Temporal responses of oxidative vs. glycolytic skeletal muscles to K+ deprivation: Na+ pumps and cell cations

Curtis B. Thompson, Cheolsoo Choi, Jang H. Youn, and Alicia A. McDonough

Department of Physiology and Biophysics, University of Southern California School of Medicine, Los Angeles, California 90033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

When K+ output exceeds input, skeletal muscle releases intracellular fluid K+ to buffer the fall in extracellular fluid (ECF) K+. To investigate the mechanisms and muscle specificity of the K+ shift, rats were fed K+-deficient chow for 2-10 days, and two muscles at phenotypic extremes were studied: slow-twitch oxidative soleus and fast-twitch glycolytic white gastrocnemius (WG). After 2 days of low-K+ chow, plasma K+ concentration ([K+]) fell from 4.6 to 3.7 mM, and Na+-K+-ATPase alpha 2 (not alpha 1) protein levels in both muscles, measured by immunoblotting, decreased 36%. Cell [K+] decreased from 116 to 106 mM in soleus and insignificantly in WG, indicating that alpha 2 can decrease before cell [K+]. After 5 days, there were further decreases in alpha 2 (70%) and beta 2 (22%) in WG, not in soleus, whereas cell [K+] decreased and cell [Na+] increased by 10 mM in both muscles. By 10 days, plasma [K+] fell to 2.9 mM, with further decreases in WG alpha 2 (94%) and beta 2 (70%); cell [K+] fell 19 mM in soleus and 24 mM in WG compared with the control, and cell [Na+] increased 9 mM in soleus and 15 mM in WG; total homogenate Na+-K+-ATPase activity decreased 19% in WG and insignificantly in soleus. Levels of alpha 2, beta 1, and beta 2 mRNA were unchanged over 10 days. The ratios of alpha 2 to alpha 1 protein levels in both control muscles were found to be nearly 1 by using the relative changes in alpha -isoforms vs. beta 1- (soleus) or beta 2-isoforms (WG). We conclude that the patterns of regulation of Na+ pump isoforms in oxidative and glycolytic muscles during K+ deprivation mediated by posttranscriptional regulation of alpha 2beta 1 and alpha 2beta 2 are distinct and that decreases in alpha 2-isoform pools can occur early enough in both muscles to account for the shift of K+ to the ECF.

sodium-potassium-ATPase isoforms; hypokalemia; soleus; white gastrocnemius


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EXTRACELLULAR FLUID (ECF) and intracellular fluid (ICF) K+ levels are tightly controlled in mammals because their ratio is the principal determinant of cell membrane potentials. When whole body K+ output exceeds K+ input over time, the plasma K+ level falls, a condition known as hypokalemia (40). This condition can occur during loop diuretic treatment, which increases K+ excretion, or if K+ intake is restricted, such as during prolonged fasting (40). In excitable tissues such as heart tissues, hypokalemia-induced disturbances in membrane potential can lead to life-threatening cardiac arrhythmias (10). K+ balance is maintained by the interplay of two key organ systems: the kidneys, which can secrete or actively reabsorb K+, and skeletal muscle, the major K+ reservoir, which can acutely control the transport of K+ between the ECF and the ICF (33, 40). Extracellular K+ loss is buffered by the transfer of muscle cell K+ to the ECF. This loss is likely mediated by the accompanying decrease in Na+ pump (Na+-K+-ATPase) levels, which would decrease active K+ transport from ECF to ICF (5, 22, 36).

The Na+ pump is a ubiquitous integral membrane P-type ion pump that pumps Na+ out of the cell and K+ into the cell, a process driven by the hydrolysis of ATP (27). It is an alpha beta heteromer composed of a catalytically active alpha -subunit (Mr approx  112,000) and a glycosylated beta -subunit (Mr approx  35,000), and recent evidence suggests that it is a tetramer (44). Multiple alpha - and beta -subunit isoforms are expressed in a tissue-specific manner (13, 29, 41, 42). In skeletal muscles, Na+ pump isoforms are expressed in a muscle fiber type-specific manner (19, 43). At the phenotypic fiber type extremes, slow-twitch oxidative soleus expresses alpha 1-, alpha 2-, and beta 1-isoforms as alpha 1beta 1 and alpha 2beta 1, whereas fast-twitch glycolytic white gastrocnemius (WG) expresses alpha 1-, alpha 2-, and beta 2-isoforms as alpha 1beta 2 and alpha 2beta 2 (43). Background information on the proportion of alpha 2- to alpha 1-type Na+ pumps in muscle is limited to two studies. For rat diaphragm membranes the fraction of alpha 2 was estimated at between 30 (in hypothyroids) and 65% (in hyperthyroids) by a backdoor phosphorylation assay that measures pumps undergoing a reaction cycle (15). In rat red (oxidative) skeletal muscle subjected to subcellular fractionation on sucrose gradients, the ratio of alpha 2 to alpha 1 ranged from 1.6 (60% alpha 2) in surface membranes to 7 (87% alpha 2) in intracellular membranes. The ratio was calculated by using ouabain binding per milligram of protein to quantitate alpha 2, and the immunoblot signal was scaled against the signal from a recombinant fragment of alpha 1 per milligram of protein to measure alpha 1 (23). In comparison, the proportion of alpha 2 to total alpha  mRNA in skeletal muscle has been reported as being between 70 and 80% (13, 37). The ratio of total alpha 2 to total alpha 1 in control muscles, either oxidative or glycolytic, has not been previously determined.

We previously reported that, when rats are fed a K+-deficient diet for 10 days, Na+ pump protein changes are isoform and muscle fiber type specific (43): in fast-twitch glycolytic WG, alpha 2 and beta 2 protein levels decrease to only 6 and 30% of control, respectively, whereas in slow-twitch oxidative soleus alpha 2 decreased to 45% of control and beta 1 did not decrease significantly. The alpha 1 protein pool size for either muscle did not change. Despite the substantially greater decrease in alpha 2 and beta 2 in WG than in soleus, the fall in whole-muscle tissue K+ level was ~20% after 10 days of K+ restriction in both muscles. These findings provoked questions about the muscle-specific mechanisms of cell K+ loss. The first aim of this study was to learn whether the decrease in alpha 2 abundance is linked to the loss of cell K+ by comparing the time courses of the two parameters at early time points in the response. The second aim was to measure both protein and mRNA levels of Na+ pump subunits over the time course of K+ depletion to understand the molecular mechanisms responsible for the decrease in expression in different muscle types. The third aim was to assess the ratio of alpha 2- to alpha 1-isoforms because it will affect the impact of alpha 2-specific regulation. The present study demonstrates that changes in alpha 2 protein levels occur early enough to account for the loss of cell K+ in both muscle types, that the decrease in alpha 2 protein is not secondary to a decrease in alpha 2 mRNA, and that the calculated percentage of alpha 2-type pumps in control muscles ranges from 40% in soleus to 55% in WG.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and diets. Male Sprague-Dawley rats, ~8 wk of age (250-300 g), were placed on a K+-deficient diet (TD 88239; Harlan Teklad, Madison, WI) for 2, 5, or 10 days and were paired to controls fed a comparable diet with K+ restored (TD 88238; Harlan Teklad). Rats were anesthetized with 0.2 ml pentobarbital sodium/100 g body wt. Soleus and WG hindlimb muscles were removed, frozen in liquid nitrogen, and stored at -80°C pending analyses. Blood samples were taken from the abdominal aorta, and the serum was separated, frozen, and stored at -20°C pending analyses.

