Inhibition of carbonic anhydrases in type I muscle

Inhibition of carbonic anhydrases in type I muscle fibers influences contractility C. COT&,'D.TREMIILAY, H. RIVERIN, P. FRBMBNT, AND P.A. ROGERS Muscle Biology Research Group, h v a l University Hospital Research Centre, Room S-750, 2705 boul. bnrurier, Ste-Foy, Que'., Canada GIV 4G2

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Received June 22, 1988 CQTB,C., TREMBLAY, B., RIVERIN,H., FRBMONT, P., and ROGERS,P.A. 1989. Inhibition of carbonic anhydrases in type I muscle fibers influences contractility. Can. J. Physiol. Pharmacol. 67: 645-649. We tested the effects of inhibiting the carbonic anhyctrase activity of rat soleus and extensor digitorum longus muscles on the isometric contractile properties and the resistance to fatigue. SOL and EBL muscles from female rats were incubated in vitro in the presence of methazolamide, a specific inhibitor of carbonic anhydrase, before determining their contractile properties. Methazolmide had no effects on the contractile properties of the soleus muscle ( l V 5 or loe3 M) and extensor digitomm longus M), except for the half-relaxation time of the soleus muscle which increased significantly. Values for half-relaxation time were significantly increased with both concentrations of the inhibitor. Muscles were then submitted to a fatigue protocol lasting 30min. During the fatigue test, no significant difference was observed between control and 1W5 M methazolamide soleus muscles. In presence of los3M methazolmide however, the soleus muscle showed a significantly increased resistance to fatigue com a d with control preparations. No significant effect was observed with the extensor digitorum longus muscle exposed to 10- M methazolaunide. Results are discussed in terms of the presence of two different isofoms of carbonic anhydrase that may he associated with calcium uptake and energy metabolic processes, respectively. Key words: carbonic anhydrase, skeletal muscle, contraction, fatigue, soleus muscle.

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C ~ T BC., , TREMBLAY, D., RIVERIN,H., FR~MONT, P., et ROGERS,P.A. 1989. Inhibition of carbonic anhydrases in type I muscle fibers influences contractility. Can. J. Physiol. Pharmacol. 67 : 645-1549, Nous avons vkrifiC si l'inhibition de 19activitCanhydrase carbonique des muscles soleus et extensor digitorum longus du rat influengait les propriktks contractiles isomktriques et la rksistance ii la fatigue. Les muscles soleus et extensor digitorum longus furent incuMs in vitro en presence de mkthazolamide, un inhibiteur spcifique de l'anhydrase carbonique, avant la dCkrmination des propriCtCs contractiles. La tension spkcifique maximale (N/cm ) n'a pas kt6 modifiCe par I'inhibiteur, et ce chez Ies deux muscles utilisks. Toutes les mesures de propriCt6s contractiles du soleus et de l'extensor digitomm longus, A l'exception du demitemps de relaxation chez le soleus, Ctaient inchangkes en pdsence de 1W5ou ~ o - ~m6thazolamide. M Las vdeurs de demi-temps de relaxation Ctaient significativement prolongkes en prksence des deux concentrations de mCthazolamide. Les muscles Ctaient ensuite soumis ii un protocole de fatigue d'une durke de 30 min au cours duquel aucune difference significative ne fut obsewke entre les muscles soleus contrdles et ceux mis en prksence de I O - ~ M mkthazolamide. En prksence de 10-% mmtazo~mide.les muscles soleus dbmontrbrent une augmentation significativede la rksistance B la fatigue comparativement aux muscles contr6les. Aucun effet significatif ne fut observk avec le muscle extensor digitomm iongbls lorsqu' expos6 A 1 0 ~ ~ hmkthazolmide. .1 Les rksultats sont discutCs en terme de l'existence de deux diffkrentes isofomes de l'anhydrase carbonique qui seraient associCes, respectivement, & la captation du calcium et au mCtabQlisme knergktique.

