Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455
Submitted 4 November 2002 ; accepted in final form 4 April 2003
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ABSTRACT |
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alveolar cell lines; protein translocation; sodium pump
Although much information about Na-K-ATPase is available, regulation of
Na-K-ATPase and the underlying mechanisms are not completely understood
(52). The many mechanisms for
regulation of Na-K-ATPase activity in a tissue-specific manner have been
reviewed (52). Long-term
regulation of Na-K-ATPase activity usually involves changes in mRNA/protein
synthesis or degradation of Na-K-ATPase subunit isoforms
(52). Short-term rapid
alternations in Na-K-ATPase activity may be mediated by changes in subcellular
distribution of pump units
(52), by reversible
noncatalytic-site phosphorylation of the 1-subunit
(21), or by changes in
intracellular Na+ concentration. Many hormones regulate
Na-K-ATPase. Insulin,
-adrenergic agonists, and dopamine can rapidly
modulate Na-K-ATPase activity
(51,
52). In some studies, dopamine
(46) or
-adrenergic
receptor stimulation by isoproterenol
(4) or cAMP analogs
(24) rapidly increased cell
surface expression of Na-K-ATPase
-subunits in rat lung epithelia,
whereas in rat proximal tubule cells, dopamine stimulated endocytosis of this
enzyme from the plasma membrane
(11,
56).
Thyroid hormones play a fundamental role in regulation of normal cell
function and differentiation by interacting with intracellular thyroid hormone
receptors and transcriptional coregulatory factors (coactivators and
corepressors) (7,
54,
55). Thyroid hormones also may
generate biological responses by nongenomic mechanisms that are independent of
nuclear receptors for 3,3',5-triiodo-L-thyronine
(T3) (13,
18,
55). Thyroid hormone
stimulates Na-K-ATPase activity in responsive tissues and differentially
regulates Na-K-ATPase isoforms
(6,
28). T3 response
elements exist in the 5'-flanking region of Na-K-ATPase - and
-subunits (20,
29). In nonlung tissues, the
T3-induced increases in Na-K-ATPase activity generally are due to
thyroid hormone-induced synthesis of Na-K-ATPase mRNA or protein
(36-39).
T3 regulates the activity and gene expression of Na-K-ATPase in
tissue- and cell type-specific manners
(17,
52). For example,
T3 upregulates Na-K-ATPase activity in rat liver, skeletal muscle,
and kidney (39) but inhibits
synaptosomal Na-K-ATPase activity in rat cerebral cortex
(48). Na-K-ATPase activity is
stimulated by T3 in K562 human erythroleukemia cells
(1), but not in human
submandibular gland cells
(34). T3 increases
significantly Na-K-ATPase activity in liver, skeletal muscle, kidney, small
intestine, cardiac muscle, and heart; however, the effect of T3 on
Na-K-ATPase in the lung has not been determined.
The clearance of alveolar fluid is essential for recovery from adult lung injury and respiratory distress syndrome. T3 pretreatment stimulates alveolar epithelial fluid clearance by 65% in normal adult rat lung (22) and exerts a synergistic effect with dexamethasone in adult rat lung (22) and with hydrocortisone in the developing sheep lung on stimulation of alveolar fluid clearance (2). Although the cellular mechanism of T3 stimulation in fluid clearance is unknown, reabsorption of fluid out of the distal air space in the lung usually is driven by active Na+ transport (16, 40), and increased edema clearance usually requires higher Na-K-ATPase activity in alveolar epithelial cells (AEC) (40, 43). T3-induced Na-K-ATPase activity may contribute to the alveolar fluid clearance stimulated by T3 in adult rat lung. Therefore, the first objective of this study was to test the hypothesis that T3 stimulates Na-K-ATPase activity in adult rat lung epithelia, and if so, the second purpose was to explore possible mechanisms involved in this effect.
