Effect of halothane on intracellular calcium oscillations in porcine tracheal smooth muscle cells

Christina M. Pabelick1, Y. S. Prakash1, Mathur S. Kannan2, Keith A. Jones1, David O. Warner1,3, and Gary C. Sieck1,3

Departments of 1 Anesthesiology, and 3 Physiology and Biophysics, Mayo Clinic and Foundation, Rochester 55905; and 2 Department of Veterinary PathoBiology, University of Minnesota, St. Paul, Minnesota 55108

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The effect of halothane on intracellular Ca2+ concentration ([Ca2+]i) regulation in porcine tracheal smooth muscle cells was examined with real-time confocal microscopy. Both 1 and 2 minimum alveolar concentration (MAC) halothane increased basal [Ca2+]i when Ca2+ influx and efflux were blocked, suggesting increased sarcoplasmic reticulum (SR) Ca2+ leak and/or decreased reuptake. In beta -escin-permeabilized cells, heparin inhibition of inositol 1,4,5-trisphosphate-receptor channels blunted the halothane-induced increase in [Ca2+]i. Both 1 and 2 MAC halothane decreased the frequency and amplitude of ACh-induced [Ca2+]i oscillations (which represent SR Ca2+ release through ryanodine-receptor channels), abolishing oscillations in ~20% of tracheal smooth muscle cells at 2 MAC. When Ca2+ influx and efflux were blocked, halothane increased the baseline and decreased the frequency and amplitude of [Ca2+]i oscillations, inhibiting oscillations in ~70% of cells at 2 MAC. The fall time of [Ca2+]i oscillations and the rate of fall of the [Ca2+]i response to caffeine were both increased by halothane. These results suggest that halothane abolishes agonist-induced [Ca2+]i oscillations by 1) depleting SR Ca2+ via increased Ca2+ leak through inositol 1,4,5-trisphosphate-receptor channels, 2) decreasing Ca2+ release through ryanodine-receptor channels, and 3) inhibiting reuptake.

airway; volatile anesthetic; muscarinic receptor; sarcoplasmic reticulum

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

VOLATILE ANESTHETICS such as halothane are potent bronchodilators, acting in part by directly depressing smooth muscle contractility (3). Elevation of free intracellular Ca2+ concentration ([Ca2+]i) plays an important role in the development and maintenance of force in tracheal smooth muscle (TSM) cells in response to agonists such as acetylcholine (ACh) (29). Halothane has been shown to reduce ACh-induced elevation in [Ca2+]i in TSM at clinically relevant concentrations (16, 17, 36). This reduction in [Ca2+]i can be explained, in part, by an inhibitory effect of halothane on Ca2+ influx (32, 35, 36). However, halothane has been shown to additionally increase Ca2+ release from the sarcoplasmic reticulum (SR), referred to as SR Ca2+ "leak," in both cardiac tissue (5) and vascular smooth muscle (36). Studies in other cell types demonstrated that anesthetic-induced SR Ca2+ leak occurs through both inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] (14) and ryanodine-receptor (RyR) channels (22).

In TSM cells, elevation of [Ca2+]i by activation of muscarinic receptors is mediated by both extracellular Ca2+ influx through either voltage-operated or receptor-operated Ca2+ channels and Ca2+ release from the SR through Ins(1,4,5)P3-receptor channels (4). More recently, it has been demonstrated that, in TSM cells, ACh induces SR Ca2+ release through RyR channels (18, 27). Furthermore, it has been suggested that cADP-ribose acts as a second messenger for SR Ca2+ release through RyR channels (26). ACh induces [Ca2+]i oscillations in porcine TSM cells (18, 21, 25, 27) and guinea pig airway smooth muscle (28). Previous studies by our laboratory (18, 25, 27) demonstrated that ACh-induced [Ca2+]i oscillations in TSM cells reflect SR Ca2+ release through RyR channels but require Ins(1,4,5)P3-induced SR Ca2+ release, at least to initiate the oscillations. In this conceptual framework, [Ca2+]i oscillations occur through a Ca2+-induced Ca2+ release (CICR) mechanism where SR Ca2+ content is reflected by the constant amplitude of the oscillations and the sensitivity for CICR is reflected by frequency and propagation velocity of the oscillations.

In the present study, we examined the effect of halothane on ACh-induced [Ca2+]i oscillations in freshly dissociated porcine TSM cells. We hypothesized that halothane inhibits ACh-induced [Ca2+]i oscillations by decreasing SR Ca2+ content via enhanced SR Ca2+ leak and decreased Ca2+ reuptake. Accordingly, the experimental protocols focused only on SR Ca2+ release and reuptake. To remove the confounding effects of halothane on Ca2+ influx and efflux, two TSM cell preparations were used: 1) intact cells where Ca2+ influx and efflux were blocked and 2) beta -escin-permeabilized cells.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell Preparation and Fluo 3 Loading

Porcine tracheae were obtained from a local abattoir. The smooth muscle layer was dissected, and TSM cells were isolated with previously described techniques (18, 25, 27). Briefly, the smooth muscle layer was excised, freed of endothelium, and minced thoroughly in Hanks' balanced salt solution (HBSS) buffered with 10 mM HEPES (pH 7.4; Life Technologies). The tissue was incubated for 2 h with 20 U/ml of papain and 2,000 U/ml of DNase (Worthington Biochemical) and subsequently for 1 h at 37°C with 1 mg/ml of type IV collagenase (Worthington Biochemical). The dissociated cells were then gently triturated, centrifuged, and resuspended in minimum essential medium (MEM) with 10% fetal calf serum. The isolated cells were plated on collagen-coated glass coverslips and incubated for 1-2 h in 5% CO2 at 37°C. Exclusion of trypan blue dye was used to assess cell viability (>90% of all cells).

