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 |
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
2 (not
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
2
can decrease before cell
[K+].
After 5 days, there were further decreases in
2 (70%) and
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
2 (94%) and
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
2,
1, and
2 mRNA were unchanged over 10 days. The ratios of
2 to
1 protein levels in both control muscles were found to be
nearly 1 by using the relative changes in
-isoforms vs.
1-
(soleus) or
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
2
1 and
2
2 are distinct
and that decreases in
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 |
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 
heteromer composed of a
catalytically active
-subunit (Mr
112,000)
and a glycosylated
-subunit
(Mr
35,000),
and recent evidence suggests that it is a tetramer (44). Multiple
-
and
-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
1-,
2-, and
1-isoforms as
1
1 and
2
1, whereas fast-twitch glycolytic
white gastrocnemius (WG) expresses
1-,
2-, and
2-isoforms as
1
2 and
2
2 (43). Background information on the proportion of
2- to
1-type Na+ pumps in
muscle is limited to two studies. For rat diaphragm membranes the
fraction of
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
2 to
1 ranged from 1.6 (60%
2) in surface membranes
to 7 (87%
2) in intracellular membranes. The ratio was calculated
by using ouabain binding per milligram of protein to quantitate
2,
and the immunoblot signal was scaled against the signal from a
recombinant fragment of
1 per milligram of protein to measure
1
(23). In comparison, the proportion of
2 to total
mRNA in
skeletal muscle has been reported as being between 70 and 80% (13,
37). The ratio of total
2 to total
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,
2 and
2 protein levels decrease to only 6 and 30% of
control, respectively, whereas in slow-twitch oxidative soleus
2
decreased to 45% of control and
1 did not decrease significantly.
The
1 protein pool size for either muscle did not change. Despite
the substantially greater decrease in
2 and
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
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
2- to
1-isoforms because it will affect the impact of
2-specific regulation. The present study demonstrates that changes
in
2 protein levels occur early enough to account for the loss of
cell K+ in both muscle types, that
the decrease in
2 protein is not secondary to a decrease in
2
mRNA, and that the calculated percentage of
2-type pumps in control
muscles ranges from 40% in soleus to 55% in WG.
 |
MATERIALS AND METHODS |
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
- and
-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
-subunit, sugar residues
were removed from
-subunits with PNGase F as previously described
(43). A constant amount of homogenate protein (100 µg for
-subunit
analysis, 50 µg for
-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
2 (45), generously provided by K. Sweadner (Harvard Medical School); anti-
1 FP (1:500), a polyclonal
antibody against
1 (43); SpET b2 (1:2,000), a polyclonal antibody
against human
2 (14), generously provided by P. Martin-Vasallo
(Universidad de La Laguna); and RNT
3 (1:2,000), a polyclonal
antibody against rat
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
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
and
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
1,
2, or
1 clones as
described by Orlowski and Lingrel (37) or from the rat
2 cDNA clone
provided by P. Martin-Vasallo (30). The
- and
-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
1- and
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 |
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
3 was expressed in muscle and regulated during
K+ deprivation was tested since
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
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
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
3-subunit at the appropriate molecular masses. We
conclude that the
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
2- and
1-isoforms (soleus) and
2-isoforms (WG) after 0, 2, and
5 days of K+ deprivation was
determined by immunoblotting. The previously reported immunoblot
results for
2 and
after 10 days of
K+ deprivation (43) are included
in this analysis. The
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
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
2 protein
expression decreased 36% as early as day
2. In soleus and WG,
1 and
2,
respectively, were depressed significantly by 23% at
day
5.

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Fig. 2.
Detection of
Na+-K+-ATPase
- and -isoforms in muscles from control (C) and
K+-deprived ( 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 2 and 50 µg
for 1 or 2) are shown. The 1- and 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
2, 1, and 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
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
2,
1, or
cell ion concentrations, whereas in WG there were significant further
decrements in both
2 and
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:
2 and
2 decreased 94 and 70%,
respectively, from control levels in WG, whereas in
soleus
2 decreased 56% and
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
2 pools during the
low-K+ diet are greater than the
decreases in
1 or
2 pools at all time points is expected because
a single
must form heteromers with not only
2 (which decreases)
but also with
1 (which is invariant). In other words, the difference
between the change in
2 and
(
1 in soleus,
2 in WG) will be
a function of the ratio of expression of
1
to
2
heteromers
in that muscle, as calculated in the next section.
Estimating the proportion of the
Na+-K+-ATPase
2-isoform in soleus and WG.
It has been difficult to directly assess the ratio of
1- to
2-type Na+ pumps in muscle.
Although absolute pool sizes of
2 vs.
1 protein subunits cannot
be determined directly with antibody probes, muscle-specific changes in
2 vs.
