Muscle Na -K - American Journal of Physiology - Endocrinology and ...

Am J Physiol Endocrinol Metab 293: E523–E530, 2007. First published May 8, 2007; doi:10.1152/ajpendo.00004.2007.

Muscle Na⫹-K⫹-ATPase response during 16 h of heavy intermittent cycle exercise H. J. Green, T. A. Duhamel, G. P. Holloway, J. W. Moule, J. Ouyang, D. Ranney, and A. R. Tupling Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada Submitted 3 January 2007; accepted in final form 30 April 2007

Green HJ, Duhamel TA, Holloway GP, Moule JW, Ouyang J, Ranney D, Tupling AR. Muscle Na⫹-K⫹-ATPase response during 16 h of heavy intermittent cycle exercise. Am J Physiol Endocrinol Metab 293: E523–E530, 2007. First published May 8, 2007; doi:10.1152/ajpendo.00004.2007.—This study investigated the effects of a 16-h protocol of heavy intermittent exercise on the intrinsic activity and protein and isoform content of skeletal muscle Na⫹-K⫹-ATPase. The protocol consisted of 6 min of exercise per˙ O2 peak) with formed once per hour at ⬃91% peak aerobic power (V tissue sampling from vastus lateralis before (B) and immediately after repetitions 1 (R1), 2 (R2), 9 (R9), and 16 (R16). Eleven untrained ˙ O2 peak of 44.3 ⫾ 2.3 ml 䡠 kg⫺1 䡠 min⫺1 participated volunteers with a V in the study. Maximal Na⫹-K⫹-ATPase activity (Vmax, in nmol 䡠 mg protein⫺1 䡠 h⫺1) as measured by the 3-O-methylfluorescein K⫹-stimulated phosphatase assay was reduced (P ⬍ 0.05) by ⬃15% with exercise regardless of the number of repetitions performed. In addition, Vmax at R9 and R16 was lower (P ⬍ 0.05) than at R1 and R2. Vanadate-facilitated [3H]ouabain determination of Na⫹-K⫹-ATPase content (maximum binding capacity, pmol/g wet wt), although unaltered by exercise, increased (P ⬍ 0.05) 8.3% by R9 with no further increase observed at R16. Assessment of relative changes in isoform abundance measured at B as determined by quantitative immunoblotting showed a 26% increase (P ⬍ 0.05) in the ␣2-isoform by R2 and a 29% increase in ␣3 by R9. At R16, ␤3 was lower (P ⬍ 0.05) than at R2 and R9. No changes were observed in ␣1, ␤1, or ␤2. It is concluded that repeated sessions of heavy exercise, although resulting in increases in the ␣2- and ␣3-isoforms and decreases in ␤3-isoform, also result in depression in maximal catalytic activity.

regular contractile activity is a potent stimulus for remodeling skeletal muscle, both with regard to its composition and function (52). It is also clear that, providing that the exercise stimulus is appropriate, numerous adaptations can occur to the muscle cell soon after the onset of training. Nowhere is this plasticity more evident than at the level of the Na⫹-K⫹-ATPase, the integral membrane protein involved in the transport of the Na⫹ and K⫹ across the sarcolemma and T-tubules (7). This enzyme, discovered in 1957 by Skou (59), occupies a central position in restoring membrane excitability following an action potential by virtue of its ability to extrude Na⫹ from the cell and to reuptake K⫹ into the cell. During voluntary activity, the ability to successfully respond to a neural command for increased force is mediated in part by increasing neural impulse frequency to the muscle and, consequently, the frequency of action potentials in the sarcolemma and T-tubules (4, 8). As a consequence, the Na⫹-K⫹-ATPase

