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J Appl Physiol 89: 200-209, 2000;
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Vol. 89, Issue 1, 200-209, July 2000

Forearm muscle metabolism studied using 31P-MRS during progressive exercise to fatigue after Acz administration

John M. Kowalchuk1,2,4, Shelly A. Smith1, Brad S. Weening1,3,4, Greg D. Marsh1,3,4, and Donald H. Paterson1,4

1 The Centre for Activity and Ageing, School of Kinesiology and Departments of 2 Physiology and 3 Medical Biophysics, University of Western Ontario, London, Ontario N6A 3K7; and 4 Lawson Research Institute, St. Joseph's Health Centre, London, Ontario, Canada N6A 4V2


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of acetazolamide (Acz)-induced carbonic anhydrase inhibition (CAI) on muscle intracellular thresholds (T) for intracellular pH (pHi) and inorganic phosphate-to-phosphate creatine ratio (Pi/PCr) and the plasma lactate (La-) threshold were examined in nine adult male subjects performing forearm wrist flexion exercise to fatigue. Exercise consisted of raising and lowering (1-s contraction, 1-s relaxation) a cylinder whose volume increased at a rate of 200 ml/min. The protocol was performed during control (Con) and after 45 min of CAI with Acz (10 mg/kg body wt iv). TpHi and TPi/PCr, determined using 31P-labeled magnetic resonance spectroscopy (MRS), were similar in Acz (722 ± 50 and 796 ± 75 mW, respectively) and Con (855 ± 211 and 835 ± 235 mW, respectively). The pHi was similar at end-exercise (6.38 ± 0.10 Acz and 6.43 ± 0.22 Con), but pHi recovery was slowed in Acz. In a separate experiment, blood was sampled from a deep arm vein at the elbow for determination of plasma lactate concentration ([La-]pl) and TLa-. [La-]pl was lower (P < 0.05) in Acz than Con (3.7 ± 1.7 vs. 5.0 ± 1.7 mmol/l) at end-exercise and in early recovery, but TLa- was higher (1,433 ± 243 vs. 1,041 ± 414 mW, respectively). These data suggest that the lower [La-]pl seen with CAI was not due to a delayed onset or rate of muscle La- accumulation but may be related to impaired La- removal from muscle.

intracellular threshold; acid-base; acetazolamide; lactate; intracellular pH


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CARBONIC ANHYDRASE (CA) catalyzes the reversible hydration of CO2 to bicarbonate and a proton. The importance of CA for the efficient transport and elimination of CO2 from tissues and the lungs is well documented (see Refs. 13 and 38), whereas the effects on muscle metabolism are less well understood. A lower plasma lactate concentration ([La-]pl) was observed during recovery from 30 s of high-intensity cycle ergometer exercise during both acute (16) and chronic (17) acetazolamide (Acz)-induced CA inhibition, as well as during constant-load, moderate, and heavy exercise intensities and recovery (29, 32). In addition, Scheuermann et al. (31) demonstrated a rightward-shift in the [La-]pl-power output relationship during progressive leg cycling exercise to fatigue after Acz administration, resulting in a lower [La-]pl at power outputs corresponding to moderate and heavy exercise intensities. The mechanism responsible for the lower [La-]pl during exercise after Acz treatment has not been established. However, as [La-]pl represents a balance between plasma La- appearance and disappearance (21, 36), the lower [La-]pl after Acz administration must be related to either 1) increased La- uptake from blood into active and inactive muscle and other tissues, 2) increased mitochondrial oxidation of pyruvate, La-, or both, 3) reduced efflux of La- from muscle and other tissues, or 4) decreased rate of glycogenolysis/glycolysis and pyruvate production (for review on La- metabolism, see Ref. 33).

It was previously shown that, during recovery from short-term, high-intensity leg cycling exercise, the arterial-venous [La-]pl difference (a-v[La-]pl) across the inactive forearm was lower after acute Acz treatment than in controls (Con; Ref. 16). Although blood flow was not determined, the lower a-v[La-]pl with Acz suggested that La- removal from plasma by inactive muscle was reduced, being related to the lower arterial [La-]pl (16). That the rates of muscle pyruvate and La- oxidation were not affected by Acz treatment was suggested by the finding that O2 uptake kinetics and exercise O2 uptake were similar in Acz and Con during 6 min of constant-load, moderate-, and heavy-intensity exercise (29). Also, Acz treatment was shown to slow muscle intracellular pH (pHi) recovery from an intracellular acid load (6). Because recovery of intracellular acid-base balance after exercise is tightly coupled with the removal of La- (11, 15), the slower Acz-induced rate of muscle pHi recovery (6) suggests that La- removal from muscle may also be impaired after Acz treatment. Recently, Scheuermann et al. (32) demonstrated that muscle glycogen breakdown during 6 min of constant-load, high-intensity exercise was similar in Acz and Con conditions, suggesting that the rate of glycogenolysis was not impaired by Acz treatment.

