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Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada
Submitted 30 November 2007 ; accepted in final form 25 January 2008
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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O2 peak 44.8 ± 2.0 ml·kg–1·min–1, mean ± SE) using a 6-day protocol (3 days of exercise plus 3 days of recovery). Tissue samples were extracted prior to (Pre) and following exercise (Post) on day 1 (E1) and day 3 (E3) and on each day of recovery (R1, R2, R3) and analyzed for changes in maximal protein (βmax) (vanadate-facilitated [3H]ouabain binding), 
- and β-isoform concentration (quantitative immunoblotting) and maximal Na+-K+-ATPase activity (Vmax) (3-O-methylfluorescein K+-stimulated phosphatase assay). For βmax (pmol/g wet wt), an increase (P < 0.05) of 11.8% was observed at R1 compared with E1-Pre (340 ± 14 vs 304 ± 17). For the -isoforms 1, 2, and 3, increases (P < 0.05) of 46, 42, and 31% were observed at R1, respectively. For the β-isoform, β1 and β2 increased (P < 0.05) by 19 and 28% at R1, whereas β3 increased (P < 0.05) by 18% at R2. With the exception of 2 and 3, the increases in the isoforms persisted at R3. Exercise resulted in an average decrease (P < 0.05) in Vmax by 14.3%. No differences were observed in Vmax at E1 - Pre and E3 - Pre or between R1, R2, and R3. It is concluded that 3 days of prolonged exercise is a powerful stimulus for the rapid upregulation of the Na+-K+-ATPase subunit isoforms. Contrary to our hypothesis, the increase in subunit expression is not accompanied by increases in the maximal catalytic activity. contractile activity; ion transport; Na+-K+ pump; adaptation
The Na-K pump in skeletal muscle is a heterodimeric protein consisting of one -subunit and one β-subunit. There are three - (1, 2, 3) and three β-isoforms (β1, β2, β3) in human muscle (33). These isoforms appear distributed in a fiber type-specific manner with regard to both the type of isoform and the - and β-subunit complex (10, 29). Each isoform is encoded by a different gene (32, 33). The -subunit is recognized as the catalytic subunit, while the β-subunit, although necessary for catalytic activity, functions to anchor and stabilize the heterodimer (4).
Given the strain imposed on the Na-K pump in protecting transmembrane electrochemical gradients, particularly during prolonged exercise where pump inactivation occurs (11, 12, 32, 41), ostensibly as a result of reactive oxygen species (ROS)-mediated damage (30), it would be expected that adaptations would occur in Na-K pump protein expression to repeated exposure to the stress of exercise. Such appears to be the case. Long-term training studies in both humans and rats have reported increases in the Na-K pump (16, 26, 31) and isoform concentration (34). Moreover, it appears that remodeling can occur soon after the onset of regular contractile activity, since increases in both muscle Na+-K+-ATPase content (18, 19, 38) and the concentration of selected isoforms (18) within the first few days of both prolonged submaximal exercise (18) and chronic low-frequency stimulation (17, 23). Curiously, the expected increase in maximal activity (Vmax) appears to be a more delayed event, not evident until after the increase in enzyme and isoform abundance has occurred (18).
There is also evidence that the increase in selected isoforms can occur within the first day of exposure to the exercise protocol. Sandiford et al. (42) have reported increases in -content in homogenates during 90 min of induced activity in the rat soleus. More recently, we have shown (20) that 16 sessions of heavy exercise in humans performed once per hour resulted in increases in both the 2- and 3-isoforms and a decrease in the β3-isoform. During the training protocol Vmax was observed to decline, a finding that could be attributed to failure of the enzyme to recover from the inhibiting effects of the previous exercise bout. More recent studies report the potency of a single exercise session for increasing transcription potential as evidenced by the increases that occur soon after the exercise in a variety of - and β-mRNA isoform transcripts (32, 33, 35, 39). Prolonged submaximal cycling exercise, as an example, resulted in increases in the respective mRNAs for 1, 3, and β2 but not the mRNAs for 2, β1, and β3, all peaking at variable time points within the first 24 h following exercise (32). Despite the increase in the mRNAs, no changes were reported in their isoform abundance, although increases in 3 and β1 were close to significance. Moreover, Vmax did not increase following exercise or during the recovery period. Given the apparent importance of transcription in regulating isoform levels, the failure to find increases in isoform concentration was unexpected. It is possible that additional sessions of prolonged exercise are needed to observe the effect of increased transcription. Moreover, the failure to find increases in Vmax with increases in protein and isoform concentration (18, 32) may be complicated by the inactivation of the enzyme that occurs with exercise.
