Journal of Muscle Research and Cell Motility 16, 611-617 (1995)

Effects of c -cyano-4-hydroxycinnamic acid on fatigue and recovery of isolated m o u s e muscle PATRICK D. CLARKE, DAVID L. CLIFT, A. M. BURNETT and N. A. CURTIN*

MOHANTHA

DOOLDENIYA,

CRAIG

Department of Physiology, Charing Cross and Westminster Medical School, Fulham Palace Rd, London W6 8RF, UK Received 25 April 1995; revised 26 June 1995; accepted 7 July 1995

Summary

Fatigue and recovery of mouse soleus and extensor digitorum longus muscles were investigated in standard saline and in saline containing the lactate + hydrogen ion transport blocker, 0~-cyano-4-hydroxycinnamic acid (cinnamate). The fatigue protocol was a series of brief isometric tetani which reduced isometric force by about 25%. Recovery was monitored by test tetani during recovery. Both muscles recovered completely in standard saline. Soleus muscle also recovered completely in the presence of cinnamate, whereas extensor digitorum longus hardly recovered at all. Force during fatigue and recovery can be described in a mathematical simulation in which force depends on intracellular inorganic phosphate and pH, and the only effect of cinnamate is to block lactate + hydrogen ion transport. The results of the simulation suggest that during the fatiguing series of tetani pH changes are small and have a negligible effect on force, but pH is a major determinant of the timecourse of recovery in extensor digitorum longus. Introduction

A l t h o u g h recovery from fatigue is a vital aspect of muscle function, it is m u c h less well u n d e r s t o o d than fatigue itself. Fatigue, the r e d u c t i o n in force production as a result of r e p e a t e d contraction, is a complex process in w h i c h the d o m i n a n t factor varies d e p e n d ing o n the muscle a n d h o w fatigue is p r o d u c e d (Westerblad et al., 1991). There is strong evidence that with m a n y fatigue protocols, the accumulation metabolic products, inorganic p h o s p h a t e (Pi) a n d h y d r o g e n ions, are the d o m i n a n t factors reducing force. P r e s u m a b l y these metabolite levels also determ i n e the timecourse of recovery, b u t this point has received little scrutiny. The N M R m e a s u r e m e n t s b y M e y e r a n d colleagues (1991) s h o w that, in cat biceps, the timecourse of force recovery is intermediate b e t w e e n that of Pi a n d p H , suggesting that both influence force. To get insight into the relative i m p o r t a n c e of Pi a n d p H in fatigue and, particularly, recovery in slow and fast m o u s e muscles, w e h a v e e x a m i n e d the effect of 0c-cyano-4-hydroxycinnamic acid, w h i c h is k n o w n to block the t r a n s p o r t of lactate + h y d r o g e n ion across the cell m e m b r a n e (Mason & Thomas, 1985, 1988; Juel, 1988). M o u s e soleus (SOL) and extensor digitorum longus (EDL) muscles w e r e fatigued to the same extent, a n d the time courses a n d extents of r e c o v e r y were *Towhom correspondence should be addressed. 0142-4319/95 © 1995 Chapman & Hall

examined. If intracellular acidification d u e to lactate a n d h y d r o g e n ion acid accumulation is an i m p o r t a n t fatiguing factor, the blockage of lactate a n d h y d r o g e n ion t r a n s p o r t w o u l d be expected to e n h a n c e fatigue a n d to p r e v e n t recovery. Materials a n d m e t h o d s

Soleus and extensor digitorum longus muscles were isolated from mice (strain 129, age 5-6 weeks) killed by cervical dislocation. Muscles were kept in standard saline containing (mM): NaC1, 137; KC1, 5; CaC12, 1; Na2HPO4, 1; NaHCO3, 24; glucose, 11. The saline was gassed with 95% 02 + 5% CO2 and maintained at 25°C (pH 7.25). The muscle was mounted in solution between an isometric force transducer and a fixed hook. Stimulus frequency was adjusted to give a fused tetanus. Stimulus voltage and muscle length were adjusted to be optimal for tetanic force production.

