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J Appl Physiol 92: 93-99, 2002;
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Vol. 92, Issue 1, 93-99, January 2002

Effect of eccentric contraction-induced injury on force and intracellular pH in rat skeletal muscles

E. W. Yeung1, J.-P. Bourreau2, D. G. Allen3, and H. J. Ballard2

1 Department of Rehabilitation Sciences, Hong Kong Polytechnic University, Hung Hom, Kowloon; and 2 Department of Physiology, University of Hong Kong, Hong Kong; and 3 Department of Physiology and Institute for Biomedical Research, University of Sydney, Sydney, New South Wales 2006, Australia


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effect of eccentric contraction on force generation and intracellular pH (pHi) regulation was investigated in rat soleus muscle. Eccentric muscle damage was induced by stretching muscle bundles by 30% of the optimal length for a series of 10 tetani. After eccentric contractions, there was reduction in force at all stimulation frequencies and a greater reduction in relative force at low-stimulus frequencies. There was also a shift of optimal length to longer lengths. pHi was measured with a pH-sensitive probe, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein AM. pHi regulation was studied by inducing an acute acid load with the removal of 20-40 mM ammonium chloride, and the rate of pHi recovery was monitored. The acid extrusion rate was obtained by multiplying the rate of pHi recovery by the buffering power. The resting pHi after eccentric contractions was more acidic, and the rate of recovery from acid load post-eccentric contractions was slower than that from postisometric controls. This is further supported by the slower acid extrusion rate. Amiloride slowed the recovery from an acid load in control experiments. Because the Na+/H+ exchanger is the dominant mechanism for the recovery of pHi, this suggests that the impairment in the ability of the muscle to regulate pHi after eccentric contractions is caused by decreased activity of the Na+/H+ exchanger.

muscle injury; eccentric exercise; pH regulation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ECCENTRIC CONTRACTION (EC), in which the muscle is stretched while contracting, results in delayed-onset muscle soreness, which peaks ~24 h after the muscle activity (6, 16). Eccentric muscle contractions have potential benefits in athletic training programs, because they result in a greater force generation at a lesser energy consumption (30). The role of EC in muscle injury and the potential benefits in training programs have stimulated interest in the mechanisms involved.

In humans, it is well documented that the development of muscle soreness is associated with reduced force production (6, 8, 9, 16) and limitation in the range of movement (6-8). In both animal and human studies, structural damage, including myofilament disruption (11, 15) and muscle swelling (12), have been observed. Muscle cells undergo some loss of the cytoskeletal proteins, such as titin and desmin, and fibronectin from the plasma is deposited inside the fibers, indicating a loss of cellular integrity (6, 19, 20). Inflammation occurs, resulting in phagocyte infiltration to the injured muscle, and protein degradation becomes elevated ~48 h after the injury (23). Intracellular proteins, such as creatine kinase and glutamic oxaloacetic acid transaminase, are released into the blood (12). It has been shown that the damage may last for as long as 14 days before full recovery (23).

The increase in the plasma concentrations of intramuscular enzymes such as creatine kinase suggests that the damage leads to leakiness of the muscle membranes (23) associated with structural damage to the sarcolemma (35). Such a loss of sarcolemmal integrity would disturb the distribution of ions across the membrane, resulting in membrane depolarization, inactivation of sodium channels, and reduced conduction and amplitude of the action potential. If action potentials are disrupted, this could contribute to the failure of the excitation-contraction coupling process, which can occur (5, 14, 35). A reduction in the sarcolemmal lactate-H+ transport capacity (29) and the glucose transporter GLUT-4 protein content (3, 4) of rat and human muscles after unaccustomed eccentric exercises has been reported. Activation of the stretch-activated ion channels producing an increase in Na+ permeability after eccentric exercise has also been reported recently (24). These studies provide evidence of possible sarcolemmal damage after exposure to EC. In addition, the reduced lactate-H+ transport raises the possibility that pH regulation may be impaired.

It is well established that cellular pH homeostasis in skeletal muscle relies on the balance between H+ accumulation (H+ influx and metabolic acid production) and H+ removal mediated by transporters located at the sarcolemma (18). In resting muscles, the Na+/H+ exchanger (NHE) accounts for 80% of the H+ efflux (2). If membrane damage is significant after EC, then the activity of pH-regulating pathways in the membrane might be affected, resulting in abnormal intracellular pH (pHi) regulation.

