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ENVIRONMENTAL, EXERCISE AND RESPIRATORY PHYSIOLOGY

Depressed Na⁺-K⁺-ATPase activity in skeletal muscle at fatigue is correlated with increased Na⁺-K⁺-ATPase mRNA expression following intense exercise

¹Muscle, Ions and Exercise Group, School of Human Movement, Recreation, and Performance, Centre for Ageing, Rehabilitation, Exercise, and Sport, Victoria University of Technology, and ²Exercise, Muscle, and Metabolism Unit, School of Health Sciences, Deakin University, Melbourne, Australia

Submitted 7 June 2004 ; accepted in final form 13 March 2005

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
We investigated whether depressed muscle Na⁺-K⁺-ATPase activity with exercise reflected a loss of Na⁺-K⁺-ATPase units, the time course of its recovery postexercise, and whether this depressed activity was related to increased Na⁺-K⁺-ATPase isoform gene expression. Fifteen subjects performed fatiguing, knee extensor exercise at 40% maximal work output per contraction. A vastus lateralis muscle biopsy was taken at rest, fatigue, 3 h, and 24 h postexercise and analyzed for maximal Na⁺-K⁺-ATPase activity via 3-O-methylfluorescein phosphatase (3-O-MFPase) activity, Na⁺-K⁺-ATPase content via [³H]ouabain binding sites, and Na⁺-K⁺-ATPase ₁-, ₂-, ₃-, ₁-, ₂- and ₃-isoform mRNA expression by real-time RT-PCR. Exercise [352 (SD 267) s] did not affect [³H]ouabain binding sites but decreased 3-O-MFPase activity by 10.7 (SD 8)% (P < 0.05), which had recovered by 3 h postexercise, without further change at 24 h. Exercise elevated ₁-isoform mRNA by 1.5-fold at fatigue (P < 0.05). This increase was inversely correlated with the percent change in 3-O-MFPase activity from rest to fatigue (%3-O-MFPase_rest-fatigue) (r = –0.60, P < 0.05). The average postexercise (fatigue, 3 h, 24 h) ₁-isoform mRNA was increased 1.4-fold (P < 0.05) and approached a significant inverse correlation with %3-O-MFPase_rest-fatigue (r = –0.56, P = 0.08). Exercise elevated ₂-isoform mRNA at fatigue 2.5-fold (P < 0.05), which was inversely correlated with %3-O-MFPase_rest-fatigue (r = –0.60, P = 0.05). The average postexercise ₂-isoform mRNA was increased 2.2-fold (P < 0.05) and was inversely correlated with the %3-O-MFPase_rest-fatigue (r = –0.68, P < 0.05). Nonsignificant correlations were found between %3-O-MFPase_rest-fatigue and other isoforms. Thus acute exercise transiently decreased Na⁺-K⁺-ATPase activity, which was correlated with increased Na⁺-K⁺-ATPase gene expression. This suggests a possible signal-transduction role for depressed muscle Na⁺-K⁺-ATPase activity with exercise.

Na⁺-K⁺ pump; signaling; muscle fatigue; ouabain binding

Relatively few studies have investigated the effects of brief, intense exercise on muscle Na⁺-K⁺-ATPase content, as fully quantified by [³H]ouabain binding (40). An upregulation is suggested by the increased muscle [³H]ouabain binding with 10 h of running (43) and training (16, 31). In contrast, no increase in [³H]ouabain binding occurred immediately after incremental or prolonged exercise (2, 27) or within 4 h after isometric contractions (12). However, these studies may have missed increases because of either the lack of a recovery biopsy (2, 27) or an insufficient recovery period (12). Acute upregulation of muscle Na⁺-K⁺-ATPase content is also suggested by the finding that 5-min fatiguing knee extensor exercise markedly increased the Na⁺-K⁺-ATPase ₂ (70%) and ₁ (26%) sarcolemmal abundance (24). Because [³H]ouabain binding did not differ between intact and cut muscle pieces in isolated rat muscles, suggesting no internal Na⁺-K⁺-ATPase stores (30), increased sarcolemmal ₂ abundance with exercise (24) implies an increased sarcolemmal Na⁺-K⁺-ATPase content. However, [³H]ouabain binding was not measured, and membrane isolation has been criticized in this context due to low Na⁺-K⁺-ATPase recovery (19). Finally, electrical stimulation of isolated rat muscles failed to elevate [³H]ouabain binding (30), suggesting that acute exercise does not elevate muscle Na⁺-K⁺-ATPase content. However, this could conceivably have been influenced by the absence of local hormonal or temperature effects. To resolve this issue, we therefore tested the second hypothesis that muscle Na⁺-K⁺-ATPase content would be unchanged immediately and within 24 h after acute, short-duration exercise.