must be able to regulate its catalytic activity over a broad range. The cation transport function of the Na⫹-K⫹-ATPase is intimately associated with its subunit composition (5). The catalytic activity and the binding sites for substrates, such as ATP, K⫹, and Na⫹, are localized to the ␣-subunit, whereas the ␤-subunit, although essential for pump function, appears to be primarily involved in insertion and assembly into the membrane. In skeletal muscle, a variety of both ␣- and ␤-isoforms exists (5). Recently, it has been demonstrated that, in humans, three ␣-isoforms (␣1, ␣2, and ␣3) and three ␤-isoforms (␤1, ␤2, and ␤3) are present as demonstrated at both the gene transcript and protein levels (44). This isoform diversity provides for numerous combinations of ␣- and ␤-heterodimers, potentially allowing for complexity in function so essential to meet the varied demands of the working muscle cell (5). The abundance of each isoform, the subcellular distribution, and the regulation of intrinsic activity are all crucial to the ability of the Na⫹K⫹-ATPase to respond appropriately to the needs for Na⫹ and K⫹ transport (6, 10). Current evidence indicates that increases in Na⫹-K⫹ATPase protein levels may be an important strategy in meeting the requirements for Na⫹ and K⫹ transport soon after the onset of contractile activity. We reported that, in chronically stimulated rabbit, the dramatic increases of the extensor digitorum longus muscle in Na⫹-K⫹-ATPase content occurred within days of the onset of stimulation (16). We have also been able to determine with the use of similar [3H]ouabain-binding techniques that increases in enzyme content also occur within the same time frame in skeletal muscles in humans in response to prolonged submaximal exercise (17, 18). One study has also reported increases in Na⫹-K⫹-ATPase content following a 100-km run (50). As might be expected, given that the [3H]ouabain technique is based on the binding to the ␣-isoform, increases in ␣-isoform composition also occur soon after the beginning of training. We have reported that within 3 days of beginning prolonged cycling, increases in the ␣2-isoform occurred that were followed by increases in the ␤1-isoform by 6 days of training (17). Although similar percent increases in the ␣1isoform were also observed by 3 days of exercise, only the increase in the ␣2-isoform was significant. Unfortunately, the additional ␣- and ␤-isoforms were not probed for. Interestingly, a single session of exercise, both prolonged (43) and intense (44, 46), can result in the elevation of several mRNA transcripts coding for the ␣- and ␤-isoforms, with some being elevated as early as 1 h following the exercise. With few

Address for reprint requests and other correspondence: H. J. Green, Dept. of Kinesiology, Univ. of Waterloo, Waterloo, ON, N2L 3G1, Canada (e-mail: [email protected]).

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sodium-potassium pump; vastus lateralis; maximal activity; content; isoforms

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REPETITIVE EXERCISE AND MUSCLE Na⫹-K⫹-ATPase REGULATION

exceptions, increases in isoform content did not accompany the increases in isoform-specific mRNAs (43, 44, 51). A peculiar finding of the short-term training study was the dissociation between the change in Na⫹-K⫹-ATPase content and the change in the catalytic activity of the enzyme (17). Increases in the maximal activity of the enzyme (Vmax) as assessed by the 3-O-methylfluorescein phosphatase assay (3-O-MFPase) were only elevated at 6 days of training. This finding suggests that increases in content of a specific combination of the ␣- and ␤-subunit isoforms are necessary to realize an increase in activity. A potential complication in addressing this possibility is an inactivation of the Vmax that occurs with exercise in humans (12, 13, 54, 55). It is possible, depending on the rate at which Vmax recovers from exercise, that adaptive increases in catalytic activity may have been obscured. Unfortunately, only limited information exists on recovery profiles. The purpose of this study was to investigate the effects of repeated sessions of heavy exercise performed during a single day on the content, isoform, and activity characteristics of the Na⫹-K⫹-ATPase. We have rationalized that the intermittent protocol of heavy exercise would repeatedly strain the catalytic activity of the enzyme given the demands for transport of the Na⫹ and K⫹ across the sarcolemma and T-tubule. Moreover, when we provide sufficient time between exercise bouts for recovery of ionic disturbances and, potentially, the catalytic activity of the enzyme, the exercise stimuli will remain persistently high. Under such circumstances, increases in Na⫹-K⫹ATPase content, isoform abundance, and Vmax should result. This research represents a continuing series of studies in which we have used a 16-h model of heavy, intermittent exercise to examine the adaptive changes that occur to a wide range of muscle properties (19, 26). METHODS

Participants Eleven healthy but untrained volunteers (8 men and 3 women) ˙ O2 peak) assessed participated in the study. Peak aerobic power (V during progressive cycle exercise to fatigue was 3.02 ⫾ 0.18 l/min. The mean age, height, and weight of the volunteers were 22.0 ⫾ 0.92 yr, 173 ⫾ 2.5 cm, and 69.0 ⫾ 3.7 kg, respectively. As required, the study was approved by the Office of Research Ethics at the University of Waterloo before obtaining written consent from each participant. Experimental Design The work protocol employed in this study involved cycling for 6 ˙ O2 peak once per hour for 16 h. The specifics of this min at ⬃91% V protocol were based on earlier research in which we have used this model to examine the adaptations that occur as measured using a standardized task at 36 to 48 h following the intermittent exercise (22, 61). In the current study, our objective was to examine the changes that occur during selected repetitions of the exercise. For the muscle properties that were examined, tissue was obtained from the vastus lateralis before (B) and immediately after (A) the exercise at the first (R1), second (R2), ninth (R9) and sixteenth (R16) repetition. The tissue was obtained by needle biopsy (3) from sites that had been prepared before the exercise. To minimize the number of tissue samples extracted in a given session, the protocol was completed on two separate visits to the laboratory. Each visit was separated by ⬃30 days. On one visit, the participant performed only the first two repetitions (R1 and R2) of the exercise. On another visit, the entire protocol was performed, but tissue samples were only extracted at R9 and R16. This meant that during a given session, four separate AJP-Endocrinol Metab • VOL

