INTRODUCTION

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THERE IS CONTROVERSY regarding the ability of red blood cells (RBCs) to regulate plasma K⁺ concentration ([K⁺]) during exercise in humans (17, 22, 24). During high-intensity leg exercise in humans, K⁺ release from contracting muscles may increase femoral venous plasma [K⁺] to 9 mmol/l within 60 s (28). RBCs provide a mass of ~3 kg that, similar to other noncontracting tissues, dynamically exchanges gases and ions with plasma (6, 14, 15). At very-high-intensity exercise [at power outputs 2-fold or more greater than that achieved at maximal O₂ consumption (O₂)], RBCs increase their [K⁺] and K⁺ content (21, 26, 27) and decrease their Na⁺ concentration ([Na⁺]) and content (2). These observations have led to the idea that RBCs may regulate plasma [K⁺] by increasing the net inward transport of K⁺ into the cytosol (J_netK) (22, 26). It is hypothesized that, with very-high-intensity exercise, stimulation of RBC K⁺ transport occurs in response to the rapidity and magnitude of changes in plasma composition, particularly increased osmolality, [K⁺], epinephrine concentration ([epinephrine]), and lactic acid concentration. It is further hypothesized that these changes result in stimulation of K⁺ transport mechanisms involved in RBC volume and pH regulation.

The human RBC transports K⁺ in both directions across the plasma membrane. Primary active transport by the Na⁺-K⁺ pump (33) and secondary active transport by the Na⁺-K⁺-2Cl cotransporter (19, 20, 34) are used to move solute (and water) into RBCs (14). A K⁺-Cl cotransporter primarily moves solute (with water) out of the cell, to decrease cell volume, in response to cell swelling. Residual K⁺ flux also occurs through various cation channels (11, 32). The main function of the Na⁺-K⁺ pump appears to be the maintenance of favorable electrochemical gradients for the operation of the cotransporters involved in cell volume and pH regulation (14).

In vitro studies of washed RBCs incubated for prolonged (up to 1-h) periods show that human RBCs have a low capacity to transport K⁺ and that K⁺ transport systems are slow to respond to extracellular changes that should stimulate K⁺ transport (14, 24). Transport is typically studied after RBCs are washed and incubated in protein-free saline solutions, often at temperatures <37°C, conditions known to alter RBC K⁺ transport (29, 36). Hespel and co-workers (13) and Maassen and co-workers (24) failed to observe an increase in RBC unidirectional influx of K⁺ (J_inK) after exercise in humans; however, RBCs obtained at rest and after exercise were incubated in identical (13) and artificial plasma (saline) solutions (13, 24).

In their experimental design, Maassen et al. (24) did, however, consider that changes in plasma composition during exercise should be primary determinants of RBC volume and pH regulatory mechanisms. Indeed, using an exercise-simulated (ES) plasma with normal pH (7.4), they showed a significant increase in unidirectional K⁺ transport. They did not report a time course of change in transport during the 60-min flux period, nor was the response determined in true exercise plasma.

An objective of the present work was to study human RBC K⁺ transport with the cells incubated in human plasma, specifically in plasma obtained from the same individual as the RBCs, to closely simulate in vivo conditions. To meet this objective, RBCs obtained at rest were studied in plasma obtained at rest and in plasma obtained after high-intensity exercise. Another objective was to determine the effect of individual plasma constituents, known to increase during high-intensity exercise, on RBC K⁺ transport. To this end, we created an ES plasma in which plasma osmolality, [epinephrine], [K⁺], and H⁺ concentration ([H⁺]) were increased to values reported in the literature after high-intensity exercise (3, 4, 21, 24, 28). When responses obtained from ES plasma were compared with those obtained from true exercise plasma, it was evident that similarly modified plasma would provide a suitable system for studying the independent effects of these plasma variables on RBC K⁺ transport.

