Pulmonary and Critical Care Medicine, Michael Reese Hospital and Medical Center, Chicago 60616; University of Illinois at Chicago, Chicago, Illinois 60612; and Instituto Nacional de Enfermedades Respiratorias, Tlalpan, Mexico 04000
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ABSTRACT |
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Previous studies
in kidney, heart, and liver cells have demonstrated that dexamethasone
regulates the expression of Na-K-ATPase. In the lungs, Na-K-ATPase has
been reported in alveolar epithelial type II (ATII) cells
and is thought to participate in active
Na+ transport and lung edema
clearance. The aim of this study was to determine whether Na-K-ATPase
would be regulated by dexamethasone in cultured rat ATII cells.
Regulation of the Na-K-ATPase by dexamethasone could lead to a greater
understanding of its role in active
Na+ transport and lung edema
clearance. Rat ATII cells were isolated, plated for 24 h, and exposed
to 107 and
10
8 M dexamethasone. These
cells were harvested at 0, 3, 6, 12, and 24 h after dexamethasone
exposure for determination of steady-state Na-K-ATPase mRNA transcript
levels, protein expression, and function. The steady-state Na-K-ATPase
1-mRNA transcript levels
increased in ATII cells 6, 12, and 24 h after dexamethasone exposure
(P < 0.05). However, the
steady-state
1-mRNA transcript
levels were unchanged. The protein expression for the
1- and
1-subunits increased in ATII
cells exposed to dexamethasone compared with controls in association
with a temporal increase in Na-K-ATPase function after dexamethasone
exposure. These results suggest that dexamethasone regulates
Na-K-ATPase in ATII cells possibly by transcriptional, translational,
and posttranslational mechanisms.
alveolar type II cells; sodium-potassium-adenosinetriphosphatase; regulation of sodium-potassium-adenosinetriphosphatase in lungs
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INTRODUCTION |
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THE NA-K-ATPASE is a transmembrane heterodimer
comprised of an - and
-subunit (16). The
-subunit is the
catalytic subunit with an intracellular ATP binding site, a
phosphorylation site, and a ouabain binding site. The
-subunit is a
smaller glycosylated subunit (26) that may play a role in the anchoring
of the enzyme to the plasma membrane (25, 29). The Na-K-ATPase plays an important role in the maintenance of osmotic and electrochemical cell
gradients (20). It couples the hydrolysis of ATP to the transport of
Na+ and
K+ against their cellular
concentration gradients and is inhibited by cardiac glycosides such as
ouabain (38, 40).
Three isoforms of the - and
-subunit coded by different genes
have been described (17, 39). In the rat lung, two isoforms of the
-subunit and one isoform of the
-subunit Na-K-ATPase have been
described (32, 35, 37). Previous studies have reported on the
Na-K-ATPase
- and
-subunits in alveolar epithelial type II (ATII)
cells and adult lung (28, 30, 31, 36).
Na-K-ATPase function has been reported to be hormonally modulated (14,
44, 45). In the lungs, glucocorticoid receptors are present in alveolar
epithelial cells (15). Corticosteroids have been shown to bind to
specific response elements in the promoter regions of the Na-K-ATPase
genes (33). In previous studies, dexamethasone has been reported to
increase the 1-mRNA of the Na-K-ATPase in rat liver cells (3). Hence, we postulated that dexamethasone would regulate the Na-K-ATPase in lung epithelial cells
and tested this hypothesis in rat ATII cells. We found that dexamethasone increased steady-state Na-K-ATPase
1-mRNA transcripts,
1- and
1-isoform proteins, and
Na-K-ATPase function in cultured ATII cells. These findings support the
hypothesis that dexamethasone regulates Na-K-ATPase by transcription
and possibly translation and posttranslational mechanisms.
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MATERIALS AND METHODS |
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Cell isolation and experimental protocol.
ATII cells were isolated from pathogen-free male Sprague-Dawley rats
(200-225 g) by standard techniques (8, 31). Briefly, the lungs
were perfused via the pulmonary artery, lavaged, and digested with 30 U/ml elastase (Worthington Biochemical). The ATII cells were purified
by differential adherence to immunoglobulin G-pretreated dishes. Cells
were suspended in Dulbecco's modified Eagle's medium (DMEM; Irvine
Scientific) containing 10% charcoal-stripped fetal bovine serum with 2 mM L-glutamine, 40 mg/ml
gentamicin, 100 U/ml penicillin, and 100 µg/ml streptomycin. For
studies of alveolar epithelial cell transport in intact cells, 1 × 106 cells were plated into
each well of 24-well tissue culture plates (Becton-Dickinson). For
preparation of membranes, assay of Na-K-ATPase activity, and mRNA
preparation, 7 ×106
cells /well were plated in
10-cm2 tissue culture plates
(Becton-Dickinson). Cells were incubated in a humidified atmosphere of
5% CO2-95% air at 37°C.
