Glucocorticoids increase sodium pump alpha 2- and beta 1-subunit abundance and mRNA in rat skeletal muscle

Curtis B. Thompson1, Inge Dorup2, Julie Ahn1, Patrick K. K. Leong1, and Alicia A. McDonough1

1 Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, California 90089; and 2 Institute of Physiology, University of Aarhus, 8000 Aarhus C, Denmark


    ABSTRACT
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DISCUSSION
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Fourteen-day adrenal steroid treatment increases [3H]ouabain binding sites 22-48% in muscle biopsies from patients treated with adrenal steroids for chronic obstructive lung disease and in rats treated with dexamethasone (Dex). Ouabain binding measures plasma membrane sodium pumps (Na+-K+-ATPase) with isoform-dependent affinity. In this study we have established the specific pattern of Dex regulation of sodium pump isoform protein and mRNA levels in muscle. Rats were infused with Dex (0.1 mg/kg per day) or vehicle for 14 days. Abundance of sodium pump catalytic alpha 1- and alpha 2-subunits and glycoprotein beta 1- and beta 2-subunits was determined by immunoblot in soleus, extensor digitorum longus, whole gastrocnemius, and diaphragm and was normalized to the mean vehicle control value. Dex increased alpha 2 and beta 1 protein in all muscle types by 53-78% and ~50%, respectively. Dex increased alpha 1 protein only in diaphragm (65 ± 7%). At the mRNA level in whole hindlimb muscle, Dex increased alpha 2 (6.4 ± 0.5-fold) and beta 1 (1.54 ± 0.15-fold) and decreased beta 2 (to 0.36 ± 0.6 of control). In summary, alpha 2beta 1 is the Dex-responsive pump in all skeletal muscles, and changes in alpha 2 and beta 1 mRNA levels can drive the 50% change in alpha 2beta 1-subunits, which can account for the reported increase in [3H]ouabain binding.

Na+-K+-ATPase isoforms; dexamethasone; soleus; extensor digitorum longus; gastrocnemius; diaphragm


    INTRODUCTION
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INTRODUCTION
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RAVN AND DORUP (34) discovered that patients treated in hospital with adrenal steroids and diuretics for chronic obstructive lung disease (COLD) had reduced concentrations of skeletal muscle potassium and elevated muscle sodium pump number, as estimated by [3H]ouabain binding. This was unexpected because a previous study (12) on patients treated with diuretics (and not steroids) showed that the accompanying K+ depletion was associated with decreased concentration of sodium pumps in muscle. In a parallel study in rats, Dorup and Clausen (11) demonstrated that 7-day treatment with dexamethasone, a potent synthetic corticosteroid, increases [3H]ouabain binding to rat skeletal muscle biopsies by 22-42%. This finding indicated that the increased muscle sodium pump number in the COLD patients was due to the glucocorticoid treatment, which could apparently override any decrease in sodium pump number due to diuretic-induced potassium depletion.

Glucocorticoids have been reported to have effects on sodium pumps in a number of tissues. These steroids return sodium pump levels to control levels in kidney cells from adrenalectomized rats (5, 35) and increase sodium pump number in heart cells (15, 21) and cultured aortic smooth muscle cells (40).

Glucocorticoids are routinely administered for a variety of medical conditions (45), and the accompanying effect on muscle sodium pump expression could precipitate potentially dangerous complications (11). For instance, the results summarized above suggest that when a COLD patient is treated with systemic glucocorticoids to reduce smooth muscle inflammation, there will be an increase in skeletal muscle sodium pump number; when an acute attack of airway constriction in these same patients is treated with a beta 2-adrenergic agonist, there will be an acute activation of the pool of sodium pumps already enlarged by glucocorticoid treatment (6-8). The activation of K+ transport into the cell could lower extracellular K+ to the point where it could reduce cardiac excitability/contractility to life-threatening levels (11).