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MATERIALS AND METHODS |
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Isolation and culture of rat AEC. ATII cells were isolated from pathogen-free adult male Sprague-Dawley rats (190-220 g) as we previously described (32). Use of the animals complied with guidelines for their ethical treatment, and the studies were approved by the Institutional Animal Care Committee. ATII cells were grown in DMEM-Ham's F-12 medium with 10% FBS and antibiotics (50 U/ml penicillin and 50 U/ml streptomycin) overnight. The cells were cultured in DMEM-Ham's F-12 medium with 5% stripped FBS for 24 h before T3 treatment.
Cell culture. The rat ATII cell line RLE-6TN (15) was purchased from American Type Culture Collection, and the MP48 cell line (44) was a gift from G. Hunninghake (University of Iowa). Both cell lines exhibit characteristics of ATII cells, such as lipid-containing inclusion bodies. RLE-6TN cells are derived from male Fischer 344 rats after simian virus 40-T antigen transformation. They stain positive for cytokeratins and lipids (phosphine 3R) and have alkaline phosphatase activity but do not express T antigen. MP48 cells are adenoviral 12S E1A immortalized diploid adult rat type II cells that resemble type II cells in situ, contain lamellar bodies, bind the lectin Maclura pomifera I, and stably express cytokeratins and the E1A gene product. MP48 cells were maintained in Waymouth's MB752/1 medium. RLE-6TN cells were grown in DMEM-Ham's F-12 medium. Both media were supplemented with 10% FBS, 40 mM HEPES, and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B). Antibiotics, including amphotericin, were included in the standard culture media for the cell lines and during T3 exposure but were not present during the Na-K-ATPase activity assay. Removal of amphotericin did not alter Na-K-ATPase activity in MP48 cells.
Experimental design. To measure the effect of T3 on the
hydrolytic activity of Na-K-ATPase, the cells were grown to 80%
confluence in the appropriate medium containing 10% FBS. In most, but not all,
experiments, the cells were T3 starved for 24 h; i.e., cells were
cultured in appropriate medium supplemented with stripped 10% FBS; then the
monolayers were exposed to T3 or inhibitors for various time
intervals in appropriate medium plus 1% stripped FBS. At the indicated times,
the cell monolayers were washed twice with ice-cold phosphate-buffered saline
(PBS) to terminate exposure to all agents. In some experiments, cells were not
preexposed to stripped serum, but after culture with 10% FBS, T3
was added to the medium with 1 or 10% FBS, and Na+ pump activity
was measured. For inhibition studies, cells were preincubated with inhibitors
for 30 min at 37°C and then treated with T3 in the continued
presence of inhibitors for various time intervals.
Preparation of crude AEC membranes. Crude cell membranes were prepared as previously described (51) with slight modification. Briefly, the cells were scraped in ice-cold PBS and centrifuged at 5,000 rpm at 4°C for 5 min. The cell pellets were resuspended in 500 µl of ATPase assay buffer containing 37.5 mM imidazole, 137.5 mM NaCl, 18.75 mM KCl, 6.25 mM sodium azide, 0.625 mM EGTA (pH 7.0), 5 mM MgCl2, and 0.005% SDS for MP48 cells and primary ATII cells and 0.01% SDS for RLE-6TN cells. The cells were sonicated on ice for 10 s with Vibra Cell (Sonics and Materials, Danbury, CT) at 45% duty cycle and 4.5 output control on ice and centrifuged at 10,000 rpm at 4°C for 10 min. The sediment was resuspended by sonication for 8 s in 350-500 µl of the ATPase assay buffer described above. Aliquots of crude cell membrane preparations were used for assay of protein contents by using the BCA protein assay kit (Pierce, Rockford, IL) and Na-K-ATPase activity. Na-K-ATPase activity assays were performed immediately after isolation of crude cell membranes.
