Changes in voltage activation of contraction in frog skeletal muscle fibres as a result of sarcoplasmic reticulum Ca2+-ATPase activity

Changes in voltage activation of contraction

in frog skeletal muscle ®bres as a result

of sarcoplasmic reticulum Ca2+-ATPase activity

W . M EÃ M E and C . L EÂ O T Y

Laboratoire de Physiologie GeÂneÂrale, CNRS EP1593, FaculteÂ des Sciences et des Techniques, 2 Rue de la HoussinieÁre,

Nantes Cedex 3, France

ABSTRACT

The effects of cyclopiazonic acid, a specific sarcoplasmic reticulum Ca2+-ATPase inhibitor, on

isometric tension were studied in response to prolonged steady-state depolarization induced by a

rapid change in extracellular potassium concentration (potassium contractures) in frog semite-

ndinosus muscle ®bres. Cyclopiazonic acid (1±10 lM) enhanced the amplitude and time-course of

relaxation of 146 mM potassium contracture. In the presence of cyclopiazonic acid 0.5 lM, the

relationship between the amplitude of potassium contractures and the membrane potential shifted to

more negative potentials, whereas the steady-state inactivation curve was unchanged. These

observations suggest that cyclopiazonic acid has no effect on voltage sensors. The difference

between potassium contractures in the absence and presence of cyclopiazonic acid in skeletal

muscle ®bres implies that the amplitude and slow relaxation of tension during prolonged steady-state

depolarization may be expected to depend not only on inactivation of the process regulating calcium

release from the sarcoplasmic reticulum but also on the ability of the sarcoplasmic reticulum to pump

calcium.

Keywords cyclopiazonic acid, frog, potassium contracture, sarcoplasmic reticulum

Ca2+-adenosinetriphosphatase.

Received 23 July 1998, accepted 8 March 1999

In the excitation±contraction coupling mechanism of

skeletal muscle, the depolarization of action potentials

in transverse tubules induces a movement of charged

voltage sensors (Schneider & Chandler 1973) within the

dihydropyridine receptors (Rios & Brum 1987). The

voltage sensors then activate the opening of Ca2+-

release channels in sarcoplasmic reticulum to generate

tension. Since the work of Hodgkin & Horowicz

(1960), potassium (K+) contractures have been widely

used as a convenient experimental model for the study

of depolarization±contraction coupling of skeletal

muscle ®bres. It is generally recognized that the voltage

dependence of tension during steady-state depolariza-

tion depends exclusively on activation of the voltage

sensor in transverse tubule membrane. Similarly, the

slow decay in tension during prolonged depolarization

is assumed to depend on inactivation of excitation±

contraction coupling (Hodgkin & Horowicz 1960,

Caputo 1972, Dulhunty 1991). Thus, the activation and

inactivation characteristics of tension re¯ect the

voltage-dependence properties of the dihydropyridine

receptors in transverse tubules (Dulhunty & Gage

1985). It is admitted that the decay in tension during

prolonged steady-state depolarization is not in¯uenced

by calcium dissociation from troponin, crossbridge

detachment, or calcium uptake by the sarcoplasmic

reticulum, which are considered to proceed at rates high

enough not to make them limiting factors (Stein et al.

1988, Dulhunty 1992).

We recently reported that cyclopiazonic acid (CPA),

a selective inhibitor of sarcoplasmic reticulum Ca2+-

ATPase in skeletal muscle (Goeger et al. 1988, Seidler

et al. 1989), directly affects the handling of intracellular

Ca2+ and force production during brief depolarization

in intact skeletal ®bres (MeÃme et al. 1998). Conse-

quently, inhibition of a fraction of sarcoplasmic

reticulum Ca2+-ATPase activity leads to a broadening

of the Ca2+-transient and a potentiation of twitch

Correspondence: Claude LeÂoty, Laboratoire de Physiologie GeÂneÂrale, CNRS EP1593, FaculteÂ des Sciences et des Techniques,