After incubation in MEM, each coverslip was washed with HBSS and incubated for 30-45 min at 37°C in HBSS containing 5 µM fluo 3-AM (Molecular Probes). The coverslip was then washed in HBSS and mounted on an open slide chamber (RC-25F, Warner Instruments) that was perfused at 2-3 ml/min and maintained at room temperature.

Real-Time Confocal Imaging

The technique for real-time confocal imaging of TSM cells has been previously published (18, 25, 27). An Odyssey XL real-time confocal system (Noran Instruments) equipped with an Ar-Kr laser and attached to a Nikon Diaphot microscope was used to visualize fluo 3-loaded TSM cells. The confocal system was controlled through a Silicon Graphics Indy workstation. A Nikon ×40/1.3 oil-immersion objective lens was used to visualize the cells. Image size was set to 640 × 480 pixels, and pixel area was calibrated with a stage micrometer (0.063 µm2/pixel). The optical section thickness for the ×40 lens was set to 1 µm by controlling the slit size on the Odyssey system. The 488-nm laser line was used to excite fluo 3 for Ca2+ imaging, and emissions were collected with a 515-nm long-pass filter and a high-sensitivity photomultiplier tube. Based on previous studies (18, 25, 27), a fixed combination of laser intensity (20% of maximum) and photomultiplier gain (1700 from a maximum of 4096) was used to ensure that pixel intensities within regions of interest (ROIs) ranged between 25 and 255 gray levels (GLs). At these settings, laser power output varied <1% across a 15-min period, and intermittent laser exposure of fluo 3 at 3 mW (<5 min) caused little detectable photobleaching (<1%).

Up to three ROIs were defined within the boundaries of individual cells. The distance between ROIs was measured and ranged between 3 and 10 µm. Propagation velocity was measured by dividing the distance between ROIs by the time difference in the peak of oscillations within each ROI.

Ca2+ Calibrations

The technique for quantifying [Ca2+]i levels from fluo 3 intensity levels has been previously described (25). Even though fluo 3 is not a ratiometric dye, [Ca2+]i can be quantified by mapping fluorescence intensity to known Ca2+ levels. A confounding factor with laser-based microscopy is that both laser intensity and photomultiplier gain can be independently varied. However, in the present study, this was not a problem because all experiments were performed at a fixed setting of laser intensity and photomultiplier gain. To calibrate fluo 3 fluorescence intensity in TSM cells to known [Ca2+]i, the cells were exposed to 10 µM A-23187 at varying levels of extracellular Ca2+ ranging from 0 (buffered with EGTA) to 1 µM. The steady-state GL values of fluo 3 fluorescence intensities were recorded, and a calibration curve relating GL to [Ca2+]i was constructed. With this calibration, the theoretical resolution for [Ca2+]i measurements was ~4 nM. However, various sources of noise such as photomultiplier output and extraneous light limited the actual resolution to ~10 nM.

Administration of Halothane

Halothane (Wyeth-Ayerst Laboratories) was added to the aerating gas mixture via a calibrated on-line vaporizer. The vaporizer was set to produce aqueous concentrations of halothane in HBSS equivalent to 1 and 2 minimum alveolar concentration (MAC) at room temperature, concentrations well within the range of clinical usage. Aqueous concentrations of halothane in the perfusion chamber were determined by gas chromatography from anerobically obtained samples with an electron capture detector (Hewlett-Packard 5880A) (31). In the solutions used to examine the effects of halothane on ACh-induced [Ca2+]i oscillations, concentrations were 0.22 ± 0.02 mM for 1 MAC and 0.48 ± 0.04 mM for 2 MAC halothane.

Experimental Protocols

Effect of halothane on SR Ca2+ release. The effect of halothane on SR Ca2+ release was first examined in the absence of ACh stimulation. Intact TSM cells were permeabilized with 25 µM beta -escin (Sigma) in a pCa 9.0 solution (6) for 60-65 s. In a previous study, Kannan et al. (18) determined that the duration of exposure to beta -escin was critical in ensuring adequate retention of fluo 3 and the ability to elicit [Ca2+]i responses. After permeabilization, the cells were immediately washed at pCa 9.0 for 2 min. The SR was then loaded by incubating the cells for 10 min at pCa 7.0.

After permeabilization and SR loading, the cells were exposed to pCa 7.0 bubbled with 1 or 2 MAC halothane for 2 min, and the change in [Ca2+]i was measured. The cells were then washed at pCa 7.0 for 15 min and reexposed to halothane to determine the reproducibility of the [Ca2+]i response. In separate experiments, cells exposed only at pCa 7.0 for a total of 19 min served as controls (n = 7).