Serum and intracellular electrolytes. Serum and muscle cell K+ and Na+ concentrations ([K+] and [Na+]) were measured by flame photometry. Muscles were thawed, blotted lightly to remove adherent fluid, and homogenized in 0.3 M trichloroacetic acid [TCA; 1:50 (wt/vol)] for 5 min with a Tissuemizer homogenizer and then centrifuged at 2,500 rpm for 20 min to remove cell debris. The K+ and Na+ contents of the muscle TCA extracts and serum samples were measured with an FLM 3 flame photometer (Radiometer, Copenhagen, Denmark), with lithium as the internal standard (21). The total muscle [K+] and [Na+] were corrected for the levels of these cations in the extracellular space.

The extracellular space of soleus and WG was determined as previously described (47). In brief, L-[3H]glucose was infused, via a cannula in a tail vein, at 0.2 µCi/min for 2 h and then rats were anesthetized with pentobarbital sodium. Soleus and WG muscles were removed from each hindlimb, immediately frozen in liquid nitrogen, and stored at -80°C. Blood samples were collected from the abdominal aorta. Muscles were homogenized in 0.3 M perchloric acid [PCA; 1:10 (wt/vol)] for 5 min with a Tissuemizer, then centrifuged at 10,000 g, 4°C, for 15 min. The supernatant was cleared of PCA with 1:4 trioctylamine-1,1,2-trichlorotrifluoroethane (Freon), 1:2 (vol/vol), and assayed for L-[3H]glucose by liquid scintillation counting. Plasma samples (20 µl) were directly assayed in scintillation fluid. Extracellular space was calculated as muscle L-[3H]glucose content divided by plasma L-[3H]glucose concentration (47). Muscle ion levels, measured by flame photometry (recorded as µmol/g wet wt and converted to µmol/ml wet wt), were corrected for the level of ions in the extracellular space (µmol/µl). Extracellular space values at day 0 were used for control ion corrections; day 10 extracellular space values were used for corrections at all low-K+ time points (days 2, 5, and 10).

Na+-K+-ATPase alpha - and beta -subunit immunoreactivities. These immunoreactivities were determined as previously described (43). In brief, skeletal muscle was homogenized 1:20 (wt/vol) in 5% sorbitol, 25 mM histidine-imidazole (pH 7.4), 0.5 mM EDTA disodium, and proteolytic enzyme inhibitors [0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 µg/ml leupeptin, and 1 mM 4-aminobenzamidine dichloride (pABAD)] with a Polytron homogenizer. To facilitate detection of the beta -subunit, sugar residues were removed from beta -subunits with PNGase F as previously described (43). A constant amount of homogenate protein (100 µg for alpha -subunit analysis, 50 µg for beta -subunit analysis) was resolved by SDS-PAGE and blotted onto Immobilon-P membranes (Millipore, Bedford, MA). Blots were incubated overnight with one of the following antibodies: McB2, a monoclonal antibody specific for alpha 2 (45), generously provided by K. Sweadner (Harvard Medical School); anti-beta 1 FP (1:500), a polyclonal antibody against beta 1 (43); SpET b2 (1:2,000), a polyclonal antibody against human beta 2 (14), generously provided by P. Martin-Vasallo (Universidad de La Laguna); and RNT beta 3 (1:2,000), a polyclonal antibody against rat beta 3 (4), provided by K. Sweadner. Blots probed with monoclonal McB2 were incubated for 2 h with rabbit anti-mouse IgG secondary antibody (Calbiochem, La Jolla, CA; 1:2,000). Antibody-antigen complexes labeled with 125I-protein A were visualized by autoradiography as described previously (28), and linearity was verified by assaying samples at multiple concentrations.

Na+ pump enzymatic activity. Na+ pump activity in crude muscle homogenates was estimated by the K+-dependent p-nitrophenylphosphatase (K+-pNPPase) reaction (35), as recommended by Kjeldsen et al. (22) because levels of ouabain-sensitive Na+-K+-ATPase in skeletal muscle homogenate cannot be reliably determined, presumably because of the ouabain resistance of alpha 1 in rats, the low abundance of Na+ pumps, and/or the high level of other ATPases in skeletal muscle. In brief, crude homogenates were freeze-thawed three times to permeabilize membranes, and 60 µg of homogenate protein were added to 500-µl sets of K+-pNPPase assay mixtures containing 100 mM KCl or 100 mM NaCl. The [Na+] carried over from the homogenization buffer was <0.5 mM. Activity is reported as micromoles of phosphate per milligram of protein per hour.

Na+-K+-ATPase alpha  and beta  mRNA analysis. Total RNA was isolated from rat skeletal muscle as described by Chomczynski and Sacchi (8) with Tri Reagent (Molecular Reasearch Center, Cincinnati, OH) according to the manufacturer's protocol. RNA concentrations were determined by measuring the optical density at 260 nm (OD260), and purity was estimated by determining the OD260/OD280 ratio. Total RNA was assayed by Northern analysis, as previously described (17, 32), on a NitroPure nitrocellulose transfer membrane (Micron Separations, Westborough, MA). Immobilized RNA was hybridized with isoform-specific restriction endonuclease fragments (~300 bp) prepared from either alpha 1, alpha 2, or beta 1 clones as described by Orlowski and Lingrel (37) or from the rat beta 2 cDNA clone provided by P. Martin-Vasallo (30). The alpha - and beta -cDNA probes were labeled to similar specific activities with [32P]dCTP probes by using a multiprimer DNA-labeling technique (12). 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. Autoradiograms of the blots were quantified by scanning densitometry.

Quantitation. Autoradiograms were quantitated by scanning with a GS670 imaging densitometer (Bio-Rad, Hercules, CA) and dedicated software. All data are expressed as means ± SE, normalized to the mean value at day 0, defined as 1. Significance was assessed by the two-tailed Student's t-test, and differences were considered significant at P < 0.05. The fractions of alpha 1- and alpha 2-isoforms in soleus and WG muscles were estimated as described in RESULTS. Parameter identifications were performed with MLAB software (Civilized Software, Bethesda, MD), implemented on an IBM-compatible computer, which uses a Marquardt-Levenberg iterative least-squares algorithm. Again, data are reported as fractions of total pumps (defined as 1.0) ± SE.

