Introduction

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In mammalian skeletal muscle, triggering of the action potential along the membrane of the T-transverse tubule system increases intracellular Ca²⁺ concentration and produces contraction through the interaction of actin and myosin. Contraction is then terminated through Ca²⁺ uptake by the sarcoplasmic reticulum (SR) via the Ca²⁺ pump. This series of events is referred to as excitation-contraction coupling. During contraction, two key proteins play a major role in the calcium release mechanism: the sarcolemmal dihydropyridine receptor (voltage sensor) and the SR Ca²⁺ release channel (ryanodine receptor). To induce relaxation, Ca²⁺ is removed from the cytosol by extrusion through the sarcolemmal Na⁺/Ca²⁺ exchanger and Ca²⁺ adenosine triphosphatase (Ca²⁺-ATPase), mitochondria, and by sequestration in the SR mediated by the Ca²⁺-ATPase. However, in slow-twitch muscle, the SR Ca²⁺ pump is the major process responsible for reducing cytosolic Ca²⁺ from a high level to a low resting level during relaxation (Leong and Maclennan, 1998; Lamb, 2000). Pharmacological tools predominantly inhibiting SR Ca²⁺ uptake, i.e., cyclopiazonic acid, thapsigargin, and 2,5-di-(tert-butyl)-1,4-hydroquinone, have long been used to separate the various cellular mechanisms that regulate the contraction-relaxation cycle in muscle. Other tools that act as specific stimulators of the SR Ca²⁺-ATPase activity of muscle cells could be useful for investigating SR Ca²⁺ uptake and storage.

Biochemical investigations have recently shown that jasmone, menthone, menthol, and methyl jasmonate are highly selective stimulators of the SR Ca²⁺-ATPase in mammalian skeletal muscle, whereas they have no effect on the properties of the phospholipid bilayer (Starling et al., 1994). Surprisingly, no study has investigated the effects of these substances on skeletal muscle contraction. Several studies have shown that two compounds of the lipoxygenase pathway, i.e., jasmonic acid and methyl jasmonate, regulate wound response in plants (Veronesi et al., 1996). Methyl jasmonate, a linolenic acid-derived cyclopentanone-base, has also been shown to trigger defense reactions in various plants and to be produced in response to wounds and elicitor treatment (Rickauer et al., 1997).

Because methyl jasmonate can stimulate ATP-dependent Ca²⁺ activity of the purified Ca²⁺-ATPase from rabbit fast-twitch skeletal muscle (Starling et al., 1994), it seemed of interest to estimate the specificity of methyl jasmonate effects on Ca²⁺ loading by the SR. Chemically skinned slow muscle fibers (soleus) were chosen for these experiments, as sarcolemmal functions can be eliminated and Ca²⁺ mobilization greatly simplified. Other experiments investigated whether methyl jasmonate in intact soleus fibers acts at different steps in the excitation-contraction coupling process.

	Materials and Methods

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General Procedure. All procedures in this study were performed in accordance with the stipulations of the Helsinki Declarations and with the European current laws for animal experimentation. Adult male Wistar rats (body weight, 400 ± 25 g) were anesthetized by ether vapor flow. After respiratory arrest, soleus was quickly excised and placed in a dissecting dish containing mammalian physiological solution at controlled room temperature (19-20°C).

Chemically Skinned Fibers. Short cut bundles containing two to five fibers (150-200 µm in diameter, 10-20 mm in length) were dissected from soleus muscle, and chemical fiber skinning was carried out immediately. For Triton-skinned fibers, preparations were incubated for 1 h in a relaxing solution (pCa 9) containing 1% Triton X-100 (v/v) to solubilize membranes and then transferred into a relaxing solution without detergent. Fibers were stored at 20°C in relaxing solution containing 50% (v/v) glycerol.

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Experimental Protocol to Study Ca²⁺ Uptake by the Sarcoplasmic Reticulum (Loading Time). Ca²⁺ uptake by the SR was studied by immersing the preparation sequentially in five different solutions to deplete (solution 1), wash (solution 2), and load (solutions 3 and 4) the SR with Ca²⁺ before releasing Ca²⁺ with caffeine (solution 5) to generate transient contracture. At the beginning of the experiments, three or four challenges of 10 mM caffeine contracture were performed. Ca²⁺ uptake was determined by an indirect method. The SR of saponin-skinned fibers was preloaded with Ca²⁺ solution for various periods (25, 35, 65, 95, 125, and 305 s). After the loading period, Ca²⁺ was released by applying 10 mM caffeine. The caffeine contracture tension that developed showed an increase in amplitude dependent on loading time, which reached its maximum after 5 min of loading. A good fit was achieved by assuming a simple system in which loading resulted from the filling of a two-compartment system (the filling rate for both compartments differed with loading time). This arrangement is roughly similar to that suggested by Chapman and Léoty (1976). For each experimental fiber, the slope of the linear fit provides an estimation of the loading rate. This parameter was evaluated in methyl jasmonate-free medium and in the presence of different methyl jasmonate concentrations (0, 100, and 200 µM).

