INTRODUCTION

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THE COMMON VAPOR ANESTHETIC agents such as halothane depress the contractile function of the heart in the clinical setting (22). Considerable research effort has been directed to the study of the mechanisms involved in the production of this depressant effect. Much of this work has been reviewed (26). Many different approaches have been used including use of the isolated intact heart, isolated intact trabeculae, papillary muscles, single myocytes, skinned muscle, and solutions of cardiac contractile proteins. Cardiac muscle samples have been obtained from species as disparate as the human and the frog. Nevertheless, despite all this work, important unresolved issues remain.

The foundation on which rests current views concerning how vapor anesthetic agents act in the heart to produce negative inotropy is that they decrease the magnitude of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) transient (7), which is of great importance in modulating force of contraction in cardiac muscle (18). It can hardly be disputed that vapor agents at high doses do depress the [Ca²⁺]_i transient, but it is not clear that this remains true in the clinically relevant dose range. Here, analysis of a large body of data yields equivocal results. This may be so in part because previous work has relied on the photoprotein aequorin, microinjected into relatively few surface myocytes, to report [Ca²⁺]_i, whereas other studies using the newer fluorescent dyes to report [Ca²⁺]_i have been done on isolated myocytes, which may have limited relevance to the physiological state.

It is also believed that vapor agents such as halothane can act by depleting the sarcoplasmic reticulum (SR) of releasable Ca²⁺ (26). The conclusion that halothane reduces the SR Ca²⁺ content of the myocardium arises from the observation that halothane decreases rapid cooling (RC) contracture, which uses contracture force as an indicator to estimate the amount of Ca²⁺ released from the SR (16). This conclusion is based on the assumption that halothane does not alter the Ca²⁺ sensitivity of the contractile apparatus. Because it has been shown that halothane does reduce the responsiveness of the contractile apparatus to Ca²⁺ (20), the commonly accepted conclusion that halothane affects SR Ca²⁺ content is, at least quantitatively, incorrect. Therefore, simultaneously monitoring both contracture force and [Ca²⁺]_i during an RC response should provide a more accurate estimate of halothane's effects on SR Ca²⁺ content.

The goal of this study was to determine the extent to which, at clinically relevant doses, halothane exerts its negative inotropic effect through reduction of the responsiveness of the contractile apparatus to [Ca²⁺]_i, rather than acting mainly through the commonly accepted mechanisms of reduction in SR Ca²⁺ content and decrease in the [Ca²⁺]_i transient. To do this, we use here an intact rat trabecula loaded with the salt form of the fluorescent dye fura 2 (14). This has made it possible to obtain new evidence about the negative inotropic action of vapor anesthetics such as halothane acting in the clinically relevant dose range. Our results indicate that, at low dose [<1 minimum alveolar concentration (MAC)], halothane markedly decreases peak twitch force mainly through a reduction of the Ca²⁺ sensitivity of the contractile system. At high dose (near 2 MAC), its effect is through both reduction in the [Ca²⁺]_i transient and reduction of the Ca²⁺ sensitivity of the contractile system. Even at the high dose of halothane, the SR Ca²⁺ content is not substantially reduced.

	METHODS

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A recent article (14) presents many important details of the methods used in this article. The following condensed version provides essential information either not provided in the above article or of particular importance here.

Dissection procedure. Male Lewis-Brown-Norway rats (150-175 g) were used in these experiments (Harlan, Indianapolis, IN). Hearts were quickly removed and placed in a cold modified Krebs solution (MKS). The hearts were then perfused retrogradely through the aorta using a Langendorff apparatus for 10 min at room temperature with the same MKS containing (in mM) 108 NaCl, 6 KCl, 1.2 MgCl₂, 2 CaCl₂, 24 NaHCO₃, 10 sodium pyruvate, 4 glucose, as well as 5 U/1,000 ml insulin, bubbled with 95% O₂-5% CO₂, final pH 7.4. Finally, the hearts were flushed with dissection solution, which is the standard MKS plus 15 mM KCl (final [KCl] = 21 mM).

