The aim of this study was to describe and compare the effects of isoflurane, sevoflurane, and halothane at selected concentrations (i.e., concentrations that led to equivalent depression of the electrically evoked Ca2+ transient) on myofilament Ca2+ sensitivity, sarcoplasmic reticulum (SR) Ca2+ content, and the fraction of SR Ca2+ released during electrical stimulation (fractional release) in rat ventricular myocytes.
Single rat ventricular myocytes loaded with fura-2 were electrically stimulated at 1 Hz, and the Ca2+ transients and contractions were recorded optically. Cells were exposed to each anesthetic for 1 min. Changes in myofilament Ca2+ sensitivity were assessed by comparing the changes in the Ca2+ transient and contraction during exposure to anesthetic and low Ca2+. SR Ca2+ content was assessed by exposure to 20 mm caffeine.
Isoflurane and halothane caused a depression of myofilament Ca2+ sensitivity, unlike sevoflurane, which had no effect on myofilament Ca2+ sensitivity. All three anesthetics decreased the electrically stimulated Ca2+ transient. SR Ca2+ content was reduced by both isoflurane and halothane but was unchanged by sevoflurane. Fractional release was reduced by both isoflurane and sevoflurane, but was unchanged by halothane.
Depressed myofilament Ca2+ sensitivity contributes to the negative inotropic effects of isoflurane and halothane but not sevoflurane. The decrease in the Ca2+ transient is either responsible for or contributory to the negative inotropic effects of all three anesthetics and is either primarily the result of a decrease in fractional release (isoflurane and sevoflurane) or primarily the result of a decrease in SR Ca2+ content (halothane).
THE volatile anesthetics, halothane, isoflurane, and sevoflurane, in addition to inducing unconsciousness, exert a potent negative inotropic effect on the heart. 1–7The mechanisms underlying the cardiac-depressant effects of these agents are yet to be fully elucidated. A decrease in contraction can only be the result of a decrease in the sensitivity of the myofilaments or a decrease in the cytosolic Ca2+transient. A decrease in the Ca2+transient could result from a decrease in the Ca2+content of the sarcoplasmic reticulum (SR) or a decrease in the fraction of the SR Ca2+content released on electrical stimulation (fractional release).
The influence of halothane and isoflurane on myofilament Ca2+sensitivity has been studied in skinned cardiac muscle, but the results of such experiments have generated conflicting data; some investigators reported little change, 8–10and others a decrease of myofilament Ca2+sensitivity. 11,12Herland et al. 13suggested that the conflicting data could result from the techniques used to skin cardiac muscle. Data have been published from intact cardiac muscle tissue (single cells or trabeculae) that are consistent with a depression of myofilament Ca2+sensitivity induced by halothane and isoflurane, 13although other investigators have reported that any contribution of reduced myofilament Ca2+sensitivity does not play a major role in the negative inotropic effect of halothane. 14–16Currently, the effects of sevoflurane on myofilament Ca2+sensitivity are undocumented. One aim of the present study was to establish the effect of sevoflurane on myofilament Ca2+sensitivity, confirm that isoflurane decreases myofilament Ca2+sensitivity, and compare the effects of sevoflurane and isoflurane on myofilament Ca2+sensitivity to that of halothane.
We have demonstrated previously that halothane decreases the SR Ca2+content in stimulated rat ventricular cells. 17Jiang and Julian 18argued that isoflurane causes a substantial decrease of SR Ca2+content in intact stimulated trabeculae. However, these investigators used both rapid cooling and caffeine to measure SR Ca2+content (because, in the presence of isoflurane, rapid cooling alone did not cause release of SR Ca2+), which is not an established technique. In contrast, Wilde et al. 19reported no significant change in SR Ca2+content measured using caffeine in isolated rat ventricular myocytes in the presence of isoflurane. The effect of sevoflurane on SR Ca2+content is unknown. Another aim of the present study was to establish the effect of sevoflurane on SR Ca2+content, reexamine the effect of isoflurane on SR Ca2+content, and compare the effects of isoflurane and sevoflurane on SR Ca2+content to that of halothane.