The purpose of this study was to investigate the effects of 3 consecutive days of prolonged submaximal cycling exercise followed by 3 days of recovery on Na+-K+-ATPase protein and isoform expression and maximal catalytic activity (Vmax) in working skeletal muscle. We have hypothesized that changes in protein and isoforms would occur with 3 days of exercise. Moreover, the effects of the changes in Na+-K+-ATPase expression on maximal enzyme activity would be observed only during the recovery days following exercise.
This study was part of a much larger study in which we investigated a wide range of cellular adaptations (9, 15) in an attempt to describe the integrated nature of the responses and functional significance (45).
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Twelve male (n = 6) and female (n = 6) recreationally active but untrained volunteers with a mean age and body mass of 19.2 ± 0.27 yr and 71.1 ± 3.4 kg, respectively, participated in the study. Peak aerobic power (O2 peak) for the group was 44.8 ± 2.0 ml·kg–1·min–1. Prior to subject recruitment, the study was approved by the Office of Research Ethics at the University of Waterloo. As a condition of approval, all potential participants were provided details of all experimental procedures and associated risks prior to obtaining written consent.
Experimental Design
Each participant was required to engage in 3 consecutive days of exercise followed by 3 consecutive days of recovery during which no exercise was permitted. The 3 consecutive days of exercise was the conditioning dose employed to investigate the malleability of the Na+-K+-ATPase. The 3 consecutive days of recovery was employed to determine whether the Na+-K+-ATPase properties exhibited a different time course in expression and/or regression following withdrawal of the exercise stimulus. For each participant, the same amount of work was performed on each day at an intensity of 
60% O2 peak. The duration of exercise for each subject was for a maximum of 2 h on each day. Where 2 h of continuous cycling could not be completed on exercise day 1 (E1), the exercise time was recorded and used for exercise on E2 and E3. Testing was performed in the morning at approximately the same time for each individual. No caffeine or alcohol was permitted for at least 24 h prior to the start of the exercise sessions or throughout the experimental period. A similar requirement was made for exercise outside that required by the experiment.
A total of seven tissue samples were extracted from the vastus lateralis muscle from separate sites, randomized between legs, using previously established procedures commonly employed in our laboratory. Tissue was extracted both prior to (Pre) and following exercise (Post) on E1 and E3 and during the first (R1), second (R2), and third (R3) day of recovery. The tissue was frozen and stored (–80°C) pending analyses. Because of concerns with the number of biopsies in such a short period, the initial exercise session (E1) was performed 3–4 wk prior to the beginning of the experiment. During this session, tissue samples were extracted. No tissue samples were extracted during the first day of the 3-consecutive-day protocol. During the period between the initial test session and the start of the 3-day protocol, volunteers were requested to maintain their normal diet and to refrain from regular exercise. An assumption with our sampling schedule is that no changes occurred in the resting cellular properties between the initial exercise session and the start of the actual experiment. This appears to be the case, since we have found no differences in a wide range of cellular properties when tissue sampling is separated by several weeks (8, 41).
Analytic Procedures: 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 β-isoforms (18, 20). Briefly, tissue (30 mg) from frozen samples stored at –80°C was homogenized (5% wt/vol) at 0–4°C for 2 x 20 s at 25,000 rpm (Polytron) in a buffer containing 250 mM sucrose, 2 mM EDTA, 10 mM Tris (pH 7.40), and a commercially prepared combination of protease inhibitors (Roche Diagnostics, Indianapolis, IN).
Na+-K+-ATPase content.
