Effect of cyclopiazonic acid on contractile responses

Effect of cyclopiazonic acid on contractile responses in slow and fast bundles of cremaster skeletal muscle from the ferret1 CONNIVE HUCIPET~ AND CLAUDE Limy

Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by Renmin University of China on 05/28/13 For personal use only.

Laboratsire de physislogie gkntmk, Unite' de recherche associde no 1340, Centre national de la recherche scient$que, Facecite' des sciences et des techniques, 2 me de la fibussinidre, 44072 Nantes C&dex 03, France Received March 3, 1994

HUCHET,C., and L~CFTY, C. 1994. Effect of cyclopiazonic acid on contractile responses in slow and fast bundles of cremaster skeletal muscle from the ferret. Can. J . Physiol. Pharmacol. 72: 833-840. The effects of cyclophzonic acid (CPA) on twitch force, calcium (Ca2+)uptake and release by the sarcoplasrnic reticulum (SR), and Ca2+ sensitivity of contractile apparatus were studied using intact and chemically skinned cremaster fibers and compared with those on the extensor digitorum longus and soleus. In cremaster muscles treated with CPA (8.5-5 pM) a potentiation of the twitch was observed, associated with an increase in time to peak and in time of relaxation. In Tritonskinned fibers, CPA, at concentrations less than 10 yM, exerted no significant effect on the contractile apparatus of either slow- or fast-twitch fibers. In slow-twitch fibers, a dose-dependent increase in Ca2+ sensitivity was associated with a decrease in maximal tension, at CPA concentrations > 18 ph4. In saponin-skinmed fibers, during the uptake phase, CPA at > 10 p M induced a dose-dependent decrease in caffeine contracture. The possibility of an action on the SR Ca2+ release channel was excluded by testing the effect of CPA during the releasing phase. The enhancing effect of CPA (0.5-5 pM) on mechanical activity could be explained by an inhibition of the SR Ca2+ ATPase in skeletal muscle cells without an effect on the contractile proteins. Our results strongly suggest that CPA (< 10 pM) has a highly specific effect on the SR Ca2+ pump in the fast- and slow-twitch fibers and therefore could be a good tool to study the mechanisms of Ca2+ regulation in skeletal muscles. Furthermore, the study of the SR properties, using CPA, has shown no significant differences in the SR fkanction of ferret cremaster fibers in comparison with extensor digitorum longus and soleus muscles. Key words : caffeine, skinned fiber, sarcoplasmic reticulum.

HUCHET,C., et L B ~ YC., 1994. Effect of cyclopiazonie acid on contractile responses in slow and fast bundles of crernaster skeletal muscle from the ferret. Can. I. Physiol. Phamacol. 72 : 833-840. On a examink les effets de l'acide cyclopiazonique (ACP) sur les contractions, sur la capture et la liberation de calcium (Ca2+)par Be rCticulum sarcoplasmique (RS) et sur la sensibilitk au Ca2+ de B'appareil contractile dans des fibres de muscles crCmasters pel6es chimiquement; on a ensuite comparC ces effets B ceux observCs sur les muscles extenseur c o m u n des orteils (eco) et sol&ires. Dans les muscles crCmasters traitks B I'ACP (0,s -5 pM),on a observC une potendialisation de la contraction associee h une augmentation du kmps de montCe B la tension maximale et du temps de relaxation. Dans les fibres pelCes au Triton, une concentration d'ACP infkrieure 3 10 yM n'a pas eu d'effets significatifs sur I'appareil contractile. Dans les fibres h contractions lentes, une concentration d'ACP supkrieure ii 10 pM a provoqu6 une augmentation dose-dependante de la sensibilitk au Ca2+associCe 21 une diminution de la tension rmaximale. Dans les fibres pelkes h la saponine, une concentration B'ACP supkrieure h 10 yM a induit, durant la phase de capture, une diminution dose-dipendante des contractions induites par la cafkine. En testant l'effet de 1'ACP durant la phase de liberation, toute action sur la liberation du Ca2+ du RS a kt6 CcartCe. On pourrait expliquer l'effet stimulant de 1'ACP (0,5 - 5 ph4) sur l'activitk mkcarnique par une inhibition de la Ca2+ ATPase, et non pas des protkines contractiles, du RS des fibres musculaires squelettiques. Nos rksultats suggB rent forternent que 1'ACP (< 10 pM) a un effet trks spkcifique sur la pompe 2 Ca2+ du RS dans les fibres a contractions Bentes et rapides, et que, par condquent, son utilisation pourrait s'avCrer utile pour Ctudier les mkcanismes de rtgulation du Ca2+ dans les muscles squelettiques. De plus, l'ktude des propriCt6s du RS, en utilisant 17ACP,n'a montrk aucune diffCrence significative dans la fonction du RS des fibres des muscles crkmasters du furet comparadivement aux muscles solCaires et extenseur commun des orteils. Msls cl&s : cafkine, fibre pelke, rkticulum sarcoplasmique. [Traduit par la RCdaction]