Introduction Evidence for the presence of carbonic mhydrase (CA; EC 4.2.1.1) isoforms in mrnmalian skeletal muscles has been accumulating in recent years (Frkmont et a%.1987; Holmes 1977; Venta et al. 1987; Zborowska-Sluis et al. 1974). The predominant isoform in mammalian muscle, CA 111, appem to be cytosolic (Frkmont et al. 1988; Vaananen et al. 1986) and differs from the CA I and CA I1 isofoms of the erythrocytes by its lower sensitivity to sulfonamides and intrinsic molecular activity (Sanyal et al. 1982). Experimental evidence exists suggesting that some CA activity is also associated with the s&coplasmic reticulum (SR) et al. 1986; Frkmont et al. 1989; Geers and Gros 1988) as well as sarcolemma (Geers et al. 1985). ~ l ' t h o u ~the h biochemical and molecular characteristics of CA III are well documented, the physiological significance of the CA activity in skeletal muscle is still unclear. The few studies investigating the functional role of CA in muscle have focussed their attention primarily on the facilitation of C02 diffusion (Barclay 1987; Zborowska-Sluis et al. 1974). Recent work in our laboratory has revealed that the cytoplasmic CA activity and content are related to the type of skeletal muscle fibers (Frkmont et d. 1987; Frkmont et al. 1988). Type I fibers have a CA III activity approximately 5 times higher than that of

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'Author for correspondence.

type IIa fibers, while no CA 111 activity can be detected in type IIb fibers using currently available methods. Considering the relative CA activity of type I and type IIa fibers and the fact that the capacity for C 0 2 production of type IIa is equal to or greater than that of type I fibers, it may be premature to conclude that CA III in muscle is associated uniquely with the diffusion of COz. We therefore investigated the effects of inhibiting the CA activity of the rat soleus (SOL) and extensor digitorum longus (EDL) muscles on the contractile properties and fatigability to acquire further information regarding the physiological role of this enzyme.

Materiab and methods Preparation of cytoplasmic extract sniideterm fmtionof CA III activity The soluble cytoplasmic fraction of the soieus muscle was prepared essentially according to Lebherz et al. (1982) with the exception that the homogenization buffer was composed of 2.5 mhl HEPES (N-12hydroxyethyl]piprazine-N1-12-ethmesulfonicacid]), 1 mM M&12, 1 mM EBTA (ethylenediaminetetraacetate), 1 mM DaT (dithiothreitol), pH 7.4. Fresh preparations were used to determine the CA 111 activity, which was measured using a procedure similar to the one used by Bruns et d. (1 986). Each individual reaction contained 50 p L of the diluted muscle extract, 50 ylb, of reaction buffer (75 rrmbl Na2CQ3, 50 mM N d C 0 3 ) , and 100 ylL of the pH indicator (2.5 mg phenol red per l 0 0 d of 5 mbf NaHC03). The reaction was initiated by the addition of 200 FL of C02-saturated H28.For each concentration of the inhibitor tested, the enzyme was in contact with methazolamide

