INTRODUCTION

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DIHYDROPYRIDINE RECEPTORS in skeletal muscle fibers sense the depolarization of action potentials in transverse tubules (24) and induce the opening of release channels in sarcoplasmic reticulum. Force production requires an increase in cytoplasmic Ca²⁺ activity, allowing Ca²⁺ to bind to troponin C and thereby activate myofilaments. In excitation-contraction coupling of fast-twitch skeletal muscle, most of the activating Ca²⁺ originates from sarcoplasmic reticulum (10, 21). For relaxation, intracellular Ca²⁺ concentration ([Ca²⁺]_i) must be lowered to its resting level. Although Ca²⁺ could be buffered by cytosolic proteins or extruded by the sarcolemmal Ca²⁺ pump (7, 13), sarcoplasmic reticulum Ca²⁺-adenosinetriphosphatase (ATPase) is generally considered to be the main system involved in the maintenance of Ca²⁺ homeostasis (7, 28). The sarcoplasmic reticulum Ca²⁺ pump removes Ca²⁺ from the cytosol, thereby dissociating it from troponin C and detaching cross bridges. In these conditions, it seemed of interest to determine whether the role of sarcoplasmic reticulum in promoting the relaxation of skeletal muscle could be further characterized by using a compound that selectively affects Ca²⁺ uptake.

Cyclopiazonic acid (CPA), a mycotoxin from Aspergillus and Penicillium, has been reported to be a specific inhibitor of sarcoplasmic reticulum Ca²⁺-ATPase in skeletal muscle (9, 16, 17). CPA inhibits the rate of sarcoplasmic reticulum Ca²⁺ uptake as well as Ca²⁺-ATPase activity but has no effect on the activities of other membrane ATPases (25) and the actomyosin-type ATPase of myofibrils (20). A recent work showed that CPA inhibited Ca²⁺ uptake in vesicles of sarcoplasmic reticulum isolated from frog skeletal muscle (6), whereas an earlier study by the same authors in mechanically skinned fibers found that sarcoplasmic reticulum Ca²⁺ uptake was inhibited by CPA (half-maximal inhibition of 7 µM and total inhibition at 50 µM) (5). However, no reports have dealt with the effect of CPA on contractile response in intact skeletal muscle cells of the frog.

The purpose of the present work was to study CPA-induced changes in contraction and the Ca²⁺ transient in intact skeletal muscle cells from frog semitendinosus muscle. Different CPA concentrations were tested for their action on isometric twitch, and nonspecific effects of CPA were searched for in skeletal muscle. Action potential duration and resting membrane potential were measured in the presence of CPA. The effect of CPA on maximum force production and the Ca²⁺ sensitivity of contractile proteins was studied in Triton-skinned fibers. Finally, we investigated the correlation between twitch and the Ca²⁺ transient, and the effect of inhibition of the sarcoplasmic reticulum Ca²⁺ pump on the kinetics of the Ca²⁺ transient and force, to assess the relative role of sarcoplasmic reticulum in promoting relaxation.

	MATERIALS AND METHODS

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General procedures. Experiments were performed at room temperature (19-21°C) on skeletal muscle cells isolated from frog (Rana esculenta) semitendinosus muscle. Frogs were killed by decapitation followed by destruction of the spinal cord. The isolated muscle was placed in a dissecting chamber containing Ringer solution, and bundles containing a few fibers were excised along their entire length under a binocular microscope. The preparation was transferred to the experimental dish on a coverslip and mounted as described by Huchet and Léoty (16). One end of the fiber 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).

Isometric tension measurements. Fibers were stretched, and resting tension was set to obtain maximal force development of the muscle length-tension curve (sarcomere length 2.9 µm). Sarcomere length was measured by analysis of video images of the fiber. Cell images were obtained with a charge-coupled device camera and digitized using a personal computer-based frame grabber system previously described (11). The flow rate of the perfusing solution in the experimental chamber was 20 ml/min. The preparation was stimulated electrically by current pulses at twice the threshold amplitude delivered at a frequency of 0.05 Hz applied between two pairs of platinum electrodes on each side of the channel.