Na-K-ATPase activity assay. The hydrolytic activity of Na-K-ATPase was measured as ouabain-sensitive ATP hydrolysis under maximal velocity conditions by measuring the release of inorganic phosphate (Pi) from ATP, as previously described (32, 37) with slight modification. Briefly, equal amounts of crude cell membrane (20, 30, and 50 µg for RLE-6TN, MP48, and primary ATII cells, respectively) for all treatments in an experiment were added to 1 ml of ATPase assay buffer with or without 10 mM ouabain. The tubes were incubated for 18 min at 37°C after the reaction was started by the addition of 10 µl of 0.439 M ATP (Sigma Chemical). The reaction was terminated by addition of 50 µl of 100% ice-cold trichloroacetic acid, and Pi was determined by the method of Fiske and Subbarow as described elsewhere (37). Na-K-ATPase specific activity was taken as the difference in Pi concentration per milligram of protein per minute in the absence and presence of 10 mM ouabain. Results were compared with controls and expressed as percent increase or inhibition of control Na-K-ATPase activity.
RNA isolation and Northern analysis. Total cellular RNA was isolated using TRI Reagent (Molecular Research Center, Cincinnati, OH) following the manufacturer's instructions. RNA was isolated, and Northern analysis was performed as previously described (30). The purity and concentration of RNA were assessed from the ratio of absorbance at 260 nm to that at 280 nm. After ultraviolet irradiation using UV Stratalinker 1800 (Stratagene), prehybridization, and hybridization were performed using the High-Efficiency Hybridization System (Molecular Research Center) following the instructions of the manufacturer. Transcripts were visualized with standard autoradiography or phosphorimaging (Bio-Rad) and quantitated. The integrated optical density of the RNA bands was determined with Densitometry Image software (Molecular Analyst). All RNA densitometry values were normalized to 18S rRNA. The experiments were performed at least in triplicate.
Western blot. Protein samples were incubated in boiling water for
10 min in loading buffer containing 2% SDS, 50 mM Tris · HCl (pH 7.5),
10% glycerol, 2% -mercaptoethanol, and 0.001% bromphenol blue. For the
1-subunit, the concentration of SDS and
-mercaptoethanol was 5.6 and 6%, respectively. Proteins were then
separated on a 7.5 or 10% denaturing SDS-polyacrylamide gel for Na-K-ATPase
1- and
1-subunits, respectively. Separated
proteins were transferred to a polyvinylidene difluoride membrane (catalog no.
IPVH00010, Millipore, Bedford, MA), which was then hybridized with primary
mouse antibodies against the Na-KATPase
1-subunit (catalog
no. 05-369, Upstate Biotechnology) or
1-subunit (catalog no.
05-382, Upstate Biotechnology) diluted 1:2,000. After incubation with a
secondary peroxidase-conjugated goat anti-mouse antibody diluted 1:2,500
(Sigma Chemical), the membranes were washed and the proteins were detected
with the ECL system (Amersham, Piscataway, NJ). The chemiluminescence of each
lane was quantified by densitometry in arbitrary units using a Bio-Rad Image
Analysis system (Molecular Analyst).
Measurement of total cell and plasma membrane Na-KATPase protein. For measurement of total cellular Na-KATPase subunit proteins, the cells were washed twice with ice-cold 1x PBS, scraped off plates, and collected in ice-cold PBS. The cells were pelleted with centrifugation at 5,000 rpm at 4°C for 5 min. To lyse the pellets, we drew them 10 times through a syringe and 25-gauge needle in 400 µl of lysis buffer containing 50 mM Tris · HCl (pH 7.5), 150 mM NaCl, 2 mM EDTA, and 1% (vol/vol) Nonidet P-40, with protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin, and 10 µg/ml each aprotinin and leupeptin). Protein concentrations were determined using the BCA protein assay kit. Equal amounts of protein were subjected to Western blot analysis as described above. The densitometry values of Na-K-ATPase subunit proteins were normalized to actin protein.
For determination of plasma membrane Na-K-ATPase, the cell surface proteins
were biotinylated as described previously
(24,
25) with minor modification.