When muscle metabolism was studied during progressive exercise to fatigue using 31P-labeled magnetic resonance spectroscopy (31P-MRS), a power output was identified at which there was a breaking point or threshold (T) in slopes of the pHi- and inorganic phosphate-to-phosphate creatine ratio (Pi/PCr) power output relationships (TpHi and TPi/PCr, respectively) (12, 20, 39). The increased muscle acidification associated with power outputs greater than TpHi, combined with the demonstrated quantitative relationship between pHi, muscle hydrogen concentration ([H+]), and muscle lactate concentration ([La-]) (14, 15, 26, 28), suggests that TpHi represents a power output or force above which there is a greater activation of intramuscular glycogenolysis/glycolysis (relative to muscle pyruvate and La- oxidation and removal), with subsequent muscle La- accumulation. That TpHi and TPi/PCr occurred at similar power outputs (12, 20) and agreed qualitatively with the threshold of blood La- accumulation (TLa-) and the gas exchange threshold (39) suggests that these measures describe similar intracellular processes.

In the present study, 31P-MRS and venous blood sampling were used to determine the effects of Acz-induced CA inhibition on the metabolic and acid-base responses to progressive forearm wrist flexion exercise to volitional fatigue in humans. Specifically, this study attempted to discern the mechanism responsible for the lower [La-]pl that is consistently seen during exercise after acute Acz administration. The following hypotheses were tested: 1) venous [La-]pl would be lower, and the plasma TLa- would occur at a higher power output after Acz administration, 2) TpHi and TPi/PCr, markers for the onset of increased muscle La- accumulation, would occur at higher power outputs after Acz administration, implying a delay in muscle La- accumulation, and 3) pHi would be more acidic at rest and throughout exercise and recovery after Acz administration because of impaired CO2 and La- removal from muscle.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Subjects. Adult male subjects (n = 9; age = 20-30 yr) participated in the study. All subjects were healthy, relatively active, and physically fit. The experimental procedures and all potential risks associated with participation in the study were explained, and informed consent was obtained from each subject. The study was approved by The University of Western Ontario Review Board for Health Sciences Research Involving Human Subjects.

General protocol. Subjects were studied twice during each of two conditions, Con and after Acz administration. Two exercise tests were performed during each condition to evaluate 1) muscle metabolism and acid-base status using 31P-MRS and 2) [La-]pl and acid-base status in venous blood draining the active forearm muscle using standard blood sampling techniques (Bld).

The subjects reported to the laboratory at least 4 hr after a light meal and after abstaining from caffeine-containing foods and beverages; exercise tests were performed at the same time of day for each subject. On arriving at the laboratory, subjects rested supine while a percutaneous 20-gauge Teflon catheter (Angiocath) was placed retrograde into the median cephalic vein of the dominant arm (toward the perforating vein) at the cubital fossa to facilitate blood sampling and Acz administration. After 15 min of rest, a preinfusion blood sample was drawn, followed by intravenous Acz infusion (10 mg/kg body wt over a 2-min period). The administration of Acz was randomly assigned for 31P-MRS and Bld. A placebo was not administered in this study because, in our experience, the side effects of Acz administration, although minor in discomfort, are recognizable by the subject. After an additional 45-min supine rest period, a postinfusion blood sample was drawn, and the subject's dominant arm was positioned in the wrist ergometer. The arm was extended and abducted to 90°, and the forearm was pronated with the hand grasping the contraction lever of the ergometer. The arm was aligned so that the pivot of the lever was centered on the wrist joint. With the arm in this position, the contracting forearm musculature was positioned at heart level, thus ensuring adequate perfusion during the relaxation phase of each contraction-relaxation cycle. The subjects remained supine throughout the protocol.

The exercise protocol was identical for each of the four tests and consisted of progressive wrist flexion exercise to fatigue performed on a specially built wrist ergometer (20). Exercise consisted of repeatedly depressing a lever at a frequency of 0.5 Hz (1-s contraction, 1-s relaxation), through a 70° range of motion, thereby raising and lowering a suspended reservoir a distance of 0.09 m. The reservoir was suspended outside the bore of the magnet and was connected to the lever by a cable-and-pulley system. The resistance on the wrist dynamometer was increased in a ramplike fashion by pumping water into the reservoir at a constant rate of 200 ml/min, by means of a roller pump (Cole-Parmer Instruments, Chicago, IL).

