Free
Meeting Abstracts  |   July 2001
Effects of Halothane on Sarcoplasmic Reticulum Calcium Release Channels in Porcine Airway Smooth Muscle Cells
Author Affiliations & Notes
  • Christina M. Pabelick, M.D.
    *
  • Yedatore S. Prakash, Ph.D.
  • Mathur S. Kannan, D.V.M., Ph.D.
  • David O. Warner, M.D.
    §
  • Gary C. Sieck, Ph.D.
    ‖‖
  • * Resident, † Associate Professor, § Professor, Department of Anesthesiology, ‖‖ Professor, Departments of Anesthesiology and Physiology and Biophysics, Mayo Clinic and Foundation. ‡ Professor, Department of Veterinary PathoBiology, University of Minnesota, St. Paul, Minnesota.
  • Received from the Department of Anesthesiology, Mayo Clinic, Rochester, Minnesota.
Article Information
Meeting Abstracts   |   July 2001
Effects of Halothane on Sarcoplasmic Reticulum Calcium Release Channels in Porcine Airway Smooth Muscle Cells
Anesthesiology 7 2001, Vol.95, 207-215. doi:
Anesthesiology 7 2001, Vol.95, 207-215. doi:
VOLATILE anesthetics such as halothane produce bronchodilation, partly by decreasing intracellular Ca2+concentration ([Ca2+]i), which plays an important role in development and maintenance of force in airway smooth muscle (ASM) cells. For example, halothane has been shown to decrease the elevation in [Ca2+]iproduced by agonists such as acetylcholine. 1–3 These effects of halothane are partly attributable to an inhibition of Ca2+influx. 3–5 Other studies in vascular smooth muscle 3 and cardiac tissue 6 have indicated that halothane depletes Ca2+stores (content) of the sarcoplasmic reticulum (SR) by increasing Ca2+“leakage.” In a recent study, Yamakage et al.  7 demonstrated that volatile anesthetic–induced depletion of SR Ca2+also occurs in canine tracheal smooth muscle. In other cell types, such anesthetic-induced SR, Ca2+leak has been shown to occur via  both inositol 1,4,5-trisphosphate (IP3) and/or ryanodine receptor (RyR) channels. 8,9 Whether a similar mechanism is involved in ASM cells remains to be determined.
In ASM cells, agonist-induced elevation of [Ca2+]iinvolves Ca2+influx as well as SR Ca2+release through IP3receptor channels. 10 More recently, it has also been demonstrated that agonist activation in ASM cells involves the novel second messenger system cyclic adenosine diphosphate ribose (cADPR) and Ca2+release through RyR channels. 11–13 In a recent study, we showed that clinically relevant concentrations of halothane inhibit acetylcholine-induced [Ca2+]ioscillations that are initiated by Ca2+release through IP3receptor channels and are sustained by repetitive SR Ca2+release through RyR channels. 14 Our results indicated that halothane inhibits [Ca2+]ioscillations by decreasing SR Ca2+content. Therefore, the purpose of the current study was to characterize the mechanisms by which halothane decreases SR Ca2+content. We hypothesized that halothane increases SR Ca2+leakage through both IP3receptor and RyR channels. To remove the confounding effects of halothane on Ca2+influx and efflux, two ASM cell preparations were used: intact cells in which influx and efflux were blocked and β-escin permeabilized cells.
Materials and Methods
Cell Preparation
After obtaining porcine tracheae from a local abattoir, the smooth muscle layer was dissected, and ASM cells were dissociated using previously described techniques. 11,12,14,15 Briefly, the endothelial layer was removed, and the smooth muscle layer was excised and minced thoroughly in Hank’s balanced salt solution (HBSS) buffered with 10 mm HEPES (pH 7.4; Life Technologies, Rockville, MD). The minced tissue was incubated for 2 h in Earle’s balanced salt solution containing 20 U/ml papain and 2000 U/ml DNase (Worthington Biochemical Corp., Lakewood, NJ), and subsequently at 37°C with 1 mg/ml type IV collagenase (Worthington Biochemical Corp.) for another hour. The cells were then gently triturated, centrifuged, and resuspended in minimum essential medium containing 10% fetal calf serum. The isolated cells were split into two batches for IP3measurements and confocal imaging.