After 24 h, nonadherent cells were removed by rinsing the monolayers.
Plating efficiency was ~60%. The cells were then cultured for 3, 6, 12, and 24 h in serum-free medium consisting of DMEM-Ham's F-12 medium containing 0.5 mg/ml bovine serum albumin (BSA)-linoleic acid, 4 ng/ml
selenic acid, 5 µg/ml transferrin, and 2 mM glutamine in the absence
or presence of 107 and
10
8 M dexamethasone.
RNA isolation and Northern analysis.
Total RNA was isolated by guanidine thiocyanate-phenol-chloroform
extraction methods as previously described (6). Quantity and purity
were determined spectrophotometrically. Five micrograms per sample were
size fractionated in 2.2 M formaldehyde-1% agarose gel by
electrophoresis at 20 V for 14-16 h. Ethidium bromide staining of
ribosomal 28S and 18S bands on the gel was visualized with ultraviolet
(UV) light and was recorded photographically to assure equal lane
loading. RNA was electrotransferred (20 V, 6 h, 4°C) to nylon
membranes. The blotting was verified as uniform across the paper by UV
transillumination of the nylon filter and by recording the 18S and 28S
ribosomal bands photographically. Membranes were baked for 2 h at
80°C and were UV cross-linked.
[32P]CTP-labeled
riboprobes (~1.0 kb) were prepared by SP6-mediated in vitro
transcription from cDNAs spanning the phosphorylation and ATP-binding
site sequences of each subunit that had been subcloned in pGEM3Z
(Promega) generously supplied by J. Emanuel (12). Membranes were
prehybridized in 50% formamide, 10% dextran sulfate, 0.5% nonfat dry
milk, 1% sodium dodecyl sulfate (SDS), 250 µg of sheared salmon
sperm DNA, and 250 µg of yeast tRNA in 6× standard sodium
citrate (SSC) overnight followed by hybridization at
57°C. They were then washed at 65°C in 0.1× SSC and 0.1%
SDS for 1 h and were exposed to X-ray film at 70°C. Multiple
exposures of the autoradiograms were made to ensure that signals were
within the linear range of the film. Bands on autoradiograms were
quantitated with a Hoeffer Scientific GS300 scanning densitometer, and
the area of the peak was determined with Lakeshore Technologies Gelscan Software. In all cases, triplicate RNA samples from
dexamethasone-treated cells and time-matched controls were analyzed
simultaneously on the same nylon membrane.
Transport measurements. Ouabain-sensitive 86Rb+ uptake was used to assess the rate of K+ transport by the Na-K-ATPase in alveolar epithelial cells. ATII cells were incubated at 37°C with and without 1 mM ouabain for 5 min. This medium was then removed, and otherwise identical fresh medium containing 1 µCi/ml 86Rb+ was added. After a 5-min incubation, uptake was terminated by aspirating the assay medium and by washing the plates in ice-cold MgCl2. Plates were allowed to air dry, and cells were solubilized in 1 N NaOH. 86Rb+ influx was quantitated in aliquots of the NaOH extract by liquid scintillation counter. Protein was quantitated in aliquots by the Bradford method. Initial influx, expressed as nanomoles of K+ per minute per milligram protein, was calculated from
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(1) |
Membrane preparation for Na-K-ATPase hydrolytic activity.
Briefly, alveolar epithelial cells were washed three times with
phosphate-buffered saline (PBS), then scraped with a rubber policeman
in 1 ml of homogenization buffer [5 mM histamine-imidazole, 2 mM
EDTA, 1 mM ethylene glycol-bis(-aminoethyl
ether)-N,N,N',N'-tetraacetic acid, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 60 µg/ml soybean trypsin inhibitor], and rinsed with an additional 1 ml of the same buffer. Alveolar epithelial cells were resuspended in
1 ml of homogenization buffer and were disrupted in a Teflon homogenizer for 20 strokes. The homogenate was centrifuged (5 min,
1,000 g, 4°C) to remove unbroken
cells and debris. The supernatant was then centrifuged at 60,000 g at 4°C for 1 h. The high-speed supernatant was designated the cytosolic fraction. The high-speed pellet was resuspended in 500 µl of homogenization buffer and was
designated membrane fraction. Total protein concentrations of cytosol
and membrane fractions were determined by Bio-Rad protein assay with
BSA as the standard.