In a second set of experiments, permeabilized cells with loaded SR were exposed to 1 or 2 MAC halothane, washed at pCa 7.0, and incubated in 0.5 mg/ml of heparin (mol wt 6,000; Sigma), an inhibitor of Ins(1,4,5)P3-receptor channels. In a previous study, Kannan et al. (18) demonstrated that this heparin concentration completely blocks [Ca2+]i responses to Ins(1,4,5)P3 in porcine TSM cells. After incubation with heparin, the cells were reexposed to halothane in the continued presence of heparin.

Effect of halothane on SR Ca2+ reuptake. The effect of halothane on SR Ca2+ reuptake in the absence of ACh stimulation was examined with the [Ca2+]i response of intact TSM cells to 5 mM caffeine. The cells were exposed to 5 mM caffeine, washed for 15 min with HBSS and 1 or 2 MAC halothane, and finally reexposed to caffeine. In control experiments, the cells were exposed two times to caffeine with no halothane present. The rate of fall in [Ca2+]i for 500 ms just after the peak caffeine response was used as an index of SR Ca2+ reuptake. The peak of the [Ca2+]i response represents the point at which caffeine has completely depleted SR Ca2+ stores. Accordingly, the rate of fall in [Ca2+]i at this point is an index of the maximum rate of SR Ca2+ reuptake. The effects of halothane on SR Ca2+ reuptake can then be examined at the peak of the caffeine response without the confounding effect of halothane on SR Ca2+ leak.

To distinguish between the effects of halothane on SR Ca2+ release versus reuptake, intact TSM cells were first exposed to zero extracellular Ca2+ and lanthanum to block Ca2+ influx and efflux. The cells were then exposed to 10 µM cyclopiazonic acid (CPA) to additionally inhibit SR Ca2+ ATPase. In the continued presence of zero extracellular Ca2+, lanthanum, and CPA, the cells were exposed to 1 or 2 MAC halothane.

To further confirm that halothane increased [Ca2+]i through Ca2+ release via Ins(1,4,5)P3-receptor channels and not exclusively by slowing reuptake, experiments were performed in permeabilized TSM cells. The permeabilized cells (at pCa 7.0) were first exposed to 10 µM CPA for 10 min to block SR Ca2+ reuptake. In the continued presence of CPA, the cells were superfused with 1 or 2 MAC halothane. The cells were then washed at pCa 7.0 to remove both CPA and halothane. Subsequently, the cells were incubated in CPA and 0.5 mg/ml of heparin to additionally block Ins(1,4,5)P3-receptor channels. In the continued presence of CPA and heparin, the cells were reexposed to halothane. In control experiments, the cells were not exposed to heparin.

Effect of halothane on ACh-induced [Ca2+]i oscillations. The effects of 1 and 2 MAC halothane on ACh-induced [Ca2+]i oscillations were studied in TSM cells in the presence of 2.5 mM extracellular Ca2+. A fixed concentration of 1 µM ACh was used for all experiments, corresponding to the concentration used in previous studies by our laboratory (18, 25, 27). The steady-state [Ca2+]i response to ACh was determined after 2 min, and after 3 min, the cells were perfused with a solution containing 1 µM ACh plus halothane. Subsequently, the effects of halothane on the ACh-induced [Ca2+]i response were evaluated between 7 and 8 min after the initial exposure to ACh. Thereafter, exposure to halothane was stopped, and the [Ca2+]i response to ACh alone was reevaluated after a 5-min washout period. Thus the effects of halothane on ACh-induced [Ca2+]i oscillations were compared for the same cell before and after exposure to halothane.

In a separate set of cells, the stability of ACh-induced [Ca2+]i oscillations was evaluated over a 15-min period (total duration of the halothane protocols). Measurements were made at three different time points corresponding to the major events in the halothane protocols: at 2 min, corresponding to the time when control values (preexposure to halothane) were obtained; between 7 and 8 min, corresponding to the time when the effects of halothane were assessed; and at 15 min, corresponding to the time when halothane washout was evaluated.

On the basis of our conceptual framework, [Ca2+]i oscillations occur via repetitive SR Ca2+ release and reuptake, and oscillation frequency and propagation velocity reflect CICR sensitivity. Accordingly, to distinguish between the effects of halothane on the frequency and propagation velocity of [Ca2+]i oscillations due to inhibition of SR Ca2+ reuptake, separate studies were performed where reuptake was inhibited with CPA. During ongoing [Ca2+]i oscillations, intact TSM cells were exposed to 10 µM CPA, and the changes in frequency and propagation velocity were measured at 30 s after the introduction of CPA.

In each of the above experiments, the baseline [Ca2+]i, oscillation amplitude, fall time, frequency, and propagation velocity were measured. Oscillation amplitude was used as an index of SR Ca2+ content, fall time as an index of SR Ca2+ reuptake, and frequency and propagation velocity as indexes of CICR sensitivity.