Bassani et al. 20introduced the concept of fractional release. Their work showed that fractional release in ferret ventricular myocytes is strongly influenced by the magnitude of the L-type Ca2+current, ICa, as well as the Ca2+content of the SR. Isoflurane, sevoflurane, and halothane have been shown to depress ICa, and halothane, at least, is known to decrease SR Ca2+content; therefore, it is possible that the volatile anesthetics may affect fractional release. Furthermore, volatile anesthetics may alter fractional release by acting directly on the SR Ca2+release channel. However, the effect on fractional release of volatile anesthetics is unknown, and the final aim of the present study was to compare the effects of isoflurane, sevoflurane, and halothane on fractional release to understand further the mechanisms underlying their negative inotropic effect.
Materials and Methods
The experiments described were conducted on Wistar rats weighing between 200 and 250 g. Animals were bred in the Biomedical Services Unit, University of Leeds, maintained under a 12-h light–dark cycle, and provided with food and water ad libitum . All procedures concerning animals conformed to the UK Home Office Animals (Scientific Procedures) Act, 1986. Animals were killed using a schedule 1 procedure sanctioned by the UK Home Office under project license 60/02087, and the heart was rapidly excised into an “isolation solution” (see below for composition), supplemented with 750 μm CaCl2and equilibrated with 100% O2. The heart was flushed of blood by retrograde perfusion via the coronary arteries with the above solution and then perfused for 4 min with the isolation solution, to which 100 μm Na2EGTA was added. 21The heart was then perfused for 9 min with the isolation solution supplemented with 1 mg/ml collagenase (type 1; Worthington Biochemical Corp., Lakewood, NJ) and 0.1 mg/ml protease (type XIV; Sigma, Poole, Dorset, United Kingdom), after which the ventricles were cut from the heart, finely chopped, and shaken in the collected enzyme solution (to which 1% bovine serum albumin was added) for 5-min intervals. Dissociated cells were collected by filtration at the end of each 5-min digestion, and the remaining tissue was returned for further enzyme treatment. The dissociated cells were centrifuged at 30 g for 40 s and resuspended in the 750 μm CaCl2solution and stored at 4°C until needed.
Single ventricular myocytes are an ideal preparation for investigating the mechanisms involved in the inotropic actions of anesthetics because equilibration is rapid throughout the entire preparation and therefore minimizes any problems associated with diffusion delays that could occur in multicellular preparations. Furthermore, single cells appear to respond to inotropic interventions in qualitatively the same way as multicellular preparations. 22,23These experiments were conducted at 30°C on unloaded rat ventricular myocytes stimulated at 1 Hz. This temperature was chosen to maximize retention of fura-2 during experimental procedures, but it should be noted that the balance between Ca2+entry and efflux from the cell would differ slightly from that at 37°C. Furthermore, as the cells are unloaded, shortening-induced alterations in the Ca2+binding to troponin-C may occur, which would not be observed in preparations during isometric conditions. However, length-dependent changes in Ca2+binding to troponin-C at physiological levels of Ca2+are likely to be minimal. 24
The isolation solution was composed of 130 mm NaCl, 5.4 mm KCl, 1.4 mm MgCl2, 0.4 mm NaH2PO4, 5 mm HEPES, 10 mm glucose, 20 mm taurine, 10 mm creatine, pH 7.1 (NaOH) at 37°C. After dissociation, cells were perfused with a physiologic salt solution of the following composition: 140 mm NaCl, 5.4 mm KCl, 1.2 mm MgCl2, 0.4 mm NaH2PO4, 5 mm HEPES, 10 mm glucose, 1 mm CaCl2, pH 7.4 (NaOH) at 30°C. Concentrations of 0.3 mm halothane, 1 mm isoflurane, and 0.6 mm sevoflurane were delivered from stock solutions made up in dimethyl sulfoxide. After dilution of the stock solutions, the final concentration of dimethyl sulfoxide in the superfusate never exceeded 0.2%, a concentration that had no significant effect on contractions (not shown). Unless stated otherwise, all solution constituents were from Sigma (Poole, Dorset, United Kingdom).
Recording Cell Length
Freshly dissociated cells were transferred to a tissue chamber (volume, 0.1 ml) attached to the stage of an inverted microscope (Nikon Diaphot; Nikon UK, Kingston-Upon-Thames, Surrey, United Kingdom). The cells were allowed to settle for several minutes onto the glass bottom of the chamber before being superfused at a rate of ∼ 3 ml/min with the physiological salt solution. Solutions were delivered to the experimental chamber by magnetic drive gear metering pumps (Micropump, Concord, CA), and solution level and temperature (30°C) were maintained by feedback circuits. 25All experiments were conducted at 30°C.