Experimental Protocol for Triton-Skinned Fibers. 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 recorded continuously on chart paper (Linear Bioblock 1200, Reno, NV), and baseline tension was established at the steady state measured in relaxing solution. The relationship between tension and the negative logarithm of [Ca²⁺], the pCa, was quantified from the Ca²⁺ sensitivity curve described by the Hill equation:
Where T is relative tension, K the Ca²⁺ concentration for half-maximal activation, and n_H the Hill coefficient.

1

Isometric Tension Measurement from Intact Fibers. After removal of connective tissue, small bundles of two to five fibers were dissected along their entire length under the microscope. The preparation was then transferred onto a coverslip in a drop of physiological solution and placed in the experimental chamber. This chamber, with an open-topped channel 1.3 × 1.3 × 25.0 mm, has at one end a four-way tap that opens directly into the channel, allowing rapid change of the perfusion solution (<0.2 s), or into a drain so that perfusion by stagnant solutions is avoided. The preparation was mounted as described by Joumaa and Léoty (2001). Briefly, one tendon of the preparation was snared under a fine silver loop and fixed in the open-topped channel. The other tendon was fixed to the tip of a force transducer (Kaman KD 2300 0.5 SU displacement measuring system, Colorado Springs, CO).

1

Experimental Protocol. The activation curve of K⁺ contracture was obtained by a rapid change from the control solution to one containing an elevated potassium concentration (20-146 mM [K⁺]₀). After spontaneous relaxation of the contracture, K⁺ solution was replaced by physiological solution in which fibers recovered for 15 min before a new contracture cycle was induced. For each experimental fiber, data points were fitted with a Boltzmann equation (Dulhunty, 1991): T = T_max · [1 + exp(E_a  E_m) · K_a¹]¹, where T is the K⁺ contracture amplitude at a given membrane potential (E_m), T_max corresponding to the amplitude of test K⁺ (146 mM), E_a the potential at which T = 0.5 T_max, and K_a a slope factor. Peak K⁺ contracture tension values were plotted against corresponding membrane potential values and normalized to maximal tension in 146 mM [K⁺]₀ solution.

1

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Membrane Potential Recording. Using a conventional glass microelectrode (10-20 M) connected to an electrometer input-negative capacitance amplifier, membrane potentials (E_m) were recorded first in physiological solution from bundles containing 20 to 30 fibers and then, after 5 min, in high-potassium solutions (20-146 mM [K⁺]₀) in the absence and presence of methyl jasmonate.

Solutions. Mammalian physiological solution contained 140 mM Na⁺, 6 mM K⁺, 3 mM Ca²⁺, 2 mM Mg²⁺, 156 mM Cl, and 5 mM HEPES. The pH was adjusted to 7.35 by addition of a trisaminomethane solution. A depolarizing solution was prepared by replacing a given amount of Na⁺ with K⁺, and the [K⁺][Cl] product was kept constant by replacing Cl with L-glutamate.

Statistical Analysis. All values are expressed as means ± S.E.M. for n observations. Statistical analysis was performed by ANOVA, and Scheffé post hoc analysis was performed when a significant F-value was obtained. A level of P < 0.05 indicates statistical significance.