1 or
2 abundance during
K+ deprivation can be employed to
calculate the ratios of
2 to
1. The calculations depend on three
assumptions. First,
1 and
2 proteins are assumed to combine with
only
1 in soleus and only
2 in WG in a 1:1 stoichiometry. Indeed,
recent evidence suggests that Na+
pumps may exist as tetramers with a 1:1
-to-
stoichiometry in
mammalian cells (44). Second, it is assumed that there are only
negligible pools of uncomplexed
- or
-subunits, a difficult assumption to test in this system but supported by evidence that
-
and
-subunits form complexes as 
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
-subunits is equal
to the total number of
-subunits (Eq.
1), the total number of
-subunits
is equal to the sum of
1- and
2-subunits
(Eq.
2), and thus that the total number
of
pumps is equal to the sum of
1 and
2 pumps
(Eq.
3)
|
(1)
|
|
(2)
|
|
(3)
|
The
number of
- or
-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)
|
(4a)
|
|
(4b)
|
|
(4c)
|
Thus
Eq. 3
becomes
Dividing
by C
Defining
A as
(C
1/C
)
and B as
(C
2/C
)
|
(5)
|
Because
the
1 signal does not change during hypokalemia,
S
1 = 1 (control defined as 1)
and
|
(6)
|
A
and B are estimated by regression analysis, and relative proportions of
1 and
2 pumps are calculated for any time point from the
immunoblot signals as follows
|
(7)
|
|
(8)
|
The fractions of pumps that are
1 and
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
1 signals at either 0 or
10 days. In the control state,
2 is calculated to make up 0.4 ± 0.12 of total
in soleus (0.6 ± 0.12
1) and 0.55 ± 0.07 of
total
in WG (0.45 ± 0.07
1). After 10 days of
K+ deprivation,
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
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|
Because the calculated ratios of
1 and
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
2 at
day 5 (assumed to equal
1 +
2) should be 45% + 55%(0.3) = 62% of the
control value, an underestimate of the 80%
2 measured by immunoblotting at this time point (Fig. 3), and the amount of
2 at
day
10 should be 45% + 55%(0.08) = 50%
of control, an overestimate of the 30%
2 measured by immunoblotting
at day
10 (Fig. 3). However, this error does
not change the prediction that there are nearly equivalent pools of
1 and
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
.
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
2 pool in this tissue. In comparison, in soleus there is only
a 20% fall in
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
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
2 in both muscles after 2 days of K+ deprivation and the
>90% decrease in WG
2 after 10 days, we tested the hypothesis
that the response was driven by decreases in
2 mRNA in both muscles.
Na+-K+-ATPase
2- and
-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
2-,
1-,
or
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
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:
2,
2
(Fig. 4C), and
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
2, 1, and 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 2- or
-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
2 mRNA species (5.3 and 3.4 kb), 2 1 mRNA species (2.7 and 2.3 kb), and 1 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
-isoform expression as hypokalemia progresses. Northern blots confirmed that even at day
10, when
2 protein had fallen 70%, WG did not express the
1 transcript. Similarly,
2 mRNA was not detected in soleus after K+
restriction (data not shown).
 |
DISCUSSION |
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
2-isoform in skeletal muscle. This report demonstrates that changes
in the
2-, not the
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
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
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
2-type pumps at
the plasma membrane) decreased 15%. That the reduction in
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
1 in soleus or that
the decrease in
2 pumps may include both surface and nonsurface
pools. Even though
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
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
2 and invariant
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
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
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
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
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
2 to
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
2 to
1 in skeletal muscle have been reported to be
between 2.5 (0.7
2 and 0.3
1) (13) and 4 (0.8
2 and 0.2
1)
(37). However, there is no a priori reason to assume that mRNA ratios
are good predictors of
protein ratios because other factors, from
isoform-specific translatability to competition for heteromer formation
with
1 or
2, will influence the protein ratio. Our estimate of
50%
2 in soleus agrees with what was observed by backdoor
phosphorylation (15), assuming that the fraction of
2 in the
euthyroid diaphragm is midway between that observed in the hypothyroid
(30%) and hyperthyroid (65%) diaphragms. The percentage of
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
2, and it has been
established that the subcellular distribution of
2 is different from
that of
1 in red muscle (18). In comparison, in this study we
calculated the
2-to-
1 ratio in total homogenate, which contains
all the cell membranes, thus averaging the ratio in membranes enriched in
2 with those enriched in
1.
Azuma et al. (5) reported that, in rats maintained on a
low-K+ diet for 14 days, hindlimb
2 mRNA decreased 35% (
1 and
1 were unchanged) and that
2
protein decreased by 82%, suggesting that changes in
2 mRNA alone
could not account for the changes in
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
2 mRNA levels in mixed gastrocnemius
muscle predicted the changes in
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
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
- and
-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
2
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
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
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
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.
 |
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