sampling sites were used, two on each leg. The order of conditions was randomly assigned. During each visit to the laboratory, the participants arrived at ⬃7:00 AM. The intermittent exercise protocol was initiated at ⬃9:00 AM. The 2-h interval was used for the preparation of the participants (blood and muscle sampling) as well as baseline measurements (respiratory gas exchange). Before reporting to the laboratory on a given experimental day, the volunteers were instructed to ingest an Ensure (250 kcal) meal replacement consisting of 9.4 g of protein, 6.7 g of fat, and 38 g of carbohydrates (Ross Products Division, Saint-Laurent, QC, Canada). The meal replacement was intended to substitute for breakfast and to standardize nutrient intake before the start of the experimental protocol. During the 16-h intermittent protocol, water (room temperature) was allowed ad libitum after the first 2 h. The volunteers were also allowed to consume selected vegetables and fruits and Gatorade on a regular basis during the 54-min period between exercise bouts. Details of nutrient intake and the schedule of ingestion appear in an earlier study (19). A more complete description of the experimental design, calculation of individual workloads used for the intermittent cycling protocol, and the specifics of the respiratory gas collection measurements has been previously published (26). As emphasized, we have used the intermittent work protocol to investigate the alterations in a wide range of muscular properties. Previous studies have examined muscle fatigue (19), sarcoplasmic reticulum Ca2⫹ cycling (26), and metabolic behavior (20). In this study, we have examined the muscle Na⫹-K⫹-ATPase pump responses, namely the maximal catalytic activity (Vmax), the pump content, and the ␣- and ␤-isoform distribution. Blood Catechalomines Blood samples were collected both before the onset of exercise (B) and near the end of exercise at R1, R2, R7, R12, and R15 from a catheter inserted in a prewarmed dorsal vein of the hand, and the plasma was extracted following standard processing and stored at ⫺20°C pending the measurement of the catecholamines epinephrine (Epi) and norepinephrine (Nor). High-performance liquid chromatography was used to measure Epi and Nor concentrations as previously described (21). Na⫹-K⫹-ATPase Properties Preparation of whole homogenates. Whole homogenate preparations were used for the measurement of Na⫹-K⫹-ATPase activity and for the protein content of the ␣- and ␤-isoforms. Briefly, tissue (⬃30 mg) from frozen samples stored at ⫺80°C was homogenized (5% wt/vol) at 0 – 4°C for 2 ⫻ 20 s at 25,000 rpm (Polytron) in a buffer containing (in mM) 250 sucrose, 2 EDTA, and 10 Tris (pH 7.40) and a commercially prepared combination of protease inhibitors (Roche Diagnostics, Indianapolis, IN). Na⫹-K⫹-ATPase activity. For the measurement of Vmax, the K⫹stimulated 3-O-MFPase was employed using previously published procedures (28, 48) but with modifications using substrate concentrations designed to produce maximal activities (14) while lowering nonspecific activity (1). To permeabilize the membrane, the homogenate (⬃25 ␮l) was freeze thawed for four cycles and diluted 1:4 in cold homogenate buffer. Following dilution, the homogenate was incubated at 37°C for 4 min in a medium containing (in mM) 5 MgCl2, 1.25 EDTA, 1.25 EGTA, 5 NaN3, and 100 Tris (pH 7.40). For a determination of K⫹-stimulated activity, 10 mM KCl and 160 ␮M 3-O-MFP were added, and the activity was determined as the difference in the slope before and after the addition of KCl using fluorescence spectroscopy (11, 14). The measure of Vmax was based on the average of three trials. Protein content of the homogenate was determined by the method of Lowry as modified by Schacterle and Pollock (57). In our laboratory, we have calculated the intra-assay variability for Vmax to be ⬃8.5%.