	Results

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Effects of Methyl Jasmonate on Ca²⁺ Uptake by the Sarcoplasmic Reticulum (Loading Time). In previous studies, the effect of jasmone and methyl jasmonate on SR Ca²⁺-ATPase was demonstrated in a series of experiments conducted on the purified Ca²⁺-ATPase from rabbit fast-twitch skeletal muscles (Starling et al., 1994). The purpose of the present study was to confirm these results in slow mammalian skeletal muscle. The effects on loading time of different methyl jasmonate concentrations (50, 100, and 200 µM) were studied on small bundles of saponin-skinned soleus fibers. The results (see Fig. 1a) show that the loading rate was not significantly changed by 50 µM methyl jasmonate, whereas an increase of 41% was found in the presence of 100 µM methyl jasmonate without change in the amplitude of maximum caffeine contracture reached after 5 min of loading. A larger methyl jasmonate concentration failed to increase the loading rate. In the presence of 200 µM methyl jasmonate, the values obtained were similar to control values (control: 48.2 × 10⁴ ± 7.5 × 10⁴ s¹, 200 µM: 46.8 × 10⁴ ± 3.9 × 10⁴ s¹, n = 6). Thus, the present results (Fig. 1b), showing a bell-shaped relationship between loading rate and methyl jasmonate effects, are in line with previous findings obtained in the presence of jasmone, menthone, menthol, and methyl jasmonate (Starling et al., 1994).

View larger version (41K): [in this window] [in a new window]   Fig. 1.   Effects of different concentrations of methyl jasmonate (50-200 µM) on the loading time. a, semilogarithmic plot of the relative tension against time during the different loading time in the absence and presence of 50, 100, and 200 µM methyl jasmonate. b, rate of the Ca2+ uptake by the sarcoplasmic reticulum before and after exposure of the fiber preparations to methyl jasmonate. Data are expressed as means ± S.E.M. for n = 6 observations. , significant differences from control values (P < 0.05). Experiments were conducted in a temperature-controlled bath (21°C).

Effects of Methyl Jasmonate on Ca²⁺-Activated Tension in Chemically Skinned Fibers. Triton-skinned rat skeletal muscle fibers from soleus developed force when immersed in solutions of increasing Ca²⁺ concentrations. In methyl jasmonate-free medium, the tension-Ca²⁺ relationship was characterized by half-maximal activation (pCa₅₀) of 6.266 ± 0.039, a Hill coefficient of 1.887 ± 0.086, and a maximal Ca²⁺-activated tension (at pCa 4.5) of 91.93 ± 7.09 mN · mm² (n = 10). There were no significant differences between the mean values found in the absence and presence of 50, 100, and 200 µM methyl jasmonate (Fig. 2). These results suggest that the reduction in loading time found in the presence of methyl jasmonate was not due to changes in the calcium sensitivity of contractile proteins.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effects of different concentrations of methyl jasmonate (50, 100, and 200 µM) on myofibrillar Ca2+ sensitivity. Isometric tension pCa (log [Ca2+]) relationships in slow-twitch rat skeletal fibers. Points represent the mean ± S.E.M. of the relative tension for n = 7 observations. Experiments were conducted in a temperature-controlled bath (21°C).

Effects of Methyl Jasmonate on Slow-Twitch Isometric Tension. In control conditions, the twitch was characterized by an amplitude of 51.5 ± 1.2 mn, a time to peak of 144 ± 7 ms, and a time constant of relaxation of 403 ± 39 ms (n = 10). The application of different methyl jasmonate concentrations (50-200 µM) induced detectable changes in contraction parameters after only 30 s. However, a 2-min exposure was generally required to reach maximal steady-state effect (Fig. 3). All of the results reported here were obtained in steady-state conditions. At methyl jasmonate concentrations between 50 and 125 µM, amplitude, time to peak, and time constant of relaxation were modified and no additional effects were found for exposures longer than 2 min. For instance, in the presence of 100 µM methyl jasmonate, twitch amplitude was decreased by 29% (36.6 ± 1.1 mn; n = 8) (Fig. 4a), and time to peak tension and the time constant of relaxation reached 62 ± 6 ms (n = 8) and 98 ± 11 ms (n = 8), respectively (Fig. 4, b and c). The effects of methyl jasmonate at these concentrations were fully reversible after a 10- to 15-min return into methyl jasmonate-free medium. The effects observed at 125 µM were not potentiated by larger concentrations of methyl jasmonate tested and were not fully reversible. The present results, indicating that twitch characteristics were modified by methyl jasmonate, suggest that this compound may act at different steps in the excitation-contraction coupling process or in the control of myofilament function.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   a, original illustration of recording of twitch tension obtained in physiological solution in the absence and presence of 50, 100, and 200 µM methyl jasmonate; b, a superimposed record after normalization of twitch tension obtained in the absence and presence of 50, 100, and 200 µM methyl jasmonate.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Effects of different concentrations of methyl jasmonate on the amplitude (a), time to peak tension (b), and time constant of relaxation (c) of twitch on slow skeletal muscle. Data are expressed as mean ± S.E.M. for n = 8 observations. , indicates significant difference from the value under the control conditions (methyl jasmonate-free medium) at P < 0.05 as determined by ANOVA statistical test.

