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LOCAL CONTROL OF CIRCULATION
1Copenhagen Muscle Research Center, Institute of Exercise and Sport Sciences, University of Copenhagen, 2100 Copenhagen; and 2Sports Medicine Research Unit, Copenhagen University Hospital, 2400 Copenhagen, Denmark
Submitted 21 January 2003 ; accepted in final form 21 March 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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0.5
mM at rest and 1.5 mM at exhaustion. The time to exhaustion was shorter in AL
than in L (2.7 ± 0.3 vs. 4.0 ± 0.3 min; P < 0.05).
Furthermore, [K+]i was higher from 0 to 1.5 min of the
intense leg exercise period in AL compared with L (9.2 ± 0.7 vs. 6.4
± 0.9 mM; P < 0.001) and at exhaustion (11.9 ± 0.5
vs. 10.3 ± 0.6 mM; P < 0.05). The dialysate content of
carnosine was elevated by exercise, but low-intensity exercise resulted in
higher dialysate carnosine concentrations than subsequent intense exercise.
Furthermore, no relationship was found between carnosine concentrations and
[K+]i. Thus the present data suggest that microdialysis
can be used to determine muscle [K+]i kinetics during
intense exercise, when low-intensity exercise is performed before the intense
exercise. The high [K+]i levels reached at exhaustion
can be expected to cause fatigue, which is supported by the finding that a
faster accumulation of interstitial K+, induced by prior arm
exercise, was associated with a reduced time to fatigue. muscle; fatigue; microdialysis; exercise; pH; potassium
During intense exercise, K+ is lost from the muscle cell through delayed rectifier K+ channels, but potassium may also be lost due to increased opening probability of the ATP-sensitive K+ (KATP) channels as a result of intracellular acidification (8, 9). This notion is supported by the results of a study in which subjects performed exhaustive one-legged knee-extension exercise with and without prior intense intermittent arm exercise (5). As a result of the arm exercise, time to exhaustion was reduced, and blood lactate and blood H+ concentrations were elevated before the exhaustive leg exercise. At exhaustion, intramuscular pH in the leg was lower when arm exercise was performed before the leg exercise. Furthermore, when corrected for exercise time, total potassium release to the blood was increased. Thus it is possible that intracellular acidification plays a role for potassium release in vivo. However, the effect of intracellular acidification on [K+]i has not been examined.
Thus the aim of the present study was to measure human muscle [K+]i during intense exercise to exhaustion and to test the hypothesis that a greater intracellular acidification leads to a higher rate of accumulation of potassium in the interstitium during exercise.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Preexperimental procedure. Leg exercise was carried out on a modified Krogh ergometer, permitting the exercise to be confined to the quadriceps femoris muscle (2). To familiarize the subjects with the exercise ergometer and to determine individual workloads resulting in fatigue after 3–5 min of exercise, all the subjects completed five to seven preexperimental sessions. During the preexperimental sessions the subjects were trained to keep a constant kicking frequency of 60 kicks/min (by visual feedback from a display showing the frequency) and to confine the exercise to the quadriceps femoris muscle (by verbal feedback based on online forcerecordings and electromyogram signals from the hamstring muscles). The subjects exercised until the point of fatigue with the objective determinant for terminating the exercise being a drop in kicking frequency to 55 kicks/min. During the preexperimental days the subjects were also familiarized to an arm cranking ergometer as described previously by Bangsbo et al. (5).
