transepithelial Na+ transport by aldosterone and vasopressin
(Bens et al., 1999;
Vandewalle et al.,
1999; Robert-Nicoud et
al., 2001; Hasler et
al., 2002),
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). Single CCDs were isolated by microdissection in
the ice-cold oxygenated incubation solution containing aprotinin (1 µg/ml)
and leupeptin (20 mg/ml) to preserve the integrity of proteins. Isolated CCDs
were incubated with or without drugs for 2 h at 37°C as described in
RESULTS. The length of tubular segments, which served as reference for
Na,K-ATPase activities and Western blotting analysis was determined from
photographs of microdissected CCDs.
Cell Culture
The mpkCCDc14 cells (passages 20–25) were grown in defined
medium (DM: DMEM:Ham's F12' 1:1 [vol/vol], 60 nM
sodium selenate, 5 µg/ml transferrin, 2 mM glutamine, 50 nM dexamethasone,
1 nM triiodothyronine, 10 ng/ml epidermal growth factor, 5 µg/ml insulin,
20 mM D-glucose, 2% vol/vol, fetal calf serum, and 20 mM HEPES, pH
7.4) at 37°C in 5% CO2/95% air atmosphere, and the medium was
changed every 2 d. Experiments were performed on confluent cells seeded on
semipermeable polycarbonate filters (Transwell, 0.4-µm pore size,
1-cm2 growth area, Corning Costar, Cambridge, MA). Cells were kept
for 6–8 d in DM medium and then placed in serum-free, hormone-deprived
medium 24 h before the experiments. Except as otherwise noted, the serum-free,
hormone-deprived medium was supplemented with 10–6
M aldosterone. For experiments, cells were preincubated for 30 min at 37°C
with or without drugs in an incubation solution (5 mM KCl, 1 mM
CaCl2, 1 mM MgSO4, 0,2 mM NaH2PO4,
0.15 mM Na2HPO4, 5 mM glucose, 4 mM NaHCO3,
12 mM essential amino acids, 2 mM nonessential amino acids, vitamins, 1 mM
pyruvic acid, 10 mM lactic acid, 20 mM HEPES, pH 7.4) supplemented with either
120 mM choline chloride (low-Na+condition; 15 mM Na+) or
120 mM NaCl (high-Na+ condition; 135 mM Na+), with or
without drugs. Incubation was carried out for 1 h at 37°C after adding 1
µg/ml amphotericin B or 0.1 U/ml nystatin to the basolateral side of the
filters. In some cases, experiments were carried out using an incubation
solution devoided of K+ (K+-free condition).
Measurement of Na,K-ATPase Activity
The hydrolytic activity of the Na,K-ATPase was determined under
Vmax conditions by the release of Pi from
[
-32P]ATP (Amersham, United Kingdom) on isolated rat CCDs
permeabilized by freeze/thawing as described
(Deschenes and Doucet, 2000).
Measurements were performed in quadruplicate on pools of four to six
microdissected rat CCDs. The Na,K-ATPase activity was taken as the difference
between the means total and Na,K-independent ATPase activities measured in the
presence (100 mM NaCl and 5 mM KCl) and in the absence of Na+ and
K+ (150 mM choline chloride, 2 mM ouabain), respectively. The
results were expressed as pmol ATP x mm–1
x h–1.
Measurement of Total and Cell Surface Na,K-ATPase
For measurement of total cellular Na,K-ATPase content, confluent
mpkCCDcl4 cells grown on filters were lysed in homogenizing buffer
(HB: 20 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA, 20 µg/ml leupeptin, 10
millitrypsin-inhibitor units/ml aprotinin, 30 mM NaF, 30 mM Na pyrophosphate,
1 mM phenylmethylsulfonyl fluoride, 1 mM AEBSF, 0.1% [wt/vol] SDS, and 1%
[vol/vol] Triton X-100), and protein content was determined using the BCA
assay (Pierce, Rockford, IL). Lysed samples were resuspended in Laemmli's
buffer (Laemmli, 1970) and
processed for SDS-PAGE. After electrophoresis on 7% polyacrylamide gels,
proteins were electrotransferred to polyvinylidene difluoride membranes
(Immobilion-P, Millipore, Waters, MA). Membranes were incubated with a
polyclonal antibody (dilution 1/10,000) raised against the 
-subunit of
Na,K-ATPase (Carranza et al.,
1996a) in Tris-buffered saline (TBS) NP-40 (150 mM NaCl, 50 mM
Tris, 0.2% NP-40, pH 7.4) with 5% (wt/vol) nonfat dried milk. After washing,
membranes were incubated with an anti-rabbit IgG antibody (dilution 1/10,000)
coupled to horseradish peroxidase (BD Transduction Laboratories, San Di-ego,
CA). The antigen-antibody complexes were detected by chemiluminescence with
the Super Signal Substrate method (Pierce) according to the manufacturer's
instructions. The protein bands revealed were quantified using a video
densitometer and Image-Quant software (Molecular Dynamics, Sunnyvalle, CA),
and the results were expressed as percent of the intensity of the protein band
from control samples.