Cells were grown on 24-mm Transwell porous cell culture inserts (0.4-µm
pore; Nucleopore polycarbonate membrane, catalog no. 3412, Costar, Cambridge,
MA). T3 and/or inhibitors were added to the apical and basolateral
sides of the filter for indicated time periods. The cells were incubated in
PBS containing 2 mM EDTA at 4°C for 5 min after being washed twice with
ice-cold 1x PBS. Then cell surface proteins were biotinylated by
addition of biotinylation buffer (10 mM triethanolamine, pH 7.5, 1 mM EDTA,
and 150 mM NaCl) containing 1 mg/ml water-soluble, cleavable sulfobiotin-X-NHS
(Calbiochem, San Diego, CA) to the apical and basolateral sides of the filter
for 30 min at 4°C. The free unreacted biotin was quenched by three washes
with 1x PBS containing 100 mM glycine. Cells were then lysed in lysis
buffer, and protein content was determined by using the BCA protein assay kit.
Equal amounts of total cell protein were precipitated with
streptavidin-agarose beads (Sigma Chemical) diluted in lysis buffer by
incubation overnight at 4°C. The beads were then washed three times with
lysis buffer, twice with high-salt buffer (5 mM EDTA, 50 mM Tris · HCl,
pH 7.4, and 500 mM NaCl), and once with 10 mM Tris · HCl (pH 7.4).
Proteins were eluted from the beads by incubation of the biotinylated
protein-streptavidin-agarose beads for 10 min in 50 µl of SDS-containing
buffer (5.6% SDS, 240 mM Tris · HCl, pH 7.5, 6% -mercaptoethanol,
16% glycerol, and 0.008% bromphenol blue) and analyzed by Western blotting.
The higher SDS concentration disrupts the avidin-biotin complexes. The amount
of protein was expressed as densitometry in arbitrary units.
Statistics. Values are means ± SD of a minimum of three experiments. Comparisons involving three or more groups were analyzed by ANOVA and post hoc pairwise comparisons. Differences between means were considered significant at P < 0.05, adjusted for the number of comparisons.
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RESULTS |
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MP48 cells were cultured for 24 h in media with 10% stripped FBS and then
exposed to T3. T3 increased the enzyme activity in a
dose-dependent fashion when presented in 1% stripped FBS or 1% FBS culture
conditions (Fig. 1A);
activity increased by >40% (P < 0.05) at concentrations as low
as 10-9 M at 6 h of incubation. The
T3-induced maximal increase was 2.2-fold at
10-5 M in the presence of stripped 1% FBS medium. The
magnitude of the increase in Na-K-ATPase activity at every T3 dose
in 1% FBS was somewhat less than that in 1% stripped FBS. Smaller increases in
Na-K-ATPase activity occurred when T3 was presented in 10% FBS
(data not shown).
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Similarly, in RLE-6TN cells, T3 increased Na-KATPase activity at 3 and 6 h and when presented in 1% stripped or 10% FBS (Fig. 1B and data not shown). At 3 h, T3 concentrations as low as 10-8 M increased Na+ pump activity in a dose-dependent fashion. The magnitude of the increase was greater at supraphysiological T3 levels but was statistically significant (30% increase, P < 0.05) at 10-8 M. In contrast to T3, 6 h of exposure to the metabolically inactive analog of T3 (50), reverse T3, at the same concentrations had no effect on Na-K-ATPase activity, indicating that the effect of T3 is specific.
Because the cell lines may not reflect the response of the primary rat ATII cells, we also assessed the effect of T3 on Na-K-ATPase activity in primary ATII isolates at 3 days of culture. The cells were T3 starved by exposure to 5% stripped FBS for 24 h before addition of T3 in 1% stripped FBS. T3 increased the Na-K-ATPase activity at 6 h of 10-8 MT3 by 131.7% and at 3 h of 10-5 M T3 by 149.3% (Fig. 1C). Because the responses to T3 were similar to those of AEC, complete dose-response curves were not determined.