Effect of halothane on [Ca2+]i during inhibition of Ca2+ influx and efflux. The effects of 1 and 2 MAC halothane were studied under conditions where Ca2+ influx and efflux were inhibited by using HBSS solutions that were nominally Ca2+ free and contained 0.1 µM nifedipine and 1 mM lanthanum (25). The combination of Ca2+-free HBSS and nifedipine ensured inhibition of Ca2+ influx through L-type channels as well as through receptor-operated and nonspecific cation channels. In addition to blocking Ca2+ efflux, lanthanum also nonspecifically inhibited Ca2+ influx. The steady-state [Ca2+]i response to 1 µM ACh was determined after 2 min, after which the cells were exposed to solutions containing 1 µM ACh plus either 1 or 2 MAC halothane. The effects of halothane on ACh-induced [Ca2+]i oscillations were evaluated between 7 and 8 min after the initial exposure to ACh.

Statistical Analysis

It was not possible to apply all the experimental protocols to every cell or to cells obtained from every animal. However, in all cases, the results were replicated in cells obtained from more than 1 animal (10 animals for each of the halothane protocols). In each experiment, at least three and up to five cells were analyzed from each coverslip. This allowed an assessment of the variability of [Ca2+]i responses across cells that had undergone similar experimental conditions (e.g., dissociation, fluo 3 loading, potential photobleaching). The variability was assessed by calculating the coefficient of variation of different [Ca2+]i oscillation parameters for cells within a coverslip and across coverslips from the same animal, as well as for cells obtained from different animals, 2 min after exposure to ACh alone.

Resting [Ca2+]i levels (before exposure to ACh) were randomly checked and varied from 100 to 125 nM. Comparisons before and after exposure to halothane for a given cell were made with a Wilcoxon signed rank test. In TSM cells where 2 MAC halothane inhibited ongoing [Ca2+]i oscillations, statistical comparisons were made for all cells concerning baseline [Ca2+]i levels. When comparing the effect of 2 MAC halothane on amplitude, fall time, frequency, and propagation velocity of ACh-induced [Ca2+]i oscillations, the statistics were done on the remaining cells where [Ca2+]i oscillations still occurred. Multiple comparisons were performed with Friedman repeated-measures ANOVA on ranks, with post hoc testing with a Wilcoxon signed rank test. Values are reported as means ± SE. In all studies, n is the number of cells. Significance was tested at the P < 0.05 level.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effect of Halothane on SR Ca2+ Release

In beta -escin-permeabilized TSM cells, both 1 (n = 10) and 2 (n = 9) MAC halothane produced transient [Ca2+]i responses that were reproducible with repeated exposures (Fig. 1). The amplitude of these [Ca2+]i responses could not be measured due to fluo 3 leakage in the permeabilized cells. The duration of these transients (measured from the initiation of the response to the return to baseline) was 23 ± 5 s for 1 MAC and 32 ± 5 s for 2 MAC. Qualitative observation suggested no obvious temporal delays in the [Ca2+]i response between different ROIs within a cell. Instead, the entire cell displayed a more or less synchronized elevation in [Ca2+]i followed by a slow decay to basal levels. In control cells (n = 7) exposed only to pCa 7.0 for a total of 19 min, no spontaneous transient [Ca2+]i responses were observed. Inhibition of Ins(1,4,5)P3-receptor channels with heparin significantly blunted the subsequent [Ca2+]i responses to both 1 (n = 6) and 2 (n = 6) MAC halothane, with the effect being greater at 1 MAC (88.3 ± 8.4% for 1 MAC and 68.3 ± 7.1% for 2 MAC; Fig. 1).


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Fig. 1.   Effect of halothane on sarcoplasmic reticulum (SR) of tracheal smooth muscle (TSM) cells. In beta -escin-permeabilized cells, exposure to 1 (A) and 2 (B) minimum alveolar concentration (MAC) halothane induced reproducible, transient SR Ca2+ release. Preexposure to heparin substantially blunted halothane-induced SR Ca2+ release (C). Heparin completely inhibited intracellular Ca2+ concentration ([Ca2+]i) response to 1 MAC halothane (data not shown). These results suggest that halothane induces SR Ca2+ release through inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-receptor channels.

Effect of Halothane on SR Ca2+ Reuptake

In intact TSM cells, 5 mM caffeine produced a large transient [Ca2+]i response. Exposure to both 1 (n = 9) and 2 (n = 8) MAC halothane decreased the peak of the [Ca2+]i response and significantly slowed the initial rate of fall of the [Ca2+]i profile. Accordingly, the rate of SR Ca2+ reuptake (normalized for amplitude) was significantly slower with both 1 and 2 MAC halothane, with the effect being greater at 2 MAC (Fig. 2). In control cells (n = 8), the [Ca2+]i response to two caffeine exposures was comparable.


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Fig. 2.   Effect of halothane on SR Ca2+ reuptake. Exposure to 1 and 2 MAC halothane significantly slowed initial rate of fall (1st 500 ms) of [Ca2+]i response to 5 mM caffeine. Because caffeine depletes SR Ca2+ stores, initial rate of fall is indicative of rate of Ca2+ reuptake by SR.