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(METH) for at least 2 miw before starting the reaction. The entire procedure was performed at 1°C and resulted in an uncatalyzed reaction time of approximately 90 s. Measurement of contrcacbile propersies and fafigabiEity Experiments were performed on whole SOL and EDL muscles carefully dissected from female Wistar rats weighing 150- 175 g. Rats were anesthetized with an i.p. injection of sodium pentobarbital (50 mg/kg). Contractile properties were measured in vitro in a buffered physiological salt solution (fiebs-Ringer) maintained at 25'C and supplemented with glucose m d curare. The pH of the bathing solution was stabilizedat 7.4 by equilibration with a gas mixture of 95% Oa-5% C02. Muscles were placed in a vertical bath and secured at each end with silk sutures. One tendon was attached to a rigid support at the bottom of the bath, while the other end was connected to an isometric force transducer (Konigsberg Jnstmments, model F5-A) though a stainless steel hook. Muscles were stimulated with 10- to 15-V square pulses sf 0.2 rns duration delivered though platinum field electrodes only when force was recorded. Current was supramaximal. Muscles were first adjusted to their optimum length, defined as the length at which maximal isometric twitch tension was produced. Muscles were then allowed to equilibrate with the incubation medium for 45 Hnin with or without METH k i n g present. For the experiments with the SOL muscle, the bathing solution contained METH at a final concentration of 10-' or B B - ~ Mwhile , only lW3h4 was used for the EDL muscle. The sulfonmide METH was selected for this study kcause its affinity for CA issfoms is comparable to that of acetazolamide (ACET), but its penetration rate into cells is 5- 15 times higher. According to Maren (1987), METH is by far the best all-purpose compound for most physiological experiments because it is able than other sulfonmides and much more specific than cyanates in general. Holder and Hayes (1965) showed that, with red blood cells at 37"G,the penetration rate of METH was such that equilibrium was reached within 30 s. Experiments in our laboratory indicatethat approximately 2 f i n are required for METH to equilibrate with the interstitial space of a 70-mg SOL muscle incubated at 25°C (C. C6t6, D. Tremblay, H. Riverin, P. Frkmont, and P. A. Rogers, unpublished data). Maximum twitch tension (P,),contraction time (CT), and 1/2 relaxation time (1/2 RT) were first obtained from a twitch followed by a frequency-force curve. Maximum betanic tension (Po) and the maimurn rate of tetanic tension development (dB/dt) were measured. A 10-min rest period preceded the fatigue protocol, which consisted sf one t e t d c contraction at 10Hz every 5 s for 3Bmin, each single contraction lasting 0.5 s, for a total sf 360 contractions. Under the experimental conditions used for these experiments, Po was stable for at least 3 h. The same general protocol was used with the EDL muscle. Because of its lower resistance to fatigue, the EDL muscle was stimulated at 26)Hz for 208 ms every I0 s for 30 min. For both muscles, the frequency of stimulation produced a partly fused tetmic contraction generating approximately 60% of B,. Following the fatigue protocol, the muscles were removed from the chamber, blotted, and weighed. The mean cross-sectional m a of each muscle was estimated by dividing muscle mass by fiber length and the density of mammalian skeletal muscle. In a separate series sf experiments, the same protocol was used with soleus muscles incubated with or without BO-'M M E W , but the fatigue protocol was interrupted briefly every 5 Knaan to obtain a twitch contraction from which isometric contractile measurements were obtained. A11 results are cxpwssed as means standard error s f the mean (SEM). Data on contractile properties were malysed by a one-way analysis of variance followed by a Fisher's PLSD test when a significant F-ratis was obtained. Data on fatigue were analysed by a two-way analysis of variance for repeated measures (split plot design) with the level of significance set at p = 0.05.

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The inhibition curve for CA 111 activity in the soleus muscle cytoplasmic extract is shown in Fig. 1. With METM, the

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20

8 0 -11

-10

-9

-8

-7

-6

-5

-4

-3

-2

log METH (M) FIG. I. In v i m inhibition of cytoplasmic carbonic anhydrase IBI activity of rat soleus muscle by smethazolmide (METH). Each point represents the mean of three separate detednations. Vertical lines represent the SEM. TABLE1. Contractile and physical properties of control and experimental soleus muscles Control ( n = 8)

IO-'M METH B B - ~ M METH ( n = 6) (n= 6 )

65.0-+0.5 Muscle mass (mg) 6 1 .2& 1.9 12.25Z0.2 Lo(mH%a) 12.420.2 68.93~1.8 65.120.8 CT (ms) 81.722.8 91.923.7" 112 RT\(ms) Pa/& 0.181 -+_0.811 0.174-+0.007 18.6-+1.1 k(~/cm') 16.821.I d ~ / d (% t P , / ~ s ) 8.$020.04 0.82a0.03