Introduction Force production in skeletal muscle is initiated by the release of calcium (Ca2+)from an internal membrane system, the sarcoplasmic reticulum (SW), in response to transverse tubule deplarization. The mechanism by whish the signal at the transverse tubular Bevel initiates the calcium release is still unknown. The use of compounds to modify the contractile responses of skeletal muscle can provide insights into the mechanisms of excitation-contraction coupling and force activation. Some drugs that have become useful tools in studying CaD release from the SW are doxombicin, ruthenium red, caffeine, and ryanodine (Fryer and Neering 1989; Fryer et al. 1989; Lee et al. 1991). However, these compounds do not spe'This study constitutes a part of a Dwtorat b-sciences (C. Huchet). 'Author for correspondence. Printed in Canada 1 Imprim6 ao Canada

cificdly affect Ca2+ uptake. By contrast, cyclopiuonic acid (CPA), a mycotoxin, has been recently reported to be a specific inhibitor of Ca2+ ATBase of the SR (Goeger et al. 1988; Goeger and Riley 1989). h imhibits not only Ca2+ ATPase activity but also the rate of SR Ca2+ uptake, while it has no effect on activities of other membrane NPases (SeidHer et al. 1989). Furthermore, in skinned strips of smooth muscle, CPA was without effect on the Ca2+ sensitivity of the contractile proteins (Uyama et al. 1992). Finally, in skeletal muscle, CBA has been shown not to affect the myofirillar ATPase, thus making it a powerful tool to explore SR function in muscle cells (Kurebayashi and Ogawa 1991). Compounds like ryanodine or caffeine have been tested in intact and skinned extensor digitorurn Bongus (edl) and soleus fibers (Fryer and Neering 1989; Fryer et al. 1989; Su 1987, 1988; Wendt and Stephenson 1983). However, in addition to

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the typical fast-twitch ( e d ) and slow-twitch (soleus) muscles, mammds possess other muscles that perform specialized functions. In ferret, the cremmter skeletal muscle is one of the components of the thermsregulatory apparatus of the testes, the cremasterico&ftovenous complex, which maintains a constant scrotd -rectal temperature difference ( B d e and Muid 1978; Ninomya 1975). It has been demonstrated by electrophysiological, mechanical, and contractile protein properties that the cremaster muscle is composed of two types of fibers, which were identified as fast and slow types (Huchet and LCoty 1993; Noireaud et d. 1992). However, the twitch time course of the fast and slow fibers s f this muscle is slower still than the twitch produced by edl and soleus muscles, respectively. The present work was undertaken to study the mechanism of action of CPA on contractile response in fast- and slowtwitch fibers from c r e m s t e r muscle. Then, the effects of different concentrations of CPA were tested on isometric force in intact bundles of fibers, on Ca2+ sensitivity of the contractile proteins in Triton-skinned fibers, and on Ca2+ uptake and release activities in saponin-skinned fibers. To compare the results obtained in cremaster with those found in typical fastand slow-twitch fibers, similar experiments were d s o carried out on ferret edl and soleus muscles.

