INTRODUCTION

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DURING ACTIVITY, the rates of Na⁺ influx and K⁺ efflux in the contracting muscles are increased due to opening of the voltage-dependent channels associated with muscle action potentials. The Na⁺-K⁺ pump has a key role in the regulation of Na⁺ and K⁺ distribution. The finding that K⁺ is accumulated extracellularly and Na⁺ intracellularly during muscle activity suggests that the activation of the pump is insufficient or that the pump capacity is limited. The extracellular accumulation of K⁺ occurs despite the fact that the pumps are adapted to the increased demand during muscle activity (5). For example, the pumps are accelerated by elevated intracellular levels of Na⁺, by catecholamines, insulin, calcitonin gene-related peptide, and the pumps may also be stimulated by the membrane excitation during activity (1, 6, 7).

It has been found in rat muscle that the cell-surface Na⁺-K⁺ pump activity can be increased by a gain in the number of pump units either by insulin stimulation or by exercise (10, 18, 22). Whether such a mechanism is of functional importance also in humans has not been investigated. This is probably due to the large amount of tissue needed for most membrane purification procedures. Thus earlier studies in rat muscle used 5-6 g of muscle tissue (18). In addition, the use of a membrane fractionation technique (homogenization and the use of discontinuous sucrose gradients during high-speed centrifugation) in these rat studies has been questioned because of the poor sarcolemmal recovery (8).

The aim of the present study was to examine if a redistribution of the Na⁺-K⁺ pump subunits takes place during muscle activity in humans. For that purpose, production of giant sarcolemmal vesicles (12), which can be obtained from needle biopsy material, was used as a membrane purification method. This method is fundamentally different from other membrane purification techniques, as the membrane separation is not based on homogenization and high-speed centrifugation.

	METHODS

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Subjects and exercise procedure. Six healthy men with a mean age of 26 (range 24-28) yr and a mean weight of 84 (71-105) kg participated. The subjects were informed about any risk and discomfort associated with the experiment before giving their consent to participate. The study was approved by the local ethics committee.

The subjects performed one-legged knee-extensor exercise (kicking frequency 1 Hz) on an ergometer in the supine position (2). After a warming-up period (10 W) and a 10-min rest period, the subjects exercised until exhaustion. The mean work rate was 90 (range 74-98) W, and the mean time to exhaustion was 4.6 (2.8-7.2) min. Three needle biopsies (total >200 mg) were obtained from the vastus lateralis muscle of the inactive leg before exercise and from the active leg immediately after exercise.

Sample preparation. Giant sarcolemmal vesicles were produced as previously described (12) and adapted to human biopsy material (14, 19). Briefly, biopsy material was treated with collagenase and high KCl concentration, and the spontaneously formed vesicles were isolated by low speed (50 g) centrifugation in a density gradient. The vesicles contain some soluble proteins. Because of the low amount of protein obtained from each sample, the soluble proteins were not removed from the membrane proteins by lysing the vesicles and washing, a procedure that could be used for further membrane purification. After vesicle production, the remaining part (99%) of the muscle material was homogenized (Polytron 2100, 2 × 30 s, setting 6) in sucrose buffer (in mM: 250 sucrose, 40 NaCl, 30 HEPES, 2 EGTA, and 2 of the protease inhibitor phenylmethylsulfonyl fluoride) and spun down (100,000 g, 60 min). Pellet and vesicles were added to TS buffer (10 mM Tris base, 0.9% NaCl, pH 7.4) + 4% SDS and homogenized 2 × 5 s. The washing procedures used during vesicle production may have removed some soluble proteins, therefore, the muscle homogenate obtained in the present study is expected to contain more membrane proteins than a normal crude muscle homogenate. Because of this fact and the presence of soluble protein in the vesicles, the purification index (pumps per mg vesicular protein/pumps per mg homogenate protein) is expected to be lower than previously reported.