Experimental procedure. On the experimental days the subjects reported to the laboratory in the morning after consuming a light meal. Intake of caffeine on the day of the experiment and heavy physical activity on the day before the experiment were avoided. Six microdialysis probes were inserted parallel to the muscle fibers of the vastus lateralis muscle in the experimental leg under local anesthesia (lidocaine, 1 ml of 20 mg/ml) as previously described (21). Three of the probes were homemade (length of microdialysis membrane 30–40 mm, OD 0.22 mm) as described by Radegran et al. (24), whereas three of the probes (length of microdialysis membrane 30 mm, OD 0.6 mm) were commercially fabricated (CMA60; CMA Microdialysis). The perfusate was Ringer acetate containing (in mM) 130 Na+, 2 Ca2+, 4 K+, 1 Mg2+ and 30 Ac-. The exact perfusate K+ concentration was determined in each experiment. In addition, the perfusate contained 1 mM of lactate, 3 mM of glucose and 201Tl (activity <7,000 Bq/ml). After insertion, the probes were flushed and connected to a pump (CMA 102). Then they were perfused at a rate of 2 µl/min for 1 h, and then the perfusion rate was increased to 5 µl/min for the remaining part of the experiment.
Exercise protocol. Three experimental days were completed. Two of the experimental days were identical (L1 and L2), and on the third day (AL) an arm exercise period was added to the protocol (Fig. 1). The following procedure was common to all three days. After insertion of the microdialysis probes, the subjects rested for at least 60 min. This was followed by submaximal one-legged kicking at 20 W for two periods of 10 min separated by 15 min of passive rest. Dialysate was collected from the probes at rest and at two time intervals (2–5 and 5–10 min) during the 20-W exercise bouts. These two submaximal exercise periods had proved to improve the reproducibility of [K+]i determinations in pilot studies. After resting for an additional 90 min, a 10-min 10-W exercise period was completed. Dialysate was collected twice during the 10-W period (2–5 and 5–10 min.). Then the subjects rested for 18 min before performing exhaustive exercise at a power output of 62.8 ± 3.0 W. Dialysate was collected from 12 to 17 min during the resting period.
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During the exhaustive exercise the first collection of dialysate was made from 0.5 to 2.0 min, representing the time interval from 0.0 to 1.5 min, when taking into account the delay due to the outlet tubing. Thereafter dialysate was collected in 1.5-min intervals with the duration of the last collection varying according to the time of exhaustion.
On day AL, the subjects performed arm cranking between the 10-W exercise period and the intense leg exercise (Fig. 1). The subjects performed the arm exercise (frequency 1 Hz) in an upright position at an
intensity of 140 W. The arm exercise started 1 min after the end of the
10-W period. It consisted of four 1-min exercise periods and one 1.5-min
period separated by 0.5-min recovery periods. This was followed by 4.5 min of
rest and another 1-min arm exercise period. Finally, the subjects rested 4 min
before the intense exhaustive leg exercise was initiated. A similar arm
cranking protocol has been shown to raise muscle and blood lactate
concentrations before the exhaustive leg exercise
(5). The experiments
L1, L2, and AL were performed in random order.
Carnosine experiment. To obtain information about the degree of sarcolemmal rupture caused by the microdialysis probes, dialysate carnosine concentrations were determined in three subjects who completed a protocol identical to L1 and L2. Two CMA60 probes were inserted in the vastus lateralis muscle of each subject, as just described. Dialysate was collected from 10 to 20 min and 60 to 70 min after insertion, 5.0 to 9.5 min during the first 20-W exercise, 80 to 90 min after the second 20-W exercise, and during the intense work period (Fig. 1).
Analysis. The perfusion rate of the individual microdialysis probes was determined throughout the experiment by weighing the sample tube before and after collection. Samples were excluded if the perfusion rate deviated by >30% from the target perfusion rate, or if there was any sign of hemoglobin in the dialysate. The K+ concentration of the samples was measured by flame photometry (FLM3, Radiometer) using lithium as internal standard. For each sample the 201Tl activity was determined in a Packard autogamma counter to determine thallium loss as previously described (13, 21). Carnosine was determined from the dialysate as performed by Gutierrez et al. (15) using an HPLC method described by Suliman et al. (27).
Calculations. The data from L1 and L2 were pooled and treated as obtained in one experiment denoted L. The relative loss (RL) of 201Tl was calculated as RLTl = (perfusate activity - dialysate activity)/perfusate activity. [K+]i was calculated from the dialysate potassium concentrations, assuming that fractional 201Tl loss from the perfusate was equal to the fractional gain of K+ to the perfusate (21): [K+]i = K+perfusate + [(K+dialysate - K+perfusate)/RLTl].