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Isoform determination. Western blot analysis performed using electrophoresis on 7.5% SDS-polyacrylamide gels (Mini-Protean II, Bio-Rad), essentially as described by Laemmli (37), was used to resolve Na⫹-K⫹-ATPase subunit isoform protein content. Detection of isoforms was based on duplicate measurements using two different aliquots from each sample and two different gels. For the analyses of ␣-subunit (␣1, ␣2, and ␣3) and ␤-subunit (␤1, ␤2, and ␤3) isoforms, the amount of protein employed was 40 and 50 ␮g, respectively. On a given analytical day, all preexercise (B) samples for a given participant and for a given isoform were probed for. A biotinylated ladder was used as a molecular weight standard (Cell Signaling Technology, Beverly, MA). For the ␤-subunits, the gels were run following deglycosylation, which was accomplished with N-glycosidase F (Boehringer-Mannheim, Indianapolis, IN) with overnight incubation at room temperature before electrophoresis. Specific details regarding the deglycosylation procedure are provided in an earlier publication from our laboratory (17). After SDS-PAGE, gels were electrophoretically transferred to polyvinyl difluoride membrane (Bio-Rad) by placing the gel in a transfer buffer and applying high voltage (20 V) for 45 min (TransBlot Cell, Bio-Rad). The transfer buffer consisted of 25 mM Tris, 192 mM glycine, and 20% (wt/vol) methanol. Nonspecific binding sites were blocked with 5% milk in Tris-buffered saline (pH 7.5) for 1 h at room temperature before the incubation with primary antibodies. The primary antibodies included ␣1, ␣2, and ␤1 (Upstate Biotechnology, Lake Placid, NY); ␣3 (Affinity Bioreagents, Golden, CO); and ␤2 and ␤3 (BD Biosciences, Mississauga, ON, Canada). Immunoblotting was performed overnight at 4°C with the antibodies in 5% milk (␣1 and ␣3, 1:1,000; ␣2, 1:500; and ␤1, ␤2, and ␤3, 1:500). Bound antibodies were detected with goat anti-rabbit IgG1 (␣2) and goat anti-mouse IgG1 (␣1, ␣3, ␤1, ␤2, and ␤3). An enhanced chemiluminescence procedure was used to detect antibody content (Amersham, Buckinghamshire, UK). Blots were analyzed by use of a Chemi Genius 2 model bioimaging system (SynGene, Frederick, MD) with SynGene software version 1.0. Additional analytical details can be found in previous publications from our laboratory (11, 17, 56). For a specific isoform, resting samples at R1, R2, R9, and R16 for a particular individual were applied to a specific gel, and the gel was run on duplicate. Each gel also contains a known protein content (1–2 ␮g) of brain standard (rat) for relative control. For each sample, data were calculated first as a relative percentage of brain standard and then with the samples obtained at R2, R9, and R16 using R1 as control (100%). The intra-assay variability as determined by the coefficients of variation ranged between 4% and 10% depending on the isoform. As in previous studies, the linearity between blot signal and the amount of protein applied was established before experimental analysis. For all assays, protein content was determined in duplicate according to Lowry as modified by Schacterle and Pollock (57) using bovine serum albumin as a standard. Na⫹-K⫹-ATPase content. Vanadate-facilitated [3H]ouabain binding was used to determine maximal Na⫹-K⫹-ATPase content based on maximal binding characteristics (Bmax) as originally described (47). Two tissue samples weighing between 2 and 8 ␮g were incubated for 10-min periods in a Tris-sucrose buffer containing (in mM) 10 Tris 䡠 HCl, 3 MgSO4, 1 Tris-vanadate, and 250 sucrose with [3H]ouabain (1.8 ␮Ci/ml) and unlabeled ouabain (l ␮M final concentration) for 2 ⫻ 60 min at 37°C. After the samples were washed (4 ⫻ 30 min in ice cold buffer), blotted, and weighed, they were soaked in l ml of 5% trichloroacetic acid for 16 h at room temperature and counted for [3H] radioactivity in a scintillation counter. The [3H] binding capacity was corrected for loss of specifically bound [3H]ouabain binding sites during washout and expressed as picomoles per gram wet weight (47). Additional details are provided in our earlier publications (17, 56). Intra-assay variability, as assessed in our laboratory, is ⬃11%. The measure of Na⫹-K⫹-ATPase content is based on the vanadate-facilitated binding of [3H]ouabain to the AJP-Endocrinol Metab • VOL

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␣-subunits, supposedly while in a functional state (7). As in previous measurements, all samples both before and after exercise at R1, R2, R9, and R16 were assessed during the same analytical session. Data Analyses The effects of time (number of repetitions) and exercise (B vs. A) on Vmax and Na⫹-K⫹-ATPase content were examined using two-way ANOVA for repeated measurements. To determine the effects of repetition on ␣- and ␤-isoform abundance, we employed one-way ANOVA for repeated measurements. Where significance was found, the Newman-Keuls technique was applied to determine which means were significantly different. Statistical significance was accepted at P ⬍ 0.05. Data are presented as means ⫾ SE. Throughout this article, statistical differences between means are expressed only as differences based on the level of probability employed. RESULTS