This study was undertaken to examine the pHi change in the early events of EC-induced injury in rat skeletal muscles. The presence of injury was established by a decrease in force production. Our hypothesis was that alterations in pHi regulation may occur as a consequence of EC-induced injury.

A preliminary account of the experiments has been communicated at the FASEB meeting (37).


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Muscle fiber dissection and mounting. Experiments were performed on soleus muscle bundles dissected from Sprague-Dawley rats weighing 200-250 g. The rats were housed two per cage in an environmentally controlled room maintained at 23°C with a 12:12-h dark-light cycle. The animal care procedures and the experimental protocol were approved by the Committee on the Use of Live Animals in Teaching and Research (No. 335-99) of the University of Hong Kong.

The rat was anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg). The skin overlying the dorsal aspect of the hindlimb was dissected open from the thigh to the ankle. Gastrocnemius and plantaris muscles were deflected and removed, exposing the soleus muscle. The proximal and the distal tendons of the soleus muscle were carefully isolated by cutting the fine connective tissues that join it to the underlying muscles. The muscle was then rapidly transferred to the dissection chamber. When both soleus muscles had been removed from the hindlimbs, the rat was killed with an overdose of pentobarbital sodium.

Soleus muscle bundles containing three to five cells were dissected under a stereomicroscope using forceps and fine-tipped ophthalmic scissors. The bundles from the soleus muscle ranged in length from 10 to 13 mm and in width from 0.1 to 0.2 mm. During dissection, point electrical stimulation was applied intermittently to identify healthy muscle bundles. Care was taken to ensure that the debris from the damaged fibers was removed from the surface of the live muscle fiber. This is important because the dead tissues at the periphery of the muscle fiber will create background problems with fluorescent dye loading, thus producing artifacts during the fluorescence measurements.

After dissection, the proximal and distal tendons were trimmed down and were gripped with aluminum foil microclips. The fiber was then transferred to an experimental chamber (capacity 1.5 ml), which allowed for simultaneous measurements of force and fluorescence. One end of the fiber was attached to a hook connected to the lever arm of a position feedback motor (300B-LC, Aurora Scientific). The motor allowed known length changes to be imposed on the muscle fiber. The other end of the muscle fiber was attached to the force transducer (BG-10g, Kulite Semiconductor Products) via a glass extension with a fine hook at the end. The force transducer was clamped to the mechanical micromanipulator (M-3333, Narishige), allowing muscle length to be adjusted to the nearest 10 µm.

Solutions. The dissection was performed at room temperature (22-24°C) in a Krebs solution with the following composition (in mM): 136 NaCl, 5 KCl, 1.8 CaCl2, 0.5 MgCl2, 11.9 NaHCO3, and 0.4 NaH2PO4. This solution was bubbled with 95% O2 and 5% CO2, maintaining a pH of 7.4.

Muscle stimulation. The bundle of muscle fibers was stimulated with adjustable platinum-plate stimulating electrodes that ran parallel to the experimental chamber. Contractions were elicited via an isolated stimulator (S48, Grass Instruments) using 0.5-ms duration at an intensity of 1.2× threshold. Tetani were 400 ms in duration, and the standard stimulation frequency was 100 Hz.

Measurement of pHi. The fluorescent pH indicator 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) AM (B1150, Molecular Probes) was used to measure pHi. BCECF is a dual-excitation, single-emission fluorescent pH indicator. pHi measurements with BCECF were made by determining the pH-dependent ratio when the dye was excited at 490 and 440 nm; the emission intensity at 535 nm was used to calculate the ratio of excitation at 490 to 440 nm. This ratio was converted to pHi using the method of Grynkiewicz et al. (13). The muscle bundle was loaded by incubation in a 10 µM solution of BCECF for 30 min at 37°C with extracellular pH maintained at 7.4. After loading, the preparation was mounted into the experimental chamber and washed with Krebs solution superfused at a flow rate of 1 ml/min for 30 min.