Effects of Methyl Jasmonate on 40 and 146 mM [K⁺]₀ Contractures. In skeletal muscle, change from normal to high [K⁺]₀ solution led to the development of a transient contractile response that reached a maximum and then relaxed in an exponential manner, even when superfusion with a depolarizing solution was maintained (Léoty and Léauté, 1982). The characteristics of the high [K⁺]₀ contractures were voltage-dependent. At more depolarized membrane potentials, its amplitude was increased, its time to peak was reduced, and its spontaneous relaxation was faster. In methyl jasmonate-free medium, 40 and 146 mM [K⁺]₀ contractures were characterized by an amplitude of 1.06 ± 0.5 and 129 ± 0.4 n, a time to peak of 31.2 ± 2.1 and 14.1 ± 1.2 s, and a time constant of relaxation of 45.2 ± 4.9 and 8.1 ± 0.6 s (n = 8), respectively. The amplitude of [K⁺]₀ contractures was decreased, and the relaxation phase was greatly reduced by methyl jasmonate in a dose-dependent manner (Fig. 5). Maximum effects after application of 50 to 125 µM methyl jasmonate were obtained in 2 min and were fully reversible after a 12-min return into methyl jasmonate-free medium. In the presence of 100 µM methyl jasmonate, 40 and 146 mM [K⁺]₀ contractures were characterized by an amplitude of 0.39 ± 0.07 N and 0.83 ± 0.05 N (Fig. 6a), a time to peak of 18.9 ± 1.5 s and 15.4 ± 1.5 s (Fig. 6b), and a time constant of relaxation of 15.3 ± 1.9 s and 5.9 ± 0.5 s (Fig. 6c) (n = 8, P < 0.05), respectively. Increasing the methyl jasmonate concentration (150 µM) produced more marked changes in the parameters of 40 and 146 mM [K⁺]₀ contractures which showed an amplitude of 0.23 ± 0.08 N and 0.41 ± 0.02 N, a time to peak tension of 21.8 ± 2.4 s and 14.9 ± 1.6 s, and a time constant of relaxation of 21.6 ± 2.7 s and 7.6 ± 0.7 s (n = 8), respectively. In fact, at concentrations between 150 and 200 µM, the effects due to methyl jasmonate application for more than 2 min were not fully reversible.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Original recording of 40 and 146 mM [K+]0 tension obtained in the same preparation in methyl jasmonate-free medium (a) and after application of different concentrations of methyl jasmonate: 50 µM (b), 75 µM (c), 100 µM (d), 125 µM (e), 150 µM (f), and 200 µM (g).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effects of different concentrations of methyl jasmonate on the amplitude (a), time to peak (b), and time constant of relaxation (c) of K+ contractures obtained for 40 and 146 mM [K+]0 on soleus muscle. Data are expressed as mean ± S.E.M. for n = 8 observations. , indicates significant difference from the value under the control conditions (methyl jasmonate-free medium) at P < 0.05 as determined by ANOVA statistical test.

Effects of 100 µM Methyl Jasmonate on Membrane Potential. Membrane potentials (E_m) were recorded on small bundles of intact soleus muscle (20-30 fibers) placed in mammalian physiological solution and high-potassium solutions (20-146 mM [K⁺]₀) in the absence or presence of methyl jasmonate (100 µM). Comparative results with control methyl jasmonate (Table 1) indicated that no significant change in membrane potential was recorded for any potassium concentration tested.

Effects of 100 µM Methyl Jasmonate on Voltage-Dependent Activation of K⁺ Contractures. The relationships between the relative amplitude of K⁺ contracture and E_m in control and methyl jasmonate conditions are shown in Fig. 7, and the parameters used to plot curves are listed in Table 2a. A comparison of the values showed that a shift to the right (more positive values) and a change in the slope of the activation curves were produced by methyl jasmonate, whereas the activation threshold was not significantly modified. Because this shift could indicate that methyl jasmonate has a direct effect on voltage-sensor properties, this possibility was investigated during voltage inactivation of the K⁺ contracture.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Effects of methyl jasmonate on the voltage-dependence activation curve. Original recording of tension, in methyl jasmonate-free medium (a) and after 2-min application of 100 µM methyl jasmonate (b). c, effects of 100 µM methyl jasmonate on the relationship between relative tension at the peak of K+ contracture and membrane potential (Em) in small bundles of soleus fibers (n = 8). Tension is expressed relative to the contracture recorded in 146 mM [K+]0. Values are expressed as means ± S.E.M. for n observations.