2 Rue de la HoussinieÁre, BP 92208, 44322 Nantes Cedex 3, France.

Acta Physiol Scand 1999, 166, 209±216

Ó 1999 Scandinavian Physiological Society 209

tension. The purpose of the present work was to use

CPA to determine the role of calcium uptake by the

sarcoplasmic reticulum on the activation and inactiva-

tion of steady-state tension in frog semitendinosus

muscle. The effects of CPA on the time-course of K+

contracture decay on the voltage sensitivity of K+

contracture activation and steady-state inactivation were

studied. The present results show that the decay of K+

contracture during prolonged depolarization depend

not only on inactivation of voltage sensors but also on

the ability of the sarcoplasmic reticulum to pump Ca2+.

MATERIALS AND METHODS

General procedures

Experiments were performed at room temperature (19±

21 °C) on skeletal muscle ®bres isolated from frog

(Rana esculenta) semitendinosus muscle. Frogs were

killed by destruction of the spinal cord followed by

decapitation. The isolated muscle was placed in a

dissecting chamber containing Ringer's solution, and

bundles with 3±5 ®bres were excised along their entire

length under a binocular microscope. The preparation

was transferred to an experimental dish on a coverslip.

One end of the bundle was immobilized by a thin loop

of silver wire fastened to the bottom of the dish with a

small hook. The opposite end was attached to the tip of

a force transducer (Kaman KD 2300 displacement

measuring system, Colorado Springs, CO, USA).

Isometric tension measurements

The preparation was stretched, and resting tension

was set to obtain maximal force development of the

muscle length±tension curve (sarcomere length

2.9 lm). Sarcomere length was measured by analysis

of video images of the ®bre. Cell images were

obtained with a charge-coupled device camera and

digitized using a personal computer-based frame-

grabber system. The ¯ow rate of the superfusing

solution in the experimental chamber was 20 mL min±1,

allowing a total bath medium change in < 0.2 s. The

preparation was stimulated electrically by current pulses

at twice the threshold amplitude delivered at a fre-

quency of 0.05 Hz applied between one pair of plati-

num electrodes on each side of the channel. The

developed tension was recorded by the force transducer

interfaced with a computer (IBM, 80486DX, 33 MHz)

via the Digi Data interface card (Digi Data Card 1200,

Axon Instruments, Foster City, CA, USA) with a

sampling frequency of 33 Hz. This computer allowed

data storage and measurement of amplitude, time to

peak which is de®ned as the delay between the

beginning of tension development and the maximal

tension, and the time constant of relaxation. The time-

course of the relaxation was evaluated by non-linear

least-squares curve ®tting of the exponential function

to experimental data.

The activation curve of the K+ contracture was

obtained by a rapid change from the control solution to

one containing an elevated potassium concentration

(10±146 mM [K+]o) in the absence of electrical stimu-

lation. In these solutions, the [K+][Cl±] product was

kept constant to allow rapid recovery of resting

potential upon return to Ringer's solution and resto-

ration of the amplitude of the tension response. [K+]oconcentrations higher than 146 mM were not used

because of hypertonicity. After spontaneous relaxation

of the contracture, K+ solution was replaced by

Ringer's solution in which ®bres recovered for 15 min

before a new contraction cycle was induced. After

control data were collected, ®bres were incubated with

CPA for 10 min to reach the steady-state maximal

effect (MeÃme et al. 1998), and K+ contractures were

generated in the presence of CPA using the same

protocol. Peak K+ contracture tension values were

plotted against the corresponding extracellular K+

concentration ([K+]o) and normalized to maximal

tension in 146 mM [K+]o solution.

The inactivation curve of K+ contracture was

obtained by measuring test 146 mM [K+]o contracture

amplitude after submaximal depolarization for 2 min in

a conditioning [K+]o. Peak tension values of test K+

contractures were plotted against the corresponding

conditioning [K+]o and normalized to maximal tension

in 146 mM [K+]o solution.

Conventional glass microelectrodes (10±20 MW)

connected to an electrometer input negative capaci-

tance ampli®er were used to measure membrane

potential. Values were obtained in separate experiments

in which corresponding membrane potentials were

measured on bundles containing 20±30 ®bres after

5 min in different high [K+]o solutions used to evoke

K+ contractures in the absence and presence of CPA.

