INTRODUCTION

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IT IS WELL ESTABLISHED that extracellular K⁺ (K⁺_o) increases during high-intensity exercise, reaching values even higher than 10 mM in the interstitial space (12, 14) and ~8 mM in plasma ([K⁺_pl]) (22, 25). K⁺_o decreases within minutes after exercise is terminated. The increase in [K⁺_pl] is caused by K⁺ release from working muscles, probably through K⁺ channels during action potentials, inadequate reuptake via the Na⁺-K⁺ pump in between action potentials (6), and/or release of cellular K⁺ through ATP- and/or Ca²⁺-sensitive K⁺ channels (27).

An excessive increase in K⁺_o reduces the excitability of muscle cells (27). Therefore, if the reuptake into the muscle cells is inadequate, additional means of K⁺ removal are required to keep the K⁺_o concentration low to warrant a normal muscle cell membrane potential and excitability. One mechanism suggested is diffusion of the released K⁺ into the plasma to be transported to inactive tissues, where it is taken up, e.g., by nonworking muscle (15, 16, 18, 21, 25, 29). However, data suggesting an involvement of red blood cells in this process are conflicting: Böning et al. (3), Bodemann et al. (2), and Juel et al. (15) found no change in red blood cell K⁺ in venous blood during incremental, exhaustive bicycle exercise. Hespel et al. (11) reported a decrease in red blood cell K⁺ concentration ([K⁺_ery]) in cubital venous blood during bicycle ergometry at 80% of maximal oxygen uptake and argued for a stress release of K⁺ from red blood cells during exercise. McKelvie et al. (20) also show a slight decrease in femoral venous blood [K⁺_ery] but an increase in that in arterial blood (20) during repeated 30-s bouts of maximal bicycle ergometry. In a different study, McKelvie et al. (21) show an increase in [K⁺_ery] from the brachial artery and antecubital veins during supramaximal bicycle ergometry. Their explanation is that K⁺ is taken up by red blood cells via active transport systems, to be transported to inactive tissues (20, 21). Similar results were reported by Lindinger et al. (17). However, in none of the studies reporting an increased [K⁺_ery] has the activity of cation transport by red blood cells been measured. Because cation transport activity is very low in human red blood cells compared with other tissues (e.g., Refs. 4, 8, 13, 19, 24), their role as a sink for K⁺ released from working muscles appears unlikely.

The present study was undertaken to clarify the role of red blood cells in K⁺ removal from working muscle. In contrast to the studies mentioned above, only small-muscle groups were exercised in an exhaustive forearm exercise test. Blood was sampled to quantify plasma and red blood cell K⁺ content and parameters to determine water shifts and changes in red blood cell volume. To obtain an estimate of the capacity of red blood cells to take up K⁺, the unidirectional ⁸⁶Rb⁺ (K⁺) uptake was measured in vitro under conditions simulating those changes in plasma composition found during exercise, which are known to activate K⁺ uptake by red blood cells. The results of both tests indicate that red blood cells do not take up K⁺ released from muscle into the interstitium and plasma during exhaustive forearm exercise. Preliminary results have been reported previously (6).

	MATERIALS AND METHODS

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Forearm exercise. Ten male subjects not specifically trained in arm exercise participated in this study after giving informed consent. Dynamic, exhaustive handgrip exercise was performed with the arm fixed in a horizontal, stretched position. In control experiments, the workload (adjusted with springs of different elasticity) was determined that caused exhaustion within ~2 min at a contraction frequency of 30 contractions/min. The tests were then performed at this exercise intensity.