Trabeculae similar to those described previously were isolated from the right ventricle (10, 19). These muscles (in mm, length 2.5-4, width 0.1-0.2, thickness 0.05) were attached to one end of each trabecula to a force transducer (model 400A, Cambridge Technology), and the other end was tied to a stainless steel wire connected to a micropositioner. In an important step in the mounting procedure, cyanoacrylate tissue adhesive (Histoacryl blau, B. Braun Melsungen) was used to fix firmly the two ends of the preparation to the force transducer wire and the micropositioner wire. We have previously shown that this method of attachment of a papillary muscle very much decreases extra end compliance as demonstrated by flash photography and use of markers and spot-follower apparatus (15). Consequently, internal movement associated with extra compliance at the ends of the preparation is minimized, thereby ensuring a contraction as close to isometric as possible.

Bath setup and electrical stimulation. The preparation was placed into a temperature-regulated chamber (8 mm × 6 mm × 5 mm) milled into a Lucite plate. The chamber had a glass coverslip floor (thickness 0.12 mm). The Lucite plate with its chamber was positioned on the stage of an inverted microscope (Nikon Diaphot 300) fit with a fluorescence system [Photon Technology International (PTI) Deltascan 4000]. Preparations were superfused at a rate of 12 ml/min with MKS. The MKS solutions were vigorously bubbled with 95% O₂-5% CO₂. The temperature in the bath was maintained at 30 ± 0.2°C, unless otherwise stated. The preparations were stimulated at 0.5 Hz with 5-ms pulses via platinum plate electrodes in the bath connected to a stimulator (Grass S88). The stimulus strength was adjusted to 1.5 times threshold, and tension was continuously monitored on both a chart recorder (Hewlett-Packard 7133A) and one channel of a digital oscilloscope (Nicolet 4094B). The optimal tension response with respect to length was obtained. Resting sarcomere length resulting in maximum tension on stimulation was always in the range of 2.18-2.30 µm. These values were measured from hard copy striation patterns sampled from the video image (using a Nikon 40× objective) with a UP1200 Mavigraph Color Video Printer (Sony, Japan).

Dye loading procedures. Before the trabeculae were loaded with fura 2, autofluorescence (emission at 510 nm) was measured at a pacing rate of 0.5 Hz and 30°C. Preparations were then loaded by iontophoretic injection of fura 2 dye by a method already described (1) in MKS with [Ca²⁺]_o of 0.5 mM at 22°C with no stimulation. Micropipettes were pulled on a Flaming/Brown micropipette puller (model P87, Sutter Instrument), and tips were filled with 1 µl of 2 mM fura 2 (pentapotassium salt, Molecular Probes, Eugene, OR). The pipettes were then back-filled with a 150 mM potassium acetate solution (pH = 7.3) and connected via a micropipette holder to the headstage of a World Precision Instruments electrometer module (model S-7071A). Tip resistances of 200-300 M were typical in micropipettes filled with the fura 2 solution before impalement of single myocytes, and stable membrane potentials of 40 to 60 mV were required for successful dye loading. After impalement, injection of the dye into the myocyte was achieved by passing a negative current of 3-5 nA. After loading was completed, the preparation was superfused again with the standard MKS, the temperature was increased to 30°C from room temperature, and the pacing was resumed at 0.5 Hz. Approximately 40 min were allowed to let the dye diffuse, yielding a complete uniformity of fluorescence. Fura 2 was loaded to levels such that the added fluorescence was about three times the autofluorescence observed at 358 nm (the isosbestic point for fura 2) before loading.

Application of halothane, 2,3-butanedione monoxime, low [Ca²⁺]_o, and ryanodine. With the use of a Dräger vaporizer, halothane was added to the superfusion solution in the treatment bottle to produce three doses (in mM): 0.18 (low), 0.37 (moderate), and 0.55 (high). The halothane concentrations were determined using a Hewlett-Packard 5890 Series II gas chromatograph with an HP 624 column and a flame ionization detector. According to a recent report, 1 MAC for halothane in non-blood-containing aqueous media at below normal body temperature in the rat should be ~0.27 mM (11), so that our high dose for halothane (0.55 mM) would be close to 2 MAC. The MAC concept is related to an intact whole body response to a noxious stimulus signaled by movement in 50% of the subjects, and dose levels up to 2 MAC are well within the clinically relevant range.

The application of 2,3-butanedione monoxime (BDM) was done by direct addition of BDM (Aldrich Chemical) into the standard MKS in the treatment bottle, yielding final concentrations (in mM) of 2, 5, or 10. When low [Ca²⁺]_o was required, the standard MKS (2 mM [Ca²⁺]) was replaced by an identical solution except that the [Ca²⁺] values were 1, 0.5, or 0.25 mM. Ryanodine (Calbiochem) treatment was performed by addition of ryanodine into the standard MKS in the treatment bottle to yield a final concentration of 10 µM, which is within the dose range that ryanodine causes SR Ca²⁺ depletion (25).