48.4-+3.0 12.45Z0.2 66.95Z 1.7 94. 1&2.5* 0.208?~0.006 16.82 1.8 8.78&-0.03

NOTE:Vdues are expressed as means r SEM. *Significantlydifferent from control value 4p < 6.05).

calculated Hm for cytopBasmic @A I%Iwas in the order sf 5 X Bo-'M, whereas B o - ~ M was required to totally inhibit the activity. No sulfonmide sensitive activity was detected in the cytoplasmic extract of this muscle, which suggests that CA 1and CA IH are not present in the soleus muscle. Physical characteristics and isometric contractile properties of soleus muscles from control and experimental groups are presented in Table B . Maximum specific tension was unaffected by the incubation with METTH. Of the two concentrations of METH selected neither induced significant changes in the contractile properties with the exception of B /2 RT,which was significantly prolonged in the presence of the inhibitor. Mean values for B /2 RT were B 12.5 md 1 B 7.6% sf the control value at concentrations 10-' and l O - ~ MMETM ,respectively. In the presence of Bo-'N METH, the soleus muscle did not show any difference compared with the control muscle at any time point during the fatigue protocol (Fig. 2). With IO-'M METH,a concentration that in vitro inhibits d l cytoplasmic @A III activity, the muscles showed an increased resistance to fatigue compared with control muscles (Fig. 21, the two fatigue curves being significant%ydifferent 4g = 0.004). The maximum difference between the two fatigue curves was observed after only 3 slain of stimulation m d seemed to decrease as a function sf t h e thereafter. When the fatigue test was intempted at various time points to obtain measurements on single twitch contractions, it was found that the difference observed in resting muscles in terns of 1/2 RT would disappear during the fatigue a s t suck that the difference between the control md exprimen-

c8~BET AL.

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6

5

10

15

20

CONTROL METH

25

30

TIME (min)

FIG.2. The effect sf methazolamide (METH)on the resistance to fatigue of soleus muscles submitted to the standardized fatigue protocol (n = 8). Muscles were incubated in presence of METH for 60 min before the fatigue protocol. Stimulation was at 16% Hz, for 500 ms, once every 5 s. Vertical bars represent the SEM. The curves obtained for control and 1W3M METH groups are significantly different (p = 0.W).

FIG. 4. Fatigue curves for the extensor digitorurn Hongus muscle incubated with or without methazolamide (METH, IO-~M)and submitted to the standardized fatigue protocol (n = 4).Muscles were incubated with METH for 60 min before the fatigue protocol. Stimulation was at 20 Hz for 200 ms once every 10 s. Vertical b a s represent the SEM. No significant difference is observed.

To provide further evidence that the effect observed with METH in the soleus muscle was due to the specific inhibition of CA III, the EDL muscle, which is essentially devoid of CA III (Jeffery et al. 198%;Shiels et d. 1984), was also incubated with 20-% METH. No change in contractile properties (Table 2) or in resistance to fatigue (Fig. 4) was observed with this muscle.

0

5

10

15

20

25

30

TIME (ms)

FIG. 3. Changes in twitch half-relaxation time 4112 WT) during the

fatigue test for soleus muscles incubated with ( n = 8) or methazolmide (METH,~ o - ~ M ( n) = 7). Muscles were incubated with METH for 60min before the fatigue protocol. Stimulation was as in Fig. 2, except that it was briefly intempted every 5 m i w to obtain a twitch contraction. The only significant difference is at time 0. TABLE 2. Contractile and physical properties of control and exprim e n d EBL muscles

-

Control (n 6 ) Muscle mass (mg) Lo qm) C'I' (ms) 112 RT (ms)

97.4r 7.1 10.9~k0.4 23.7k0.9 23.9+-1.5 Pt/& 0.248*0.010 Po 4N/cm2B 18.8k 1.0 dP/dt(%PP,/ms) 3.720.4

10-'M METH (n= 6 ) 99.494.6 10.8*0.3 24.720.5 23.6+-1.2 0.287k8.006 17.5ko.8 3.8+-0.2

td (10-'M METH) groups was not statistically significant anymore after 2-3 rnin into the test (Fig. 3). On the other hand, the relative decline in maximum twitch tension (P,)was almost identical to the decline observed for tension production at 18Hz during the fatigue protocol (results not shown).