The present study tested the hypotheses that 1) RBCs sampled from subjects during and within 30 s after the completion of high-intensity exercise will have an elevated J_inK, due to increases in Na⁺-K⁺ pump and Na⁺-K⁺-2Cl cotransporter activity, compared with RBCs sampled from subjects before the exercise; 2) RBCs sampled from subjects at rest, before exercise, will show a stimulation of J_inK when placed into plasma from blood sampled at the end of high-intensity exercise; 3) increases in plasma constituents (listed above) that occur with high-intensity exercise, independently and in combination, increase J_inK in human RBCs by stimulating the Na⁺-K⁺ pump and the Na⁺-K⁺-2Cl cotransporter. The results of the experiments reported below support these hypotheses.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Twenty-six healthy subjects (24 men and 2 women), 25 ± 1 yr old, 80 ± 5 kg mass, and 177 ± 4 cm height, participated in the study. Written informed consent was obtained after the procedures and potential risks were described to the subjects. The study was approved by Human Ethics Committees at both universities.

Exercise Studies

A 20-gauge, 2.5-cm-long Vialon catheter (Intima, Becton Dickinson, Mississauga, ON, Canada) was placed into an antecubital vein and secured using surgical tape. From each subject at rest before exercise, 20 ml of blood were sampled using 10-ml syringes fitted with 16-gauge needles. The needle was removed from the syringe, and the sample was expelled into a 10-ml Vacutainer tube containing lithium heparin as anticoagulant (Becton Dickinson). Microhematocrit was determined in duplicate by centrifugation (15,000 g for 15 min) of blood-filled capillary tubes.

Each subject performed two 30-s bouts of supramaximal-intensity leg-cycling exercise separated by a 4-min rest period. The protocol was a repeated Wingate test in which a modified Monark cycle ergometer was used. The first 10 ml of blood were sampled during the last 10 s of the second exercise bout; a further 15 ml were sampled during the first 20-30 s after exercise. The blood was transferred from syringes to Vacutainer tubes (Becton Dickinson) containing lithium heparin and mixed by rocking for 1 min. A 1.5-ml aliquot was dispensed into a 1.8-ml plastic tube and sealed for later analysis. Blood remaining in the Vacutainer tubes was centrifuged (10 min at 5,000 g) within 5 min of collection. The buffy coat layer was completely removed and discarded. The separated plasma and RBCs were stored in capped vials and allowed to cool to room temperature (~22°C).

Plasma [Na⁺], [K⁺], Cl concentration ([Cl]), glucose, lactate, pH, and PCO₂ were measured in sealed (to minimize gas exchange) blood samples within 1 h of sampling with ion-, metabolite-, and gas-sensitive electrodes (Statprofile 9⁺ analyzer, Nova Biomedical, Waltham, MA). Plasma protein was measured by refractometry (Atago clinical refractometer). Aliquots (200 µl) of plasma and whole blood were dried to a constant weight in preweighed plastic vials to determine plasma water content (liters H₂O/liter plasma) and whole blood water content [liters H₂O/liter whole blood (WB)]. A 1.2-ml blood sample within a sealed plastic vial was repeatedly frozen (4 times) in liquid nitrogen with thawing in warm water; when it was warmed, the sample was vigorously mixed. This procedure results in complete lysis of RBCs. The samples were immediately analyzed for lysate [K⁺] with the Nova analyzer. Units for electrode measurements of plasma and lysate ion concentrations are millimoles per liter water.

RBC J_inK in True Exercise Plasma

Resting RBCs in resting plasma. The purpose of measuring resting RBCs in resting plasma was to determine the control J_inK, and to test the hypothesis that J_inK, and remained constant over the period of incubation (Table 2).

Resting RBCs in true exercise plasma. The hypothesis is that the J_inK of RBCs obtained from the subject at rest before exercise is increased (compared with control) when RBCs are incubated in plasma obtained after high-intensity exercise (Table 2).

Resting RBCs in exercise plasma + 0.1 mmol/l bumetanide. Bumetanide at 0.1 mmol/l fully inhibits Na⁺-K⁺-2Cl cotransport activity (7, 20). This experiment tested the hypothesis that plasma obtained after exercise results in a stimulation of Na⁺-K⁺-2Cl cotransport activity.