Rapid cooling procedure. Rapid cooling was used to assay SR Ca²⁺ content using both contracture force as well as the [Ca²⁺]_i signal reported by fura 2. A simple procedure was devised using a fixed-volume (2,000 µl) tip ejector pipetter to deliver ejectate into the muscle chamber (250-µl volume). The superfusate for both control and treatments in the pipetter tip was precooled to 0°C by placing the tip in a solution of ice and distilled water. The temperature near the preparation was monitored using a thermistor needle probe, with a response time of ~0.15 s. This showed that the technique could produce a temperature change in the chamber solution from 30°C to 3°C in ~1 s. The cold injectates could be reliably delivered at ~2 s after the last electrical stimulation. Just before injection, flow of the perfusate as well as electrical stimulation was interrupted and then resumed after the cold response was recorded (both fura 2 signal response and contracture force). The peak contracture force and the peak [Ca²⁺]_i were used to estimate the amount of Ca²⁺ released from the SR in response to an RC as previously reported (5, 23).

Fluorescence system. The PTI Deltascan 4000 system was used in these experiments, and a 75-W xenon lamp was the source for excitation light. The light passed through a scanning monochromator that was programmed to select wavelengths of 345 and 380 nm for excitation of fura-2. Light exiting the monochromator was carried by a quartz fiber optic bundle to a filter cassette coupled to the microscope objective. On entering the cassette, the light was first short-pass filtered (<470 nm) to remove the residual 510-nm light before reflecting off a dichroic mirror (400-nm long pass) en route to the objective, an Olympus 10× (Dapo UV/340; NA 0.4). Fluorescence from the preparation passed back through the objective and dichroic mirror to the side-port of the microscope. Here, a second dichroic mirror, with a rectangular region-of-interest adjustment, sent 92% of the light to photomultiplier A and 8% to photomultiplier B. Just before reaching the photomultiplier tubes, light signals were passed through emission filters, which transmitted wavelengths of 510 ± 20 nm.

Data collection and analysis. Fluorescence signals were collected in either photon counting mode (entrance and exit slit-widths set to 2 nm) or analog mode (entrance and exit slit-widths set to 10 nm). When analog mode was used, the voltage output from photomultiplier A was sent to a low-pass analog filter (Stanford Research Systems, model SR650) with a 500-Hz cutoff and then to a four-channel digital oscilloscope (Nicolet 4094B) sampling at a frequency of 2 kHz. Photomultiplier B was used only to monitor dye loading in the photon counting mode. The fluorescence system in the analog mode was controlled by PTI software "Oscar" running on an IBM-compatible 486 computer (American Megatrends) used to control shutter openings, monochromator settings, and other automated data acquisition programs. The fluorescence and twitch force signals were recorded using the oscilloscope, and all traces presented here are averages of nine sweeps. The autofluorescence at excitation wavelengths 345 nm (F_345a) and 380 nm (F_380a) were interleaved in time so that a sweep at 345 nm was followed by one at 380 nm and so on until a total of 18 sweeps was recorded. These were then averaged in two groups of nine. In this way, a noise reduction by a factor of three was achieved. The fluorescence values after dye loading were collected in the same way as for the autofluorescence yielding total fluorescence at the two excitation wavelengths (F_345t and F_380t). The F_345a and F_380a were then subtracted from the F_345t and F_380t, respectively, yielding the dye-related fluorescence F₃₄₅ and F₃₈₀. The R signal was formed by dividing F₃₄₅ by F₃₈₀. This made it possible to use the R signal to greatly minimize the effect of motion artifact on fura 2 reporting of [Ca²⁺]_i. Fluorescence and twitch force were analyzed using Vu-Point 3 data analysis software (Maxwell Laboratories).

When photon counting mode was used, the excitation wavelengths 345 and 380 nm were alternated by a chopper at a rate of 200/s, and the "Felix" program of PTI was used to record the fluorescence intensities of F₃₄₅ and F₃₈₀ from photomultiplier A. Dye loading was monitored also using photomultiplier A. The autofluorescence values of F₃₄₅ and F₃₈₀ were obtained before dye loading, and these were subtracted from the raw fluorescence signals after dye loading, thus yielding corrected fluorescence signals in real time. The R signal and the corresponding force trace were also recorded and displayed using the same program.