The data presented for contractile properties are in good agreement with previously published reports using rat SOL and EDL muscles (Close 1944; Close 1972). Maximum specific tetmic tension was well within the physiological range, which indicates ahat the integrity of the preparation was adequate. Furthermore, the fact that this same measurement was unchanged following incubation with METH indicates that the inhibitor selected does not interfere with the contractile process per se. No sulfonamide-sensitive CA activity was detected in the cytosolic fraction of the ssleus muscle, which is composed predominantly of type I fibers (Akano et al. 2973). This suggests that no significant level of the soluble CA I a d CA II isofoms is present in type I fibers. In the presence of l ~ - ~ n / l METH, the mean value for 1/2 RT was significantly prolonged. This observation is in good agreement with the data of Geers and Gros (1988) who also observed an increased relaxation time of SOL muscles incubated with CA inhibitors. These results support the existence of a sulfonarnide-sensitive CA isofonn associated with the SR. FrCmont et al. (1989) have shown that sulfonamide-sensitive CA activity, with an IS0 for ACET in the mder of 7.5 x 10-'h/1 and which is totdly inhibited by IO-~PVI ACET, is present in purified SW preparations from rat soleus muscle. This activity is d s o totally inhibited by IQ-~AIIMETW (results not shown). Sirnilah results were obtained by Bruns et d . (1986) with SR preparations from rabbit muscle. Together, these studies strongly suggest that a sulfoniunide-sensitive CA isofom is associated with the SR of mammalian type I skeletal muscle fibers. Experimental evidence exists showing that Hf and other cations act as counterions during ca2+ movements across the SR membrane (Meissner 1983). Moreover, protons compete with ca2+ for the high affinity binding sites on the ca2+ATPase (Inesi and Hill 1983; Levitsky and Benevolensky 1986) and are


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CAN. J . PWYSBBL. PHARMACBL. VOL. 67, 1989

ejected from the SR during Ca2f uptake (Yamaguchi and Kanazawa 1985). It is still unclear, however, if countertransport of H+ is essential for optimal function of the ca2' ATPase (Tanford 1984). It has been suggested that a CA isofom in association with the SR could provide a rapid source and sink for H+ during the contractile process (Bruns et al. 1986). The present results are consistent with such a role and suggest that the availability and (or) adequate buffering of H' may be essential for optimal ca2+ATPase activity in the SR of intact slow-twitch skeletal muscle. This does not appear to be the case for fast-twitch muscle, since no change was observed for the 182 RT of the EDL muscle. Accordingly, data from Bmns et al. (1986) and preliminary results from our laboratory suggest that pure fast muscles may have significantly less CA activity associated with the SR fraction, if any. The EDL muscle was included in the experimentalprotocol to fkrther substantiate our contention that the effect observed during the fatigue protocol with the soleus muscle was specifically related to the inhibition of CA I11 activity. Radioimmunoassay determinations have revealed that the CA I11 content of the rat EDL muscle is approximately 60-fold lower than the value found in the soleus muscle (Jeffery et al. 1982; Shiels et al. 1984). Since no effect was obsewed in terms of fatigability when the EDL muscle was incubated with I O - ~ MMETH, we conclude that the effect observed with the soleus is most likely related to the inhibition of the CA activity. Geers et al. (1985) have reported that a sarcolemmal form of CA with an extracellular active site is present in rabbit skeletal muscle. Their results indicate that this CA isofom may be involved in facilitating C02 diffusion. Hn rat soleus muscle, analysis of the effect of ACET on transient surface pH changes suggested the presence of a similar CA isoform (De Hemptinne et al. 1987).To test the possibility that the effect obsewed on the fatigability of the SOL muscle could be related to the inhibition of such m isoform, we repeated the same fatigue protocol on SOL muscles with the difference that B0-3M ACET was added just prior to initiating the fatigue protocol. ACET is a sulfona i d e with CA affinity characteristics that are almost identical to METH, but its rate of penetration is much lower (Holder and Hayes 1965). Under the experimental conditions used, it would require at least 30-60 min before equilibrium with the intracellular space is reached. Ceers et al. (1985) have shown that, using ACET, full inhibition of the putative sarcolemmd CA isoform is reached within 30 s of perfusion. In this protocol with ACET, one would expect to see the same difference in fatigability if the effect is related to the sarcolemmal @"A isoform. However, no difference was observed under such conditions (Fig. 5) suggesting that methazolamide has to penetrate the intracellular space to produce its effect. In s u m q , since the effect on resistance to fatigue (i) does not appear to be linked to the inhibition of a sarcolemal CA isoform, (ii) is absent in the presence of 10-'M METH, a concentration that totally inhibits the SR CA isoform, and ( i f i ) is not present in the EDL muscle incubated with 10-% M T H , we conclude that the effect observed in terns of resistance to fatigue is due to the inhibition of the cytosolic CA I11 isoform. Very few studies have investigated the physiological effects of inhibiting CA activity in skeletal muscle. Barclay (1987), using mouse SOL muscles, was unable to detect any effect under normocapnic conditions. Scheid and Siffert (1985), using frog hindlimb muscles, concluded that the CA isoform found in frog was acting on the neuromuscular junction. However, in both cases the intensity of the stimulation protocol selected was