The isoform dependence of glucocorticoid regulation of sodium pumps in muscle has not been previously studied. The sodium pump exists as an alpha -catalytic, beta -glycoprotein heteromer, and muscle expresses two isoforms of each subunit in a muscle fiber type-specific manner (18, 42). Slow oxidative soleus expresses alpha 1-, alpha 2- and beta 1-isoforms (as alpha 1beta 1 and alpha 2beta 1 heteromers), while at the opposite phenotypic extreme, fast glycolytic white gastrocnemius expresses alpha 1-, alpha 2-, and beta 2-isoforms (presumably as alpha 1beta 2 and alpha 2beta 2) (42). Extensor digitorum longus (EDL), a fast metabolically mixed glycolytic and oxidative-glycolytic muscle, expresses all four isoforms. alpha 3- and beta 3-isoforms are not expressed at significant levels in adult skeletal muscle (28, 32, 39, 41). alpha 2 abundance is about the same, per muscle protein, in all muscle types examined, while alpha 1 abundance is twice as high in oxidative soleus and diaphragm as in fast glycolytic white gastrocnemius (41). We have calculated that the ratio of alpha 1 to alpha 2 is around 1:1, on the basis of relative changes in alpha 1, alpha 2, and beta 1 in soleus, and beta 2 in white gastrocnemius, during K+ deprivation (41).

[3H]ouabain binding can be used as a measure of the concentration of sodium pumps because the cardiac glycoside binds to the extracellular face of alpha -catalytic subunits of plasma membrane-localized sodium pumps. However, there are two caveats to this use of ouabain in rat muscle biopsies: 1) the [3H]ouabain will not bind to pumps located in endosomes (17); and 2) in rat, the alpha 1-isoform has far less affinity for ouabain [dissociation constant (Kd) = 5 × 10-5 M] than the alpha 2-isoform (Kd = 10-7-10-9 M). Because of the low affinity for alpha 1-isoform, it has been proposed that the [3H]ouabain binding assay only accurately detects the higher affinity alpha 2-isoform in muscle (11).

On the basis of these observations, we aimed to determine whether the previously reported increase in [3H]ouabain binding to skeletal muscle driven by dexamethasone treatment could be accounted for by an increase in the alpha 1 and/or alpha 2 protein abundance and, if so, whether the changes were driven by changes in the mRNA levels of the subunits. We report that alpha 2 and beta 1 protein and mRNA levels increase after glucocorticoid treatment in all muscles examined, enough to account for the observed increase in [3H]ouabain binding.


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Animals and diets. Two series were conducted, one for protein and another for mRNA analysis, both as previously described (11). In short, female Wistar rats, ~10 wk of age (~200 g), were maintained on standard chow at constant temperature (21°C) under a 12:12-h artificial light cycle with unlimited access to water. Dexamethasone (0.1 mg/kg per day) or vehicle (polyethylene glycol 400) was continuously infused for 14 days via dorsally implanted osmotic minipumps (model 2002; Alzet, Palo Alto, CA). For protein analysis, day 14 rats (n = 5 treated and 5 controls) were anesthetized and decapitated, and the soleus, EDL, whole gastrocnemius, and diaphragm were removed, frozen in liquid nitrogen, and stored at -80°C pending analyses. For RNA analysis (n = 5-6 in each group), whole hindlimb muscle was removed and either extracted directly or frozen in liquid nitrogen pending extraction.

Na+-K+-ATPase subunit protein analysis. Skeletal muscle was homogenized with a Polytron homogenizer 1:20 (wt/vol) in 5% sorbitol, 25 mM histidine imidazole (pH 7.4), 0.5 mM Na2EDTA, and the proteolytic enzyme inhibitors phenylmethylsulfonyl fluoride (PMSF; 0.5 mM), leupeptin (1 µl/ml), and 4-aminobenzamidine dichloride (pABAD; 1 mM), as previously described (42). Protein concentration was determined after TCA precipitation of 10-µl aliquots of the homogenate in triplicate, resuspension of the pellet in NaOH, and assay by the Lowry method (26).