pHi within the muscle fiber using BCECF was measured with the use of the fluorescence spectrometer system (DeltaScan 4000, Photon Technology International). The fluorescence ratio and force data were collected at a sampling rate of 20 Hz using the data-acquisition software of the spectrometer (Photon Technology International). The signals were displayed on-line for visual inspection during the experiment. The raw data were then stored for later analysis. A manual-controlled shutter was available to prevent photobleaching of the indicator. The two other sources of fluorescent light are background fluorescence and autofluorescence. Background fluorescence was determined by moving the muscle fiber out of the field of view at the beginning of the experiment. This was automatically subtracted from the raw signal values before calculation of the fluorescence ratio. The background values remained constant throughout the experiment. Autofluorescence arises from fluorescent cellular constituents such as NADH. Autofluorescence was measured in three muscle bundles before loading of BCECF and was found to be <4% of the fluorescent signal after loading. This was too small to significantly affect the signals.

Calibration of pHi and force signals. In situ calibration of BCECF was achieved by exposing the muscle fiber to nigericin in the presence of a K+ concentration similar to the intracellular K+ concentration (17). Nigericin, an ionophore, abolished the transmembrane intracellular H+ concentration gradient and was used to set the pHi equal to the extracellular pH. The muscle fiber was first exposed to 10 µM nigericin in the following solution (in mM): 160 KCl, 0.5 MgCl2, 1.2 KH2PO4, and 10 HEPES at 22°C. The muscle fiber was then exposed to solutions with a variety of pH values ranging from 5.0 to 9.0, and the fluorescence signals were obtained once the ratio had stabilized. A calibration curve with the fluorescence ratio plotted against the pHi of the solution was obtained.

Muscle contraction protocol. The muscle bundles were set to the length that gave optimal tetanic tension [optimal length (Lo)]. Force signals were converted to force per unit area (Pa or N/m2). The muscle cross-sectional area was estimated with the aid of the eyepiece micrometer (MBM12100, Nikon), and the force per area was expressed in kilopascals. Length changes made during the eccentric experiments were determined with respect to Lo.

A force-frequency curve was constructed by stimulating the fiber at frequencies of 10, 20, 40, 70, and 100 Hz. Each fiber then received a control series of ten 100-Hz isometric contractions (IC) at 4-s intervals. Another force-frequency curve was then constructed. In the EC protocol, the muscle fiber was stretched by 30% Lo over a 150-ms period starting 250 ms after the start of each tetanus. The fiber was then returned to its original length over 400 ms after the end of the tetanus. This was repeated for 10 tetani. To account for any shift in the length-tension relationship, we remeasured Lo after the EC protocol and then reset the muscle to the new Lo. The force-frequency relation post-IC was determined at the new Lo. Normalized force-frequency relations (see Fig. 2B) were normalized to the maximum force at the current Lo.

pHi regulation. The mechanism of pHi regulation was studied by perturbing the steady conditions with a standard acid load. NH4Cl (20-40 mM) was applied for 3-5 min, causing a transient alkalosis; the NH4Cl was then removed, causing a substantial acidosis from which the muscle then recovered. The rate of pHi recovery from this acidosis was measured. pHi recovery after an acid load was examined twice: after the IC protocol and then again in the same fiber after the EC protocol when the fiber had been reset to the new Lo. To avoid photobleaching, measurement of pHi after removal of NH4Cl was recorded for 10 min and then at 5-min intervals.

Intracellular buffering power (beta ) was estimated by using the technique described by Vaughan-Jones and Wu (33). This is defined as
&bgr;(mM<IT>/</IT>pH unit)<IT>=&Dgr;</IT>[NH<SUP>+</SUP><SUB>4</SUB>]<SUB>i</SUB><IT>/&Dgr;</IT>pH<SUB>i</SUB>
where Delta [NH<UP><SUB>4</SUB><SUP>+</SUP></UP>]i refers to the amount of intracellular NH<UP><SUB>4</SUB><SUP>+</SUP></UP> at the end of the NH4Cl exposure, and Delta pHi is the difference between pHi at the end of the NH4Cl exposure and immediately after NH4Cl removal. [NH<UP><SUB>4</SUB><SUP>+</SUP></UP>]i was calculated as
[NH<SUP>+</SUP><SUB>4</SUB>]<SUB>i</SUB><IT>=</IT>[NH<SUP>+</SUP><SUB>4</SUB>]<SUB>o</SUB><IT>×</IT>10<SUP>(pH<SUB>o</SUB><IT>−</IT>pH<SUB>i</SUB>)</SUP>
where subscript o denotes extracellular. Acid extrusion rate was calculated by multiplying the buffering power by the rateof pHi recovery
Acid extrusion rate<IT>=&bgr;×</IT>(dpH<IT>/</IT>d<IT>t</IT>)
where dpH/dt is the slope of pHi recovery measured at various pHi over the range 6.75-6.95 (see Fig. 7).