Inositol Triphosphate Measurements
Measurements of IP3in ASM samples were performed using a radioreceptor assay. 16,17 ASM cell suspensions (106cells/ml) were placed in test tubes and aerated with 95% O2and 5% CO2. The test tubes were placed on ice to minimize protein degradation. Cells were taken through one of the following protocols: (1) HBSS (vehicle control) for 2 min; (2) 1 μm acetylcholine in HBSS for 2 min; (3) 2 minimum alveolar concentration (MAC) halothane for 2 min; (4) 1 μm acetylcholine and 2 MAC halothane for 2 min; (5) 10 μm U73122 (Sigma Chemicals, St. Louis, MO), an inhibitor of phospholipase C (PLC), for 5 min, followed by 1 μm acetylcholine for 2 min; (6) 10 μm U73122 for 5 min followed by 2 MAC halothane; and (7) 10 μm U73122 for 5 min. A high concentration of U73122 was used to ensure maximum inhibition of PLC. One set of control experiments was performed to determine whether halothane by itself interfered with the IP3assay. HBSS with no ASM cells was bubbled with halothane, and the solution was processed as for the ASM cells.
After one of the aforementioned exposures, reactions were terminated by addition of equal volume of ice-cold 1 m trichloroacetic acid. The trichloroacetic acid was extracted from the medium using trioctylamine and trichloro-trifluoroethane in a 1:3 ratio. The cell-free extract was then used to measure IP3concentrations, using a commercially available radioreceptor assay (NEN Research Products, Boston, MA). 16,17 The measurement technique is similar to that used by other investigators. 7 A Lowry protein assay was used to measure protein concentrations for normalization of IP3concentrations. 18 
Real-time Confocal Imaging
Freshly dissociated cells were plated on collagen-coated glass coverslips and incubated in 5% CO2at 37°C for 1–2 h. Exclusion of trypan blue was used to assess cell viability (>90% of all cells). After incubation in minimum essential medium, each coverslip was washed with HBSS. The coverslip was then transferred to HBSS containing 5 μm of the cell-permeant form of the fluorescent Ca2+indicator fluo-3 AM (Molecular Probes, Eugene, OR) and incubated for 30–45 min at 37°C. The coverslip was then washed in HBSS and mounted on an open slide chamber (RC-25F; Warner Instruments, Hamden, CT). Cells were perfused at 2–3 ml/min and maintained at room temperature.
The technique for real-time confocal imaging of ASM cells has been previously described in detail. 11,12,15 Briefly, cells were visualized using an Odyssey XL real-time confocal system (Noran Instruments, Middleton, WI) equipped with an Ar-Kr laser and attached to a Nikon Diaphot microscope. A Nikon 40X/1.3 oil-immersion objective lens (Melville, NY) was used for Ca2+imaging. Image size was set to 640 × 480 pixels, and pixel area was calibrated using a stage micrometer (0.063 μm2/pixel). The optical section thickness for the 40× lens was set at 1 μm by controlling the slit size on the confocal system. The 488-nm laser line was used to excite fluo-3, and emissions were collected using a 515-nm long-pass filter and a high-sensitivity photomultiplier tube. Based on previous experience, 11,12,15 a fixed combination of laser intensity (20% of maximum) and photomultiplier gain (1,700 from a maximum of 4,096) was used to ensure that pixel intensities within cells ranged between 25 and 254 gray levels. At these settings, laser power output at 3 mW varied less than 1% across a 15-min period, and only intermittent laser exposure of fluo-3 was required (<5 min), causing little detectable photobleaching (<1%). Measurements of [Ca2+]iwere obtained by defining a large region of interest around an entire cell. Software limitations allowed measurement from a maximum of eight regions of interest within a field.
Ca2+Calibrations
Although fluo-3 is a nonratiometric Ca2+indicator, several studies have used in vitro  calibrations where fluorescence levels are measured at known Ca2+concentrations; however, the dissociation constant (Kd) of the fluorescent dye differs in vitro  versus  in vivo  (see Takahashi et al.  19 for a review). Therefore, as in previous studies, 15,20 we used an empirical in vivo  calibration technique based on measurement of intracellular fluorescence levels at known [Ca2+]ilevels. Based on previous experience, a fixed combination of laser intensity (20% of maximum) and photomultiplier gain (1,700 from a maximum of 4,096) was set a priori  to ensure pixel intensities between 25 and 255 gray levels. ASM cells loaded with 5 μm fluo3 AM were sequentially exposed to solutions containing 10 known Ca2+concentrations (0 nm to 1.25 μm; Calcium Calibration Buffer Kit, Molecular Probes) and 10 μm of the Ca2+ionophore A-23187. This technique allowed equilibration of [Ca2+]iand extracellular [Ca2+].