Measurements of cell surface Na,K-ATPase were performed on isolated rat
CCDs and cultured mpkCCDcl4 cells as described
(Gonin et al., 2001)
using EZ-Link sulfosuccinimidobiotin (Sulfo NHS-S-S-Biotin, Pierce) to label
cell surface proteins. After lysis in HB buffer, equal amounts of protein were
precipitated in the presence of streptavidin-agarose beads (Immunopure
immobilized streptavidin, Pierce) diluted in an antiprotease-supplemented
buffer solution (TLB: 50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, 20
µg/ml leupeptin, 1 µg/ml aprotinin). The beads were then washed three
times with the TLB solution and once with 10 mM Tris-HCl, pH 7.4. After
resuspension in Laemmli's buffer (Laemmli,
1970), samples were processed for SDS-PAGE and Western blotting as
described above.
Measurement of Intracellular cAMP Content
After incubation under various experimental conditions, the cAMP content of
individual microdissected rat CCDs was measured using a previously described
radioimmunoassay (Chabardès et
al., 1984). Results were expressed as fmol cAMP x
mm–1.
Confluent mpkCCDcl4 cells grown on filters under various experimental conditions were scraped off the filters and homogenized in a buffer containing 50 mM Tris, 4 mM EDTA, pH 7.4. IBM-X (10–5 M) was added during the incubation period and in the homogenization buffer to prevent cAMP degradation by phosphodiesterases. Cellular cAMP levels were then measured using the cyclic AMP (3H) system (Amersham, United Kingdom) according to the manufacturer's instructions. Results were expressed as a pmol cAMP x µg protein–1.
Measurement of PKA Activity
Confluent mpkCCDcl4 cells grown on filters and incubated under
various experimental conditions were scraped off and homogenized in an
extraction buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM
-mercaptoethanol, 1 µg/ml leupeptin, and 1 µg/ml aprotinin).
Protein Kinase A activity was then measured using the SignaTECTR
cAMP-Dependent Protein Kinase (PKA) Assay System (Promega, Madison, WI)
according to the manufacturer's instructions. Results were expressed as pmol
ATP x min–1 x µg
protein–1.
Statistics
Results are given as the mean ± SE from (n) independent experiments.