Resting RBCs in exercise plasma + 0.1 mmol/l bumetanide + 0.1 mmol/l ouabain. Ouabain at 0.1 mmol/l fully inhibits Na⁺-K⁺ pump activity (33). The experiment tested the hypothesis that residual J_inK, i.e., the J_inK remaining after inhibition of the Na⁺-K⁺ pump and the Na⁺-K⁺-2Cl cotransporter, is increased in RBCs that are incubated in plasma obtained after exercise. The hypothesis that Na⁺-K⁺ pump activity is increased in RBCs that are incubated in plasma obtained after exercise was also tested. These responses were quantitatively compared with those obtained from resting RBCs in exercise plasma + 0.1 mmol/l bumetanide.

Exercise RBCs in resting plasma. This experiment tested the hypothesis that J_inK will decrease when RBCs obtained after exercise are incubated in plasma obtained at rest before exercise.

Exercise RBCs in exercise plasma. This experiment tested the hypothesis that J_inK will be increased compared with control when RBCs obtained after exercise are incubated in plasma obtained after exercise.

Exercise RBCs in exercise plasma + 0.1 mmol/l bumetanide. The hypothesis was that cotransporter activity was increased in exercise RBCs incubated in exercise plasma.

Exercise RBCs in exercise plasma + 0.1 mmol/l bumetanide + 0.1 mmol/l ouabain. The hypothesis was that increased Na-K pump and Na-K-2Cl cotransporter activity were increased in exercise RBCs incubated in exercise plasma.

RBC J_inK in ES Plasma

These experiments were performed over a 3-wk period on nine resting men. Forty milliliters of blood were sampled from a 22-gauge butterfly catheter placed in an antecubital vein. The blood was transferred to 10-ml Vacutainer tubes containing lithium heparin as anticoagulant. Microhematocrit was determined in duplicate. Plasma was separated from cells by centrifugation for 15 min at 5,500 g. The plasma was removed and stored in polycarbonate vials until used. The buffy coat layer was completely removed and discarded.

A portion of the plasma sample was analyzed for [Na⁺], [K⁺], [Cl], and osmolality. Radioactive ⁸⁶Rb (Amersham, Little Chalfont, Buckinghamshire, UK) was added to the plasma to give an activity of 2 µCi/ml and specific activity with respect to K⁺ of ~8 × 10⁵ cpm/mmol. Plasma osmolality was increased by adding 40 µl of 1 M NaCl to 1 ml of plasma. Plasma [K⁺] was increased 5 mmol/l by addition of 5 µl of 1 M KCl to 1 ml of plasma (Table 1). Plasma [H⁺] and lactate concentration ([lactate]) were increased together by 50 nmol/l and 30 mmol/l, respectively, by addition of 30 µl of 1 M lactic acid (in 0.9% NaCl) to 1 ml of plasma. Plasma [H⁺] was increased by addition of 15 µl of 2 N HCl to 1 ml of plasma. Plasma [lactate] was increased by addition of 30 µl of 1 M sodium lactate (in 0.9% NaCl) to 1 ml of plasma to increase plasma [lactate] by 30 mmol/l.

Each aliquot was placed in a 15-ml, 2-cm-diameter, glass flat-bottom reaction vessel. Each aliquot and a vial with RBCs were warmed for 15 min at 37°C in a water bath with an orbital rotation cycling with a frequency of 200/min. The reaction was started by pipetting 1 ml of RBCs into the vial containing plasma. The vial was vortex mixed for 7 s, and 200-µl blood samples were removed at 10 s and 2, 5, 15, and 30 min of the reaction; some experiments were extended, with samples taken at 45 and 60 min. Microhematocrit was determined at each time point and corrected for trapped plasma of 2.5% (10).

RBC J_netK in ES Plasma

Chemicals

Sample Processing

Calculations

(1)

(2)

From the resting and postexercise blood samples, [K⁺] in the cell water phase of RBCs ([K⁺]_RBC, mmol/l H₂O) was calculated from the following equation [after Maassen et al. (24)]

(3)

where lysate and plasma [K⁺] have units of millimoles per liter of water, plasma water has units of liters of water per liter of plasma, lysate water (i.e., WB H₂O content) has units of liters of water per liter of whole blood, and RBC water has units of liters of water per liter of whole blood. RBC water was calculated from the measured wet and dry weights of whole blood and plasma (34) as follows

(4)

The remaining calculations are pertinent to the in vitro studies. The blood sample obtained immediately after RBCs were mixed with plasma (10-s sample) was used to estimate extracellular radioactivity trapped in the cell pellets, and this value was subtracted from each sample obtained at subsequent time points (18).