Materials. Chemicals were reagent-grade, spectroquality, or electrophoresis purity reagents. SDS-PAGE reagents were from Bio-Rad. Leupeptin, PMSF, pABAD, and SDS-PAGE molecular weight standards were from Sigma Chemicals (St. Louis, MO). PNGase F (N-glycanase) was from Genzyme Corporation (Cambridge, MA). 125I-protein A and [32P]dCTP were from ICN (Costa Mesa, CA). L-[3H]glucose was from DuPont NEN (Boston, MA).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Time course of change in extracellular [K+] during K+ deprivation. In 8-wk-old rats placed on a K+-deficient diet, serum [K+] fell significantly from a control value of 4.6 ± 0.08 to 3.7 ± 0.09 mM by day 2 and continued to fall to 3.3 ± 0.2 and 2.9 ± 0.2 mM by days 5 and 10, respectively (Fig. 1). The fall in serum [K+] during the first 2 days (0.9 mM) is greater than or equal to the fall during the subsequent 8 days (0.8 mM), the time when renal and muscle adjustments to hypokalemia come into play (26, 48). Serum [Na+] did not change throughout the course of the study. The fall in serum [K+] is consistent with previous reports (22, 36) and demonstrates that K+ output exceeds input in this K+ deprivation model.


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Fig. 1.   Time course of change in extracellular (serum) K+ and Na+ concentrations ([K+] and [Na+]) during K+ restriction. Measurements were made by flame photometry, as described in MATERIALS AND METHODS. Results are means ± SE. * P < 0.05 (significant result). Sample sizes for each time point are in parentheses above abscissa.

Time course of change in Na+-K+-ATPase isoform abundance vs. cell [K+] in soleus and WG muscles during K+ deprivation. To determine whether changes in Na+ pump expression precede the decrease in cell [K+] and to assess the relative contributions of distinct muscles in the adaptive responses to hypokalemia, two muscles at phenotypic extremes were chosen for study: soleus, with 87% slow-twitch oxidative fibers and some fast-twitch glycolytic-oxidative fibers, and WG with 84% fast-twitch glycolytic fibers and some fast-twitch oxidative fibers (2, 3).

The possibility that beta 3 was expressed in muscle and regulated during K+ deprivation was tested since beta 3 has been detected in skeletal muscle microsomes from both 7-day postnatal and adult rats (4). Immunoblot studies of both glycosylated and deglycosylated soleus and WG homogenates were conducted (not shown), and no beta 3 signal that shifted from a predicted glycosylated molecular mass of 55 kDa to a deglycosylated band at 35-38 kDa and/or that showed a positive signal at the appropriate molecular mass for deglycosylated beta 3 (35-38 kDa) was detected in adult soleus or WG. Adult rat testis homogenate, run on the same blots as a positive control, did yield glycosylated and deglycosylated signals for the beta 3-subunit at the appropriate molecular masses. We conclude that the beta 3-subunit, if present in adult rat skeletal muscle, is below the level of detection obtained with the currently available antibody on crude homogenate.

The relative expression of Na+-K+-ATPase alpha 2- and beta 1-isoforms (soleus) and beta 2-isoforms (WG) after 0, 2, and 5 days of K+ deprivation was determined by immunoblotting. The previously reported immunoblot results for alpha 2 and beta  after 10 days of K+ deprivation (43) are included in this analysis. The alpha 1-subunit has been shown to be unchanged during 10 days of hypokalemia (5, 43). Typical autoradiograms of a subset of the samples are shown in Fig. 2. Control and K+-deprived samples at a given time point were run on the same gel and processed identically; samples for beta  detection were deglycosylated before analysis. The relative levels of expression, determined by scanning densitometry, are summarized in Fig. 3, A and B, top. In both muscles alpha 2 protein expression decreased 36% as early as day 2. In soleus and WG, beta 1 and beta 2, respectively, were depressed significantly by 23% at day 5.


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Fig. 2.   Detection of Na+-K+-ATPase alpha - and beta -isoforms in muscles from control (C) and K+-deprived (down-arrow K) rats at 2, 5, and 10 days (2d, 5d, and 10d, respectively). Representative autoradiograms of immunoblots of homogenate samples from soleus (A) and white gastrocnemius (WG; B) (100 µg for alpha 2 and 50 µg for beta 1 or beta 2) are shown. The beta 1- and beta 2-subunit immunoreactivities were measured after removal of sugar residues with N-glycanase, as described in MATERIALS AND METHODS. Samples from control and K+-restricted animals at a time point were run on the same gel and processed identically. Thus comparisons should be made at a given time point between results for control and K+-deprived rats (i.e., horizontally), not between results for control or K+-deprived rats at different times (i.e., vertically), because of variable incubation and exposure times of different autoradiograms. The antibody-antigen complexes were detected with 125I-protein A.



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Fig. 3.   Time courses of change in Na+ pump subunits and cell cation concentrations during K+-restricted diet. Na+-K+-ATPase alpha 2, beta 1, and beta 2 immunoreactivities and intracellular [K+] and [Na+] in soleus (A) and WG muscles (B) are shown. A, top, and B, top: summaries of subunit immunoreactivities, which were assessed by scanning densitometry as described in MATERIALS AND METHODS. Data are means ± SE and are expressed relative to mean control values, defined as 1. * P <0.05 (significant result). Sample sizes: 5 for days 0, 2, and 5; 7 for day 10. A, bottom, and B, bottom: summaries of intracellular [K+] and [Na+], determined by flame photometry and corrected for extracellular space as described in MATERIALS AND METHODS. Sample size was 5 for all data points. Data are means ± SE. * P < 0.05 (significant result).

To compare the time course of change in Na+ pump subunits to that of cell [K+], muscle [K+] and [Na+] were measured after 0, 2, 5, and 10 days of K+ deprivation. Extracellular space (expressed as µl/g wet wt) in the two muscles was measured with L-[3H]glucose, and that in soleus was found to be greater than that in WG (142 ± 6 vs. 72 ± 8 µl/g), as expected on the basis of the known difference in the respective levels of vascularization. K+ depletion for 10 days did not cause a significant change in extracellular space in either soleus (125 ± 11 µl/g) or WG (65 ± 10 µl/g). These findings agree with previous estimates of ECF space in muscle (1, 22). Intracellular [K+] and [Na+] were calculated as described in MATERIALS AND METHODS. The changes in these cation concentrations in soleus and WG ICF are summarized in Fig. 3, A and B, bottom. For controls, ICF [K+] was higher, and ICF [Na+] was lower, in WG than in soleus, a difference which may reflect a lower level of muscle activity in fast-twitch glycolytic WG (thus less K+ leak out and Na+ leak into the cell) compared to slow-twitch oxidative soleus (9, 20). After 2 days of K+ deprivation, soleus [K+] fell 9% (116 ± 3 to 106 ± 2 mM), and, although WG [K+] did not fall significantly (126 ± 3 to 120 ± 4 mM), WG [Na+] did increase (19.6 ± 1.2 to 23.2 ± 1.6 mM). From these results we conclude that the decrease in Na+ pump alpha 2-isoform expression, 35-40% in both soleus and WG at day 2, occurs early enough in the time course of K+ deprivation to account for the changes in cell [K+]. Between days 2 and 5, the two muscles responded quite differently: in soleus there were no further changes in alpha 2, beta 1, or cell ion concentrations, whereas in WG there were significant further decrements in both alpha 2 and beta 2 protein, which decreased 70 and 22%, respectively, and in cell [K+] which decreased ~10% to 115.2 mM. Between days 5 and 10 there were far greater decrements in Na+ pump expression in WG than in soleus: alpha 2 and beta 2 decreased 94 and 70%, respectively, from control levels in WG, whereas in soleus alpha 2 decreased 56% and beta 1 decreased no further. Despite these differences, the decrements in cell [K+] for the two muscles were not significantly different by day 10: [K+] fell 16 ± 2% in soleus and 19 ± 2.5% in WG. Because [Na+] increased as [K+] fell, the sum of cell [K+] and [Na+] was not significantly altered.