Each experiment was performed on microdissected tubules from the same animal
or on cultured cells from the same passage. Statistical differences in
Na,K-ATPase activity, PKA activity, and cellular cAMP content measured under
various conditions were done using the unpaired Student t test or by
analysis of variance for comparison of two or more than two groups,
respectively. Statistical analysis of Na,K-ATPase -subunit
immunoreactivity was done using the Mann-Whitney U test or the
Kruskal-Wallis test for comparison of two or more than two groups,
respectively. p < 0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Similar experiments were performed in confluent mpkCCDcl4 cells grown on
filters and incubated with high (135 mM) or low (15 mM) Na+
solutions. In this case, cultured cells were incubated with two different
Na+ ionophores, nystatin (0.1 U/µl) or amphotericin B (1
µg/ml), in order to enhance plasma membrane permeability to Na+
and to decrease the concentration gradient between intracellular and
extracellular Na+. Experiments were also performed using a
K+ free solution, which reversibly inhibits Na,K-ATPase and
therefore disrupts the physiological Na+ concentration gradient
across the plasma membrane (Blot-Chabaud
et al., 1990). Under these conditions,
mpkCCDc14 cells incubated in a solution containing 135 mM
Na+ in order to increase [Na+]i, exhibited a greater
cell surface expression of Na,K-ATPase as compared with the same set of cells
incubated in a solution containing 15 mM Na+
(Figure 2, A and B). The
increase in Na,K-ATPase cell surface expression occurred after a lag time of
30 min and was maximal after 1-h incubation at 37°C (our unpublished
results). In contrast, a rise in [Na+]i did not alter Na,K-ATPase
total cellular content (Figure 2, C and
D). A graded increase in [Na+]e from 15–135 mM
produced a Na+ concentration-dependent increase in Na,K-ATPase cell
surface expression in amphotericin B-permeabilized mpkCCDcl4 cells
(Figure 3). Control experiments
were performed to assess the effect of amphotericin B permeabilization on
mpkCCDc14 cell viability. Addition of
10–9 M vasopressin during the last 10 min of the
1-h incubation in the presence of amphotericin B produced a close to sixfold
increase in cellular cAMP content (see
Figure 7) indicating that
cellular ATP content was not depleted by ionophore treatment. In addition,
although transepithelial resistance and potential fell to zero in amphotericin
B permeabilized confluent cells, a full recovery was observed 24 h after
ionophore washout. Moreover, addition of vasopressin
(10–9 M) for 24 h after ionophore washout
decreased transepithelial resistance from 4928 ± 142 to 3500 ±
214 Omhs/cm2 (n = 4), increased transepithelial potential from 41
± 2 to 53 ± 2 mV and induced aquaporin-2 water channel
expression (our unpublished results), as previously described in
nonpermeabilized cells (Vandewalle
et al., 1999;
Hasler et al., 2002).
Therefore, short-term (1 h) amphotericin B permeabilization did not
significantly alter the viability and vasopressin responsiveness of mpkCCDcl4
cells. These results indicate that cultured mpkCCDcl4 cells
represent a suitable ex vivo cell system for extensive analysis of
mechanism(s) underlying the Na,K-ATPase recruitment to the cell surface in
response to high [Na+]i.
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The Increase in Na,K-ATPase Cell Surface Expression Induced by a Rise
in [Na+]i Is Enhanced by Corticosteroids and Independent of Ongoing
Transcription and De Novo Translation
The recruitment of active Na,K-ATPase units induced by a rise in
[Na+]i has been shown to require the presence of aldosterone in
isolated mammalian CCDs (Barlet Bas et al., 1990;
Blot-Chabaud et al.,
1990). Experiments were performed on confluent
mpkCCDc14 cells grown in serum-free, hormone-deprived medium and in
the absence or presence of 10–6 M aldosterone for
72 h to assess the aldosterone-dependency of [Na+]i-induced
increase in cell surface expression of Na,K-ATPase. The
[Na+]i-dependent increase in Na,K-ATPase cell surface expression
measured on amphotericin B-permeabilized cells was significantly reduced in
mpkCCDc14 cells grown in hormone-free medium (as percentage of
control ± SE; 111 ± 1.6%) compared with aldosterone-treated
cells (as percentage of control ± SE; 126 ± 0.3; n = 6; p
<0.05; our unpublished results). Therefore, these results indicate that
corticosteroid hormones potentiate Na,K-ATPase cell membrane recruitment
caused by a rise in [Na+]i in mpkCCDcl4 cells.
Because the time course of [Na+]i-induced recruitment of Na,K-ATPase units to the cell surface (30–60 min) is compatible with de novo protein synthesis, we assessed the effects of actinomycin D and cycloheximide, two classical inhibitors of transcription and protein synthesis, respectively. In amphotericin B–permeabilized cells, neither 5 µM actinomycin D nor 20 µM cycloheximide prevented the effect of [Na+]i on Na,K-ATPase cell surface expression (Figure 4). These results indicate that recruitment of Na,K-ATPase by a rise in [Na+]i does not rely on transcriptional activity or on de novo protein synthesis.
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The [Na+]i-dependent Increase of Na,K-ATPase Cell Surface
Expression Relies on PKA Activation
We next examined the involvement of serine/threonine kinase in
[Na+]i-dependent Na,K-ATPase cell surface expression. In
amphotericin B-permeabilized mpkCCDcl4 cells incubated in low- or
high-Na+ media, the broad-range serine/threonine kinases inhibitor
H-7 (10–5 M) abolished the
[Na+]i-dependent increase of Na,K-ATPase cell surface expression
(Figure 5).