Measurement of the Ca²⁺ transient. The experimental chamber (the bottom consisted of a glass coverslip) was placed on the stage of an inverted microscope (Nikon Diaphot). Fluorescence measurements were monitored using the Ca²⁺-sensitive indicator indo 1 (Sigma Chemical, St. Louis, MO). Cells were loaded, including the salt form of the dye, by microinjection with a glass microelectrode. When the microinjection was performed properly, fiber swelling subsided at the injection site within a few seconds after pressure release, leaving no visible sign of impalement. Intracellular diffusion was allowed to proceed for 15 min. After several isometric contractions, twitch was compared with the control before microinjection. Care was taken to perform experiments on preparations in which tension was not modified by microinjection. Excitation wavelength (365 nm) was provided via a 75-W xenon arc lamp with a monochromator. The excitation light beam was directed into the microscope equipped for epifluorescence measurements. Ultraviolet light was reflected toward a fluorescence objective (Nikon Neofluor ×40) by a 380-nm dichroic mirror. Fluorescence from the cells crossed the dichroic mirror and was reflected by the prism toward a second dichroic mirror (455 nm) where the light beam was split. Wavelengths of 405 and 480 nm were selected by interference filters (10-nm bandwidth) placed in front of the two photomultiplier tubes. The microscope field was restricted to the bundle by means of an adjustable window, and a manual shutter was used for fiber illumination only during light signal readings to reduce photobleaching. Optical signals were acquired as photon counts per second and stimultaneously stored with the output of the force transducer in a data acquisition and analysis system. After control data were collected, CPA was incubated with fibers for 10 min, and twitches were generated in the presence of the inhibitor. No attempts were made to calibrate fluorescence signals in terms of absolute Ca²⁺ concentrations.

Chemically skinned fibers. Short cut bundles containing 5-10 fibers (150-250 µm in diameter, 10-20 mm in length) were dissected from freshly harvested semitendinosus muscles. The fibers were incubated for 1 h in a relaxing solution (pCa 9, see composition below) containing 1% Triton X-100 (vol/vol) to solubilize membranes and then transferred into relaxing solution without detergent. After skinning, some fibers were stored at 20°C in relaxing solution containing 50% (vol/vol) glycerol.

Experimental protocol. The tension-pCa relationship (pCa = log [Ca²⁺]) was obtained by exposing the fiber sequentially to solutions of decreasing pCa until maximal tension was reached (in pCa 4.5), after which the fibers were returned to pCa 9. Isometric tension was continuously recorded on a chart recorder (Linear Bioblock 1200, Reno, NV), and baseline tension was established in steady state and measured in relaxing solution. Data for relative tensions above 10% and below 90% were fitted using a modified Hill equation where T is relative tension, K is the Ca²⁺ concentration for half-maximal activation, and n_H is the Hill coefficient.

Solutions. The normal physiological solution contained (in mM) 105 NaCl, 2.5 KCl, 2 CaCl₂, 5 C₃H₃NaO₃ (pyruvic acid sodium salt), 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 (BDH volumetric standard Analar grade) to a concentration of 2 mM Ca²⁺. Relaxing (pCa 9, solution A) and activating (pCa 4.5, solution B) solutions were prepared using the computer program of Godt and Nosek (8). All solutions were calculated to contain (in mM) 10 EGTA, 30 imidazole, 30.6 Na⁺, 1 Mg²⁺, 3.16 MgATP, 12 phosphocreatine, and 0.3 dithiothreitol. Ionic strength was adjusted to 160 mM with KCl, and pH was adjusted to 7.10. Solutions of intermediate Ca²⁺ concentrations were prepared by mixing two solutions of extreme concentrations (A and B) in suitable proportions. All chemical products were purchased from Sigma Chemical.

Statistical analysis. All values are expressed as means ± SE for n observations. Student's paired and unpaired t-tests were used to compare (when appropriate) the parameters between groups. Statistical significance was reached when P < 0.05.