Immunoblotting. Vesicle and homogenate protein contents were determined with a BSA standard (DC protein assay, Bio-Rad). Samples (15 µg protein) were run on SDS-PAGE (8-18% gradient gel) and electroblotted to a polyvinylidene difluoride membrane (Millipore Immobilon-P). Membranes were blocked in TS buffer (added 5% BSA + 2.5% dry milk), washed, and incubated overnight at 4°C in primary antibodies diluted 1:1,000 in TS buffer containing 5% BSA. The membranes were then washed twice in TS buffer and incubated in secondary antibodies (horseradish peroxidase goat anti-rabbit immunoglobulins, DAKO) 1:1,000 in TS buffer containing 5% BSA. After washing (TS buffer + 0.5% Triton X-100) membranes were treated with enhanced chemiluminescent reagents (Amersham) and visualized on film. The quantification of the - and -proteins was performed by scanning the film and by analyzing band intensities (arbitrary units) with the SigmaGel software.

Antibodies. A polyclonal rabbit anti--antibody was raised against the recombinant large cytoplasmic loop (AA 330-767) of the Na⁺-K⁺-ATPase from pig and a polyclonal rabbit anti-₁-antibody was raised against the recombinant extracellular part (AA 58-302) of ₁ from pig. Both antigens were produced with a histidine tag in Escherichia coli. Antibodies were affinity purified before use in Western blots. The antibodies were kindly provided by Dr. P. Amstrup Pedersen, University of Copenhagen. The anti--antibodies do not discriminate between the ₁-, ₂-, and ₃-isoforms and therefore quantify total -subunit content. The anti--antibodies are specific for ₁. Specific ₁-, ₂-, and ₂- antibodies, all produced from the rat sequence, were obtained from Upstate Biotechnology, Lake Placid, NY.

Calculations and statistics. The samples obtained from biopsies taken before and after exercise were run on the same gel. The relative changes in isoform content were given as means ± SE and analyzed by Students t-test. A P value < 0.05 was considered statistically significant.

	RESULTS

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The total protein content of the muscle homogenates before and after exercise was 12,530 ± 2,310 and 9,033 ± 1,410 µg, respectively. The total (membrane + soluble) protein yield of the vesicles obtained before and after exercise was 108 ± 48 and 110 ± 20 µg, respectively.

The total - and total -subunit contents were more than 10 times higher in sarcolemmal vesicles compared with muscle homogenates (Fig. 1). The anti ₁-, anti ₂-, and unspecific anti--antibodies all labeled a 100- to 110-kDa protein. In addition, by using a long-lasting film exposure, both the unspecific -antibodies and the specific ₁-antibodies labeled a weak 65-kDa band, which was not included in the density measurements. The anti-₁-antibody labeled a wide band at 50-55 kDa and in some preparations a narrow band at 65 kDa. Only the 55-kDa band was included in the density measurements. The specific anti-₂-antibodies failed to recognize any protein band in human muscle, but clearly recognized a 55-kDa band in rat muscle homogenates (Fig. 1).

View larger version (55K): [in this window] [in a new window]   Fig. 1.   Na+-K+ pump - and -subunits visualized by immunoblotting. Top: comparison between total - and total -subunit content in vesicles and muscle homogenates. Same amount (15 µg) of vesicular and homogenate protein was run on a gel. Arrows indicate approximate molecular mass obtained with molecular mass standards run on same gel. Bottom: examples of immunoblot labeling obtained with unspecific -, specific 1- and 2-, specific 1-, and specific 2-antibodies applied to human sarcolemmal vesicles. Right: rat sarcolemmal vesicles labeled with 2-antibodies.

The vesicular membrane contents of the ₂-, unspecific -, and ₁-subunits were 70 ± 29 (n = 5), 35 ± 10 (n = 5), and 26 ± 5% (n = 6), respectively, higher (P < 0.05) after exercise (Fig. 2). The ₁-subunit content in vesicles obtained after exercise was not significantly different from pre-exercise. The ratio of vesicular to crude muscle homogenate content of the ₂- and total -subunit increased (P < 0.05) by 67 ± 33 and 23 ± 6%, respectively, during exercise. In addition, exercise caused a trend (P = 0.06) toward an increase by 40 ± 14% in the vesicular to crude homogenate content of the ₁-subunit. The effect did not reach statistical significance, because one subject had a lower value after exercise. The homogenate ₁-, ₂-, total -, and ₁-subunit content after exercise was 102 ± 4 (n = 5), 102 ± 5 (n = 5), 96 ± 9 (n = 5), and 99 ± 12% (n = 6), respectively, of the preexercise levels. Thus exercise did not affect the total amount of subunit proteins in the muscle homogenates.