J_inK (in µmol · h¹ · l cells¹) by the RBCs was calculated as follows

(5)

where and represent the radioactivity (in cpm) of m liters of cells, a is the specific activity of the plasma (cpm/µmol), and t is the incubation period (in h). All calculations were based on the initial volume of cells. For the experiments in which ES plasma was used, the plasma [K⁺] used in Eq. 3 averaged 1.6 mmol/l lower than that measured before the incubation of RBCs; this was based on the decrease in plasma [K⁺] that occurred in the first 2 min of incubation, measured continuously with a K⁺ electrode (see RESULTS). The uptake of K⁺ at each point in time was measured as /a (34).

Statistics

In the experiments that continuously measured plasma [K⁺] with a K⁺ microelectrode, the peak rate of net K⁺ flux into RBCs was calculated for each incubation by using linear regression analysis of plasma [K⁺] against time during the initial 20 s of incubation. These relationships yielded P < 0.001 and r² = 0.76-1.00.

Statistical significance was accepted at P  0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In Vivo Exercise Responses

The exercise resulted in significant increases in plasma [H⁺], [Na⁺], [K⁺], and [lactate], as well as blood lysate [K⁺] and [lactate] (Table 2). Lysate [K⁺] increased 14.6 ± 1.7% compared with the decrease in blood volume of 12.9 ± 0.8%; thus most of the increase in lysate [K⁺] with exercise was accounted for by the loss of water from the vascular compartment with exercise. Calculated RBC [K⁺] increased significantly (+3.4 ± 0.9%) from 116.8 ± 1.7 to 120.8 ± 1.4 mmol/l RBC H₂O. Total vascular RBC content of K⁺ was 219 ± 14 mmol at rest and unchanged at 212 ± 17 mmol at the end of exercise.

RBC J_inK in True Exercise Plasma

J_inK in resting RBCs in resting plasma (controls) was 1,236 ± 256 µmol · h¹ · l cells¹ at 2 min and did not change significantly during 15 min of incubation (Fig. 1). The addition of resting RBCs to exercise plasma increased J_inK to 1,426 ± 209 µmol · h¹ · l¹ at 2 min (Fig. 1A); this was associated with an increase in K⁺ uptake (Fig. 2A). Similarly, when exercise RBCs were incubated in exercise plasma, J_inK increased to 1,538 ± 192 µmol · h¹ · l¹ at 2 min (Fig. 1B), and K⁺ uptake was also increased compared with control (Fig. 2B). In these experiments, J_inK remained above control values during the 15 min of incubation. The addition of exercise RBCs to resting plasma resulted in J_inK similar to controls (not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Unidirectional transport of K+ (JinK, at 37°C) into human erythrocytes (RBCs). , JinK in absence of transport inhibitors; RBCs from resting subjects incubated in plasma from resting subjects. There was no change over time. , JinK in absence of transport inhibitors: RBCs obtained at rest incubated in plasma obtained after high-intensity exercise (A) and RBCs obtained after high-intensity exercise incubated in plasma obtained after high-intensity exercise (B). , JinK after addition of 0.1 mmol/l bumetanide to plasma: RBCs obtained at rest incubated in plasma obtained after high-intensity exercise (A) and RBCs obtained after high-intensity exercise incubated in plasma obtained after high-intensity exercise (B). , JinK after addition of 0.1 mmol/l ouabain and 0.1 mmol/l bumetanide to plasma: RBCs obtained at rest incubated in plasma obtained after high-intensity exercise (A) and RBCs obtained after high-intensity exercise incubated in plasma obtained after high-intensity exercise (B). Values are means ± SE (n = 10); some error bars have been omitted for clarity. * Significantly different from control (100%), P  0.05;  significantly different from JinK in absence of inhibitors (), P  0.05.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   K+ uptake (at 37°C) by human RBCs. , K+ uptake in absence of transport inhibitors: RBCs from resting subjects incubated in plasma from resting subjects. There was no change over time. , K+ uptake in absence of transport inhibitors: RBCs obtained at rest incubated in plasma obtained after high-intensity exercise (A) and RBCs obtained after high-intensity exercise incubated in plasma obtained after high-intensity exercise (B). , K+ uptake after addition of 0.1 mmol/l bumetanide to plasma: RBCs obtained at rest incubated in plasma obtained after high-intensity exercise (A) and RBCs obtained after high-intensity exercise incubated in plasma obtained after high-intensity exercise (B). , K+ uptake after addition of 0.1 mmol/l ouabain and 0.1 mmol/l bumetanide to plasma: RBCs obtained at rest incubated in plasma obtained after high-intensity exercise (A) and RBCs obtained after high-intensity exercise incubated in plasma obtained after high-intensity exercise (B). Values are means ± SE (n = 10); some error bars have been omitted for clarity. * Significantly different from control (100%), P  0.05;  significantly different from JinK in absence of inhibitors (), P  0.05.