The observation that the decreases in alpha 2 pools during the low-K+ diet are greater than the decreases in beta 1 or beta 2 pools at all time points is expected because a single beta  must form heteromers with not only alpha 2 (which decreases) but also with alpha 1 (which is invariant). In other words, the difference between the change in alpha 2 and beta  (beta 1 in soleus, beta 2 in WG) will be a function of the ratio of expression of alpha 1beta to alpha 2beta heteromers in that muscle, as calculated in the next section.

Estimating the proportion of the Na+-K+-ATPase alpha 2-isoform in soleus and WG. It has been difficult to directly assess the ratio of alpha 1- to alpha 2-type Na+ pumps in muscle. Although absolute pool sizes of alpha 2 vs. alpha 1 protein subunits cannot be determined directly with antibody probes, muscle-specific changes in alpha 2 vs. beta 1 or beta 2 abundance during K+ deprivation can be employed to calculate the ratios of alpha 2 to alpha 1. The calculations depend on three assumptions. First, alpha 1 and alpha 2 proteins are assumed to combine with only beta 1 in soleus and only beta 2 in WG in a 1:1 stoichiometry. Indeed, recent evidence suggests that Na+ pumps may exist as tetramers with a 1:1 alpha -to-beta stoichiometry in mammalian cells (44). Second, it is assumed that there are only negligible pools of uncomplexed alpha - or beta -subunits, a difficult assumption to test in this system but supported by evidence that alpha - and beta -subunits form complexes as alpha beta heteromers before leaving the endoplasmic reticulum and that increasing the synthesis of one subunit increases the stability of the other, implying that uncomplexed subunits are less stable (discussed in Ref. 25). Third, the abundance or pool size is a linear function of the autoradiographic signal, a fact established in previous investigations (6, 28, 46). It follows from these assumptions that the total number of beta -subunits is equal to the total number of alpha -subunits (Eq. 1), the total number of alpha -subunits is equal to the sum of alpha 1- and alpha 2-subunits (Eq. 2), and thus that the total number of beta  pumps is equal to the sum of alpha 1 and alpha 2 pumps (Eq. 3)
&bgr;-subunits = &agr;-subunits (1)
&agr;-subunits = &agr;1-subunits + &agr;2-subunits (2)
&bgr;-subunits = &agr;1-subunits + &agr;2-subunits (3)
The number of alpha - or beta -subunits is equal to the measured immunoblot autoradiographic signal (S), expressed as fraction of control, times a constant (C) related to antibody efficiency (Eqs. 4a-4c)
&agr;1-subunits = C<SUB>&agr;1</SUB> × S<SUB>&agr;1</SUB> (4a)
&agr;2-subunits = C<SUB>&agr;2</SUB> × S<SUB>&agr;2</SUB> (4b)
&bgr;-subunits = C<SUB>&bgr;</SUB> × S<SUB>&bgr;</SUB> (4c)
Thus Eq. 3 becomes
C<SUB>&bgr;</SUB> × S<SUB>&bgr;</SUB> = C<SUB>&agr;1</SUB> × S<SUB>&agr;1</SUB> + C<SUB>&agr;2</SUB> × S<SUB>&agr;2</SUB>
Dividing by Cbeta
S<SUB>&bgr;</SUB> = (C<SUB>&agr;1</SUB>/C<SUB>&bgr;</SUB>) × S<SUB>&agr;1</SUB> + (C<SUB>&agr;2</SUB>/C<SUB>&bgr;</SUB>) × S<SUB>&agr;2</SUB>
Defining A as (Calpha 1/Cbeta ) and B as (Calpha 2/Cbeta )
S<SUB>&bgr;</SUB> = A × S<SUB>&agr;1</SUB> + B × S<SUB>&agr;2</SUB> (5)
Because the alpha 1 signal does not change during hypokalemia, Salpha 1 = 1 (control defined as 1) and
S<SUB>&bgr;</SUB> = A + B × S<SUB>&agr;2</SUB> (6)
A and B are estimated by regression analysis, and relative proportions of alpha 1 and alpha 2 pumps are calculated for any time point from the immunoblot signals as follows
fraction of pumps that are &agr;1 pumps = &agr;1-subunits/(&agr;1-subunits + &agr;2-subunits)
= C<SUB>&agr;1</SUB> × S<SUB>&agr;1</SUB>/(C<SUB>&agr;1</SUB> × S<SUB>&agr;1</SUB> + C<SUB>&agr;2</SUB> × S<SUB>&agr;2</SUB>) (7)
= S<SUB>&agr;1</SUB>/[S<SUB>&agr;1</SUB> + (C<SUB>&agr;2</SUB>/C<SUB>&agr;1</SUB>) × S<SUB>&agr;2</SUB>]
= S<SUB>&agr;1</SUB>/[S<SUB>&agr;1</SUB> + (B/A) × S<SUB>&agr;2</SUB>]
fraction of pumps that are &agr;2 pumps (8)
 = 1 − fraction of &agr;1 pumps
The fractions of pumps that are alpha 1 and alpha 2 were calculated by simple linear regression (Eq. 6) and are summarized in Table 1. Similar values were obtained by employing multiple regression analysis (Eq. 5) using alpha 1 signals at either 0 or 10 days. In the control state, alpha 2 is calculated to make up 0.4 ± 0.12 of total alpha  in soleus (0.6 ± 0.12 alpha 1) and 0.55 ± 0.07 of total alpha  in WG (0.45 ± 0.07 alpha 1). After 10 days of K+ deprivation, alpha 1 is estimated at 0.76 ± 0.09 of the total pumps in soleus and 0.92 ± 0.09 of the total pumps in WG.

