Although malignant hyperthermia after application of sevoflurane has been reported, little is known about its action on intracellular calcium homeostasis of skeletal muscle. The authors compared the effect of sevoflurane with that of isoflurane and halothane on Ca2+ release of mammalian sarcoplasmic reticulum and applied a novel method to quantify Ca2+ turnover in permeabilized skeletal muscle fibers.
Liquid sevoflurane, isoflurane, and halothane at 0.6 mM, 3.5 mM, and 7.6 mm were diluted either in weakly calcium buffered solutions with no added Ca2+ (to monitor Ca2+ release) or in strongly Ca2+ buffered solutions with [Ca2+] values between 3 nM and 24.9 microm for [Ca+]-force relations. Measurements were taken on single saponin skinned muscle fiber preparations of BALB/c mice. Individual [Ca2+]force relations were characterized by the Ca2+ concentration at half-maximal force that indicates the sensitivity of the contractile proteins and by the steepness. Each force transient was transformed directly into a Ca2+ transient with respect to the individual [Ca2+]-force relation of the fiber.
At 0.6 mM, single force transients induced by sevoflurane were lower compared with equimolar concentrations of isoflurane and halothane (P < 0.05). Similarly, calculated peak Ca2+ transients of sevoflurane were lower than those induced by equimolar halothane (P < 0.05). The Ca2+ concentrations at half maximal force were decreased after the addition of sevoflurane, isoflurane, and halothane in a concentration-dependent manner (P < 0.05).
Whereas sevoflurane, isoflurane, and halothane similarly increase the Ca2+ sensitivity of the contractile apparatus in skeletal muscle fibers, 0.6 mM sevoflurane induces smaller Ca2+ releases from the sarcoplasmic reticulum than does equimolar halothane.
VOLATILE anesthetics induce Ca2+release from the sarcoplasmic reticulum (SR). This effect has been shown in functionally skinned skeletal muscle fibers [1–3] and in skinned cardiac muscle [4–8] with classical volatile anesthetics such as halothane, isoflurane, and enflurane. So far, little information exists about the effect on the SR of sevoflurane. This could be of clinical relevance because the Ca2+release channel has been associated with malignant hyperthermia (reviewed in MacLennan and Phillips  and MacLennan and Chen ), and already case reports about malignant hyperthermia after administration of sevoflurane have been published. [11–13]
Force measurements are used as an indirect tool in skinned skeletal muscle fibers to measure calcium release from the SR. However, the extent of force induced by the Ca2+release may be influenced by the Ca (2+) sensitivity of the regulatory proteins (troponin-tropomyosin complex) and the actin-myosin interaction. Therefore, in addition to the force measurements, the evaluation of the direct influence of the anesthetics on Ca (2+) sensitivity is also important. In skeletal muscle, reports about the effects on Ca2+sensitivity by inhalational anesthetics have been controversial: Tavernier et al.  observed no effect of halothane on the [Ca2+]-force relation of fast skeletal muscle, whereas Ca2+sensitivity of the contractile apparatus of slow-twitch skeletal muscle was decreased. In contrast, Su and Bell  observed an increase in Ca2+sensitivity when isoflurane or halothane was added to skinned fibers of fast-twitch muscle. However, both volatile anesthetics caused no change in slow-twitch skeletal muscle. 
For a more quantitative assessment of Ca2+release from the SR, we developed a novel method to derive Ca2+transients directly from Ca2+-inducedforce transients. [15,16] This method is based on measuring the force induced by calcium release of skinned muscle together with the Ca2+sensitivity of the contractile proteins of the same muscle fiber to transform subsequently force transients into Ca2+transients. The aim of our study was to compare the effect of sevoflurane with those of isoflurane and halothane on Ca2+release from the SR and to consider the effects of the volatile anesthetics on the Ca2+sensitivity of the contractile apparatus.