Subjects accommodated to the exercise protocol with 4 min of wrist flexion against zero load. The empty reservoir (mass = 1.07 kg) was then attached to the apparatus, and contractions continued for an additional minute. After this initial accommodation period, the exercise continued as the roller pump was started, and the load increased in relation to the volume of the reservoir. Wrist flexion exercise continued until volitional fatigue. The exercise was then stopped, and the subjects were monitored over an additional 15-min resting recovery period. Power output was calculated as the product of the mass of the reservoir plus water [1.07 kg + (flow rate × time)], the contraction frequency (0.5 Hz), and the vertical distance traveled by the reservoir per contraction (0.09 m). Thus the exercise was initiated at a work rate of 0.47 W (empty reservoir) and increased at ~0.09 W/min until fatigue. The rate of water flow delivered by the roller pump was calibrated before the start of each protocol using timed volume collection. In addition, the actual rate of water flow into the reservoir was calculated as the total water volume added to the reservoir during the exercise test divided by the time to fatigue; the average rate of flow for each of the exercise tests was similar in Con and Acz (204 ± 2 and 201 ± 3 ml/min, respectively).

31P-MRS. Forearm muscle metabolism was studied using 31P-MRS with a replica of the wrist flexion ergometer positioned in the bore of the magnet. 31P-MRS data were accumulated using a 20-cm bore, 1.89-T superconducting magnet interfaced with an SMIS console (Surrey Medical Imaging Systems, Guilford, UK). While in the supine position, the subject was positioned so that the arm extended into the bore of the magnet, and the hand grasped the lever of the wrist ergometer as described in General protocol. A 4-cm, dual-tuned surface coil was placed under the belly of the forearm, ~7-9 cm distal to the medial epicondyle of the humerus. In this position, the 31P-MRS signal obtained during the wrist flexion protocol was primarily from the flexor digitorum superficialis muscle. Proper position of the surface coil was ensured by imaging the forearm before the start of the exercise protocol using a multislice gradient echo sequence. The proton signal was used to shim the magnet homogeneity and improve spectral resolution. Homogeneity was adjusted until the full-width half-maximum of the water peak was <0.4 PPM and the peak was Lorentzian in shape.

Spectra were collected sequentially throughout the resting, accommodation, ramp exercise, and recovery periods. All spectra were acquired using a 3-ms adiabatic 90°-RF pulse, 12 µs delay time, 3.33 kHz receiver bandwidth, and 2,048 complex data points. The initial nine excitations were used to establish steady-state T1 (spin-lattice relaxation) saturation, with a total of four resting spectra collected to establish a baseline before the start of exercise. Each spectrum consisted of eight averages to give a sampling time of 24 s. As a consequence of the time required to store the acquired data, the minimum time resolution of the protocol was ~25-26 s. The 1-s pulse repetition rate ensured that the signals associated with the high-energy phosphate compounds were significantly saturated; correction factors were not applied because only the ratios of metabolites were used.

Data analysis. Quantification of metabolite data was performed in the time domain by fitting the free induction decay data to a sum of components. Each component corresponded to a resonant frequency in the spectrum and was modeled with an exponentially damped sinusoid that could be varied in amplitude, phase, time delay, damping constant, and frequency. The quantification software (1, 22) used a priori knowledge and the Levenberg-Marquardt algorithm (19) to iteratively reduce the difference between the data and the exponential model. All spectra were fit to the same template after apodization with a 2 Hz Lorentzian filter. To eliminate the very broad phosphorus components (full-width half-maximum > 100 Hz) originating from regions with large inhomogeneities or bone, the first 1.5 ms of data were not used. The area of each peak in the frequency domain, and thus its corresponding relative concentration, was taken as the amplitude of the exponential model function at time zero. beta -ATP, PCr, Pi, and Pi/PCr were calculated from these areas. The pHi was determined from the chemical shift of Pi relative to PCr (40).

The logarithms of Pi/PCr (log Pi/PCr) and pHi were each plotted against power output, and a piecewise linear regression analysis was applied to individual data plots (42). The program estimated the parameters of two regression functions and determined the threshold at which the slopes of the two lines diverged. TPi/PCr and TpHi were also visually determined by three investigators. With the investigators blinded to subject and condition, a threshold was identified by visual inspection of the log Pi/PCr- and pHi-power output plots as the power output corresponding to the point of intersection of two straight lines fit through the data. The computer- and investigator-derived thresholds were compared statistically and found to be similar; the actual T value reported represents the mean T derived from the visual inspection method. A previous study reported high test-retest correlations of 0.92 and 0.98 for identifying TPi/PCr and TpHi, respectively, demonstrating the reliability and reproducibility of these threshold measurements (20).