                              
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Table 1.   Fractions of catalytic subunits of Na pumps in soleus and white gastrocnemius muscles at early stages of K+ deprivation

Because the calculated ratios of alpha 1 and alpha 2 depend on a linear regression analysis of measurements made at all four points in the time course, there is a finite error between the calculated and measured values of variability from one specific time point to the other. For example, the calculated amount of beta 2 at day 5 (assumed to equal alpha 1 + alpha 2) should be 45% + 55%(0.3) = 62% of the control value, an underestimate of the 80% beta 2 measured by immunoblotting at this time point (Fig. 3), and the amount of beta 2 at day 10 should be 45% + 55%(0.08) = 50% of control, an overestimate of the 30% beta 2 measured by immunoblotting at day 10 (Fig. 3). However, this error does not change the prediction that there are nearly equivalent pools of alpha 1 and alpha 2 at the zero time point. Adding additional time points to the linear regression analysis would help to bring the calculated estimates closer to measurements of beta .

Na+ pump enzymatic activity. Na+-K+-ATPase activity in muscle homogenates of soleus and WG was measured via the K+-pNPPase reaction as micromoles of Pi per milligram of protein per hour. Activities in soleus and WG were 0.034 ± 0.004 and 0.033 ± 0.002 µmol Pi · mg protein-1 · h-1, respectively (n = 6 for each). After 10 days of a K+-restricted diet, activity was 0.028 ± 0.002 µmol Pi · mg protein-1 · h-1 in soleus, which was not significantly different from control, and 0.026 ± 0.002 µmol Pi · mg protein-1 · h-1 (P < 0.05) in WG (n = 6). This 19% decrease in WG activity falls short of the 70% decrease predicted from the decrease in the beta 2 pool in this tissue. In comparison, in soleus there is only a 20% fall in beta 1, which comes close to the measured change in total activity. We conclude that enzymatic activity is not a direct function of abundance. For example, the change in cell Na+ that ensues during K+ deprivation may provoke a modification of the alpha 1, in WG which persists through homogenization, that changes activity per pump.

Na+-K+-ATPase mRNA expression in soleus and WG muscles during K+ deprivation. Considering the 35-40% decreases in alpha 2 in both muscles after 2 days of K+ deprivation and the >90% decrease in WG alpha 2 after 10 days, we tested the hypothesis that the response was driven by decreases in alpha 2 mRNA in both muscles. Na+-K+-ATPase alpha 2- and beta -isoform mRNA levels in both muscles were measured after 0, 2, 5, and 10 days of K+ deprivation. Northern blot results indicate no change in alpha 2-, beta 1-, or beta 2-isoform mRNA expression in either muscle during 10 days of K+ deprivation (Fig. 4, A and B). Although distinct bands at the appropriate sizes were observed by this analysis, the quality of the RNA was compromised by the time it takes to dissect the muscles. To circumvent this problem and to reexamine our previous report of a decrease in whole hindlimb alpha 2 mRNA in rats deprived of K+ (5), we repeated the mRNA analysis in whole hindlimb muscle isolated as quickly as possible. The results confirmed the findings for the individual muscles: alpha 2, beta 2 (Fig. 4C), and beta 1 (not shown) mRNA levels in whole hindlimb did not change during 10 days of K+ deprivation.


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Fig. 4.   Northern hybridization analysis of Na+-K+-ATPase alpha 2, beta 1, and beta 2 mRNA during K+-restricted diet for 2 or 10 days. Total RNA (15 µg/lane) was fractionated on formaldehyde agarose gels, blotted onto nitrocellulose, and hybridized with 32P-labeled alpha 2- or beta -isoform-specific probes as described in MATERIALS AND METHODS. Representative autoradiograms of soleus (A), WG (B), and whole hindlimb (C) Northern blots are shown. Two alpha 2 mRNA species (5.3 and 3.4 kb), 2 beta 1 mRNA species (2.7 and 2.3 kb), and 1 beta 2 mRNA species (2.8 kb) are shown. In C, whole hindlimb control (C) vs. 2-day and control vs. 10-day results are from 2 separate autoradiograms.

Finally, we tested the possibility that there was a shift in beta -isoform expression as hypokalemia progresses. Northern blots confirmed that even at day 10, when beta 2 protein had fallen 70%, WG did not express the beta 1 transcript. Similarly, beta 2 mRNA was not detected in soleus after K+ restriction (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The existence of Na+ pump isoforms suggests the potential for isoform-specific function, expression, and regulation (31). The need to shift K+ from intracellular stores to the ECF is satisfied by tissue-specific expression and regulation of the Na+-K+-ATPase alpha 2-isoform in skeletal muscle. This report demonstrates that changes in the alpha 2-, not the alpha 1-, isoform occur early enough during K+ deprivation to drive the shift of cell K+ to the ECF, that there are distinct patterns of regulation in slow-twitch oxidative vs. fast-twitch glycolytic muscles, and that the regulation of alpha 2 occurs by posttranscriptional regulation. As muscle cell [K+] falls during K+ deprivation, cell [Na+] reciprocally increases to as high as 37 mM in WG, which is a classical stimulus for increasing Na+ pump activity in most tissues (11). However, skeletal muscle has an altruistic response to elevated cell [Na+]: Na+ pump number is decreased, which leads to a further loss of K+ and gain in Na+ in order to contribute intracellular K+ to the K+-depleted ECF. Extracellular [Na+] does not change reciprocally with [K+] during this early phase of hypokalemia because the amount of Na+ in the ECF is a primary determinant of the ECF volume, a condition that keeps the concentration fairly constant.

In the first 2 days of dietary K+ restriction there is a more rapid decrease in ECF [K+] (a 20% fall) than that over the subsequent 8 days (an additional 22% fall) (Fig. 1). The reduced rate of ECF K+ loss beyond day 2 is not unexpected because it takes ~48 h for rats on K+-restricted diets to induce mechanisms to actively reabsorb K+ and maximally reduce urinary K+ excretion, mediated by the induction of renal collecting duct H+-K+-ATPase activity (26, 48).

The responses in soleus and WG are indistinguishable during the initial 2 days of K+ restriction (Fig. 3): both muscles lose over 36% of the initial alpha 2-subunits, and changes in muscle [K+] and [Na+] are similar. Kjeldsen et al. (22) reported that after 3 days of K+ deprivation ouabain binding to whole soleus muscle (a measure of active alpha 2-type pumps at the plasma membrane) decreased 15%. That the reduction in alpha 2 Na+ pump pool sizes in soleus is greater than the decrease in surface ouabain binding sites suggests that there is some ouabain binding by alpha 1 in soleus or that the decrease in alpha 2 pumps may include both surface and nonsurface pools. Even though alpha 2 levels in both muscles fall 36%, intracellular [K+] is only beginning to fall in soleus and does not fall significantly in WG, evidence that the decrease in alpha 2 precedes, and likely accounts for, the decrease in muscle K+ stores. The finding also indicates that a loss of 36% of the muscles' Na+ pumps is not associated with a rapid change in cell K+ stores. That is, the remaining alpha 2 and invariant alpha 1 pumps are capable of nearly matching active K+ influx to passive K+ outflux, so that cell K+ stores change slowly and progressively during the 10 days of the K+-restricted diet. In previous studies, losses in total muscle K+ levels as high as 11% in whole gastrocnemius muscles after 3 days of K+-deficient fodder have been reported (22).