Previous studies have used the equation:MATHto calculate [Ca2+]ilevels from fluorescence values (F), where Fminis the fluorescence at minimal [Ca2+]iconcentrations (0 nm in this study) and Fmaxis the fluorescence at saturating concentrations, determined using a buffer and ionophore technique similar to the one described above. 19 Using the Fmin, Fmax, and gray level values from our measurements in ASM cells as previously described, we calculated the apparent Kdfor fluo-3 in our system to be 455 ± 89 nm (mean of 10 calibrations), which is comparable to the 400 nm reported in previous studies. 19,20 
Administration of Halothane
A calibrated online vaporizer was used to deliver halothane (Wyeth-Ayerst Laboratories, St. Davids, PA) to the aerating gas mixture (95% O2, 5% CO2). The vaporizer setting produced aqueous concentrations of halothane in the HBSS equivalent to 2 MAC at room temperature. The halothane concentration in the perfusion chamber (in aqueous solution) was determined by gas chromatography from anerobically obtained samples using an electron capture detector (Hewlett-Packard 5880A, Palo Alto, CA), as described previously. 21 In the solutions used to examine the effects of halothane, the concentrations for 2 MAC halothane were 0.47 ± 0.03 mm.
Experimental Protocols
Effect of Halothane on Intracellular Ca2+Concentration.
In the first set of experiments, intact ASM cells were preexposed to zero-Ca2+HBSS and 1 mm lanthanum chloride (Sigma) to nonspecifically inhibit both Ca2+influx and efflux across the plasma membrane. 14,15 During these conditions in which the SR was effectively isolated, cells were exposed to 2 MAC halothane, and the [Ca2+]iresponse was recorded.
In a second set of experiments, ASM cells were first permeabilized with 25 μm β-escin (Sigma) as described previously. 11,14 This permeabilization procedure allows for entry of large-molecular-weight compounds into the cytosolic compartment and for control of cytosolic Ca2+concentrations via  externally applied solutions of known Ca2+concentration (pCa, negative logarithm of Ca2+concentration). However, the confounding effects of Ca2+fluxes across the cell membrane are removed. Furthermore, unlike Triton-X permeabilization, β-escin does not destroy receptor and G-protein structures in the plasma membrane and allows for agonist stimulation if necessary. The pCa solutions were prepared as described by Fabiato, 22 with stabilization constants described by Godt and Lindley. 23 
After β-escin permeabilization, cells were exposed to 9.0 pCa for 2 min, and the SR was then loaded by exposure to 6.3 pCa for 15 min. The cells were then exposed to 2 MAC halothane in 6.3 pCa, and the [Ca2+]iresponse was recorded.
Effect of Inhibition of Inositol Triphosphate Receptor Channels.
Intact ASM cells were first exposed to 2 MAC halothane in zero-Ca2+HBSS and lanthanum chloride as previously described. The cells were then washed for 10 min and then for an additional 5 min with medium containing 20 μm Xestospongin D (XeD; Calbiochem, San Diego, CA), a potent cell-permeant inhibitor of IP3receptor channels. 24 In the continued presence of XeD, cells were reexposed to 2 MAC halothane. In control experiments, cells were exposed twice to halothane with an intervening 15-min wash period.
In a second set of experiments, β-escin–permeabilized cells were exposed to 2 MAC halothane in 6.3 pCa. The cells were then washed for 10 min in 6.3 pCa, and for an additional 5 min in 20 μm XeD in 6.3 pCa. They were then reexposed to halothane. Other cells were exposed to 0.5 mg/ml heparin instead of XeD, and then to halothane. We previously demonstrated that heparin can enter β-escin–permeabilized cells and inhibits IP3receptor channels. 11,14 In the current study also, inhibition of IP3receptor channels by XeD or heparin was confirmed by a lack of an [Ca2+]iresponse to 10 μm IP3(Sigma) at the end of the experimental protocol, after a reloading of the SR with 6.3 pCa. In corresponding control experiments, cells were exposed twice to exogenous IP3with just an intervening wash. In other control experiments, cells were exposed twice to halothane in 6.3 pCa with an intervening 15-min wash period.
In a third set of experiments, the direct effect of halothane on IP3receptor channels independent of elevation of IP3was examined. Intact ASM cells were first exposed to 2 MAC halothane in zero-Ca2+HBSS and lanthanum chloride as previously described. The cells were then washed for 10 min and then for an additional 5 min with medium containing 10 μm U73122 (Sigma). In the continued presence of U73122, cells were reexposed to halothane. To further verify the direct effect of halothane on IP3receptor channels, cells were washed, exposed to U73122 as well as XeD, and then exposed for a third time to halothane.
In a fourth set of experiments, the interactions between halothane and exogenous IP3on SR Ca2+release were examined in β-escin–permeabilized ASM cells. After the first exposure to 10 μm IP3in 6.3 pCa, cells were washed in 6.3 pCa and exposed simultaneously to 2 MAC halothane and IP3.