Vanadate-facilitated [3H]ouabain binding was used to determine total Na+-K+-ATPase content based on maximal binding characteristics (βmax), as originally described (36). Two tissue samples weighing between 2 and 8 µg were incubated for 10 min periods in a Tris-sucrose buffer containing 10 mM Tris·HCl, 3 mM Mg SO4, 1 mM Tris-vanadate, and 250 mM sucrose with [3H]ouabain (1.8 µCi/ml) and unlabeled ouabain (l µM final concentration) for 2 x 60 min at 37°C. After the samples were washed (4 x 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 (36). Additional details are provided in our earlier publications (18, 42). 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 -subunits, supposedly while in a functional state (6). Samples obtained prior to exercise on E1 and E3 and at R1, R2, and R3 were assessed during the same analytic session.
Na+-K+-ATPase activity.
The K+-stimulated 3-O-methylfluorescein phosphatase assay (3-O-MFPase) for the measurement of Vmax was employed using previously published procedures (24, 37) but with modifications utilizing substrate concentrations designed to produce maximal activities (13, 20) while lowering nonspecific activity (1). To permeabilize the membrane, the homogenate (25 µl) was freeze-thawed for four cycles and diluted 1:4 in cold homogenate buffer. We have previously shown that this protocol results in optimization of enzyme activity, supposedly by making all pumps accessible for measurement (1). Following dilution, the homogenate was incubated at 37°C for 4 min in a medium containing 5 mM MgCl2, 1.25 mM EDTA, 1.25 mM EGTA, 5 mM NaN3, and 100 mM Tris (pH 7.40). For determination of K+-stimulated activity, 10 mM KC1 and 160 µM 3-O-MFP were added, and the activity was determined as the difference in the slope before and after addition of KC1 using fluorescence spectroscopy (10, 13). 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 (43). In our laboratory, we have calculated the intra-assay variability for Vmax to be 8.5%. All samples for a given subject, which included tissue obtained both pre- and postexercise at E1 and E3 and at R1, R2, and R3 were measured for Vmax during the same analytic session.
Isoform Determination
To resolve Na+-K+-ATPase subunit isoform protein content, Western immunoblotting performed using electrophoresis on 7.5% SDS-polyacrylamide gels (Mini-Protean II, Bio-Rad), essentially as described by Laemmli (27), was used. Detection of isoforms was based on duplicate measurements using two different aliquots from each sample and two different gels. For the analyses of -subunit isoforms (1, 2, 3) and β-subunit isoforms (β1, β2, β3) the amount of protein employed was 40 and 50 µg, respectively. On a given analysis day, all samples for a given participant and for a given isoform were assessed. A biotinylated ladder was used as a molecular weight standard (Cell Signaling Technology, Beverly, MA). For the β-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 (18).
After SDS-PAGE, gels were electrophoretically transferred to polyvinylidene difluoride membrane (Bio-Rad) by placing the gel in a transfer buffer and applying high voltage (20 V) for 45 min (Trans-Blot 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 incubation with primary antibodies. The primary antibodies included 1, 2, and β1 (Upstate Biotechnology, Lake Placid, NY) 3 (Affinity Bioreagents, Golden, CO), and β2 and β3 (BD Biosciences, Mississauga, ON, Canada). Immunoblotting was performed overnight at 4°C with the antibodies in 5% milk (1, 3, 1:1,000; 2, 1:500; and β1, β2, and β3, 1:500). Bound antibodies were detected with goat anti-rabbit IgG1 (2) and goat anti-mouse IgG1 (1, 3, β1, β2, β3).
An enhanced chemiluminescence procedure was used to detect antibody content (Amersham, Buckinghamshire, UK). Blots were analyzed by use of a Chemi Genius2 model bioimaging system (SyngGene, Frederick, MD) with SynGene software version 1.0. Additional analytic details can be found in previous publications from our laboratory (10, 18, 42).
For a specific isoform, samples at E2 (Pre), E3 (Pre), and R1, R2, and R3 for a particular individual were applied to a specific gel, and the gel was run on duplicate. Each gel also contained a known protein content (1–2 µg) 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 E1, E3, R1, R2, and R3 using E1 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 prior to experimental analysis. For all assays, protein content was determined in duplicate according to Lowry as modified by Schacterle and Pollock (43), using bovine serum albumin as a standard.
