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J Appl Physiol 103: 494-503, 2007. First published April 19, 2007; doi:10.1152/japplphysiol.00049.2007
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Effects of extracellular HCO3– on fatigue, pHi, and K+ efflux in rat skeletal muscles

Martin Broch-Lips,1 Kristian Overgaard,2 Helle A. Praetorius,1 and Ole Bækgaard Nielsen1

1Institute of Physiology and Biophysics, 2Department of Sport Science, University of Aarhus, Denmark

Submitted 11 January 2007 ; accepted in final form 16 April 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Elevated plasma HCO3– can improve exercise endurance in humans. This effect has been related to attenuation of the work-induced reduction in muscle pH, which is suggested to improve performance via at least two mechanisms: 1) less inhibition of muscle enzymes and 2) reduced opening of muscle KATP channels with less ensuing reduction in excitability. Aiming at determining whether the ergogenic effect of HCO3– is related to effects on muscles, we examined the effect of elevating extracellular HCO3– from 25 to 40 mM (pH from 7.4 to 7.6) on fatigue, intracellular pH (pHi), and K+ efflux in isolated rat skeletal muscles contracting isometrically. Fatigue induced by 30-Hz stimulation at 30 and 37°C was similar between soleus muscles incubated in high and normal HCO3– concentrations. In extensor digitorum longus muscles stimulated at 60 Hz, elevated HCO3– did not affect fatigue at 30°C. In soleus muscles, 30-Hz stimulation induced a ~0.2 unit reduction in pHi, as determined by using the pH-sensitive probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. This reduction in pHi was not affected by elevated HCO3–. Estimation of K+ efflux using 86Rb+ showed that elevated HCO3– did not affect K+ efflux at rest or during contractions. Similarly, other modifications of the intra- and extracellular pH had little effect on K+ efflux during contraction. In conclusion, elevated extracellular HCO3– had no significant effect on muscle fatigue, pHi, and K+ efflux. These findings indicate that alternative mechanisms must be considered for the ergogenic effect of HCO3– observed in integral exercise studies.

muscle fatigue

THE INFLUENCE OF THE HYDROGEN ion (H+) on muscle fatigue and exercise performance has been investigated extensively [for review, see Fitts (17) and Allen et al. (2)]. Muscle activity leads to changes in intracellular pH (pHi) in response to increased metabolic demand. At the beginning of activity, muscles alkalinize due to catabolism of creatine phosphate, only to acidify as glycolysis leads to production of H+ (17). In human subjects, pHi has been found to decrease from ~7.1 at rest to values as low as ~6.4 during intense exhaustive exercise (36). This reduction in pHi has been suggested to be a major cause of muscle fatigue (17). Several authors have investigated the effect of acidification on muscle performance, and numerous mechanisms for the acidification-induced muscle fatigue have been proposed, including 1) reduced glycolysis through inhibition of glycolytic enzymes such as phosphofructokinase, 2) reduction of crossbridge activation resulting from the competitive inhibition of H+ on Ca2+ binding to troponin C, 3) inhibition of the sarcoplasmatic reticulum (SR) Ca2+-ATPase, which reduces the Ca2+ reuptake and subsequently Ca2+ release (2, 6, 11, 17, 55). Furthermore, reduced muscle pHi has been associated with an increased release of K+ from working muscles (3) and a larger accumulation of K+ in the muscle interstitium (40). It was suggested that this relationship between pH and the contraction-induced K+ loss was caused by a pH-dependent activation of ATP-sensitive K+ channels (KATP channels), which, in isolated muscles, is known to increase K+ efflux (31). The increase in the extracellular K+ concentration might depolarize the muscle membrane, causing slow inactivation of the voltage-dependent Na+ channels (44) and thereby excitation failure (12).

Based on the notion that intracellular acidification is a major cause of fatigue, several groups have studied the effect of ingested bicarbonate salts on exercise performance in humans. HCO3– ingestion is reported to significantly increase both extracellular buffer capacity and extracellular pH (2, 5, 19, 35, 36), which may lead to an increased H+ gradient across the sarcolemma. In support of a role of low pHi in fatigue, several of these studies have shown a clear ergogenic effect of HCO3– loading. These findings have been interpreted as improved muscle function due to enhanced H+ handling and better maintenance of pHi (5, 26, 32, 33, 35, 42, 45, 54). The reduced pHi decline after HCO3– loading is possibly mediated by H+ transport over the sarcolemma, such as the monocarboxylate cotransport and Na+/H+ exchange (23, 53). In support of this suggestion, ingestion of HCO3– has been found to accentuate the release of lactate from muscles during exercise (8, 19, 21, 36, 41, 47, 49), with lactate being followed stoichiometrically by H+ ions (24). These findings are, however, still subject of debate since a number of studies have been unable to verify ergogenic effects of HCO3– loading (9, 26, 28, 46, 47, 49, 50). Moreover, the role of low pHi in muscle fatigue has recently been questioned [see Lamb et al. (25) for a discussion].