There was no signi®cant difference between the average

values for control and CPA (Table 1).

Solutions

Ringer's solution contained (in mM) 110 NaCl, 2.5 KCl,

2 CaCl2, and 8 Tris(hydroxymethyl)aminomethane

(Tris)±HCl. pH was adjusted to 7.5 with a Tris solution.

Ca2+ was added as a 1 M CaCl2 solution to a concen-

tration of 2 mM Ca2+. Depolarizing solutions were

prepared by replacing a given amount of NaCl with

KCl, and the [K+][Cl±] product was kept constant by

replacing Cl± with L-glutamate. A stock solution of

CPA (20 mM) was prepared in dimethylsulphoxide

(DMSO). The ®nal concentration of DMSO for 2 lM

Role of SR in EC coupling � W MeÃme and C LeÂoty Acta Physiol Scand 1999, 166, 209±216

210 Ó 1999 Scandinavian Physiological Society

CPA was 0.1%. All chemical products were purchased

from Sigma Chemical, St Louis, MO, USA.

Statistical analysis

All values are expressed as mean � SE for n observa-

tions. Student's paired and unpaired t-tests were used to

compare (when appropriate) the parameters between

groups. Statistical signi®cance was reached when

P < 0.05.

RESULTS

Effects of CPA on 146 mM [K+]o contracture

The effects of CPA on 146 mM [K+]o contractures

were studied on small bundles of frog semitendinosus

muscle. Figure 1 shows K+ contractures tested with

different concentrations of CPA (0.5±10 lM). In con-

trol conditions (CPA-free), 146 mM [K+]o induced a

rapid transient contracture which reached a peak and

then relaxed to resting level even when superfusion

with 146 mM [K+]o solution was maintained. The

response was characterized by an amplitude of

1.28 � 0.06 mN, a time to peak of 2.9 � 0.2 s and a

time constant of relaxation of 2.1 � 0.2 s (n� 14),

when relaxation was ®tted to a single exponential

function. The results showed that, following 10 min

exposure to CPA, the amplitude of the K+ contracture

was increased and the relaxation phase greatly pro-

longed in a dose-dependent manner (Fig. 1). CPA

0.5 lM had no statistically signi®cant effect on tension,

whereas CPA 5 lM signi®cantly increased tension by

35 � 11% (1.73 � 0.21 mN; n� 6; P < 0.05). Time to

peak and the time constant of relaxation reached

4.4 � 0.9 s and 12.0 � 3.7 s (n� 6, P < 0.05), respec-

tively. Increasing the CPA concentration to 10 lM

produced a more marked change in the parameters of

146 mM [K+]o contracture. In view of these results, it

seemed of interest to determine whether the activation

and inactivation curves of K+ contractures were

affected by CPA. However, the rates of rise and decay

of tension in high potassium solutions were voltage

dependent and thus slower at low membrane potential

level (Fig. 4c). Consequently, at high CPA concentra-

tions (1±10 lM), the relaxation phase was greatly pro-

longed and ®bres failed to relax spontaneously during

submaximal depolarization (data not show). Accord-

ingly, submaximal depolarizations were subsequently

performed at a CPA concentration (0.5 lM), which

allowed full relaxation of tension to its resting level.

Effects of CPA on voltage-dependent activation of K+

contractures

Figure 2 shows K+ contractures of a muscle prepara-

tion tested with different concentrations of [K+]o(10±146 mM) in the absence and presence of 0.5 lM

CPA. In Ringer's solution, small contractures devel-

oped in 10 mM [K+]o, and tension increased with

higher potassium concentrations. The results showed

that CPA 0.5 lM increased the amplitude and prolonged

the relaxation phase of tension response at all K+

concentrations tested. However, CPA produced a more

Table 1 Effects of cyclopiazonic acid on membrane potential in high

potassium solutions

Membrane potential (mV)