A constant amount of homogenate protein (20 µg for alpha 1- and alpha 2-subunit analysis; 15 µg for beta 1- and beta 2-subunit analysis) was resolved by sodium dodecyl sulfate-polyacrylamide mini-gel electrophoresis (SDS-PAGE) and blotted onto Immobilon-P membranes (Millipore, Bedford, MA). On every blot, one sample from vehicle-treated animals and one sample from dexamethasone-treated animals were also run at one-half the amount of homogenate (10 µg for alpha ; 7.5 µg for beta ) for assessment of linearity of the detection system. To facilitate detection of the sodium pump beta 1- and beta 2-subunits, we removed sugar residues with peptide-N-glycosidase F (PNGase F; Oxford GlycoSciences, Seattle, WA), as previously described (3, 42). Blots were incubated overnight with one of the following antibodies at the dilutions indicated: C464.6 (1:100), a monoclonal antibody specific for alpha 1 (20, 33), provided by M. Kashgarian (Yale Medical School, New Haven, CT); McB2 (1:200), a monoclonal antibody specific for alpha 2 (43), provided by K. Sweadner (Harvard Medical School, Cambridge, MA); anti-beta 1 FP (1:500), a polyclonal antibody against beta 1 (42); and SpET b2 (1:2,000), a polyclonal antibody against human beta 2 (13), provided by P. Martin-Vasallo (Universidad de La Laguna, Tenerife, Spain). To validate consistent protein loading, we subsequently probed a subset of blots with a monoclonal antibody specific for muscle calsequestrin, VIIID12 (Affinity Bioreagents, Golden, CO) (22). Blots probed with monoclonal antibodies were incubated with rabbit anti-mouse IgG secondary antibody (Calbiochem, La Jolla, CA) (1:2,000). Antibody-antigen complexes were visualized by using 125I-labeled protein A and autoradiography, as described previously (27), and linearity was verified by assaying samples at multiple concentrations. Approximate molecular masses were determined by running molecular mass protein standards on each gel (SDS-PAGE Protein Standards; Bio-Rad, Hercules, CA).

Na+-K+-ATPase activity. Whole hindlimb muscle was homogenized by using a procedure specific for the measurement of Na+-K+-ATPase activity in muscle (1), distinct from that used for the immunoblot assay. Specifically, hindlimb muscle was dissected, weighed, and minced with scissors in ice-cold homogenization medium (250 mM mannitol, 30 mM L-histidine, 5 mM Tris-EDTA, and 0.1% sodium deoxycholate, brought to pH 6.8 with NaOH at room temperature). The minced suspension was homogenized with Ultra Turrax T25 (IKA-Labortechnik) at 22,000 rpm for 15 s twice before it was transferred to a glass homogenizer tube and further homogenized for six full strokes with a motor-driven Teflon pestle (setting 5; Dyna-Mix, Fisher Scientific). The homogenate was centrifuged at 3,000 g for 20 min (at 4°C) and frozen at -80°C overnight. The next day, the Na+-K+-ATPase activity in the supernatant was measured in triplicate as the ouabain-sensitive ATPase activity: homogenate was preincubated with or without 2 mM ouabain for 30 min, and the reaction was started with the addition of ATP. Na+-K+-ATPase activity was calculated as the difference in inorganic phosphate (Pi) generated over 15 min in the absence (total activity) and presence (Mg-ATPase activity) of 2 mM ouabain and expressed as micromoles of Pi liberated per milligram of protein per hour. In addition, the samples were reassayed for establishment of reproducibility on another day.

Na+-K+-ATPase subunit mRNA analysis. At day 14 of dexamethasone or vehicle treatment, whole hindlimbs were removed, and either RNA was isolated immediately or muscles were flash frozen in liquid nitrogen and stored at -80°C until RNA isolation; no effects of freezing on degradation of RNA were evident when muscles from the same animal were compared. Total RNA was isolated from rat skeletal muscle and probed as previously detailed (41). In short, RNA was isolated with Tri Reagent (Molecular Research Center, Cincinnati, OH) according to the manufacturer's protocol, concentrations were determined by optical density OD260, and purity was estimated by OD260/OD280. Total RNA was assayed by Northern analysis on NitroPure nitrocellulose transfer membrane (Micron Separations, Westborough, MA). Immobilized RNA was probed with either isoform-specific restriction endonuclease fragments (~300 bp) of alpha 1, alpha 2, and beta 1 or the rat beta 2 cDNA clone (provided by P. Martin-Vasallo) labeled for similar specific activity with [32P]dCTP probes with the use of a multiprimer DNA-labeling technique, as described previously (3, 41). Blots were washed three times in 2× SSC (0.3 M NaCl-0.03 M sodium citrate, pH 7.0) with 0.05% SDS at room temperature for 10 min each and then twice for 20 min each in 0.1× SSC with 0.1% SDS at 55°C.

Quantitation. Film exposure time was adjusted to a range wherein the samples run at one-half the amount of protein on each gel had one-half the autoradiographic density relative to the same samples run at twice that amount of protein. Autoradiograms were quantitated with the Bio-Rad GS670 Imaging Densitometer and dedicated software. All data are expressed as means ± SE. Significance was assessed by using the two-tailed Student's t-test, and differences were considered significant at P < 0.05.