The aim of this study was to investigate the effect of increased buffer HCO3– on fatigue, pHi, and K+ efflux in isolated rat skeletal muscles. Furthermore, we performed a detailed study of the influence of intra- and extracellular pH alterations on K+ efflux.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animal handling and muscle preparation.   All experiments were performed using 4-wk-old male or female Wistar rats of own breed weighing 60–75 g. Young rats were preferred because of their relatively small muscle size (~20–25 mg). This minimizes the diffusional barriers to substrates, ions, and oxygen and thus the risk of ischemia during the experiments. Animals were fed ad libitum and kept in a thermostatic environment at 21°C with a 12:12-h light-dark cycle. The rats were killed by cervical dislocation followed by decapitation, and intact soleus [predominantly slow-twitch (type I); 85–93% (7)] muscles were dissected out. Additionally, a small number of contractile force measurements were done on isolated extensor digitorum longus [EDL; predominantly fast-twitch (Type II); >97% (7)] muscles. All handling of the animals was in accordance with the Danish law on animal welfare, and the procedures were supervised by the University Animal Officer.

At the beginning of the experiments, muscles were incubated in standard Krebs-Ringer bicarbonate (KR) buffer containing (in mM): 122 NaCl, 25 NaHCO3–, 2.8 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.3 CaCl2, and 5.0 D-glucose (pH = 7.4 at 30°C). After an initial 30-min equilibration period, buffers were changed to either a high HCO3– buffer or a control buffer. In high HCO3– buffer, HCO3– was increased to 40 mM and 15 mM NaCl was omitted (pH = 7.6 at 30°C). In control buffers, 15 mM NaCl was omitted and 15 mM sodium methanosulfonate was added to obtain the same Cl– concentration and osmolarity as in the high HCO3– buffer (pH = 7.4). All buffers were equilibrated with a 95% O2-5% CO2 mixture, except in a few experiments where the fraction of CO2 was changed to investigate the effect of CO2-induced alkalinization and acidification. The pH of buffers was determined using a pH meter (model PHM 92 Lab, Radiometer, Copenhagen, Denmark). In experiments using high HCO3–, muscles were preincubated in high HCO3– buffer for at least 30 min.

Isometric force.   Muscles were mounted on isometric force transducers at optimal length and exposed to field stimulation across the central part of the muscle through platinum electrodes. Muscles were stimulated tetanically using continuous 30-Hz stimulation with supramaximal pulses (12 V and duration of 0.2 ms) for soleus muscles and 60-Hz stimulation for EDL muscles, and force was recorded continuously. The frequencies of stimulation were chosen to ensure the development of a smooth tetanic force in both types of muscles. In other experiments, soleus muscles were stimulated intermittently with a duty cycle of 0.25 (1 s on 3 s off) using 30-Hz stimulation during the contraction periods as described above. Contractile force was recorded continuously, and the maximal rate of force development and relaxation was estimated as the maximal numerical slope-value of the force development during contraction and force decline during relaxation, respectively. Force was measured using force displacement transducers (Grass FTO3, W. Warwick, RI) and recorded digitally at 1,000 Hz with PowerLab data acquisition system (ADI Instruments). During experiments, the temperatures of the incubation buffers were either 30 or 37°C as indicated. In experiments where muscles were stimulated at 37°C, the temperature was increased from 30 to 37°C just before the stimulation began. The initial force development was not significantly affected by the presence of high HCO3–. Thus, at 30°C, the initial force was 380 ± 9 and 346 ± 14 mN (n =12) with and without high HCO3– in soleus muscles (P = 0.15) and 462 ± 32 (n = 4) and 432 ± 29 mN (n = 8) with and without high HCO3– in EDL muscles (P = 0.52). In soleus muscles at 37°C, the initial force was 363 ± 15 and 352 ± 12 mN in high HCO3– and control buffer, respectively (n = 12, P = 0.38).