TLa- was identified by visual inspection of the [La-]pl-power output curve. The investigators were blinded to subject and condition, and TLa- was identified as the power output corresponding to a 1.0 mmol/l increase in [La-]pl above resting levels.

Blood sampling. During Bld studies, blood was drawn from the deep arm vein at the following times: pre- and postinfusion in Acz, preaccommodation in Con, 4 min (after zero-load contractions) and 5 min (after reservoir-only contractions) of the accommodation period, 30-s intervals during ramp protocol, the point of fatigue (0 min recovery), 1 min of the recovery period, and at 2-min intervals during recovery to 15 min. Blood was drawn into syringes containing lithium heparin, mixed, placed in ice water, and analyzed after a short delay. Whole blood samples (200 µl) were analyzed at 37°C for plasma pH, PCO2, and [La-]pl using selective electrodes (StatProfile 9 Plus blood gas-electrolyte analyzer, Nova Biomedical Canada, Mississauga, ON); the electrodes were calibrated before each test and at regular intervals during analysis. Plasma [H+] was calculated from the measured pH.

Statistical analysis. Statistical analyses were performed using SigmaStat statistical program for the PC (Jandel Scientific, San Rafael, CA). Muscle and plasma metabolic data were analyzed for main Con, Acz, and time effects using a two-way repeated-measures ANOVA. Intracellular and plasma thresholds were analyzed for main Con or Acz and 31P-MRS or Bld effects using a two-way repeated-measures ANOVA. In both instances, a significant F ratio was further analyzed using Student-Newman-Keuls post hoc analysis. Statistical significance was accepted at P < 0.05. Data are reported as means ± SD.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Exercise performance. Two series of exercise tests were performed to allow separate collection of muscle and blood metabolite data. The protocol for each series was identical in every respect except for the use of 31P-MRS to collect muscle metabolic and acid-base data and venous catheterization for blood sampling and collection of blood metabolite and acid-base data. Resistance increased in a ramplike fashion, with no difference in the rate of ramp increase (~0.09 W/min; ~200 ml/min H2O) between 31P-MRS and Bld or Acz and Con. The time to exhaustion and peak power output were greater (P < 0.05) during the ramp tests performed during Bld than during 31P-MRS; however, there was no difference between Acz and Con (Table 1) in either instance.

                              
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  		Table 1.   Time to fatigue and peak power output during ramp exercise to fatigue in the 31P-MRS and blood sampling exercise series during control and acetazolamide administration

Plasma lactate and acid-base status. The effect of Acz on the equilibrated forearm venous [La-]pl during the forearm ramp protocol is presented in Fig. 1. [La-]pl increased with increasing power output, with end-exercise [La-]pl being lower (P < 0.05) in Acz than Con (3.7 ± 1.7 vs. 5.0 ± 1.7 mmol/l), despite the similar peak power output between conditions. [La-]pl remained lower (P < 0.05) in Acz than Con during the first 3 min of recovery, after which no difference was observed between conditions. End-recovery [La-]pl in Acz and Con (1.94 ± 0.33 and 1.93 ± 0.52 mmol/l, respectively) remained elevated (P < 0.05) above Acz and Con resting levels (1.23 ± 0.36 and 1.01 ± 0.43 mmol/l, respectively). Plasma TLa- occurred at a higher (P < 0.05) absolute power output (1,433 ± 243 vs. 1,041 ± 414 mW) and percentage of peak power output (76 ± 17 vs. 53 ± 19%) in Acz than in Con, respectively (Table 2).


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  		Fig. 1.   The effect of acetazolamide (Acz) administration on forearm venous plasma lactate concentration (plasma [La-]) during incremental wrist flexion exercise to fatigue and recovery. Plasma [La-] during exercise is expressed relative to power output (A) and as a percentage of time to fatigue (B). Solid symbols, control (Con); open symbols, Acz.  and , Rest.  and open circle , Exercise and recovery (each point corresponds to mean value calculated based on 5 or more subjects in A or 8 subjects in B). black-triangle and triangle , Fatigue (each point corresponds to mean value calculated based on 8 subjects). Arrows indicate power output corresponding to lactate thresholds for CON (TLa-CON) and ACZ (TLa-ACZ).