In addition to Na⁺/K⁺ exchange, Na⁺-K⁺-ATPase also acts as a signal transducer in cardiac muscle (for review, see Ref. 53) and in liver, where low K⁺ inhibition of Na⁺-K⁺-ATPase activity increased Na⁺-K⁺-ATPase mRNA abundance (44). Whether exercise-induced Na⁺-K⁺-ATPase inhibition also exerts a similar signaling function in skeletal muscle is unknown. Acute exercise increases the mRNA expression for each of Na⁺-K⁺-ATPase ₁-, ₂-, ₃-, ₁-, ₂-, and the ₃-isoforms (34). Thus acute inhibition of Na⁺-K⁺-ATPase activity with exercise might then be linked to increased gene expression (34) and also increased Na⁺-K⁺-ATPase content with chronic training (16, 31). This may occur via direct protein-protein interaction between Na⁺-K⁺-ATPase and its neighboring proteins, triggering a signaling cascade that culminates in increased gene transcription (53). Alternately, ionic disturbances with depressed Na⁺-K⁺-ATPase activity may stimulate synthesis of new Na⁺-K⁺-ATPase enzymes to reestablish favorable Na⁺ and K⁺ gradients (5, 52). We therefore tested the third hypothesis that the reduction in Na⁺-K⁺-ATPase activity immediately after an acute bout of exhaustive exercise is correlated with increases in the gene transcripts of the Na⁺-K⁺-ATPase catalytic -isoforms.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Subjects and Overview of Exercise Tests

Fifteen healthy volunteers (8 men, 7 women) participated in our study. Subject characteristics were as follows [mean (SD)]: age, 24.7 (6.7) yr; height, 174.5 (6.8) cm; body mass, 73.2 (11.4) kg; and peak O₂ consumption, 50.5 (2.8) ml·kg^–1·min^–1. Each subject refrained from vigorous exercise, alcohol, and caffeine for 24 h before each exercise test. Subjects consumed standardized meals for 24 h before and after the invasive trial and were informed of all possible risks before giving written consent. This study was approved by the Human Research Ethics Committee at Victoria University of Technology. Our group has recently (34) reported the muscle function, aerobic power, and muscle Na⁺-K⁺-ATPase isoform gene and protein expressions before and after exercise in these subjects. Here, we report new data on the acute effects of exercise on muscle Na⁺-K⁺-ATPase content and maximal activity, new data on the recovery time course of Na⁺-K⁺-ATPase maximal activity, and novel correlations between depressed muscle maximal Na⁺-K⁺-ATPase activity and gene transcription changes.

Knee Extensor Muscle Fatigue Test

The knee extensor strength and fatigue tests were performed on an isokinetic dynamometer (Cybex Norm 770, Henley HealthCare), and have been described in detail elsewhere (34). In brief, subjects performed an isokinetic knee extensor muscle strength test, which involved three consecutive maximal contractions at 180°/s. The muscle fatigue test involved continuous one-legged knee extensions at 180°/s, repeated every 1.5 s, until fatigue. The work rate was 40% of the total work produced during the maximal isokinetic knee extensor strength test. Fatigue was defined as an inability to maintain more than 90% of the target work rate for three successive contractions. This exercise model was chosen because it is similar to one that showed an apparent increase in sarcolemmal Na⁺-K⁺-ATPase ₂- and ₁-subunits following knee extension exercise (24).

Blood Sampling and Processing

A 20-gauge catheter was inserted into a dorsal hand vein and covered by a waterproof patch (Tegaderm); the hand was then sheathed in a plastic bag. The hand was heated in a 45°C water bath throughout the duration of the sampling period to enable arterialized samples to be drawn at rest, during exercise, at fatigue, and at 1, 2, 5, and 10 min postexercise. The catheter was kept patent by periodic infusions of heparinized isotonic saline. The blood sample was transferred to a tube containing lithium heparin; 1 ml of blood was analyzed in duplicate for Hb concentration and Hct (K-800, Sysmex, Kobe, Japan), and another 1.5 ml was centrifuged at 4,500 rpm for 2 min with the plasma removed and frozen until later duplicate analysis of plasma K⁺ concentration ([K⁺]) using an ion-selective electrode (Ciba Corning 865pH/blood-gas analyzer, Bayer, MA).

Muscle Biopsies

A muscle needle biopsy was taken from the middle third of the vastus lateralis muscle at rest, fatigue, and 3 and 24 h postexercise. After injection of a local anesthetic into the skin and fascia (1% xylocaine), a small incision was made; we took a muscle sample (120 mg) using a Stille biopsy needle. A small portion of muscle was homogenized and then frozen in liquid N₂ for later analysis of maximal Na⁺-K⁺-ATPase activity (3-O-MFPase). The remaining sample was immediately frozen in liquid N₂ until later analysis of Na⁺-K⁺-ATPase content ([³H]ouabain binding site content) and gene expression (real-time RT-PCR).

