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 β-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 α-subunit and a β-subunit. Four different isoforms of the α-subunit, α₁, α₂, α₃, and α₄, and three separate isoforms of the β-subunit, β₁, β₂, and β₃, have been cloned and sequenced (30, 31, 33, 46, 49). Skeletal muscle of the mature rat expresses α₁- and α₂-subunit, with α₂ being the more abundant subunit (18, 29, 42, 49), and all three β-subunit isoforms have been reported in this tissue (2, 21). The α₁ and β₁ are more abundant in fast and slow oxidative rich fibers than in fast glycolytic fibers, whereas the opposite is true for β₂ (21,51). Distribution of the recently identified β₃-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 α₁-isozyme is a better substrate for phosphorylation by kinase, and therefore its activity may be modified (6, 12), insulin appears to selectively translocate α₂-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 α₁- and β₁-subunit, downregulation of α₂- and β₂-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.

MATERIALS AND METHODS

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 α-subunit isoforms, equal amounts of homogenates (80 μg) were electrophoresed in 5 or 7.5% acrylamide gels. For analysis of the β-subunits, equal amounts of homogenate preparations (200 or 260 μg) were deglycosylated withN-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 α-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 α₁-, α₂-, and β₂-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 α₁-, α₂- (generous gift from Dr. K. Sweadner, Harvard University), or β₂-subunits (5, 43). To detect actin, blots were blocked with TBST plus 5% dry milk (Blotto-Tween) and incubated with a monoclonal antibody against α-sarcomeric actin (Sigma Chemical, St. Louis, MO). To detect the β₁-subunit, membranes were blocked in Blotto-Tween and incubated with an anti-β₁-antiserum (Upstate Biological, Lake Placid, NY) or with a monoclonal anti-β₁-antibody (Affinity BioReagents, Golden, CO). Bound monoclonal antibodies were detected with rabbit anti-mouse IgG, followed by¹²⁵I-labeled Protein A (ICN, Costa Mesa, CA), whereas bound polyclonal antibodies were detected with¹²⁵I-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).

Because of the low abundance of β₁ in white gastrocnemius, a chemiluminescent detection method was used. Blots were incubated with monoclonal anti-β₁-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 (B_max) of [³H]ouabain to high-affinity ouabain binding site, the α₂-isozyme, is calculated according to the equation: B_max = B +K_d(B/O_f), where B is the measured binding;K_d = 50 nM and is the estimated dissociation constant for ouabain binding to α₂-isozyme in rat tissue (1); and O_f is the concentration of free ouabain at which binding is measured (100 nM).

Analysis of MHC isoforms. For separation of the four known MHC isoforms in SDS gel, a procedure similar to that described by Talmadge and Roy (50) was used, except that total tissue homogenates were used without the extraction of myofibrillar proteins. The separating gel contained 8% acrylamide and 30% glycerol. Gels were stained with silver (Bio-Rad, Melville, NY) to visualize the MHC isoforms.

Statistical analysis of data. Results are expressed as means ± SE. One-way ANOVA was used to compare group means, and the Duncan test was used for post hoc analysis. A value of P < 0.05 was considered statistically significant.

RESULTS

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).

Expression of the α- and β-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 α- and β-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, α₂ 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 α₁-subunit markedly increased in both types of muscle in the old rats. In red gastrocnemius, α₁ 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, α₁ 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.

In soleus, similar to that observed in gastrocnemius, levels of α₁ increased by ∼100% in 30-mo-old rats, compared with 6-mo-old rats, whereas levels of α₂ 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 α₂in 30-mo-old rats, although the difference did not reach statistical significance (P = 0.057).

Levels of expression of β-subunit isoforms were examined. In red gastrocnemius, levels of β₁-subunit in 30-mo-old rats were increased about threefold, compared with 6- and 18-mo-old rats (Fig. 3). The β₂, 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 β₁in 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, β₂ 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 β₁, which has been shown to be the predominant β-subunit isoform in this skeletal muscle (51), did not change among the three age groups examined.

To determine whether relative proportions of muscle/nonmuscle tissues are altered in skeletal muscle of older rats, especially in gastrocnemius muscles, which demonstrated muscle atrophy, we examined expression of α-sarcomeric actin, a muscle-specific protein, in white and red gastrocnemius muscles of young and old rats. The data show that relative expression of α-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.

[³H]ouabain binding sites.

To evaluate the effect of decreased α₂-subunit expression and changing expression of β₁(increased) and β₂ (decreased) on the abundance of α₂-isozyme in red and white gastrocnemius of rats with age, the amount of α₂-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 α₂-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).

To evaluate possible contribution of low-affinity ouabain binding sites (α₁-isozyme) in the [³H]ouabain binding observed above, homogenate of rat kidney, which expresses almost exclusively the α₁-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 (α₁- and α₂-isozyme) as well as to kidney (α₁-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 α₁ than does the kidney homogenate (Fig. 5B). Therefore, in red gastrocnemius, α₁-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 α₁-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.

DISCUSSION

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 α₂-subunit decreased, whereas that of α₁-subunit markedly increased in red and white gastrocnemius and in soleus muscles;3) β₁-subunit increased, whereas β₂-subunit decreased in red and white gastrocnemius, and in soleus β₁-subunit remained unchanged; and 4) despite decreased levels of α₂-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 α₁ and β₁, and the decrease in β₂, 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 α₂ 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 α₁-subunit in skeletal muscle of older rats probably contributes to the increased K⁺-dependent 3-O-MFPase activity. This increased expression of the α₁-subunit is somewhat unexpected, especially in view of the fact that α₁ 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 α₁ remains relatively unaltered, despite marked changes in the expression of the α₂-subunit. However, in an earlier study (37), our laboratory showed moderate increases in abundance of α₁-subunit in mixed hindlimb skeletal muscle of streptozotocin-induced diabetic rats. Collectively, these results suggest that expression of α₁ in skeletal muscle may be more dynamically regulated than previously recognized.

Cellular mechanisms underlying increased expression of the α₁-subunit with advancing age are unclear at present. McDonough and co-workers (51) demonstrated a correlation between the abundance of α₁-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 α₁. 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 α₂-subunit with advancing age, high-affinity [³H]ouabain binding sites, a measure of the α₂-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 α₁-subunit during the aging process (Fig. 5B), because α₁-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 α₂-subunit, despite the large increase in α₁-subunit. These data further suggest that binding of [³H]ouabain to the α₂-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 β-subunit appears to regulate overall Na⁺-K⁺-ATPase activity in subcellular membranes of rat skeletal muscle (29), such that membrane fractions with higher α/β 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 α₂- and β-subunit, especially in view of the large increase in β₁-subunit. Future studies will examine interactions between the α- and β-subunit isoforms in aged skeletal muscle.

It is generally accepted that skeletal muscle expresses more α₂- than α₁-subunit isoform (49); the α₂-to-α₁ratio has been estimated to be between 2 and 4 (18, 29, 42). In the aging F-344/BN rats, as a result of decreased α₂-subunit and a marked increase in α₁-subunit, α₁-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 α₂-subunit from intracellular membrane to plasma membrane (20), and that α₁ appears to be a much better substrate than α₂ and α₃ for phosphorylation by protein kinase C (6), it is possible that the large increase in α₁-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.

FOOTNOTES

• 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.