Na⫹-K⫹-ATPase Properties 3-O-MFPase activity. Measurement of the maximal 3-OMFPase activity (Vmax) indicated the main effects of both exercise and repetitions for the 16-h intermittent exercise protocol (Fig. 1). For exercise, it was found that Vmax was generally lower following exercise than before. This was a main effect that applied to all repetitions. In the case of the repetition effect, the first two repetitions of the exercise (R1 and R2) displayed a larger Vmax than R9 and R16. From R9 to R16, no further changes in Vmax were observed. [3H]ouabain binding. The maximal [3H]ouabain binding (Bmax) was altered with the number of repetitions but not with exercise per se (Fig. 2). In the case of the repetition effect, the first repetition of the exercise (R1) was lower than the R9 and R16. No differences were observed between R2, R9, and R16. Na⫹-K⫹-ATPase isoforms. Na⫹-K⫹-ATPase isoforms (␣1, ␣2, ␣3, ␤1, ␤2, and ␤3) were assessed by Western blot analysis, and the changes during the repetitive exercise

Fig. 1. Maximal Na⫹-K⫹-ATPase activity as assessed by 3-O-methylfluorescein phosphatase (3-O-MFPase) before (B) and after (A) exercise at selected repetitions (Reps). Values are means ⫾ SE; n ⫽ 11 participants. R1, R2, R9, and R16 represent 1st, 2nd, 9th, and 16th repetition of the exercise, respectively. Main effects (P ⬍ 0.05) were found for both exercise and repetition. For exercise, B ⬎ A. For repetition, R1 and R2 ⬎ R9 and R16.

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Blood Measurements Blood Epi and Nor. The first repetition of the exercise resulted in a greater than ninefold increase in Epi and a greater than fourfold increase in Nor (Table 1). For blood Epi, no further increases at the end of exercise were observed at R2 or at R7, R12, and R15. For Nor, at E, an increase first occurred at R2 followed by further increases at R15. For Epi, no changes were observed at B between repetitions. DISCUSSION

Fig. 2. Na⫹-K⫹-ATPase content as measured by the vanadate-facilitated [3H]ouabain binding technique before and after exercise at selected repetitions. Values are means ⫾ SE; n ⫽ 11 participants. A main effect (P ⬍ 0.05) of repetition was found. For repetition, R1 ⬍ R9 and R16.

protocol were expressed relative to the first repetition (R1) (Figs. 3 and 4). For the ␣-isoforms, both ␣2 and ␣3 were observed to change. For ␣2, R2, R9, and R16 were elevated over R1. For ␣3, increases were first observed at R9 and persisted through R16. No changes were found to occur in either ␤1 or ␤2 with the 16-h protocol. However, at R16, ␤3 was lower than at R2 and R9.

The changes that we have observed on the properties in the Na⫹-K⫹-ATPase with the 16-h heavy intermittent work model reveal several novel features of the enzyme not previously acknowledged. With regard to the catalytic behavior of the Na⫹-K⫹-ATPase, the 6-min session of cycle exercise resulted in significant reductions in Vmax regardless of the number of repetitions. It was also shown that the 54-min period of rest between repetitions was insufficient for full recovery of Vmax. The incomplete recovery accumulated over repetitions such that, by R9 and extending to R16, Vmax was lower than at R1 and R2. The reduction in Vmax that was observed at R9 and R16 was a main effect determined with the two-way ANOVA. In a follow-up analysis using a one-way ANOVA for repeated measures applied to B samples only, lower values were also observed at R9 and R16 compared with R1 and R2. Surprisingly, the decreases in Vmax were accompanied by an increase in ␣-subunit content as measured by the [3H]ouabain binding technique. Western blot analysis showed that for the ␣-subunits, increases in content of both ␣2 and ␣3 were observed. For the ␤-subunit, the intermittent protocol resulted in a de-

Fig. 3. A and B: representative Western blots (A) and relative changes in the ␣-isoform distribution of the Na⫹-K⫹-ATPase before exercise for selected repetitions (B). Values are means ⫾ SE; n ⫽ 11 participants. Note that the relative values were first calculated against a standard (STD) and that the repetition effects at R2, R9, and R16 were calculated against R1, which was set at 100%. ␣1-, ␣2-, ␣3 represent isoform subunits. *P ⬍ 0.05, significantly different from R1; #P ⬍ 0.05, significantly different from R2. AJP-Endocrinol Metab • VOL

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Fig. 4. A and B: representative Western blots (A) and relative changes in the ␤-isoform distribution of the Na⫹-K⫹-ATPase before exercise for selected repetitions (B). Values are means ⫾ SE; n ⫽ 11 participants. Note that the relative values were first calculated against a standard and that the repetition effects at R2, R9 and R16 were calculated against R1, which was set at 100%. ␤1, ␤2, and ␤3 represent ␤-isoform subunits. #P ⬍ 0.05, significantly different from R2; {P ⬍ 0.05, significantly different from R9.