	RESULTS

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Effects of CPA on isometric tension. The effects of CPA (0.5-50 µM) on isometric tension were studied in intact skeletal fibers of frog semitendinosus muscle. In control conditions, contractions by fast-twitch fibers were characterized by a tension of 1.04 ± 0.09 mN (n = 6), a time to peak of 25.7 ± 0.4 ms (n = 6), and a time constant of relaxation of 14.4 ± 1.5 ms (n = 6), where relaxation was fitted to a single exponential function. In Ringer solution, the application of different CPA concentrations (0.5, 1, 2, 5, and 10 µM) induced a detectable change in contraction after only 60 s. However, 10-min exposure was generally required to reach the steady-state maximal effect. A longer exposure in this concentration range gave no additional effects. The results showed that CPA affects amplitude, time to peak, the time constant of twitch relaxation, and resting tension. Representative twitches elicited in control conditions and in the presence of CPA after 10 min are shown in Fig. 1. Low concentrations (0.5 and 1 µM) had little (and nonsignificant) effect on twitch parameters, whereas exposure to 2 µM CPA significantly increased isometric tension by 39 ± 9% (1.45 ± 0.12 mN; n = 6). Time to peak and the time constant of relaxation reached 35.9 ± 2.8 ms (n = 6) and 22.2 ± 1.7 ms (n = 6) (P < 0.05), respectively. Increasing the concentration of CPA from 2 to 10 µM produced a more marked change in twitch parameters (Figs. 1 and 2) and resting tension. The variation in tension values corresponded to 1.1 ± 0.2, 2.7 ± 0.3, and 7.8 ± 0.6% (n = 6) of the total increase in force with 2, 5, and 10 µM CPA, respectively. At these concentrations, the effects of CPA were fully reversible after 10-20 min in a CPA-free medium.

View larger version (8K): [in this window] [in a new window]   Fig. 1.   Effect of cyclopiazonic acid (CPA) on twitch force. Original recordings showed an increase in force, time to peak, and relaxation time after 10-min application of CPA (0.5-10 µM) to frog semitendinosus bundles. Resting tension was increased after addition of 5 and 10 µM CPA. Dashed line represents resting tension in control conditions (absence of CPA).

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Dose-dependent effects of CPA on time to peak (A), time constant of relaxation (B), and isometric tension (C) (n = 6). * Significant differences from control values (P < 0.05).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Time-dependent effects of 50 µM CPA on twitch force. Preparation was stimulated every 20 s, but twitches are represented only every minute for greater clarity. Twitches are superimposed in control conditions (trace A) and in presence of 50 µM CPA for 5 min (traces B-F). Note development of contracture.

Effects of CPA on resting and action potentials. Although CPA has been reported to act on specific Ca²⁺ pools (9), previous studies on electromechanical coupling of smooth muscle cells showed that CPA depolarized the membrane and prolonged the duration of the action potential and the contractile phase of the contraction-relaxation cycle (22). This led us to investigate whether the change in force production in the presence of CPA was correlated with changes in resting membrane and action potentials. The results show that the duration of action potentials measured at 50% of full repolarization was not significantly altered in the presence of 2, 5, and 10 µM CPA (control, 2.43 ± 0.12 ms; 10 µM CPA, 2.48 ± 0.12 ms; n = 6; P > 0.05). After 10-min exposure to 10 µM CPA, a significant hyperpolarization was observed (control, 86.6 ± 0.2 mV; 10 µM CPA, 89.8 ± 0.2 mV; n = 107; P < 0.01), which disappeared within 15-20 min after the preparation was returned to Ringer solution.

Effect of CPA on Ca²⁺-activated tension in Triton-skinned fibers. The enhancement of twitch force and resting tension in CPA-treated muscles could be related to an increase in myofibrillar Ca²⁺ sensitivity. This hypothesis was tested using Triton-skinned fibers, which allowed us to study the properties of contractile proteins in the absence of functional sarcoplasmic reticulum. Solutions ranging from pCa 7 to 4.5 were used to determine the relative pCa-tension curves, which were then fitted to the Hill equation. Maximal Ca²⁺-activated tension, the Hill coefficient, and the pCa for half-maximal activation were calculated for each experiment in the absence or presence of CPA (2, 5, 10, and 50 µM) (Table 1). With respect to apparent Ca²⁺ sensitivity, the relative pCa-tension curves were not significantly affected for any of the CPA concentrations in the Ca²⁺ solutions tested. Figure 4 shows the pCa-tension curves plotted in control conditions and in the presence of 50 µM CPA. These results suggest that the effects of CPA on twitch and resting tensions in the range of concentrations tested (0.5-50 µM) were not due to the action of the mycotoxin on the Ca²⁺ sensitivity of contractile proteins.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Effect of CPA on myofibrillar Ca2+ sensitivity of semitendinosus skinned fibers. Isometric tension-pCa (log [Ca2+]) relationships in presence () and absence () of 50 µM CPA are expressed as a percentage of maximal force at pCa 4.5. Curves were fitted by modified Hill equation (see text) (n = 6).

