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1 Department of Pharmacology, The Milton S. Hershey Medical Center, College of Medicine, The Pennsylvania State University, Hershey, Pennsylvania 17033-0850; and 2 Division of Biochemistry, School of Biological Sciences, Royal Holloway University of London, Egham, Surrey TW20 0EX, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Skeletal muscle expresses multiple isoforms of
the
Na+-K+-ATPase.
Their expression has been shown to be differentially regulated under
pathophysiological conditions. In addition, previous studies suggest
possible age-dependent alterations in
Na+-K+
pump function. The present study tests the hypothesis that advancing age is associated with altered
Na+-K+-ATPase
enzyme activity and isoform-specific changes in expression of the
enzyme subunits. Red and white gastrocnemius (Gast) as well as soleus
muscles of male Fischer 344/Brown Norway (F-344/BN) rats at 6, 18, and
30 mo of age were examined.
Na+-K+-ATPase
activity, measured by
K+-stimulated
3-O-methylfluorescein phosphatase
activity, increased by ~50% in a mixed Gast homogenate from
30-mo-old compared with 6- and 18-mo-old rats. Advancing age was
associated with markedly increased
1- and
1-subunit, and decreased
2- and
2-subunit in red and white
Gast. In soleus, there were similar changes in expression of
1- and
2-subunits, but levels of
1-subunit were unchanged.
Functional
Na+-K+-ATPase
units, measured by
[3H]ouabain binding,
undergo muscle-type specific changes. In red Gast, high-affinity
ouabain-binding sites, which are a measure of
2-isozyme, increased in
30-mo-old rats despite decreased levels of
2-subunit. In white Gast, by
contrast, decreased levels of 2-subunit were accompanied by
decreased high-affinity ouabain-binding sites. Finally, patterns of
expression of the four myosin heavy chain (MHC) isoforms (type I, IIA,
IIX, and IIB) in these muscles were similar in the three age groups
examined. We conclude that, in the skeletal muscles of F-344/BN rats,
advancing age is associated with muscle type-specific alterations in
Na+-K+-ATPase
activity and patterns of expression of - and -subunit isoforms.
These changes apparently occurred without obvious shift in muscle fiber
types, since expression of MHC isoforms remained unchanged. Some of the
alterations occurred between middle-age (18 mo) and senescence (30 mo),
and, therefore, may be attributed to aging of skeletal muscle.
aging; immunoblotting; ouabain binding; isozyme; sodium-potassium pump
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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THE NA+-k+ pump plays a critical role in maintaining Na+-K+ homeostasis, and skeletal muscle contains one of the largest pools of this pump in the body (9). In skeletal muscle, in addition to keeping a low intracellular Na+ concentration, the pump is important in clearance of K+ from extracellular space and in restoration of K+ loss after muscle use (4, 8, 38). Indeed, contractile function of skeletal muscle is associated with Na+-K+-pump activity (17, 38), consistent with the notion that accumulation of extracellular K+ contributes to muscle fatigue (39, 47). Thus exercise training increases expression and activity of the Na+-K+-ATPase (23, 24), the functional unit of the Na+-K+ pump, resulting in attenuated rise in plasma K+ during physical activity (34, 35). On the other hand, when K+ intake is low, expression of Na+-K+-ATPase is decreased such that the uptake of K+ into skeletal muscle is reduced, and plasma K+ levels are preserved (40, 51).
With advancing age, fatigue of muscles occurs at lower intensity of
physical activity (45). Previous reports (14, 15) have
provided evidence that in humans and in rats extrarenal
K+ adaptation, which, to a large
extent, is modulated by K+
handling by the skeletal muscle, may be impaired with advancing age. In
elderly men, the rate of increase in plasma
K+ concentration during a bout of
exercise was greater than in young men (14). Elderly subjects are also
less responsive to -adrenergic receptor-mediated increases in net
flux of K+ in the forearm (15), as
measured by arterial and venous K+
differences, suggesting possible impairment of
K+ uptake mechanisms. Finally, the
number of [3H]ouabain
binding sites, a measure of functional
Na+-K+-ATPase
units, in human vastus lateralis muscle showed a tendency to decrease
with age (24, 41). Together, these studies suggest possible
age-dependent alterations in
Na+-K+
pump function.