Na-K-ATPase hydrolytic activity.
Na-K-ATPase activity was determined in membrane fractions of alveolar
epithelial cells as previously described (16, 21). Briefly, the total
activity reaction mixture contained (in a final volume of 500 µl; in
mM) 130 NaCl, 20 KCl, 3 ATP, 3 MgCl2, and 30 imidazol, the
ouabain-sensitive reaction mixture contained the same with 3 mM
ouabain. Na-K-ATPase activity was determined by preincubating the
microsomal fraction at 37°C in buffer for 30 min. The results were
corrected for spontaneous hydrolysis of ATP. The reaction was stopped
with 1% trichloroacetic acid, and the precipitated proteins were
pelleted. Pi was determined by the
method (11) that utilized
molybdate-H2SO4
solution with a Fiske reducing agent. The specific activity of the
enzyme is expressed as nmol
Pi · mg
protein1 · h
1.
Membrane preparation for Western blot analysis. Membranes were prepared from ATII cells the same way as mentioned above but were centrifuged first at 10,000 g for 10 min at 4°C, and the supernatant was centrifuged at 100,00 g for 1 h at 4°C.
Western blot analysis.
Abundance of Na-K-ATPase subunits was determined by Western blot
analysis of ATII plasma membranes. In brief, 5 µg protein/lane of
homogenate for 1 and 50 µg
for
1 were size fractionated on SDS containing 7.5 or 10% polyacrylamide gels (23) and were transferred to nylon membranes. The membranes were quenched overnight in Blotto [5% dry milk and 0.05% Tween 20 in
tris(hydroxymethyl)aminomethane-buffered saline
(TBS)] followed by incubation with Na-K-ATPase
subunit antibodies for 12 h. The antibodies used were
1-specific mouse monoclonal
anti-rat antibody C464-6B, generously provided by M. Caplan, and
rabbit polyclonal anti-rat
1-antibody
FP
1, generously provided by
Alicia McDonough. Blots were washed three times with wash buffer (TBS
and 0.05% Tween 20) and then were incubated with horseradish
peroxide-conjugated goat anti-mouse or anti-rabbit secondary antibody
(Bio-Rad) for 1 h. Blots were washed three times with wash buffer,
developed as previously described using the enhanced chemiluminescence
detection system (Amersham), and analyzed by densitometry (4).
Data analysis. When multiple comparisons were made, a one-way analysis of variance and Duncan's means comparison test were used. The results were expressed as means ± SE. Results were considered significant at P < 0.05.
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RESULTS |
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Effect of dexamethasone on Na-K-ATPase steady-state mRNA
transcript levels in ATII cells.
The steady-state Na-K-ATPase
1-mRNA transcript levels in
ATII cells exposed to dexamethasone were not different from
time-matched control ATII cells at either dexamethasone concentration
(Fig. 1A).
However, the steady-state Na-K-ATPase
1-mRNA transcript levels were
significantly increased after incubation with
10
8 M dexamethasone at 6, 12, and 24 h compared with control levels (Fig.
1B). A representative Northern blot
is shown for the
1 Na-K-ATPase
mRNA (Fig.
2A).
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Effect of dexamethasone on Na-K-ATPase protein levels.
The subunit protein levels were evaluated in ATII cells exposed to
dexamethasone by Western blot analysis. The signal at ~96 kDa
(corresponding to the Na-K-ATPase
1-subunit) increased in ATII
cells after 6 h of exposure to dexamethasone compared with time-matched
ATII control cells (Fig.
3A). A
representative autoradiogram is shown for the
1-protein expression in Fig.
3B. Evaluation of the Na-K-ATPase
1-subunit protein revealed a
band of ~55 kDa that increased by 6, 12, and 24 h after incubation of
ATII cells with dexamethasone compared with time-matched controls (Fig.
4A). A
representative autoradiogram is shown for the
1-protein expression (Fig.
4B).
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Effect of dexamethasone on Na-K-ATPase hydrolytic activity and
86Rb+
uptake.