	RESULTS

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A typical time-dependent response of an isolated trabecula to 0.55 mM halothane is shown in Fig. 1. Halothane caused a small initial increase in the peak [Ca²⁺]_i transient (Fig. 1A), which was associated with an obvious transient increase in peak twitch force (Fig. 1B). This is similar to recent findings from rat myocytes using indo 1 (27). After the initial potentiation, both the peak [Ca²⁺]_i transient and peak twitch force declined gradually and reached their steady-state levels ~100 s from the beginning of halothane treatment. For this preparation, halothane (0.55 mM, 2 MAC) in the steady state depressed the peak twitch force by ~80%, whereas the peak [Ca²⁺]_i transient was depressed by only ~35%. There was no detectable effect on either diastolic force or diastolic [Ca²⁺]_i. Washing out halothane led to a complete recovery of both twitch force and [Ca²⁺]_i transient (data not shown). This result shows that treatment with halothane causes a profound time-dependent reduction in peak twitch force that is not accompanied by a proportional decrease in the peak [Ca²⁺]_i transient.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Time-dependent effects of halothane on intracellular Ca2+ concentration ([Ca2+]i) transients and twitch force. Treatment with halothane (0.55 mM) began at time 0. A: [Ca2+]i transients. B: corresponding twitch force to transients in A. The trabecula was paced at a rate of 0.5 Hz at 30°C, extracellular Ca2+ concentration ([Ca2+]o) of 2 mM. The data for the R signal were collected in the photon counting mode at a rate of 200/s. [Ca2+]i was obtained by transformation of the R signal (not shown) using the minimum R value (Rmin), maximum R value (Rmax), and Kd ×  obtained from an in vivo calibration (14). Note that halothane caused a characteristic early small transient increase in peak twitch force, which was accompanied by a less obvious increase in the peak [Ca2+]i transients. In the steady state, halothane markedly reduced the peak twitch force, whereas the peak [Ca2+]i transients were not proportionally depressed.

Figure 2 shows the effects of halothane in the steady state at different doses [low (0.18), moderate (0.37), and high (0.55 mM)] on [Ca²⁺]_i transients (Fig. 2A) and twitch forces (Fig. 2B). Halothane (0.18 mM) diminished peak twitch force by 44% (compared with precontrol value) with only a small reduction in the peak [Ca²⁺]_i transient. Increasing the halothane doses to 0.37 and 0.55 mM caused further reductions in both the peak [Ca²⁺]_i transient and peak twitch force. Washing out halothane resulted in nearly complete recovery of both peak twitch force and peak [Ca²⁺]_i transient as shown in the postcontrol. The reductions in peak twitch force are larger than the reductions in the corresponding peak [Ca²⁺]_i transient. This suggests that halothane depresses cardiac muscle contraction through both reduction of the peak [Ca²⁺]_i transient and reduction of the Ca²⁺ sensitivity of the contractile system.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Steady-state effects of varying halothane concentrations on [Ca2+]i transients (A) and the corresponding twitch force (B). The preparation was paced at 0.5 Hz at 30°C, [Ca2+]o of 2 mM. The data were collected in the analog mode at a rate of 2,000/s. For this preparation, halothane was introduced in the order 0.18, 0.37, and 0.55 mM after the precontrol twitch was obtained. For each dose of halothane, data were obtained in the steady state of maximal depression of peak twitch force. The postcontrol twitch was obtained after washing out halothane and allowing the twitches to recover to steady-state values. Note that in the steady state of the halothane action, reduction of peak twitch force is much more prominent than is depression of the corresponding peak [Ca2+]i transient. The [Ca2+]i was obtained by transformation of the R signal (not shown) using the Rmin, Rmax, and Kd ×  obtained from an in vivo calibration (14).

To establish the factors that contribute to its depressant influence, the effects of halothane were compared with those of two other agents with better defined mechanisms of negative inotropic action. One intervention was by lowering [Ca²⁺]_o and thus lowering the peak [Ca²⁺]_i transient, which should reduce contractility by decreasing the [Ca²⁺]_i transient for force generation without significant alteration of the Ca²⁺ sensitivity of the contractile system. The other intervention was by using BDM, which is an agent that markedly reduces muscle force generation by a peripheral mechanism interfering with cross-bridge formation with little effect on the peak [Ca²⁺]_i transient (2).