Fatigue + recovery protocol Each muscle performed a fatiguing series of isometric tetani, which rapidly reduced force by about 25%. Soleus muscle performed 25 0.6 s tetani at 1.0 s intervals, and EDL performed 15 0.5 s tetani at 5.0 s intervals. Each fatigue series was followed by a recovery period with test tetani to monitor recovery; test tetani were given at 1.0, 2.5, 5.0, 7.5, 10.0, 12.5 and 15.0min after the end of the fatiguing series or until recovery was complete. The fatigue + recovery protocol was done in standard saline and in cinnamate-saline. Cinnamate-saline was like standard saline but contained 5 mM 0~-cyano-4-hydroxycin-

C L A R K E etal.

612 namic acid (Sigma), which blocks the lactate + hydrogen ion transporter in the surface membrane (Mason & Thomas, 1985, 1988; Juel, 1988; Westerblad & Allen, 1992); the blocker replaced 5 mM NaCI. The p H of the two salines was the same. Each muscle was equilibrated in the appropriate solution for 20 rain before proceeding with the fatiguing series. Peak tetanic force was measured to assess fatigue. The mean value of peak force for the first three tetani in the fatigue series was used as the unfatigued value. The means for three consecutive tetani at later stages in the fatigue series are also reported, along with the test tetani during the recovery period.

The change in PC concentration is the net effect of PC synthesis by oxidation and by glycolysis, and PC breakd o w n due to contraction. (APC = APCox + APCg - RAt

where R is the rate of PC splitting due to contraction, assumed to be constant during the fatiguing series and 0 during the recovery period. The rates of oxidative and glycolytic recovery, APCox and APCg, are proportional to the displacement of PC from its initial, prefatigue value, and are calculated as follows:

Simulation of fatigue and recovery time course The force during fatigue and recovery was compared to force calculated from a simple mathematical model in which force depends on Pi concentration and pH inside the cells. The model calculated the force at 5 s intervals (At = 5 s) during fatigue and recovery.

Abbreviations: PC, phosphocreatine: Pi, inorganic phosphate; H, hydrogen ion; LA, lactate ion and hydrogen ion (lactic acid is fully ionized at the p H ' s involved here); pH, intracellular pH; A, change in the specified parameter within time At.

APCox = kl(PC0 - PC)At

(4)

APCg = k2(PC0 - PC)At

(5)

where kl and k2 are the relevant rate constants.

Intracellular pH Change in intracellular p H is due to hydrogen ion involvement in the phosphocreatine reaction [see (a) above], in glycolytic reactions [see Values (b) above], and the action of the intracellular buffers. ApH = {AHa - ALA}/BP

AHa = APCfil + 10PH-&88)

(a) For each PC split, 1.0 Pi is formed and an amount of H is absorbed due to H binding to the newly formed Pi. The amount depends on the p H and the pK of the reaction, and was calculated using pK 6.68 for the dissociation constant for H from Pi (25 ° C, ionic strength 170 mM, Bates & Acree, 1945). (b) During glycolysis, 1.5 PC is formed per 1.0 LA formed (Woledge et al., 1985). (c) During oxidation, 18 PC's are formed per LA used; if no LA is present other substrate(s) are used (Woledge et al., 1985).

Force Force was taken to decrease exponentially with increasing Pi, towards a minimum value of 50% (Cooke & Pate, 1985; Bowater & Sleep, 1988), and to be a linear function of p H (Chase & Kushmerick, 1988, and references therein). The sensitivity of force to pH and to Pi are adjustable parameters: F/F0 = {1 - [0.5(1 - exp [S~(Pi0 - Pi)]} {1 - [SpH(pH0 - pH)]}

(1)

where subscript 0 refers to the start of the fatiguing series, F is force, Pi is inorganic phosphate concentration, p H is intracellular pH, and S~ and SpH are the sensitivities of force to Pi and pH, respectively.