Na+-K+-ATPase
consists of a transmembrane catalytic -subunit and a -subunit.
Four different isoforms of the -subunit,
1, 2,
3, and
4, and three separate isoforms
of the -subunit, 1,
2, and
3, have been cloned and
sequenced (30, 31, 33, 46, 49). Skeletal muscle of the mature rat
expresses 1- and 2-subunit, with
2 being the more abundant
subunit (18, 29, 42, 49), and all three -subunit isoforms have been
reported in this tissue (2, 21). The
1 and
1 are more abundant in fast and
slow oxidative rich fibers than in fast glycolytic fibers, whereas the
opposite is true for 2 (21,
51). Distribution of the recently identified
3-subunit has not yet been
determined. Physiological functions of the isozymes remain incompletely
understood, although they differ in their affinities for
Na+ and
K+ (10, 22) and digitalis
glycosides (36, 49). Whereas
1-isozyme is a better substrate
for phosphorylation by kinase, and therefore its activity may be
modified (6, 12), insulin appears to selectively translocate
2-isozyme from intracellular
site to the plasma membrane (20). Thus changes in expression of the isozymes under certain pathophysiological conditions may affect Na+-K+
transport in skeletal muscle cells.
Relative expression of
Na+-K+-ATPase
subunit isoforms in skeletal muscle with advancing age is completely
unknown. Elucidation of potential alterations in these subunits may
provide insight into the functional changes in skeletal muscle during
aging. Therefore, in this study, we examined activity and expression of
the
Na+-K+-ATPase
isozymes in gastrocnemius and soleus muscles of rats with advancing
age. Male Fischer 344/Brown Norway (F-344/BN) rats at 6, 18, and 30 mo
of age, were studied, representing mature adult, middle-aged, and
senescent rats (26, 32), respectively. Because aging of skeletal muscle
has been shown to cause transition from fast to slow fiber types (27,
28), which differ in their expression of
Na+-K+-ATPase
subunit isoforms (51), possible switching of fiber types in the muscles
was evaluated by examining expression of myosin heavy chain (MHC)
isoforms. The results show differential regulation of the subunit
isoform with advancing age. In skeletal muscles of older rats, there is
a muscle-type-specific marked upregulation of the
1- and
1-subunit, downregulation of
2- and
2-subunit, and an overall
increase in K+-dependent
3-O-methylfluorescein phosphatase
(3-O-MFPase) activity, a
measure of
Na+-K+-ATPase
activity. Interestingly, altered expression of the
Na+-K+-ATPase
subunit isoforms during aging is not associated with any obvious
fiber-type switching.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Animals. Male F-344/BN rats at 6, 18, and 30 mo of age were purchased from the National Institute on Aging.
The rats were housed in our animal-care facility for 2 wk before being
killed. They were deeply anesthetized with ether, and the chest was
opened to remove the heart. Hindlimb skeletal muscles, including red and white gastrocnemius and soleus muscles, were dissected, weighed, and frozen in liquid nitrogen. The tissues were stored at
70°C until use.
Preparation of tissue homogenates.
Skeletal muscles (~100 mg) were pulverized and homogenized with a
Polytron (Brinkmann Instruments, Westbury, NY) at a speed of
6.5 (11.0 full scale) for three 20-s periods at 4°C in
a buffer containing Tris · HCl (10 mM, pH 7.5), EDTA
(1 mM), protease inhibitors (phenylmethylsulfonyl fluoride; 500 µM),
leupeptin (1 µM), pepstatin (1 µM), and E-64 (10 µM). In
experiments in which enzyme activity was measured, homogenates were
prepared without protease inhibitors, stored at 70°C, and used within 8 days. Protein concentrations were determined by the
Bio-Rad protein assay (Bio-Rad, Melville, NY).
Western blotting of
Na+-K+-ATPase
isoforms.