Materials and Methods
Muscle Fiber Preparation
After we received approval from the local animal care committee, BALB/c mice were anesthetized for 2–3 min with carbon dioxide and subsequently killed by cervical dislocation. The extensor digitorum longus muscle was isolated in paraffin oil at 4 [degree sign]C, and a small single-fiber bundle containing one to three fibers (between 80 and 150 [micro sign]m in diameter) was dissected. The fiber preparation was glued to a force transducer pin (AE801; Senso-Noras, Horten, Norway) and a micrometer-adjustable screw using a collagen glue.
(Table 1) shows the concentrations of the experimental solutions. The high-activating solution and the high-relaxing solution contained 30 mM EGTA to "clamp" free Ca2+, whereas the low-relaxing solution contained 0.3 mM EGTA and 29.7 mM 1,6-diamino exane-N,N,N,N-tetraacetic acid (HDTA), which, in contrast to EGTA, has a very low affinity to Ca2+. The addition of 50 [micro sign]g/ml saponin to the low-relaxing solution created the skinning solution. Free ion concentrations were calculated using the REACT program from Smith and Miller.  Solutions were adjusted to pH 7.0, and ionic strength was calculated to 157 mM. All measurements were performed at room temperature (23 [degree sign]C). The release solution consisted of the low-relaxing solution plus caffeine (30 mM), liquid sevoflurane, isoflurane, or halothane. The solutions to measure the [Ca2+]-force relation were obtained by mixing appropriate amounts of high-activating solution with high-relaxing solution and adding 30 mM caffeine, liquid sevoflurane, isoflurane, or halothane.
Liquid sevoflurane, isoflurane, and halothane (0.5–5 [micro sign]l) was diluted in 5 ml weakly calcium-buffered release solution, high- activating solution, or high-relaxing solution; solubilized with an ultrasound bath for 120 s; and thereafter stirred with a magnet for at least 60 min in an air-tight glass syringe. The anesthetic solution was added into a vial shortly before the muscle fiber was immersed in the solution. For gas chromatographic analysis of the concentration of the anesthetic agents, 400-[micro sign]l aliquots of the working solutions were transferred into 5-ml vials for head space injections. After the addition of 10 [micro sign]l chloroform-saturated water (internal standard), the vials were capped with septa with aluminum foil placed beneath them. The vials were stored frozen at -20 [degree sign]C for further analysis.
Before the analysis, the samples were incubated for 30–45 min in a thermal block at 75 [degree sign]C to achieve a stable equilibrated gas phase. For gas chromatography mass selective analysis, 100-[micro sign]l aliquots were collected using a gastight 500-[micro sign]l glass syringe. Analysis by gas-liquid chromatography was performed on a Hewlett Packard gas chromatograph (6890 series) combined with a mass selective detector (HP 5973 series; Hewlett Packard, Bad Homburg, Germany) equipped with a Hewlett Packard 5-MS capillary column, 30 m x 0.25 mm with a film thickness of 0.25 [micro sign]m. Separation conditions included an injector temperature of 120 [degree sign]C and a detector temperature of 280 [degree sign]C with helium as the carrier gas at a starting flow of 0.5 ml/min and increasing after 3 min to a rate of 2 ml/min2to 1 ml/min. The temperature gradients were 33 [degree sign]C at 0–2 min and 33–55 [degree sign]C at 2–5.7 min. For quantification, substance-specific ions were used.
The corresponding anesthetic concentrations in volume percentages at 37 [degree sign]C were calculated using the reported Ostwald water-gas partition coefficients in aqueous solutions at 37 [degree sign]C (0.26 for sevoflurane, 0.57 for isoflurane, and 0.67 for halothane),  as described by Franks and Lieb. 