Statistics. Force generation and Lo were compared by the use of Student's paired t-tests. To compare the recovery of pHi after NH4Cl after isometric and eccentric tetani, the rate of recovery was determined over the same pHi interval. The significance level was set at P = 0.05. The values are expressed as means ± SE.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In all of the experiments, measurements on force-frequency relation and pHi recovery from acid load were performed 10 min post-EC and after the muscle had been stretched to the new Lo.

Contractile properties of the muscle fibers. Figure 1 shows force records at different stimulus frequencies from one fiber post-IC (Fig. 1A) and post-EC (Fig. 1B). Compared with the IC protocol, the EC protocol resulted in a reduction in tension at all frequencies. Figure 2A shows average force data and illustrates that the reduction of force was significant at all stimulation frequencies (10, 20, 40, 70, 100 Hz) (P < 0.01, n = 14). When tension was plotted as a ratio of the 100-Hz stimulation, the relative reduction of force was greater at low-stimulus frequencies after EC. Figure 2B shows the relationship of tension generation normalized to 100 Hz as 100% of control.


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  		Fig. 1.   Force-frequency relationship after the isometric contraction (IC; A) and eccentric contraction (EC; B) protocols. Tracings are of the force signals for tetanic stimulation at 10, 20, 40, 70, and 100 Hz.



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  		Fig. 2.   A: force-frequency relationship post-IC and post-EC protocols (n = 14). B: relative tension plotted against different stimulus frequency normalized to 100% for the 100-Hz point. Significant differences in the force-frequency relationship between control and EC, * P < 0.01.

To determine whether any of the reduction in force after EC was due to muscle fatigue, we performed two sets of 10 IC tetani and measured for any reduction of force after each set of tetani. There was no significant difference in the force-frequency relation observed (P ~ 0.73, n = 4). Furthermore, stretching an inactive muscle fiber did not result in any change in force generation.

As a result of EC protocol, the Lo of the muscle fibers was increased. Figure 3 shows the length-tension relationship of one muscle bundle before and after the EC protocol. It is clear that Lo was greater after the EC protocol, and the maximum tension generated was also reduced. In the 15 fibers studied (Fig. 4), there was a significant shift in Lo of 14.8 ± 2.0% (P < 0.0001) after the EC protocol.


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  		Fig. 3.   Relative force of an individual muscle fiber plotted against the relative fiber length, where the new optimal length was redefined post-EC protocol.



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  		Fig. 4.   Optimal length of the muscle fiber post-EC was significantly longer than before the experiment, * P < 0.0001 (n = 15).

pHi regulation. The mean pHi recorded in Krebs solution buffered with 5% CO2 under control conditions was 6.98 ± 0.04. Five minutes after IC, pHi was 6.97 ± 0.04 and was relatively stable, whereas, after EC, the pHi was significantly more acidic (after 5 min, pHi = 6.80 ± 0.06; P < 0.05; n = 17). pHi recovery after an acid load produced by NH4Cl was examined after IC and then again in the same fibers after EC. The rate of recovery of pHi from an acid load after EC was significantly slower (P < 0.05) than after IC. pHi recovered at an average rate of 0.022 ± 0.003 U/min after IC and 0.013 ± 0.002 U/min after EC. In the muscle fibers studied, pHi recovery from an acid load was 50% complete in 15.9 ± 1.7 and 23.3 ± 1.9 min post-IC and post-EC, respectively. Figure 5 shows the time course of pHi changes and recovery post-IC and post-EC conditions.


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  		Fig. 5.   Time course of intracellular pH (pHi) recovery from NH4Cl acid load post-IC (A) and post-EC (B) protocols.