When resting RBCs were incubated in exercise plasma, Na⁺-K⁺-2Cl cotransport inhibition (0.1 mmol/l bumetanide) abolished the increase in J_inK seen during the first 5 min of incubation (Fig. 1A). In comparison, when exercise RBCs were in exercise plasma, bumetanide had no effect on J_inK (Fig. 1B). Combined inhibition of the Na⁺-K⁺ pump (0.1 mmol/l ouabain) and the Na⁺-K⁺-2Cl cotransporter decreased J_inK in resting RBCs by 71 ± 6% and by 75 ± 5% in exercise RBCs, resulting in similarly low J_inK values (Fig. 1) and K⁺ uptakes (Fig. 2) in both series. Peak inhibition occurred within 10 min to 205 ± 63 µmol · h¹ · l¹ in both series. After 15 min of incubation of resting RBCs in exercise plasma, the ouabain-sensitive, bumetanide-sensitive, and residual J_inK accounted for 71 ± 5, 12 ± 1, and 27 ± 7% of total J_inK, respectively. The comparable values for exercise RBCs in exercise plasma were 67 ± 17, 8.1 ± 5.6, and 21 ± 9%, respectively.

Plasma [K⁺] was measured at 30 s and 4 min of incubation in five samples from the series in which resting RBCs were incubated in true exercise plasma. Plasma [K⁺] decreased by 0.5 ± 0.37 mmol/l in 30 s, with minimal further decrease at 4 min. The average J_netK during the initial 30 s was 1.43 ± 0.57 mmol K⁺ · min¹ · l plasma¹ or, with a mean Hct of 40%, 2.15 mmol K⁺ · min¹ · l cells¹.