In intact TSM cells exposed to zero extracellular Ca2+, lanthanum, and CPA, exposure to both 1 (n = 6) and 2 (n = 7) MAC halothane induced a sustained elevation in [Ca2+]i (Fig. 3A). In permeabilized cells exposed to CPA, both 1 (n = 7) and 2 (n = 8) MAC halothane induced an elevation in [Ca2+]i (Fig. 3B). After washout and reintroduction of CPA, a similar [Ca2+]i response was observed in control cells. Heparin significantly blunted the elevation in [Ca2+]i in the presence of CPA (Fig. 3C).


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Fig. 3.   Effects of halothane on SR Ca2+ release under conditions where Ca2+ reuptake was blocked. In intact TSM cells when Ca2+ influx and efflux were blocked with Ca2+-free Hanks' balanced salt solution (HBSS) and La3+ (A), exposure to 10 µM cyclopiazonic acid (CPA) resulted in a gradual increase in [Ca2+]i. Under these conditions, 2 MAC halothane still induced an elevation in [Ca2+]i, suggesting an effect on SR Ca2+ release. In permeabilized TSM cells, a similar effect of halothane on [Ca2+]i was observed in presence of CPA, which was reproducible when CPA was washed out to allow refilling of SR and then reintroduced (B). However, inhibition of Ins(1,4,5)P3-receptor channels by heparin blocked halothane-induced elevation in [Ca2+]i in presence of CPA (C).

Effect of Halothane on ACh-Induced [Ca2+]i Oscillations

As previously reported (18, 21, 25, 27), ACh induced [Ca2+]i oscillations in porcine TSM cells. These ACh-induced [Ca2+]i oscillations displayed a biphasic pattern, with an initial period of more rapid oscillations and elevated baseline [Ca2+]i, followed by a prolonged steady-state period of slower oscillations and stable lower baseline [Ca2+]i (Fig. 4). Once a steady state was reached, there was very little change in ACh-induced [Ca2+]i oscillations with time (Fig. 4).


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Fig. 4.   ACh-induced [Ca2+]i oscillations in TSM cells. Oscillations show a biphasic pattern, with an initial period of rapid oscillations of smaller amplitude followed by a sustained phase of slower oscillations of larger amplitude. Times corresponding to measurements are shown.

The coefficients of variation for the cellular responses within a single coverslip were 40.3 ± 3.4% for the steady-state (measured 2 min after ACh exposure) baseline [Ca2+]i, 60.5 ± 3.3% for oscillation amplitude, 10.1 ± 1.0% for oscillation frequency, and 14.2 ± 1.3% for propagation velocity (n = 6). The coefficients of variation for the cellular responses within a single trachea were 33.6 ± 2.1, 45.5 ± 3.3, 15.3 ± 1.4, and 18.3 ± 1.4% for these parameters, respectively (n = 6). Furthermore, the coefficients of variation for cells obtained across all animals were 24.6 ± 2.2, 45.3 ± 3.0, 15.1 ± 1.0, and 21.4 ± 2.3%, respectively (n = 88). Because the distribution of these parameters was not significantly different, data from different coverslips and from different animals were pooled for statistical analyses.

At 2 min after ACh exposure, the average baseline [Ca2+]i level was 223 ± 8 nM, the average oscillation amplitude was 531 ± 35 nM, the average oscillation frequency was 9.0 ± 0.2/min, and the average propagation velocity was 27.3 ± 2.4 µm/s (n = 11). Across the 15-min period evaluated, these parameters did not change significantly. Compared with steady-state values at 2 min after ACh exposure, baseline [Ca2+]i levels increased by 5.5 ± 4.9% after 6 min and by 10.7 ± 7.3% after 15 min. The oscillation amplitude decreased by 6.0 ± 2.6% after 6 min and by 5.4 ± 4.0% after 15 min compared with the oscillation amplitude at 2 min. The oscillation frequency increased by 1.6 ± 4.3% after 6 min and decreased by 7.5 ± 1.3% after 15 min compared with the values at 2 min. The propagation velocity increased by 5.1 ± 3.1% after 6 min and decreased by 11.4 ± 2.4% after 15 min.

In TSM cells exposed to 2.5 mM extracellular Ca2+, halothane did not significantly change basal [Ca2+]i levels at 1 (5.1 ± 3.0%; n = 21) or 2 (7.0 ± 3.1%; n = 23) MAC in comparison to basal [Ca2+]i values before halothane exposure (control values). During exposure to 1 MAC halothane, the amplitude of ACh-induced [Ca2+]i oscillations was significantly reduced by 37.2 ± 3.1% (P < 0.01; Fig. 5). In a subset of the cells under this protocol, where exposure to 1 MAC halothane was maintained for up to 10 min, halothane did not cause complete inhibition of ACh-induced [Ca2+]i oscillations. Exposure to 2 MAC halothane decreased the amplitude of ACh-induced [Ca2+]i oscillations by 25.0 ± 4.1% (P < 0.01). In 17% of the cells studied, ongoing [Ca2+]i oscillations were abolished.