To rule out the possibility that the rate of pHi recovery may become slower in subsequent exposures, intracellular acid load with 40 mM NH4Cl was performed on four muscle fibers twice, each time after 10 isometric tetani. The rate of pHi recovery was almost identical, recovering at a rate of 0.022 ± 0.002 U/min after the second IC protocol (P ~ 0.7). Furthermore, the rate of pHi recovery from acid load was measured and compared when the fiber was held at Lo and at 1.3 Lo, both after 10 IC. The rate of recovery at 1.3 Lo (0.023 ± 0.003 U/min) was not significantly different from that at Lo (P ~ 0.51, n = 4).

We next studied the effect of an NHE inhibitor on the rate of pHi recovery from acid loading by exposing the fiber to 0.1 mM amiloride. As a result of the amiloride application, the rate of pHi recovery from acidosis was significantly inhibited to a rate of 0.0012 ± 0.0006 pH U/min (P < 0.005, n = 4). On removal of amiloride, pHi recovered back fully to 7.09 ± 0.03 at a maximum rate of 0.022 ± 0.003 pH U/min, comparable to muscle fibers under normal conditions (Fig. 6).


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  		Fig. 6.   pHi recovery in the presence of 0.1 mM amiloride in control experiments. The addition of amiloride after the removal of NH4Cl significantly slowed pHi recovery. On removal of amiloride, the muscle fiber was able to recover back to resting pHi.

The acid extrusion rate (mmol · l-1 · min-1) was calculated for each fiber within the pHi range of 6.5-7.2 and plotted as a function of pHi (Fig. 7). The intracellular buffering power was assessed for each individual muscle fiber. There was no significant difference (P ~ 0.23, n = 17) in the estimated buffering power post-IC (73.1 ± 11.2 mM) and post-EC (76.8 ± 12.2 mM). The acid extrusion rate was smaller at all pHi measured (Fig. 7). At the mean pHi of 6.94, the acid extrusion rate post-IC (2.27 ± 0.43) was significantly greater (P < 0.05, n = 17) than that post-EC (1.71 ± 0.23).


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  		Fig. 7.   Acid extrusion rate of an individual muscle fiber recovering from an intracellular acidosis after 10 IC () and EC (black-triangle) protocols.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of this study was to determine whether EC affected pHi regulation. We show that EC protocol causes a significant acidosis from which recovery is very slow. Furthermore, recovery from an applied acid load was also slowed. We also confirm earlier studies in which eccentric damage leads to a reduction in force generation (5, 21, 22, 28) and a shift of the Lo (28, 32, 36).

Contractile properties of the muscle. For all of the fibers studied, tension was reduced at all stimulation frequencies post-EC. This reduction in tension was not caused by fatigue, because the same force-frequency relation was observed with two sets of 10 isometric tetani. Furthermore, stretching unstimulated muscle fibers did not affect the force. Thus it seems reasonable to suggest that the EC were responsible for the force deficit. We observe a greater force deficit at low-stimulation frequencies. However, the magnitude of deficit was smaller compared with other human (16) and animal (25) studies. The greater force reduction in these studies may be a consequence of shorter length of active sarcomeres, where the shift of Lo was not taken into consideration (27). In our experiments, the shift in the length-tension relationship was accounted for by readjusting to the new Lo, and this may be the possible explanation of such difference.

To account for the contribution of the shift of Lo, we measured the new Lo again after EC. This shift by 14.8% in our experiments is similar to that reported previously in one study on frog single-muscle fibers (28). Morgan (26) proposed the "popping sarcomere hypothesis" in which EC on the descending limb of the length-tension curve can cause selected half-sarcomeres to be preferentially lengthened when they exceed their yield point. During subsequent contractions, the disrupted sarcomeres are stretched to longer lengths before the attainment of peak tension, thus explaining the shift in the length-tension relationship (28). Furthermore, extra load is placed on adjacent sarcomeres because of the disruption of these half-sarcomeres, more likely causing them to overextend (pop) in the subsequent EC. If the disruption is large enough (27), it may lead to tearing of sarcolemma, T tubules, or sarcoplasmic reticulum, and this may be the cause of the failure of Ca2+ release observed in some eccentrically damaged fibers (5, 14, 35). The failure of the damaged sarcomeres to produce tension may explain the force deficit after EC, even after the muscle is readjusted to the new Lo. The possibility that EC induces disruption to the membrane systems involved in excitation-contraction coupling is supported by a recent study (31) showing distortion of the T tubules after eccentric muscle damage.