Citrate Synthase Activity
Citrate synthase (CS) used as a measure of oxidative potential was measured in tissue that had been frozen and stored at –80°C. A sample of the frozen tissue was hand homogenized (0–4°C) in a phosphate buffer (pH 7.4) containing 5 mM mercaptoethanol, 0.5 mM EDTA, and 0.2% BSA and then diluted in 20 mM imidazole buffer with 0.2% BSA. Enzyme measurements were performed at 24–25°C according to the procedures of Henriksson et al. (22) as modified in our laboratory (28). On a given analytic day, all samples for a given individual participant were assessed in duplicate. Protein was determined with the Lowry techniques as modified by Schacterle and Pollack (43).
Statistical Procedures
The data were analyzed using a one-way analysis of variance for repeated measures. For the measurement of the Na+-K+-ATPase and isoform content and CS activity, rest samples for E1 and E3 and each of the three recovery days were used. For Na+-K+-ATPase activity, both pre- and postexercise samples on E1 and E3 and the three recovery day samples were grouped for analyses. Where significant differences were found, Newman-Keuls procedures were used to locate differences between specific means. Significance was set at the 0.05 level. Where a difference between means is indicated in the text, significance is implied.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Total Na+-K+-ATPase concentration, as measured by the [3H]ouabain binding procedure, increased 12% at R1 following 3 consecutive days of prolonged exercise (Fig. 1). No change in concentration was observed following the first 2 days of exercise. By R3, the third day of inactivity, the exercise-induced increases were reversed such that there were no differences with E1, the point at which the exercise began.
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- and β-isoforms were observed to increase with our exercise protocol (Figs. 2 and 3). In the case of the -isoforms, increases of 46, 42, and 31% were observed at R1 for 1, 2, and 3, respectively, compared with preexercise at E1. For the β-isoforms, β1 and β2 increased by 19 and 28%, respectively, at R1, but for β3 the increase was more delayed, not occurring until R2, the second day of recovery. With exception of 1, the increase in the -isoforms was rapidly reversed, with no differences observed between R3 and E1 for either 2 and 3. For 1, the increase observed at R1 persisted at R3. Unlike the -isoforms, the increases in all three β-isoforms persisted by the third day of recovery.
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CS, used as representative enzyme for oxidative potential, was not altered either during the days of consecutive exercise or during the following 3 days of recovery (Fig. 5).
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Since both males and females were included in the study, we also examined for differences in the response to the exercise and recovery intervention. No significant differences (P < 0.05) were found between the sexes for any of the Na+-K+-ATPase properties investigated.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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- and β-subunits adapt to the strain imposed by prolonged submaximal exercise. Increases in protein levels for the three - and three β-isoforms were observed with just 3 consecutive days of exercise and, with the exception of β3, the increases were fully manifested within the first 24 h of recovery. Although, there is a suggestion that β3 responded in a similar fashion, the increase was not significant (P > 0.05) until 48 h of recovery. As expected, we found that exercise reduced maximal Na+-K+-ATPase activity, an effect that was abolished with 24 h of recovery. Since no differences were observed between maximal Na+-K+-ATPase activity between preexercise at E1 and any of the recovery days, it would appear that a dissociation occurs between increases in protein levels, expressed in the upregulation in the - and β-isoforms, and maximal catalytic activity. In this regard, our results failed to support our hypothesis, namely that during recovery increases in maximal Na+-K+-ATPase activity would accompany the increase in subunit protein expression.
The increase in Na+-K+-ATPase protein that was observed following the 3 days of exercise, as assessed by the vanadate-facilitated [3H]ouabain binding procedure, was expected on the basis of several short-term training studies published from our laboratory (5, 18, 19) and elsewhere (23, 38). It is also evident that the increase in Na+-K+-ATPase protein was accomplished by increases in all of the - and β-isoforms. In the case of the -isoforms, the full magnitude of the increase was observed by the first day of recovery, some 24 h after the third consecutive day of prolonged exercise. Increases in one or more of the -isoforms was expected at this time point, since the [3H]ouabain binding technique used to measure Na+-K+ pump concentration is based on the binding of the vanadate to the -isoform (6). Vanadate is believed to bind to the -isoform on the intracellular side at the same aspartyl residue as ATP and, as such, forms the active charged intermediate complex (2). Since the /β-heterodimer is needed for activation, the vanadate-facilitated ouabain binding technique is believed to provide a measure of total Na+-K+-ATPase protein concentration (21). In this regard, it is important to emphasize that all of our measurements of Na+-K+ pump concentration were performed on resting tissue that had not been activated by exercise. The fact that we have also found increases in all three β-isoforms during the recovery from our short-term exercise program, may also contribute to the increases in Na+-K+ pump levels that were observed.