crease of ␤3. No changes were observed in the other isoforms, namely, ␣1, ␤1, and ␤2. The reduction in Vmax during R1 was expected given the numerous studies that have been published documenting similar effects with heavy exercise (12, 13, 55). The fact that similar changes were observed with each of the other repetitions assessed would suggest that the mechanism responsible is the same. It is apparent that the restoration of maximal activity occurs during the interval between bouts early in the protocol as indicated by the nonsignificant difference at R1 and R2 for B. Later in the protocol, restoration of Vmax is incomplete at B. This occurs even though the metabolic disturbances in the cell induced by the exercise return to preexercise levels (20). The response of Vmax to repetitive intermittent exercise would be expected to represent the net effect of both stimulating and inhibitory factors. The catalytic activity of enzyme is subject to complex regulatory control, with a number of primary and secondary factors involved in intrinsic regulation via the ␣-subunits. With the onset of exercise, large increases in

in vivo activity occur to meet the demands for increased transmembrane exchange of Na⫹ and K⫹. The rapid activation of the enzyme is believed to be due to increased intracellular Na⫹ concentration (7). As exercise progresses, a variety of hormones, particularly the catecholamines, is believed to exert a strong stimulatory role by binding to ␤2-adrenergic receptors on the sarcolemma and activating protein kinase A via adenosine 3⬘,5⬘-cyclic monophosphate, resulting in increased phosphorylation of the ␣-subunit, possibly with the involvement of a small regulatory protein phospholemman (15). In this study, this intermittent exercise should be a potent stimulus in increasing Vmax given the large increases in Epi and Nor that occurred. Moreover, since the majority of the increase in the blood catecholamines occurred during the first repetition, the stimulatory effect on Vmax would be expected to occur throughout the protocol. The fact that Vmax decreased with exercise when measured in vitro would suggest that inhibitory factors predominate. Several possibilities exist. In another study from our laboratory

Table 1. Response of blood catacholamines to intermittent heavy exercise Repetitions 1

Epi B E Nor B E

25.4⫾5.2 239⫾43 357⫾32 1,764⫾166*

2

40.8⫾7.9 313⫾64 369⫾42 2,193⫾343*†

7

12

31.3⫾5.6 316⫾60

41.1⫾4.2 322⫾57

361⫾27 1,920⫾267*

382⫾37 2,189⫾267*†

15

54.5⫾10 358⫾58 438⫾39 2,553⫾326*†‡

Values are means ⫾ SE; n ⫽ 10 (in pg/ml). Repetitions, number of repetitions of the exercise. B, before exercise; E, end of exercise; Epi, epinephrine; Nor, norepinephrine. *P ⬍ 0.05, significantly different from B; †P ⬍ 0.05, significantly different from the end of exercise at repetition 1; ‡P ⬍ 0.05, significantly different from the end of exercise following 7 repetitions (for Epi, a main effect of condition was found). For condition, E ⬎ B. AJP-Endocrinol Metab • VOL