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We next refined our analysis of protein kinases involved in the
[Na+]i-induced upregulation of cell surface-expressed Na,K-ATPase
by investigating the effects of various narrow-range serine/threonine kinase
inhibitors. The results obtained indicate that neither GF109203X
(10–6 M), a protein kinase C inhibitor, nor ML-7
(10–5 M), a myosin light chain kinase inhibitor,
altered the stimulatory effect of increased [Na+]i on cell surface
expression of Na,K-ATPase (Figure
5). In contrast, H89 (5 x 10–5
M), a preferential but not fully specific protein kinase A (PKA) inhibitor
(Davies et al.,
2000), prevented the [Na+]i-dependent recruitment of
Na,K-ATPase to the cell surface of mpkCCDcl4 cells
(Figure 6, A and B).
Figure 6, C and D, shows that
50 µM myristoylated PKI, a cell-permeable peptide inhibitor of PKA, also
prevented the effect of increased [Na+]i on cell surface expression
of Na,K-ATPase. In addition, H89 also abolished the increase of Na,K-ATPase
activity induced by nystatin in isolated rat CCDs (as pmol x
mm–1 x h–1
± SE; Control: 501 ± 83; Nystatin: 844 ± 64*; H89: 570
± 40; H89 + Nystatin: 569 ± 21; n = 4*; p <0.05 vs. control).
Because we have previously shown that cAMP, a classical PKA activator, induced
the translocation of an intracellular pool of Na,K-ATPase to the plasma
membrane (Gonin et al.,
2001), we studied the additivity of Na,K-ATPase cell surface
expression induced by both [Na+]i and cAMP. In amphotericin
B–permeabilized mpkCCDcl4 cells incubated in a low
Na+ (15 mM) solution, addition of db-cAMP
(10–3 M), a cell-permeant cAMP analog, for 10 min
at 37°C increased Na,K-ATPase cell surface expression by 25 ± 5%.
In contrast, db-cAMP did not further increase Na,K-ATPase cell surface
expression in amphotericin B–permeabilized cells incubated in a
high-Na+ (135 mM) solution
(Figure 6, E and F). These
results strongly suggest that PKA activity is stimulated by a rise in
[Na+]i.
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To further confirm that the stimulatory effect of high [Na+]i on Na,K-ATPase cell surface expression was dependent on PKA activation, PKA activity was measured in amphotericin B–permeabilized mpkCCDcl4 cells. As shown in Figure 7A, incubation of permeabilized cells for 30–60 min at 37°C in the presence of 135 mM Na+ induced a sustained increase in PKA activity. Because PKA activation is classically related to an increase in cellular cAMP content, cellular cAMP content was measured in amphotericin B–permeabilized mpkCCDc14 cells. Unexpectedly, the amount of cellular cAMP content did not change in cells incubated in mediums containing either 15 mM or 135 mM Na+ (Figure 7B). Similarly, increasing [Na+]ii did not alter the cAMP content of cells preincubated with the phosphodiesterase inhibitor IBMX (10–4 M; our unpublished results). In contrast, 10–9 M vasopressin, a classical adenylate cyclase activator, added during the last 10 min of incubation, induced a large increase in cellular cAMP content in amphoterincin B–permeabilized cells (Figure 7B). This AVP-induced increase in cellular cAMP content was not influenced by increased [Na+]i. Therefore, amphotericin B permeabilization did not induce a large leak of cAMP out of mpkCCDcl4 cells and increasing [Na+]i did not alter the adenylate cyclase responsiveness to AVP. Furthermore, cellular cAMP content was not altered in nystatin-permeabilized isolated rat CCDs (Figure 7C). Altogether, these results indicate that a rise in [Na+]i induces PKA activation independently of any detectable increase in cellular cAMP content.
Inhibitors of the Proteasomal Degradation Pathway Prevent
[Na+]i-induced PKA Activation and Increase in Na,K-ATPase Cell
Surface Expression
It has been reported that cytokines may trigger cAMP-independent activation
of PKA (Zhong et al.,
1997) through a pathway that requires proteasomal activity
(Dulin et al., 2001).