The time course of the T3 effect on Na-K-ATPase activity in MP48 cells is shown in Fig. 2. Surprisingly, the T3-induced increase in Na-K-ATPase activity occurred rapidly at 10-5 or 10-6 M T3 in 1% stripped FBS. The increase in Na-K-ATPase activity was significant at 1 h of T3 exposure and peaked at 6 h (Fig. 2A). Pharmacological levels of T3 (10-4 M) also rapidly increased Na-K-ATPase activity in RLE-6TN cells in 10% FBS (Fig. 2B), with the maximal effect at 6 h. The rapid T3-induced increase in Na-K-ATPase activity suggested that the T3-induced increase may not require transcription or translation of Na-K-ATPase.
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T3 does not alter expression of
Na-K-ATPase 1- and
1-subunit mRNA and total cellular protein.
The principal mechanism for action of T3 on Na-K-ATPase usually is
transcriptional (7,
55), but in our study, the
rapid increase in Na-K-ATPase activity observed within 0.5-1 h of exposure
suggested that T3 stimulation was not transcriptional. To test this
hypothesis, the change of Na-K-ATPase mRNA and protein levels was determined
after cells were subjected to T3 treatments for various periods.
Although the ATII cells expressed some quantities of the Na-K-ATPase
1-,
2-,
3-,
1-, and
3-isoforms, the predominant
Na-K-ATPase isoforms that we detected in adult rat primary ATII cells and in
the cell lines are
1,
1, and
3 (data not shown). Therefore, we investigated the effect of
T3 on
1- and
1-subunit mRNA and
proteins to identify whether T3 affects the Na-K-ATPase activity in
a fast manner by regulating the gene expression of
1- and
1-subunits in adult alveolar cells.
In MP48 cells, T3 did not significantly alter the steady-state
contents of Na-K-ATPase 1- and
1-subunit
mRNA at 6 h of exposure (P > 0.05;
Fig. 3). The Northern blot for
the
1-subunit mRNA revealed two bands just below the 18S rRNA
marker, with the signal much stronger for the lower band.
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T3 also did not significantly change the protein levels of
1- and
1-subunits at
10-9-10-5 M T3 at 6 h
of exposure (Fig. 4, A and
B) and at 10-5 M at 1-6 h of
exposure (Fig. 4, C and
D).
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In RLE-6TN cells, Northern blot quantification of the steady-state mRNA
levels of the 1-,
2-,
3-,
1-, and
3-subunits was
performed to determine whether T3 induced a shift in Na-K-ATPase
isoform expression. At 6 h of T3 exposure, there was no significant
change in
1- and
1-subunit levels in each
of three experiments (data not shown). At 24 h,
10-8-10-6 M T3 did not
increase the levels of any of the subunits (n = 3, mean change
<10% for each subunit at 10-4 M T3). High
T3 concentrations (10-5 M) led to small
increases in the mRNA
1-subunit (mean 30% increase,
n = 3) but not of other mRNA subunit levels during 24 h of exposure.
Thus T3 did not increase the total cellular mRNA and protein levels
of Na-K-ATPase subunits during the period with significantly increased
Na-KATPase activity. These data suggested that the T3-induced
increase in Na-K-ATPase activity was independent of transcription.
Actinomycin D does not block the
T3-induced increase in Na-K-ATPase activity.
Because the principal action of T3 is transcription and
T3 could affect transcription of other Na-K-ATPase genes beyond the
1- and
1-subunits, we wished to test more
directly whether transcription was required for T3 stimulation of
Na-KATPase. The general inhibitor of transcription, actinomycin D, was used to
determine the role of transcription in T3 stimulation. MP48 cells
were T3 starved for 24 h in 10% stripped FBS medium, and then cells
were exposed to 10-6 M T3 with or without 10
µg/ml actinomycin D for 6 h in 1% stripped FBS. T3 stimulation
of Na-K-ATPase activity was not diminished by actinomycin D
(Fig. 5A). Similarly,
actinomycin D did not inhibit T3 stimulation in MP48 cells at 3 or
6 h in the presence of 1% FBS (data not shown). Similarly, in RLE-6TN cells,
actinomycin D did not inhibit T3 stimulation of Na+ pump
activity at 3 or 6 h, even at very high T3 concentrations
(10-4 M; Fig.