RBC J_inK in ES Plasma

In the control series [incubation of RBCs in true (unmodified) plasma, except for added ⁸⁶Rb], J_inK was 1,691 ± 223 µmol · h¹ · l¹ at 2 min and did not change significantly during 60 min of incubation (Fig. 3). Na⁺-K⁺ pump inhibition with 0.1 mM ouabain reduced J_inK to 33 ± 18% of control values during the first 15 min of incubation (Fig. 3A). Combined Na⁺-K⁺ pump and Na⁺-K⁺-2Cl cotransport inhibition further reduced J_inK to 15 ± 3% of the 2-min control value and to only 7 ± 3% of control values at 30 min (residual J_inK, Fig. 3A). RBC K⁺ uptake increased linearly with time in true plasma and was significantly inhibited with ouabain and with combined ouabain and bumetanide (Fig. 4A). In this control series conducted using true plasma, 67 ± 11% of J_inK was due to Na⁺-K⁺ pump activity (ouabain-sensitive), 25 ± 6% was due to Na⁺-K⁺-2Cl cotransport activity (bumetanide-sensitive), and the residual (8.1 ± 3.3%) was insensitive to ouabain and bumetanide (Fig. 4).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   JinK (at 37°C) into human RBCs from resting subjects incubated in plasma from resting subjects (A) and exercise-simulated (ES) plasma (B). There were no changes over time unless indicated. , JinK of RBCs incubated in plasma from resting subjects in absence of transport inhibitors. , JinK of RBCs incubated in ES plasma in absence of transport inhibitors for 30 min; at 30 min, ouabain was added to incubation to create plasma ouabain concentration of 0.1 mM (+O); at 45 min, bumetanide was added to incubation to create plasma bumetanide concentration of 0.1 mM (+B), so ouabain and bumetanide were present. , JinK in presence of 0.1 mM ouabain to inhibit Na+-K+ pump activity. , JinK in presence of 0.1 mmol/l ouabain and 0.1 mmol/l bumetanide in plasma. Values are means ± SE (n = 9). * Significantly different from control (), P  0.05;  significantly different from ouabain trial (), P  0.05;  significantly different from 30-min value in ES trial without drugs (), P  0.05.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   K+ uptake (at 37°C) into human RBCs from resting subjects incubated in plasma from resting subjects (A) and ES plasma (B). , K+ uptake of RBCs incubated in plasma from resting subjects in absence of transport inhibitors. , K+ uptake of RBCs incubated in ES plasma in absence of transport inhibitors for 30 min; at 30 min, ouabain was added to incubation to create plasma ouabain concentration of 0.1 mM. At 45 min, bumetanide was added to incubation to create plasma bumetanide concentration of 0.1 mM, so ouabain and bumetanide were present. , K+ uptake in presence of 0.1 mM ouabain to inhibit Na+-K+ pump activity. , K+ uptake in presence of 0.1 mmol/l ouabain and 0.1 mmol/l bumetanide in plasma. Values are means ± SE (n = 9). * Significantly different from control (), P  0.05;  significantly different from ouabain trial (), P  0.05.

Incubation of RBCs in ES plasma (Table 3) resulted in a rapid increase in J_inK to 2,330 ± 191 µmol · h¹ · l¹ (+34 ± 8%) compared with control values at 5 min (Fig. 3B). RBC K⁺ uptake was linear until the addition of ouabain to the reaction at 30 min (Fig. 4B), which reduced J_inK by 57 ± 12% of the 30-min value at 45 min (Fig. 3B). The further addition of bumetanide at 45 min reduced J_inK to 33 ± 13% of the 30-min value (residual J_inK, Fig. 3B). Calculated bumetanide-sensitive J_inK in this series was 10 ± 6% of the 30-min value. The presence of ouabain in the ES plasma before addition of RBCs resulted in a J_inK that was 53 ± 14% of control values (Fig. 3B) and greatly attenuated K⁺ uptake (Fig. 4B). The presence of both ouabain and bumetanide resulted in a residual J_inK that was 16 ± 3% of control values (Fig. 3B) with very low K⁺ uptake (Fig. 4B).

The presence of epinephrine alone rapidly stimulated J_inK by 25 ± 8% at 2 min (Fig. 5A). There was minimal effect of epinephrine on K⁺ uptake (Fig. 5B). The effect of epinephrine was transient, and by 30 min J_inK was similar to the control values. A combination of epinephrine and high [K⁺] maintained J_inK at values above that seen with epinephrine alone (Fig. 5A) and resulted in increased K⁺ uptake compared with controls (Fig. 5B). If it is assumed that the epinephrine and raised plasma [K⁺] effects were purely additive, then raised [K⁺] alone was calculated to increase J_inK to 10 ± 8 and 47 ± 8% above the control values at 2 and 30 min of incubation, respectively. The effect of increased osmolality was similar to that of increased epinephrine and [K⁺], with J_inK increased 17 ± 9% above control values at 2 min (Fig. 5A) and increased K⁺ uptake compared with controls (Fig. 5B). J_inK remained elevated until after 15 min of incubation and was followed by a decrease to control values by 30 min.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   JinK (at 37°C; A) and K+ uptake (B) of human RBCs from resting subjects modified as follows. , RBCs incubated in true plasma from resting subjects. , Plasma epinephrine concentration increased to 10 nmol/l. , Plasma K+ concentration raised to 8.6 ± 0.2 mmol/l and epinephrine concentration increased to 10 nmol/l. , Plasma osmolality increased to 339 ± 3 mosmol/kg by addition of NaCl. , Plasma lactate and H+ concentrations increased to 30 mmol/l and 180 nmol/l, respectively, by addition of lactic acid. Values are means ± SE (n = 9); some error bars have been omitted for clarity. * Significantly different from control (), P  0.05.