Muscle fibers were kept in the skinning solution for 5 min. The sarcomere length was adjusted from the difraction pattern of a helium-neon laser to 2.6 [micro sign]m.  Before loading the SR of the fibers with Ca2+for 1 min in the loading solution, which contained 0.41 [micro sign]M Ca2+, they were placed for 3 s in the release solution and 3 s in high-relaxing solution, and they were equilibrated for 2 min in the low-relaxing solution. Subsequently, they were transferred for 2–4 s into the high-relaxing solution and for 2 min in the low-relaxing solution. The fibers were exposed to the release solution containing 30 mM caffeine or liquid isoflurane-sevoflurane in the low-relaxing solution. The maximum force was measured in the high-relaxing solution at 24.9 [micro sign]M Ca2+.
As a quality control for permeabilization of the fibers, and because saponin skinning causes some reduction of the Ca2+loading ability of the SR in rat skeletal muscle,  the initial caffeine (30 mM) release had to be at least 30% of maximal force; otherwise the fiber was discarded.
To reveal the time constant of decay, a fit by a single exponential function was applied, with 1/[Greek small letter tau] in the exponent reflecting the time constant of decay.
The [Ca2+]-force relation was measured with at least six different Ca2+concentrations ranging from 0.003 [micro sign]M to 24.9 [micro sign]M. By nonlinear regression, a Hill curve was fitted to the measured data points by applying the following equation: y = 10-x[middle dot] H/(10-H[middle dot] EC50 + 10-H[middle dot] x), where x =-log[Ca2+], which is a modified version of the Hill Equation describedby Fink et al.  The Hill coefficient (H) gives an indication of the maximum steepness of the sigmoidal curve. The EC50value indicates the Ca2+concentration for half-maximal isometric force activation as a measure of the sensitivity of the contractile proteins to Ca (2+). The correlation coefficients (r values) were calculated to determine the accuracy of the fit. The individual EC50values and Hill coefficients (H) were subsequently applied to transform each force transient directly into a Ca2+transient, by using the individual [Ca2+]-force relation as a Ca2+indicator and reversing each point of the force transients into the corresponding free Ca2+level. [15,16] The sensitivity of the Ca2+-regulatoryproteins and the corresponding force development directly measures the free myofibrillar [Ca2+] and relates free Ca2+and force. Thus, the [Ca2+]-force relation can be used as a bioassay to convert the rather slow force transients from the Ca2+release of the SR into apparent Ca2+transients.
To compare the effects seen with different concentrations and different volatile anesthetics, the effects of caffeine were measured as a control and for standardization in all experiments. The normal distributions of each group were confirmed by applying the Kolmogorov-Smirnov test. One-way analysis of variance was applied for comparisons between groups. When differences between the groups were greater than would be expected by chance, the Bonferroni t test was applied. A significant difference was defined as P < 0.05. Data are presented as the mean +/- SD.
All measurements were evaluated at three levels of concentrations: The lowest dose of 0.6 mM can be calculated to correspond to 5.87 vol% sevoflurane, 2.68 vol% isoflurane, and 2.28 vol% halothane at 37 [degree sign]C.  Higher levels (3.5 mM and 7.6 mM) were chosen to evaluate the extent and direction of effects.
Representative Ca2+-inducedforce transients, [Ca2+]-force relations, and the derived SR Ca2+transients from an extensor digitorum longus muscle fiber preparation are shown for sevoflurane in Figure 1, for isoflurane in Figure 2, and for halothane in Figure 3at 3.5 mM. These recordings show that peak force transients induced by either anesthetic were lower compared with the standard transient of 30 mM caffeine and that force transients of isoflurane and sevoflurane also revealed a slower decay when compared with caffeine (Figure 1A and Figure 1C, Figure 2A and Figure 2C, and Figure 3A and Figure 3C). The individual [Ca2+]-force relation revealed greater EC50values for the volatile anesthetics compared with 30 mM caffeine and lesser EC50values when compared with the control, suggesting a Ca2+-sensitizingeffect of the contractile proteins by the volatile anesthetics. Hill coefficients of sevoflurane, isoflurane, and halothane were greater compared with caffeine, resulting in a steeper Ca2+dependence of isometric force (Figure 1B, Figure 2B and Figure 3B). The peak Ca2+transients derived from the force curves were less with sevoflurane, isoflurane, and halothane than were those induced by caffeine comparable to the reduced peak force transients (Figure 1C, Figure 2C and Figure 3C).