                              
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  		Table 2.   Plasma lactate threshold and intracellular threshold for pHi and log Pi/PCr during control and acetazolamide

The effect of Acz on the equilibrated forearm venous plasma pH during the forearm ramp protocol is presented in Fig. 2. Plasma pH was not affected by Acz infusion before exercise (7.48 ± 0.13 in Acz and 7.41 ± 0.04 in Con, with [H+] = 35 ± 9 and 40 ± 4 nmol/l, respectively). Plasma pH decreased (P < 0.05) with increasing power output and reached a similar end-exercise pH in Acz (pH = 7.31 ± 0.06, [H+] = 50 ± 6 nmol/l) and Con (pH = 7.29 ± 0.05, [H+] = 51 ± 7 nmol/l-1). Plasma pH returned to preexercise values during recovery, with no consistent differences seen between conditions.


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  		Fig. 2.   Effect of Acz administration on forearm venous plasma pH during incremental wrist flexion exercise to fatigue and recovery. Plasma pH during exercise is expressed relative to power output (A) and as a percentage of time to fatigue (B).  and , Rest.  and open circle , Exercise and recovery. black-triangle and triangle , Fatigue. See Fig. 1 for other details.

The effect of Acz on the equilibrated forearm venous plasma PCO2 is presented in Fig. 3. Plasma PCO2 was similar at rest in Acz and Con (42 ± 10 and 47 ± 6 Torr). Plasma PCO2 increased (P < 0.05) abruptly at the onset of exercise with little change during moderate-intensity exercise, but a further increase occurred during heavy-intensity exercise, reaching similar end-exercise values between Acz and Con (60 ± 11 vs. 59 ± 11 Torr, respectively). Plasma PCO2 decreased to resting levels during the 15-min recovery period and showed no difference between conditions.


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  		Fig. 3.   Effect of Acz administration on forearm venous plasma PCO2 (PVCO2) during incremental wrist flexion exercise to fatigue and recovery. Plasma PCO2 during exercise is shown relative to power output (A) and as a percentage of time to fatigue (B).  and , Rest.  and open circle , Exercise and recovery. black-triangle and triangle , Fatigue. See Fig. 1 for other details.

Intramuscular metabolic and acid-base status. The effect of Acz on pHi during the forearm protocol is presented in Fig. 4. The pHi decreased (P < 0.05) with increasing power output, reaching similar end-exercise pHi in Acz and Con (pH = 6.38 ± 0.10 and 6.43 ± 0.22, [H+] = 432 ± 109 and 424 ± 186 nmol/l, respectively). The slope of the pHi-power output relationship was less (P < 0.05) below TpHi than above, but in neither instance was there any difference between conditions (Table 3). Recovery of pHi was slower in Acz than Con; pHi was lower (P < 0.05) in Acz during min 1-7 of early recovery, returning to preexercise values after 15 min in Acz and after 9 min in Con. The pHi at end-recovery was similar between Acz and Con (7.04 ± 0.06 and 7.02 ± 0.06, respectively) and to preexercise values (Acz, 7.07 ± 0.05; Con, 7.05 ± 0.06). TpHi occurred at similar absolute power outputs (722 ± 50 and 855 ± 211 mW) and percentage of peak power outputs (46 ± 7 and 49 ± 11%) in both Acz and Con, respectively (Table 2). In Acz, TpHi occurred at a lower power and percentage of peak power output than did TLa-, but there was no difference in the Con results (Table 2).


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  		Fig. 4.   Effect of Acz administration on forearm muscle intracellular pH (pHi) during incremental wrist flexion exercise to fatigue and recovery. Muscle pHi during exercise is expressed relative to power output (A) and as a percentage of time to fatigue (B). Arrows indicate power outputs corresponding to intramuscular pHi thresholds for Con (TpHi-CON) and Acz (TpHi-ACZ).  and , Rest.  and open circle , Exercise and recovery. black-triangle and triangle , Fatigue. See Fig. 1 for other details.


                              
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  		Table 3.   Regression analysis of slopes, intercepts, and correlation coefficients for pHi-power output relationship and log Pi/PCr relationship below and above the TpHi and TPi/PCr, during Con and Acz

The effects of Acz on log Pi/PCr are presented in Fig. 5. Log Pi/PCr increased with increasing power output, reaching similar end-exercise values in both Acz and Con (0.91 ± 0.54 and 0.56 ± 0.51, respectively). The slope of the log Pi/PCr-power output relationship was less (P < 0.05) below than above TPi/PCr but was similar between conditions (Table 3). Log Pi/PCr decreased to preexercise values during recovery with no difference between conditions. TPi/PCr, expressed as either absolute power output (Acz, 796 ± 75 mW; Con, 835 ± 235 mW) or percentage of peak power output (Acz, 51 ± 6%; Con, 53 ± 11%), was similar between conditions and was similar to TpHi within each condition. During Acz, TPi/PCr occurred at a lower (P < 0.05) power output and percentage of peak power output than TLa-; there was no difference between TPi/PCr and TLa- in Con (Table 2).