Effects of CPA on the Ca²⁺ transient. Fluorescence measurements were monitored using the Ca²⁺-sensitive indicator indo 1. The time course of the Ca²⁺ transient and the development of isometric tension during twitch were recorded simultaneously. In the six preparations studied, no changes were observed in time to peak force and the time constant of relaxation after microinjection of Ca²⁺ dye. Thus impalement was not deleterious to cells, and the amount of indo 1 loaded was large enough to sense the change in [Ca²⁺]_i without modifying contractile response. Figure 5 shows the mean determinations for four fluorescence transients and twitches in a frog skeletal fiber (fluorescence intensity occurred at 405 and 480 nm). During electrically stimulated twitch, intensities of emitted light at 405 and 480 nm increased and decreased, respectively, due to the Ca²⁺ transient, which was expressed as a change in the fluorescence ratio at wavelengths optimal for bound (405 nm) and unbound (480 nm) forms. Recordings of force and Ca²⁺ transients were superimposed and normalized to determine the relationship between the fluorescence intensity peak and the time course of the tension response during twitch. The Ca²⁺ transient preceded the increase in force, attaining a peak within 13.1 ± 1.2 ms and gradually returning to its resting level as tension rose. After reaching its maximal level, the fluorescence signal decreased, with a time constant of 7.1 ± 1.2 ms, and had nearly returned to baseline when twitch force became maximal. Tension began to rise 3.8 ± 1.8 ms after onset of the Ca²⁺ transient, and the average time to peak for twitch was 30.1 ± 4.2 ms, corresponding to a time delay from the peak fluorescence signal of 16.9 ± 3.2 ms (n = 5).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Fluorescence transients at 405 nm (A), at 480 nm (B), and as 405-to-480 nm ratio (C), and twitch force from an indo 1-loaded skeletal muscle cell. Fluorescence ratio and tension recordings were superimposed and normalized with respect to peak amplitude. AU, arbitrary units.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effect of CPA on Ca2+ transient and force in an isometric twitch. A: superimposed records of fluorescence in control Ringer solution (C) and in presence of 2 µM CPA. B: tension traces in absence and presence of 2 µM CPA.

	DISCUSSION

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This study shows that CPA had distinct dose-dependent effects on the twitch contraction of frog skeletal muscle cells. Within a range of 0.5-10 µM, CPA increased contractile force and prolonged time to peak and the relaxation phase, whereas 10-50 µM CPA depressed twitch and induced sustained contracture. If it is assumed that CPA has a selective effect on sarcoplasmic reticulum Ca²⁺-ATPase in skeletal muscle, Ca²⁺ content may be expected to decrease with large CPA concentrations (>10 µM). In this case, Ca²⁺ released at each contraction cycle would not be taken up, leading to a progressive decrease in force development. Moreover, Ca²⁺ in the cytosol would probably remain bound to troponin C, leading to the development of sustained contracture. With low CPA concentrations (<10 µM), only a fraction of sarcoplasmic reticulum Ca²⁺-ATPase activity would be inhibited (5). On stimulation, the Ca²⁺ released from sarcoplasmic reticulum was removed from the cytosol by sarcoplasmic reticulum Ca²⁺-ATPase, and the rate of uptake became lower. Thus CPA (0.5-10 µM) induced an increase in twitch force and relaxation phase duration but had a minimal effect on the Ca²⁺ content of sarcoplasmic reticulum. Under these conditions, the preparation could be stimulated repeatedly without depletion of the sarcoplasmic reticulum, and further exposure to CPA had no additional effects after ~10 min. These results are in agreement with observations previously reported (5) in frog skeletal skinned fibers in which the CPA effect was dependent on the concentration used and on the time allowed for the mycotoxin to diffuse into the fiber. Thus, for a diffusion time of 4-6 min, 50% inhibition was achieved by a CPA concentration of 7 µM and total inhibition by 50 µM.