Inorganic phosphate

(6)

where BP is buffer power. The hydrogen ion involvement in the phosphocreatine reaction (AHa, see Values (a)) is calculated as follows:

Values The following net "stoichiometric'" values were assumed:

(3)

(7)

Change in LA concentration is the net effect of production by glycolysis [see Values (b)], use by oxidation [see Values (c)], and transport out of the cell. We assume that the initial lactate concentration is zero and that the concentration outside the cell remains negligible. ALA = (aPCg/1.5) - (APCox/18) - k3(LA)At

(8)

where 1.5 and 18 are the stoichiometric factors relating LA to phosphocreatine in glycolysis and in oxidation (see Values (b) and (c)). k3 is the rate constant for the transport of LA out of the cell.

Fixed and fitted values The following values from the literature were used as fixed values in the simulation: Pi0 = 6 ~tmolg -1 wet weight in SOL and 0 ~tmolg -1 wet weight in EDL (Kushmerick et al., 1992) PC0 -- 11.4 ~tmolg -1 wet weight in SOL and 21 ~tmolg -1 wet weight in EDL (Kushmerick et al., 1992) pH0 = 7.0 in both SOL and EDL (Phillips et al., 1993) BP = 34 mequiv per litre muscle volume per p H unit (based on 56 mequiv per litre of intracellular water per pH unit measured in the presence of 5% CO2 by Aickin and Thomas (1977) and 0.58 of total muscle volume is intracellular water (Baylor et al., 1982)). The value agrees with that measured by Westerblad and Allen (1992). Literature values of Sr~, SpH, R, kl, k2, k3 (if available) were used as starting points and then adjusted by trial and error to simulate the force results.

Statistics

Pi and PC concentrations are related: aPi = - A P C

(2)

Values were compared using Student's paired t-test unless stated otherwise, p = 5% taken as statistically significant.

Fatigue of mouse muscle

613

Results and discussion T h e m e a s u r e m e n t s of force in Table 1A s h o w that c i n n a m a t e did n o t affect force p r o d u c t i o n b y n o n fatigued EDL, a n d slightly e n h a n c e d that of SOL. C i n n a m a t e did not alter the extent of fatigue prod u c e d b y the fatigue protocols u s e d h e r e (Table 2B a n d W e s t e r b l a d a n d Allen (1992)). Like W e s t e r b l a d a n d Allen, w e conclude that intracellular acidification is n o t the m a j o r factor causing force decline d u r i n g fatigue b y i n t e r m i t t e n t stimulation in m o u s e muscle, It is, of course, possible that w i t h a different fatigue protocol that p H m a y be a significant factor causing force decline d u r i n g fatigue. RECOVERY FROM FATIGUE Table 1B s h o w s results of the fatigue protocol, w h i c h p r o d u c e d a b o u t 25% r e d u c t i o n of force in all cases. Figure 1 s h o w s fatigue a n d the time course of force recovery. Both SOL a n d EDL r e c o v e r e d c o m p l e t e l y in s t a n d a r d saline (Fig. 1A a n d C). R e c o v e r y h a d a r o u g h l y e x p o n e n t i a l t i m e c o u r s e for b o t h m u s c l e s , b u t w a s c o n s i d e r a b l y faster in SOL t h a n EDL (note the difference in time scale). In SOL it w a s c o m p l e t e in a b o u t 5 m i n w h e r e a s EDL h a d o n l y r e c o v e r e d to 91.2% (SEM 1.3%, n = 14) of its initial v a l u e after 5 rain. In cinnamate-saline, SOL r e c o v e r y w a s essentially the s a m e as in s t a n d a r d saline (Fig. 1A a n d B). The fact t h a t force r e c o v e r y of SOL in n o r m a l in cinna-

Table 1A. Force produced by non-fatigued muscle in cinnamate saline (CINN), and in standard saline (CONTROL). Force expressed relative to that produced in standard saline as indicated.

mean SEM P n

Soleus (Cinn/Control)

EDL (Cinn/Control)

1.058 0.027 5% 22

0.997 0.024 NS 14

n, number of muscles. Paired comparisons. p, probability based on comparing the mean with 1.000. Table lB. Force at the end of the fatiguing series of tetani expressed relative to the unfatigued value in the same solution.