Cells were stimulated electrically at a frequency of 1 Hz (stimulus duration, 2 ms) via two platinum electrodes situated in the sides of the chamber. Cell length was recorded continuously using an optical system based on a photodiode array 26and displayed on a chart recorder (Gould 2600S; Gould Electronics Ltd., Ilford, Essex, United Kingdom). A sample and hold circuit 27was used to display active shortening of a cell during each contraction (twitch shortening) on the chart recorder. This circuit has the effect of excluding changes in resting cell length, although the time course of the twitch is recorded faithfully.
Cells were loaded with fura-2 by gentle agitation of a 2-ml aliquot of cell suspension with 6.25 μl of 1 mm fura-2 acetoxy methyl ester in dimethyl sulfoxide for 12 min. After centrifugation as before, the supernatant was removed by suction, and the pellet of cells was resuspended in 750 μm Ca2+solution. The fura-2–loaded cells were left for at least 30 min before use to allow de-esterification of the dye to take place. These cells were then transferred to the tissue chamber and stimulated as above. To record Ca2+transients, the fura-2–loaded cells were excited alternately with light at two wavelengths (340 and 380 nm), and fluorescence was detected at 510 nm using a spectrophotometer (Cairn Research Ltd., Faversham, Kent, United Kingdom). The ratio of fluorescence at 510 nm in response to excitation at 340 and 380 nm was used as a measure of the intracellular Ca2+concentration.
Cell length, Ca2+transients, and twitch shortening were recorded using a pulse code modulator (Neuro-Corder DR-890; Neuro Data Instruments Corp., New York, New York) coupled to a standard VHS video recorder. Averaged cell contraction and Ca2+transients were digitized at 0.2 kHz and displayed using an Axoscope (Axon Instruments, Foster City, CA).
Data are presented as mean values ± SEM, and statistical comparisons were conducted with the Student paired t test or Wilcoxon signed rank test as appropriate, using SigmaStat 2.0 (Jandel Scientific, Erkrath, Germany). All graphs were prepared using SigmaPlot 4.0 (Jandel Scientific).
Effects on Ca2+ Transient and Contraction
This study focuses on the mechanisms contributing to the inhibitory action of isoflurane, sevoflurane, and halothane on myocyte contractility. The approach taken here was to use concentrations of each anesthetic that led to a similar level of inhibition of the electrically evoked Ca2+transient to compare the involvement of various mechanisms that could contribute to the negative inotropic effects of these agents. The concentrations chosen were 1 mm isoflurane, 0.6 mm sevoflurane, and 0.3 mm halothane, which reduced the Ca2+transient to approximately 70–80% of control. However, as these concentrations differ in their anesthetic potency, other experiments were also conducted with 0.6 mm of each anesthetic, a concentration that approximates to twice the minimum alveolar concentration [MAC50] for each anesthetic.
Figure 1shows paired recordings of Ca2+transients and contractions before, during, and after a 1-min exposure to anesthetic. Figure 1Ashows that on application of 1 mm isoflurane, both the Ca2+transients and contractions decreased. During the 1-min exposure there was a small degree of recovery of both Ca2+transients and contractions toward control. On wash-off of isoflurane, contractions increased to a level slightly greater than that observed during control conditions before returning to control levels. Figure 1Bshows that the Ca2+transients and contractions were rapidly reduced in the presence of 0.6 mm sevoflurane before returning toward control levels during the exposure. On wash-off of sevoflurane, both the Ca2+transients and contractions increased to levels in excess of those observed during control conditions before returning to control levels. Figure 1Cshows that on application of 0.3 mm halothane, there was an initial increase in contraction, followed by a decrease that was sustained until wash-off. On removal of halothane, there was a small decrease of both Ca2+transients and contractions before they returned to control levels.
These results illustrate that the negative inotropic effects of volatile anesthetics are caused, at least in part, by a reduction in the intracellular Ca2+transient. Further experimental work was conducted to assess whether the effects of the agents were also the result of a decrease in myofilament Ca2+sensitivity.