5B). Actinomycin D alone had no significant effect on
Na-K-ATPase activity in MP48 and RLE-6TN cells. It is not surprising that
actinomycin D also had little inhibitory effect on Na-K-ATPase, because the
half-life of the Na+ pump protein is long in primary ATII cells and
Madin-Darby canine kidney cells (unpublished data). Thus these data strongly
support the concept that T3 stimulation of Na-K-ATPase activity is
principally through a nontranscriptional mechanism.
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T3 increases Na-K-ATPase subunit protein
levels in plasma membrane of MP48 cells. The short-term
posttranscriptional regulation of Na-K-ATPase activity by hormones may involve
direct effects on the kinetic behavior of the enzyme or translocation of the
enzyme between the plasma membrane and intracellular stores
(52). On the basis of the
minimal effect of T3 on mRNA and protein expression of Na-K-ATPase
subunits in MP48 and RLE-6TN cells, we hypothesized that T3
stimulates rapid recruitment of Na-K-ATPase proteins to the plasma membrane in
MP48 and RLE-6TN cells. To investigate the effect of T3 on cell
surface expression of Na-K-ATPase, intact cells treated with T3
were reacted with a membrane-impermeable biotinylation reagent
(sulfobiotin-X-NHS, water soluble, cleavable) that labels proteins exposed on
the cell surface but not proteins in intracellular membranes. T3
increased the amount of Na-K-ATPase 1- and
1-subunit proteins at the cell surface in MP48 cells
(Fig. 6). T3 at
10-5 M stimulated a significant increase in
1-and
1-subunit proteins at the cell
surface after 6 h of T3 exposure; the maximal increase was 1.7-fold
for the
1-subunit and 2-fold for the
1-subunit. Significant augmentation (P < 0.05) of
1-subunit (133.9% of control, n = 4) and
1-subunit (131.5% of control, n = 3) proteins at the
plasma membrane also was induced at 1 h of T3 exposure
(Fig. 6). T3 did not
significantly change the total cell protein level of the Na-K-ATPase
1-subunit at 6 h of T3 treatment
(Fig. 4); however, it markedly
increased the amount of Na-K-ATPase
1-and
1-subunit protein at the cell surface. Taken together with
the prior findings, these data suggest that the T3-induced increase
in Na-K-ATPase activity and protein at the membrane is due to increased plasma
membrane recruitment of this enzyme and not increased de novo protein
synthesis.
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Brefeldin A decreases T3-induced plasma
membrane expression and activity of Na-K-ATPase. Brefeldin A disassembles
and redistributes the Golgi complex into the endoplasmic reticulum within
minutes of application (8) to
disrupt cellular protein trafficking. Brefeldin A inhibits protein transport
at various concentrations. Brefeldin A at 1-10 µg/ml completely inhibited
protein secretion in cultured rat hepatocytes
(42), and at 20 µg/ml, it
prevented the dibutyryl-cAMP-dependent increase in cell surface expression and
activity of Na-KATPase in renal epithelia
(24). The effect of 10
µg/ml brefeldin A on the T3-induced increase in protein content
of Na-K-ATPase in the plasma membrane was determined.