The effects of the three negative inotropic interventions (halothane, low [Ca²⁺]_o, and BDM) at three different doses on peak twitch force, time to peak, half-width, and diastolic force can be summarized as follows. The peak twitch forces were decreased using the three different doses of each intervention to about the same levels (~60, 40, and 20% of precontrol values). The peak twitch forces completely recovered after the removal of the treatments. At each of the three levels of reduction, the pairwise differences of peak twitch forces caused by the three interventions were not statistically significant. The time to peak of twitch force decreased with reduction of peak twitch force caused by halothane and BDM, whereas it increased as peak twitch force decreased with low [Ca²⁺]_o treatment. When peak twitch force was reduced to 40 and 20% of the precontrol values, time to peak of twitch force with low [Ca²⁺]_o treatment was significantly increased compared with treatments with halothane or BDM. The difference in the time to peak of twitch force between the halothane-treated and BDM-treated groups is not significant when peak twitch force is reduced to a similar degree. The effects of these interventions on the half-width of twitch force followed a similar pattern to that of time to peak of twitch force. The more peak twitch force was decreased by either halothane or BDM, the shorter the twitch half-widths were. When peak twitch force was reduced to 40 and 20% of the precontrol values, the half-width of twitch force with treatment by halothane fell between those with low [Ca²⁺]_o and BDM treatments, and the pairwise differences among the three groups were statistically significant. Concerning diastolic force, at any given level of peak twitch force reduction, there were no pairwise statistically significant differences among the groups with the three treatments.

The corresponding changes in peak [Ca²⁺]_i transients of the twitch forces presented above are summarized as follows. The peak values of the [Ca²⁺]_i transients were hardly reduced, whereas peak twitch forces were markedly reduced as BDM dosage was increased. Low [Ca²⁺]_o caused a linear decrease in the peak [Ca²⁺]_i in proportion to the reduction of [Ca²⁺]_o. At any level of peak twitch force depression, the peak [Ca²⁺]_i transients of the low-[Ca²⁺]_o-treated group were significantly smaller than those of the BDM-treated groups. With peak twitch force reduction to 60% of the precontrol values, the peak value of the [Ca²⁺]_i transient of the halothane-treated group was no different from that of the BDM-treated group, but significantly higher than that of the low-[Ca²⁺]_o-treated group. When peak twitch force was diminished to 40 and 20% of the precontrol values, the peak [Ca²⁺]_i transients of the halothane-treated groups were significantly smaller than those of groups with a similar degree of peak twitch force reduction caused by BDM and were significantly larger than those of low-[Ca²⁺]_o-treated groups. Halothane and BDM did not significantly alter the time to peak and half-width of the [Ca²⁺]_i transients. Low [Ca²⁺]_o prolonged time to peak and half-width of the [Ca²⁺]_i transient and reduced diastolic [Ca²⁺]_i, but these changes were not statistically significant compared with those of the halothane- or BDM-treated groups.

A plot of peak twitch force as a function of the corresponding peak [Ca²⁺]_i transient produced by the three interventions at varying doses as well as the precontrol and postcontrol values is shown in Fig. 3. A decrease in the peak [Ca²⁺]_i transient was accompanied by a reduction in the peak twitch force in the groups treated with low [Ca²⁺]_o. BDM caused little decrease in the peak [Ca²⁺]_i, whereas the peak twitch force fell steeply. Both the low [Ca²⁺]_o and BDM groups could be fit using linear regressions as indicated in the legend to Fig. 3, although there was a pronounced difference in slopes. The data from the halothane-treated groups fell between those of the low [Ca²⁺]_o- and BDM-treated groups and required a nonlinear regression to obtain an adequate fit, as also indicated in the legend to Fig. 3. It is clear from the plots shown in Fig. 3 that treatment with halothane produced a curvilinear response intermediate between the linear declines produced by low [Ca²⁺]_o and BDM.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Plot of peak twitch force as functions of the corresponding peak [Ca2+]i transients in the absence and presence of various doses of halothane, [Ca2+]o, and 2,3-butanedione monoxime (BDM). The data from treatments with low [Ca2+]o and BDM were fit using a linear regression (SigmaPlot). For low [Ca2+]o, the slope was 1.14, whereas for BDM it was 15.71. The data for treatment with halothane were fit using a two-parameter, single nonlinear exponential growth regression equation y = 0.2522 × exp(0.0596x). The slopes of the halothane curve at the upper (near 0 dose) and lower (at the high dose) ends are 5.78 and 0.66, respectively.