CPA could have an indirect action on the electrophysiological properties of skeletal fibers through its effect on intracellular Ca²⁺ transport, as reported for smooth muscle cells (22). However, our results showed that CPA effects in frog skeletal muscle were not associated with changes in resting membrane and action potentials.

Because force production is not only correlated with [Ca²⁺]_i but also depends on myofibrillar Ca²⁺ sensitivity and the ability of cross bridges to produce force, these two points were studied to determine whether CPA has any nonspecific effects. Previous results in mammalian skeletal muscle indicated that CPA increases the Ca²⁺ sensitivity of contractile proteins of slow-twitch (CPA >10 µM) but not fast-twitch fibers (15). Studies in skinned skeletal muscle fibers also showed that CPA inhibits Ca²⁺-ATPase activity without affecting actomyosin-type ATPase (20). In the present study, the pCa-tension curve of skinned fibers without functional sarcoplasmic reticulum was only slightly affected by CPA, indicating that Ca²⁺ binding to the specific site on troponin C was not altered and that CPA (5-50 µM) had little influence on myofibrillar Ca²⁺ sensitivity.

Changes in the ability of cross bridges to produce force were estimated from changes in maximal force, i.e., the force at [Ca²⁺]_i levels in which troponin C was saturated with Ca²⁺. Our results showed that, in the presence of 2, 5, 10, and 50 µM CPA, the capacity of contractile proteins to develop force when maximally activated by Ca²⁺ was reduced by 2.0, 10.2, 15.5, and 22.2%, respectively. Although the mechanisms underlying the decrease in tension remain unclear, CPA probably affects the biochemical states of the cross bridges during force activation, resulting in a reduction in the number of their interactions (12). This mechanism should be taken into account in estimating twitch tension in the presence of CPA (2-10 µM). In intact skeletal muscle fibers, near-maximal activation was elicited by depolarization of the membrane in high-K⁺ solution (190 mM K⁺). In amphibian semitendinosus fibers, the twitch-to-contracture tension ratio was 0.4, and the peak [Ca²⁺]_i reached during twiches was ~4 µM, whereas during maximal K⁺ contracture (190 mM K⁺), the peak was 10.4 µM (3). Thus it may be assumed that during a twitch the mechanisms underlying decreased tension in skinned fibers were less important than for maximal activation. However, this mechanism would have operated at a CPA concentration >5 µM, whereas at 2 µM, changes because of cross-bridge alterations were not significantly different from control values (Table 1). Consequently, 2 µM CPA was used in fluorescence measurements to avoid changes in myofibrillar Ca²⁺ sensitivity and the ability of cross bridges to produce force. The modifications in twitch parameters observed with 2 µM CPA differed significantly from control values, whereas resting tension and resting and action potentials were unchanged.