Subunits of
Na+-K+-ATPase
were resolved by SDS-PAGE according to the method of Laemmli (25), with
slight modifications, as previously described (36). For analysis of
-subunit isoforms, equal amounts of homogenates (80 µg) were
electrophoresed in 5 or 7.5% acrylamide gels. For analysis of the
-subunits, equal amounts of homogenate preparations (200 or 260 µg) were deglycosylated with
N-glycosidase F (Boehringer Mannheim,
Indianapolis, IN) for 18-20 h at 37°C, heated for 10 min at
60°C, and electrophoresed in 7.5% acrylamide gels according to the
method of Smith et al. (48), as previously described (7). For analysis
of -sarcomeric actin, equal amounts of homogenates (13 µg) were
separated on 8.75% acrylamide gel. Gels were electrophoretically
transferred to Immobilon-P membrane (Millipore, Bedford, MA) or
PVDF membrane (Bio-Rad, Melville, NY). To detect
1-,
2-, and
2-subunits, membranes were
blocked in a Tris-buffered saline solution (10 mM Tris, 150 mM NaCl, pH
7.4) containing 0.2% Tween-20 (TBST) and subsequently incubated with
monoclonal antibodies against 1-,
2- (generous gift from Dr. K. Sweadner, Harvard University), or
2-subunits (5, 43). To detect
actin, blots were blocked with TBST plus 5% dry milk (Blotto-Tween)
and incubated with a monoclonal antibody against -sarcomeric actin
(Sigma Chemical, St. Louis, MO). To detect the
1-subunit, membranes were
blocked in Blotto-Tween and incubated with an
anti-1-antiserum (Upstate Biological, Lake Placid, NY) or with a monoclonal
anti-1-antibody (Affinity
BioReagents, Golden, CO). Bound monoclonal antibodies were detected
with rabbit anti-mouse IgG, followed by
125I-labeled Protein
A (ICN, Costa Mesa, CA), whereas bound polyclonal antibodies were detected with
125I-Protein A alone. The blots
were subjected to autoradiography for the purpose of displaying the
images. Subsequently, band signal intensities were quantitated by
scanning the blots using a phosphor imager (Molecular Dynamics,
Sunnyvale, CA). The transferred gels and portions of some of the
membranes were stained with Coomassie blue to verify, respectively,
efficient transfer of proteins and equal loading (data not shown).
1 in white gastrocnemius, a
chemiluminescent detection method was used. Blots were incubated with
monoclonal anti-1-antibody and,
subsequently, with rabbit anti-mouse IgG antibody and goat anti-rabbit
IgG antibody conjugated with horseradish peroxidase (Sigma Chemical,
St. Louis, MO). Chemiluminescent substrate from Pierce (SuperSignal;
Rockford, IL) was used, and blots were exposed to multiple films to
ensure that signals were within the linear range of the film.
K+-dependent 3-O-MFPase activity. Enzyme activity in total skeletal muscle homogenates was determined according to the method of Huang and Askari (19). Briefly, homogenates (60 µg) were incubated in a buffer for 5 min at 37°C, containing (in mM) 50 Tris, 1 EDTA, 3 MgCl2, and 10 KCl. Reaction was started by the addition of 3-O-methylfluorescein phosphate (10 µM), and fluorescence was collected for 5 min during linear increase in signals by using a SPEX spectrophotometer (Edison, NJ). Nonspecific enzyme activity was assayed in the absence of KCl and in the presence of 2 mM ouabain. Specific activity was defined as the difference between total and nonspecific activities. Homogenates were not treated with detergent, because preliminary study showed that detergent treatment did not increase enzyme activity in our protocol (data not shown).
[3H]ouabain binding.
Specific binding of
[3H]ouabain to
high-affinity ouabain binding sites was estimated as described
previously (36). Briefly, tissue homogenates were incubated in a medium
containing 1.5 mM MgCl2, 1 mM
phosphate, 10 mM Tris · HCl buffer (pH 7.5), and 100 nM [3H]ouabain. Bound
and free [3H]ouabain
was separated after a 60-min incubation at 37°C by filtering the
mixture through a nitrocellulose filter (Millipore, Boston, MA).