TIME (min)

FIG.5. Fatigue cwves for soleus muscles submitted to the same standardized fatigue protocol as in Fig. 2. Acetazolamide (ACET, 1W3h4) was added 10 s before initiating the fatigue test ( n = 4). Verticd bars represent the SEM. No significant difference is observed. inadequate for a significant recruitment of the oxidative metabolic pathways, which makes a comparison between these experiments and those reported here relatively difficult. In the present study, the SOL muscle showed a significant increase in resistance to fatigue over the 30-min stimulation ~ a concentration protocol when incubated with 1 o s 3 METH, that totally abolishs CA I11 activity in vitro. The production of C02 by oxidative metabolism gradually increases during restwork transition. In highly oxidative muscles this C02 prduction could modify the CA reaction equilibrium toward an increased production of HCOT and H+ ions. It is therefore likely that for an equal C02 production, one would observe a lower intracellular pH in the control SOL as compared with one in which CA 111 activity has been inhibited. Connett (1987) recently showed using dog gracilis muscles, which have high oxidative metabolic capacity, that continuous low intensity isometric contractions resulted in a decrease of cytosolic pH to a value of 6.5 within 3 min of stimulation. In view of this interpretation, the effect of CA IIH inhibition on resistance to fatigue is consistent with the fact that intracellular acidosis is partially responsible for the reduction of tension production associated with muscle fatigue (Mainwood and Renaud 1985; Metzger and Fitts 1987). Assuming that fatigue is at least partly related to some disturbance of the excitation-contraction coupling mechanism leading to a decreased ca2+ release (Mman and Mattiazzi 1981), one could also postulate that, in the presence of METH, a decrease in ca2+ uptake may occur that would increase the cytosolic ca2+ concentration between and during contraction, without inducing my contractwe between contractions. However, this explanation appears unlikely ia? view of %hefact that no difference was observed between the two groups during most of the fatigue test in t e r n of 112

RT. Since tension production in a partly fused tetanic contraction, like the one induced in ow fatigue protocol, is basically determined by the height of the twitch and by the rate of relaxation, one could postulate that the increased tension produced during the fatigue protocol in the presence of l o - % ¶ MErPH was due totally or partially to a prolongation of the twitch relaxation in the METH group. However, it is clearly not the case because in both groups the values for twitch 112 RT become similar once the fatigue test is initiated. 'khe difference between both groups in terms of tension production is therefore related to a smaller twitch in the control soleus muscles when