There is a provocative divergence in the responses of soleus and WG to K+ deprivation after 2 days (Fig. 3). In soleus, neither total alpha 2 Na+ pump abundance nor cell K+ level decreases further between days 2-5, but both decrease between days 5 and 10. Thus changes in soleus [K+] mimic the changes in soleus alpha 2 immunoreactivity, evidence for a causal link between the two. The lack of change in cell [K+] or [Na+] between days 2 and 5 of K+ restriction in soleus may be influenced by the fact that slow-twitch oxidative soleus muscle is more sensitive to changes in intracellular [Na+] than fast-twitch muscles (11), that is, the increases in muscle [Na+] might stimulate Na+ pumps and retard the loss of K+ more in slow-twitch oxidative soleus than in fast-twitch glycolytic WG. In WG, total alpha 2 Na+ pump abundance falls dramatically throughout the 10 days of K+ restriction. By day 5, the fall in WG is twofold greater than that in soleus, and by day 10 only 6% of the total alpha 2 pools remain in WG. Despite this large difference between the muscles, they both lose about the same percentage of cell K+. One hypothesis is that both muscles lose the same fraction of Na+ pumps expressed in the plasma membrane, with soleus storing pumps in endosomal pools during K+ restriction.

What is the magnitude of the physiological impact of the shift of K+ from the muscle ICF to the ECF? ECF [K+] would fall precipitously without the skeletal muscle adjustment because the amount of K+ in the ECF is small. In fact, the amount of K+ shifted from ICF to ECF is more than seven times the amount of K+ contained in the ECF of a control animal, a result calculated as follows (assumptions from Ref. 40). If we assume that ECF is 20% of the body weight, then a 280-g rat would have an ECF of 0.056 liters. Because ECF [K+] is 4.5 mM (Fig. 1), the control ECF contains ~0.25 mmol K+. Assuming that ICF is 40% of the body weight, the same rat would have an ICF of 0.112 liters. Because muscle contains ~80% of the ICF (0.090 liters) and muscle ICF [K+] is 120 mM (Fig. 3), muscle ICF contains 10.8 mmol of K+, roughly 40 times more than that in the ECF pool. After 10 days of the low-K+ diet, muscle loses an average of 17% of the intracellular stores (Fig. 3), equivalent to 1.84 mmol, which is more than seven times the amount contained in the ECF at day 0 (0.25 mM). In other words, the extracellular K+ has been replaced seven times over with K+ from the muscle stores after 10 days of a K+-restricted diet, evidence that this regulatory adjustment is critical.

The calculated ratio of alpha 2 to alpha 1 of near 1:1 in both muscles is not what one would predict from the relative RNA levels in control muscles. RNA ratios can be estimated directly with isoform-specific cDNA probes of similar lengths and labeled to similar 32P specific activities. mRNA ratios of alpha 2 to alpha 1 in skeletal muscle have been reported to be between 2.5 (0.7 alpha 2 and 0.3 alpha 1) (13) and 4 (0.8 alpha 2 and 0.2 alpha 1) (37). However, there is no a priori reason to assume that mRNA ratios are good predictors of alpha  protein ratios because other factors, from isoform-specific translatability to competition for heteromer formation with beta 1 or beta 2, will influence the protein ratio. Our estimate of 50% alpha 2 in soleus agrees with what was observed by backdoor phosphorylation (15), assuming that the fraction of alpha 2 in the euthyroid diaphragm is midway between that observed in the hypothyroid (30%) and hyperthyroid (65%) diaphragms. The percentage of alpha 2 in soleus, calculated by Lavoie et al. (23) at between 60% in the surface membrane and 87% in intracellular membranes, is higher than our estimate. This is not unexpected because the subcellular membrane fractions assayed are expected to be enriched in alpha 2, and it has been established that the subcellular distribution of alpha 2 is different from that of alpha 1 in red muscle (18). In comparison, in this study we calculated the alpha 2-to-alpha 1 ratio in total homogenate, which contains all the cell membranes, thus averaging the ratio in membranes enriched in alpha 2 with those enriched in alpha 1.

Azuma et al. (5) reported that, in rats maintained on a low-K+ diet for 14 days, hindlimb alpha 2 mRNA decreased 35% (alpha 1 and beta 1 were unchanged) and that alpha 2 protein decreased by 82%, suggesting that changes in alpha 2 mRNA alone could not account for the changes in alpha 2 protein (5). Because the whole hindlimb in rats is a composite of different muscles, it was conceivable that the mRNA changes were muscle specific. We have previously determined that during the transition from euthyroid to hypothyroid states, changes in alpha 2 mRNA levels in mixed gastrocnemius muscle predicted the changes in alpha 2 protein levels (both decreased 45%) (6). However, unlike what was found for regulation by thyroid status, mRNA levels in either soleus, WG, or whole hindlimb measured in this study did not change during 10 days of K+ deprivation (Fig. 4). Thus we do not confirm the observations in our previous study (5) and hypothesize that alpha 2 mRNA levels decrease only after a K+ deprivation of more prolonged duration. Alternatively, it is possible that the K+-deficient diet used in the present study is better matched to the control diet than in the previous report, in which the weights of the K+-deprived rats were significantly lower than the controls after 14 days of the low-K+ diet. We conclude that the decreases in Na+ pump expression during K+ deprivation can be explained by either a decrease in alpha - and beta -transcript translatability and/or increased protein degradation. A rapid increase in protein degradation in muscle is not without precedent. The ubiquitin-proteasome pathway plays a role in increasing the rate of muscle protein degradation during denervation, fasting, or insulinopenia (34). Plasma membrane proteins are also degraded by internalization to endosomal pools and routing to lysosomes. The alpha 2beta 1-type heteromers are known to shuttle between endosomal pools and the plasma membrane with insulin stimulation in oxidative muscles such as soleus but not in glycolytic muscles (24). These findings suggest the hypothesis that when ECF [K+] falls the rate of Na+ pump internalization increases in both muscle types, that a portion of the internalized pumps are stored in endosomal pools in the soleus and are available for return to the plasma membrane with K+ restoration or insulin stimulation, and that pumps are routed for degradation in the lysosomes in both muscles. Such a pattern would account for the smaller decrease in alpha 2 in soleus than in WG, along with the similar rates of loss of K+ from both muscle types (Fig. 3).

How tissues like muscle or kidney sense the fall in extracellular [K+] and then effect tissue-specific changes such as the decrease in muscle Na+-K+-ATPase alpha 2 levels remains unclear. One theory is that there are K+ sensors in the gut, portal circulation, and/or liver that respond to local changes in extracellular [K+], secondary to enteric changes (39). This suggestion complements the recent identification of the calcium-sensing receptor (CaR) (7). Indeed, Quarles et al. (38) have theorized that CaRs may represent one member of a family of "cation-sensing" cell surface receptors. Further, Hevener et al. (16) localized glucosensors to the portal vein. When these results are taken together, it is tempting to speculate that both glucose and K+ sensors in the hepatic portal vein or liver may respond to dietary intake, stimulating the release of humoral factors (such as insulin when glucose and K+ levels are elevated) that alter muscle and kidney K+ handling by regulating transporter levels.