⁸⁶Rb⁺ uptake into red blood cells. Heparinized blood (1 U/ml) was drawn from antecubital veins of six healthy male volunteers. The red blood cells were washed twice with washing solution (150 mM NaCl, 2 mM HEPES, pH 7.3, at room temperature). Contaminating leukocytes and platelets were removed by aspiration of the buffy coat. Because leukocytes have a high transport activity that might be modified by various stimuli, it was important to ensure that the results of flux measurements were not affected by leukocyte and platelet contamination. Therefore, in control experiments, fluxes were also measured in red blood cells after removal of platelets together with the plasma after spinning of whole blood at 50 g at room temperature for 10 min. The remaining cells were filtered through a cellulose column to remove leukocytes (1). The comparison of both methods revealed that careful removal of the buffy coat was sufficient to avoid obscuring of results by leukocyte and platelet contamination of the red blood cell population.

Statistical evaluation. Results are presented as means ± SD. When the time course of changes of parameters during forearm exercise was evaluated, analysis of variance for repeated measures was performed. Scheffé's tests were used for post hoc testing. Control values and values after termination of exercise as well as ⁸⁶Rb⁺ fluxes under control and simulated exercise conditions were compared by using paired t-tests, respectively. The level of significance was P  0.05.

	RESULTS

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Forearm exercise. Forearm exercise was performed to avoid an increase in K⁺_o in the systemic circulation and to test whether the red blood cells collected in the venous outflow had taken up K⁺ as they passed the maximally working muscles and thus would participate in K⁺ removal from working muscles.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   K+ concentration in red blood cells () and plasma () before and during dynamic, exhaustive forearm exercise. Plasma K+ concentration was corrected for volume of plasma proteins. Red blood cell K+ concentration was calculated, as indicated in text, after correction for volume of Hb and red blood cell stroma. Values are means ± SD. cw, Cell water; pw, plasma water. * Significant differences between control and exercise, P  0.05.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Changes in red blood cell water () and K+ content () during dynamic, exhaustive forearm exercise. Cell water was calculated from mean cellular Hb concentration (MCHC) and specific volume of Hb and cell stroma (see MATERIALS AND METHODS). Ratio of red blood cell K+ to MCHC is given as a measurement of red blood cell K+ content that is independent of changes in cell volume. Values are means ± SD.

⁸⁶Rb⁺ uptake by red blood cells. Unidirectional Rb⁺ (K⁺) uptake measurements were performed under control conditions and after stimulation in a medium that was composed to simulate those exercise-related changes in plasma composition that cause an increase in unidirectional K⁺ uptake by activating ion-specific transport systems. Under these conditions, MCHC was 335 ± 7 and 356 ± 6 g/l, respectively. Figure 3 summarizes the results of the unidirectional ⁸⁶Rb⁺ (K⁺) uptake measurements. Figure 3 shows that, under control conditions, ~78% of the total ⁸⁶Rb⁺ uptake (2.1 mmol · l cells¹ · h¹) was mediated by the Na⁺-K⁺ pump and that Na⁺-K⁺-2Cl cotransport contributed ~15%. Under stimulated conditions, the total ⁸⁶Rb⁺ uptake was increased significantly by ~15%. Most of this increase can be attributed to a stimulation of Na⁺-K⁺-2Cl cotransport. Catecholamines activated Na⁺-K⁺ pump activity significantly but only under the stimulated conditions, whereas Na⁺-K⁺-2Cl cotransport and leaks were not affected by these hormones. The increase in the total unidirectional ⁸⁶Rb⁺ uptake achieved on stimulation with increased K⁺_o, osmolarity, and catecholamines was ~20% of the flux under control conditions, or 0.42 mmol · l cells¹ · h¹.

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Effects of simulated-exercise condition on unidirectional 86Rb+ (K+) uptake by red blood cells. 86Rb+ uptake into red blood cells was measured in red blood cells after removal of contaminating leukocytes and platelets in media composed to simulate control condition and changes in plasma composition, known to stimulate K+ uptake, as they occur during exercise (see MATERIALS AND METHODS). Na+-K+ pump was taken as portion of 86Rb+ uptake that was inhibited by 0.1 mM ouabain. Na+-K+-2Cl cotransport (CT) was bumetanide (10 µM)-sensitive flux component. Leaks indicate ouabain+bumetanide-insensitive 86Rb+ uptake. Values are means ± SD from 6 independent experiments with flux measurements in triplicate (mmol/l red blood cells of original cell volume). * P  0.05 between control and simulated-exercise conditions. # P  0.05 for effects of catecholamines.