pHi measurements.   Changes in pHi were measured using pHi-dependent fluorescence spectroscopy. The soleus muscles were mounted horizontally and connected to a force transducer coupled to a chart recorder, and placed on an inverted microscope (TE-2000, Nikon, BBT-LifeScience, Denmark) equipped with low-light level fluorescence provided via a xenon lamp and monochromator (Visitech International, Sunderland, UK). The microscope was focused on the surface fibers of the muscle and imaging was performed with a long-distance Plan Fluo x20, 0.45 normal aperture (Nikon, Copenhagen, Denmark), an intensified SVGA CCD camera, and imaging software (Quanticell 2000/Image Pro, Visitech). The muscles were incubated with membrane permeant intracellular H+ indicator 2'-7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM; Invitrogen) and loading was monitored continuously. After load, the muscles were washed extensively in standard KR buffer. Excitation of the intracellular BCECF molecules with light of rapidly alternating wavelengths (440 and 488 nm) induced a fluorescence light-emitting reaction and the emitted light was then measured above 520 nm. The pH-dependent signal was derived from the ratio of fluorescence intensities measured at two different excitation wavelengths (488 nm/440 nm). When a stable ratio was obtained, the standard KR buffer was replaced by experimental buffer and the muscles rested for another 30 min. Hereafter the temperature in the chamber was increased from 30 to 37°C and the muscle was stimulated at a frequency of 30 Hz (pulse duration 0.2 ms at 12 V) continuously for 2 min. During contraction, cellular fluorescence was sampled at a rate of 1 Hz. The emission ratio was calibrated to pHi following experimentation using the nigericin method (52). The method takes advantage of a buffer in which intra- and extracellular pH becomes the same [for details on skeletal muscle, see Nielsen et al. (38)] and therefore allows pHi to be determined by measuring buffer pH. Buffer pH was changed by adding HCl or NaOH, and linear regression was used to establish the relationship between pHi and the recorded emission ratio from the muscle preparation (r2 ≥ 0.95 for all calibrations).

K+ efflux.   K+ efflux was estimated from measurements of fractional 86Rb+ loss. 86Rb+ is qualitatively a satisfactory marker for K+ movement through several K+ channels, including the KATP channel (10). The method used was essentially as previously described by Clausen and Kohn (13). To load muscles with 86Rb+, the muscles were placed in perforated plastic containers and incubated for 60 min in standard KR buffer containing 86Rb+ (2 µCi/ml). The muscles were then mounted between electrodes at optimal length. The efflux of 86Rb+ from each muscle was then monitored over time by transferring the muscle through a series of 12 tubes containing KR buffer without the isotope. The first three tubes constituted a washing period of 20 min/tube. In the following experimental periods, muscles were transferred through a series of up to nine tubes with 10-min incubation in each tube. Muscles rested during the experimental period except for one 10-min interval in which the muscles were stimulated for 5 min either continuously or intermittently (1 s on, 1 s off) at 30 Hz using the stimulation protocol described earlier. At the end of the experiment, the muscle was blotted on filter paper, weighed, and soaked in 2 ml of 5% trichloracetic acid (TCA). The 86Rb+ activity of the TCA extract of the muscle and the washout tubes was then determined by Cerenkov radioactive counting. The 86Rb+ content in the muscle at each transfer between tubes was calculated by adding successively the 86Rb+ activity in the washout tubes to the 86Rb+ activity in the TCA extract of the muscle. On the basis of these values, the fractional 86Rb+ loss was calculated for each washout interval. Estimation of the excitation-induced fractional 86Rb+ loss was made by subtracting the fractional 86Rb+ loss determined during resting conditions in the interval preceding contraction from the fractional 86Rb+ loss in the interval with contraction.

All efflux experiments were carried out at 30°C. Using the method and procedure described above, the effects of pinacidil (100 µM), glibenclamide (20 µM), HCl (20 mM), lactic acid (20 mM), high HCO3– (40 mM), and increased CO2 (15% CO2, 85% O2 in a 70 mM HCO3– buffer to keep extracellular pH constant) on fractional 86Rb+ loss were investigated.

Statistics.   All data are expressed as means ± SE. The statistical significance of any difference between groups was ascertained using Student's two-tailed t-test for non-paired observations (2 groups) or two-way ANOVA followed by a Bonferroni post hoc test where appropriate (more than 2 groups). Significance was accepted at a post hoc level with P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Effects of high extracellular HCO3– on force production during continuous or intermittent tetanic contraction.   Soleus and EDL muscles were incubated in high HCO3–, whereas the contralateral muscle was incubated in control buffer. Muscles were stimulated to fatigue by different protocols and all force measurements are displayed relative to the initial tetanic force. Figure 1A shows that at 30°C, muscles incubated in high HCO3– showed a slight tendency for better force maintanance during tetanic stimulation than controls. The relative force production was, however, not significantly different at any time point. In the same experiments, the recovery of force after the cessation of the continuous 30-Hz stimulation was examined by short tetanic stimulations. These experiments did not reveal any difference in the force production between control and high HCO3– muscles (data not shown). To test if high extracellular HCO3– affected the force development in contractions induced by nerve stimulation at 30°C, the pulse duration was decreased to 0.02 ms (12 V, 30 Hz). This has previously been shown to excite the motor nerves without directly affecting the sarcolemma (39). The results were similar to those described above (data not shown) and, thus, did not depend on the mode of stimulation.