Soleus mean SEM n EDL mean SEM n

Control

Cinn

0.704 0.020 22

0.757 0.014 22

0.739 0.017 14

0.703 0.018 14

Paired comparisons n, number of muscles.

Table 2. Lines in Figs 1 and 2 were calculated from the model using the values shown below. See text for the details of the model. Muscle weight in g wet weight. Soleus

EDL

Parameter

Control

Cinn

Control

Cinn

Sensitivity to phosphate, Sei (~tmo1-1 g)

0.080

0.080

0.050

0.050

Sensitivity to pH, SpH (pH unit -1)

0.300

0.300

0.500

0.500

0.52

0.38

0.28

0.867

3.800

3.800

PC split per s of the fatigue series R 0xmol g-~ s -1) 0.52 PC split per s stimulation (~tmol g-1 s-l) 0.867 Rate constant for oxidation, kl (s -1)

0.070

0.070

0.001

0.001

Rate constant for glycolysis k2 (s-l)

0.007

0.007

0.090

0.090

Rate constant for LA transport k3 (s -1)

0.038

0.0004

0.030

0.0004

CLARKEetal.

614

B. S o l C i n n

A. Sol Control

1.2

1.2 (3)

¢-

II

,._° 1.0

£

f

o

>

~ 0.8 rr

o

o

1.0

(1) >

~

FF

0.8 i

0.6

I'

i

i

0

5

10

C. EDL

0.6 0

D. E D L

Control

1.2

I

1

10

I

I

I

q3

(1)

o 1.0

oo 1.0

->~

i

5 Cinn

1.2

I

I

t

>

.i

-~ 0,8

0.8

i

f12

f_E

t

0.6

i 0

i 5

I 10 time (min)

I 15

0.6 20

0

i 5

"L"

i 10 time (min)

T

T

I 15

20

Fig. 1. Timecourse of force production during fatiguing protocol (filled symbols, between vertical broken lines) and recovery (open symbols). Mean + SEM. (A) SOL muscle in standard saline, results for 22 muscles. (B) SOL muscles in cinnamate-saline, results for same 22 muscles as in (A). (C) EDL muscle in standard saline, results for 14 muscles. (D) EDL muscle in cinnamate-saline, results for same 14 muscles as in (C). Solid lines show the force calculated from the model described in the text and values shown in Table 2. mate shows that cinnamate does not directly interfere with metabolic reactions during recovery, for example, by blocking enzymes catalyzing metabolic reactions, nor does it inhibit pyruvate transport into mitochondria. While cinnamate did not affect force recovery in SOL, it was very effective at preventing recovery of EDL (Fig. 1D). What might the basis of the difference between SOL and EDL be? It is well established that cinnamate blocks the lactate + h y d r o g e n ion transporter in both slow a n d fast muscle (Juel, 1988; Mason & Thomas, 1988). Can the force results presented here be explained if this is the ONLY effect of cinnamate? We have considered the following possible explanation. The intracellular p H of EDL acidifies during recovery due to accumulation of h y d r o g e n ion from

glycolysis, a major recovery process in this muscle; cinnamate prevents transport of h y d r o g e n ions out of the cell. In SOL cinnamate does not prevent recovery of force, even though the cinnamate-sensitive transporter exists in this muscle (Juel, 1988; Mason & Thomas, 1988; Westerblad & Allen, 1992) and force in SOL m u s d e is sensitive to pH (Chase & Kushmerick, 1988). Cinnamate has little effect on force recovery in SOL because lactate and hydrogen ions do not accumulate during recovery of this muscle. SIMULATION OF FORCE DURING FATIGUE AND RECOVERY The mathematical simulation was done to see h o w well these ideas could quantitatively match the force results. The solid lines in Fig. 1 were calculated from the