Material and methods General procedures Animal experimentation throughout was conducted in accordance with the guidelines of the Canadian Council on Animl Care. Adult male ferrets weighing 1.81 f 0.06 kg (n = 26) were heavily anesthetized by an ether vapour flow. After respiratory arrest, the cremaster muscle and the testis were excised and placed in oxygenated Hepesbuffered physiological solution at room temperature. The cremaster muscle was removed and pinned in the dissecting dish (Noireaud et ale 1992). The quiescent cremaster muscle is divided into two wellseparated areas, each consisting of thin bundles arranged in a single muscle pattern. To identify the type of bundles under study, before skinning, the characteristics of the contraction (time to peak and relaxation time) were measured (see Results). For comparative experiments on saponin-skinned fibers, edl and soleus muscles were also removed. Twitch tension measurements For the contractile experiments, bundles of 5 - 10 fibers of cremaster muscle were excised along their entire length under a binocular microscope. The preparation was transferred on a cover slip in a drop of physiological solution to the experimental chamber. The two ends of the muscle were carefaally snared by fine platinum-wire loops, one fixed in the experimental dish, the other to the tip of a force transducer (displacement measuring system Kaman KD 2300, Kaman Sciences Corporation, Colorado Springs, Colo.). The flow rate of solution in the experimental chamber was 20 mllnain. The twitch was displayed and analysed on a digital oscilloscope (Nicolet 3091, Nicolet Instrument Corporation, Madison, Wisc .), and the time to peak (ms), the time of relaxation (ms), and the force in the absence or in the presence of CPA (0.5 -5 pM) were measured. Data were expressed as the percentage increase of the force relative to control. Skinned-fiber preparation Small bundles 0.15-0.25 m in diameter and 10-20 m in length were dissected from freshly isolated soleus, edl, and cremaster. Chemical skinning was carried out immediately aker disswtion. For Triton-skinned fibers, preparations were incubated for I h in a relaxing solution (pCa 9, see composition below) containing 1% Triton X-106 (vlv) to solubilize membranes and then transferred to relaxing solution without detergent. Following skinning, some fibers were stored at -20°C in relaxing solution containing 50% glycerol

(vlv). Sagonin-skinning was performed by incubating the bundles for 30 min in relaxing solution containing 50 pg saponinlml. By this treatment, the ability of the SR to accumulate and release Ca2' is preserved (Fano et al. 1989). After the skinning procedure the fibers were mounted between two stainless-steel tubes. One of these tubes was connected to a force transducer (Kaman KD 2300). The diameter and the length of skinned muscles were estimated visually under the microscope. The preparation was adjusted to slack length and then stretched step by step until the tension developed in pCa 4.5 became maximal. The maximal tension was generally reached when the resting length was increased by 20%. All experiments were performed at 22°C. Experimental protocol The tension-pCa relationships (pCa = -log [Caw]) were obtained by exposing the Triton-skinned fiber sequentially to sslutions of decreasing pCa until maximal tension (T,,,) was reached at pCa 4.5, and then fibers were returned to pCa 9. The isometric tension was continuously recorded on chart paper (Linear Bioblock 1208, Reno, Nev.), and the baseline tension was established at the steady state measured in relaxing solution. Data for the relative tensions above 10 and below 90 % were fitted using a modified Hill equation (Huchet et Uoty 1993): Relative tension = [Ca2+]"HY/(# f [Ca2+1 ) " ~ The Hill coefficient, n,, and the pCa for half-maximal activation, pCa,, = (-log,o Kln,) were calculated for each experiment, using linear regression analysis. The Hill coefficient of each type of fiber was calculated as the slope of the fitted straight lines. Resting tension was the tension in pCa 9, and maximal tension was obtained in pCa 4.5. Tension is expressed as mN mm-=.