Nonspecific binding of
[3H]ouabain was
assayed concurrently in the presence of 2 mM ouabain. Maximum binding
(Bmax) of
[3H]ouabain to
high-affinity ouabain binding site, the
2-isozyme, is calculated
according to the equation: Bmax = B + Kd(B/Of), where B is the measured binding;
Kd = 50 nM and is
the estimated dissociation constant for ouabain binding to
2-isozyme in rat tissue (1);
and Of is the concentration of
free ouabain at which binding is measured (100 nM).
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Skeletal muscle atrophy with advancing age. In F-344/BN rats, gastrocnemius muscle weight decreased by 23.1 and 16.6% in 30-mo-old rats compared with 6- and 18-mo-old rats, respectively (6-mo-old rats = 3.98 ± 0.04 g; 18-mo-old rats = 3.67 ± 0.20 g; and 30-mo-old rats = 3.06 ± 0.09 g). Although there was a trend for decreasing muscle mass from 6 to 18 mo of age, the difference was not statistically significant. In soleus, muscle mass did not change among the animals in the three age groups (6-mo-old rats = 0.29 ± 0.01 g; 18-mo-old rats = 0.28 ± 0.02 g; and 30-mo-old rats = 0.30 ± 0.01 g).
K+-dependent
3-O-MFPase activity with age.
To determine the
Na+-K+-ATPase
activity in skeletal muscle of rats with advancing age,
K+-dependent
3-O-MFPase activity was measured in
tissue homogenate of mixed gastrocnemius muscle.
K+-dependent activity increased by
55.6 and 49.3% in 30-mo-old rats, compared with 6- and 18-mo-old rats,
respectively (Fig. 1).
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- and -subunit
isoforms. To elucidate cellular mechanisms underlying
increased K+-dependent
3-O-MFPase activity in gastrocnemius
of older animals, expression of the
Na+-K+-ATPase
- and -subunit isoforms was determined by immunoblotting. Expression of the subunits in white and red gastrocnemius and in soleus
was examined to determine possible fiber type-specific differential
expression of the subunits. As shown in Fig.
2,
2 levels in red and white
gastrocnemius decreased by 30-40% in 18- and 30-mo-old rats,
compared with 6-mo-old rats. By contrast, levels of
1-subunit markedly increased in
both types of muscle in the old rats. In red gastrocnemius,
1 in 30-mo-old rats increased ten- and sevenfold, compared with 6- and 18-mo-old rats, respectively. No significant increase occurred between 6- and 18-mo-old rats. Similarly, 1 in white
gastrocnemius of 30-mo-old rats increased five- and twofold, compared
with 6- and 18-mo-old rats, respectively. Differences between 6- and
18-mo-old rats were not statistically significant.
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1 increased by ~100% in
30-mo-old rats, compared with 6-mo-old rats, whereas levels of
2 decreased by 23.2% in
18-mo-old rats, compared with 6-mo-old rats (Fig. 2). There was also a
trend for decreased levels of 2
in 30-mo-old rats, although the difference did not reach statistical
significance (P = 0.057).
Levels of expression of -subunit isoforms were examined. In red
gastrocnemius, levels of
1-subunit in 30-mo-old rats
were increased about threefold, compared with 6- and 18-mo-old rats (Fig. 3). The
2, by contrast, decreased by
57.3% between 6 and 30 mo of age; the decrease between 6- and
18-mo-old animals was not statistically significant. In white
gastrocnemius, expression of 1
in 30-mo-old rats increased by ~14- and 3.5-fold, compared with 6- and 18-mo-old rats, respectively. Similar to that in red gastrocnemius,
2 decreased by 62.4 and 36.3%
in 30-mo-old rats, compared with 6- and 18-mo-old rats, respectively.
In soleus muscle, expression of
1, which has been shown to be
the predominant -subunit isoform in this skeletal muscle (51), did
not change among the three age groups examined.
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-sarcomeric actin, a muscle-specific protein, in white
and red gastrocnemius muscles of young and old rats. The data show that
relative expression of -sarcomeric actin remained unchanged among
the different age groups (Fig. 4). This
result suggests that the amounts of nonmuscle tissue did not change
significantly in older rats. Thus, in the Western blots, it is valid to
normalize expression of the
Na+-K+-ATPase
subunit isoforms by the amounts of protein loaded.
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[3H]ouabain binding sites.