It is clear that halothane, particularly at high doses, causes a reduction in the peak [Ca²⁺]_i transient reported by fura 2 as indicated above. The reduction could be due to decreasing the Ca²⁺ content of the SR or to lowering the Ca²⁺ current through the L-type Ca²⁺ channels during an action potential or to both. To estimate to what extent the reduction in peak [Ca²⁺]_i transient was caused by a decrease in SR Ca²⁺ content, RC responses were obtained in the absence and presence of 0.55 mM halothane. Typical RC responses from a trabecula are shown in Fig. 4. In Fig. 4A, an RC response before halothane treatment (control 1) was elicited 2 s after the last stimulus while pacing at 0.5 Hz. The top trace in Fig. 4A is the [Ca²⁺]_i response, whereas the bottom trace is the corresponding force. The RC produced a transient rise in [Ca²⁺]_i to a higher level as rapidly as did the preceding twitch response. The corresponding force level of the RC response was close to that of the preceding twitch. Halothane (0.55 mM) reduced markedly both the peak twitch force and the contracture force, while decreasing the peak [Ca²⁺]_i of both the twitch and the RC response to a lesser extent (Fig. 4B). The peak force and peak [Ca²⁺]_i of the twitch and RC response after washing out halothane (Fig. 4C, control 2) were nearly identical to those of the initial control (Fig. 4A). The RC technique used here was tested to determine if it could detect a reduction in Ca²⁺ content of SR, and to do this the trabecula was treated with ryanodine. The results are also shown in Fig. 4D. Ryanodine reduced the peak twitch force and peak [Ca²⁺]_i transient by 85 and 74% of the control values, respectively. The RC produced a barely detectable increase in [Ca²⁺]_i. These results show that the RCs used here provide a reliable way to estimate SR Ca²⁺ content.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Rapid cooling (RC) responses for control and treatment with halothane or ryanodine. The data for the R signals (not shown) were collected in the photon counting mode at a rate of 200/s. The [Ca2+]i was obtained by transformation of the R signal using the Rmin, Rmax, and Kd ×  obtained from an in vivo calibration (14). The preparation was paced at a rate of 0.5 Hz, at 30°C, [Ca2+]o 2 mM. The RC was initiated after the stimulator was turned off and 2 s from the last stimulus. Top trace in A-D: [Ca2+]i transient of the preceding twitch response followed by the RC response. Bottom trace in A-D: corresponding force responses are shown. All forces were vertically shifted downward the same amount (0.75 U) to avoid overlap of the 2 traces. Note that the relative peak amplitudes of the 2 control twitches are nearly identical. Data are from a single trabecula and were obtained in the order: control 1, halothane (0.55 mM), control 2, and ryanodine (10 µM). For halothane treatment, data were collected after the twitch force had declined to reach a steady-state level. The RC in the presence of ryanodine was initiated 30 min after application of ryanodine when twitch amplitude was diminished by ~85%. Each RC lasted for 15 s. Between two adjacent RCs, a 10-min recovery period was allowed. Note that in comparing A and B, if contracture force were used as the index for SR Ca2+ content, the erroneous conclusion would be drawn that the SR Ca2+ content was markedly reduced by halothane.

The effects of halothane (0.55 mM) on RC-induced peak [Ca²⁺]_i response and RC-induced peak contracture force were further studied. As shown in Fig. 5A, halothane (0.55 mM) caused significant (21 and 16%) reductions in the peak [Ca²⁺]_i RC responses compared with precontrol and postcontrol values. The effect of halothane on the corresponding RC contracture force is shown in Fig. 5B. Here, the peak RC contracture force was reduced significantly (47 and 53%), compared with precontrol and postcontrol values, much more than the peak RC [Ca²⁺]_i shown in Fig. 5A. There were no statistically significant differences between pairs of control values in any of the panels.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Effect of halothane on RC response. A: peak values of [Ca2+]i elicited by RC in the presence of halothane (79%) and for the postcontrol (94%) are given as a percentage of the precontrol value (100%). B: corresponding contracture force in response to RC in the presence of halothane (53%) and for the postcontrol (111%) are also given as a percentage of the precontrol value (100%). * Difference between the adjacent groups is significant (P < 0.05). Values are means ± SE; n = 6. No statistically significant differences were found between pretreatment and posttreatment controls in both the RC-induced Ca2+ responses and the RC-induced contractures.