3-O-MFPase Assay

We measured maximal in vitro Na⁺-K⁺-ATPase activity using the K⁺-stimulated 3-O-MFPase assay (13, 14). Approximately 20 mg of muscle were blotted on filter paper, weighed, and then homogenized on ice for 2 x 20 s at 20,000 rpm (Omni 1000, Omni International) in a homogenate buffer containing 250 mM sucrose, 2 mM EDTA, and 10 mM Tris (pH 7.4). Muscle homogenates were then rapidly frozen and stored in liquid nitrogen until later analyses. Before analyses, homogenates were thawed, diluted one-fifth with ice-cold homogenate buffer, and then freeze-thawed a further three times. Thirty microliters of the diluted, freeze-thawed homogenate were incubated in 2.5 ml of assay medium containing 100 mM Tris, 5 mM MgCl₂, 1.25 mM EDTA, and 80 nM 3-O-methylfluorescein (pH 7.4) for 5 min at 37°C, before addition of 40 µl of 10 mM 3-O-MFP (final concentration of 156 µM) to start the reaction. The reaction was measured for 80 s before addition of 10 µl of 2.58 M KCl (final concentration 10 mM) to stimulate K⁺-dependent phosphatase activity. All assays were performed at 37°C, with continuous stirring, in a spectrofluorometer (Aminco Bowman AB2 SLM, Urbana, IL). Excitation wavelength was 475 nm, and emission wavelength was 515 nm, with 4-nm slit widths. The K⁺-stimulated 3-O-MFPase activity was calculated by subtracting the initial activity from the activity obtained after addition of 10 mM KCl.

[³H]ouabain Binding Site Content

Skeletal muscle total Na⁺-K⁺-ATPase content was determined by vanadate-facilitated [³H]ouabain binding site content analyses (40, 41). Muscle samples were cut into 2- to 5-mg pieces and washed for 2 x 10 min in 37°C vanadate buffer containing 250 mM sucrose, 10 mM Tris, 3 mM MgSO₄, and 1 mM NaVO₄ (pH 7.2–7.4). Muscle samples were then incubated for 120 min at 37°C in the above buffer with the addition of [³H]ouabain (10^–6 M, 2.0 µCi/ml). After incubation, muscle samples were washed for 4 x 30 min in ice-cold vanadate buffer to remove any unbound [³H]ouabain, blotted on filter paper, and weighed before being soaked overnight in vials containing 0.5 ml of 5% trichloroacetic acid and 0.1 mM ouabain. The following morning, 2.5 ml of scintillation cocktail (Opti-Fluor, Packard) were added before liquid scintillation counting of the ³H activity. The content of [³H]ouabain binding sites was calculated on the basis of the sample wet weight and the specific activity of the incubation medium and samples (expressed as pmol/g wet wt). The final [³H]ouabain binding site concentration was then calculated by subtracting the nonspecific [³H]ouabain uptake (2.5%) (40) and multiplying by a correction factor of 1.13 to allow for impurity of the [³H]ouabain (1.05) (measured by supplier; Amersham Pharmacia Biotech, Castle Hill, Australia), loss of specifically bound [³H]ouabain during washout (1.05) (40), and incomplete saturation (1.025) (T. Clausen, personal communication).

Real-Time RT-PCR Measurement of mRNA

We extracted total RNA from 5–10 mg of muscle with the FastRNA reagents (BIO 101, Vista, CA) using methods previously employed in our laboratory (35). The resulting RNA pellet was dissolved in EDTA-treated water, and total RNA concentration was determined spectrophotometrically at 260 nm. We transcribed RNA (1 µg) into cDNA using the Promega avian myeloblastosis virus reverse transcription kit (Promega, Madison, WI), and the resulting cDNA was stored at –20°C for subsequent analysis.

Real-time PCR (GeneAmp 5700 sequence detection system) was run for 1 cycle (50°C for 2 min, 95°C for 10 min) and 50 cycles (95°C for 15 s, 60°C for 60s). Primer sequences were designed for the Na⁺-K⁺-ATPase ₁, ₂, ₃, ₁, ₂, and ₃ genes as described elsewhere (34). All samples were run in triplicate, and measurements included a no-template control as well as a human skeletal muscle sample endogenous control. Cyc (cyclophilin) mRNA expression was unchanged with exercise (data not shown) and was therefore used as a control (housekeeping gene) to account for any variations in the amount of input RNA and the efficiency of reverse transcription. We quantified gene expression using a cycle threshold (C_T) method, whereby the relative expression of the genes compared with resting samples was made with the expression 2^–CT, in which the expression of each gene was normalized for input cDNA using the housekeeping gene Cyc. Muscle mRNAs are presented for 14 subjects (7 men, 7 women) because of insufficient sample for one subject.