Materials. Chemicals were reagent grade, spectroquality, or electrophoresis purity reagents. SDS-PAGE reagents were from Bio-Rad (Hercules, CA). Leupeptin, PMSF, pABAD, and SDS-PAGE molecular weight standards were from Sigma (St. Louis, MO). PNGase F was from Genzyme (Cambridge, MA). 125I-labeled protein A and [32P]dCTP were from ICN (Costa Mesa, CA).


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Because skeletal muscle is a heterogeneous tissue consisting of different muscle fiber types and because there is a muscle fiber-specific pattern of sodium pump isoform expression, the effects of 14-day dexamethasone treatment (0.1 mg/kg per day) were examined in a panel of distinct skeletal muscles including soleus, a predominantly slow oxidative (type I) muscle; EDL, a classically fast muscle that is metabolically both glycolytic (IIB) and oxidative-glycolytic (IIA); gastrocnemius, a mixed muscle type; and diaphragm, also a highly mixed muscle type (2, 30, 37).

Na+-K+-ATPase isoform protein and activity regulation by dexamethasone. To determine whether dexamethasone regulated the total pool size of the alpha 1-, alpha 2-, beta 1-, or beta 2-subunits expressed in skeletal muscle, we evaluated subunit pool size by immunoblotting a constant amount of total homogenate protein. Samples for quantitation of beta 1- or beta 2-subunit were deglycosylated before analysis, as described in METHODS, because glycosylation appears to interfere with antibody binding to both beta -subunits (38). Typical autoradiograms of skeletal muscle homogenate samples, probed with isoform-specific antibodies against alpha 1, alpha 2, beta 1, and beta 2, are shown in Fig. 1. alpha 1 and alpha 2 were detected at ~110 kDa. Deglycosylated core beta 1 and beta 2 proteins were detected at 32-35 kDa. In Fig. 1, the relative autoradiographic densities reflect the subunit expression levels when control vs. dexamethasone-treated samples are compared within a specific muscle for a specific isoform because these samples were run on the same blot, in which autoradiographic exposure time was adjusted to assure the linearity of signal density with the amount of sample loaded (as described in METHODS). In comparison, the relative densities of signals from different muscles or isoforms cannot be compared because these were obtained from different films with different exposure times and because each antibody has a unique affinity for its subunit epitope.


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Fig. 1.   Effect of 14-day dexamethasone (Dex) treatment (0.1 mg/kg per day) on Na+-K+- ATPase subunit abundance in skeletal muscle. Representative autoradiograms of immunoblots of homogenate samples from soleus, extensor digitorum longus (EDL), gastrocnemius (Gastroc), and diaphragm (20 µg/lane for alpha -subunits and 15 µg/lane for beta -subunits). beta 1- and beta 2-subunit immunoreactivities were measured after the removal of sugar residues with peptide-N-glycosidase F (PNGase F), as described in METHODS. The antibody-antigen complexes were detected with 125I-labeled protein A. Because dexamethasone- and vehicle-treated control (C) samples were run on the same gel and processed identically, comparisons should be made horizontally (dexamethasone vs. control), not vertically (soleus vs. EDL) because of the variable incubation and exposure times between different representative autoradiograms. nd, Not determined.

The effect of dexamethasone treatment on relative pool size of sodium pump subunit isoforms was determined by analyzing samples from dexamethasone- and vehicle-infused animals on the same gel and immunoblot, thus processed identically. Scanning densitometry results, summarized in Fig. 2, were normalized to the mean value of the vehicle-infused samples, defined as 1.0. Also included in Fig. 2, for comparison, are previously reported [3H]ouabain binding results from 14-day dexamethasone-infused rats (10). Dexamethasone infusion did not change alpha 1 abundance in soleus or EDL but did increase alpha 1 protein pool size in diaphragm to 65 ± 7% over vehicle control (P < 0.001). In comparison, dexamethasone significantly increased alpha 2 to 53-78% over vehicle controls in all muscles analyzed and, thus, was independent of fiber type. This change in alpha 2-isoform is larger than the 22-48% increase in [3H]ouabain binding to muscle biopsies from rats treated with the same protocol, summarized in Fig. 2B (10). Dexamethasone also increased the abundance of the beta 1-isoform, which is associated with both oxidative and mixed oxidative-glycolytic fibers (18, 42), by ~50% in all muscles tested; however, the increase was not significant in soleus, where the expression of beta 1 previously has been shown to be highly variable (42). The beta 2-isoform, which is associated with both glycolytic and mixed oxidative-glycolytic fibers (18, 42), had a tendency to increase in dexamethasone-treated EDL compared with vehicle-treated control but was unchanged in gastrocnemius and was not detected in oxidative soleus and mixed diaphragm muscle. Together, these results demonstrate that the response of Na+-K+- ATPase subunit expression to dexamethasone treatment is muscle specific. The results suggest that alpha 2beta 1 is the dexamethasone-responsive pump in all skeletal muscles; that the alpha 1beta 1 heteromer is upregulated specifically in diaphragm, analogous to the previously reported dexamethasone regulation of renal alpha 1beta 1 sodium pumps (24, 35); and that there may be a tendency for an increase in alpha 2beta 2 heteromer in EDL.