As shown in Fig.
5A, the
Na-K-ATPase hydrolytic activity increased in ATII cells 12 and 24 h
postincubation with 108 M
dexamethasone (P < 0.05). Figure
5B depicts that the
86Rb+
uptake in ATII cells increased two- and fourfold when incubated for 12 h with 10
7 and
10
8 M dexamethasone,
respectively (P < 0.05).
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DISCUSSION |
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The Na-K-ATPase has been shown to be regulated by corticosteroids in
different organs in vivo and in vitro. In the thick ascending limb of
Henle of the developing rat kidney, increased levels of serum
corticosterone were associated with increased Na-K-ATPase activity and
increased Na+ concentration
capacity (34). Also, 108 M
dexamethasone restored the Na-K-ATPase activity in the medullary thick
ascending limbs of Henle's loop from adrenalectomized rats (9). This
effect was inhibited by cyclohexamide and actinomycin D, indicating the
participation of transcriptional and translational mechanisms. In
adrenalectomized rats, corticosteroids increased the
1 and
1 Na-K-ATPase mRNA transcripts
and renal Na-K-ATPase activity (22).
In the lungs, glucocorticoids have been shown to modulate the timing of lung development and to prevent respiratory distress syndrome in premature infants (2). Also, glucocorticoids have been shown to modulate fibroblasts to stimulate ATII cell surfactant secretion (12) and to decrease lung damage induced by hyperoxia in piglets (7).
Glucocorticoid receptors have been reported in lung fetal fibroblasts
and epithelial cells, suggesting that both cell types could be
potential targets of these hormones and that corticosteroids may play a
role in the maturation of the lung (15). Orlowski and Lingrel (33)
reported a rapid increase in Na-K-ATPase
1- and
1-mRNA transcript levels in the
rat lung during neonatal development; mRNA transcript levels continued
to increase until 2 days after birth and then declined to basal levels.
These changes possibly respond to the need of the lung to clear fluid
during the neonatal stage and to maintain the dry alveoli for adequate gas exchange (30, 33). In a recent study, dexamethasone upregulated alveolar epithelial liquid clearance in anesthetized ventilated rats,
which supports our present findings that these effects may be a result
of increased Na-K-ATPase activity in ATII cells (13). Finally, steady-state Na-K-ATPase
1- and
1-mRNA transcript levels and
Na-K-ATPase protein expression and activity in rat ATII cells increased
in two different models of hyperoxic injury in parallel with changes in
lung edema clearance (28, 31, 41).
In the present study, the steady-state Na-K-ATPase
1-mRNA transcript levels
increased by 2-, 3-, and 1.5-fold after 6, 12, and 24 h of incubation
with dexamethasone without change in the steady state of
1 mRNA transcript levels. These
results are consistent with previous studies in liver cells of
adrenalectomized rats, in which an increase in
1 Na-K-ATPase mRNA was observed
after stimulation with dexamethasone without modifying the expression of the
-subunit (3). Another example of differential regulation of
the
1-subunit mRNA was
demonstrated in
LLC-PK1/Cl4
cells incubated in a low concentration of
K+; the
1 Na-K-ATPase mRNA transcript
levels increased 1.9-fold over control levels at 6 h, resulting in a
2-fold increase of the
- and
-proteins and increased Na-K-ATPase
hydrolytic activity (24). These studies suggest that the
-subunit
could be rate limiting for the assembly of the Na-K-ATPase heterodimer.
Other studies also support the hypothesis that the
1-subunit is critical for the
intracellular transport of the newly synthesized
Na+ pump molecules to the plasma
membrane and that it plays an important role in the correct assembly of
the
-subunit into the membrane for the formation of the Na-K-ATPase
heterodimers (29) and normal functional properties (1). The
dexamethasone-mediated regulation of the
1 Na-K-ATPase mRNA observed in
this report is compatible with the previous studies and could be due to
increased gene transcription as shown in infant rat kidney in which
glucocorticoids directly stimulated transcription of the
1 and
1 Na-K-ATPase subunits (45).
However, interpretation of the effect of glucocorticoids in isolated
ATII cells is limited by changes that may have been induced during the
isolation procedure and cell culture.