	DISCUSSION

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The major new findings in this study are as follows. At low dose (<1 MAC), halothane markedly diminishes twitch force while producing much less decrease in the peak [Ca²⁺]_i transient. At high dose (near 2 MAC), halothane very much decreases both twitch force and the [Ca²⁺]_i transient. At the moderate dose, the effects are intermediate. Remarkably, at all dose levels, we find small influence on the SR Ca²⁺ content as indicated by an RC-induced Ca²⁺ release, so that even at the high-dose SR, Ca²⁺ content is only reduced by 21%. These findings are not easy to reconcile with those of others owing mainly to the diverse ways this problem has been addressed in the past, i.e., different preparations, different temperatures, different experimental protocols, different ways of obtaining and expressing relevant vapor agent doses, and so forth. In the following, we make an attempt at a reconciliation, particularly with respect to noting that there have been hints in earlier reports confirming our present view.

Earlier work has been done concerning the effects of halothane on [Ca²⁺]_i transients and papillary muscle twitch force, and it was reported that halothane produced a dose-dependent parallel decrease of both contractile force and the [Ca²⁺]_i transient (9). Note that it was also suggested that halothane might also decrease myofilament sensitivity to Ca²⁺ (9). Further work was done using isolated intact papillary muscles from guinea pigs and, again, aequorin, injected into superficial myocytes as before, to report [Ca²⁺]_i (8). The results also indicate that for halothane there is a parallel depression of both peak tension development and peak aequorin signal for low- and high-dose halothane (8). These results seem to be different from those we present here. One possible explanation would be that aequorin preferentially reports [Ca²⁺]_i from a restricted space within the cellular cytoplasm, whereas fura 2 reports [Ca²⁺]_i from a space more nearly similar to the bulk of the cytoplasm (24).

Results from work using fura 2 to report [Ca²⁺]_i, in contrast to the use of aequorin, and the influence of halothane have been reported in rat single myocytes loaded with fura 2-acetoxymethyl ester (28). The authors indicate that halothane caused a dose-dependent depression of the Ca²⁺ transient. However, a relationship between reductions in peak twitch forces and corresponding peak [Ca²⁺]_i transients could not be made, as we have done, because isolated myocytes were used. Nevertheless, significant depressions of the [Ca²⁺]_i signals were observed only at higher dose levels of halothane (28), similar to what we report here. These results do not seem to be completely supportive of those mentioned above for papillary muscles to establish that halothane produces a strictly parallel dose-dependent depression of both twitch peak force and peak [Ca²⁺]_i transient. By comparing our results to these, and to those above, the general conclusion can be drawn that halothane depresses peak twitch force to a greater degree than it does the corresponding peak [Ca²⁺]_i transient. It is unlikely that fura 2 is not able to report the actual reduction in the peak [Ca²⁺]_i caused by halothane, since it reported the reduction in the [Ca²⁺]_i transient caused by lowering [Ca²⁺]_o, as shown in Fig. 3.

Regarding the influence of halothane on cardiac SR function, previous results indicate that a vapor agent such as halothane reduces the SR Ca²⁺ content, possibly by making this organelle leaky to Ca²⁺ (17). Under our conditions, contracture force and particularly the [Ca²⁺]_i response to an RC were monitored. Our results suggest that SR Ca²⁺ still remains near 80% of the control value, even when the peak twitch force is depressed to 20% of the control level by the high dose of halothane. This implies that SR Ca²⁺ content should be more than 80% of the control value at moderate and low doses of halothane even though the peak twitch forces are reduced by ~40 and 60% of the control values, respectively. However, if the contracture force, not the [Ca²⁺]_i response, is used as an indicator of the amount of Ca²⁺ released from the SR, the reduction of SR Ca²⁺ content caused by halothane would be 47% (Fig. 5B), instead of 21% estimated by the change in [Ca²⁺]_i (Fig. 5A). Apparently, the reduction of contracture force induced by an RC results from a decrease in both [Ca²⁺]_i and responsiveness of the contractile system to [Ca²⁺]_i. The [Ca²⁺]_i response to an RC is a more accurate indicator for determination of SR Ca²⁺ content when Ca²⁺ sensitivity of the contractile system is altered, as seems to be the case for halothane. The true reduction of SR Ca²⁺ content caused by halothane may not be as much as that previously reported in which contracture force was used as the indicator (17).