Figure 1
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  		Fig. 1. Effect of high HCO3– on force development during continuous tetanic stimulation of rat soleus muscles. Pairs of contralateral muscles were mounted in force transducers and incubated in standard Krebs-Ringer (KR) buffer equilibrated with 5% CO2 in O2. After initial force in standard KR buffer was determined, one of the muscles was exposed to either 40 mM HCO3– or 4% CO2 (bullet) and the other was used as control and was incubated in control KR buffer ({circ}). The muscles were stimulated continuously with use of 0.2-ms pulses of 12 V at 30 Hz when incubated at 30°C and 5% CO2 (A), when incubated at 30°C and 4% CO2 (B), and when incubated at 37°C and 5% CO2 (C). Values are represented as percentage of initial force. Post hoc test showed no significant difference at any time point. Each data point shows the mean ± SE of observations on 6 muscles, except for the low CO2 experiment which was n = 4.

 
To test if an alkalinization of the muscles by reduced CO2 tension in the buffer could produce an ergogenic effect the experiment shown in Fig. 1A was repeated with muscles incubated in standard KR buffer equilibrated with 4% CO2, 96% O2 gas (buffer pH ~7.5; Fig. 1B). This type of alkalinization did not increase the force production during 5 min of 30-Hz stimulation despite an expected rise in pHi. Additional alkalinization of the muscles by reducing CO2 to 3% (pH ~7.6) resulted in a large drop in the ability to maintain force (data not shown) and a large reduction in absolute force. The initial force at 3% CO2 was 276 ± 25 mN (n = 4) compared with 375 ± 14 mN (P = 0.013) at 5% CO2.

To investigate if an ergogenic effect of HCO3– was present in other muscle fiber types, we measured tetanic force development in the highly glycolytic EDL muscle. In this muscle type, no significant effect of high HCO3– on force production when stimulated continuously at 60 Hz at 30°C was detected (data not shown).

The experimental temperature is known to be extremely important when investigating effects of pH on force development in isolated muscle. Westerblad et al. (56) found that at lower temperatures muscles bathed in alkaline solutions develop more force compared with controls. This effect was much attenuated as temperature was increased. Based on these findings, we determined if high HCO3– affected force production in soleus muscles at a higher and more physiological temperature. At 37°C, high HCO3– had no effect on force development during continuous stimulation (Fig. 1C). Likewise, when muscles were stimulated intermittently at 37°C, the maximal force production during each contraction was similar with and without high HCO3– (Fig. 2A). All muscles incubated at 37°C developed contracture ~10 min into the recovery period, and recovery was therefore not analyzed in these experiments. The experiments on EDL muscles were not performed at 37°C as they, in pilot experiments, showed signs of reduced viability.


Figure 2
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  		Fig. 2. Effect of high HCO3– on contractile parameters during intermittent stimulation in rat soleus muscles at 37°C. Contralateral muscles were mounted in force transducers and incubated in standard KR buffer equilibrated with 5% CO2 in O2. After determination of initial force, one of the contralateral muscles was exposed to 40 mM HCO3– (bullet), and the other was used as control ({circ}). The muscles were stimulated intermittently with 0.2-ms pulses of 12 V at 30 Hz (1 s on 3 s off). A: peak force during each train of pulses; B: rate of force development; and C: rate of force relaxation during each train of pulses. Values are represented as percentage of initial rate of force development or relaxation. Post hoc test showed no significant difference at any time point. Each data point indicates the mean ± SE of observations on 6 muscles.

 
The data obtained from the intermittent stimulation protocol were also analyzed for rate of force development and relaxation. The maximal rate of force development in the first contraction in the intermittent stimulation protocol amounted to 10.9 ± 1.7 N/s in muscles incubated in high HCO3– and 9.0 ± 0.6 N/s in controls (n = 6). The difference was not statistically significant (P = 0.329). The maximal rate of force development from each successive stimulation was plotted as percent of the initial rate of force development. The muscles incubated in high HCO3– showed no significant difference from control muscles (Fig. 2B). Likewise, the maximal rate of force relaxation calculated from the first contraction in the intermittent stimulation protocol did not differ between muscles incubated with and with out high HCO3–. The values were –9.3 ± 0.3 N/s and –8.9 ± 0.3 N/s, respectively (P = 0.426). When the rate of force relaxation from each stimulation was plotted as percent of the initial value, again there was no detectable difference between muscles incubated in high HCO3– compared with control (Fig. 2C). Finally, an analysis of force-time integrals from experiments with and with out high HCO3– (data represented in Figs. 1, A and C, and 2A) did not reveal any significant difference in area under the curves (data not shown).

Effects of HCO3– on pHi during fatigue.   To evaluate whether elevated extracellular HCO3– improved the regulation of pHi during contractions, we measured the magnitude and the rate of the reduction in pHi in surface fibers of muscles stimulated to fatigue. Stimulation of soleus muscles initially led to an intracellular alkalinization of the muscles fibers, which was then followed by a large reduction in pHi (Fig. 3). A similar two-phasic pH response to intense muscle activity has been reported by others and reflects an initial metabolic expenditure of H+ during breakdown of creatine phosphate followed by a large metabolic production of H+ caused by anaerobic catabolism of glucose (17).