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Fig. 2.   A: effect of dexamethasone on Na+-K+-ATPase subunit abundance in skeletal muscles. Na+-K+-ATPasealpha 1-, alpha 2-, beta 1-, and beta 2-subunit immunoreactivities were measured in soleus (Sol), EDL, gastrocnemius, and diaphragm (Diaph) muscles in vehicle controls (n = 5 samples) and in response to dexamethasone infusion (n = 5 samples). Subunit immunoreactivity was assessed by scanning densitometry as described in METHODS. Data are normalized to mean control values, defined as 1 ± SE. *P <=  0.05. B: effect of dexamethasone on [3H]ouabain binding to skeletal muscle biopsies [data from Dorup (10)]. Left: binding expressed as pmol/g wet weight. Right: data normalized to control mean, defined as 1.0 ± SE. *P <=  0.05.

To determine whether glucocorticoid treatment increased sodium pump enzymatic activity in line with the 50% increases in alpha 2 and beta 1 pool size and the 25-50% increases in [3H]ouabain binding, we assayed ouabain-sensitive ATPase activity. Rat alpha 1-isoform has far less affinity for ouabain (Kd = 5 × 10-5 M) than the alpha 2-isoform (Kd = 10-7-10-9 M), but it is still difficult to add enough ouabain to inhibit all of alpha 2 without inhibiting some of alpha 1. Therefore, samples were preincubated with 2 mM ouabain for 30 min, which would inhibit both alpha 1- and alpha 2-type pumps. In initial assays of muscles homogenized in the same buffer used for the immunoblot analysis (after debris was removed by a low-speed spin), there was no significant ouabain-inhibitable fraction of ATPase activity detected. However, when the samples were prepared as described in METHODS (1), significant ouabain-sensitive activity was detected, and it made up 60% of the total ATPase activity. Nonetheless, the ouabain-sensitive ATPase activity (in µmol Pi · mg protein-1 · h-1) was not significantly increased in animals infused with dexamethasone for 14 days (1.065 ± 0.039, n = 5) compared with that in vehicle-infused controls (0.985 ± 0.123, n = 5), and the total ATPase was not different between dexamethasone-infused (1.624 ± 0.105) and control groups (1.805 ± 0.224). What fractional change in total Na+-K+-ATPase activity is predicted from the measured change in alpha 1 vs. alpha 2 protein levels? If the ratio of alpha 1 to alpha 2 in muscle is ~1:1, as we have previously predicted (41), then a 50% change in alpha 2 pool size would increase total Na+-K+-ATPase pool size by 25% at the most, assuming efficient alpha beta heteromer assembly and assuming that all of the pumps are enzymatically active. Given these predictions and constraints, as well as the difficulty in detecting Na+-K+-ATPase activity in skeletal muscle, it is not too surprising that a significant increase was not observed. A measurement of active K+ uptake in response to insulin or catecholamine stimulation in intact muscles would provide a better assay of the physiological impact of the increases in alpha - and beta -isoform pools on muscle K+ uptake.