Calculations

Details of the time course of changes in Na⁺-K⁺-ATPase isoform mRNA response to exercise in these individuals have been reported elsewhere (34). The mRNA in resting muscle was contrasted against mRNA at fatigue for each isoform, as well as against the average postexercise mRNA expression (average of fatigue, 3 h postexercise, and 24 h postexercise) for each of the Na⁺-K⁺-ATPase ₁-, ₂-, ₃-, ₁-, ₂-, and ₃-isoforms. The average postexercise mRNA expression of each isoform was determined to account for variability in the individual time response of mRNA upregulation postexercise (34). Correlations were calculated between the percent change in 3-O-MFPase activity from rest to fatigue (%3-O-MFPase_rest-fatigue) and the percent changes in both mRNA at fatigue and the average postexercise mRNA (34).

Statistics

Data are presented as means (SD). A one-way ANOVA with repeated measures was used to analyze all variables except mRNA, with post hoc analyses using Fisher's least significant difference test. Correlations were determined by least squares linear regression. We tested single comparisons (e.g., rest vs. fatigue mRNA) using a paired t-test. Statistical significance was accepted at P < 0.05.

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
Exercise Time, Plasma Volume, and [K⁺]

Knee extensor time to fatigue was 352 (SD 267) s. Plasma volume decreased by 4.3 (SD 2-1)% after 1 min of exercise (P < 0.05), by 12.3 (SD 4-5)% at fatigue (P < 0.05), and remained 3.3 (SD 4-2)% below rest at 10 min postexercise (P < 0.05). Arterialized venous plasma [K⁺] increased (P < 0.05) after 1 min of exercise and increased further (P < 0.05) at fatigue (Fig. 1). In recovery, plasma [K⁺] fell rapidly by 1 min postexercise (P < 0.05), dropped below preexercise values at 2 and 5 min postexercise (P < 0.05), but had recovered by 10 min postexercise.

View larger version (18K): [in this window] [in a new window]   Fig. 1. Arterialized venous plasma K+ concentration ([K+]) at rest, during single-leg knee extensions continued to fatigue (Fat), and for 10 min after exercise (recovery). Values are means (± SE); n = 15 subjects, except at fatigue and at 1 min, where n = 14, and at 10 min, where n = 13. Shaded bar represents exercise bout. *Different from rest, P < 0.05.

Exercise decreased muscle in vitro maximal 3-O-MFPase activity by 10.7 (SD 8)% (P < 0.05). This decline occurred in 11 of 12 subjects. Muscle 3-O-MFPase activity did not differ significantly from rest at either 3 or 24 h postexercise (Fig. 2A).

View larger version (33K): [in this window] [in a new window]   Fig. 2. Maximal in vitro K+-stimulated 3-O-methylfluorescein phosphatase (3-O-MFPase) activity (A) and [3H]ouabain binding site content (B) measured in vastus lateralis muscle at rest, at fatigue induced by repeated isokinetic single-leg knee extensions, and at 3 and 24 h postexercise. Values are means (± SE); for 3-O-MFPase activity, n = 12 subjects, for [3H]ouabain binding site, n = 14 subjects. *Less than rest, P < 0.05.

Muscle [³H]ouabain binding site content was not affected by the exercise bout; that is, it was unchanged from rest at fatigue and at 3 and 24 h postexercise (Fig. 2B).

Correlations Between Changes in Muscle Na⁺-K⁺-ATPase mRNA and 3-O-MFPase Activity

₁-Isoform. Exercise elevated ₁ mRNA by 1.5-fold at fatigue (P < 0.05, Fig. 3A). The percent change in ₁ mRNA at fatigue was inversely correlated with %3-O-MFPase_rest-fatigue [–10.7 (SD 8)%] (r = –0.60, P < 0.05, Fig. 3B). The average postexercise (average of fatigue and 3 and 24 h postexercise) ₁ mRNA was increased by 1.4-fold above rest (P < 0.05, Fig. 3A). The percent change in average postexercise ₁ mRNA approached a significant inverse correlation with %3-O-MFPase_rest-fatigue (r = –0.56, P = 0.08, Fig. 3C).