To evaluate the effect of decreased
2-subunit expression and
changing expression of 1
(increased) and 2 (decreased)
on the abundance of 2-isozyme
in red and white gastrocnemius of rats with age, the amount of
2-isozyme was estimated by
[3H]ouabain binding
assay. [3H]ouabain was
used at 100 nM, such that only high-affinity ouabain binding sites,
i.e., the 2-isozyme, were
detected. The number of
[3H]ouabain binding
sites in red gastrocnemius of 30-mo-old rats was 118.3 and 49.3%
higher than that of 6- and 18-mo-old rats, respectively (6 mo = 0.43 ± 0.11; 18 mo = 0.63 ± 0.26; 30 mo = 0.95 ± 0.06 pmol/mg
protein; Fig.
5A);
differences between 6-mo-old and 18-mo-old rats did not reach
statistical significance. By contrast, in white gastrocnemius,
[3H]ouabain binding
sites in 18- and 30-mo-old rats were ~30% less than those in
6-mo-old rats (Fig. 5A).
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1-isozyme) in the
[3H]ouabain binding
observed above, homogenate of rat kidney, which expresses almost
exclusively the 1-subunit (49),
was used to estimate such binding. As shown in Fig.
5B, at a concentration of 100 nM,
[3H]ouabain binds to
red gastrocnemius (1- and
2-isozyme) as well as to kidney
(1-isozyme) homogenate
(skeletal homogenate = 1,471 cpm/0.3 mg protein; kidney homogenate = 499 cpm/0.3 mg protein). By performing a Western blot analysis, we
further estimated that on a per milligram protein basis gastrocnemius
muscle homogenate contains ~300-fold less
1 than does the kidney
homogenate (Fig. 5B). Therefore, in
red gastrocnemius, 1-isozyme
will contribute an amount of
[3H]ouabain binding
that is roughly equal to ~1/300 of the binding observed in kidney
homogenate, or 1.7 cpm (499 cpm/300), if it is assumed that all
1-subunits form enzyme units
capable of binding to ouabain. This amount of
[3H]ouabain binding is
only ~0.12% of the total binding observed in skeletal muscle (1.7 cpm/1,471 cpm). These data demonstrate that the low-affinity ouabain
binding sites contribute very little to the observed
[3H]ouabain binding in
skeletal muscle homogenate.
Expression of MHC isoforms. Previous
studies have demonstrated age-associated motor unit transformation, as
determined by MHC isoforms composition (27, 28). Specifically, Larsson
and co-workers (27, 28) demonstrated in albino Wistar rats a transition from the faster type IIB to the slower IIX motor unit in the
fast-twitch extensor digitorum longus and tibialis anterior muscles.
Because expression of
Na+-K+-ATPase
isoforms appears to be correlated with muscle fiber types (51), we
examined whether significant changes in fiber types occurred in
skeletal muscle of the F-344/BN rats with advancing age. In 6-mo-old
rats, red gastrocnemius expresses mainly type IIX MHC, with lesser
amounts of IIA, IIB, and I (Fig. 6). By
contrast, white gastrocnemius expresses similar amounts of type IIX and IIB. The data show that patterns of expression of MHC isoforms in each
muscle type remained relatively unchanged between 6 and 30 mo of age,
suggesting a lack of significant switching in fiber types in
gastrocnemius muscles of the F-344/BN rats in the age groups examined.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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The major findings in the present study are that, with advancing age,
1)
K+-dependent
3-O-MFPase activity in gastrocnemius
muscle of F-344/BN rats increased;
2) expression of
Na+-K+-ATPase
subunit isoforms was regulated differentially; the amount of
2-subunit decreased, whereas
that of 1-subunit markedly
increased in red and white gastrocnemius and in soleus muscles;
3)
1-subunit increased, whereas
2-subunit decreased in red and
white gastrocnemius, and in soleus
1-subunit remained unchanged;
and 4) despite decreased levels of
2-subunit, high-affinity
ouabain binding sites in red gastrocnemius increased. These data
demonstrate for the first time dynamic regulation in expression of the
Na+-K+-ATPase
subunit isoforms and
Na+-K+-ATPase
activity in skeletal muscles with age.