Figure 3
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  		Fig. 3. Representative figure showing changes in pHi in a soleus muscle before, during, and after contraction during incubation in a high HCO3– buffer. The symbols show pHi calculated every second from the ratio of fluorescence excited by light at 488 and 440 nm in a muscle loaded with the pH-sensitive fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Top: changes in pHi during contraction and recovery. Vertical line (VL) 1 marks the start of a 2-min contraction induced by continuous stimulation at 30 Hz (0.2-ms pulses of 12 V), which ends at VL 2. The decrease in pHi at time point 35 min was caused by an increase in temperature from 30 to 37°C. Bottom: data from the contraction part (from VL 1 to VL 2) of Fig. 3, top, on an expanded time scale. Arrows in bottom indicated data points used in subsequent data analyses.

 
Figure 3, top, shows a representative experiment in which pHi in a soleus muscle was measured before, during, and after contraction elicited by 30-Hz continuous stimulation at 37°C. Figure 3, bottom, shows an expansion of the interval in which the muscle contracted as indicated by vertical lines 1 and 2 in Fig. 3, top. From experimental data obtained from 16 such traces of pHi, the rate of the decline in pHi during contraction was approximated with linear regression and included data points from the peak of alkalinization through the following 10 s (r2 > 0.90). In addition, the magnitude of the initial increase in pHi [{Delta}pHi(peak–rest)] and the following reduction in pHi during the contraction [{Delta}pHi(end–rest)] was calculated as the difference between peak pHi and pHi at rest (average of values from the last 10 s before contraction) and the difference between end pHi (average of pHi in the last 10 s of contraction) and pHi at rest, respectively (Fig. 3, bottom). Table 1 displays the summarized results obtained from analysis of the 16 pHi traces. None of the differences between high HCO3– and control were statistically significant; however, the alkalinization at the beginning of contraction tended to be larger in muscles incubated in control buffers (P = 0.06).


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  		Table 1. High HCO3– does not affect the rate of acidification or the change in pHi during a 2-min contraction

 
Effects of high extracellular HCO3– concentration and other manipulations of intra- and extracellular pH on K+ efflux in resting and contracting muscles.   To examine for possible effects of increased extracellular HCO3– and changes in pH on the muscular K+ handling and the possible involvement of KATP channels, we determined the efflux of K+ in resting and contracting muscles. To ensure that the method had a sensitivity that was sufficient to detect an opening of the KATP channels in the muscles, we first determined the effect of 100 µM pinacidil on the fractional 86Rb+ loss. At this concentration, pinacidil has been shown to cause opening of the KATP channels in the surface membranes of the muscles fibers (4, 31). In resting muscles, incubation with pinacidil increased fractional 86Rb+ loss slightly, but this was only significant at time interval 10–20 min (P < 0.05), as shown in Fig. 4, A and B. When all resting muscles shown in Fig. 4, A and B, were grouped and analyzed, resting fractional 86Rb+ loss was significantly increased by pinacidil at all time intervals (Fig. 5A, P < 0.05). Calculation of the excitation-induced fractional 86Rb+ loss showed that during continuous and intermittent stimulation the excitation-induced 86Rb+ efflux in pinacidil-treated muscles was increased to 132 ± 5% (P = 0.017) and 150 ± 20% (P = 0.10), respectively, of the excitation-induced fractional 86Rb+ loss in contralateral control muscles. Together, these results demonstrate that the 86Rb+ efflux assay was sufficiently sensitive to detect the opening the KATP channels in isolated muscle.


Figure 4
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  		Fig. 4. Effect of pinacidil and high HCO3– on the fractional 86Rb+ loss from soleus muscles at rest and during stimulation. Muscles were loaded with 86Rb+ and then washed in the experimental buffer for 3 times 20 min. Following wash, the muscles were incubated 7 times for 10 min in similar buffers and the fractional loss of K+ was determined for each 10-min interval. A and B: pairs of contralateral muscles were incubated in standard KR buffer with or without pinacidil (100 µM). C and D: pairs of contralateral muscles were incubated in standard buffer (25 mM HCO3–) or high HCO3– buffer (40 mM HCO3–) equilibrated with 5% CO2 in O2. Muscles rested during all time intervals except in interval 30–40 min in A and C where the muscles were stimulated continuously for 5 min at 30 Hz (0.2-ms pulses of 12 V) and in B and D, where muscles were stimulated intermittently for 5 min (1 s on, 1 s off) at 30 Hz (0.2-ms pulses of 12 V). A 2-way ANOVA test followed by a post hoc test (*) showed that fractional 86Rb+ loss in muscles stimulated in the pinacidil buffer was significantly higher than in muscles stimulated in standard KR buffer (P < 0.001). {dagger}Post hoc difference (P < 0.05). Each point indicates the mean ± SE of observations on 3 muscles (A and B) or on 6 muscles (C and D).