View larger version (18K): [in this window] [in a new window]   Fig. 3. Na+-K+-ATPase 1-isoform mRNA expression and relationship with 3-O-MFPase activity. A: Na+-K+-ATPase 1-isoform mRNA expression at fatigue induced by repeated isokinetic single-leg knee extensions and the average postexercise 1 mRNA expression (average of fatigue, 3 h, and 24 h postexercise) expressed as percent change relative to rest. Values are means (± SE); n = 14 subjects. *Greater than rest, P < 0.05. B and C: scatter plots of percent change in 3-O-MFPase activity from rest to fatigue induced by repeated isokinetic single-leg knee extensions against percent change in 1 mRNA expression from rest to fatigue (B; n = 11 subjects, r = –0.68, P < 0.05, y = –14.7x + 19.6) and against percent change in average postexercise 1 mRNA expression relative to rest (C; n = 11 subjects, r = –0.56, P = 0.08, y = –12.7x + 57.1). Dotted curves indicate 95% confidence intervals; dashed lines indicate mRNA expression at rest.

₂-Isoform.

View larger version (20K): [in this window] [in a new window]   Fig. 4. Na+-K+-ATPase 2-isoform mRNA expression and relationship with 3-O-MFPase activity. A: Na+-K+-ATPase 2-isoform mRNA at fatigue induced by repeated isokinetic single-leg knee extensions and average postexercise 2 mRNA expression (average of fatigue, 3 h, and 24 h postexercise) expressed as percent change relative to rest. Values are means (± SE); n = 14 subjects. *Greater than rest, P < 0.05. B and C: scatter plots of percent change in 3-O-MFPase activity from rest to fatigue induced by repeated isokinetic single-leg knee extensions against percent change in 2 mRNA expression from rest to fatigue (B; n = 11 subjects, r = –0.60, P = 0.05, y = –24.8x + 52.7) and against percent change in average postexercise 2 mRNA expression relative to rest (C; n = 11 subjects, r = –0.60, P < 0.05, y = –18.8x + 66.8). Dotted curves indicate 95% confidence intervals; dashed lines indicate mRNA expression at rest.

₃-Isoform.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Na+-K+-ATPase 3-isoform mRNA expression and relationship with 3-O-MFPase activity. A: Na+-K+-ATPase 3-isoform mRNA at fatigue induced by repeated isokinetic single-leg knee extensions and average postexercise 3 mRNA expression (average of fatigue, 3 h, and 24 h postexercise) expressed as percent change relative to rest. Values are means (± SE); n = 14 subjects. *Greater than rest, P < 0.05. B and C: scatter plot of percent change in 3-O-MFPase activity from rest to fatigue induced by repeated isokinetic single-leg knee extensions against percent change in 3 mRNA expression from rest to fatigue (B; n = 11 subjects, r = –0.32, P = 0.34, y = –8.7x + 135.2) and against percent change in average postexercise 3 mRNA expression relative to rest (C; n = 11 subjects, r = –0.34, P = 0.31, y = –6.8x + 71.9). Dotted curves indicate 95% confidence intervals; dashed lines indicate mRNA expression at rest.

_1-, ₂-, and ₃-Isoforms.

	DISCUSSION

TOP ABSTRACT METHODS RESULTS DISCUSSION REFERENCES

 
There are three major findings of this study. First, brief, exhaustive muscle contractions only transiently depressed skeletal muscle maximal Na⁺-K⁺-ATPase activity, as measured by maximal K⁺-stimulated 3-O-MFPase activity, with no significant difference in 3-O-MFPase activity evident from rest after 3 h and until 24 h recovery. Second, exhaustive muscle contractions did not modify muscle Na⁺-K⁺-ATPase content, as measured by [³H]ouabain binding site content, either immediately or for up to 24 h after exercise. Third, and our most novel findings, significant inverse relationships were found between %3-O-MFPase_rest-fatigue and the percent increase in mRNA expression at fatigue, for both the ₁ and ₂ Na⁺-K⁺-ATPase isoforms and for the percent increase in the average postexercise ₂ mRNA expression. These findings suggest that this reversible depression in muscle 3-O-MFPase activity with fatiguing exercise, which is independent of muscle [³H]ouabain binding site content, may exert a modulatory role in muscle Na⁺-K⁺-ATPase gene expression.

Reversible Depression in Muscle Maximal Na⁺-K⁺-ATPase Activity With Exercise

The 3-O-MFPase activity measurements were performed on crude muscle homogenates with no membrane purification, to ensure maximal Na⁺-K⁺-ATPase recovery (19). This assay is the preferred method for measuring Na⁺-K⁺-ATPase activity in small muscle biopsy samples due to its high sensitivity and specificity for Na⁺-K⁺-ATPase (14). Maximal K⁺-stimulated 3-O-MFPase activity was reduced by 11% immediately after fatiguing exercise, consistent with several previous studies (2, 12, 13, 27, 45). We unequivocally demonstrate that this depression in human muscle was transient, with activity not significantly different from rest at 3 or 24 h after the exercise bout. Thus our work clarifies a previous finding that also suggested an early postexercise recovery but that had internally inconsistent results and was thus inconclusive (12).