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Fig. 5.   Effect of 1 (A) and 2 (B) MAC halothane on ACh-induced [Ca2+]i oscillations. Exposure to halothane caused a decrease in amplitude and frequency of oscillations without a significant change in baseline [Ca2+]i level. Exposure to 2 MAC halothane inhibited ongoing [Ca2+]i oscillations in 17% of cells. Baseline [Ca2+]i levels during ACh exposure alone were 240 nM in A and 230 nM in B.

Exposure to 1 MAC halothane decreased the frequency of ACh-induced [Ca2+]i oscillations by 21.5 ± 3.1% (P < 0.01). Propagation velocity was reduced by 38.4 ± 3.1% (P < 0.01). In cells where the oscillations were not completely abolished, exposure to 2 MAC halothane decreased the frequency of ACh-induced [Ca2+]i oscillations from 9.3 ± 0.6 (control) to 6.6 ± 0.5/min (halothane; P < 0.01). The propagation velocity was reduced by 63.5 ± 3.8% (P < 0.01).

In intact TSM cells exposed to 10 µM CPA (n = 8), both oscillation frequency and propagation velocity were significantly increased at 30 s after the introduction of CPA (39 ± 5 and 27 ± 4%, respectively). With continued CPA exposure, [Ca2+]i levels increased such that oscillations were no longer observed.

Effect of Halothane on [Ca2+]i During Inhibition of Ca2+ Influx and Efflux

In contrast to maintained Ca2+ influx and efflux (see Effect of Halothane on Ach-Induced [Ca2+]i Oscillations), when Ca2+ influx and efflux were inhibited by using extracellular free Ca2+ HBSS, nifedipine and lanthanum, both 1 (n = 17) and 2 (n = 16) MAC halothane significantly increased basal [Ca2+]i (1 MAC: 17.1 ± 3.0%, P < 0.01; 2 MAC: 36.4 ± 9.2%, P < 0.01; Fig. 6). The presence or absence of Ca2+ influx and efflux did not have a significant effect on basal [Ca2+]i level before halothane exposure (223 ± 8 nM under conditions of intact Ca2+ influx and efflux vs. 248 ± 7 nM under conditions of inhibited influx and efflux).


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Fig. 6.   Effect of 1 (A) and 2 (B) MAC halothane on ACh-induced [Ca2+]i oscillations in nominally Ca2+-free HBSS in presence of La3+. Exposure to halothane caused a significant increase in baseline [Ca2+]i level and reduced amplitude and frequency of oscillations. In 69% of cells exposed to 2 MAC halothane, [Ca2+]i oscillations were completely abolished. Baseline [Ca2+]i levels during ACh exposure alone were 260 nM in A and 240 nM in B.

As previously shown (25), under conditions where Ca2+ influx and efflux were inhibited, ACh-induced [Ca2+]i oscillations continued without significant changes in baseline [Ca2+]i level, oscillation amplitude, frequency, or propagation velocity over at least a 15-min period. During exposure to 1 MAC halothane, the amplitude of ACh-induced [Ca2+]i oscillations was significantly reduced by 45.0 ± 5.1% (P < 0.01; Fig. 6). Continued exposure to 1 MAC halothane did not cause complete inhibition of ACh-induced [Ca2+]i oscillations. Exposure to 2 MAC halothane abolished the oscillations in ~70% of the cells. In cells where the oscillations were not completely abolished, exposure to 2 MAC halothane decreased the amplitude of [Ca2+]i oscillations by 37.3 ± 5.0% (P < 0.01; Fig. 6).

Exposure to 1 MAC halothane decreased the frequency of ACh-induced [Ca2+]i oscillations by 27.1 ± 5.2% (P < 0.01). The propagation velocity was reduced by 44.2 ± 3.3% (P < 0.01). For the five cells where [Ca2+]i oscillations persisted, exposure to 2 MAC halothane did not significantly change oscillation frequency [4.9 ± 0.4 (control) to 3.6 ± 0.2/min (halothane)]. The propagation velocity was also unaffected in these cells by 2 MAC halothane.

In the absence of halothane, the fall time of [Ca2+]i oscillations (normalized for oscillation amplitude) under conditions of blocked Ca2+ influx and efflux averaged 5.1 ± 0.04 nM/s. Exposure to 1 MAC halothane increased the fall time by 110 ± 15%, whereas exposure to 2 MAC halothane increased the fall time by 97 ± 11% in cells where oscillations were not completely inhibited (P < 0.05; Fig. 7).


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Fig. 7.   Effect of halothane on fall time of ACh-induced [Ca2+]i oscillations. Fall time, normalized for oscillation amplitude, represents rate of SR Ca2+ reuptake. Exposure to 1 and 2 MAC halothane significantly slowed fall time, indicating inhibition of SR Ca2+ ATPase.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

The results of the present study demonstrate that clinically relevant concentrations of halothane affect [Ca2+]i in TSM cells by decreasing SR Ca2+ content and reducing the sensitivity for CICR. In the absence of agonist stimulation, both 1 and 2 MAC halothane increased SR Ca2+ release through Ins(1,4,5)P3-receptor channels. The substantial increase in the fall time of ACh-induced [Ca2+]i oscillations under conditions of blocked Ca2+ influx and efflux, as well as the initial rate of fall in the [Ca2+]i response to caffeine, indicates that SR Ca2+ reuptake is slowed, additionally contributing to the reduction in SR Ca2+ content. These halothane-induced decreases in SR Ca2+ content were reflected by a decreased amplitude of [Ca2+]i oscillations under conditions of intact as well as blocked Ca2+ influx and efflux. In keeping with our conceptual framework for ACh-induced [Ca2+]i oscillations in TSM cells, the reduction in the frequency and propagation velocity of [Ca2+]i oscillations suggests that halothane decreases CICR sensitivity.