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  		Fig. 5.   Effect of Acz administration on forearm muscle logarithm of inorganic phosphate-to-phosphate creatine ratio [log Pi/PCr)] during incremental wrist flexion exercise to fatigue and recovery. Log (Pi/PCr) during exercise is shown relative to power output (A) and as a percentage of time to fatigue (B). Arrows indicate power outputs that correspond to intramuscular log(Pi/PCr) thresholds for Con (TPi/PCr-CON) and ACZ (TPi/PCr-ACZ).  and , Rest.  and open circle , Exercise and recovery. black-triangle and triangle , Fatigue. See Fig. 1 for other details.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study examined the muscle and forearm venous plasma metabolic and acid-base responses to progressive forearm wrist flexion exercise to fatigue before and after Acz-induced CA inhibition using a combination of 31P-MRS and blood sampling techniques. The major findings of this study were: 1) the [La-]pl-power output relationship was shifted rightward in Acz such that the venous [La-]pl was lower at fatigue and during early recovery in Acz compared with Con; 2) TLa- occurred at higher power and percentage of peak power outputs in Acz compared with Con; 3) TpHi and TPi/PCr were similar in Con and Acz and similar to each other; 4) TpHi and TPi/PCr were similar to TLa- in Con but occurred at a lower power output and percentage of peak power output compared with Acz; and 5) recovery of pHi after progressive exercise to fatigue was slower in Acz than Con.

Although the Acz infusion protocol (30-45 min before exercise) and dose (10 mg/kg body wt) used in this study were similar to those used previously (16, 17, 29, 31, 32), they did not allow us to distinguish the location of the CA isozymes that were inhibited. However, at the dose used, the calculated in vivo Acz concentration is ~1.8×10-4 M [assuming an 80-kg subject and equal distribution throughout the extracellular compartment (~25% total body water)], which should provide >99% inhibition of erythrocyte CA isozymes (CA I, CA II) and the CA isozymes associated with the muscle sarcolemma and capillary endothelium (CA IV) (7, 43). The relative insensitivity of CA III to Acz inhibition (Ki ~ 3.1×10-4 M) and the low membrane solubility and slow cellular uptake of Acz suggest that the muscle cytosolic CA III and sarcoplasmic reticular CA IV isozymes are minimally affected by this protocol (7, 43).

[La-]pl during exercise and recovery. The forearm venous [La-]pl was reduced during heavy exercise and recovery after Acz administration. This agrees with previous studies demonstrating that Acz induces a lowering of [La-]pl during various exercise protocols, including short-term, high-intensity exercise (16, 17), progressive leg cycling exercise to fatigue (31), and constant-load exercise below and above the ventilatory threshold (29, 32). The lowering of the venous [La-]pl in Acz resulted in a rightward shift of TLa- similar to that found during progressive leg cycling exercise to fatigue (31).

In the present study, venous plasma was sampled from a deep vein at the elbow in an attempt to restrict our sampling to blood drained from the exercising forearm musculature and to minimize contamination from other sources. Although the rise in venous [La-]pl appears appropriate for progressive exercise using small muscle groups, the extent that the plasma measures were influenced by blood flowing from nonworking tissues cannot be established with the techniques used in this study.

The rise in [La-]pl that occurred during exercise was determined by an imbalance between La- appearance in plasma from working muscle and other tissues and La- removal by various tissues, including active and inactive skeletal muscle, heart, liver, and kidneys (21, 36). Whereas we interpreted the appearance and increase in forearm venous [La-]pl as being due to La- release from the exercising forearm muscles, it is possible that La- released from other nonworking tissues may have also contributed to the increase in venous [La-]pl. Recently, Brooks et al. (4) demonstrated that, during constant-load leg cycling exercise, arterial [La-]pl rose at exercise onset as a consequence of an increase in leg muscle net La- release and that, with time in exercise, the arterial [La-]pl remained elevated despite a decrease toward zero in muscle La- release. These findings suggest that tissues other than working muscle were contributing to the elevated [La-]pl with continued exercise. The mechanism responsible for La- release for nonworking tissues is unknown, but a rise in circulating catecholamine levels and beta -adrenergic stimulation of muscle glycogenolysis may contribute, in part, to this response (3, 34, 35). However, with the small muscle mass exercise, relatively low power outputs, and presumably low levels of circulating catecholamines during exercise, it was suggested that stimulation of La- release from nonworking muscle would only minimally contribute to the rise in forearm venous [La-]pl seen in this study; however, this hypothesis requires further investigation.