The transient nature of this Na⁺-K⁺-ATPase impairment after exercise suggests that this phenomenon reflects enzyme inactivation as part of an exercise-induced process and/or some other cellular regulatory process, rather than being due to muscle damage. That this depression in Na⁺-K⁺-ATPase activity is an obligatory response to exercise is further suggested by having been demonstrated across a wide range of exercise intensities and durations (2, 12, 13, 27, 45). During prolonged cycling, muscle 3-O-MFPase activity was progressively decreased with increased exercise time to fatigue (27), suggesting that the decrease in 3-O-MFPase activity was due to progressive exercise-induced changes in the cellular milieu. Previous studies have speculated that increased intracellular Ca²⁺ concentration and free radical damage are the most likely mechanisms for the reduced maximal in vitro 3-O-MFPase activity (2, 12, 13, 45). Although the exact mechanisms remain to be determined, they appear to be rapidly reversible, evidenced by recovery of maximal 3-O-MFPase activity within 3 h postexercise.

It has been suggested that depressed maximal Na⁺-K⁺-ATPase activity may contribute to fatigue by contributing to reduced transcellular Na⁺ and K⁺ gradients and membrane inexcitability (2, 27).

There is considerable evidence that intense muscle contractions are accompanied by muscle K⁺ loss, elevated interstitial [K⁺], membrane depolarization of between 10 and 20 mV, and reduced M-wave area [see reviews by Sejersted and Sjøgaard (46), Clausen (6), and Nielsen and Clausen (36)]. However, so far, there is no direct evidence that depressed maximal Na⁺-K⁺-ATPase activity exacerbates these ionic changes.

Data on the effects of decreased Na⁺ and K⁺ gradients on muscle excitability in humans are limited. Calculations of membrane potential in exercising humans suggest a depolarization of 14 mV at fatigue (47), whereas animal studies have shown both hyperpolarization (21) and depolarization (3) following exercise. Measurements of the compound muscle action potential (M wave) in humans have shown decreased (4, 12, 15, 26), unchanged (32, 45), and increased (4) area or amplitude after exercise. Therefore, whether membrane inexcitability occurs during exercise in humans is equivocal, and it is not yet possible to determine whether a decrease in 3-O-MFPase activity during exercise affects excitability.

These in vitro measures of depressed maximal Na⁺-K⁺-ATPase activity most likely do not reflect in vivo changes. First, in vivo Na⁺-K⁺-ATPase activity is highly likely to be substantially elevated with exercise, based on the 18- to 22-fold increase in Na⁺-K⁺-ATPase activity that occurs with electrical stimulation in isolated rat muscles (6, 30, 36, 37), the rapid postexercise reversal of arteriovenous K⁺ differences across exercising muscles, and the rapid postexercise decline in [K⁺] observed here. All of these provide evidence of increased in vivo Na⁺-K⁺-ATPase activity in human muscles. Second, our findings indicate that the maximal attainable Na⁺-K⁺-ATPase activity is decreased after exercise, whereas evidence based on ion-selective electrodes and arteriovenous K⁺ differences suggest that the in vivo Na⁺-K⁺-ATPase activity in human muscles is considerably below the levels achieved in isolated rat muscles [see Sejersted and Sjøgaard (46) and McKenna (29)]. The in vivo pump activity may also be adversely affected during exercise due to localized declines in glycogen, phosphocreatine, or ATP, but this has not yet been demonstrated. Further studies with more sophisticated techniques are required to determine the exact relationship between depressed maximal 3-O-MFPase activity and in vivo Na⁺/K⁺ exchange in human muscles with exercise.

The relatively small depression in Na⁺-K⁺-ATPase activity found in this study (11%) may cast doubt on the functional significance of these findings. Interestingly, the percent decline in Na⁺-K⁺-ATPase activity at fatigue is similar to the percent gain in total Na⁺-K⁺-ATPase content ([³H]ouabain binding) with intense exercise training in humans and the percent decline with inactivity (for review, see Ref. 6). It is apparent that these interventions therefore produce only relatively small changes in Na⁺-K⁺-ATPase in human muscles. Thus the 11% decline in maximal activity with fatigue here might also be expected to have important implications for the muscle. It is also unknown whether this depression reflects a similar decline in Na⁺-K⁺ pump activity in all muscle fibers or a more marked depression in Na⁺-K⁺-ATPase activity in some fibers.