In the present study, we used freshly dissociated TSM cells to examine the effects of halothane on [Ca2+]i oscillations. A potential limitation of this technique is that variations in dissociation and fluo 3 loading and inherent cellular differences may introduce variability in the observed [Ca2+]i responses between cells obtained from the same animal or from different animals. However, because there were no significant differences in the coefficients of variation of [Ca2+]i responses of TSM cells within or across animals and, in the experimental design, each cell served as its own control, cellular variability was not an issue.

Agonist-induced oscillations have been observed in a variety of other smooth muscle types including vascular (2, 11, 15), colonic (23), and uterine (19) smooth muscles. [Ca2+]i oscillations may involve different mechanisms such as SR Ca2+ release and reuptake and/or cyclical Ca2+ influx (see Ref. 1 for a review). Recent studies by our laboratory (18, 27) in porcine TSM cells have demonstrated that ACh-induced [Ca2+]i oscillations reflect an all-or-none release of SR Ca2+ through RyR channels but require SR Ca2+ release through Ins(1,4,5)P3-receptor channels for initiation. Although a number of studies (20, 24) have attributed the steady-state phase of the ACh-induced [Ca2+]i response to Ca2+ influx, our studies in porcine TSM demonstrate that even during the steady-state phase, [Ca2+]i oscillations represent repetitive SR Ca2+ release and reuptake. Ca2+ influx appears to be essential for the replenishment of SR Ca2+ stores because inhibition of Ca2+ influx leads to an eventual blockade of oscillations but does not prevent initiation of oscillations (25). However, under conditions where Ca2+ influx and efflux are inhibited, [Ca2+]i oscillations, once initiated, continue indefinitely (25), again demonstrating that the oscillations arise from SR Ca2+ release even during the steady-state phase. The results of the present study are consistent with these previous observations.

Based on the results of previous studies by our laboratory (18, 25, 27), we propose a conceptual framework (Fig. 8A) where 1) the baseline [Ca2+]i level represents a balance between Ca2+ influx and efflux across the cell membrane and SR Ca2+ release and reuptake, 2) the oscillation amplitude represents the size of the SR Ca2+ pool (SR Ca2+ content), and 3) the oscillation frequency and propagation velocity reflect the sensitivity for SR Ca2+ release through RyR channels (CICR sensitivity). This framework facilitates the identification of potential mechanisms by which halothane modulates [Ca2+]i oscillations in TSM cells and decreases [Ca2+]i (Fig. 8B). The present study focuses only on halothane effects at the level of the SR while recognizing that additional effects on mechanisms such as Ca2+ influx and efflux are entirely possible. Indeed, there is already considerable evidence in the literature that the decrease in [Ca2+]i by halothane involves inhibition of Ca2+ influx (16, 17, 36) through voltage-gated L-type Ca2+ channels (35, 36). The little information on volatile anesthetic effects on Ca2+ efflux in brain tissues and cardiac muscle suggests that anesthetics actually inhibit plasma membrane Ca2+-ATPase (8, 9) and Na+/Ca2+ exchange (13). Both of these effects would only lead to an elevation in [Ca2+]i, and, therefore, their contribution to the effects on reduced [Ca2+]i is unlikely.


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Fig. 8.   A: conceptual framework for ACh-induced [Ca2+]i oscillations in porcine TSM cells. Each [Ca2+]i oscillation represents all-or-none release via ryanodine-receptor (RyR) channels from an SR Ca2+ pool, represented by amplitude of oscillations (SR Ca2+ content). SR Ca2+ release through Ins(1,4,5)P3 (IP3)-receptor channels is required, at least for initiation of oscillations. Frequency and propagation velocity of oscillations represent sensitivity for Ca2+-induced Ca2+ release (CICR) through RyR channels. cADPR, cADP-ribose; CaM, calmodulin. Effects of halothane on ACh-induced [Ca2+]i oscillations (B) may result from increased SR Ca2+ leak, especially through Ins(1,4,5)P3-receptor channels, decreased SR Ca2+ content, and decreased sensitivity of CICR.

The transient [Ca2+]i response of beta -escin-permeabilized TSM cells to halothane is entirely consistent with previous evidence from other tissues such as pituitary cells (14), cardiac muscle (5, 33), and vascular smooth muscle (7, 30), indicating that halothane increases [Ca2+]i levels by releasing Ca2+ from the SR, thereby depleting SR Ca2+ stores. The present study extends these results by demonstrating that halothane increases SR Ca2+ release through Ins(1,4,5)P3-receptor channels. Halothane-induced SR Ca2+ release through Ins(1,4,5)P3-receptor channels has been previously demonstrated in pituitary cells (14). However, the results of the present study are the first demonstration of this effect in smooth muscle.