	DISCUSSION

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The results show that, despite an exercise-induced increase in [K⁺_pl] and [K⁺_ery], the amount of K⁺ in red blood cells does not change significantly as they pass through the maximally working forearm muscle. In vitro experiments show that the unidirectional ⁸⁶Rb⁺ (K⁺) uptake by red blood cells is significantly increased when fluxes were measured in an artificial medium that simulates some changes in plasma that occur during exercise, changes known to activate transport. However, under both control and stimulated conditions, the magnitude of even the unidirectional Rb⁺ (K⁺) uptake is far too small to explain any significant increase in red blood cell K⁺ content within short time periods. This indicates that red blood cells are unable to take up significant amounts of K⁺ as they pass through the exercising forearm muscle and seem therefore not to be involved in the removal of K⁺ released from there for delivery to nonworking muscles.

K⁺ release from working small-muscle groups. In contrast to other experiments on the issue of K⁺ removal from working muscle (3, 11, 20), our in vivo experiments were performed on a small-muscle group. In this experimental setup, the amount of K⁺ released from the maximally exercising forearm muscles was high enough to cause a significant increase in the [K⁺_pl] of the venous outflow (Table 1); it was about as high as, or even higher than, values reported from bicycle ergometry (e.g., Refs. 3, 17, 22, 26, 29). In contrast to whole body exercise, in this setup the amount of released K⁺ was too small to increase the [K⁺] in the entire circulating blood. Therefore, the gradient for the diffusion of K⁺ from the interstitium to the blood in the exercising muscle remains high throughout the experiment. When large-muscle groups were exercised, the [K⁺_pl] is increased not only in the venous effluent of working muscles but also systemically. In this case, the diffusion of K⁺ from the interstitium into plasma might be impaired because of a decrease in the concentration gradient. It is important to distinguish between these two different experimental setups to answer the question of whether red blood cells play any significant role as a sink for K⁺ and as a K⁺-transport vehicle. If red blood cells are involved in the redistribution of K⁺, they should take up K⁺ where its concentration is highest, i.e., during the passage through the exercising muscle and in venous drainage.

Mechanisms of K⁺ removal. An elevated K⁺_o concentration disturbs the muscle cell membrane potential and might impair muscle contractility. Because the elevation of K⁺_o was discussed as one mechanism that causes muscular fatigue (25, 27), it is important to remove any K⁺ released from the interstitial space surrounding working muscles.

Cation fluxes in red blood cells. Unidirectional ⁸⁶Rb⁺ uptake measurements were performed to obtain a measure of the capacity for K⁺ uptake by red blood cells and to see whether this flux would be high enough to mediate the uptake of amounts of K⁺ from plasma large enough to contribute significantly to the removal of K⁺ from the interstitium of exercising muscle. These fluxes represent unidirectional K⁺ uptake but are no measure of the net K⁺ flux into or out of the red blood cells, the magnitude of which is determined by the net electrochemical driving force, the state of activation of the respective transport pathways, as well as by outward K⁺ leaks. To achieve an estimate of transport capacity, unidirectional ⁸⁶Rb⁺ (K⁺) uptake was measured in a medium with an increased [K⁺] and osmolality similar to what is found in plasma during exercise. Fluxes were deliberately measured at normal pH because acidosis is known to inhibit red blood cell Na⁺-K⁺-2Cl cotransport (8), which would reduce ⁸⁶Rb⁺ uptake. Catecholamines were added because they increase during exercise, although their effects on red blood cell ion transport are unclear. The results shown in Fig. 3 indicate an increase in unidirectional ⁸⁶Rb⁺ (K⁺) uptake from 2.1 mmol · l cells¹ · h¹ under control conditions to ~2.5 mmol · l cells¹ · h¹ under stimulated conditions. Even if these fluxes represented the net K⁺ uptake by red blood cells, this increased flux is too small to account for a millimolar increase in the K⁺ content of red blood cells even during prolonged exposure to this environment.