Figure 7, A and
B, shows that brefeldin A alone did not significantly modify the
basal quantity of cell surface Na-K-ATPase 1- and
1-subunit proteins but completely abolished the
T3-induced increment in plasma membrane expression of Na-K-ATPase
1- and
1-subunits in MP48 cells during 6 h
of exposure. Thus T3 increased cell surface expression through a
brefeldin A-dependent process in MP48 cells, suggesting that T3
stimulation of Na-K-ATPase trafficking is related to the Golgi complex. As
expected, 10 µg/µl brefeldin A also completely blocked the
T3-induced increment in Na-K-ATPase activity but did not alter
basal Na-KATPase activity in MP48 cells
(Fig. 7C). Abolition
of the T3-induced plasma membrane expression and activity of
Na-K-ATPase by brefeldin A established that T3-dependent
translocation of Na-K-ATPase is responsible for upregulation of the enzyme
activity.
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DISCUSSION |
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T3 specifically stimulates Na-K-ATPase activity in primary ATII cells and two alveolar cell lines. This finding is important for several reasons. Although Na-K-ATPase is a ubiquitous and essential cell enzyme that has been studied intensively, tissue-specific regulation of Na-K-ATPase and the underlying mechanisms are not completely understood (52). Specifically, little is known about the T3 effect on Na-K-ATPase function in the lung. Because Na-K-ATPase is necessary for active Na+ and fluid transport across the alveolar epithelia (43) and T3 stimulates the alveolar fluid in adult rat lung (22), we hypothesized that T3 would stimulate AEC Na-K-ATPase. However, no prior studies have investigated whether the thyroid hormone activates AEC Na-K-ATPase. In the present study, we investigated T3 effects on Na-K-ATPase in primary ATII cells and two adult rat AEC lines, MP48 and RLE-6TN. We demonstrated that T3 stimulated alveolar epithelial Na-K-ATPase activity in a dose- and time-dependent fashion, as T3 does in rat liver cells (27). The inactive isomer reverse T3 had no significant effect on Na-K-ATPase activity in RLE-6TN cells, indicating that the T3 effect is specific.
In contrast to many other tissues or cell types, the stimulatory effect of
T3 on Na-K-ATPase activity in AEC is not derived from
transcriptional augmentation. Our data show that T3 stimulation of
Na-K-ATPase activity was detected within 60 min in MP48 cells and within 30
min in RLE-6TN cells, with maximal stimulation at 6 h. Similarly,
T3 rapidly activates Na-KATPase in liver of the Anabas
fish (49). The mechanism
underlying the short-term effect of T3 on Na-K-ATPase remains to be
clarified. T3 is a well-known transcriptional regulator for
Na-K-ATPase genes in an isoformspecific fashion
(17,
29). There are positive
thyroid hormone response elements in the 5'-flanking region of the
Na-K-ATPase 1-subunit
(20) and negative thyroid
hormone response elements in the 5'-flanking region of Na-K-ATPase
2- and
3-subunits
(29). T3 stimulates
Na-K-ATPase activity and isoform mRNA levels in various mammalian tissues
(17,
23,
29). However, in our AEC,
T3 did not change the steady-state levels of
1-
and
1-subunit mRNAs (Fig.
3). Moreover, the T3-induced Na-K-ATPase activity was
not sensitive to a general inhibitor of gene transcription, actinomycin D. The
failure of actinomycin D to block the T3-induced Na-K-ATPase
activity directly demonstrated that T3 stimulated the Na-K-ATPase
activity in a transcription-independent manner. Virtually all thyroid hormone
effects previously were believed to be transcriptional; however, nongenomic
effects of thyroid hormone are gaining some recognition
(13,
18,
55).
The mechanisms by which thyroid hormone regulates Na-K-ATPase vary among
tissues. T3 increases the protein level of Na-K-ATPase in kidney,
heart, and skeletal muscle tissues
(17); however, the effects of
T3 on lung Na-K-ATPase subunit protein have not been reported. Our
data indicate that the T3-induced increase in Na-K-ATPase activity
was not accompanied by an increase in total cell Na-K-ATPase
1- and
1-subunit protein, suggesting that
T3 also does not affect translation of Na-K-ATPase mRNA at 6 h.