Effect of Inhibition of Ryanodine Receptor Channels.
Intact ASM cells were first exposed to 2 MAC halothane in zero-Ca2+HBSS and lanthanum chloride and then washed for 10 min. The cells were then exposed for an additional 5 min to 20 μm ryanodine (Sigma) to inhibit RyR channels. 11,15 In the continued presence of ryanodine, cells were reexposed to 2 MAC halothane.
In a second set of experiments, β-escin–permeabilized cells were exposed to 2 MAC halothane in 6.3 pCa. The cells were then washed for 10 min in 6.3 pCa and then for an additional 5 min in 20 μm ryanodine in 6.3 pCa. The cells were then reexposed to halothane.
Effect of Simultaneous Inhibition of Inositol Triphosphate and Ryanodine Receptor Channels.
Intact ASM cells were first exposed to 2 MAC halothane in zero-Ca2+HBSS and lanthanum chloride and then washed for 10 min. The cells were then exposed for an additional 5 min to 20 μm XeD and 20 μm ryanodine to simultaneously inhibit both IP3and RyR channels. In the continued presence of these inhibitors, cells were reexposed to 2 MAC halothane.
In a second set of experiments, β-escin–permeabilized cells were exposed to 2 MAC halothane in 6.3 pCa, washed for 10 min in 6.3 pCa, and then exposed for an additional 5 min to 20 μm XeD and 20 μm ryanodine in 6.3 pCa. The cells were then reexposed to halothane.
Statistical Analysis
Inositol triphosphate measurements were compared using the independent Student t  test. In these studies, n refers to the number of samples. For Ca2+imaging experiments, it was not possible to apply all of the experimental protocols to every cell or to cells obtained from every animal. In each experiment, at least three and up to five cells were analyzed from each coverslip. If not otherwise stated, comparisons before and after halothane and/or inhibitor exposure were made using paired t  tests. Bonferroni correction was used for pairwise comparisons. A P  value < 0.05 was considered statistically significant. In all studies relating to single cells, n refers to the number of cells. Cells were obtained from eight animals; however, no attempt was made to determine interanimal variability in Ca2+imaging studies. Values are reported as mean ± SD.
Results
Inositol Triphosphate Measurements
In ASM cells that were exposed to HBSS only for 2 min (vehicle control), IP3concentrations were 1.92 ± 0.07 pmol/106cells or 17.12 ± 11.56 pmol/mg protein (n = 5). Exposure to 1 μm acetylcholine resulted in an approximately twofold increase in IP3concentrations (fig. 1;P  < 0.05). Preexposure to 2 MAC halothane alone for 2 min also resulted in significantly elevated IP3concentrations (P  < 0.05) that were almost fourfold higher than that obtained with exposure to acetylcholine alone. Simultaneous exposure to both acetylcholine and halothane increased IP3concentrations beyond those observed with acetylcholine or halothane alone (P  < 0.05). Inhibition of PLC by U73122 significantly blunted the acetylcholine-induced IP3response to approximately 25% of that observed with acetylcholine alone (P  < 0.05). In contrast, halothane-induced elevation of IP3was decreased by U73122 to only approximately 95% of that observed with halothane alone (fig. 1;P  < 0.05). In control experiments, U73122 alone slightly decreased IP3concentrations in unstimulated cells (97 ± 4%), but this decrease was not significant. Exposure of HBSS to halothane did not result in any detectable levels of IP3in the assay.
Fig. 1. Effect of halothane on inositol triphosphate (IP3) concentrations in porcine airway smooth muscle cells. IP3concentrations were measured using radioimmunoreceptor assay. Compared with unstimulated control, IP3concentrations were significantly (P  < 0.05) elevated by exposure to acetylcholine, 2 minimum alveolar concentration (MAC) halothane, and a combination thereof. The IP3response to simultaneous application of acetylcholine and halothane was significantly greater than that induced by either agent alone. Inhibition of phospholipase C by U73122 significantly blunted the IP3response to both acetylcholine and halothane, but the effect on halothane was considerably smaller. *Significant (P  < 0.05) difference from unstimulated control. †Significant difference from acetylcholine exposure alone. §Significant difference from halothane exposure alone.
Fig. 1. Effect of halothane on inositol triphosphate (IP3) concentrations in porcine airway smooth muscle cells. IP3concentrations were measured using radioimmunoreceptor assay. Compared with unstimulated control, IP3concentrations were significantly (P 
	< 0.05) elevated by exposure to acetylcholine, 2 minimum alveolar concentration (MAC) halothane, and a combination thereof. The IP3response to simultaneous application of acetylcholine and halothane was significantly greater than that induced by either agent alone. Inhibition of phospholipase C by U73122 significantly blunted the IP3response to both acetylcholine and halothane, but the effect on halothane was considerably smaller. *Significant (P 
	< 0.05) difference from unstimulated control. †Significant difference from acetylcholine exposure alone. §Significant difference from halothane exposure alone.