We assumed that La- uptake by inactive muscle and other tissues was reduced in the present study because it was shown previously that, during recovery from short-term, high-intensity leg cycling exercise, La- uptake into the inactive forearm, as determined by the a-v[La-]pl difference, was reduced during acute Acz treatment (16). A reduced rate of La- uptake into other tissues would be expected because La- uptake into tissues is dependent, in part, on the La- gradient across the muscle membrane (2, 11, 24); the lower [La-]pl observed in this and other studies during acute Acz administration (29, 31, 32) would presumably contribute to a lowering of the plasma-muscle [La-] gradient.

Release of La- from muscle into blood is impaired by an extracellular acidosis (10, 11, 18, 24, 37). In the present study, Acz was administered acutely to avoid any significant plasma acidosis (16, 29, 31, 32). No difference was found in the venous plasma [H+] between conditions in our study, which agrees with previous studies using a similar drug treatment protocol that showed either no difference in the arterial plasma [H+] (32), or a slight increase in arterial plasma [H+] (~2-3 nmol/l) during acute Acz treatment (29). In the studies of Jones et al. (10) and Sutton et al. (37), [La-]pl was reduced during exercise in an NH4Cl-induced acidosis compared with a CaCO2 placebo trial, but, in those studies, the difference in plasma [H+] between the placebo and acidosis trials was ~20 nmol/l, much greater than found in this or our other studies. Thus the lower [La-]pl found in this and our other studies is probably not related to impaired La- efflux from active muscle as a consequence of an Acz-induced plasma (or bulk extracellular) acidosis.

Relationship between TpHi, TPi/PCr , and TLa-. TpHi and TPi/PCr, determined from 31P-MRS data, were used in this study to indicate a power output or exercise intensity beyond which there was significant muscle accumulation of protons (or H+ equivalents) and were assessed as an increase in the slope of the muscle pHi-power output relationship. In this study, no difference was found in the power output or percentage of peak power output corresponding to TpHi or TPi/PCr between conditions. Also, no differences were found in the slope of the pHi-power output relationship above TpHi, or in pHi at the point of fatigue between conditions. These findings suggest that the power output that corresponds to an increased accumulation of protons (or H+ equivalents) and the rate of proton accumulation, once initiated, were not affected by Acz within the conditions of this study. Because the increase in muscle [H+] during exercise is mainly associated with an increase in muscle [La-] (15, 27, 28), we interpreted the increase in slope in the pHi-power output relationship seen using 31P-MRS techniques as reflecting enhanced muscle La- accumulation with increasing power output. These data also suggest that muscle La- accumulation was not affected by Acz treatment; however, we were unable to determine whether Acz treatment had specific effects on muscle La- production, oxidation, or efflux (i.e., transport) or affected the relationships between these processes. Scheuermann et al. (32), using a similar drug protocol, demonstrated that after 6 min of heavy-intensity, constant-load cycling exercise muscle glycogen breakdown and muscle La- (and pyruvate) accumulation were similar in Con and Acz conditions, suggesting that muscle glycogenolysis and pyruvate production were not affected by Acz administration. In contrast, Rose et al. (23) observed a decrease in glycogen breakdown during heavy exercise after 3 days of Acz-induced CA inhibition in horses. However, in that study, Acz was administered chronically for 3 days and was associated with a metabolic acidosis before the start of exercise. In addition, muscle glycogen levels were reduced by ~50% before exercise began, which also may have contributed to a lower rate of glycolysis because the glycolytic rate may be influenced by the initial muscle glycogen content (9).

In the present study, TpHi, TPi/PCr, and TLa- occurred at a similar power and percentage of peak power outputs during Con. However, during Acz, TLa- occurred at higher power and percentage of peak power outputs than either TpHi or TPi/PCr, suggesting a dissociation between the intra- and extracellular markers of glycogenolysis and La- accumulation. The similarities of TpHi and TPi/PCr between conditions, the similarities in the slopes of the pHi- and log Pi/PCr-power output relationships above their respective thresholds, and the muscle biopsy data of Scheuermann et al. (32) showing a similar glycogen breakdown between conditions strongly suggest that the onset and rates of muscle glycolysis and La- accumulation were not affected by Acz treatment. Whereas an effect on intramuscular pyruvate and La- oxidation would contribute to differences in La- production, accumulation, and release from muscle, Scheuermann et al. (29) demonstrated that the kinetics of O2 uptake at the onset of moderate- and heavy-intensity exercise were not affected by Acz administration, suggesting similar oxidation rates between conditions. The delayed accumulation of plasma La-, the higher TLa-, and the slower rate of pHi recovery in Acz observed in this study, along with the previous findings of a lower a - v [La-]pl across an inactive forearm muscle (16), suggest that La- (and H+) transport and efflux from muscle may be impaired by Acz and/or CA inhibition, although this awaits further investigation.