Effects on Myofilament Ca2+Sensitivity
Figure 2Ashows Ca2+transients and contractions from a cell during control conditions (1 mm Ca2+), in the presence of reduced Ca2+(0.5 and 0.8 mm Ca2+) and in the presence of 1 mm isoflurane. As expected, reducing extracellular Ca2+decreased the Ca2+transient and contraction. Isoflurane decreased the Ca2+transient to an equivalent extent to 0.5 mm Ca2+, but the contraction was depressed to a greater degree. Figure 2Bshows representative fast time base Ca2+transients and contractions during control conditions (1 mm Ca2+) and in the presence of 0.5 mm Ca2+and 1 mm isoflurane. In this example, 0.5 mm Ca2+and 1 mm isoflurane depressed the peak of the Ca2+transient to a similar extent, but the contraction was depressed to a greater degree by isoflurane. These data illustrate that isoflurane caused a greater depression of contraction than can be explained by the depression of the Ca2+transient alone, suggesting that myofilament Ca2+sensitivity was reduced by isoflurane.
Figure 3shows similarly acquired data for 0.6 mm sevoflurane and 0.3 mm halothane. In the example shown in figure 3A, 0.6 mm sevoflurane and 0.5 mm Ca2+depressed the Ca2+transient to a similar degree; however, the contraction was slightly larger in the presence of sevoflurane than in the presence of 0.5 mm Ca2+. This might suggest that in this example, myofilament Ca2+sensitivity was increased by sevoflurane; however, this result was not typical, and on balance, the result from a group of cells were consistent with little or no effect of sevoflurane on myofilament Ca2+sensitivity (see below). In the example shown in figure 3B, halothane and 0.5 mm Ca2+reduced the peak of the Ca2+transient to similar levels, whereas halothane depressed the contraction to a much greater extent. This result supports previous data that halothane causes a depression of myofilament Ca2+sensitivity. 17It should be noted that with both isoflurane and halothane, but not sevoflurane, diastolic cell length increased during application of anesthetic with no change in diastolic Ca2+; these observations also suggest that myofilament Ca2+sensitivity is reduced by isoflurane and halothane but not by sevoflurane.
Figure 4shows mean data summarizing the results from the experiments shown in figures 2 and 3. Each panel illustrates the amplitude of contraction (expressed as a percentage of control) plotted against the amplitude of the Ca2+transient (expressed as a percentage of control). The solid lines represent the observed changes of the Ca2+transient and contraction when the extracellular Ca2+concentration was altered from 1 mm to 0.8 or 0.5 mm. The same data are shown in each panel and represent mean responses from 27 cells. The dashed lines represent the relation between the magnitude of the Ca2+transient and contraction in the presence of the chosen concentration of anesthetic. If myofilament Ca2+sensitivity was unchanged by the anesthetics, then the relation between the magnitude of the Ca2+transient and contraction would also be expected to be unchanged, i.e. , the data for each anesthetic should lie on the same trajectory as that obtained by varying extracellular Ca2+concentration. The vertical dotted lines in figures 4A and 4Cshow that in the presence of isoflurane and halothane, there was deviation from the relation between the contraction and the Ca2+transient when extracellular Ca2+was varied. The deviation is consistent with a depression of myofilament Ca2+sensitivity induced by these concentrations of anesthetic. In figure 4B, the data for sevoflurane lie on the same trajectory as the data obtained in 1, 0.8, and 0.5 mm Ca2+, suggesting that sevoflurane has little or no effect on myofilament Ca2+sensitivity.
Figure 5shows the relation between cell length and intracellular Ca2+during a single contraction during control conditions and in the presence of each anesthetic. Spurgeon et al. 28argued that during the relaxation phase of a contraction, the myofilaments come into quasi-equilibrium with cytosolic Ca2+. Therefore, by plotting cell length against the fura-2 fluorescence ratio during the relaxation phase of a contraction, it is possible to obtain an index of the Ca2+sensitivity of the myofilaments. For a given contraction, the data proceed in an anticlockwise direction. Figure 5illustrates such plots from contractions recorded in the absence and presence of isoflurane, sevoflurane, and halothane. The inset in each panel shows data throughout the Ca2+transient and contraction. The main plot of each panel shows these data during the final phase of relaxation. The solid lines are the result of linear regression of these data. In figures 5A and 5C, the relation between the fura-2 fluorescence ratio and cell length in the presence of both isoflurane and halothane is less steep (n = 7 and 11 cells;P = 0.008 and P = 0.031, respectively) and is shifted down. The decrease in the gradient of the regression lines and the downward shift is suggestive of a depression in myofilament Ca2+sensitivity. Figure 5Bshows that in the presence of sevoflurane there was no significant change in the gradient of the regression line (n = 9 cells;P = 0.22) and no substantial downward shift, suggesting that there was little or no change in the myofilament Ca2+sensitivity.