Acidification of plasma with lactic acid before addition of RBCs resulted in no change at 2 min and then a marked stimulation of J_inK that peaked at 5 min (to 156 ± 11% of control value) and remained above control values for the first 15 min of incubation (Fig. 5A). The separate effects of increased lactate and acidity on J_inK were further studied. Plasma modified by the addition of sodium lactate (to result in plasma [lactate] of 30 mmol/l) showed no stimulation of J_inK (open circles, Fig. 6). In contrast, in plasma acidified with HCl (calculated to increase plasma [Cl] by 30 mmol/l and [H⁺] from 10 to 180 nmol/l at PCO₂ of 10 Torr), J_inK was increased to 129-150% of the control values between 5 and 30 min of incubation. The time course and magnitude of the response to HCl acidification (Fig. 6) was similar to that seen with lactic acid (Fig. 5A), including the delayed stimulation of J_inK. The data show that lactate per se had no effect on RBC K⁺ transport, whereas plasma acidosis had a pronounced, although delayed (by >2 min), stimulatory effect on J_inK.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   JinK (at 37°C) into human RBCs obtained from subjects at rest, expressed as percentage of control value (100% data from Fig. 4, ). , Plasma in which lactate concentration was increased to 30 mmol/l by addition of sodium lactate. , Plasma in which lactate and H+ concentrations were increased to 30 mmol/l and 180 nmol/l, respectively, by addition of hydrochloric acid. * Significantly different from control and sodium lactate values, P  0.05.

RBC J_netK in ES Plasma

1

1

1

1

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Plasma K+ concentration ([K+]) measured continuously with a K+-selective electrode (in vitro, 37°C) for 10 min before addition of RBCs (arrow at 10 min, to give hematocrit of ~40%) and for 5 min after addition of RBCs. * Significantly less than 9.5-min value, P  0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This appears to be the first study to show an increase in unidirectional K⁺ transport into RBCs from blood sampled from human subjects at the end of high-intensity exercise. Furthermore, it has been shown that results obtained using ES plasma were comparable to those obtained using true exercise plasma. With ES plasma used as a model system, it was demonstrated that increased plasma [H⁺], [epinephrine], [K⁺], and osmolality independently stimulated J_inK in human RBCs. The magnitude of difference in RBC J_inK incubated in resting vs. exercise plasma within 2 min of incubation indicated that initial (0- to 2-min) transport rates may have been higher than those measured at 2 min. Subsequent examination of net K⁺ uptake by RBCs incubated in true exercise plasma and in ES plasma showed that virtually all J_netK occurred within the first 2 min of incubation.

Limitations

The equation of Decker and Rosenbaum (5) used previously (21, 26, 27) to calculate intracellular ion concentrations is incorrect, because it does not consider the different proportions of water in the plasma and cellular fractions of the blood. As a result, earlier studies (4, 21, 26, 27) reported RBC [K⁺] that are ~10 mmol/l cell water too low and [Na⁺] that are ~20 mmol/l cell water too high.

Stimulation of J_inK

Na⁺-K⁺ Pump Activation

In resting RBCs incubated in true exercise plasma, the ouabain-sensitive component was 709 ± 50 µmol · h¹ · l¹ (71%) compared with 819 ± 66 µmol · h¹ · l¹ (82%) in ES plasma. In comparison, when RBCs were incubated in ES plasma, the addition of ouabain decreased J_inK by 570 ± 189 µmol · h¹ · l¹, thus accounting for 57 ± 12% of the 1,031 ± 214 µmol · h¹ · l¹ ES-induced increase in J_inK. When exercise RBCs were incubated in true exercise plasma, the ouabain-sensitive component was 922 ± 154 µmol · h¹ · l¹ or 92% of control J_inK. These results are consistent with in vitro studies attributing 70-85% of J_inK to the Na⁺-K⁺ pump (12, 31, 33).