[K+]o (mM) Control CPA 0.5 lM n

2.5 )89.2 � 0.9 )88.7 � 1.1 34

10 )48.1 � 0.4 )47.8 � 0.5 26

20 )40.3 � 1.9 )39.9 � 1.8 10

25 )35.1 � 0.5 )35.2 � 0.4 18

30 )31.9 � 1.6 )32.1 � 1.8 10

50 )25.2 � 0.8 )24.9 � 0.7 20

80 )16.6 � 1.9 )16.3 � 2.0 11

146 )9.1 � 0.4 )9.2 � 0.4 39

Membrane potentials were measured in the presence and absence of

cyclopiazonic 0.5 lM in control and after 5 min in high potassium

solutions. Values in the presence of cyclopiazonic acid were not

signi®cantly different (P < 0.05). Values are mean � SE.

Figure 1 Effects of CPA on 146 mM [K+]o contracture. Original

recordings showed an increase in peak tension and relaxation time

after 10-min application of CPA (0.5±10 lM) to frog semitendinosus

muscle. Small bundles containing 3±5 ®bres were allowed to rest for

15 min in 2.5 mM [K+]o solutions (Ringer or Ringer + CPA) between

contractures. Dashed lines show baseline tension in control

conditions (absence of CPA).


Acta Physiol Scand 1999, 166, 209±216 W MeÃme and C LeÂoty � Role of SR in EC coupling

marked change in tension at low depolarization levels.

Tension was 0.51 � 0.06 mN (control) and

0.82 � 0.08 mN (CPA 0.5 lM) (n� 5; P < 0.05) in

20 mM [K+]o, whereas tension was not signi®cantly

changed in the absence (1.26 � 0.07 mN) and presence

of CPA 0.5 lM (1.30 � 0.07 mN; n� 5; P > 0.05) in

146 mM [K+]o. The relationship between the relative

amplitude of the K+ contracture and the extracellular

K+ concentration, in the absence and presence of

0.5 lM CPA, is shown in Fig. 3. CPA induced little

change in the contractile threshold but caused a shift to

the left and a change in the slope of the tension±[K+]ocurve. The corresponding potassium concentration

calculated for half-maximal activation was 23.9 � 1.3 mM

and 17.2 � 1.5 mM in the absence and presence of CPA

0.5 lM (n� 5; P < 0.05). In separate experiments,

membrane potentials were measured to investigate

whether the change in the relative tension of K+ con-

tractures in the presence of CPA could be correlated with

changes in membrane potential (Table 1). The results

show that compared with control, CPA 0.5 lM induced

no change in membrane potential at all potassium con-

centrations tested. Thus, the observed negative shift of

the potassium concentration for half-maximal activation,

which corresponded to a shift of ±8.3 mV, was not

correlated with any change in resting membrane

potential.

Effects of CPA 0.5 lM on the time-course

of K+ contracture decay

It is generally admitted that the slow decay in tension

during prolonged steady-state depolarization depends

solely on inactivation of excitation±contraction cou-

pling and is not in¯uenced by the kinetics of contractile

protein response and the rate of calcium uptake by the

sarcoplasmic reticulum (Dulhunty 1992). In Ringer's

solution, membrane depolarization induced a rapid and

transient contracture which reached a peak and then

relaxed to resting level even when superfusion with

high K+ solution was maintained. The rates of rise and

decay of tension in high potassium solutions were

voltage dependent and faster at more depolarized

membrane potentials (Fig. 4). Figure 4(a) clearly shows

that the time to peak and the duration of the relaxation

phase were prolonged at 25 mM [K+]o compared with

tension response in 146 mM [K+]o. Exposure of the

bundle of ®bres to 0.5 lM CPA induced an increase in

the amplitude of the K+ contractures and prolonged

the relaxation phase in 25 mM [K+]o. In Fig. 4(b), the

decay of K+ contracture tension was ®tted to a single

exponential function, and the time constant of relax-

ation was plotted against [K+]o (Fig. 4c). The results

show that the time constant of relaxation was not sig-

ni®cantly affected by CPA in 146 mM [K+]o (control,

1.9 � 0.2 s; CPA, 2.3 � 0.4 s; n� 5; P > 0.05) but

greatly increased at lower membrane depolarizations.