Cd' uptake by and release from the SR For the experiments on saponin-skinned fibers, the preparation was immersed sequentially in five different solutions, initially to load the SR with Ca2' and subsequently to release it with caffeine. The application of 10 mM caffeine generates a transient contracture (Su 1987, 1988). The ionic composition of solutions was the same as that of the relaxing solution except that free magnesium and the concentration of EGTA and Ca2+ varied as described below. Solution 1, pCa 9, was a high-EGTA (10 M), high-Mg2+(1 &), and high-caffeine (25 mM) solution used to deplete the SR of calcium. Solution 2 was a caffeine-free wash solution similar to solution 1. Solution 3, pCa 7, was a high-EGTA (10 mh4) and high-Mg2+ (1 mM) solution used to load the SR with calcium. Solution 4, pCa 7, was a low-EGTA (0.1 M)and low-Mg2' (0.1 mM) solution used to prepare the release of calcium. Solution 5 was similar to solution 4 but contained 10 mM caffeine to release calcium from the SR. The duration in each solution was 2 min, except for solution 5, where the time was based on the duration of the contracture. When the effects of CPA were tested during the releasing phase, solution 3 was applied for only 60 s. The application of caffeine induced a transient contracture, whose area was measured. At the beginning of the experiments, three or four challenges of the 18 mM caffeine contramre were done. The experimental protocol consisted of performing a control cycle in the absence of CBA. The dose-dependent effect of CBA (18, 20, 50,70,and 108 pM) was studied by using a protocol where CPA was added rando~dyeither in the uptake (solution 3) or in the release (solution 5) medium. ~eversibilit~ of the effects and the absence of deterioration due to CBA were tested by performing a subsequent control cycle after all concentrations. The area of the 10 mM caffeine contracture in each of these conditions was measured. The decrease of the contracture area in the presence of different CPA concentrations was normalized with respect to the preceding 10 mA4 control. Data were expressed as the curve area versus log [CPA]. The 10 mM caffeine control contracture was very similar in the two types of cremaster muscle fibers. To elicit a consistent contracture and to limit


FIG. 2. Concentration-dependent effects of CPA on (A) time to and slow peak and (El) time of relaxation of twitch force in fast (0) (m) cremaster bundles (n = 4 for each group). Points represent mean ( f SEM). Temperamre 22°C. FIG. 1. Effect of CPA on twitch force of cremaster muscle. (A) Original traces showing the increase in force due to the application of CPA (0, 1,2, and 5 pM) in fast ( @ ) and slow ( @ ) cremaster bundles. (El) Effects of CPA, on fast (a)and slow (H) cremaster bundles, expressed as mean percent increase in force (f SEM) compared with control (n 4 for each group). Temperature 22°C.

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the sensitizing effect of caffeine on contractile proteins, 10 mM caffeine was used instead of 25 mM caffeine (Wendt and Stephenson 1983). Without any further Ca2+ loading step, subsequent application of caffeine did not elicit any contracture. Solutism The control physiological solution contained (in mha) NaCl, 140; KC1, 6; CaCl,, 3; MgCl,, 2; glucose, 5; Mepes, 5 (pH 7.4 adjusted with Tris base). The solutions were equilibrated with 100%8, and all experiments were conducted at room temperature (19-22°C). CPA was added to physiological solutions at different concentrations (0.5-5 pM). All chemicals were prepared in distilled, deionized water.

Skinned-$her solutions The calcium concentration of relaxing (pCa 9, solution A) and activating (pCa 4.5, solution B) solution was calculated using the computer program of Godt and Nosek (1986). AH solutions were calculated to contain 10 mhl EGTA, 30 mM imiclazole pH 7.10, 30.6 rrmhiI Na+, 1 m M Mg2+, 3.16 rnM Mg ATP, 12 mM phosphocrmtine, and 0.3 mM dithiothreitol (ionic strength, 160 mM adjusted with KCl). In saponh-skinned fiber experiments, solutions also contained phosphscreatine kinase (1%W/mL) and sodium azide (1 mM). Solutions with intermediate Ca2+concentrations (solution 3), for Triton- and saponin-skinned fiber experiments, were obtained by mixing solution A with El in appropriate proportions. EGTA, phosphocreatine, and CPA were obtained from Sigma Chemical Company (St. Louis, Mo.). CPA was prepared as a 20 mM stock solution in DMSB. The drug was present in loading or in releasing

solution to obtain a final concentration of 1- 100 pM. At 188 ph4 CPA, DMSB was equivalent to 0.5%, which in control experiments, did not affect contractile proteins and SR knction. Statistical analysis All values were expressed as mean f standard error of the mean (fSEM). An unpaired t test was used to compare the different parameters between groups. Differences were considered statistically significant at p < 0.05.