Peak values of isometric force transients induced by sevoflurane, isoflurane (7.6 mM, 3.5 mM, and 0.6 mM), and halothane (3.5 mM and 0.6 mM) are given in Figure 4A. Apart from the highest concentrations (7.6 mM), all peak values obtained by application of the volatile anesthetics were less than those of the caffeine transients. At 0.6 mM, the effect induced by halothane was more pronounced than that of isoflurane and sevoflurane, and the effect of isoflurane was greater than that of sevoflurane. The corresponding calcium transients (derived from the force transients based on the [Ca2+]-force relation) revealed similar findings compared with the isometric force transients for all given experimental conditions (Figure 4B). The rate of decay (1/[Greek small letter tau]) was more reduced after the addition of 3.5 mM sevoflurane and isoflurane compared with equimolar halothane (Figure 5).
The [Ca2+]-force relation was fitted to the Hill Equation withmean correlation coefficients (r values) greater than 0.993 (SD < 0.05). Mean EC50values under different experimental conditions are summarized in Figure 6. In contrast to the changes of EC50, no effect of the volatile anesthetics was observed on the Hill coefficient when compared with the control. However, for 30 mM caffeine (H = 1.46 +/- 0.31; n = 10; P < 0.05), the Hill coefficient was reduced compared with the control value (H = 2.66 +/- 0.51; n = 7) or compared with that observed with the high or low concentrations of sevoflurane (7.6 mM: H = 2.60 +/- 0.40; 3.5 mM: H = 2.42 +/- 0.78, n = 5; 0.6 mM: H = 2.84 +/- 0.35, n = 3), isoflurane (7.6 mM: 2.75 +/- 0.48; 3.5 mM: H = 2.44 +/- 0.71, n = 5; 0.6 mM: H = 3.00 +/- 0.34 mM, n = 3) and halothane (3.5 mM: H = 2.39 +/- 0.15, n = 5; 0.6 mM: H = 3.16 +/- 0.19 mM, n = 3); mean +/- SD.
The maximal Ca2+-activatedforce in the presence of the volatile anesthetics revealed increased maximal values for all three volatile agents at the lowest concentration (0.6 mM) and increased values for 3.5 mM and 7.6 mM sevoflurane compared with the control (Figure 7).
Little is known about the effect of newer volatile anesthetics on skeletal muscle. However, there have been case reports about malignant hyperthermia after administration of sevoflurane, [11–13] and the Ca2+-releasechannel of the SR has been associated with malignant hyperthermia. [9,10]
Our goal was to evaluate the direct effect of sevoflurane on the SR compared with isoflurane and halothane. By measuring the Ca2+sensitivity of the contractile apparatus of the same muscle fiber, we also calculated the individual Ca2+release of the fiber compared with caffeine. This was possible by using a novel approach developed in our laboratory to transform force transients directly into Ca2+transients. [15,16] It is based on the fact that the sensitivity of the Ca2+regulatory proteins directly measures the free myofibrillar [Ca2+] and relates free Ca2+and force (see also Materials and Methods section). All experiments were performed at room temperature and can be extrapolated only tentatively to the experimental condition expected at 37 [degree sign]C.
Interestingly, equimolar concentrations of the more recent agent sevoflurane behaved differently compared with isoflurane and halothane. All agents induced force transients by releasing Ca2+from the SR coupled with a simultaneous increase in Ca2+sensitivity of the contractile proteins. However, at the lowest concentration of 0.6 mM, Ca2+-inducedisometric force measurements revealed a significantly reduced peak force transient with sevoflurane when compared with isoflurane and halothane. This finding supports previous data, which were, however, derived from myocardial preparations. [23–25]
To our knowledge, the effect of sevoflurane on Ca2+release from the SR has not been described. It is known, however, that isoflurane and halothane induce Ca2+release. This had been shown indirectly by demonstrating that isoflurane or halothane increase as submaximal caffeine transient. [1–3] In our study, we recorded Ca2+-dependentforce transients that were induced by the volatile agents alone.