In conclusion, our results demonstrate a temporal relationship between the decreases in alpha 2 Na+ pump pools and a coincident or subsequent decrease in muscle [K+] during K+ deprivation in both slow-twitch oxidative and fast-twitch glycolytic muscles. The study establishes there are muscle type-specific mechanisms in place to effect the shift of K+ from ICF to ECF and that in both muscles the changes are independent of changes in mRNA levels.


    ACKNOWLEDGEMENTS

This work was supported by National Science Foundation Grant IBN 9S13958 to A. A. McDonough and J. N. Youn and by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-34316 to A. A. McDonough.


    FOOTNOTES

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. §1734 solely to indicate this fact.

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 90033.

Received 24 August 1998; accepted in final form 25 March 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akaike, N. Contribution of electrogenic sodium pumps to membrane potential in mammalian skeletal muscle fibres. J. Physiol. (Lond.) 245: 499-520, 1975[Abstract].

2.   Ariano, M. A., R. B. Armstrong, and V. R. Edgerton. Hindlimb muscle fiber populations of five mammals. J. Histochem. Cytochem. 21: 51-55, 1973[Medline].

3.   Armstrong, R. B., and R. O. Phelps. Muscle fiber type composition of the rat hindlimb. Am. J. Anat. 171: 259-272, 1984[Medline].

4.   Arystarkhova, E., and K. J. Sweadner. Tissue-specific expression of the Na,K-ATPase beta 3 subunit. J. Biol. Chem. 272: 22405-22408, 1997[Abstract/Free Full Text].

5.   Azuma, K. K., C. B. Hensley, D. S. Putnam, and A. A. McDonough. Hypokalemia decreases Na+-K+-ATPase alpha 2- but not alpha 1-isoform abundance in heart, muscle, and brain. Am. J. Physiol. 260 (Cell Physiol. 29): C958-C964, 1991[Abstract/Free Full Text].

6.   Azuma, K. K., C. B. Hensley, M.-J. Tang, and A. A. McDonough. Thyroid hormone specifically regulates skeletal muscle Na+-K+-ATPase alpha 2- and beta 2-isoforms. Am. J. Physiol. 265 (Cell Physiol. 34): C680-C687, 1993[Abstract/Free Full Text].

7.   Brown, E. M., G. Gamba, D. Riccardi, M. Lombardi, R. Butters, O. Kifor, A. Sun, M. A. Hediger, J. Lytton, and S. C. Hebert. Cloning and characterization of an extracellular Ca2+-sensing receptor from bovine parathyroid. Nature 366: 575-580, 1993[Medline].

8.   Chomczynski, P., and N. Sacchi. Single step method of RNA isolation by acid guanidinium thiocyanate phenol chloroform extraction. Anal. Biochem. 162: 156-159, 1987[Medline].

9.   Clausen, T., K. Kjeldsen, and A. Norgaard. Effects of denervation on sodium, potassium and [3H]ouabain binding in muscles of normal and potassium-depleted rats. J. Physiol. (Lond.) 345: 123-134, 1983[Abstract].

10.   Clausen, T. G., K. Brocks, and H. Ibsen. Hypokalemia and ventricular arrhythmias in acute myocardial infarction. Acta Med. Scand. 224: 531-537, 1988[Medline].

11.   Everts, M. E., and T. Clausen. Activation of the Na-K pump by intracellular Na in rat slow- and fast-twitch muscle. Acta Physiol. Scand. 145: 353-362, 1992[Medline].

12.   Feinberg, A. P., and B. Vogelstein. A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 137: 266-267, 1984[Medline].

13.   Gick, G. G., M. A. Hatala, D. Chon, and F. Ismail-Beigi. Na,K-ATPase in several tissues of the rat: tissue-specific expression of subunit mRNAs and enzyme activity. J. Membr. Biol. 131: 229-236, 1993[Medline].

14.   Gonzalez-Martinez, L. M., J. Avila, E. Marti, E. Lecuona, and P. Martin-Vasallo. Expression of the beta -subunit isoforms of the Na,K-ATPase in rat embryo tissues, inner ear, and choroid plexus. Biol. Cell 81: 215-222, 1994[Medline].

15.   Haber, R. S., and J. N. Loeb. Selective induction of high-ouabain-affinity isoform of Na+-K+-ATPase by thyroid hormone. Am. J. Physiol. 255 (Endocrinol. Metab. 18): E912-E919, 1988[Abstract/Free Full Text].

16.   Hevener, A. L., R. N. Bergman, and C. M. Donovan. Novel glucosensor for hypoglycemic detection localized to the portal vein. Diabetes 46: 1521-1525, 1997[Abstract].

17.   Horowitz, B., C. B. Hensley, M. Quintero, K. K. Azuma, D. Putnam, and A. A. McDonough. Differential regulation of Na,K-ATPase alpha 1, alpha 2, and beta subunit mRNA and protein levels by thyroid hormone. J. Biol. Chem. 265: 14308-14314, 1990[Abstract/Free Full Text].

18.   Hundal, H. S., A. Marette, Y. Mitsumoto, T. Ramlal, R. Blostein, and A. Klip. Insulin induces translocation of the alpha 2 and beta 1 subunits of the Na+/K+-ATPase from intracellular compartments to the plasma membrane in mammalian skeletal muscle. J. Biol. Chem. 267: 5040-5043, 1992[Abstract/Free Full Text].

19.   Hundal, H. S., A. Marette, T. Ramlal, Z. Liu, and A. Klip. Expression of beta subunit isoforms of the Na+,K+-ATPase is muscle type-specific. FEBS Lett. 328: 253-258, 1993[Medline].

20.   Ianuzzo, C. D., and B. Dabrowski. Na+/K+-ATPase activity of different types of striated muscles. Biochem. Med. Metab. Biol. 37: 31-34, 1987[Medline].

21.   Kjeldsen, K., M. E. Everts, and T. Clausen. The effects of thyroid hormones on 3H-ouabain binding site concentration, Na,K-contents and 86Rb-efflux in rat skeletal muscle. Pflügers Arch. 406: 529-535, 1986[Medline].

22.   Kjeldsen, K., A. Norgaard, and T. Clausen. Effect of K-depletion on 3H-ouabain binding and Na-K-contents in mammalian skeletal muscle. Acta Physiol. Scand. 122: 103-117, 1984[Medline].

23.   Lavoie, L., R. Levenson, P. Martin-Vasallo, and A. Klip. The molar ratios of alpha  and beta  of the Na+-K+-ATPase differ in distinct subcellular membranes from rat skeletal muscle. Biochemistry 36: 7726-7732, 1997[Medline].