The average time constant of relaxation was 5.1 � 0.5 s

and 13.3 � 1.1 s (n� 5; P < 0.05) in 25 mM [K+]o, in

the absence and presence of CPA 0.5 lM, respectively.

On the other hand, the time to peak of the amplitude of

K+ contracture was not affected by CPA 0.5 lM

(Fig. 4c). If CPA has a selective effect on sarcoplasmic

reticulum Ca2+-ATPase in skeletal muscle (Seidler et al.

1989), these results suggest that the slow decay in

Figure 2 Effects of cyclopiazonic acid 0.5 lM on K+ contractures

evoked at different bath K+ concentrations ([K+]o). The bars under

the tension traces indicate the periods of exposure to high [K+]osolutions, and the number below each tension record indicates [K+]oconcentrations in mM. Small bundles were allowed to rest for 15 min

in 2.5 mM [K+]o solution (Ringer or Ringer + 0.5 lM CPA) between

contractures.

Figure 3 Effects of cyclopiazonic acid 0.5 lM on the relationship

between relative tension at the peak of K+ contracture and membrane

potential. Tension is expressed relative to the maximal contracture

recorded in 146 mM [K+]o, in the presence (.) and absence (n) of

CPA 0.5 lM. Values are means � SE, n� 5.



tension during prolonged depolarization may be

expected to depend not only on inactivation of voltage

sensors but also on the ability of the sarcolasmic

reticulum to pump Ca2+. However, the shift of the

activation curve towards a more negative membrane

potential could indicate that CPA has a direct action on

the properties of voltage sensors. This possibility led us

to investigate whether CPA could affect the onset of

steady-state inactivation.

Effects of CPA 0.5 lM on the inactivation curve

A general model for depolarization±contraction

coupling suggests that depolarization sequentially

converts a fraction of the resting voltage sensors to an

active state and then to an inactive state, and that K+

contracture tension is proportional to the fraction of

voltage sensors in the active state (Caputo 1972,

Dulhunty 1991). Thus, the fraction of resting activator

available for conversion to the active state is reduced

by inactivation. If the action of CPA were considered

to increase the fraction of voltage-sensitive molecules

converted to the active state upon depolarization, the

fraction of resting voltage sensors available after CPA

treatment would be reduced. In the present experi-

ments, the fraction of resting voltage sensors available

after CPA treatment was determined from the

amplitude of test 146 mM [K+]o contracture, which

re¯ects maximal activation in frog skeletal muscle.

Figure 5 illustrates the protocol used to investigate the

inactivation of K+ contracture tension by measuring

test 146 mM [K+]o contracture amplitude after

submaximal depolarization in 20 or 25 mM [K+]o for

2 min. The amplitude of K+ contractures for maximal,

submaximal and test depolarizations was increased by

CPA. The inactivation curve was obtained by plotting

peak tension values of test 146 mM [K+]o contractures

against the corresponding conditioning [K+]o and

normalizing to maximal tension in 146 mM [K+]osolution. The present experiments showed that CPA

0.5 lM induced no signi®cant changes in the inacti-

vation curves (Fig. 5). This suggests that CPA 0.5 lM

did not increase the fraction of voltage-sensitive

molecules converted to active state upon depolariza-

tion and thus had no effect on the voltage depen-

dence of inactivation of K+ contracture. If it is

assumed that CPA has a selective effect on sarco-

plasmic reticulum Ca2+-ATPase in skeletal muscle, it is

likely that, on the steep ascending part of the

Figure 4 Effects of CPA 0.5 lM on the time-

course of K+ contracture decay. (a) Records

obtained in 25 and 146 mM [K+]o, before and after

exposure of the muscle preparation to 0.5 lM CPA.