Statistics. Values are means ± SE. For each subject an average [K+]i was calculated from the individual measurements from each probe. Mean [K+]i and SE were determined from these individual means. Possible differences in time to fatigue were tested using a paired Student's t-test. Possible differences in [K+]i were tested using a two-way ANOVA for repeated measurements. In case of significant main effects, a Student-Newman-Keuls post hoc test was used to identify the points of difference. In the carnosine experiment, data from the two probes from each of the three subjects were treated as individual data points. Possible differences were analyzed by a one-way ANOVA for repeated measurements, and the Student-Newman-Keuls post hoc test was used to identify points of difference. Significance was set at the 0.05 level.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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[K+]i. There were no detectable differences in the [K+]i response between L1 and L2 (Fig. 2). The mean difference between [K+]i determined during L1 and L2 at various time points was -0.6 ± 0.4 mM at rest, -0.3 ± 0.4 mM after warmup, 1.1 ± 0.7 mM during exercise, and 1.5 ± 2.2 mM at exhaustion. Individual differences in [K+]i determined during L1 and L2 are depicted in Fig. 3.
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No differences in [K+]i between AL (4.9 ± 0.3 mM) and L (4.2 ± 0.2 mM) were observed at rest before the intense exercise. In the period from 0 to 1.5 min of the intense exercise, [K+]i was higher (P < 0.001) in AL than in L (9.2 ± 0.7 and 6.4 ± 0.9 mM; Fig. 4). [K+]i at exhaustion was also higher (P < 0.05) in AL compared with L (11.9 ± 0.5 vs. 10.3 ± 0.6 mM). The [K+]i at exhaustion ranged from 8.8 to 13.7 mM between subjects, and a large variation was apparent for individual measurements. When two 20-W exercise periods separated by 15 min were performed (Fig. 5), a larger increase in [K+]i was apparent in the first 20-W period compared with the second (P < 0.001).
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Carnosine in dialysate. In the period from 10 to 20 min after insertion of the microdialysis probes, the carnosine concentration in the dialysate was higher (P < 0.05) than during the two following resting periods (Fig. 6). The dialysate carnosine concentration was 105 ± 14 µM during the first 20-W exercise period and lower (50 ± 11 µM; P < 0.05) during the intense exercise.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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In vitro studies have provided substantial evidence indicating that membrane depolarization caused by accumulation of extracellular potassium leads to development of muscular fatigue (6,
19). We found
[K+]i at exhaustion to be 11 mM, with individual
values from 9 to 14 mM. These values correspond to previous reports on
[K+]i determined in human skeletal muscle during
exercise (14). It should be
noted that because [K+]i was determined in 1.5-min
intervals, the true peak [K+]i may have been even
higher. [K+]i in the range found in the present study
drastically reduces peak tetanic force in rat soleus muscle
(6). Thus the
[K+]i found in the present study would depolarize rat
skeletal muscle to between -65 and -50 mV
(6). In the rat, a
depolarization to -60 mV at physiological temperatures greatly reduces the
Na+ current (25)
due to slow inactivation of Na+ channels, ultimately leading to
inexcitability of the muscle cells. Thus it seems plausible that the
exercise-induced elevation of [K+]i found in the present
study results in reduced excitability of the active muscle fibers.
During the first 1.5 min of exercise, [K+]i increased more when leg exercise was preceded by arm exercise, whereas no difference was present in the increase from the first to the last measurement during exercise in L and AL. In accordance with this, Bangsbo et al. (5) found that the release of K+ from the working muscles to the bloodstream was higher in AL than L and more pronounced at the beginning of the exercise period. One possible explanation could be that a substantial drop in muscle pH is required before the open probability of the KATP channels increases (8). The increased extracellular lactate and H+ concentration in AL (5) probably caused a reduced efflux of H+ from the active muscle cells (20), leading to a more rapid drop in muscle pH in AL than in L. As the exercise period progresses, the difference in the intracellular H+ between AL and L was probably reduced (22), and thus the possible difference in potassium release via H+ activation of the KATP channels is likely to have been reduced.