Our study also confirms the results of several recent studies and, in particular, the study of Murphy et al. (32), who reported that a single session of prolonged cycle exercise, although resulting in increases in the mRNA levels of several isoforms (1, 3, and β2), did not significantly increase Na+-K+-ATPase isoform protein levels when measured periodically over 24 h of recovery. On the basis of the measurements that we made prior to exercise at E3, two sessions of exercise also appears insufficient to promote the upregulation in - and β-isoform distribution.
Our results also indicate that elevations in all - and β-isoforms can occur if the exercise sessions are repeated over 3 days. This was a surprising observation given the results of a previous study using a similar exercise stimulus that demonstrated that the mRNA of several isoforms did not change with 1 day of exercise (32). Since increases in protein levels represent the net difference between synthesis and degradation, it is not clear what dominated to promote the change or lack of change in the isoforms that we observed with our exercise protocol.
Early increases in at least some of the - and β-isoforms was to be expected given the publication of several recent studies demonstrating increases in selected mRNA isoforms in human vastus lateralis with just a single exercise session (32, 33). It is interesting that collectively these studies show increases in the mRNAs of all three - and all three β-isoforms but not in the same study. Given the wide variability in the type of exercise protocol employed, it is conceivable that the type of mRNA that is elevated depends on the nature of the strain imposed on transmembrane Na+ and K+ gradients in the working muscle.
Considering the rapidity with which increases in mRNA isoform can be induced with exercise, the question arises whether protein levels can also be altered with different exercise regimes. We addressed this question using a 16-h model of intermittent exercise where participants performed heavy exercise for 6 min followed by a 54-min rest (20). On the basis of the results of tissue samples extracted prior to exercise at repetitions 2, 9, and 16, we reported elevations in 2 (at repetition 2), and 3 (at repetition 9). For the β-isoforms, there was a strong trend for β2 to increase at repetition 2, whereas β3 decreased at repetition 16. This work clearly demonstrates that the protein levels of selected isoforms can increase within the first hours of exposure to the exercise, the nature of the isoforms expressed probably dependent on the nature of the exercise stress. Tsakirides et al. (47) were the first to demonstrate the precocious nature of the mRNA, reporting that increases can occur with just 60 min of treadmill exercise in rats. We have also reported increases in 1 protein levels in rat soleus muscle subjected to 90 min of electrical stimulation (42).
In an earlier study, we found increases in Na+-K+-ATPase concentration at 3 days of a 6-day training protocol using the same vanadate-facilitated [3H]ouabain binding technique and a 6-day period of training using an exercise protocol similar to that used in the present study (18). We also reported that increases in 2 and possibly 1 (P = 0.06) occurred by 3 days. The β1-isoform was not observed to increase until 6 days of training. No measurements were made of the 3-, β2-, and β3-isoforms. A curious finding was that increases in Vmax were not observed until the sixth day of training. We speculated that the delayed increase might have occurred in conjunction with the increase the β1-isoform or that the adaptation in catalytic activity might be complicated by the inactivation that occurs to the enzyme as an acute effect of the exercise (18). The present study was, in part, an attempt to address this issue. We rationalized that, by providing 3 days of recovery, the adaptive effects would be evident; such was not the case. Although we report the expected reductions in Vmax with exercise, the restoration of Vmax was complete by 24 h after the last exercise session. Although there is a trend for Vmax to increase on day 2 and day 3 of recovery, the value at day 3 of recovery was not different from that prior to exercise on day 1. This represents a clear dissociation between the increases in - and β-protein levels and the catalytic activity of the enzyme.