The modifications in twitch tension in the presence of CPA could have been because of changes in Ca²⁺ transient amplitude resulting from sarcoplasmic reticulum release during stimulation. The amplitude of the Ca²⁺ transient depends on three factors: the rate of Ca²⁺ release from sarcoplasmic reticulum, myoplasmic Ca²⁺ buffering, and the rate of Ca²⁺ uptake by sarcoplasmic reticulum (7, 29). Our results show that CPA induced a sustained contracture (for large concentrations of CPA) and potentiated Ca²⁺ transient amplitude and the tension associated with twitch. CPA may affect passive Ca²⁺ permeability or the Ca²⁺ release channel of sarcoplasmic reticulum, thereby increasing the amount of Ca²⁺ released (or leaked) into the myoplasm when the sarcoplasmic reticulum Ca²⁺ pump is inhibited. Studies in skinned fibers from skeletal muscle of the ferret (16) and frog (5) showed that caffeine-induced force development remained virtually unchanged when CPA was applied after Ca²⁺ loading. This result was based on a specific protocol in which tension response kinetics were limited by caffeine diffusion (a diffusion coefficient of 2.4 cm²/s in muscle fibers) (1), and the relaxation phase was the result of Ca²⁺ diffusion from skinned fiber to the washing medium. However, peak amplitude and the global transient of caffeine-induced contracture are usually used to assay the content of sarcoplasmic reticulum. Thapsigargin is known to be a potent inhibitor of ATP-dependent Ca²⁺ uptake by isolated sarcoplasmic reticulum vesicles of the frog (27), and the inhibition of endo(sarco)plasmic reticulum Ca²⁺-ATPase by thapsigargin results in a steady passive Ca²⁺ leak from internal stores. Thapsigargin (300 µM) applied after Ca²⁺ loading in frog skinned fibers reduced caffeine response by releasing some of the Ca²⁺ load. Conversely, in skinned fibers, CPA at a concentration of up to 100 µM appeared to have no major effect on the passive Ca²⁺ permeability of sarcoplasmic reticulum (5). Moreover, we used fluorescence measurements to monitor changes in myoplasmic Ca²⁺ activity during isometric twitch. Modifications in the spatially average Ca²⁺ transient were affected by all Ca²⁺ translocations associated with an action potential and gave the net balance between Ca²⁺ release and Ca²⁺ uptake. However, as during activation Ca²⁺ release is much greater than Ca²⁺ uptake (otherwise activation would be impossible), the initial value of d[Ca²⁺]/dt was practically equivalent to average Ca²⁺ release. The present experiments showed that the initial rising phase of the Ca²⁺ transient was not changed. Thus it is likely that CPA (2-10 µM) in intact fibers did not affect average Ca²⁺ release or passive Ca²⁺ permeability from intracellular stores during a twitch.

Ca²⁺ released from sarcoplasmic reticulum by muscle stimulation rapidly binds to various myoplasmic Ca²⁺ buffers such as troponin C and parvalbumin. Our results show that Ca²⁺ binding to the specific site on troponin C was not changed in the presence of CPA (see above). Parvalbumin is present in high concentrations in fast-contracting skeletal muscle of the frog (0.76 mM) (13) as compared with troponin C (0.09 mM) (25). Low temperature (13, 14) and pharmacological inhibition of the sarcoplasmic reticulum Ca²⁺ pump (18) revealed that parvalbumin, like sarcoplasmic reticulum, sequesters Ca²⁺ and promotes relaxation by exchanging Mg²⁺ for Ca²⁺. This reaction was rate limited by the Mg²⁺ dissociation from parvalbumin. It was assumed that in skeletal muscle parvalbumin has its greatest relative effect at low temperatures. Moreover, the Mg²⁺ dissociation rate from parvalbumin was less sensitive to temperatures of 10-20°C than the rate of Ca²⁺ uptake by sarcoplasmic reticulum (14). Q₁₀ values were 1.9 for the Mg²⁺ dissociation rate from parvalbumin (14) and 3 for the rate of Ca²⁺ uptake by sarcoplasmic reticulum (23), which is very similar to the Q₁₀ between 2.5 and 3 for relaxation (7). Thus, in the present experiments conducted at room temperature (19-21°C), relaxation was mainly due to sarcoplasmic reticulum. Furthermore, at 20°C, the dissociation of Mg²⁺ from parvalbumin exhibited a rate constant of 3.42 s¹ (14). The rate constant of Ca²⁺ decay obtained here, i.e., 156.8 ± 22.6 s¹ in control conditions and 94.5 ± 14.8 s¹ (P < 0.05, n = 5) in the presence of 2 µM CPA, was too rapid to have been due to the Mg²⁺ dissociation rate from parvalbumin. Thus parvalbumin seems to have contributed little to CPA-induced broadening in the Ca²⁺ transient.