We also found that the Na-K-ATPase
1-isoform protein was increased
in ATII cells incubated with dexamethasone for 6 h after incubation and
that the
1-subunit increased at
6, 12, and 24 h (see Figs. 3 and 4). The increase in Na-K-ATPase
1-protein was not associated
with increases in the
-isoform mRNA, suggesting that
posttranslational events may be involved in the regulation of
Na-K-ATPase in this model.
The dynamics of the events that occur in the Na-K-ATPase synthesis and
transport to the plasma membrane have been previously studied. After
their synthesis in polysomes that become bound to the membrane of the
rough endoplasmic reticulum, the - and
-subunits of the
Na-K-ATPase are transported through a network of membrane compartments
to their functional site in the cell surface. Tamkun and Fambrough
(42), in pulse chase experiments with cultured chick neurons, found
that this process is achieved within 50 min of synthesis. The delivery
of newly synthesized Na-K-ATPase to the plasma membrane continues for
several hours, and 20% of the cells' pulse-labeled
Na+ pump appears to be retained
intracellularly. The synthesis of proteins that we analyzed by Western
blot in the membranes obtained from ATII cells was done without a
subcellular fractionation, so we could not differentiate between
endoplasmic reticulum, Golgi, or plasma membrane. Thus a limitation of
our study is the uncertainty of whether the Na-K-ATPase protein
subunits are in the plasma membrane or in the intracellular pool (19,
27). Conceivably, the increase in
-isoform protein in our model
could be due to mobilization of this protein from intracellular pools
by translocation to the plasma membrane or other posttranslational
events (19).
Our study shows an increase in the expression of
1-mRNA that corresponds to an
increase in the protein expression of the
1-subunit at the same time
points. The
1-mRNA did not
increase when ATII cells were incubated with dexamethasone; however,
the
1-protein abundance
increased at 6 h. Therefore, we reason that dexamethasone could have
increased the Na-K-ATPase function in ATII cells as a result of
transcription, translation, and posttranslational events. This is shown
in Figs. 3 and 4. The
86Rb+
uptake in ATII cells was increased after 12 h of exposure compared with
control ATII cells and decreased to control values by 24 h after
exposure (see Fig. 5A), whereas the
hydrolytic activity of the Na-K-ATPase was increased for up to 24 h
after exposure to dexamethasone (see Fig.
5B). The observed differences
between the two Na-K-ATPase assays can be explained by the somewhat
different aspects of the Na-K-ATPase function that these assays
measure. The
86Rb+
uptake measures transport across the ATII cell, whereas the hydrolytic assay measures the release of Pi
under maximal velocity conditions in isolated microsomes
from ATII cells. Finally, the effect of dexamethasone in ATII cells
appears to be biphasic. This may be a result of dexamethasone
consumption or the glucocorticoid receptors may have become
desensitized before the end of the incubation period.
The increase in Na-K-ATPase activity induced by dexamethasone in ATII cells could be explained as an increase of transport efficiency or as an increase in the number of active pumps in the surface of the cell. Chapman et al. (5) demonstrated that the cation transport by rabbit lung epithelial cells changes at birth and that ouabain-sensitive Rb uptake is greater in lung epithelial cells obtained from newborn rabbits than in cells obtained from pups. There is also a postnatal increase in ouabain-sensitive Rb uptake that reached adult values at 30 days after birth. They calculated the turnover rate of the enzyme, concluding that there is an increase in the Na-K-ATPase turnover near birth, whereas the number of Na+ pumps increases postnatally (5).
In summary, this study demonstrates that the exposure of rat ATII cells
in culture to dexamethasone results in increased steady-state Na-K-ATPase 1-mRNA transcript
levels and increased
1- and
1-protein abundance that is
associated with increased Na-K-ATPase function measured by two
functional assays. We thus reason that corticosteroids could be
utilized as a strategy to regulate Na-K-ATPase in the alveolar
epithelium.
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ACKNOWLEDGEMENTS |
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We thank M. Passos, G. Pelluffo, and B. Gonzalez-Flecha for help in this work.
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FOOTNOTES |
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This study was supported in part by National Heart, Lung, and Blood Institute Grant HL-48129, the American Heart Association of Metropolitan Chicago, the American Lung Association Career Investigator Award (to J. I. Sznajder), and Michael Reese Hospital and Medical Center.
Address for reprint requests: J. I. Sznajder, Michael Reese Hospital and Medical Center, Pulmonary Medicine, RC 216, 2929 S. Ellis Ave., Chicago, IL 60616.
Received 30 December 1996; accepted in final form 2 July 1997.
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