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(20), we have shown that the heavy exercise results in a severe metabolic strain as indicated by the large reduction in phosphorylation potential and increase in metabolic by-products. It is possible that one or more of these metabolites could be inhibiting to the enzyme activity early in the protocol (35). However, given the dissociation between the metabolic and catalytic activity late in this protocol, other mechanisms appear involved. Increases in reactive oxygen species (ROS) represent another inviting possibility to explain the exercise-induced depressions in Vmax. The type of exercise employed would be expected to result in large productions of ROS, such as superoxide and hydrogen peroxide (30, 58). The Na⫹-K⫹-ATPase is known to be sensitive to ROS attack, leading to alterations, most notably in the region of adenine nucleotide binding (36). In a recent study (42), with the use of prolonged exercise, it has been reported that the ingestion of the antioxidant N-acetylcysteine attenuated the decline in Na⫹-K⫹-ATPase activity. It is worth noting that another cation pump, namely the sarcoplasmic reticulum Ca2⫹-ATPase, has been shown to be inactivated during repetitive contractile activity as a result of structural alterations to the enzyme, secondary to direct oxidation and nitrosylation by ROS (34, 39, 40). Since recovery of normal catalytic activity is a relatively slow process, it is possible that at least some of the decline in resting Vmax over the 16 h could occur by similar mechanisms. In this regard, it is interesting that the Vmax for Ca2⫹-ATPase shows essentially the same response to the 16-h protocol, namely, a reduction with exercise and an incomplete recovery between repetitions. However, it must be emphasized that changes in Vmax should be more demonstrable in vivo given the changes that occur with exercise in the intracellular environment. In our study, Vmax was measured in vitro using K⫹ to maximally stimulate the catalytic activity of the enzyme (14). Accordingly, it is not clear, given the conditions under which the homogenate was prepared, if the high level of metabolic by-products and the second messengers involved would be reflected in the values obtained. It should be noted that the vastus lateralis consists of a mixture of fiber types (53), and although extensive recruitment of both the slow and fast twitch fibers would be expected during the heavy exercise task (62), it is unclear whether the reduction in Vmax observed in this study was specific to one or both fiber types. The [3H]ouabain binding procedure was used to estimate the muscle Na⫹-K⫹-ATPase content (49). With this procedure, we found no differences in content with exercise regardless of the number of repetitions. This was unexpected given the reductions in Vmax that occurred with the exercise task. Moreover, in contrast to the decrease in Vmax that occurred late in the protocol, we found an increase in Na⫹-K⫹-ATPase content. The measurement of Na⫹-K⫹-ATPase content, which is based on the vanadate-facilated [3H]ouabain binding to the ␣-subunits, has been assumed to represent the functional enzyme given the close correspondence with measurements of maximal transmembrane Na⫹ fluxes in muscle (7). Our results would dispute this assumption. There may be several mitigating reasons, some of which revolve around the measurements employed. In this study, the catalytic activity of the enzyme was based on K⫹-stimulated 3-O-MFPase that is based on the terminal AJP-Endocrinol Metab • VOL

step in ATP hydrolysis and not the total hydrolytic activity (27). The measurement of phosphatase activity is based on the K⫹-dependent hydrolysis of the chronogenic substrate, 3-Omethylfluorescein phosphate, which is used as a substitute for the aspartylphosphate intermediate of the Na⫹-K⫹-ATPase (27). Although previous work has shown that the measured activity is specific to the Na⫹-K⫹-ATPase as indicated by the near-complete suppression that occurs with ouabain (48), it must be acknowledged that the assay provides only an estimation of the maximal hydrolytic activity. Not only is the sensitivity of the assay compromised given the high nonspecific activity (48), but even under maximal activating conditions, the assay only yields a fraction of the maximal hydrolytic activity (11, 27, 29). In contrast to 3-O-MFP activity, which was measured in homogenates, Na⫹-K⫹-ATPase content was measured directly in muscle samples based on [3H]binding. Whereas the Vmax is based on the total cellular population of the Na⫹-K⫹-ATPase pool, it has been proposed that the [3H]ouabain binding procedure measures only the enzymes of the plasma membrane (41). It is generally accepted based on subunit abundance in isolated fractions that translocation of ␣-subunits, at least ␣1 and ␣2 to the plasma membrane, occurs during the contractile activity (32, 33, 56, 60). If such is the case, increases in Na⫹-K⫹-ATPase content, as indicated by increased ␣-subunits in the plasma membrane, might be expected. However, the issue appears to be more complex. There is evidence to indicate that the [3H]ouabain binding measurements represent the active state of the enzyme that depends on the ␣-␤ heterodimer (2, 23). It also appears that ouabain binding in itself induces internalization of the ␣-subunits of the cell (24, 38). These factors in conjunction with the possibility that [3H]ouabain binding may not be specific to the plasma membrane may explain why McKenna et al. (42) failed to observe increases in Na⫹-K⫹-ATPase content with different schedules of contractile activity in rat muscles. However, it does appear that, under some circumstances, increases in Vmax do parallel increases in Na⫹-K⫹-ATPase content (9, 31, 32, 56). Based on the increase in ␣ content observed with [3H]ouabain in our intermittent work protocol, it is to be expected that increases in one or more of the ␣-isoforms measured by Western blot analysis would occur. This is essentially what we have found. The ␣2-isoform was increased by ⬃26% following the first repetition of the exercise, whereas the ␣3-isoform was increased by 29% by R9. In contrast to the effects of exercise on ␣2- and ␣3-isoforms, ␤3-isoform content was reduced. No change was detected in either the ␣1-, ␤1-, or ␤2-isoform. These findings are significant in that they demonstrate that increases in ␣-isoform protein abundance can, in fact, occur in a relatively short period of time. These findings contradict previous studies in which increases in isoformspecific RNAs were observed with a single exercise session without changes in protein level (43, 44, 51). Collectively, our finding might suggest that in response to the severe challenge imposed by our intermittent protocol, two opposing effects are induced, one resulting in decreases in the catalytic activity of the Na⫹-K⫹-ATPase and one promoting either protein synthesis or reduced degradation as indicated by the increase in the ␣-isoforms. The reduction in enzyme activity has been postulated to promote the increase in the isoform mRNAs (51).