We have not yet investigated the mechanism by which halothane affects SR Ca²⁺ movements. Our observations indicate that halothane (0.55 mM equivalent to ~2 MAC) reduces by a moderate extent (20%) SR Ca²⁺ content. However, it seems that halothane at concentrations near 2 MAC does not make the SR leaky enough to markedly reduce its Ca²⁺ content. The supporting evidence is that the diastolic [Ca²⁺]_i level was not elevated during the time-dependent early phase of halothane application, as well as during the steady state of halothane effect. An elevation of diastolic [Ca²⁺]_i was observed, but only at very high doses of halothane (1.4 mM, data not shown). We conclude that at concentrations equivalent to or less than 2 MAC, halothane does not make the SR very leaky, or, if it does, this may be compensated for by SR Ca²⁺ sequestration. A reduction in SR Ca²⁺ content probably is not the main cause of the depression of twitch force caused by halothane at or below 2 MAC.

The small reduction in both SR Ca²⁺ content and in the peak [Ca²⁺]_i transient caused by the low dose of halothane, which caused a large decrease in peak twitch force, suggests that here the negative inotropic effect of halothane may be mainly through reduction of the responsiveness to Ca²⁺ of the contractile system. This seems similar to the way BDM acts, since BDM, in the dose range up to 10 mM, appears to slow the cross-bridge cycling rate (2). This was tested by comparison of the dynamic, i.e., not in steady state, force-[Ca²⁺]_i relationship at different doses of halothane with those of BDM. Prior work (2) has shown that in concentrations up to 10 mM BDM produces very little effect on the [Ca²⁺]_i transient while profoundly depressing the peak twitch force in rat trabeculae, and our results are in agreement with this. We are also in agreement with this earlier work concerning the lack of any effect of BDM on diastolic [Ca²⁺]_i in this dose range. It should be noted that there is no evidence indicating that BDM influences binding of Ca²⁺ to troponin C (13, 29). Since the dynamic force-[Ca²⁺]_i relationship generated with varying doses of halothane resembles that of BDM, particularly at low doses, the negative inotropic effect of halothane in this dose range may also be mainly through a similar kind of slowing of cross-bridge cycling rate manifested as an apparent reduction of the responsiveness of the contractile system to [Ca²⁺]_i.

The amplitude of the L-type Ca²⁺ current (I_Ca) depends on the level of [Ca²⁺]_o, and if the I_Ca is abolished there is no force generation (21). However, the peak [Ca²⁺]_i transient is not related directly to peak I_Ca (6), although the I_Ca and force generation have a common voltage threshold (3). It seems the I_Ca acts as a trigger to cause a much amplified release of Ca²⁺ from the SR, and in this way enough Ca²⁺ is made available to both fill intracellular buffers and cause force generation (12). In the chain [Ca²⁺]_o  I_Ca  SR Ca²⁺ release  force generation, each element must have acted in a proportional linear manner. Reduction of the peak twitch force should be proportional to that of the peak [Ca²⁺]_i transient when [Ca²⁺]_o is lowered, as shown in Fig. 3, presumably without a significant alteration of the Ca²⁺ sensitivity of the contactile system. If an agent such as halothane acts mainly by producing a dose-dependent inhibition of I_Ca with consequent reduction of the [Ca²⁺]_i transient, as decreasing [Ca²⁺]_o does, then the dynamic force-[Ca²⁺]_i relationship generated by varying [Ca²⁺]_o should resemble that produced by halothane. The results shown in Fig. 3 clearly show that the halothane responses are well separated from the low [Ca²⁺]_o ones, and this leads us to believe that the primary target for halothane action, particularly at low and moderate doses, is not on I_Ca and the [Ca²⁺]_i transient. Rather, at these dose levels, halothane exerts negative inotropic action mainly through a reduction in the responsiveness of the contractile system to [Ca²⁺]_i.

The above could be tested by comparing the force-[Ca²⁺]_i relationship in the absence and presence of halothane in a tetanized condition, as was done for BDM (2). BDM depresses force generation by decreasing cross-bridge cycling rate, and it depresses peak twitch force much more than it does the maximal force during a tetanus (2). This is also true for halothane, since halothane has a much more depressant effect on twitch force than it does on maximally activated tetanic force (4). Determination of halothane effects on the force-[Ca²⁺]_i relationship in the steady state, e.g., during a tetanus, would yield additional information; however, the effects of halothane on the dynamic force-[Ca²⁺]_i relationship during twitches may more closely resemble the physiological condition.