Na+-K+-ATPase isoform mRNA regulation by dexamethasone. RNA was extracted from whole hindlimb, a mixture of different fiber types, because RNA isolated from distinct muscles may be degraded during the dissection process, while hindlimb muscle can be very quickly removed and extracted or frozen (41). As summarized in Fig. 3A, alpha 1 mRNA was detected as a single band at 3.7 kb, alpha 2 was detected as two bands at 5.3 and 3.4 kb, beta 1 was detected primarily as two main bands at 2.7 and 2.3 kb (which were scanned for quantitation), and beta 2 was detected as a single band at ~2.8 kb. Kidney RNA was loaded as a control sample that contains high levels of alpha 1 and beta 1 but little or no alpha 2 or beta 2 (Fig. 3A, first lane), and brain RNA was added as a control sample, also enriched in alpha 1 and beta 1 but containing alpha 2 and beta 2 as well (Fig. 3A, last lane). To verify the linearity of the autoradiographic signal with the amount of RNA loaded, we loaded one-half the amount for two of the samples (Fig. 3, 1/2C and 1/2D). As summarized in Fig. 3B, 14-day dexamethasone treatment did not significantly increase alpha 1 mRNA but increased alpha 2 mRNA more than sixfold in whole hindlimb. Dexamethasone also increased Na+-K+-ATPase beta 1 mRNA levels 1.54 ± 0.15-fold, in line with the change in alpha 2 and beta 1 protein levels and ouabain binding, but also decreased beta 2 levels in hindlimb to 0.36 ± 0.6 of levels for vehicle-treated controls. Thus, on the whole, the changes in alpha 2 and beta 1 mRNA levels in hindlimb can account for the changes in these subunit protein levels and ouabain binding measured in individual muscles.


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Fig. 3.   Northern hybridization analysis of Na+-K+-ATPase alpha 1, alpha 2, beta 1, and beta 2 mRNA extracted from whole hindlimb after 14 days of dexamethasone or vehicle infusion. Total RNA (15 µg/lane) was fractionated on formaldehyde agarose gels, blotted onto nitrocellulose, and hybridized with 32P-labeled isoform-specific probes, as described in METHODS. A: representative autoradiograms of Northern blots. Each lane represents a sample from a separate animal (n = 5-6 samples), except for the 1/2C and 1/2D lanes, which contain 7.5 µg of the control or dexamethasone-treated sample shown to the right to verify linearity of signal with amount loaded. One alpha 1 transcript (3.7 kb), two alpha 2 mRNA transcripts (5.3 and 3.4 kb), two beta 1 mRNA transcripts (2.7 and 2.3 kb), and one beta 2 mRNA transcript (2.8 kb) were detected. K, total RNA from kidney; B, total RNA from brain. B: relative subunit mRNA levels assessed by scanning densitometry, as described in METHODS. Data are normalized to the mean control values, defined as 1 ± SE; n = 56 samples for dexamethasone treatment and 5 samples for control. *P <=  0.05.


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In the previous study by Dorup and Clausen (11), the infusion of graded doses of dexamethasone (0.02-0.1 mg/kg per day) increased the binding of the cardiac glycoside [3H]ouabain to skeletal muscle biopsies by 22-48%. However, the molecular mechanism responsible for this change in ouabain binding was not determined. Ouabain is not likely to bind to all the pumps in a muscle biopsy preparation because it will have access only to active sodium pumps in the sarcolemma (25) and not to the endosomal pools of alpha 2-type pumps identified in oxidative muscle fibers (29). These sequestered pumps are known to shuttle between sarcolemma and endosomal pools in response to insulin (23, 29), so they are physiologically relevant. In addition, the fairly ubiquitous alpha 1 has such a low affinity for ouabain that it is termed "ouabain resistant" and may not significantly bind [3H]ouabain under these assay conditions (25). In addition, there are likely to be significant pools of alpha 1 in muscle: we recently calculated that the ratio of alpha 1 to alpha 2 protein levels was near 1 by using regression analysis of the relative changes in alpha 1- and alpha 2-isoforms vs. beta 1-isoform in soleus or vs. beta 2-isoform in white gastrocnemius in response to potassium deprivation (41). These observations prompted us to determine the effect of dexamethasone treatment on specific alpha -catalytic and beta -glycoprotein pool sizes in skeletal muscle.