Indo 1 is a high-affinity Ca²⁺ indicator that acts as a myoplasmic Ca²⁺ buffer and may reduce the Ca²⁺ transient. Studies in frog skeletal muscle using a high concentration of fura 2, another high-affinity Ca²⁺ dye (2), indicated that the amplitude and decay half time of the Ca²⁺ transient were reduced, which could have influenced related force development. However, the concentration of Ca²⁺ dye in the cell after microinjection was not known in these studies, but twitches were compared with controls, which showed no significant changes. Thus, although parvalbumin and indo 1 are essential myoplasmic Ca²⁺ buffers, it is unlikely that they contributed to a change in the Ca²⁺ transient during our experiments. This suggests that changes in [Ca²⁺]_i amplitude in the presence of CPA depended on the rate of Ca²⁺ uptake by sarcoplasmic reticulum, since the rate of Ca²⁺ release from sarcoplasmic reticulum and the conditions of myoplasmic Ca²⁺ buffering were unchanged.

This hypothesis was investigated using the fluorescence indicator indo 1 to monitor changes in myoplasmic Ca²⁺ activity during isometric twitch of single frog muscle fibers (sarcomere length 2.9 µm; 19-21°C). Application of 2 µM CPA increased the Ca²⁺ transient and isometric force, whereas the rate of Ca²⁺ decay and force declined during relaxation. In a recent study (18), it was reported that twitch force, in the presence of 2,5-di-(tert-butyl)-1,4-benzohydroquinone (TBQ), another inhibitor of Ca²⁺-ATPase, was increased and that the relaxation phase was prolonged. There was also a concomitant slowing of the decay phase of the Ca²⁺ transient, with no significant increase in the amplitude of the Ca²⁺ signal. These authors did not exclude a possible elevation of the Ca²⁺ signal upon TBQ inhibition of the sarcoplasmic reticulum and suggested that it may not have been detectable with the Ca²⁺ indicators used (fluo 3 and mag-fura 2). Our results with indo 1 confirm this hypothesis, since a significant increase (10%) in the amplitude of the Ca²⁺ transient was detected with 2 µM CPA. It has recently been reported that the time during which the contractile system is exposed to an increase of [Ca²⁺]_i, rather than the peak amplitude of the Ca²⁺ transient, is the most important determinant of mechanical response (26). This implies that there is an inverse relationship between peak twitch force and the rate constant of Ca²⁺ decay among individual muscle fibers. CPA-induced changes in the Ca²⁺ transient amplitude were slight compared with the increases in time to peak (59%) and the time constant of decay (65%). However, this slight change in the amplitude of the Ca²⁺ signal produced a larger increase (35%) in twitch force. The fact that CPA inhibited the sarcoplasmic reticulum Ca²⁺ pump confirms the results of Sun et al. (26). Thus twitch potentiation would appear to be correlated with a broadening, rather than an increase, in the amplitude of the Ca²⁺ transient. It was shown that Ca²⁺ binding to the specific sites of troponin C is a very rapid process, whereas the removal of Ca²⁺ from regulatory sites is a slow process, limited only by the rate of Ca²⁺ diffusion from troponin C (19). Thus, when sarcoplasmic reticulum Ca²⁺-ATPase activity is inhibited by CPA, the Ca²⁺ transient is prolonged, so that Ca²⁺ should remain bound to troponin C. As a result, twitch force, time to peak, and relaxation time were increased in our study.

This study used complementary approaches to analyze the effect of CPA in fast-twitch skeletal muscle cells of frog semitendinosus muscle. CPA, depending on the concentration used, had distinct effects on the twitch contraction cycle. The results clearly show that 2 µM CPA did not change the resting and action potentials and had no effect on myofibrillar Ca²⁺ sensitivity and the ability of cross bridges to produce force. Changes in [Ca²⁺]_i elevation were not associated with any modification in the rate of Ca²⁺ release and myoplasmic Ca²⁺ buffering. Our experimental results indicate that CPA at a concentration of up to 2 µM does not alter any system other than sarcoplasmic reticulum Ca²⁺-ATPase. Changes observed in the Ca²⁺ transient and force development may have been related to inhibition of the sarcoplasmic reticulum Ca²⁺ pump. Although parvalbumin appears to accelerate relaxation at low temperature, the sarcoplasmic reticulum Ca²⁺ pump at 20°C seems to be one of the rate-limiting steps in brief contraction-relaxation.