Aging of skeletal muscle is associated with marked decline in its
function, yet, underlying mechanisms remain incompletely understood.
The present study demonstrates that some of the age-associated changes,
such as the increase in
K+-dependent
3-O-MFPase activity,
[3H]ouabain binding,
the expression of 1 and
1, and the decrease in
2, occurred between middle-age
(18-mo-old) and senescence (30-mo-old) and thus may be attributed to
aging of the F-344/BN rats. It can be speculated that these changes may
contribute to altered skeletal muscle function during aging. On the
other hand, some age-associated changes were clearly detectable in
18-mo-old rats, and, importantly, without further significant changes
thereafter. For example, the decrease in
2 in red and white
gastrocnemius and soleus muscles and the decrease in ouabain binding
sites in white gastrocnemius all occurred between 6 and 18 mo of age.
These changes cannot be attributed to senescence per se, although they do not appear to be the result of developmental growth, because 6-mo-old F-344/BN rats are considered mature adults (26). Underlying mechanisms for these changes remain unclear at present.
In agreement with earlier studies (13, 16), gastrocnemius muscle of F-344/BN rats showed significant muscle atrophy with advancing age. Although there appears to be a trend for reduced muscle mass between young adult and middle-aged rats, significant muscle atrophy was detected only in senescent rats. By contrast, no significant muscle loss was detectable in soleus muscle with advancing age, similar to a previous report in which the F-344/BN rats were used (13). The muscle-type dependent changes in muscle weight may be related to the fact that gastrocnemius muscle is used in strenuous exercise, whereas soleus is postural, being used most of the time.
The present study shows that K+-dependent 3-O-MFPase activity, a measure of Na+-K+-ATPase activity, is increased in gastrocnemius muscle of older rats. Such a result is unexpected, since aging is associated with lower levels of spontaneous physical activity (53), a condition known to reduce Na+-K+-ATPase units (9). The increase in enzyme activity could indicate an unexpected adaptation of the Na+-K+-ATPase during the aging process. It is possible that sarcolemmal membrane may become more leaky to Na+ and/or K+ with age and, therefore, requires more Na+-K+ pump units to maintain Na+-K+ balance. Nevertheless, it remains to be determined whether transport activity of the sarcolemmal Na+-K+ pump is increased in skeletal muscle cells of aging rats. For example, in soleus muscle of spontaneously hypertensive rats, Na+-K+-pump activity was found to be decreased, despite increased Na+-K+-ATPase number (44). Furthermore, subcellular distribution of the subunit isoforms with advancing age is not known; the possibility remains that the changes occur intracellularly and thus do not result in increased Na+-K+ pump transport activity on the sarcolemmal membrane.
The marked increase in
1-subunit in skeletal muscle of
older rats probably contributes to the increased
K+-dependent
3-O-MFPase activity. This increased
expression of the 1-subunit is
somewhat unexpected, especially in view of the fact that
1 has often been referred to as
the "housekeeping" isoform. Indeed, previous studies examining
expression of the
Na+-K+-ATPase
subunit isoforms in skeletal muscle have consistently shown that under
different pathophysiological conditions, such as hyper- and hypothyroid
states (3) and hypokalemia (18, 51), expression of
1 remains relatively unaltered,
despite marked changes in the expression of the
2-subunit. However, in an
earlier study (37), our laboratory showed moderate increases in
abundance of 1-subunit in mixed
hindlimb skeletal muscle of streptozotocin-induced diabetic rats.
Collectively, these results suggest that expression of
1 in skeletal muscle may be
more dynamically regulated than previously recognized.