 

Figure 5
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  		Fig. 5. Fractional 86Rb+ loss from soleus muscles at rest during incubation in either pinacidil or high HCO3–. Experimental conditions as in Fig. 4. Pairs of contralateral muscles were mounted between electrodes and incubated either with pinacidil, high HCO3–, or in standard KR buffer equilibrated with 5% CO2 in O2. A: incubation with pinacidil significantly increases the fractional 86Rb+ loss due to opening of KATP channels (post hoc difference, *P < 0.05 and **P < 0.01, n = 6). B: fractional 86Rb+ loss with and without HCO3–. A post hoc test showed no significant difference between control and high HCO3– at any specific time point (n = 18). Each point indicates the mean ± SE.

 
The assay was hereafter used to evaluate the effect of extracellular HCO3– on the fractional 86Rb+ loss from muscles. Figure 4, C and D, shows that 40 mM HCO3– was without effect on the fractional 86Rb+ loss in the intervals with excitation. The excitation-induced fractional 86Rb+ loss in control and HCO3–-treated muscles was 0.0039 ± 0.0002 and 0.0038 ± 0.0001 min–1, respectively, during continuous stimulation (n = 6, P = 0.19) and 0.0041 ± 0.0003 and 0.0040 ± 0.0002 min–1 during intermittent stimulation (n = 6, P = 0.79). Also, in resting muscles no effect of 40 mM HCO3– was detected in any of the experimental series (Fig. 4, C and D). To further investigate if the fractional 86Rb+ loss in resting muscles incubated in 40 mM HCO3– differed from controls, all data were grouped and analyzed (Fig. 5B). This revealed no post hoc difference between the groups at any time point.

The data presented in Figs. 4, C and D, and 5B clearly show that elevation of HCO3– did not influence 86Rb+ loss from resting or working muscles. These findings, however, do not exclude that pHi influences the K+ extrusion in isolated muscles. To further evaluate the importance of pHi and the H+ gradient for the fractional 86Rb+ loss from muscles we determined the effect of manipulations of intra- and extracellular pH on the resting and excitation-induced fractional 86Rb+ loss and compared the results to the effect of 100 µM pinacidil or 20 µM glibenclamide (an inhibitor of the KATP channels; Ref. 20, 31). Table 2 summarizes all data of the experiments. The table shows that 20 mM HCl, which acidifies the buffer (15), had very little effect on pHi. Lactic acid (20 mM) acidifies both the buffer and cytosol (38) and, finally, increased CO2 (15%) together with an elevation in HCO3– (70 mM) reduce pHi but does not effect extracellular pH (15). Values for the fractional 86Rb+ during treatments were in all cases expressed relative to the fractional 86Rb+ loss in contralateral control muscles. The resting level was defined as the fractional 86Rb+ loss in the efflux interval preceding the interval with stimulation, and the excitation-induced fractional 86Rb+ loss was determined as the fractional 86Rb+ loss during the interval with stimulation minus the fractional 86Rb+ loss at rest. Table 2 reveals that despite the large variations in extra- and intracellular pH, there were no consistent effects on either the resting or the excitation-induced 86Rb+ loss. The only significant effect was a reduction in the resting fractional 86Rb+ loss induced by the decrease in pHi as a result of increased CO2 tension and an increased excitation-induced fractional 86Rb+ loss from muscles incubated in lactic acid.


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  		Table 2. Effects of pinacidil, glibenclamide, and pHi on the resting and excitation-induced fractional 86Rb+ loss in soleus muscles

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Effects of HCO3– on the contractility of rat skeletal muscles.   Most human studies that demonstrate an ergogenic effect of HCO3– loading evaluate performance by measuring time to fatigue (16, 42, 45) or power output (33, 41), as opposed to performance time for a specific task (26). The greatest ergogenic effect was observed in exercise regimens that exhausted the subjects in 1–7 min (26, 42). In the present study, these conditions are mimicked in an in vitro system. Isolated soleus muscles were stimulated continuously or intermittently at 30 Hz and the decline in force production and the rate of force development and relaxation during stimulation was determined. Here we use this in vitro system to investigate if the above described ergogenic effect of HCO3– ingestion is a direct consequence of increased extracellular HCO3– on the muscles. Groups of muscles were preincubated for 30 min in buffer containing 40 mM HCO3–, a level close to the plasma HCO3– observed in many studies after ingestion of HCO3– in humans (36, 42). The use of 30-min preexposure of muscles to elevated HCO3– is short compared with human studies where ingestion of HCO3– often starts hours before the beginning of exercise. In the human studies, however, the exposure of the muscles to elevated HCO3– is delayed by the time it takes for HCO3– to accumulate in the blood stream after ingestion.