Results Efleets of CPA on isometric twitch of skeletal cremster muscle In twitch contraction experiments, two fiber types could be distinguished in cremaster on the basis of contractile response (Fig. 1). Thus, the fast-twitch fibers are characterized by a time to peak vdue of 153 & 5 ms and time of relaxation of 472 f 16 ms (n = 4). These times are shorter than in the slowtwitch fibers, whose time to peak is 192 & 23 ms and time of relaxation is 1170 & 64 ms (n = 4). Figure 1 illustrates the increase in force due to different concentrations of CPA for fast and slow bundles sf cremaster muscle. The three parmeters measured, in the two types of fibers, were differently affected without any variation in resting tension. Exposure sf the fast-twitch muscle to 1 p M CPA significantly increased twitch force by 48.5 % and time to peak and time of relaxation by 48.5 and 42.4% (n = 4; p < 0.001), respectively. Encreasing the concentration of CPA from E to 5 pM led to a more pronounced increase in the twitch parmeters observed (Fig. I). In contrast, in slow-twitch fibers at 8.5 or 1 pM CPA, a decrease in time to peak and time of relaxation (Fig. 2) was observed with no significant change in force (Fig. 1B). At 5 pM CBA, a significant increase in force and time to peak was observed, while the time of relaxation was not significantly affected

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FIG. 3. Effect of various CPA csncentrations on Ca2+-activated tension of slow-twitch fibers from cremaster muscle. Experimental &-aces showing the effect of CPA (100 yM) on Ca2+-activatedtension in Triton-skinned slow fibers (diameter 200 ym; length 1 m). The fiber was bathed in submaximal Ca" concentration (a, pCa 6.625; b, pCa 6.375; or c, pCa 5.$75) and maximal Ca2+csncentration (d, pCa 4.5)(indicated by the arrows). After steady state was reached in control, the fiber was immersed in the same Ca2+ concentration containing CPA (a', b', c', d', respectively) and then returned to the Ca2+ concentration without CPA, to test for reversibility. Next, the preparation was exposed to the relaxing solution P C 9). ~

(Fig. 2). N 1 the modifications observed were fully reversible. For example, h e inotropic effects due to 0.5 - 1 and 1 -5 pM CPA were reversed following 10 and 20 min, respectively, in CPA-free medium. Therefore experiments in intact ferret cremaster bundles have shown that CPA ( < 10 pM) has clear differentid effects on the two types of fibers. The enhancement sf twitch force in CPA-treated muscles could tentatively k accounted for by an increase in the Ca2+ sensitivity of the contractile proteins. This possible effect was tested by using Triton-skinned fibers. Eflects of CPA on Ca2 -activated tension in Triton-skinned fibers 'khe effect of CPA on the maximal Ca2+-activatedtension and the apparent Ca2' sensitivity s f the contractile proteins was tested at five concentrations (5, 10, 20, 50, and 100 pM) in the two types of fibers from cremaster muscle. To explain the positive inotropic effect of CPA on the twitch force by a possible effect on the contractile apparatus we tested 5 and 10 pM CPA on Triton-skinned fibers. The last three concentrations of CPA (20, 58, and 100 pM)were dso chosen to cornpare the results obtained in experiments with saponin-skinned fibers (see below).

Fro. 4. Effect of various CPA concentrations on myofibrilhr Ca2+sensitivity of fast and slow fibers from cremaster muscle. ZSQmetric tension - pCa (-log [Ca2+])relationships in twitch fibers in the absence ( 0 ) or in presence of increasing CPA concentrations: 50 (A) and 100 (B) pM. Typical tension-pCa curves were obtained with (A) fast-twitch and (B) slow-twitch fibers. Force is expressed as the percentage of maximal tension at pCa 4.5 for each concentration of CPA tested. The curves were fitted by the modified Hill equation (see text). Temperature 22 "C .