Fig. 1. Effect of halothane on inositol triphosphate (IP3) concentrations in porcine airway smooth muscle cells. IP3concentrations were measured using radioimmunoreceptor assay. Compared with unstimulated control, IP3concentrations were significantly (P  < 0.05) elevated by exposure to acetylcholine, 2 minimum alveolar concentration (MAC) halothane, and a combination thereof. The IP3response to simultaneous application of acetylcholine and halothane was significantly greater than that induced by either agent alone. Inhibition of phospholipase C by U73122 significantly blunted the IP3response to both acetylcholine and halothane, but the effect on halothane was considerably smaller. *Significant (P  < 0.05) difference from unstimulated control. †Significant difference from acetylcholine exposure alone. §Significant difference from halothane exposure alone.
×
Intracellular Ca2+Concentration Measurements
Effect of Halothane on Intracellular Ca2+Concentration.
In intact ASM cells where both Ca2+influx and efflux across the plasma membrane were nonspecifically inhibited by preexposure to zero-Ca2+HBSS and lanthanum chloride, basal [Ca2+]iranged from 90 to 130 nm (108 ± 49 nm; n = 12). These values were not significantly different from [Ca2+]iwhen cells were perfused with normal HBSS (80–125 nm; 94 ± 42 nm). During these conditions, exposure to 2 MAC halothane induced a transient [Ca2+]iresponse after an approximately 10-s delay (fig. 2A). The profile of the [Ca2+]iresponse to halothane was comparable to that observed with halothane in previous studies from our laboratory. 14 The rate of increase of the [Ca2+]iresponse (measured over a 1-s interval) was 20 ± 17 nm/s, peak amplitude was 500 ± 42 nm, and rate of decrease (also measured over 1 s) was 75 ± 14 nm/s. After washout, reexposure to halothane produced another transient [Ca2+]iresponse with a profile that was not significantly different from the first response (fig. 2A), with a rate of increase of 23 ± 17 nm/s, peak amplitude of 485 ± 45 nm, and rate of decrease of 82 ± 17 nm/s.
Fig. 2. Representative examples of the intracellular Ca2+concentration ([Ca2+]i) response of intact (A  ) and β-escin–permeabilized (B  ) airway smooth muscle cells to 2 minimum alveolar concentration (MAC) halothane. In intact cells, Ca2+influx and efflux across the plasma membrane were nonspecifically blocked using zero extracellular Ca2+and lanthanum chloride. During these conditions, exposure to halothane induced a transient [Ca2+]iresponse that was reproducible after an intervening washout. A similar effect was elicited with cells in which plasma membrane effects were eliminated by permeabilization. Amplitude bar for (A  ) only.
Fig. 2. Representative examples of the intracellular Ca2+concentration ([Ca2+]i) response of intact (A 
	) and β-escin–permeabilized (B 
	) airway smooth muscle cells to 2 minimum alveolar concentration (MAC) halothane. In intact cells, Ca2+influx and efflux across the plasma membrane were nonspecifically blocked using zero extracellular Ca2+and lanthanum chloride. During these conditions, exposure to halothane induced a transient [Ca2+]iresponse that was reproducible after an intervening washout. A similar effect was elicited with cells in which plasma membrane effects were eliminated by permeabilization. Amplitude bar for (A 
	) only.
Fig. 2. Representative examples of the intracellular Ca2+concentration ([Ca2+]i) response of intact (A  ) and β-escin–permeabilized (B  ) airway smooth muscle cells to 2 minimum alveolar concentration (MAC) halothane. In intact cells, Ca2+influx and efflux across the plasma membrane were nonspecifically blocked using zero extracellular Ca2+and lanthanum chloride. During these conditions, exposure to halothane induced a transient [Ca2+]iresponse that was reproducible after an intervening washout. A similar effect was elicited with cells in which plasma membrane effects were eliminated by permeabilization. Amplitude bar for (A  ) only.