The faster accumulation of K+ in AL was associated with a more rapid development of fatigue and supports the hypothesis that accumulation of K+ in the muscle interstitium is causatively linked to muscle fatigue (26). However, the finding of different [K+]i at exhaustion in AL and L indicates that K+ is not the only agent acting to induce muscular fatigue during intense exercise. It is likely that accumulation of extracellular K+, intracellular H+, and/or intracellular Pi in combination acts to induce fatigue (10, 29). It is also possible that other factors are causing the reduced intracellular calcium release observed during muscular fatigue (1).
At exhaustion a pronounced variation in [K+]i was observed. There may be several explanations for this finding. First, physiological spatial heterogeneity caused by varying degrees of muscle fiber activation in different regions of the muscle is one likely reason. Second, differences in the fiber type composition around the microdialysis probe could be a possibility, because different fiber types in the mouse are known to have different capacities for maintaining ion homeostasis (7). It should be noted, however, that differences in Na+-K+-ATPase density in relation to fiber types have not been demonstrated in humans.
The use of microdialysis allows determination of [K+]i in exercising human skeletal muscle (21). Determination of [K+]i has previously only been possible from
calculations using several assumptions
(16). Nevertheless, it should
be discussed whether [K+]i determined by microdialysis
can be considered valid. To investigate if rupture of the sarcolemma occurs
when the microdialysis probes are inserted in human skeletal muscle, the
dialysate was analyzed for carnosine concentrations. Carnosine is found in
high concentrations inside the muscle cell
(12) but is undetectable in
blood plasma (15). High
carnosine concentrations were observed in the dialysate during the first 20-W
exercise period, indicating that rupture of the sarcolemma did occur. However,
the low carnosine concentrations in the dialysate during the intense exercise
period suggest that multiple exercise periods result in gradual reduction of
sarcolemmal rupture. The higher dialysate carnosine concentrations during
exercise compared with rest are probably not only due to a greater degree of
sarcolemmal rupture, because the recovery of carnosine is most likely higher
during exercise. No data exist on the recovery of carnosine, but the recovery
for adenosine has been shown to change from 35% at rest to 60%
during exercise (11). However,
such an increase in recovery during exercise cannot explain the entire
difference in the carnosine concentrations between rest and exercise. No
relationship existed between [K+]i and dialysate
carnosine concentrations (Fig.
7). This suggests that muscle rupture does not have a considerable
effect on the examined concentrations of [K+]i. Taken
together, the reproducibility of [K+]i in L1
and L2, the finding of a difference between AL and L, as well as
the lack of correlation between [K+]i and carnosine,
suggest that microdialysis can be used for determination of interstitial
K+ kinetics in exercising human skeletal muscle.
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In summary, the present study showed that muscle interstitial K+ kinetics can be determined by microdialysis, but we propose that the subjects perform a period of low-intensity exercise before measurement of [K+]i is made during exercise. This study showed that [K+]i reached levels during intense exercise that can be expected to cause fatigue. In support of this notion is the observation that the faster accumulation of interstitial K+ induced by prior arm exercise was associated with a reduced time to fatigue. However, an increase in [K+]i during intense exercise appears not to be the only cause of development of muscular fatigue during high-intensity exercise, because [K+]i at exhaustion was different when leg exercise was performed with or without previous arm exercise.
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ACKNOWLEDGMENTS |
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This study was supported by grants from Team Danmark and Danish National Research Foundation Grant 504–14.
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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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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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2-adrenoceptor activation on ion-shifts and fatigue in mouse
soleus muscles stimulated in vitro. Acta Physiol Scand
134: 209–216,
1988.
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, Collection of dialysate for carnosine determination; 
,
collection of dialysate for potassium determination.
) or without (L; 
) arm exercise before leg exercise. *Significant difference between AL and L (P < 0.05).
), during 20-W exercise (
), and during
intense exercise (