In the present study, acute changes in buffer HCO3– at 30°C did not significantly improve the maintenance of force in rat soleus muscles. Likewise, we found no ergogenic effect of HCO3– in EDL muscles stimulated at 60 Hz. When we in the present study investigated the effect of increased buffer HCO3– on pHi, we found little effect. To examine if an intracellular alkalinization could have an ergogenic effect, the buffer and the muscle were alkalinized by a decreased CO2 fraction in the gas bubbling the buffer and the stimulation regimen was repeated. This, however, also failed to produce an ergogenic effect. Indeed, when the CO2 fraction was decreased to the extent that it produced the same changes in buffer pH as 40 mM HCO3–, absolute force and force maintenance was very depressed compared with control.

An experimental temperature of 30°C was chosen to limit hypoxia in the muscles. Westerblad et al. (56) found, however, that the effect of pH on contractile performance decreased with rising temperature. Thus we repeated the experiments on soleus muscles where muscles were stimulated continuously at 37°C. In agreement with these findings, no ergogenic effect of high HCO3– was detected when the temperature was risen to 37°C. Furthermore, neither rate of force development nor rate of force relaxation were affected by the increase in extracellular HCO3–, which also supports that HCO3– is without effect on the fatigability in isolated muscles.

The lack of effect in both EDL and soleus muscles on force production indicates that this is the case for both type I and type II fibers. Similar results have been observed in previous studies on skeletal muscle, using different muscle preparations and other stimulations protocols and measurements. Thus our study supports the findings of Spriet et al. (46) and Lindinger et al. (28), who failed to find any increase in tetanic force production with increased HCO3– in a perfused rat hindlimb preparation. Likewise, Mainwood and Cechetto (30) could not demonstrate an ergogenic effect of high HCO3– in rat diaphragm muscles stimulated in vitro.

Effects of HCO3– on the regulation of pHi.   Studies in both human subjects and on isolated muscles have shown that although increased extracellular HCO3– concentration leads to an increase in extracellular pH, it has no or only a very limited effect on pHi in resting muscles (22, 36, 49). In agreement with this, it has been shown that HCO3– does not readily cross the plasma membrane of the skeletal muscle (26, 57). Despite the lacking effect of HCO3– on pHi at rest, it has been hypothesized that the increased H+ gradient across the muscle fiber membrane and the larger extracellular buffer capacity, which results from elevated extracellular HCO3–, attenuate the reduction in pHi during intense work by facilitating the extrusion of intracellular H+ via transport proteins (2, 19, 35, 36). In agreement with this, Roos and Boron (43) report an increase in the acid extrusion rate with increased extracellular HCO3– in giant barnacle muscles during intracellular acid load. Moreover, HCO3– loading increase the lactate concentration in the extracellular space during work (19, 21, 36, 41, 47, 49), and HCO3– loading has been shown to attenuate the decline in pHi during the last minute of dynamic fatiguing forearm exercise using nuclear magnetic resonance (NMR; Ref. 36). It should be noted however, that whereas the effects of increased extracellular HCO3– on lactate extrusion may indeed be the result of improved conditions for lactate transport out of the muscle fibers, others have argued that when metabolic alkalosis is imposed on skeletal muscles it creates a mismatch between glycogenolysis and the maximal pyruvate dehydrogenase activity (21). The necessary regeneration of cytosolic NAD+ in the face of the increased glycogenolysis leads to an increase in the lactate production, which also may explain the elevated lactate efflux during work.

In the present study, the effect of elevation of extracellular HCO3– on pHi was determined in the surface fibers of intact muscle. Since these fibers are readily exposed to the incubation medium, the elevation of buffer HCO3– can be assumed to improve the conditions for the pHi regulation of these fibers. Despite this, the increase in buffer HCO3– did not affect either the magnitude of the initial increase in pHi or the magnitude of the following reduction in pHi during contractions. This indicates that in rat soleus muscles the extrusion of intracellular H+ was not significantly affected by the increase in the H+ gradient and the extracellular buffer capacity induced by high extracellular HCO3–. Likewise the rate of the decrease in pHi was independent of buffer HCO3–. This finding supports a study by Linossier et al. (29) who reported that alkalinization after citrate ingestion in human subjects preceding supramaximal exercise resulted in similar pHi as the control before and after exercise. Likewise a study by Bouissou et al. (8) found no differences in intramuscular pHi during dynamic exercise in metabolically alkalinized human subjects compared with control. This conclusion is, however, at some variance with studies by Nielsen et al. (36) and Forbes et al. (18) who, in NMR studies on humans, both found that HCO3– loading led to an attenuated pHi decline during the last minute of dynamic forearm exercise. The reason for this difference may be related to differences between species, muscle preparations, and the fatigue protocol.