(b) Semilogarithmic plot of the relative tension

against time during the relaxation phase of K+

contractures shown in (a) (tension was normalized

to maximal peak tension, and t� 0 was taken as the

peak tension). The relaxation of K+ contractures

decay as a single exponential in 25 mM [K+]o (jh)

and 146 mM [K+]o (ds) in the absence (sh) and

presence (dj) of CPA 0.5 lM. (c) The time to

peak and the time constant of relaxation of the K+

contracture were plotted against [K+]o, in the

absence (sh) and presence (dj) of CPA 0.5 lM.

*Signi®cant differences from control values

(P < 0.05). Values are mean � SE, n� 5.



activation curve, cytoplasmic Ca2+ will remain higher

for longer. As a result, the amplitude of tension and

the relaxation phase will increase.

DISCUSSION

The present work was carried out to study the mem-

brane potential dependency of tension relaxation during

prolonged depolarization and to determine the extent to

which sarcoplasmic reticulum Ca2+-ATPase may be

involved in the time-course of K+ contractures in

skeletal muscles. The main results show that a low

concentration of CPA increased the amplitude of K+

contractures and prolonged the relaxation phase in frog

semitendinosus muscle. In addition, the relationship

between the relative amplitude of K+ contracture and

membrane potential was shifted towards more negative

values, whereas the inactivation curve remained

unchanged.

The time-course of K+ contracture tension during

prolonged depolarization is generally considered in

terms of the activation and inactivation of the process

regulating the release of calcium from the sarcoplasmic

reticulum. This process depends on the conformational

states of voltage-sensitive dihydropyridine receptor

molecules in the transverse tubule membrane (Caputo

1972, Dulhunty 1991). In addition, a close similarity

between the voltage dependence of tension and charge

movement was observed (Chandler et al. 1976,

Rakowski 1981). In this context, it was suggested that

the voltage-sensing process involved in the excitation±

contraction coupling mechanism could be indirectly

assessed by studying the voltage dependence of tension

(Dulhunty & Gage 1985, Brum & Rios 1987).

Does CPA have other effects than inhibition

of the sarcoplasmic reticulum Ca2+-ATPase?

It is possible that the CPA-induced change of tension

and shift of the activation curve could be caused by a

modi®cation of the EC coupling process similar to the

effects of perchlorate ions (ClO4±) which potentiate

submaximal K+ contractures in amphibian and mam-

malian skeletal muscles and shift the voltage depen-

dence of tension to more negative membrane potentials

(Gomolla et al. 1983, Dulhunty et al. 1992). However,

the relative fraction of the voltage sensor converted in

the active state during submaximal depolarization did

not change in the presence of CPA, which suggests that

CPA had no effect on the voltage-dependence activa-

tion and steady-state inactivation of the voltage sensors.

On the other hand, the enhancement of voltage-

dependent tension by CPA 0.5 lM could be related to

an increase in the Ca2+ sensitivity of the contractile

proteins, which may be expected to shift the activation

curve to more negative membrane potential values.

However, 50 lM CPA had no effect on myo®brillar

Ca2+ sensitivity (MeÃme et al. 1998). In addition, CPA

effects at a concentration of up to 2 lM were not

associated with an increase in resting intracellular

calcium concentration (MeÃme et al. 1998).

Alternatively, previous studies on electromechanical

coupling of smooth muscle cells showed that secondary

to the inhibition of sarcoplasmic reticulum Ca2+-AT-

Pase by CPA, the cell was depolarized and the

Figure 5 The effects of cyclopiazonic acid 0.5 lM on steady-state

inactivation of K+ contracture. (a) Original recordings of tension, in

the absence or presence of CPA, from experiments designed to

measure inactivation of test 146 mM [K+]o contracture tension after

2-min conditioning depolarization in 20 or 25 mM [K+]o. (b) Peak

tension values of test 146 mM [K+]o contracture after submaximal

depolarization for 2 min were plotted against the corresponding

conditioning extracellular K+ concentration and normalized to

maximal tension in 146 mM [K+]o in the presence (.) and absence (n)

of CPA 0.5 lM. Values are mean � SE, n� 5.