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Maximal Ca" -activated tension The capacity of the contractile apparatus of chemically skim& fibers from fast- and slow-twitch cremaster muscle to develop force when maximally activated by Ca2+ (pCa 4.5) was not affected in the presence sf low concentrations of CPA (5, 10, and 20 pM) in CaD solutions (not illustrated). However, in slow-twitch fibers, 108 pM CPA slightly decreased the steadystate tension (Fig. 3). Ca2 sensitivity No significant difference could be observed in the relative tension - pCa curves in the presence of either 5 or 10 ph4 CPA +

between fast- and slow-twitch fibers. However, in slow-twitch fibers, as illustrated in Fig. 4, where the experiments are plotted as pCa -tension concentration curves, it is clear that pCaSO is different for 50 and 100 pM CPA. The increase in pCaSo a b v e the control values (ApCaSo)for 50 and 100 pM CPA was 0.044 -b 0.005md 0.070 f 0.005,respectively, where the corresponding values of n~ were l .!XI f 0.03 and 1.90 f 0.03 (82 = 5). In fast-twitch fibers, far dl the concentrations of CBA (5 - 1663 pM) tested, the relative tension - pCa curves were not significantly affected. These results suggest that the positive inotropic effect of low CPA concentrations (0.5 -5 pM) observed on twitch force in intact fast and slow cremaster fibers was not due to an action of the substance on the contractile proteins. However, in slow cremaster fibers, concentrations of CPA lager than 10 pM lead to an increase in Ca2+ sensitivity sf the contractile proteins and to a decrease in maximal Ca2+-activated tension (Fig. 3). Eflects of CPA on SR finction in saponin-skinned skeletal muscle fibers Figure 5 illustrates the effects of CPA on the contracture induced by 10 mM caffeine in saponin-skinned slow and fast


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FIG. 5. Effect of CPA on SIX content in fast and slow cremaster fibers (assessed by contractures elicited with 10 m M caffeine) when CPA is applied during the Ca2' uptake phase. Records of isometric tension from (A) fast and (B) slow cremaster bundles. a, control response to 10 rnM caffeine. In the following run, the same fiber was exposed to a range of CPA concentrations: b, 20; c, 50; d, 70; and e , 100 ph4, added in the Ca2+ loading solution. Temperature 22 "C.

cremaster fibers. Identical experiments were carried out in the two types of fibers from ferret edl and soleus muscles (Fig. 6). Under the present experimental conditions, concentrations of CPA larger than 10 pM were tested because lower concentrations were without effect on 10 mM caffeine contracture in saponin-skinned fibers. When 20 pM CPA was present during the Ca2+ loading step, the caffeine responses in the two types of fibers were decreased. These effects were more pronounced if larger concentrations of CPA were used. Thus, CPA inhibited the caffeine contracture of saponin-skinned fibers in a dose-dependent manner. The inhibition by CPA can be fitted by a sigmoidd relationship, which allowed estimation of the concentration of CPA that produced 50% of the maximal inhibition (ICS0) and a slope coefficient. The fitted curves are illustrated in Fig. 7 for cremaster, edl, and soleus muscles, and calculated values are given in Table 1. The data show that IC50 is significantly smaller in fast than in slow fibers but similar in fast cremaster and edl. Ca2+ uptake for edl and fast cremaster appears to be more sensitive to CPA than slow cremaster and soleus muscle. A similar result was obtained when the effect of CPA was tested on twitch force. The observed reduction in the caffeine contracture could be interpreted as the result of a decrease in SR Ca2+ content caused by an inhibitory effect on SR Ca2+ ATPase by CPA. Another possible explanation would be that CPA affects the SR Ca2+ release channel, thereby reducing the amount of Ca2+ released into the myoplasm. We tested this possibility by the study of the release of the SR Ca2+ content with caf-