Effects on Sarcoplasmic Reticulum Ca2+Content and Fractional Release
The aim of these experiments was to determine the extent to which anesthetic-induced changes in SR Ca2+content and fractional release contributed to the ∼ 20–30% decrease in the electrically evoked Ca2+transient that was observed during application of the chosen concentrations of anesthetic. The left panels of figure 6show records of electrically stimulated Ca2+transients during control conditions, whereas the right panels illustrate the corresponding data in the presence of isoflurane (fig. 6A), sevoflurane (fig. 6B), and halothane (fig. 6C). Both during control conditions and in the presence of anesthetic, stimulation was stopped, and 20 mm caffeine was applied rapidly to release Ca2+from the SR. The peak of the caffeine-evoked Ca2+transient is a measure of the Ca2+content of the SR. 29Once the caffeine-evoked Ca2+transient had decayed, the caffeine was washed off with anesthetic-free control solution, and stimulation was recommenced. In the left panel of figure 6A, the dashed line represents the mean peak of the electrically stimulated Ca2+transient, and the arrows show how fractional release was measured: the peak of the electrically stimulated Ca2+transient was expressed as a percentage of the peak of the caffeine-evoked Ca2+transient; in this example, fractional release was 61%. The horizontal lines in the right panels in figure 6represent the peaks of the electrically and caffeine-evoked Ca2+transients during control conditions. Figure 7represents mean data from these experiments. The first three bars of each panel in figure 7show mean data (n = 7) in the presence of isoflurane, sevoflurane, and halothane. Figure 7Ashows the sustained effects of the anesthetics on the electrically stimulated Ca2+transient, figure 7Bshows the effects on SR Ca2+content, and figure 7Cshows the effects on fractional release.
Figures 6 and 7Aconfirm that the three anesthetics decreased the Ca2+transient significantly by 20–30%. Figures 6 and 7Bshow that the effects on SR Ca2+content varied between the anesthetics—isoflurane caused a modest but significant decrease of SR Ca2+content to 90 ± 3.9% of control (P = 0.045), sevoflurane caused no change (SR Ca2+content, 99 ± 1.1% of control), and halothane caused a substantial and significant decrease to 72 ± 1.3% of control (P < 0.001).
Other experiments were performed to assess the effects of 0.6 mm isoflurane and halothane on the Ca2+transient, SR Ca2+content, and fractional release for comparison with data for 0.6 mm sevoflurane; 0.6 mm is approximately equivalent to 2 × MAC50for all three anesthetics. As might be expected, the measured parameters changed in a dose-dependent manner: with 0.6 mm isoflurane, the Ca2+transient, SR Ca2+content, and fractional release were reduced to a lesser extent (to 85 ± 3%, 95 ± 2%, and 90 ± 4%, respectively; n = 9) than with 1 mm isoflurane. In 16 cells with 0.6 mm halothane, the Ca2+transient and SR Ca2+content were reduced to 68 ± 2% and 72 ± 3%, respectively, but fractional release was maintained at 97 ± 3%. Data at equi-anesthetic concentrations as well as at concentrations that induce a similar depression of the electrically evoked Ca2+transient (figs. 6 and 7C) show that fractional release was reduced significantly in the presence of isoflurane and sevoflurane but was maintained by halothane despite a large reduction in the SR Ca2+content.
To assess the possible mechanisms underlying the action of these anesthetics, experiments such as those in figure 6were conducted using 0.5 μm nifedipine and 0.25 mm caffeine. The data obtained are summarized in figure 7. Nifedipine, a dihydropyridine antagonist, is known to selectively block ICa. Volatile anesthetics are also known to reduce ICa(see Discussion). To assess the effects of partial blockade of ICaalone on SR Ca2+content and fractional release, 0.5 μm nifedipine, a concentration that depressed the electrically stimulated Ca2+transient to a similar degree as the chosen concentration of each anesthetic, was used. These data showed that 0.5 μm nifedipine caused significant reductions in the Ca2+transient (P < 0.001), SR Ca2+content (P = 0.008), and fractional release (P < 0.001;fig. 7).