contractile phase of the contraction±relaxation cycle

was prolonged (Maggi et al. 1995). Thus, CPA could

have an indirect action on the electrophysiological

properties of skeletal ®bres through its effects on

membrane potential. However, our results show that

CPA 0.5 lM did not affect resting membrane potential

in high potassium solutions (Table 1). The rate of

change of Vm is also important in terms of the peak K+

contracture tension as a change in the rate of depo-

larization would affect the amplitude of the K+ con-

tracture. Complementary measurements of tension and

membrane potential were carried out. Microelectrode

recordings in control solutions (absence of CPA), have

shown that, according to [K+]o, depolarization was 90±

95% complete after 3±4 s and reached a steady level in

5±8 s. Similar measurements in the presence of CPA

0.5 lM have shown that the rate of depolarization was

not signi®cantly changed. As a result, the time to peak

of K+ contracture was identical in the absence and

presence of CPA (Fig. 4c). It could be then proposed

that the shift of the tension curve to the left was not

associated with a change in resting membrane potential

or an acceleration of depolarization by CPA.

It is now well established that CPA is a speci®c

inhibitor of sarcoplasmic reticulum Ca2+-ATPase in

skeletal muscle (Goeger et al. 1988, Seidler et al. 1989).

Then, the fact that, after small depolarization, the

amplitude of potassium contracture was increased and

the time course of relaxation was prolonged, suggests

that when cytoplasmic Ca2+ uptake becomes slower

than inactivation of the voltage sensor, the Ca2+ uptake

could be rate limiting in the decay of tension in parallel

with the process regulating Ca2+ release from the

sarcoplasmic reticulum.

It is interesting to note that, in the presence of

CPA, the slower relaxation is easier to observe with

low than with high potassium concentrations. It has

been shown that, in the absence of CPA, the ampli-

tude of the Ca2+ transient is smaller and the Ca2+

transient decay is greatly prolonged when ®bres are

depolarized with low compared with high potassium

concentrations (Caputo & Bolanos 1994). Then, our

results suggest that CPA may have its greatest effect

on contractures that decay more slowly possibly

because of the lower calcium transients. However, it

should be noticed that tension relaxation in 146 mM

[K+]o can be slowed if larger CPA concentrations

(1±10 lM) were used (Fig. 1).

In conclusion, the difference between K+-contrac-

tures in the absence and presence of CPA in skeletal

muscle ®bres implies that the amplitude and slow decay

of tension during prolonged depolarization may be

expected to depend not only on inactivation of voltage

sensors but also on the ability of the sarcolasmic

reticulum to pump Ca2+.

In view of our results, care must be taken in inter-

preting changes that occur when the functional capacity

of the sarcoplasmic reticulum is modi®ed. For example,

hindlimb unweighting is considered to elicit changes in

metabolic and contractile properties of skeletal muscle.

Reports have shown that the activation and inactivation

curves calculated from K+ contracture experiments were

shifted towards more positive membrane potentials in

slow-twitch muscle (soleus) from suspended rats,

becoming similar to those determined in fast-twitch

muscle (edl) (Khammari & Noireaud 1994). These

changes were correlated with a modulation of dihydro-

pyridine receptor gene expression (Kandarian et al.

1992). However, in soleus muscle, a marked upregulation

of the fast isoform of the sarcoplasmic reticulum Ca2+-

pump gene at the mRNA and protein levels has been

demonstrated (Schulte et al. 1993), which was consistent

with increased Ca2+-dependent ATPase activity and the

speeding up of muscle relaxation properties. Moreover,

we recently demonstrated that the sensitivity to CPA of

intact and skinned soleus ®bres following hindlimb

unweighting became similar to that of fast-twitch muscle

(Huchet-Cadiou et al. 1996). Thus, in parallel with

modulation of dihydropyridine receptors, changes in the

capacity of the sarcoplasmic reticulum to pump calcium

could account for the shift in the activation curve of

soleus muscle following hindlimb unweighting.

This work was supported by the Foundation Langlois and, as part of

the PhD studies of W. MeÃme, by The French Ministry of Education

and Research.

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Changes in voltage activation of contraction in frog skeletal muscle fibres as a result of sarcoplasmic reticulum Ca2+-ATPase activity