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Exercise-induced changes in sarcolemmal content of Na+-K+ pump subunit. Left: exercise-induced changes (%) in density of total  (tot)-, 1-, 2-, and 1-subunits in sarcolemmal vesicles obtained postexercise compared with vesicles obtained preexercise. Right: exercise-induced changes in sarcolemmal Na+-K+-pump density calculated as ratio of vesicles to homogenate subunit content in postexercise samples relative to ratio in preexercise samples. Values are means ± SE. * Significantly (P < 0.05) different from control; # P = 0.06.

	DISCUSSION

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The present study demonstrated that the muscle membrane Na⁺-K⁺ pump content is increased by a short, high intensity bout of exercise. Thus both total - and total -subunit content was elevated by ~30% at the end of exercise. Because the functional unit of the Na⁺-K⁺ pump is the /-subunit heterodimer, it is likely that the increase in pump unit density has a functional role. Therefore, the present results suggest that part of the increase in pump activity in response to exercise is mediated by translocation of - and -subunits to the sarcolemmal membrane.

The exercise-induced increase in -subunits was restricted to the ₂-isoform. This is in agreement with the finding that the ₁-isoform in human skeletal muscle is mainly located in sarcolemma, whereas ₂ is found in sarcolemma as well as in internal membranes (11). Thus ₂ likely serves as a pool of subunits available for translocation. Of the -subunits, only the ₁-isoform was identified, which is consistent with previous findings that ₁ is the only -isoform in human muscle (11).

The present study was only possible because giant sarcolemmal vesicles can be produced from the limited amount of tissue available from needle biopsy material. It is a prerequisite for the conclusions that the vesicles represent sarcolemma. The vesicular membranes from rat and human muscle have been characterized in a number of studies. In the light microscope it can be seen that the vesicles are budding out from single fibers (21). Ouabain binding experiments (4) and patch-clamp analysis of rectifying ion channels (20) demonstrated that the orientation of the vesicular membrane is right side out. The plasma membrane enzyme K⁺-stimulated phosphatase is enriched 16- to 37-fold in vesicles compared with crude homogenate. Vesicular membranes have a low content of the mitochondrial enzymes succinic dehydrogenase and cytochrome-c oxidase and a low or absent activity of the sarcoplasmic reticulum marker Ca²⁺-ATPase (20, 21, 25). These findings all suggest that sarcolemma is the main constituent of the vesicles. The T-tubule markers dihydropyridine receptors and nitrendipine binding sites are not present in the vesicles (21, 24). The vesicular contents of the vesicle-associated membrane protein 2 and GLUT-5 proteins are high, whereas GLUT-1 is not detectable in vesicles, although it is present in muscle homogenates mainly due to the presence of nerve cells and vascular tissue (9, 15, 16, 21). Furthermore, vesicles have been used to demonstrate GLUT-4 translocation and there is a positive correlation between muscle glucose uptake and density of GLUT-4 proteins in the vesicles (17). On the basis of these findings, it can be concluded that the giant vesicles represent sarcolemma.

The present study does not allow a final discrimination between the possibility of recruitment of pre-existing subunits or synthesis of new proteins. However, the finding that there was no exercise-induced change in subunit content in muscle homogenates speaks against the possibility of de novo protein synthesis. Furthermore, the signal involved in the translocation of pump subunits cannot be determined from the present study. It is known that a 30-min exposure to insulin increases the plasma membrane content of pump subunits in rat skeletal muscle (10, 18). The insulin level was not measured in the present study, but during intense one-legged exercise the insulin concentration was observed to be unchanged (3). Therefore, insulin can be excluded as mediator of the exercise-induced translocation. Other mechanisms, such as catecholamine stimulation, are possible, as these hormones are known to stimulate pump activity (6). However, the changes in concentration are moderate during exhaustive one-legged knee-extensor exercise (2).

Perspectives