After 14 days of dexamethasone infusion, the increases in [3H]ouabain binding (40-48%) and in the ouabain-sensitive alpha 2-isoform abundance (53%) were similar in soleus and EDL (Fig. 2). This finding suggests that there are similar changes in the pools of alpha 2-type pumps present at the plasma membrane and in endosomes. In contrast, in gastrocnemius and diaphragm, the increase in alpha 2 protein abundance exceeded the increase in [3H]ouabain binding by 39% and 53%, respectively. One interpretation is that in these muscles, dexamethasone stimulates a larger increase in the ouabain-inaccessible internal pools of alpha 2 than in the ouabain-accessible sarcolemmal pool. Another possibility is that there are pools of unassembled inactive alpha 2 in these muscles. In fact, in diaphragm, the change in expression of beta 1 (1.39 ± 0.07) protein after dexamethasone treatment is less than the increase in changes in alpha 1 or alpha 2 levels (1.65 ± 0.07 and 1.78 ± 0.09, respectively), suggesting the possibility that beta 1 (not the alpha -subunits) is rate limiting for the production of heterodimers capable of moving to the cell surface and binding [3H]ouabain and suggesting the corollary that there may be spare unassembled alpha 1- and alpha 2-subunits that would not bind ouabain.

Dexamethasone stimulates an increase in alpha 2 protein independent of fiber type. Conversely, alpha 1 abundance is increased significantly only in diaphragm (to 1.65 of control). There was also an increase in the mean alpha 1 expression in gastrocnemius, but the variability of the response was large. We verified that these muscle specific changes were not loading artifacts by reprobing the blots with calsequestrin and glucose transporter 4 (19), neither of which showed an analogous increase after glucocorticoid treatment (not shown).

The glucocorticoid-mediated increase in relative abundance of sodium pump subunits, whether direct or indirect, is a result of either increased subunit synthesis or decreased subunit degradation. The analysis of the mRNA levels in whole hindlimb indicates that the change in alpha 2 mRNA, >6-fold, can more than account for the change in the alpha 2 protein levels observed in the individual muscles and that the change in beta 1 mRNA, ~1.5 fold, is equivalent to the change in beta 1 protein levels. Thus we conclude that the changes in alpha 2 and beta 1 protein levels are mediated by increased synthesis driven by increases in their mRNA levels.

The relative changes in alpha 2 and beta 1 mRNA vs. protein levels demonstrate that the large induction of alpha 2 is not translated into a similarly enlarged stable pool of alpha 2 and suggest that the changes in beta 1 synthesis may limit the magnitude of increase in functional alpha 2beta 1 protein heteromers. In other words, these comparisons suggest that formation of alpha 2beta 1 heteromers stabilizes alpha 2 during or after translation and that unassembled alpha 2-subunits are degraded. We have previously described similar discoordinate changes in sodium pump subunit protein vs. mRNA levels in heart and muscle in response to thyroid hormone (T3) treatment (3, 16). In rat muscle, T3 increased alpha 2 and beta 2 mRNA five- and fourfold but increased protein levels only three- and twofold, respectively, suggesting an inefficiency in the translation and assembly of the alpha beta heteromers or an accompanying T3-driven increase in alpha beta degradation rate (3). In the rat heart, 8- to 16-day thyroid hormone treatment increased alpha 1 mRNA and protein levels 2- to 3-fold but increased alpha 2 mRNA and protein levels 6- and 15-fold and beta 1 mRNA and protein levels 15- and 3-fold, respectively. The very discoordinate changes in alpha 2 mRNA vs. protein levels are not easily explained but suggest that beta 1 levels are rate limiting in the heart and that the 15-fold increases in beta 1 mRNA stabilize nascent alpha 2 proteins by alpha beta assembly. This effect would decrease the degradation rate of alpha 2 during T3 treatment and thus amplify the 6-fold increase in alpha 2 mRNA into a 15-fold increase in alpha 2 protein levels during T3 treatment (16). Similarly, if beta 1 mRNA is rate limiting for the synthesis of sodium pumps in muscle, the dexamethasone-driven increase could also explain the tendency for alpha 1 protein increase in gastrocnemius and diaphragm in the absence of a change in alpha 1 mRNA by increasing alpha 1beta 1 heteromer formation and decreasing alpha 1 degradation rate. Additional support for this theory comes from experiments in rat alveolar epithelial cells, where dexamethasone increases only beta 1 mRNA expression but increases both alpha 1 and beta 1 protein levels relative to controls (4), suggesting increased stabilization of alpha 1beta 1 during or after translation by assembly of nascent alpha 1 with an increased pool of newly synthesized beta 1.