Effects of 100 µM Methyl Jasmonate on the Time Constant of Relaxation of K⁺ Contracture. Figure 8 clearly shows that the duration of the relaxation phase was reduced at all membrane depolarization after 2 min of exposure to 100 µM methyl jasmonate. Because the decay of K⁺ contractures could be fitted to a single exponential function, the time constant of relaxation was plotted against [K⁺]₀. The results indicate that the time constant of relaxation was significantly reduced by methyl jasmonate and that the effect was more marked at lower membrane depolarization.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Effects of methyl jasmonate on the time constant of relaxation of [K+]0 contractures. The time constant of relaxation was plotted against the membrane potential (Em) in the absence (n = 8) and presence of 100 µM methyl jasmonate (n = 8). , significant difference from the value under the control conditions at P < 0.05 as determined by ANOVA statistical test. Values are expressed as means ± S.E.M. for n observations.

Effects of 100 µM Methyl Jasmonate on the Inactivation Curve. Steady-state inactivation was assessed from the amplitude of test 146 mM K⁺ contractures (that reflects maximal activation in soleus muscle) (Fig. 9) after 3-min equilibration in conditioning solutions containing 20 to 110 mM [K⁺]₀. The relationships between the relative amplitude of K⁺ contracture and E_m in control and methyl jasmonate conditions are shown in Fig. 4, and the parameters used to plot curves are listed in Table 2b. A comparison of the values calculated showed that exposure to 100 µM methyl jasmonate induced no significant change in inactivation curves. This suggests that methyl jasmonate did not modify the fraction of voltage-sensitive molecules converted to active state upon depolarization and had no effect on the voltage dependence of the activation of K⁺ contractures.

View larger version (14K): [in this window] [in a new window]   Fig. 9.   Effects of methyl jasmonate on the inactivation of 146 mM [K+]0 contracture. Original recording of tension, in the absence (a) and presence of 100 µM methyl jasmonate (b). c, effects of 100 µM methyl jasmonate on the steady-state inactivation of K+ contracture. Peak tension values of test 146 mM [K+]0 contracture after submaximal depolarization were plotted against the corresponding membrane potential (Em) and normalized to tension in 146 mM [K+]0. Values are expressed as means ± S.E.M. for n = 8 observations.

	Discussion

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To our knowledge, the results reported here provide a first description of the effects of methyl jasmonate on the contractile responses of intact mammalian skeletal muscle fibers. Soleus muscle was chosen because a stimulating effect of SR Ca²⁺-ATPase was likely to be observed more easily on a slow- than a fast-twitch muscle. Moreover, it has been shown that SR Ca²⁺-ATPase plays a rate-limiting role in the twitch relaxation of single slow skeletal muscle. (Chua and Dulhunty, 1988).

Our results for saponin-skinned fibers show that methyl jasmonate concentrations of up to 100 µM reduced the time required to load the SR with calcium, whereas this effect was less marked at higher concentrations. These results are similar to those previously reported on the ATPase activity of the purified Ca²⁺-ATPase from rabbit fast skeletal muscle (Starling et al., 1994), except that in our experiments, methyl jasmonate concentrations were 100-fold lower. The fact that comparable effects were obtained for calcium loading in both types of muscle suggests that the application of methyl jasmonate resulted in an increase of ATPase activity in slow muscle, with a maximum response at 100 µM.