~6T6 ET AL. compared with the CA I11 inhibited muscles. Whatever the cellular mechanism leading to the decrease in excitation-contraction coupling, the fact remains that both P, and tension production at 10 Hz are significantly decreased in the presence of CA activity during the fatigue test. The observation of a decreased tension production during the fatigue protocol when CA I11 is active does not appear by itself physiologically beneficial to type I fibers. In this type of fiber, oxidation of fatty acids and glycogen is the major metabolic pathway used for ATB production during work of long duration and moderate intensity. Although the intracellular stores of lipids are generally not limiting, depletion of the glycogen stores is closely related to the appearance of muscle fatigue and exhaustion (Newsholme 1981). Interestingly, a positive linear relationship is found between the CA I11 activity of a muscle and its triacylglycerol lipase activity ( r = 6.98; Frernont et al. 1988; Miller et al. 1987). In that respect, it is conceivable that the effect of CA I11 activity o n muscle homeostasis could influence substrate utilization to prevent rapid depletion of the glycogen reserves and thereby prevent or delay the appearance of exhaustion. In summary, our results support the existence of a CA isofom associated with the SR, which could play a role in c a 2 + release and (or) uptake mechanisms. It is also concluded that inhibition of the C A 111 isoform in SOL muscle may influence the resistance to fatigue during long duration, low intensity stimulation protocol.

Acknowledgements This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada m d the Fonds concerti5 d' aide h la recherche (Quebec). Methazolamide was generously provided by Cymamid Canada Inc. Pierre Frkmont is 'the recipient of a scholarship from the Natural Sciences and Engineering Research Council s f Canada. Thanks are expressed to Mrs Lucie Turcotte for typing the manuscript. ARIANO, M. A,, ARMSTRONG, R. B., and EBGERTON, V. R. 1973. Hindlimb muscle fiber populations of five mammals. J. Histochem. Cytochem. 21: 51-55. BARCLAY, J. K. 1987. Carbonic anhydrase I11 inhibition in normocapnic and hypercapnic contracting mouse soleus. Can. J. Ph ysiol. Pharmacol. 65: 180- 104. BRUNS, W., DERMFTZEL, R., and GROS,G. 1986. Carbonic a n h y b in the sarcoplasmic reticulum of rabbit skeletal muscle. J. Physiol. (London), 371: 351-364. CLOSE,R. 1964. Dynamic properties of fast and slow skeletal muscles of the rat during development. 9. Physiol. (London), 173: 74-95. CLOSE,R. I. 1972. Dynamic properties of mammalian skeletal muscles. Physiol. Rev. 52: 129- 197. CONNETT, R. J. 1987. Cytosolic pH during a rest-to-work transition in red muscle: application of enzyme equilibria. 9. Appl. Physiol. 63: 2360-2365. DE H E ~ N N EA.,, MARRANNES, R., and VANHEEL, B. 1987. SurfacepH and the control of intracellular pH in cardiac and skeletal muscle. Can J. Physiol. Phmacol. 65: 970-977. EDMAN, K. A. P., and M A ~ I A Z ZA.I ,R. 198 1 . Effects of fatigue and altered pH on isometric force md velocity of shortening at zero load in frog muscle fibres. J. Muscle Res. Cell Motil. 2: 321-334. MMONT, P., LAZURE,C., TREMBLAY, R. R., C H ~ T I E N M., , and ROGERS,P. A. 1987. Regulation of carbonic anhydrase ILI by thyroid hormone: opposite modulation in slow- and fast-twitch skeletal muscle. Biochem. Cell Biol. 65: 798-797. FRI~IONT, P., CHAREST, P. M., COI-6, C., and ROGERS, P. A. 1988. Carbonic anhydrase LU in skeletal muscle fibers. An immunocyto-

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