Na⁺-K⁺-2Cl Cotransporter Activation

1

1

1

1

Variables That Independently Increased J_inK

Increased plasma osmolality resulted in a significant increase in RBC J_inK. Exercise at intensities greater than ~70% of peak O₂ is accompanied by increased plasma osmolality that is primarily attributed to the osmotic movement of water into contracting skeletal muscle, resulting in 5-20% decreases in PV (16, 23). Despite large increases in plasma osmolality, high-intensity running exercise in humans at ~200% of peak O₂ for 2 min resulted in minor, although significant, 1-3% decreases in RBC volume that were evident from the end of exercise until the 2nd min of recovery (35). A similar magnitude of decrease in RBC volume was observed during prolonged leg cycling at 75% of peak O₂ (23). Importantly, RBC volume does not decrease to the extent dictated by the increase in plasma osmolality (4, 23, 35). Thus it may be postulated that ion transport mechanisms are evoked to maintain volume in the presence of increased extracellular tonicity. In support of this hypothesis, Sejersted et al. (35) reported that RBCs that have lost volume during high-intensity exercise subsequently swell by 1-2% between 2 and 6 min of recovery. Furthermore, in a study examining increases in plasma and intra-RBC osmolality during graded exercise, Buono and Faucher (4) concluded that human RBCs have the ability to increase osmolality in vivo and that this mechanism allows RBC volume to remain relatively unchanged. These studies provide evidence that volume regulatory mechanisms are activated during the period of exercise.

In the present study the magnitude and rapidity of stimulation of J_inK by 10 nmol/l epinephrine (20-30% increase during the first 5 min of incubation) were similar to the 20% increase in Na⁺-K⁺ pump-mediated ⁸⁶Rb influx in human platelets (37) and leukocytes (1) exposed to 10 nmol/l epinephrine or salbutamol, a ₂-adrenoceptor agonist. Salbutamol also reportedly increased human RBC K⁺ uptake in vitro (25, 30); however, it was not known whether this was mediated by the Na⁺-K⁺ pump or by the cotransporter. Furthermore, administration of the -adrenergic agonists fenoterol or hexoprenaline to resting humans resulted in a significant lowering of RBC [Na⁺] and Na⁺ content within 1 h (25). These decreases were similar to the decrease in RBC [Na⁺] and Na⁺ content at the end of 90 s of high-intensity leg-cycling exercise (4). Taken together, these studies suggest activation of the Na⁺-K⁺ pump by high-intensity exercise and by catecholamines and -adrenergic agonists.

Consistent with the literature, increased plasma [H⁺], by addition of lactic acid or hydrochloric acid to plasma, resulted in a delayed stimulation of J_inK. The RBC responses to increased plasma [H⁺] are complex and appear to result initially from an increase in intracellular acidification that produces an increase in RBC volume from a net influx of Cl and water into the cell (8, 9). Acidification appears to result in a direct and simultaneous activation of the outwardly directed K⁺-Cl cotransporter (8, 9), such that the resultant loss of intracellular K⁺ may provide a stimulus for increased K⁺ transport into the cell via the Na⁺-K⁺ pump, as occurs in cultured human embryonic kidney cells (P. B. Dunham, personal communication). Such a sequence could account for the delayed (5-min) stimulation of J_inK seen in response to plasma acidification. Alternatively, intracellular acidification may result in a stimulation of Na⁺ influx through the amiloride-sensitive Na⁺ channel (12), with a rise in intracellular [Na⁺] providing the stimulus to increase Na⁺-K⁺ pump activity.

It is known that an increase in external [K⁺] or Rb⁺ concentration results in increased uptake of K⁺ by RBCs by the Na⁺-K⁺ pump (12, 31, 33) and the Na⁺-K⁺-2Cl cotransporter (7) that is proportional to the increase in external [K⁺]. In the present study, a 5 mmol/l increase in plasma [K⁺] to 9 mmol/l (in the presence of 10 nmol/l epinephrine) was calculated to have increased J_inK by 12-47% above that seen in the presence of 10 nmol/l epinephrine alone. The 9 mmol/l plasma [K⁺] used in our in vitro experiments is consistent with that found in femoral venous plasma at the end of a 1-min bout of high-intensity exercise (28). The lower 1 mmol/l increase in antecubital venous plasma [K⁺] is due to net K⁺ movement into noncontracting tissues (27).

Conclusions