However, the detailed effects of T3 on translation of Na-KATPase
subunits and rate of translation need further investigation.
T3 increased plasma membrane Na-K-ATPase enzyme in AEC, likely
because of stimulation of translocation. T3 previously has been
reported to affect sorting and trafficking of proteins. T3
stimulates the translocation of Trip230, a coactivator of thyroid hormone
receptor, from the Golgi complex to the nucleus
(10), of choline
phosphotransferase from cytosol to mitochondria
(9), and of type II
iodothyronine 5'-deiodinase from plasma membrane to endosomes
(19). In our study,
T3 did not change the total cell content of Na-K-ATPase
1- and
1-subunit proteins but increased the
plasma membrane expression. The T3-induced increase of Na-K-ATPase
protein at the cell surface was abolished by brefeldin A, a potent inhibitor
of translocation that disassembles the Golgi complex and redistributes it into
the endoplasmic reticulum
(10). Because brefeldin A also
may alter recycling of plasma membrane proteins, these data strongly suggest,
but do not prove, that T3 stimulates delivery of Na-KATPase to the
cell surface via the endoplasmic reticulum-Golgi complex constitutive pathway
in AEC. It is somewhat surprising that brefeldin A did not affect basal
Na-K-ATPase activity or membrane protein quantity. The effects of
T3 on internalization of Na-KATPase and the mechanism(s) underlying
the control of T3 on trafficking of Na-K-ATPase remain to be
determined.
The T3-stimulated translocation of Na-K-ATPase enzyme to the cell surface is necessary for T3-induced Na-K-ATPase activity in both AEC lines. Lo and Edelman (38) reported that the T3-induced increase in Na-K-ATPase activity in rat kidney in vivo appeared to occur through stimulation of enzyme synthesis, as they measured incorporation of labeled methionine into the protein in a membrane-rich fraction. However, because their assayed proteins came from the membrane-rich fraction, the observed increase in enzyme protein may have resulted from translocation of Na-K-ATPase enzyme units to the membrane, rather than increased synthesis. Indeed, their subsequent study also verified that T3 augmented the number of membrane-bound Na-K-ATPase units in rat kidney (39). Thus we propose that the T3-induced rapid increase of Na-KATPase activity depends on translocation of Na-KATPase to the cell plasma membrane but cannot exclude effects on recycling.
This rapid increase in plasma membrane Na-KATPase is similar to the
augmentation of ATII cell Na-K-ATPase activity by dopamine and
-adrenergic agonists reported by Sznajder and colleagues
(4,
26,
45,
46). In their studies,
increased Na+ pump activity resulted from a rapid translocation of
Na-K-ATPase to the plasma membrane from a late endosomal compartment and from
the slower effects of a mitogen-activated protein kinase pathway. Protein
kinase C and phosphoinositide 3-kinase also regulate Na-K-ATPase protein
trafficking, as occurs with dopamine in the kidney
(56). In our studies of
T3, it is likely that other processes, such as phosphorylation of
Na-K-ATPase, and other T3-activated signals are required for this
short-term stimulation by T3 along with translocation to the plasma
membrane. For example, thyroid hormone stimulates the mitogen-activated
protein kinase in 293T cells, CV-1 cells, and HeLa cells
(14,
35). Therefore, T3
stimulation of Na-K-ATPase function may also be linked to T3
activation of other kinases, such as phosphoinositide 3-kinase or
mitogen-activated protein kinase.
In summary, T3 directly stimulates ATII cell and AEC Na+ pump activity through posttranscriptional activation, likely by increasing translocation of Na+ pump molecules to plasma membrane. Because T3 augments alveolar fluid clearance in vivo (22), we hypothesize that increased activity of Na-K-ATPase is at least part of the in vivo stimulatory mechanism. However, the effects of T3 on apical proteins involved in Na+ transport, such as the epithelial Na+ channel, have not been defined.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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