×
In β-escin–permeabilized cells, exposure to 2 MAC halothane in 6.3 pCa also resulted in a transient [Ca2+]iresponse that was qualitatively similar in profile to that observed in intact cells (fig. 2B). As in previous studies, we did not attempt to quantify the amplitude of the [Ca2+]iresponse in permeabilized cells because of indeterminate amounts of fluo-3 leakage after exposure to β-escin. 14 To ensure that there was minimal continued leakage of dye through the course of the protocol, control experiments were performed in which the cells were exposed to the same agent (e.g.  , halothane) twice with an intervening washout period (fig. 2B). In these studies, we found the amplitude of the second [Ca2+]iresponse to be 94 ± 9% of the first response (n = 10). Furthermore, in subsequent protocols, comparisons of anesthetic–drug effects were made only within a cell and not across cells.
Effect of Inhibition of Inositol Triphosphate Receptor Channels.
In intact ASM cells with blocked Ca2+influx and efflux, exposure to 2 MAC halothane produced a transient [Ca2+]iresponse as previously described. Subsequent exposure to 20 μm XeD did not significantly alter resting [Ca2+]i(110 ± 46 nm; n = 11). However, in the continued presence of XeD, exposure to 2 MAC halothane produced a transient [Ca2+]iresponse that was significantly slower (rate of increase, 136 ± 30%; rate of decrease, 154 ± 36% control;P  < 0.05) and smaller (amplitude, 61 ± 20% control;P  < 0.05) in profile compared with the first response (fig. 3A).
Fig. 3. Effect of halothane on inositol triphosphate (IP3) receptor channels in intact (A  ) and β-escin–permeabilized (B  ) airway smooth muscle (ASM) cells. During conditions of blocked Ca2+influx and efflux, halothane induced a transient intracellular Ca2+concentration ([Ca2+]i) response even when IP3receptor channels were blocked by a membrane permeant inhibitor, Xestospongin D (XeD). These data indicate that halothane increases sarcoplasmic reticulum Ca2+release also through ryanodine receptor (RyR) channels. The decrease in the [Ca2+]iresponse to halothane during these conditions is consistent with figure 3, indicating the IP3receptor component. Amplitude bar for (A  ) only. MAC = minimum alveolar concentration.
Fig. 3. Effect of halothane on inositol triphosphate (IP3) receptor channels in intact (A 
	) and β-escin–permeabilized (B 
	) airway smooth muscle (ASM) cells. During conditions of blocked Ca2+influx and efflux, halothane induced a transient intracellular Ca2+concentration ([Ca2+]i) response even when IP3receptor channels were blocked by a membrane permeant inhibitor, Xestospongin D (XeD). These data indicate that halothane increases sarcoplasmic reticulum Ca2+release also through ryanodine receptor (RyR) channels. The decrease in the [Ca2+]iresponse to halothane during these conditions is consistent with figure 3, indicating the IP3receptor component. Amplitude bar for (A 
	) only. MAC = minimum alveolar concentration.
Fig. 3. Effect of halothane on inositol triphosphate (IP3) receptor channels in intact (A  ) and β-escin–permeabilized (B  ) airway smooth muscle (ASM) cells. During conditions of blocked Ca2+influx and efflux, halothane induced a transient intracellular Ca2+concentration ([Ca2+]i) response even when IP3receptor channels were blocked by a membrane permeant inhibitor, Xestospongin D (XeD). These data indicate that halothane increases sarcoplasmic reticulum Ca2+release also through ryanodine receptor (RyR) channels. The decrease in the [Ca2+]iresponse to halothane during these conditions is consistent with figure 3, indicating the IP3receptor component. Amplitude bar for (A  ) only. MAC = minimum alveolar concentration.
×
In β-escin–permeabilized cells (n = 9) first exposed to 2 MAC halothane in 6.3 pCa, subsequent exposure to halothane in the presence of 20 μm XeD also significantly blunted the second [Ca2+]iresponse (fig. 3B; 56 ± 22% control). In other cells exposed to 0.5 mg/ml heparin instead of XeD (n = 5), the second exposure to halothane also resulted in a diminished [Ca2+]iresponse (54 ± 31% control). In both protocols, exposure to 10 μm IP3at the termination of the protocol did not produce a significant elevation in [Ca2+]i, confirming complete inhibition of IP3receptor channels by XeD or heparin. In control experiments, where cells were exposed twice to IP3with just an intervening wash, there was no significant difference in the two [Ca2+]iresponses (second exposure was 95 ± 8% of first exposure). These control data are consistent with our previous study. 11,14 
In a third set of experiments, the direct effect of halothane on IP3receptor channels was examined in the presence of U73122, which inhibited PLC. During these conditions, the [Ca2+]iresponse to halothane was 80 ± 15% of control (first exposure to halothane;P  < 0.05). Further inhibition of IP3receptor channels with XeD, in the continued presence of U73122, resulted in a response to halothane that was 58 ± 10% of the first exposure (P  < 0.05) but was not significantly different from the response in the presence of XeD alone (61 ± 20%; see above).