Red blood cell volume during exercise. Results on red blood cell volume changes during exercise are conflicting. On the basis of the increase in plasma osmolality during forearm exercise shown in Table 1, a decrease in cell volume in the range of ~10% might be expected. However, we found the cell water content to be practically unaltered (Fig. 2). The lack of shrinkage of the red blood cells in this environment indicates that cell volume perturbations must have been balanced. Because the K⁺ content of the cells did not change, the cell volume has to be kept stable by means other than K⁺ uptake. One possible explanation is the exercise-induced acidosis (Table 1) that causes red blood cells to swell because of shifts in Donnan equilibrium (3, 9). This indicates that, in this situation, there is actually no need for the red blood cells to regulate their cell volume by K⁺ uptake via specific cation-transport systems.

Discrepancies with other studies. The question arises, Why do different studies report different results on changes in red blood cell K⁺ during exercise that lead to different conclusions on the role of red blood cells in K⁺ removal from working muscle? Hespel et al. (11) reported a decrease in [K⁺_ery] during submaximal bicycle exercise and explained it by K⁺ leakage because they found no exercise-related change in the activity of cation-transport systems. To measure [K⁺_ery], they washed the cells with a medium containing 140 mM choline chloride. This medium is slightly hypotonic relative to plasma osmolality under control conditions but significantly hypotonic relative to exercise conditions, when plasma osmolality is elevated (see Table 1). Therefore, when washed with this medium, red blood cells can be expected to swell, which will dilute red blood cell K⁺. In this case, [K⁺_ery] (but not the cells' K⁺ content) will be lower than reported normal values (e.g., Ref. 25). On the contrary, McKelvie et al. (20, 21) reported increased red blood cell K⁺ during exercise and argued for a role of red blood cells in removing K⁺ released from the exercising muscle. In the work of McKelvie et al., [K⁺_ery] was calculated from [K⁺_pl] and [K⁺_ly] measured with ion-selective electrodes on the basis of Hct. McKelvie et al. appear not to have considered that the ratio of the volume of water in both compartments differs from Hct because of the specific volume of proteins in plasma and red blood cells. This explains the underestimation of the [K⁺_ery] per liter of cell water, which they found to be in the range of 110 to 120 mmol/l cell water (20, 21), whereas these values are typically ~130 mmol/l cell water (e.g., Ref. 31). Also, considering these specific volumes of red blood cell and plasma constituents, correcting Hct reduces the increase in [K⁺_ery] reported (21) during exercise to ~2 mmol/l cell water, which is within the experimental error of the measurements. The results of other studies cannot be reevaluated because Hb and plasma protein concentrations are not available. It appears, therefore, that the main reason for the diverging results reported on changes in red blood cell K⁺ comes from differences in the methods used to evaluate the red blood cell K⁺ content. The results of the studies noted above (11, 20, 21) agree very well with ours, when a cell volume-independent measurement of [K⁺_ery] (i.e., red blood cell K⁺ content) is introduced. This supports our notion of no increased red blood cell K⁺ content during exercise. Red blood cells seem, therefore, not to serve as transport vehicles to remove K⁺ released from the working muscle to inactive tissues. This transport appears to be mediated solely by plasma. They also do not use muscle K⁺ to restore their cell volume when they are exposed to increased plasma osmolality during exercise. For both actions, K⁺ uptake for removal and for cell volume regulation, even the unidirectional uptake rates for K⁺ into red blood cells, is far too slow (24).