It is not possible to state that phosphatase activity measured enzymatically directly reflects functional pump activity. Nonetheless, the 3-O-MFPase assay is specific for Na⁺-K⁺-ATPase, as shown by ouabain inhibition (14), and correlates with [³H]ouabain binding site content (13). The 3-O-MFPase assay is the preferred method for measuring Na⁺-K⁺-ATPase activity in small muscle biopsy samples because of its high specificity, sensitivity, and inability to utilize more traditional assays for monitoring ATP hydrolysis rate in muscle (14).

Lack of Effect of Exercise on Muscle [³H]ouabain Binding Site Content

Our [³H]ouabain binding measurements were conducted on cut muscle pieces in the presence of vanadate, thus enabling full quantification of muscle Na⁺-K⁺-ATPase content (7, 30). Because [³H]ouabain binding measurements did not differ when measured in either muscle pieces or intact muscles, it was suggested that all functional Na⁺-K⁺-ATPase units in muscle are measured with this technique (7, 30). We show that fatiguing exercise lasting 6 min did not affect muscle [³H]ouabain binding site content, either immediately or for up to 24 h postexercise. The lack of change in [³H]ouabain binding with fatigue confirms previous findings in studies of acute dynamic or isometric exercise in humans (2, 12) and in studies of 10-s to 240-min electrical stimulation of isolated rat muscles (30). Conversely, an increase in [³H]ouabain binding site content was observed after a 100-km run of 641-min duration (43), most likely due to Na⁺-K⁺-ATPase synthesis during exercise. The lack of change for up to 24 h postexercise extends earlier findings (2, 12, 27, 30), as these failed to follow changes for 24 h after exercise and may therefore have missed any postexercise upregulation. It should be sufficient to detect increased Na⁺-K⁺-ATPase synthesis within 24 h, as this has a half-time of 18 h (52). Because [³H]ouabain selectively binds to Na⁺-K⁺-ATPase in both the sarcolemma and T-tubular system (7), our previous findings argue against a translocatable intracellular Na⁺-K⁺-ATPase pool (30). Therefore, our findings of unchanged [³H]ouabain binding with 6 min of exercise are inconsistent with an apparent translocation of Na⁺-K⁺-ATPase ₂- and ₁-isoforms from undefined intracellular stores to the sarcolemma with exercise (24).

Correlation Between Depressed Na⁺-K⁺-ATPase Activity and -Isoform Gene Expression

The most novel finding of this study is the significant inverse relationship found between %3-O-MFPase_rest-fatigue and the percent change from rest to fatigue for both ₁ and ₂ mRNA expression. This finding is reinforced by the significant inverse correlation between the %3-O-MFPase_rest-fatigue and the average postexercise percent change in both ₂ and ₁ mRNA expression. This is the first time such a relationship has been demonstrated in contracting skeletal muscle. This suggests a possible modulatory role of depressed Na⁺-K⁺-ATPase activity in muscle Na⁺-K⁺-ATPase gene expression. This is consistent with findings in rat liver cells, in which reduction of Na⁺-K⁺-ATPase activity by incubation in low [K⁺] medium for 6 h resulted in a 60% increase in Na⁺-K⁺-ATPase - and -subunit mRNA expression (44). These findings strongly suggest that acutely depressed maximal Na⁺-K⁺-ATPase activity might be one factor underlying the chronic exercise-induced upregulation of Na⁺-K⁺-ATPase content in skeletal muscle, as shown with exercise training (16, 31). However, we are unable to unequivocally demonstrate this link here, as no upregulation of [³H]ouabain binding site content was observed within 24 h after this short exercise bout. We have previously reported that the Na⁺-K⁺-ATPase isoform protein expression was also unchanged (34). Further studies with either very prolonged exercise (43) or chronic training (16, 31) are required to verify this possibility.