Experiments were performed to investigate the possible roles of the
proteasomal and lysosomal protein degradation pathways in Na,K-ATPase
recruitment to the mpkCCDcl4 cell surface induced by a rise
[Na+]i. Proteasomal activity was inhibited by addition of
10–6 M MG132 or lactacystin, two specific and
structurally unrelated proteasome inhibitors. To analyze the contribution of
the lysosome, we used either 2 x 10–6 M
leupeptin, an inhibitor of cysteine proteases, or
10–7 M chloroquine, a weak base that increases
lysosomal pH and thereby inhibits the proteolytic activity of lysosomal
enzymes. We first checked that these drugs did not modify the high
transepithelial electrical resistance of confluent mpkCCDC14 cells
(our unpublished results). Figure
8 shows that the presence of proteasomal inhibitors completely
abolished the [Na+]i-induced increase of Na,K-ATPase expressed at
the cell surface and blocked the stimulation of PKA activity in amphotericin
B–permeabilized mpkCCDcl4 cells. In contrast, none of the
lysosomal inhibitors used altered the increase in Na,K-ATPase cell surface
expression caused by a rise in [Na+]i
(Figure 9). These results
indicate that the proteasomal but not the lysosomal degradation pathway is
involved in [Na+]i-dependent recruitment of Na,K-ATPase to the cell
surface of mpkCCDcl4 cells.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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[Na+]i, aldosterone, and vasopressin
(Gonin et al., 2001;
Summa et al., 2001)
all control Na,K-ATPase cell surface expression in collecting duct principal
cells. [Na+]i is the major limiting factor for Na,K-ATPase activity
in intact cells (Skou, 1998).
In resting renal epithelial cells, [Na+]i is maintained below
levels required for half-maximal Na,K-ATPase activation (K0.5),
which explains why this enzyme works at only 20–30% of its maximal rate
(Cheval et al., 1990;
Féraille et al.,
1995). It has therefore been assumed that the only rate limiting
step for Na+ reabsorption in collecting duct principal cells was
the Na+ influx occurring through the apical epithelial
Na+ channel (ENaC) and that the kinetic control of Na,K-ATPase by
[Na+]i was sufficient to maintain the balance between the apical
entry and basolateral exit of Na+. However, several studies have
shown that aldosterone and vasopressin enhance both apical and basolateral
steps of vectorial Na+ transport in collecting duct principal cells
through the coordinated stimulation of ENaC and Na,K-ATPase
(Palmer et al., 1982;
Schafer and Troutman, 1990;
Bens et al., 1999;
Gonin et al., 2001;
Summa et al., 2001).
Such coordinated control seems to be of special importance in the renal
collecting duct. Under normal conditions, luminal Na+
concentrations in CCD are quite low and any increase in [Na+]i
secondary to increased Na+ influx should theoretically reduce the
electrochemical driving force for apical Na+ entry
(Silver et al.,
1993). However, because the apparent affinity of Na,K-ATPase for
Na+ is twice as high in the collecting duct than in the proximal
tubule and thick ascending limb (Barlet-Bas
et al., 1990a; Féraille et al.,
1994,
1995), the kinetic reserve for
a [Na+]i activation of Na,K-ATPase is much lower in the collecting
duct than in the more proximal nephron segments. In view of ENaC
downregulation by high [Na+]i
(Kellenberger et al.,
1998; Awayda,
1999), the observed increase of Na,K-ATPase cell surface
expression that allows a more efficient decrease in [Na+]i suggests
that the restoration of a normal [Na+]i is a priority with respect
to Na+ reabsorption in collecting duct principal cells. Therefore,
acute fluctuations of [Na+]i may exert a rapid kinetic effect on
Na,K-ATPase activity whereas sustained changes in [Na+]i would also
control the number of active Na pumps present at the cell surface of CCD
cells.
Our results also show that high [Na+]i induces a proportional
increase in maximal hydrolytic activity and Na,K-ATPase cell surface
expression in mammalian CCDs. Therefore, the increase in the number of
specific ouabain binding sites, i.e., the number of active Na-pump units at
the cell surface, previously observed in response to high [Na+]i
(Barlet-Bas et al.,
1990b; Blot-Chabaud et
al., 1990) most likely relies on an increase in Na-pump cell
surface expression. This increase is observed in the absence of a variation of
the total cellular pool of Na,K-ATPase and is independent of transcriptional
regulation and de novo protein synthesis
(Barlet Bas et al.,
1990b), strongly suggesting that translocation of intracellular
Na-pumps to the plasma membrane occurs in response to increased
[Na+]i. This interpretation is further supported by the PKA
dependency of this process because we have recently shown the existence of an
intracellular pool of Na,K-ATPase, which can be recruited rapidly to the
plasma membrane in response to cAMP in collecting duct principal cells
(Gonin et al., 2001).