CA inhibition and acid-base balance. In this study, pHi values at rest and during exercise were similar between conditions, but recovery of pHi after exercise was slowed in Acz. A lower pHi at rest was observed previously in isolated muscle preparations after CA inhibition (5, 8). That pHi at rest was not significantly different between conditions in this study may be related to the somewhat lower plasma PCO2 resulting from a mild hyperventilation that is often seen at rest in these studies (30). Slower recovery of pHi suggests that removal of H+ equivalents (i.e., acid-base-independent variables, including La- and CO2) was slowed after Acz administration. CA inhibition was shown to slow pHi recovery in cardiac muscle after 10 min of ischemia, regardless of whether a membrane-permeant or -impermeant CA inhibitor was used (41), suggesting that a CA isozyme with activity directed to the extracellular compartment was involved. In addition, De Hemptinne et al. (6), using an isolated muscle preparation, demonstrated that pHi recovery from a propionic acid-induced intracellular acid load was slowed after incubation with Acz and that muscle surface pH acidified to a greater extent during recovery, independent of the constant acid-base status of the bulk solution perfusing the muscle. The greater decrease in surface membrane pH during recovery in Acz was attributed to the accumulation of protons on the muscle surface because inhibition of CA impaired the facilitated removal of protons as CO2.

Recovery of intracellular acid-base status is dependent, in part, on removal of intracellular La- and restoration of intracellular strong-ion difference (15). Lactate removal from muscle occurs via diffusion of undissociated lactic acid and La- transport via the monocarboxylate transporter found in the sarcolemma (11, 24, 25). It is likely that the muscle surface membrane pH, rather than the pH of the bulk extracellular fluid, is "sensed" by the monocarboxylate transporter. Inhibition of the muscle sarcolemmal CA IV isozyme with Acz may contribute to a greater acidification of the muscle surface as H+ equivalents (i.e., La- and CO2) are removed from muscle, similar to that demonstrated by De Hemptinne et al. (6). In control conditions, the rise in muscle surface membrane pH during coupled H+-La- cotransport or undissociated lactic acid diffusion may be attenuated by the CA-catalyzed dehydration of carbonic acid. Inhibition of CA with Acz may interfere with this process, leading to an accumulation of protons on the membrane and thereby impairing the removal of both La- and protons from the muscle. Thus the slow pHi recovery, combined with the lower venous [La-]pl in Acz observed in the present study, may be attributed to impaired efflux of La- from muscle that could also contribute to the rightward shift of TLa- and the lower [La-]pl during Acz.

In summary, in the present study, the acute administration of Acz to inhibit CA was associated with 1) a rightward shift in the [La-]pl-power output relationship, resulting in a lower [La-]pl at fatigue and during early recovery; 2) a rightward shift in the plasma TLa- to higher power and %peak power outputs; 3) no effect in the intracellular TpHi and TPi/PCr; and 4) a slowing of pHi recovery after exercise. It was concluded that the lower [La-]pl consistently observed after Acz administration was not related to a reduced onset or rate of muscle La- accumulation, which was similar between conditions, but may have been related to an impaired removal of La- (and H+) from muscle.


    ACKNOWLEDGEMENTS

We thank the participants of this study; B. S. Ahluwalia for technical support; and Dr. R. T. Thompson and colleagues in the Imaging Division of the Lawson Research Institute for providing research time on the MRS unit.


    FOOTNOTES

Financial support was provided by an operating grant from the Natural Sciences and Engineering Research Council of Canada.

Research was performed at The Centre for Activity and Ageing (affiliated with the School of Kinesiology, the Faculty of Health Sciences, and the Faculty of Medicine at the University of Western Ontario), and the Imaging Division of the Lawson Research Institute, St. Joseph's Health Centre.

Address for reprint requests and other correspondence: J.M. Kowalchuk, School of Kinesiology, 3M Centre, Univ. of Western Ontario, London, Ontario, Canada N6A 3K7 (E-mail: jkowalch{at}julian.uwo.ca).

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. §1734 solely to indicate this fact.

Received 8 March 1999; accepted in final form 9 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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