24.   Lavoie, L., D. Roy, T. Ramlal, L. Dombrowski, P. Martin-Vasallo, A. Marette, J. L. Carpentier, and A. Klip. Insulin-induced translocation of Na+-K+-ATPase subunits to the plasma membrane is muscle fiber type specific. Am. J. Physiol. 270 (Cell Physiol. 39): C1421-C1429, 1996[Abstract/Free Full Text].

25.   Lescale-Matys, L., D. S. Putnam, and A. A. McDonough. Na+-K+-ATPase alpha 1- and beta 1-subunit degradation: evidence for multiple subunit specific rates. Am. J. Physiol. 264 (Cell Physiol. 33): C583-C590, 1993[Abstract/Free Full Text].

26.   Linas, S. L., L. N. Peterson, R. J. Anderson, G. A. Aisenbray, F. R. Simon, and T. Berl. Mechanism of renal potassium conservation in the rat. Kidney Int. 15: 601-611, 1979[Medline].

27.   Lingrel, J. B., and T. Kuntzweiler. Na,K-ATPase. J. Biol. Chem. 269: 19659-19662, 1994[Free Full Text].

28.   Magyar, C. E., J. Wang, K. K. Azuma, and A. A. McDonough. Reciprocal regulation of cardiac Na-K-ATPase and Na/Ca exchanger: hypertension, thyroid hormone, development. Am. J. Physiol. 269 (Cell Physiol. 38): C675-C682, 1995[Abstract].

29.   Malik, N., V. A. Canfield, M. C. Beckers, P. Gros, and R. Levenson. Identification of the mammalian Na,K-ATPase beta 3 subunit. J. Biol. Chem. 271: 22754-22758, 1996[Abstract/Free Full Text].

30.   Martin-Vasallo, P., W. Dackowski, J. R. Emanuel, and R. Levenson. Identification of a putative isoform of the Na,K-ATPase beta subunit. Primary structure and tissue-specific expression. J. Biol. Chem. 46: 13-18, 1989.

31.   McDonough, A. A., K. K. Azuma, L. Lescale-Matys, M. J. Tang, F. Nakhoul, C. B. Hensley, and Y. Komatsu. Physiologic rationale for multiple sodium pump isoforms. Differential regulation of alpha 1 vs. alpha 2 by ionic stimuli. In: Ion Motive ATPases, edited by A. Scarpa, E. Carafoli, and S. Papa. New York: NY Acad. Sci., 1992, p. 156-168.

32.   McDonough, A. A., T. A. Brown, B. Horowitz, R. Chiu, J. Schlotterbeck, J. Bowen, and C. A. Schmitt. Thyroid hormone coordinately regulates Na+-K+-ATPase alpha - and beta -subunit mRNA levels in kidney. Am. J. Physiol. 254 (Cell Physiol. 23): C323-C329, 1988[Abstract/Free Full Text].

33.   McDonough, A. A., and C. B. Thompson. Role of skeletal muscle sodium pumps in the adaptation to potassium deprivation. Acta Physiol. Scand. 156: 295-304, 1996[Medline].

34.   Mitch, W. E., and A. L. Goldberg. Mechanisms of muscle wasting. N. Engl. J. Med. 335: 1897-1905, 1996[Free Full Text].

35.   Murer, H., E. Ammann, J. Biber, and U. Hopfer. The surface membranes of the small intestinal epithelial cell. I. Localization of adenyl cyclase. Biochim. Biophys. Acta 433: 509-519, 1976[Medline].

36.   Norgaard, A., K. Kjeldsen, and T. Clausen. Potassium depletion decreases the number of 3H-ouabain binding sites and the active Na-K transport in skeletal muscle. Nature 293: 739-741, 1981[Medline].

37.   Orlowski, J., and J. B. Lingrel. Tissue-specific and developmental regulation of rat Na,K-ATPase catalytic alpha  isoform and beta  subunit mRNAs. J. Biol. Chem. 263: 10436-10442, 1988[Abstract/Free Full Text].

38.   Quarles, L. D., J. E. Hartle, S. R. Siddhanti, R. Guo, and T. K. Hinson. A distinct cation-sensing mechanism in MC3T3-E1 osteoblast functionally related to the calcium receptor. J. Bone Miner. Res. 12: 393-402, 1997[Medline].

39.   Rabinowitz, L., R. L. Sarason, and H. Yamauchi. Sheep renal K+ excretion: efferent kaliuretic regulatory factors. Am. J. Physiol. 247 (Renal Fluid Electrolyte Physiol. 16): F520-F526, 1984[Medline].

40.   Seldin, D. W., and G. Giebisch. The Regulation of Potassium Balance. New York: Raven, 1989.

41.   Shamraj, O. I., and J. B. Lingrel. A putative fourth Na+,K+-ATPase alpha-subunit gene is expressed in testis. Proc. Natl. Acad. Sci. USA 91: 12952-12956, 1994[Abstract/Free Full Text].

42.   Sweadner, K. J. Isozymes of the Na+/K+-ATPase. Biochim. Biophys. Acta 988: 185-220, 1989[Medline].

43.   Thompson, C. B., and A. A. McDonough. Skeletal muscle Na,K-ATPase alpha  and beta  subunit protein levels respond to hypokalemic challenge with isoform and muscle type specificity. J. Biol. Chem. 271: 32653-32658, 1996[Abstract/Free Full Text].

44.   Tsuda, T., S. Kaya, T. Yokoyama, Y. Hayashi, and K. Taniguchi. ATP and acetyl phosphate induces molecular events near the ATP binding site and the membrane domain of Na+,K+-ATPase. The tetrameric nature of the enzyme. J. Biol. Chem. 273: 24339-24345, 1998[Abstract/Free Full Text].

45.   Urayama, O., H. Shutt, and K. J. Sweadner. Identification of three isozyme proteins of the catalytic subunit of the Na,K-ATPase in rat brain. J. Biol. Chem. 264: 8271-8280, 1989[Abstract/Free Full Text].

46.   Wang, J., R. H. Schwinger, K. Frank, J. Muller-Ehmsen, P. Martin-Vasallo, T. A. Pressley, A. Xiang, E. Erdmann, and A. A. McDonough. Regional expression of sodium pump subunits isoforms and Na+-Ca++ exchanger in the human heart. J. Clin. Invest. 98: 1650-1658, 1996[Abstract/Free Full Text].

47.   Wi, J. K., J. K. Kim, and J. H. Youn. Reduced glucose clearance as the major determinant of postabsorptive hyperglycemia in diabetic rats. Am. J. Physiol. 274 (Endocrinol. Metab. 37): E257-E264, 1998[Abstract/Free Full Text].

48.   Wingo, C. S., and B. D. Cain. The renal H-K-ATPase: physiological significance and role in potassium homeostasis. Annu. Rev. Physiol. 55: 323-347, 1993[Medline].


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