In a fourth set of experiments, where the interaction between halothane and exogenous IP3on SR Ca2+release was examined, the [Ca2+]iresponse to simultaneous application of IP3and halothane was 126 ± 14% of the response to IP3alone (P  < 0.05).
Effect of Inhibition of Ryanodine Receptor Channels.
In intact ASM cells with inhibited Ca2+influx and efflux, exposure to 20 μm ryanodine slightly elevated basal [Ca2+]i(150 ± 11 nm;P  < 0.05; n = 13). In the continued presence of ryanodine, exposure to 2 MAC halothane resulted in transient [Ca2+]iresponse that was also considerably slower (rate of increase, 146 ± 43%; rate of decrease, 175 ± 50% control;P  < 0.05) and smaller (amplitude, 43 ± 29% control;P  < 0.05) compared with the first response in the absence of ryanodine (fig. 4A). Compared with the [Ca2+]iresponse to halothane in the presence of XeD, the response in the presence of ryanodine was significantly smaller when expressed as percentage of control (P  < 0.05). However, it must be emphasized that these experiments were conducted in separate cells.
Fig. 4. Effect of halothane on ryanodine receptor channels in intact (A  ) and β-escin–permeabilized (B  ) airway smooth muscle cells. During conditions of blocked Ca2+influx and efflux, halothane induced a transient intracellular Ca2+concentration ([Ca2+]i) response even when ryanodine receptor channels were blocked by high concentrations of ryanodine. These data indicate that halothane increases sarcoplasmic reticulum Ca2+release through inositol triphosphate receptor channels. The decrease in the [Ca2+]iresponse to halothane during these conditions suggests a ryanodine receptor component. Amplitude bar for (A  ) only. MAC = minimum alveolar concentration.
Fig. 4. Effect of halothane on ryanodine receptor channels in intact (A 
	) and β-escin–permeabilized (B 
	) airway smooth muscle cells. During conditions of blocked Ca2+influx and efflux, halothane induced a transient intracellular Ca2+concentration ([Ca2+]i) response even when ryanodine receptor channels were blocked by high concentrations of ryanodine. These data indicate that halothane increases sarcoplasmic reticulum Ca2+release through inositol triphosphate receptor channels. The decrease in the [Ca2+]iresponse to halothane during these conditions suggests a ryanodine receptor component. Amplitude bar for (A 
	) only. MAC = minimum alveolar concentration.
Fig. 4. Effect of halothane on ryanodine receptor channels in intact (A  ) and β-escin–permeabilized (B  ) airway smooth muscle cells. During conditions of blocked Ca2+influx and efflux, halothane induced a transient intracellular Ca2+concentration ([Ca2+]i) response even when ryanodine receptor channels were blocked by high concentrations of ryanodine. These data indicate that halothane increases sarcoplasmic reticulum Ca2+release through inositol triphosphate receptor channels. The decrease in the [Ca2+]iresponse to halothane during these conditions suggests a ryanodine receptor component. Amplitude bar for (A  ) only. MAC = minimum alveolar concentration.
×
In β-escin–permeabilized cells first exposed to 2 MAC halothane in 6.3 pCa, addition of 20 μm ryanodine also resulted in a significantly smaller [Ca2+]iresponse on reexposure to halothane (fig. 4B; 50 ± 24% control;P  < 0.05; n = 8).
Simultaneous Inhibition of Inositol Triphosphate and Ryanodine Receptor Channels.
In intact ASM cells (n = 11) where Ca2+influx and efflux were inhibited and a [Ca2+]iresponse to halothane was first verified, simultaneous addition of 20 μm XeD and 20 μm ryanodine completely abolished the subsequent [Ca2+]iresponse to halothane (fig. 5).
Fig. 5. Effect of simultaneous inhibition of both inositol triphosphate (IP3) and ryanodine receptor (RyR) channels on the intracellular Ca2+concentration ([Ca2+]i) response of intact airway smooth muscle (ASM) cells to halothane. In the presence of blocking concentrations of both Xestospongin D and ryanodine, halothane did not elevate [Ca2+]i, indicating that the effect of halothane on the sarcoplasmic reticulum is mediated entirely via  the two receptor channels. MAC = minimum alveolar concentration.
Fig. 5. Effect of simultaneous inhibition of both inositol triphosphate (IP3) and ryanodine receptor (RyR) channels on the intracellular Ca2+concentration ([Ca2+]i) response of intact airway smooth muscle (ASM) cells to halothane. In the presence of blocking concentrations of both Xestospongin D and ryanodine, halothane did not elevate [Ca2+]i, indicating that the effect of halothane on the sarcoplasmic reticulum is mediated entirely via 
	the two receptor channels. MAC = minimum alveolar concentration.