pHi regulation. The resting pHi after EC is more acidic than the pHi in postisometric controls. As observed, the pHi was ~0.17 lower after EC than IC. There was also significant slowing of the rate of recovery from acidosis induced by application and removal of NH4Cl. In some experiments, the acid load post-EC was performed by using 20 mM instead of 40 mM of NH4Cl to ensure that the pHi recovery could be measured over the same pH range as that of the IC. The Na+-H+ transport system is the dominant mechanism for the recovery of pHi (18), and we confirmed this in the present experiments by showing that amiloride, an established NHE1 inhibitor, could greatly reduce the rate of recovery of pHi after an acid load.

The present estimate of buffering power (75 mM at pHi 6.94, 22°C) is higher than the values reported in mammalian skeletal muscles (58 mM at pHi 7.23, 28-37°C) (1), which may be ascribed to the difference in temperature, cell preparation, and methodology. Pilegaard and Asp (29) showed that the lactate-H+ transport activity and the buffer capacity were reduced 2 days after EC. However, in our experiments, the EC did not have a significant effect on the buffering power. A possible reason is that, in our experiment, the buffering power was measured only 10 min after EC. The reduction in buffering power described after 2 days (29) is probably a consequence of delayed loss of proteins. In the present study, we have not tested the involvement of lactate transport using inhibitors such as cinnamate. Given that the lactate transport plays an important role for proton extrusion under fatigue conditions (18), the present contraction protocol is unlikely to cause significant accumulation of lactic acid. The question of whether lactate transport becomes ineffective at the early phase of eccentric injury will need to be tested further.

The underlying cause for the acidosis after EC is not known. There are two main possibilities: one is the production of acid products, e.g., lactic acid is increased after EC, and the other is that the mechanism(s) that extrudes protons from the cell has become less effective. The fact that the rate of pHi extrusion after an exogenous acid load is reduced points to the second explanation. Because it is established, and we have confirmed, that the NHE is the main pathway for acid extrusion, this suggests that its activity has been reduced. There are many possible reasons for the reduction in NHE activity. 1) There may be a direct reduction of NHE activity by a local metabolite or paracrine or autocrine influence (for review of regulation of NHE, see Ref 34). 2) There may be damage to T tubules, resulting in sealing over of some T tubules so that some NHEs are no longer in contact with extracellular solution. 3) There may be increased permeability of the surface membrane to protons, so that the efflux of the NHE is effectively reduced by an inward leak of protons. 4) There may be an increase in intracellular Na+ associated with membrane damage so that the inward Na+ gradient is reduced, and, hence, there is a reduction in proton efflux. Presently, we have no evidence to distinguish among these and other possibilities.

This study is concerned with the initial phase of eccentric muscle injury, and we show that EC lead to intracellular acidosis and affect pHi regulation. How these changes in pHi regulation contribute to the early functional changes observed after EC is unclear at present; furthermore, the change in pHi may be implicated in the later development of the injury. For instance, an interesting possibility is that the reduced Ca2+ sensitivity of the myofibrillar proteins (5) might be caused by a direct effect of the acidosis on the myofibrillar proteins (10). The susceptibility to EC-induced injury might potentially be modified by alterations to the pHi. Further work is required to understand the mechanisms that lead to EC-induced injury.

In conclusion, we show that both resting pHi and recovery from an acid load are modified in rat soleus muscle after EC. Our evidence suggests that the impairment is caused by decreased activity of the NHE. As a consequence, the ability of the muscle to regulate its pHi is impaired.


    ACKNOWLEDGEMENTS

This study was supported by Hong Kong Polytechnic University Department Research Fund Grant G-S497.


    FOOTNOTES

The work described will be submitted to the University of Hong Kong by E. W. Yeung as part of her doctoral thesis.

Address for reprint requests and other correspondence: E. W. Yeung, Dept. of Rehabilitation Sciences, Hong Kong Polytechnic Univ., Hung Hom, Kowloon, Hong Kong (E-mail: rsella{at}polyu.edu.hk).

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.

Received 10 May 2001; accepted in final form 29 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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J APPL PHYSIOL 92(1):93-99
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