Caffeine is known to cause a sensitization of Ca2+-induced Ca2+release (CICR) from the SR. 30Volatile anesthetics may also affect the sensitivity of CICR 6; therefore, to assess the effects of a change (increase) in the sensitivity of CICR alone on SR Ca2+content and fractional release, 0.25 mm caffeine, a concentration known to sensitize CICR 30but not to deplete the SR of Ca2+fully, was applied in experiments such as that shown in figure 6. These data showed that 0.25 mm caffeine caused a small but significant reduction in the Ca2+transient (P = 0.036), a significant reduction in SR Ca2+content (P = 0.009) but a significant increase in fractional release (P < 0.001;fig. 7). The significance of these findings is considered in the Discussion.
The majority of experiments conducted in the present study used concentrations of anesthetic that induced a 20–30% reduction in the magnitude of the electrically evoked Ca2+transient to further investigate the mechanisms underlying their negative inotropic effect. A reduction in the Ca2+transient would reduce contractility; however, at anesthetic concentrations that led to a similar depression of the Ca2+transient, isoflurane and halothane, but not sevoflurane, also reduced myofilament Ca2+sensitivity, which would contribute to the negative inotropic effect of isoflurane and halothane.
In the present study, 0.6 and 1 mm isoflurane caused a modest decrease in SR Ca2+content, which conflicts with previous data from Jiang and Julian, 18whereas sevoflurane had little or no effect on SR Ca2+content despite decreasing the electrically stimulated Ca2+transient. On the other hand, 0.3 mm halothane reduced the SR Ca2+content to a greater extent than isoflurane. Both isoflurane and sevoflurane caused a decrease in fractional release, whereas halothane did not. The effects of sevoflurane on myofilament Ca2+sensitivity and SR Ca2+content are novel findings, as are the effects of all three anesthetics on fractional release.
Previous reports concerning the effects of halothane and isoflurane on myofilament Ca2+sensitivity are contradictory (see Introduction). The findings of Herland et al. 13suggest that the presence of the cell membrane is needed for the effect of halothane on myofilament Ca2+sensitivity to be observed. It follows from this that the effect of halothane is mediated by the cell membrane, although how this occurs is not known. However, in intact cardiac muscle tissue, some investigators report data that are consistent with a depression of myofilament Ca2+sensitivity by both halothane and isoflurane, 8,10,12,17,31–33whereas others suggest that a reduction in myofilament Ca2+sensitivity plays little or no role in the negative inotropic effects of these agents. 14–16In the present study, 1 mm isoflurane and 0.3 mm halothane depressed myofilament Ca2+sensitivity, whereas sevoflurane did not affect myofilament Ca2+sensitivity.
Sarcoplasmic Reticulum Ca2+Content and Fractional Release
The reported effects of isoflurane on SR Ca2+content are inconsistent. Wilde et al. 19reported a modest but not significant decrease in SR Ca2+content to 88% of control by 1.5% isoflurane. Although Jiang and Julian 18recorded a significant decrease to 71% of control by 0.62 mm isoflurane, the method used to measure SR Ca2+content was not standard (see Introduction). In the present study, we observed a modest but significant decrease in SR Ca2+content to 90 ± 3.9% in the presence of 1 mm isoflurane, no effect of sevoflurane on SR Ca2+content, whereas halothane reduced the SR Ca2+content to a greater extent, as has been observed previously. 34Finally, isoflurane and sevoflurane, but not halothane, reduced fractional release. Data from experiments using equivalent concentrations of each anesthetic (0.6 mm) gave a broadly similar picture; the decrease in the Ca2+transient with isoflurane and sevoflurane appeared to result from a decrease in fractional release, whereas with halothane, was primarily a result of a reduction in SR Ca2+content as fractional release was maintained.