Similarly, short-term aldosterone induces the redistribution of intracellular
Na-pumps to the cell surface (Summa et
al., 2001). The aldosterone-dependency of both high
[Na+]i- and cAMP-induced increase in cell surface expression of
Na,K-ATPase (Barlet-Bas et al., 1990,
Blot-Chabaud et al.,
1990, Gonin et al.,
2001) may suggest the requirement of (an) aldosterone-induced
regulatory protein(s) exerting a permissive effect.
The results of this study also strongly support that PKA activation is
required for the [Na+]i-induced recruitment of Na-pumps to the cell
surface in collecting duct principal cells: first, pharmacological inhibition
of PKA prevented the [Na+]i-induced increase in Na,K-ATPase cell
surface expression and activity; second, high [Na+]i concomitantly
stimulated PKA activity and increased Na,K-ATPase cell surface expression;
third, the effects of [Na+]i and cAMP on Na,K-ATPase cell surface
expression are not additive; and finally proteasome inhibitors prevented both
[Na+]i-induced PKA activation and Na,K-ATPase increased cell
surface expression. It remains to be determined whether PKA directly induces
Na,K-ATPase redistribution of, e.g., though phosphorylation of the Na,K-ATPase
-subunit (Beguin et al.,
1994; Feschenko and Sweadner,
1994; Fisone et al.,
1994; Carranza et al.,
1996b,
1998), and/or whether other
signaling intermediate(s) is(are) involved.
The PKA holoenzyme is a heterotetramer consisting of two catalytic (PKAc)
subunits associated with two regulatory subunits
(Scott, 1991;
Taylor et al., 1990;
Francis and Corbin, 1994).
Dissociation of the holoenzyme is induced by binding of cAMP to the regulatory
subunits, which consequently alleviates autoinhibitory contacts and releases
active PKAc. Our results clearly indicate that the [Na+]i-induced
activation of PKA is independent of a rise in cellular cAMP content, because
even in the presence of phosphodiesterase inhibitor or AVP, intracellular cAMP
concentration is not altered by high [Na+]i in CCD cells. The large
increase in cellular cAMP content observed in Na+-loaded
amphotericin B–permeabilized cells treated with AVP indicate that cAMP
did not leak out the cells and that high [Na]i did not interfere with
adenylate cyclase activity. On the other hand, Na+-sensitive
adenylyl cyclase stimulation (Cooper
et al., 1998) does not seem to be involved in
[Na+]i-induced PKA activation. An alternate cAMP-independent
mechanism of PKA activation has been recently described. In response to
cytokines, free active PKAc is released upon dissociation of a multiprotein
complex containing PKA, I
B, and NF-B p65
(Zhong et al., 1997;
Zieger et al., 2001).
Dissociation of the PKAc/IB/NF-B p65 complex is triggered
by phosphorylation (Karin,
1999) and subsequent proteasomal degradation of IB
(Dulin et al., 2001).
In addition, the association of a discrete pool of PKAc with the
tonicity-responsive enhancer binding protein (TonEBP) and cAMP-independent
activation of PKA in response to extracellular hypertonicity have been
recently described (Ferraris et
al., 2002). Incubation of cells under hypertonic conditions
rapidly stimulates the transcriptional activity of TonEBP
(Miyakawa et al.,
1999) in a PKA-dependent manner
(Ferraris et al.,
2002). Our results show that PKA activation by [Na+]i
is independent of cAMP and requires proteasomal activity, suggesting that
[Na+]i controls the degradation rate of a regulatory protein that
maintains a discrete cellular pool of PKAc in an inactive state.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Abbreviations used: PKA, protein kinase A; CCD, cortical collecting duct; AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride.
¶ Corresponding author. E-mail address: Eric.Feraille{at}medecine.unige.ch.
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