The significant dexamethasone-driven decrease in beta 2 mRNA was unexpected given the unchanged levels of beta 2 in gastrocnemius and the tendency for beta 2 increase in EDL, the two muscles in which beta 2 was detected at the protein level. By employing the reasoning developed to explain the discoordinate changes in alpha 2 and beta 1, we can postulate that beta 2 mRNA is not rate limiting for the formation of sodium pump heteromers and that beta 2 mRNA may be in such excess in the control state that decreases in beta 2 mRNA levels by 50% by dexamethasone may not impact the formation of stable alpha 1beta 2 or alpha 2beta 2 heteromers in these muscles. While both EDL and gastrocnemius are represented in the hindlimb sample used for preparation of the RNA, it will be important to measure the beta 2 mRNA levels and protein levels in the same specific muscles to further test this idea that beta 2 mRNA is not rate limiting for the formation of sodium pumps in muscle.

Dexamethasone regulation of sodium pump subunit mRNA levels appears to be quite tissue or cell dependent. Orlowski and Lingrel (31) determined in primary cultures of myocardiocytes that dexamethasone treatment induced alpha 2 mRNA levels but did not influence alpha 1, alpha 3, or beta 1 mRNA levels (beta 2 not tested) (31). While the increase in alpha 2 (but not alpha 1) mRNA agrees with the finding in this study, the lack of an effect on beta 1 mRNA is in contrast to most other studies, including the current study. The increase in beta 1 is predicted because there are glucocorticoid receptors in muscle, and the Na+-K+-ATPasebeta 1 gene promoter contains a glucocorticoid-responsive element that is specifically activated by glucocorticoid receptors (9). In addition, while this study in skeletal muscle as well as the others discussed (4, 31) found no direct dexamethasone regulation of alpha 1, there are a number of reports in the literature that do support specific glucocorticoid activation of Na+-K+-ATPasealpha 1 expression, especially in epithelia. Wang et al. (44) provided evidence that glucocorticoids regulate alpha 1 transcription in infant rat kidney. Lee et al. (24) also observed a glucocorticoid-driven increase in alpha 1 and beta 1 mRNA and protein in cultured adult proximal tubule and, furthermore, provided evidence that the response is indirect, requiring ongoing protein synthesis.

When examining regulators of sodium pump expression, it is always important to ask whether the effects are mediated indirectly by changes in cell [Na+] or [K+]. In the previous, related study (11), 7-day dexamethasone infusion did not significantly change plasma [Na+] or [K+] and had no, or minute, effects on tissue cations in this same panel of muscles. Furthermore, no direct in vitro effects of dexamethasone on 22Na influx were detected, indicating that glucocorticoid-driven increases in sodium pump isoform mRNAs and proteins in skeletal muscle are not driven by primary changes in cell cations. These results agree with those of earlier studies in rat heart (21) and outer medullary kidney tubules (36), in which investigators demonstrated that glucocorticoid-mediated increases in sodium pump activity were not mediated by changes in intracellular Na+ or K+ levels.

Our previous studies of ionic and hormonal regulation of sodium pump isoform expression in muscle have established that alpha 2 is highly regulated while alpha 1 expression is not, that hypokalemia and hypothyroid status depresses alpha 2 but not alpha 1 abundance, and that thyroid hormone increases alpha 2 but not alpha 1 abundance (3, 41, 42). Similarly, glucocorticoid treatment increases alpha 2 expression in all muscles tested but increases alpha 1 only in diaphragm. These findings provide the rationale for future investigations on how multiple stimuli affect muscle sodium pump expression. For example, chronic obstructive lung disease patients are often given diuretic therapy that is known to provoke hypokalemia, which depresses muscle alpha 2 expression, along with glucocorticoids, which are shown here to raise muscle alpha 2 expression. Because muscle sodium pump expression is a critical regulator of plasma potassium and an important determinant of muscle endurance, the net influence of these multiple therapies may have important health consequences that should be understood.


    ACKNOWLEDGEMENTS

This work was supported by National Science Foundation Grant IBN 9S13958, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34316, and an American Heart Association-Western Affiliate Grant-in-Aid (to A. A. McDonough) as well as grants from the Nordic Insulin Foundation, The NOVO Industry Research Foundation, the Alfred Benzons Foundation, and the Danish Biomembrane Research Center (to I. Dorup).


    FOOTNOTES

Address for reprint requests and other correspondence: A. A. McDonough, Dept. of Physiology and Biophysics, Univ. of Southern California School of Medicine, 1333 San Pablo St., Los Angeles, CA 90089-9142.

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.

Received 16 September 1999; accepted in final form 18 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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