A previous study (12) demonstrated that the Ca2+ pumping rate of SR Ca2+-ATPase is proportional to the state of depletion of the SR stores. Accordingly, the peak of the [Ca2+]i response to caffeine represents a time point when the SR stores are more or less emptied. Therefore, the initial rate of fall in the [Ca2+]i response can be used to estimate the maximum rate of SR Ca2+-ATPase. This measurement was particularly useful in the present study for avoiding the confounding influence of halothane-induced SR Ca2+ leak. At the peak of the caffeine response, the SR is empty, and, therefore, the SR Ca2+ leak is minimized and the effects of halothane on SR Ca2+ reuptake can be assessed in isolation. Thus the slowing of the initial rate of fall in the [Ca2+]i response to caffeine is consistent with an inhibition of SR Ca2+ reuptake by halothane. Indeed, previous studies (30, 34) suggested that volatile anesthetics inhibit SR Ca2+-ATPase. In contrast, one study (10) in isolated myocardial SR found that halothane actually increases SR Ca2+-ATPase activity. However, this effect may be a secondary consequence of the halothane-induced increased SR Ca2+ leak because the depletion of SR stores can lead to stimulation of Ca2+-ATPase (12).

From our conceptual framework presented above, the amplitude of a [Ca2+]i oscillation represents the SR Ca2+ content. Accordingly, the decrease in the amplitude of ACh-induced [Ca2+]i oscillations induced by halothane under conditions of blocked Ca2+ influx and efflux is consistent with a decrease in SR Ca2+ content, most likely mediated via increased SR Ca2+ leak through Ins(1,4,5)P3-receptor channels as indicated by the data from permeabilized cells. This is further substantiated by the fact that in both intact and permeabilized cells, halothane induces [Ca2+]i elevations even when SR Ca2+ reuptake is blocked and that heparin (in permeabilized cells) blunts the [Ca2+]i response in the presence of CPA. The decrease in the amplitude of [Ca2+]i oscillations could additionally result from a reduction in SR Ca2+ reuptake, which would also lead to decreased SR Ca2+ content. Indeed, the prolongation of the fall time of the [Ca2+]i oscillations is suggestive of inhibited SR Ca2+ reuptake.

The decrease in the frequency and propagation velocity of ACh-induced [Ca2+]i oscillations under conditions of blocked Ca2+ influx and efflux suggests that CICR sensitivity for SR Ca2+ release through RyR channels is decreased by halothane. Although similar effects were observed under conditions of intact Ca2+ influx and efflux, the additional inhibition of Ca2+ influx by halothane (16, 17, 35, 36) is a confounding factor. Another factor to be considered is the effect of halothane on Ca2+ reuptake, which would also lead to eventual depletion of the SR and a slowing of oscillation frequency. However, the effects of halothane on the oscillations cannot be explained by its effects on reuptake alone. As reported in the present study, as well as in a recent publication by Prakash et al. (25), we demonstrated that during ongoing ACh-induced [Ca2+]i oscillations, inhibition of reuptake elevates [Ca2+]i but first leads to an acceleration of oscillation frequency and propagation velocity. These previous observations are in contrast to the slowing of frequency and propagation velocity with halothane, suggesting that additional mechanisms are involved, i.e., decreased CICR sensitivity.

It was beyond the scope of the present study to examine the mechanisms underlying decreased CICR sensitivity, but these need to be explored in future studies. One potential target for halothane is cADP-ribose, which has been recently demonstrated to modulate Ca2+ release through RyR channels via an indirect, receptor-mediated mechanism (26). Halothane might decrease cADP-ribose levels or inhibit receptor binding. Halothane may additionally inhibit the RyR channel itself. However, studies (5, 22, 33) in cardiac muscle suggested that, if anything, volatile anesthetics actually increase SR Ca2+ leak through RyR channels. Therefore, it is likely that halothane inhibition of Ca2+ release through RyR channels involves additional mechanisms.

In summary, halothane affects ACh-induced [Ca2+]i oscillations in porcine TSM cells by decreasing the amplitude, frequency, and propagation velocity of oscillations. The effect on oscillation amplitude can be explained, at least in part, by decreased SR Ca2+ content mediated via enhanced SR Ca2+ leak through Ins(1,4,5)P3-receptor channels and inhibited Ca2+ reuptake. Decreased sensitivity for Ca2+ release through RyR channels may underlie the reduction in oscillation frequency and propagation velocity.

    ACKNOWLEDGEMENTS

We thank Thomas Keller for technical assistance in cell preparation.

    FOOTNOTES

This work was supported by Deutsche Forschungsgemeinschaft Research Training Grant Pa 668-1 to C. Pabelick; National Heart, Lung, and Blood Institute Grants HL-57498, HL-45532, and HL-54757; the Mayo Foundation (Rochester, MN); and the University of Minnesota (St. Paul).

Address for reprint requests: G. C. Sieck, Anesthesia Research, Mayo Clinic, Rochester, MN 55905.

Received 17 December 1997; accepted in final form 12 October 1998.

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Discussion
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