615

Fatigue of mouse muscle simulation in which force d e p e n d s on intraceUular inorganic phosphate concentration and pH. The agreement with the observations of force (symbols in Fig. 1) is good, indicating that the simple description embodied in the model is sufficient to account for the observations. Table 2 shows the set of values for sensitivities of force to Pi and pH, rate of PC splitting and rate constants for recovery processes and lacate + hydrogen ion transport. These values were adjusted to fit the calculated and observed force values. The parameters had different values for EDL and SOL, reflecting the different sensitivities and metabolic profiles of the two muscles. The only parameter affected by cinnamate was the rate constant lactate + hydrogen ion transport (k3). All the final values of the adjusted parameters are reasonable compared with values in the literature based on experimental evidence.

Recovery metabolism In the simulation the values for the rate constants for metabolic recovery processes (kl and k2) are very different for the two muscles. In SOL oxidation is much more important than glycolysis for the resynthesis of PC during recovery, whereas in EDL glycolysis is more important than oxidation. This agrees with the finding by Spurway (1981, 1985) that there is an inverse relation between quantitative histochemical markers in fast and slow fibres for glycolytic and oxidative pathways in mammalian muscle.

LA transporter (k3) Juel and colleagues (1991) measured flux through the transporter in isolated membrane from fast and slow muscle and concluded that there are about 39% more transporters per unit area of membrane from slow muscles than from fast muscles. Total LA flux depends on the total surface membrane area as well as transporter density. The simulations shown in Figs 1 and 2 and values in Table 2 are for the extreme case of maximum difference in flux between SOL and EDL (ratio 1.39:1.00); that is, assuming the total surface area of membrane is the same in SOL and EDL. However, good fits of calculated force to observed force are also achieved if we take account of differences in surface areas of different fibres types. As explained below this factor counteracts the effect of the difference in transporter density. EDL contains some small diameter II-A fibres (30 I~m) (Rosenblatt & Parry, 1992); the other fibres in EDL and all fibres in SOL are of similar diameter (50 ktm) (Parry, personal communication). If the small diameter fibres are taken into account, the total membrane area in EDL is larger than in SOL and this diminishes the difference in flux due to the difference in density of transporter.

Rate of PC splitting during contraction In the simulation the rates of PC splitting during simulation (R/duty cycle) is lower in SOL than in EDL, in agreement with the results of Kushmerick and Crow (1983). Our ratio for SOL/EDL, 0.23, is lower than that found by Kushmerick and Crow (1983), 0.41. The difference between our ratio and that of Kushmerick and Crow is not surprising in the light of the difference in experimental design; we used a series of brief tetani, whereas they used continuous tetanization. The energy consumption per unit of force has been s h o w n to be higher during intermittent stimulation than during continuous stimulation in fast fibres (de Haan et al., 1986).

Sensitivity of force to Pi (Spi) The fractional loss of force during the fatiguing series was the same for the two muscles. In the simulation SOL used less PC (and thus formed less Pi) during the fatiguing series than EDL, as explained above. We found that to obtain a good fit to the force results, SOL had to be more sensitive to changes in Pi than EDL. In the simulation the dependence of force on Pi in SOL is similar to that found in skinned fast and slow rabbit fibres (Nosek et al., 1990, taking account of the relatively high Pi in resting SOL and the intracellular volume fraction). However, unlike our simulation, experimental results for skinned fibres from rabbits by Nosek and colleagues (1990) show that the Pi sensitivities of fast and slow muscles are the same. This may be a species difference or may reflect a difference in the behaviour of skinned and intact fibres.