FIG. 6. Effect of CPA during the uptake phase on SR Ca2+ content in edl and soleus (assessed by 10 m M caffeine). Records of isometric tension from (A) d l and (B) ssleus bundles. a, control response to 10 m M caffeine. In the following run, the same fiber was exposed to a range s f CPA concentrations: b, 20; c, 50; d, 70; e , 100 pM, added in the Ca2+ loading solution. Temperature 22°C.

feine in the presence of CPA, One of these experiments is illustrated in Fig. $A, where it can be noticed that in the presence of CPA, caffeine-induced force development of cremaster muscle was slightly affected. It can also be observed, in Fig. $B, that Ca2+ release in soleus was not affected and was only slightly modified in edl muscle by 20 - 100 pM CPA.

Discussion The present study has shown that CPA (0.5-5 p M ) enhanced, in a dose-dependent manner, the mechanical activity of cremaster skeletal muscle and that both types of fibers displayed a progressive slowing of contraction and relaxation. Although these changes are consistent with the known effects of CPA inhibition of SR Ca2+ ATPase (Seider et al. 1989), it is also possible that CPA has other effects that could contribute to these changes. Previous reports have shown that CPA depleted the sarcoplasmic reticulum - endoplasrnic reticulum Ca2+ stores by inhibiting the ATP-driven Ca2+ sequestration in smooth and nonsmooth muscle cells (Uyama et al. 1992). Furthermore, in SR vesicles from rabbit skeletal muscle, Seidler et al. (1989) reported that CPA acted on the Ca2+ ATPase of the SR,and this effect on Ca2+ uptake and Ca2+-dependent ATPase activity was fairly specific for the SR pump. If CPA is indeed blocking SR Ca2+ ATPase, a decrease in SR Ca2+ content could be expected because Ca2+ released at each contraction cannot be taken up, thus leading to a decrease in force development. Indeed, CPA at micromolar concentrations reversibly

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LOG [CBA]

PIG. 7. Dose-response curves of CPA effects during the Ca2+ uptake phase. (A) ed8 (m) and soleus ( a ) fibers. (B) Fast (m) and slow ( a ) cremaster muscle. Data were expressed as the percentage of decrease of the 10 mM caffeine contracture area for each concentration of CPA, compared with control. Points are connected by a curve that fits a~Hillrelation. Mill coefficient, n,, and IC, are given for each type of fiber in Table 1. Vertical bars represent f SEM.

PIG. 8. Effects of CPA on the Ca2+release phase of the caffeineinduced contracture of saponin-skinned skeletral fibers from 41, soleus, and cremaster. (A) C1, contracture control before the administration of CPA; C2, second caffeine transient after administration sf 20, 50, and 100 pM CPA. (B)Dose-response curves of CPA effects during the Ca2+ release phase. Data are expressed as the percentage of area of C2 compared with C1 for each concentration tested and each type sf muscle, soleus (o),eel1 (@I, and cremaster (m). Vertical bars represent f SEM. Temperature 22 "C.

TABLE1. Effect of CPA on the uptake phase

dl Soleus Cre~mster Fast Slow

25.45f0.85 58.90f 0.90

2.03k0.20 1.55f0.25

5 4

28.20f 0.85 60.53 k0.85

2.05 k0.13 1.78fO. 10

5 4

NOTE:The dose-response curves could be fitted using the Hill equation, thereby yielding the Hill toeacient, n ~and , the CPA concentration that gave 50% of the inhibitory action, ICSO.Values are mean (%!EM) obtained for the three types sf muscks. n, number of experiments.