Three possible mechanisms have been identified that may link decreased Na⁺-K⁺-ATPase activity to increased Na⁺-K⁺-ATPase gene transcription. The first is via ion concentration-dependent regulation, whereby decreased maximal Na⁺-K⁺-ATPase activity allows greater Na⁺ and K⁺ net fluxes across the cell membrane and, consequently, increased muscle intracellular Na⁺ concentration and extracellular [K⁺]. These ionic disturbances may stimulate synthesis of new Na⁺-K⁺-ATPase enzymes to reestablish favorable Na⁺ and K⁺ gradients, as evidenced by a 44% increase in rat gastrocnemius muscle [³H]ouabain binding site content after 7 days of diet-induced hyperkalemia (5). Also, activation of voltage-sensitive Na⁺ channels with veratridine increased sarcolemmal Na⁺-K⁺-ATPase by 60% in cultured chick skeletal muscle (52). The second proposed mechanism is via decreased Na⁺-K⁺-ATPase activity-induced, direct protein-protein interaction of the Na⁺-K⁺-ATPase and its neighboring proteins, which triggers a signaling cascade that culminates in increased Na⁺-K⁺-ATPase gene transcription (53). The third mechanism is via increased cytosolic Ca²⁺ concentrations. Tetanic and resting cytosolic Ca²⁺ concentrations increase during fatiguing muscle contractions (51). Incubation of mouse diaphragm in 10 mM Ca²⁺ completely and reversibly inhibits Na⁺-K⁺-ATPase activity (48). Ca²⁺ has also been established as a regulator of gene transcription, often acting via the Ca²⁺ receptor calmodulin and Ca²⁺/calmodulin-dependent protein kinases (8). It cannot be ruled out that the relationships between maximal 3-O-MFPase activity and mRNA observed in this study were merely coincidental. It is possible that the factors responsible for depressing maximal 3-O-MFPase activity are also involved in the regulation of Na⁺-K⁺-ATPase mRNA expression. Further studies are required to determine the factors responsible for depression of maximal 3-O-MFPase activity and upregulation of Na⁺-K⁺-ATPase mRNA expression with exercise.

Isoform-Specific Relationships

An important finding was that only changes in the ₁- and ₂-isoforms mRNA were related to the change in 3-O-MFPase activity with fatigue. The catalytic -subunit contains the substrate (Na⁺, K⁺, Mg²⁺, and ATP) and inhibitor (ouabain) binding sites (10, 18, 28). The mRNAs of the ₁- and ₂-isoforms are the most abundantly expressed of the -isoforms in skeletal muscle (42), consistent with the dominant protein abundance of the ₁- and ₂-isoforms (17, 49). Hence, the ₁- and ₂-isoforms probably play the most important role of the -subunit isoforms in skeletal muscle. Thus their regulation is presumably the most tightly controlled and sensitive to possible regulatory stimuli, such as ionic concentration changes caused by decreased maximal Na⁺-K⁺-ATPase activity. The Na⁺ affinity of the ₃-isoform in humans is 30- and 10-fold lower than that for the ₁- and ₂-isoforms, respectively (9), and the rat ₃-isoform is three times less sensitive to Na⁺ activation than the ₁- and ₂-isoforms (33). The lower sensitivity of ₃-isoform to Na⁺ may therefore make the ₃-isoform less sensitive to exercise-induced changes in intracellular Na⁺ concentration and explain the lack of a relationship between 3-O-MFPase activity and mRNA expression for this isoform. There was also no relationship between the change in 3-O-MFPase activity and the change in mRNA expression for any of the -subunit isoforms, which are needed for the correct configuration, maturation, transport, and K⁺ kinetics of the Na⁺-K⁺-ATPase enzyme (1, 9, 39). This may be related to an overabundance of - compared with -subunits (25), suggesting that there would always be adequate -subunits available to form new functional -complexes. Finally, several studies have found that the ₁- and ₂-isoform abundances in plasma membranes were elevated in both oxidative and glycolytic muscle fibers after treadmill running in rats (23, 50).

In conclusion, we have demonstrated that acute short-duration exercise depressed maximal in vitro 3-O-MFPase (Na⁺-K⁺-ATPase) activity in skeletal muscle, which recovered within 3 h postexercise. The decrease in maximal 3-O-MFPase activity was not due to a decline in [³H]ouabain binding site (Na⁺-K⁺-ATPase) content, and brief exercise did not upregulate Na⁺-K⁺-ATPase content over the following 24 h, as [³H]ouabain binding site content was not changed after exercise. There were significant inverse relationships between the decline in 3-O-MFPase activity from rest to fatigue and the increase in ₁ and ₂ mRNA expression at fatigue, as well as when averaged over 0, 3, and 24 h postexercise. This suggests a possible signal transduction role of depressed Na⁺-K⁺-ATPase activity in the exercise-induced upregulation of Na⁺-K⁺-ATPase in skeletal muscle.

	ACKNOWLEDGMENTS

 
We thank our subjects, without whom this study would not have been possible, and Ivan Medved and Anula Costa for assistance on some trial days. We also thank Professor Torben Clausen, rhus University, Denmark, for assistance with establishing the [³H]ouabain binding assay.

	FOOTNOTES

 

Address for reprint requests and other correspondence: M. J. McKenna, School of Human Movement, Recreation and Performance (FO22), Victoria Univ. of Technology, PO Box 14428, MCMC, Melbourne, Victoria, Australia 8001 (e-mail: michael.mckenna{at}vu.edu.au)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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