The effects of the anesthetics on SR Ca2+content and fractional release could be the result of a decrease in ICaor a direct effect on the SR Ca2+release channel. Isoflurane, sevoflurane, and halothane at concentrations greater than 1.65%, 351%, 36and 1.2%, 37respectively, decrease ICa. There is evidence that halothane affects the SR Ca2+release channel: halothane has been shown to increase both the open probability and the open time, but not the conductance of Ca2+release channels (whereas isoflurane does not show this behavior). 34In quiescent preparations, halothane, but not isoflurane, has been shown to increase intracellular free Ca2+, 31,38whereas in electrically stimulated myocytes, halothane, but not isoflurane or sevoflurane, results in a brief increase in contractions and Ca2+transients 6,17,32,39,40; these effects of halothane have been suggested to be the result of an enhancement of CICR from the SR by halothane. 6,34,41,42
To assess the effect of a reduction of ICaalone on the SR Ca2+content and fractional release, the effects of the anesthetics were compared with those of 0.5 μm nifedipine, a known Ca2+channel antagonist. Figure 7shows that nifedipine decreased both the SR Ca2+content and fractional release. The decrease in the SR Ca2+content after block of ICais an expected consequence of a decrease in Ca2+influx into the cell. 43Both a decrease in ICa(the trigger for SR Ca2+release) and a decrease in SR Ca2+content would be expected to reduce fractional release, 20and either or both of these factors could be the cause of the decrease in fractional release in the presence of nifedipine. In figure 7, the mean data for isoflurane and nifedipine are essentially very similar. Both depressed the Ca2+transient, SR Ca2+content, and fractional release to a similar degree. This is consistent with the possibility that the effects of isoflurane on the Ca2+transient, SR Ca2+content, and fractional release are the result of a decrease in ICaalone.
The profile of changes with sevoflurane is unlike that with nifedipine (fig. 7): although sevoflurane resulted in a decrease in fractional release, the SR Ca2+content remained unchanged. An alternative possibility is that sevoflurane inhibits CICR from the SR, as does tetracaine. 44However, although this would explain the decrease in fractional release, such an effect would be expected to result in an increase in SR Ca2+content similar to that with tetracaine. 44Such an increase in SR Ca2+content was not observed in the present study. We propose that the effects of sevoflurane are the result of inhibition of ICaand an inhibition of CICR from the SR (or suppression of Ca2+extrusion via Na+–Ca2+exchange). This could explain why there is a decrease in fractional release and no change in SR Ca2+content with sevoflurane.
The profile of changes with halothane is also unlike that with nifedipine (fig. 7); although halothane resulted in a decrease in SR Ca2+content, fractional release remained unchanged. As discussed previously, there is evidence that halothane enhances CICR from the SR. To assess the effect of an enhancement of CICR alone, the effect of 0.25 mm caffeine, which is known to enhance CICR, 30was studied. As expected, caffeine increased fractional release and decreased SR Ca2+content (fig. 7). Although halothane decreased SR Ca2+content like caffeine, it did not increase fractional release. We propose that the effects of halothane (at this concentration) are the result of inhibition of ICaand an enhancement of CICR from the SR. This could explain why there is a decrease in SR Ca2+content and no net change in fractional release with halothane.
The data from the present study are summarized in table 1. It should be noted that these data refer to concentrations of anesthetic that induce a similar depression of the electrically evoked Ca2+transient and are not equi-anesthetic concentrations. These data show that approximately one third of the negative inotropic effect of 1 mm isoflurane is the result of a decrease in myofilament Ca2+sensitivity and approximately two thirds is the result of a decrease in the Ca2+transient. The decrease in the Ca2+transient is the result of decreases in both the SR Ca2+content and fractional release (table 1); we have argued that these effects are the result of an inhibition of ICa. The data summarized in table 1show that the negative inotropic effect of 0.6 mm sevoflurane is the result of a decrease in the Ca2+transient, and the decrease in the Ca2+transient results from a reduction in fractional release. We have argued that the decrease in fractional release could be the result of an inhibition of ICa. Finally, the data summarized in table 1suggest that half of the negative inotropic effect of 0.3 mm halothane results from a decrease in myofilament Ca2+sensitivity, and half results from a decrease in the Ca2+transient, which in turn reflects the decrease in SR Ca2+content. We have argued that the decrease in the SR Ca2+content is the result of both an inhibition of ICaand an enhancement of the CICR from the SR.
The authors thank Luke Blumler, Andy O’Brien, and Dave Johanson, technicians, School of Biomedical Sciences, University of Leeds, United Kingdom, for expert technical assistance.