Sensitivity of force to pH (Spn) In the simulation EDL is more sensitive to p H than SOL. The values we used in the model are the same as those found by Chase and Kushmerick (1988) for skinned fast and slow mammalian fibres in the range of p H values calculated in the model, pH 6.81 and 7.11. The SOL value agrees with that found by Westerblad and Allen (1992) for intact fibres from mouse foot muscle. EDL being more sensitive than SOL also agrees with the results of Metzger and Moss (1990). Pi AND pH DURING FORCE RECOVERY Figure 2 shows results from the simulation for the contributions of Pi and of pH to determining force during fatigue and recovery. In none of the four cases does p H contribute to the reduction in force during the fatiguing protocol of stimulation. As noted earlier, this is consistent with Westerblad and Allen's (1992) direct measurements of p H during a fatiguing series of tetani of mouse fibres. After the end of the fatiguing series, force recovery

CLARKEetal.

616

B. S o l C i n n

A. S o l C o n t r o l 1.2

o o

1.2

]

1.0

-[

1-

5

10

o~ 1.0 O

.>_

>

-$ 0.8

0.8

rr

n"

0.6

l 5

0

0.6

10

0

C. E D L C o n t r o l

D. E D L C i n n

1.2

~jo --,

1.2

i

>

1.0

I/I-/i

I

/i

o

1.0

I

O O

ili,I

(D >

r r 0.8

.....

i

rr

0.6

0.6 0

5 10 time (min)

15

20

0

~

I

l

5

10

15

20

Fig. 2. Results of model calculation, showing separately the contribution from pH (dotted line) and the contribution from Pi (broken line) to force (solid line, same as in Fig. 1). Force is the product of the contributions from pH and Pi. The vertical lines mark the start and end of the fatiguing series of tetani. reflects both decrease of Pi and "he effects of pH. The results from the simulation of untreated EDL (Fig. 2C) are very similar to the NMR measurements of Meyer and colleagues (1991). Additional acidification occurred at the beginning of recovery, whereas Pi decreased quickly to its prefatigue value. Force recovery started before pH recovery, but lagged behind Pi recovery. In the simulation of cinnamatetreated EDL (Fig. 2D), the pH continued to acidify (tending to depress force) while Pi decreased to its pre-fatigue value (tending to increase force). The net effect was that force changed very httle after the end of the fatiguing series, that is, negligible recovery of force occurred. The following main points emerge from the simulation. For the fatiguing protocol used here, pH is not an important determinant of force in either SOL or EDL during the fatiguing series of tetani; it is also unimportant during recovery of SOL. However pH

does play a large part in setting the time course of recovery of force in EDL. Thus we conclude that pH should not be completely dismissed as irrelevant to fatigue, since recovery from fatigue has a functional role which is as important in vivo as fatigue itself. Direct measures of pH during recovery could test this prediction based on the model.

Acknowledgements We thank Drs R. Godt, D. Parry, and A. Rowlerson for information about fibres types, and Mrs Barbara Coen for technical assistance.