The characteristics of the twitch generated by short electrical stimulation were modified by methyl jasmonate. In fact, our results indicate that methyl jasmonate has distinct dose-dependent effects on the contraction of skeletal soleus muscle cells. Within the range of 50 to 125 µM, methyl jasmonate reversibility decreased twitch amplitude, reduced the time to peak, and accelerated the relaxation phase, whereas 150 to 200 µM methyl jasmonate depressed twitch irreversibly, without producing any further effects on time to peak and the time constant of relaxation. These results may indicate that methyl jasmonate, like jasmone menthone and menthol (Starling et al., 1994), has a dose-dependent effect on SR Ca²⁺-ATPase in skeletal muscle. Thus, Ca²⁺ in the cytosol would probably decrease with methyl jasmonate exposure as a result of activation of the SR Ca²⁺ pump, reducing the Ca²⁺ ions bound to troponin C. This effect would produce acceleration of the relaxation phase in association with a reduction in twitch amplitude. This scenario is consistent with the results obtained for concentrations of up to 100 µM. However, because the changes due to methyl jasmonate were irreversible at larger concentrations, other mechanisms involved in excitation-contraction coupling may have been affected. Depolarization-contraction coupling can be studied with the [K⁺]₀ contracture model. Our results show a great similarity in the effects of methyl jasmonate on twitch and [K⁺]₀ contractures. In particular, methyl jasmonate decreased tension and reduced time to peak tension and the relaxation phase in the 50 to 125 µM range. With larger concentrations (150-200 µM), methyl jasmonate induced a more marked decrease in tension but prolonged time to peak tension and the relaxation phase, which were still faster than controls. It was then shown that the time course of K⁺ contracture tension during prolonged depolarization depends exclusively on activation and inactivation of the process regulating Ca²⁺ release from the SR. This process relates to the conformational states of voltage-sensitive dihydropyridine receptor molecules in the transverse tubule membrane (Caputo, 1972; Dulhunty, 1991). Moreover, a close similarity between the voltage dependence of tension and charge movement has been observed (Chandler et al., 1976; Rakowski, 1981). A general model for depolarization-contraction coupling has suggested 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). In our experimental conditions, the effect of 100 µM methyl jasmonate was tested at a concentration producing a maximal effect on twitch and K⁺ contracture characteristics. The results showed that the relative fraction of the voltage sensor converted to the active state during submaximal depolarization did not change in the presence of 100 µM methyl jasmonate, which suggests that methyl jasmonate had no effect on the voltage-dependence activation and steady-state inactivation of the voltage sensor.

Although the excitation-contraction coupling mechanism was not affected by methyl jasmonate, the shift in the activation curve to more positive values, resulting from exposure to methyl jasmonate, could be related to changes in membrane potential and/or the depolarization rate and/or Ca²⁺ sensitivity for contractile proteins.

No significant differences in membrane potential, in different depolarization [K⁺]₀ solutions, were found for values recorded in methyl jasmonate-free medium and those in the presence of 100 µM methyl jasmonate. Moreover, the rate of change in membrane potential is an important factor in terms of the kinetics of K⁺ contractures, particularly because it affects response amplitude. With respect to [K⁺]₀, measurements of tension and membrane potential in depolarizing solutions showed that the depolarization rate was similar in the presence or absence of methyl jasmonate. Thus, it is likely that the shift of the activation curve to the right observed in the present study was not associated with a methyl jasmonate-induced change in membrane potential or depolarization.

Nevertheless, it is possible that the shift in the activation curve in the presence of methyl jasmonate was related to a change in the Ca²⁺ sensitivity of contractile proteins. Yet, experiments performed on Triton-skinned fibers showed that the application of different methyl jasmonate concentrations (50, 100, and 200 µM) had no significant effect on maximal activated Ca²⁺ force and the Ca²⁺ sensitivity of contractile proteins.

To date, it is recognized in mammalian skeletal muscle that the slow decay in tension during prolonged steady-state depolarization depends on the inactivation of excitation-contraction coupling and is not influenced by the kinetics of contractile protein response and the rate of calcium uptake by the SR (Dulhunty, 1992). However, it has recently been reported that the slow decay of tension in frog skeletal muscle during prolonged steady-state depolarization depends not only on inactivation of the process regulating Ca²⁺ release from the SR, but also on the ability of the SR to pump Ca²⁺. Experiments involving potassium contractures showed that the activation curve was shifted to the left by cyclopiazonic acid (CPA), whereas the inactivation curve remained unchanged (Même and Léoty, 1999). It is now well established that CPA is a specific inhibitor of sarcoplasmic Ca²⁺-ATPase in skeletal muscle (Seidler et al., 1989). Thus, the fact that the effect of methyl jasmonate on the activation curve was opposite to that of CPA suggests that methyl jasmonate is a stimulator of sarcoplasmic Ca²⁺-ATPase.

In summary, our data show that methyl jasmonate has a similar effect on the characteristics of twitch tension and [K⁺]₀ contractures of slow-twitch mammalian skeletal muscle fibers and that these effects are fully reversible for concentrations less than 125 µM. Moreover, the difference between the contractile responses obtained in the presence and absence of methyl jasmonate could be related to the acceleration of the SR to pump Ca²⁺ in the absence of any detectable modification in the excitation-contraction coupling process. Finally, methyl jasmonate appears to be a convenient tool for selective activation of SR Ca²⁺-ATPase in slow-twitch skeletal muscle.