Cellular mechanisms underlying increased expression of the
1-subunit with advancing age
are unclear at present. McDonough and co-workers (51) demonstrated a
correlation between the abundance of
1-subunit and muscle types,
such that its abundance was highest in slow oxidative muscle and lowest
in fast glycolytic muscle. Because aging has been shown to be
associated with preferential reduction of muscle cross-sectional area
and, perhaps, the number of fast glycolytic fibers (11, 45, 52), a
change in the composition of fiber types in aged muscles could result
in relative increase in the levels of
1. However, in F-344/BN rats,
our result showed no significant changes in relative amounts of the
four MHC isoforms in red and white gastrocnemius muscles among the three age groups examined. Of particular interest is the apparent lack
of transition from the faster type IIB to the slower type IIX fiber, a
transition that was demonstrated in tibialis anterior and extensor
digitorum longus muscles of F-344 rats between 3 and 24 mo of age (27,
28). The reason for the apparent difference is not clear but may be due
to differences in the strains of rats, muscle types, and/or age of the
animals being studied. Nevertheless, it seems clear that altered
expression of the
Na+-K+-ATPase
isoform in these aging F-344/BN rats is not associated with any obvious
shift in muscle fiber types. Because we did not quantitate relative
expression of the MHC isoforms, the possibility cannot be excluded that
minor changes in fiber types contribute in small parts to an altered
expression of the
Na+-K+-ATPase isoforms.
Another interesting finding in the present study is that in red
gastrocnemius, despite decreased levels of
2-subunit with advancing age,
high-affinity
[3H]ouabain binding
sites, a measure of the
2-isozyme, increased. Cellular
mechanisms responsible for these seemingly contradictory observations
are unclear at present. Nevertheless, our data indicate that the
increased [3H]ouabain
binding is unlikely to be due to the large increase in
1-subunit during the aging
process (Fig. 5B), because
1-isozyme appears to contribute
very little to the observed
[3H]ouabain binding.
In addition, in white gastrocnemius, high-affinity binding sites
decreased, correlating with decreased expression of
2-subunit, despite the large
increase in 1-subunit. These data further suggest that binding of
[3H]ouabain to the
2-isozyme is specific and
demonstrate distinct patterns of expression of the
Na+-K+-ATPase
isoforms and ouabain binding in red and white gastrocnemius muscles.
A recent report demonstrated that abundance of -subunit appears to
regulate overall
Na+-K+-ATPase
activity in subcellular membranes of rat skeletal muscle (29), such
that membrane fractions with higher / ratio have higher enzyme
activity. Thus it may be speculated that the increase in high-affinity
[3H]ouabain binding
sites could be the result of increased association between
2- and -subunit, especially
in view of the large increase in
1-subunit. Future studies will
examine interactions between the - and -subunit isoforms in aged
skeletal muscle.
It is generally accepted that skeletal muscle expresses more
2- than
1-subunit isoform (49); the
2-to-1
ratio has been estimated to be between 2 and 4 (18, 29, 42). In the
aging F-344/BN rats, as a result of decreased
2-subunit and a marked increase
in 1-subunit,
1-isozyme may become the
predominant Na+-K+-ATPase
isozyme in aged skeletal muscle. Physiological significance of such a
change in the ratio of the isozymes remains to be explored, since
functional differences of the isoforms have yet to be clearly defined
(30). In light of the recent findings that insulin preferentially translocates the 2-subunit from
intracellular membrane to plasma membrane (20), and that
1 appears to be a much better
substrate than 2 and
3 for phosphorylation by
protein kinase C (6), it is possible that the large increase in
1-subunit could affect the
fraction of the
Na+-K+-ATPase
that can be translocated or phosphorylated.
In summary, in F-344/BN rats, advancing age is associated with marked differential alterations in expression of the Na+-K+-ATPase subunit isoforms in skeletal muscle. It remains to be determined whether these changes are the result of compensatory adaptation in response to other age-related changes or maladaptation of the Na+-K+-ATPase in aged skeletal muscle. Pathophysiological significance of these changes in skeletal muscle function during the aging process is being examined.
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ACKNOWLEDGEMENTS |
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We thank Dr. K. Sweadner (Harvard University) for providing the
1- and
2-specific antibodies. We also
thank Linghong Kong for her excellent technical assistance.
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FOOTNOTES |
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Present address of X. W. Sun: Dept. of Medicine, Division of Cardiothoracic Surgery, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17033-0850.
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: Y.-C. Ng, Dept. of Pharmacology H078, The Milton S. Hershey Medical Center, The Pennsylvania State Univ., Hershey, PA 17033-0850 (E-mail: ycn1{at}psu.edu).
Received 23 July 1998; accepted in final form 25 May 1999.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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