Effects of high extracellular HCO3– concentration and pH on K+ efflux in resting and contracting muscles.   It has been argued that the ergogenic effect of HCO3– found in some studies on humans is caused partly by an attenuation of the exercise-induced rise in extracellular K+. This suggestion is based on the finding that ingestion of citrate or HCO3– significantly reduces the accumulation of K+ in the interstitium (51) and the venous blood of working muscles (45). Moreover, a relationship between the reduction in muscle pH and the increase in interstitial K+ during intense exercise has been observed in humans (34). The mechanism that leads to this relation between pH and the contraction-induced K+ loss is unknown but a key role for the abundant muscular KATP channels has been proposed repeatedly. KATP channel opening depends on, among other things, the intracellular ATP concentration and on pHi. ATP inhibits KATP channel opening, whereas H+ decreases the inhibitory effect of ATP on the channels, thereby increasing channel open probability (14, 48). In addition, changes in the intracellular concentration of H+ have been shown to affect the open probability of the channel directly (1).

In the present study, we examined the effect of HCO3– on the efflux of K+ from muscles and the possible involvement of KATP channels by determining the fractional loss of 86Rb+. This loss is the result of a unidirectional 86Rb+ flux, which depends on the K+ permeability of the fibers and the driving force for 86Rb+. Thus, theoretically, changes in the open probability of the KATP channels should cause a change in the fractional 86Rb+ loss, as hypothesized by several authors (45, 51). Indeed, as shown in Fig. 4, opening of the KATP channels by incubation with pinacidil increased the fractional 86Rb+ loss, especially in the contracting muscles. Similar observations were made by Matar et al. (31). In contrast, closure of the KATP channels by incubation with glibenclamide had no effect on the fractional 86Rb+ loss from resting muscles (Table 2), suggesting that the KATP channels were closed at rest. This observation tallies with studies on isolated muscles but is inconsistent with a study on perfused rat hindlimb muscles where the majority of the K+ efflux was sensitive to glibenclamide (27) and a study on humans where the interstitial K+ concentration of muscles could be reduced by infusion of the inhibitor (37). Importantly, however, the present study also showed that glibenclamide was without effect on the fractional 86Rb+ loss from contracting isolated muscles (Table 2). This suggests that KATP channels are not involved in the contraction-induced K+ efflux from muscles, which tallies the conclusions of both studies on isolated muscles and humans performing intense exercise (37).

To evaluate whether the relationship between pH and the accumulation of extracellular K+ observed in humans performing intense exercise (34, 45, 51) was related to an increased efflux of K+, the fractional 86Rb+ loss was examined in muscles where intra- and extracellular pH had been modified. These experiments showed that HCO3– had no effect on the fractional 86Rb+ loss, indicating that the reduced work-induced accumulation of extracellular K+ observed in humans after HCO3– or citrate loading (45, 51) was not related to a reduction in the efflux of K+ from the muscles. In accordance with this, Sostaric et al. (45) concluded that HCO3– loading actually increased the efflux of K+ from the muscles but at the same time improved their reuptake of the ion.

The only significant effect of manipulations of pH on the fractional 86Rb+ loss (Table 2) was observed in contracting muscles when both the intra- and extracellular compartments were acidified using lactic acid, and in resting muscles where only the pHi was acidified using high PCO2 in combination with high extracellular HCO3–. However, lactic acid increased the fractional 86Rb+ loss, whereas high PCO2 decreased the 86Rb+ loss. Furthermore, the increase in the 86Rb+ loss with lactic acid was small compared with the effect of pinacidil. In these experiments, the actual pHi during contractions was not measured. Since, however, the acidification produced by lactic acid or 15% CO2 preceded the contraction of the muscles, it is most likely that the treatments led to substantially lower pHi than after contraction alone. Together these findings indicate that even large reductions in pHi, as those seen during exhaustive exercise, only produce minor effects in the excitation-induced K+ loss.

To summarize, increased extracellular HCO3– concentration did not have ergogenic effects in isolated skeletal muscles. Moreover, exposure to high HCO3– did not affect the pHi changes that occurred during contractions. In addition, HCO3– did not decrease the K+ efflux either at rest or during stimulation. A further investigation of the effects of pH on K+ efflux revealed no consistent conclusion, but did suggest that the KATP channels could not be opened by a reduction in muscle pH within the physiological range. These findings indicate that other mechanisms than those proposed to occur in the muscles may constitute the ergogenic effect of HCO3– observed in integral exercise studies.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This study was supported by a grant from the Danish Medical Research Council (j.nr. 272-05-0304).


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We thank Marianne Stürup Johansen, Tove Lindahl Andersen, Vibeke Uhre, and Ann-Charlotte Andersen for skilled technical assistance. We also thank Prof. Christian Aalkjær for advice and helpful discussion about measurements of intracellular pH.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Broch-Lips, Institute of Physiology and Biophysics, Ole Worms Allé 1163, 8000 Aarhus C, Univ. of Aarhus, Denmark (e-mail: mbl{at}fi.au.dk)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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