diminished the contractures induced by 10 m M caffeine. Surprisingly, CPA induced an increase in twitch force, in cremaster skeletal muscle (Fig. 1) as well as in rat edl and soleus muscles (C. Huchet and C. Uoty, unpublished results). This is probably due to the effects of CPA on the inhibition of the Ca2+ uptake and to the difference in the quantity of Ca2+

released during caffeine contracture compared with a twitch. The application of caffeine (10 mM) leads to the release of a large amount of Ca2+ that can be lost by the SR in the presence of CPA. By contrast during a twitch it will take a longer time to deplete the SR because only a small fraction of the Ca2+ is released. Thus CPA induced an increase in twitch force because it slows down the Ca2+ uptake and minimally affects the Ca2+ content of the SR. Furtkermore the increase in time to peak presently observed could be accounted for by the slowing of SR Ca2+ pumps, prolonging the interaction between Ca2+ and troponin C. Our results are consistent with the ability of CPA to block calcium uptake. However, we found a larger value for ICSo, 25.45 f 0.85 pM for edl and 58.90 f 0.09 pM for soleus (a = 5), than in isolated SR vesicles, where CPA inhibited Ca2+uptake with an ICSoof 0.2 pM (Kurebayashi and Ogawa 1991). This has been dready described for other molecules acting at the SR level, e.g., procaine or ryanodine, and different explanations can be proposed; first, factors or cytoarchitecturd features necessary for CPA sensitivity may be lost or


HUCWET AND L ~ C ~ T Y

disrupted on removal of the cell membranes and, second, the experiments used for in vitro assay may not reproduce conditions inside cells necessary for CaD regulation (Vites and Pappano 1992). The experiments in Triton-skinned fibers excluded the possibility of an increase in CaB sensitivity by a direct effect of CPA (0.5-5 pM) on the contractile proteins. However, in soleus muscles, at concentrations of CPA > 10 pM, an increase in calcium sensitivity probably due to a direct effect of CPA on the regulatory proteins was observed. The concentration threshold of CPA that produces a detectable increase in twitch force was lower in fast-twitch than in slow-twitch cremaster muscle fibers. This result suggests that fast-twitch fibers were more sensitive to CPA than the slow ones. Furthermore, the difference in CPA sensitivity observed between muscles was also found in saponin-skinned fibers. ICS0values determined in slow-twitch fibers were larger than in fast-twitch cremaster fibers. A similar difference was also found between soleus and edl. This difference in sensitivity to pharmacological tools, between slow and fast muscles, has already been observed using caffeine (Fryer and Neering 1989) or ryanodine (Fryer et al. 1989). Various explanations can account for the differences in the effects of CPA on SR CaD uptake. (i) A difference in CPA sensitivity of the Ca2+ ATPase pump may exist between the muscle fiber types because the molecular basis of the SR Ca2+ ATPase was not identical in fast and slow muscles (Brand1 et al. 1987; Campbell et al. 1991; Grover and Khan 1992). In fact, three distinct isoforms of Ca2 -transporting ATPase residing in intracellular organellar stores of mammdian skeletal muscle cells have been described. Their expression was tissue dependent and developmentally regulated. These isoforms can all be recognized as alternatively processed products of two separate but homologous genes, SEBCA P and SEBCA 2 (Grover and Khan 1992). (ii) There may be a higher pump density in fast- than in slow-twitch muscles: biochemical studies have demonstrated that the fasttwitch muscle relaxes faster than slow-twitch muscles because it contains 2.2 times more Ca2+ ATPase per milligram of SR protein and the maximal rate of CaD uptake as well as the maximal Ca2+ capacity are higher than in slow-twitch muscles (Gillis 1985; Ferguson and Franzini-Armstrong 1988; Riiegg 1987). It is generally accepted that the properties of the SR of the different types of muscles are similar and that the characteristics of the SR are in close relation with the species and the type and the function of the muscles. However, in the ferret cremaster the presence of a slow contraction could not be accounted for by the present results, which have shown no significant differences in the SR properties. Furthermore, on the basis of the sensitivity to CPA in cremaster saponin-skinned fibers, the results suggest that the SR properties are similar to those of ferret edl and soleus muscles. The present observations in association with the results previously obtained from the study of Ca" sensitivity in cremaster fiber (Huchet and Uoty 1993) support the hypothesis that this muscle is composed of fibers similar to those found in edl and soleus.

Acknowledgements This work was supported by the Association fran~aisede lutte contre les myopathies and by the Fondation Eanglois. The authors thank J.Y. Su for reading and discussing the manuscript.

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