There are several possibilities to explain our failure to find increases in Vmax with our exercise schedule. It is possible that increases in the appropriate combination of - and β-isoforms, necessary to increase Vmax, did not occur. However, this appears remote since all isoforms for both and β were observed to be elevated particularly on the second day of recovery. Moreover, the increases that we observed were much more emphasized than in our earlier study, where increases in Vmax occurred by the sixth day of training. Given that our assessment of the subunit isoforms was performed on whole homogenates, it is not clear whether the increases were localized to the plasma membrane or occurred intracellularly, the distribution of which could affect Vmax (42). Another probable reason has to be with the expression of one or more accessory proteins necessary to realize the full catalytic capacity of the enzyme. A probable candidate is PLM, a small protein distributed in the plasmalemma that belongs to an FXYD family of proteins involved in ion transport (7, 14). This protein is found in heart and skeletal muscle and is known to coprecipitate with the -subunit (7) and to alter Vmax (7). There is also evidence, at least in heart muscle, that increases in PLM phosphorylation can result in increases in Vmax (48). Increases in PLM expression in skeletal muscle have been reported to occur with exercise training in rats (40), whereas incubation of tissue homogenates with an anti-PLM antibody decreased Na+-K+-ATPase activity by 50% (40). Collectively, these observations suggest that the increased expression of PLM and/or increase in PLM phosphorylation may be necessary for the effect of increases in the protein levels of the - and β-isoforms on catalytic activity to be realized. Future studies are needed to examine the time course of changes in PLM in association with the changes in - and β-isoforms and to determine whether the phosphorylation level of PLM has been altered. Protein kinase A and protein kinase C are known to lead to phosphorylation of SER68 and SER68/63, respectively (14).
It should be noted that, although Vmax was not increased by our training and recovery protocol, it is possible that the substrate affinity of the enzyme could have been altered. Unfortunately, measurements of Km defined as the substrate concentration necessary to elicit 50% Vmax, was not possible in humans due to assay variability. However, we have addressed this issue in a related study using rodents. In this study, we found that 5 days of training, although increasing Na+-K+-ATPase content as measured by the [3H]ouabain binding procedure, did not result in an increase in either Vmax or the Km (K+), regardless of the fiber composition of the locomotor muscle studied (D. Barr, H. J. Green, and J. Ouyang unpublished observations).
Perspectives and Significance
In summary, it is instructive to consider the adaptations that we observed in the Na+-K+-ATPase to the other properties examined with our short-term training model. We observed that the inactivation that occurs in Vmax of the Na+-K+-ATPase with exercise is quickly followed by increases in the abundance of all - and β-isoforms of the Na+-K+-ATPase in skeletal muscle soon after the onset of regular prolonged exercise. However, the increase in subunit concentration is not accompanied by increases in maximal catalytic activity of the enzyme during the days of either training or recovery. We also report that our training protocol failed to alter the loss of membrane excitiability that occurs with the exercise (45), which might be expected given the failure to alter the Vmax of the enzyme. Exercise also results in an inactivation of the other cation pump examined, namely the sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) (9). As with the Na+-K+-ATPase, we have found an upregulation in the SERCA1a but not the SERCA2a isoform but not the Vmax of the enzyme (9). We have also found that our short-term training protocol failed to attenuate the reduction in the phosphorylation potential that occurs with exercise (unpublished observations), an adaptation that has been clearly documented with training. What was conspicuous was the rapid increase that occurred in the glucose (GLUT4) and lactate [monocarboxylate transporter (MCT)1 and MCT4] transporters (unpublished observations), which would be expected to promote a greater entry of the substrate glucose and removal of the hydrogen and lactate byproducts from the working muscle cell. The increase in the transporters was not accompanied by increases in oxidative potential, as assessed by the maximal activities of several mitochondrial enzymes (unpublished observations). Collectively, these results suggest that, in response to prolonged submaximal exercise, the management of substrate and byproduct appears to be a priority.
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FOOTNOTES |
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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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REFERENCES |
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-subunits and β-subunit assembly. J Biol Chem 273: 24921–24931, 1998.This article has been cited by other articles:
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  H. J. Green, E. Bombardier, T. A. Duhamel, R. D. Stewart, A. R. Tupling, and J. Ouyang Metabolic, enzymatic, and transporter responses in human muscle during three consecutive days of exercise and recovery Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2008; 295(4): R1238 - R1250. [Abstract] [Full Text] [PDF]
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Significantly different (P < 0.05) from E3; 
Significantly different (P < 0.05) from R1; 
Significantly different (P < 0.05) from R2.