We analyzed the Ca2+transients of the SR derived from the force transients and the [Ca2+]-force relation of the same fiber. These results revealed an increased peak Ca2+transient of halothane compared with sevoflurane and isoflurane. Our results suggest a possible direct interaction of sevoflurane with the calcium release channel of the SR. Although the amino acid sequence and three-dimensional structure of the ryanodine receptor have been resolved in part, [26,27] little is known about possible interaction sites between volatile anesthetics and this receptor. Our results suggest that the volatile anesthetics interact differently with their binding region of the release channel; however, future studies defining these sites of action will be necessary.
The shape of the force transient and the corresponding Ca2+transients show that the initial decay during relaxation induced by isoflurane and sevoflurane is slower compared with that of caffeine. One possible factor that influences the decrease in [Ca2+] is the altered activity of the Ca2+adenosine triphosphatase (ATPase) of the SR (SERCA), which pumps the released Ca2+from the cytosol back into the lumen of the SR.  Volatile anesthetics have been shown to stimulate the SR Ca2+ATPase activity [28,29] but also to inhibit Ca2+uptake into the SR.  Under our experimental conditions, the slower initial relaxation of sevoflurane and isoflurane suggests an inhibition compared with caffeine. A slow initial relaxation has been described when the Ca2+ATPase was blocked with the SR Ca2+ATPase inhibitor cyclopiazonic acid,  which revealed a reduced decay similar to that seen in the presence of sevoflurane and isoflurane.
Whereas the influence of sevoflurane on Ca2+sensitivity of the contractile proteins has not been evaluated before, our finding of a significant increase of Ca2+sensitivity after the addition of isoflurane or halothane supports similar observations by Su and Bell.  In contrast, when a slow-twitch skeletal muscle (soleus) was evaluated, no effect of halothane on the [Ca2+]-force relation was detected. [2,31] Little is known about the detailed mechanisms of the Ca2+-sensitizingeffect. Possible targets include a direct interaction with the tropomyosin-troponin complex, resulting in altered calcium binding to troponin C or in a different shift of tropomyosin. In addition, a direct interaction of isoflurane or sevoflurane with the contractile proteins actin or myosin may be involved. This suggestion is speculative at this stage and more studies are needed for a precise definition of the target protein or proteins. In rat myocardium, however, the method of skinning has been described to be important for the Ca2+sensitivity.  An increase in Ca2+sensitivity by isoflurane has been observed, although to a different extent both with saponin skinning and mechanical skinning. Under otherwise identical experimental conditions, a decrease in Ca2+sensitivity was found after Triton X-100 skinning, which also disrupts the SR membrane. Whether the influence of different skinning methods can be transferred easily from cardiac to skeletal muscle is yet to be resolved.
The minimum alveolar anesthetic concentration is used to describe anesthetic potency in an intact organism (in vivo), whereas our in vitro experiments describe the effect of an anesthetic dose on a single organelle, the SR. Comparison of sevoflurane, isoflurane, and halothane using minimum alveolar anesthetic concentrations may be misleading because the anesthetic concentration in the SR during anesthesia and its proportion to the minimum alveolar anesthetic concentration have not been described in detail yet and remain open because lipid solubility is likely to be important. Thus, we compared isoflurane, sevoflurane, and halothane on an equimolar basis.
When interpreting the action of volatile anesthetics on calcium activation of muscle, the study of sensitivity changes of calcium-activated force and the effects on calcium release and uptake properties of the SR are important and potentially clinically relevant.
The authors thank H. Schmidt-Gayk and F. T. Weiland for their support, M. Gautel for critical reading of the manuscript, D. Uttenweiler for critical discussion, and A. Stucke for help with the gas chromatography measurements.