References AICKIN, C. C. & THOMAS, R. C. (1977) M i c r o - e l e c t r o d e

measurement of the intraceUular pH and buffering

Fatigue of mouse muscle power of mouse soleus muscle fibres. J. Physiol, 267, 791-810. BATES, R. G. & ACREE, S. F. (1945) p H of aqueous mixtures of potassium dihydrogen phosphate and disodium hydrogen phosphate at 0° C to 60° C. J. Res. Natl Bur, Stand. 34, 371-94. BAYLOR, S. M., CHANDLER, W. K. & MARSHALL, M. W. (1982) Optical m e a s u r e m e n t s of intracellular p H a n d m a g n e s i u m in frog skeletal muscle fibres. J. Physiol. 331, 105-37. BOWATER, R. & SLEEP, J. (1988) D e m e m b r a n a t e d muscle fibres catalyze a more rapid exchange between phosphate a n d adenosine triphosphate than actomyosin subfragment 1. Biochem. 27, 5314-23. CHASE, P. B. & KUSHMERICK, M. J. (1988) Effect of pH on contraction of rabbit fast a n d slow skeletal muscle fibers. Biophys. J. 53, 935-46. COOKE, R. & PATE, E. (1985) The effects of ADP and phosphate on the contraction of muscle fibers. Biophys. J. 48, 789-98. DE HAAN, A., DE JONG, J., VAN DOORN, I. E., HUIJING, P. A., WOITTIEZ, R. C. & WESTRA, H. G. (1986) Muscle economy of isometric contractions as a function of stimulation time and relative muscle length. Pfl~igers Arch. 407, 445-50. JUEL, C. (1988) IntraceUular p H recovery a n d lactate efflux in mouse soleus muscles stimulated in vitro: the involvement of s o d i u m / p r o t o n exchange a n d a lactate carrier, Acta Physiol. Scand. 132, 363-71. JUEL, C., HONIG, A. & PILEGAARD, H. (1991) Muscle lactate transport studied in sarcolemmal giant vesicles: dependence on fibre type and age. Acta Physiol. Scand. 143, 361-5. KUSHMERICK, M. J. & CROW, M. T. (1983) Regulation of energetics a n d mechanics by myosin light chain phosphorylation in fast-twitch skeletal muscle. Fed. Proc. 42, 14-20. KUSHMERICK, M. J., MOERLAND, T. S. & WISEMAN, R. W. (1992) Mammalian skeletal muscle fibers distinguished by contents of phosphocreatine, ATP a n d Pi. Proc. Natl Acad. Sci. USA 89, 7521-5. MASON, M. J. & THOMAS, R. C. (1985) Evidence for facilitated diffusion of L-lactate across frog skeletal muscle

617 membranes. J. Physiol. 361, 25P. MASON, M. J. & THOMAS, R. C. (1988) A microelectrode study of the mechanisms of L-lactate entry into a n d release from frog sartorius muscle. J. Physiol. 400, 459-79. METZGER, ]. M. & MOSS, R. L. (1990) p H modulation of the kinetics of a Ca2+-sensitive crossbridge state transition in m a m m a l i a n single skeletal muscle fibres. ]. Physiol. 428, 751-64. MEYER, R. A., ADAMS, G. R., FISHER, M. J., DILLON, P. F., KRISANDA, I. M., BROWN, T. R. & KUSHMERICK, M. J. (1991) Effect of decreased pH on force a n d phosphocreatine in m a m m a l i a n skeletal muscle. Can. J. Physiol. Pharmacol. 69, 305-10. NOSEK, T. M., LEAL-CARDOSO, J. H., MCLAUGHLIN, M. & GODT, R. E. (1990) Inhibitory influence of phosphate and arsenate on contraction of skinned skeletal and cardiac muscle. American J. Physiol. 259 (Cell Physiol. 28), C933-9. PHILLIPS, S. K., WISEMAN, R. W., WOLEDGE, R. C. & KUSHMERICK, M. J. (1993) Neither changes in phosphorus metabolite levels nor myosin isoforms can explain the weakness in aged mouse muscle. J. Physiol. 463, 157-67. ROSENBLATT, J. D. & PARRY, D. J. (1992) G a m m a irradiation prevents compensatory hypertrophy of overloaded mouse extensor digitorum longus muscle. J. Appl. Physiol. 73, 2538-43. SPURWAY, N. C. (1981) Objective characterization of cells in terms of microscopical parameters: an example from muscle histochemistry. Histochem. J. 13, 269-317. SPURWAY, N. C. (1985) Positive correlation between oxidative and glycolytic capacities in frog muscle fibres. IRCS Med. Sci. 13, 78-9. WESTERBLAD, H. & ALLEN, D. A. (1992) Changes of intracellular p H d u e to repetitive stimulation of single fibres from mouse skeletal muscle. J. Physiol. 449, 49-71. WESTERBLAD, H., LEE, J. A., L.~NNERGREN, J. & ALLEN, D. A. (1991) Cellular mechanisms of fatigue in skeletal muscle. Am. J. Physiol. 261, C195-209. WOLEDGE, R. C., CURTIN, N. A. & HOMSHER, E. (1985) Energetic Aspects of Muscle Contraction. London: Academic Press.