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The increase in the ␣2-subunit that we have observed with our 16-h model of intermittent exercise has also been observed as an early training response to prolonged exercise (17). However, in contrast to our early training study employing prolonged exercise, we did not find any change in the ␤1-isoform. The reduction in the ␤3-isoform was unexpected. It appears that, providing the exercise stimulus is appropriate, an upregulation in all isoforms, both ␣ and ␤, can occur, the expression of which appears to be regulated in both a muscle- and fiber-specific manner (25, 45). In summary, we have demonstrated that a 16-h protocol of heavy intermittent exercise can result in an increase in Na⫹K⫹-ATPase content as measured by the [3H]ouabain binding technique. Moreover, the increase in Na⫹-K⫹-ATPase content is accompanied by increases in ␣2 and ␣3 protein abundance. In contrast, the maximal catalytic activity of the enzyme, as measured by 3-O-MFP activity, is depressed with exercise, an effect that accumulates with the number of repetitions of the exercise. It must be concluded that increases in Na⫹-K⫹ATPase content, which is frequently reported as an early adaptation to increased contractile activity, need not be accompanied by increases in the functional capabilities of the enzyme. REFERENCES 1. Barr D, Green H, Fowles J. Factors affecting specific and non-specific activity in the measurement of Na⫹-K⫹-ATPase by 3-O-methylfluorescein phosphatase. Med Sci Sports Exerc 32, Suppl: S103, 2000. 2. Beguin P, Hasler U, Beggah A, Horisberger JD, Geering K. Membrane integration of Na,K-ATPase alpha-subunits and beta-subunit assembly. J Biol Chem 273: 24921–24931, 1998. 3. Bergstro¨m J, Hultman E. A study of the glycogen metabolism during exercise in man. Scand J Clin Lab Invest 19: 218 –228, 1967. 4. Bigland-Ritchie B, Garland SJ, Garland SJ, Walsh ML. Task-dependent factors in fatigue of human voluntary contractions. Adv Exp Med Biol 384: 361–380, 1995. 5. Blanco G, Mercer RW. Isozymes of the Na⫹-K⫹-ATPase: heterogeneity in structure, diversity in function. Am J Physiol Renal Physiol 275: F633–F650, 1998. 6. Chibalin AV, Kovalenko M, Ryder JW, Fe´raille E, WallbergHenriksson H, Zierath JR. Insulin and glucose-induced phosphorylation ␣-subunits in rat skeletal muscle. Endocrinology 142: 3474 –3482, 2001. 7. Clausen T. Na⫹-K⫹ pump regulation and skeletal muscle contractility. Physiol Rev 83: 1269 –1324, 2003. 8. DeLuca CJ. Control properties of motor units. J Exp Biol 115: 125–136, 1985. 9. Everts ME, Clausen T. Excitation-induced activation of the Na⫹-K⫹ pump in rat skeletal muscle. Am J Physiol Cell Physiol 266: C925–C934, 1994. 10. Ewart SH, Klip A. Hormonal regulation of the Na⫹-K⫹ ATPase: mechanism underlying sustained changes in pump activity. Am J Physiol Cell Physiol 269: C295–C311, 1995. 11. Fowles JR, Green HJ, Ouyang J. Na⫹-K⫹-ATPase in rat skeletal muscle: content, isoform, and activity characteristics. J Appl Physiol 96: 316 –326, 2004. 12. Fowles JR, Green HJ, Tupling R, O’Brien S, Roy BD. Human neuromuscular fatigue is associated with altered Na⫹-K⫹-ATPase activity following isometric exercise. J Appl Physiol 92: 1585–1593, 2002. 13. Fraser SF, Li JL, Carey MF, Wang YN, Sangkabutra T, Sostaric S, Selig SE, Kjeldsen K, McKenna MJ. Fatigue depresses maximal in vitro skeletal muscle Na⫹-K⫹-ATPase activity in untrained and trained individuals. J Appl Physiol 93: 1650 –1659, 2002. 14. Fraser SF, McKenna MJ. Measurement of Na⫹-K⫹-ATPase activity in human skeletal muscle. Anal Biochem 258: 63– 67, 1998. 15. Geering K. FXYD proteins: new regulators of Na-K-ATPase. Am J Physiol Renal Physiol 290: F241–F250, 2006. 16. Green HJ, Ball-Burnett M, Chin ER, Pette D. Time-dependent increases Na⫹-K⫹-ATPase content of low-frequency-stimulated rabbit muscle. FEBS Lett 310: 129 –131, 1992. AJP-Endocrinol Metab • VOL

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