Fig. 5. Effect of simultaneous inhibition of both inositol triphosphate (IP3) and ryanodine receptor (RyR) channels on the intracellular Ca2+concentration ([Ca2+]i) response of intact airway smooth muscle (ASM) cells to halothane. In the presence of blocking concentrations of both Xestospongin D and ryanodine, halothane did not elevate [Ca2+]i, indicating that the effect of halothane on the sarcoplasmic reticulum is mediated entirely via  the two receptor channels. MAC = minimum alveolar concentration.
×
In β-escin–permeabilized cells (n = 7) first exposed to 2 MAC halothane in 6.3 pCa, addition of 20 μm XeD and 20 μm ryanodine also completely abolished the [Ca2+]iresponse to a subsequent reexposure to halothane (not shown). The effects of various inhibitors and their interactions with halothane in intact ASM cells are summarized in figure 6.
Fig. 6. Summary of the effects of halothane on inositol triphosphate versus  ryanodine receptor channels. Statistical comparisons were made to the intracellular Ca2+concentration ([Ca2+]i) response to the first exposure to halothane alone. Accordingly, the control for these interactions was a second exposure to halothane alone (first bar). § Significant (P  < 0.05) difference from first halothane exposure. ‡ Significant difference from exposure to Xestospongin D or ryanodine exposure alone.
Fig. 6. Summary of the effects of halothane on inositol triphosphate versus 
	ryanodine receptor channels. Statistical comparisons were made to the intracellular Ca2+concentration ([Ca2+]i) response to the first exposure to halothane alone. Accordingly, the control for these interactions was a second exposure to halothane alone (first bar). § Significant (P 
	< 0.05) difference from first halothane exposure. ‡ Significant difference from exposure to Xestospongin D or ryanodine exposure alone.
Fig. 6. Summary of the effects of halothane on inositol triphosphate versus  ryanodine receptor channels. Statistical comparisons were made to the intracellular Ca2+concentration ([Ca2+]i) response to the first exposure to halothane alone. Accordingly, the control for these interactions was a second exposure to halothane alone (first bar). § Significant (P  < 0.05) difference from first halothane exposure. ‡ Significant difference from exposure to Xestospongin D or ryanodine exposure alone.
×
Discussion
The results of the current study demonstrate that a clinically relevant concentration of halothane affects [Ca2+]iin ASM cells by decreasing SR Ca2+content via  increased leak through Ca2+channels in the SR. Even in the absence of agonist activation, halothane increases IP3concentrations, partly by activating PLC in the plasma membrane. However, independent of this effect on IP3, halothane also increases SR Ca2+leak through IP3receptor channels, which contributes to the depletion of SR Ca2+content. Additional leak is induced by halothane effects on RyR channels.
Methodologic Issues
We used freshly dissociated ASM cells to examine the effects of halothane on [Ca2+]iregulation. Variations in cell dissociation and dye loading, and inherent cellular differences, may introduce potential variability in the observed [Ca2+]iresponses between cells and/or animals. However, in previous studies we determined that there were no significant differences in the coefficient of variation of [Ca2+]iresponses of ASM cells within or across animals. 14 Furthermore, in the current experimental design, each cell served as its own control. Therefore, cellular variability was not considered to be a confounding issue.
A potential concern with the use of a nonratiometric Ca2+indicator such as fluo-3 is that dye compartmentalization or bleaching may affect the observed [Ca2+]iresponses. Furthermore, the apparent Kdof the dye may differ in vitro  versus  in vivo  and furthermore may be cell-specific. Therefore, it was essential to perform an empiric calibration using ASM cells and the confocal microscope used in the current study. The reliability of the calibration technique is indicated by relatively small variations in basal [Ca2+]iacross cells obtained on different days.
The current study focused only on halothane effects at the level of the SR, while recognizing that additional effects on mechanisms such as Ca2+influx and efflux are possible. Indeed, there is already considerable evidence in the literature that the decrease in [Ca2+]iby halothane involves inhibition of Ca2+influx 1–3 through voltage-gated L-type Ca2+channels. 3,5 However, these effects could not have contributed to the observed [Ca2+]iresponses because all experiments were conducted during conditions of blocked influx and efflux. 3,5 
Effect of Halothane on Inositol Triphosphate Levels
In ASM cells, muscarinic receptors are coupled to G proteins that activate plasma membrane PLC, which catalyzes hydrolysis of membrane-associated phosphatidylinositol bisphosphate to IP3and diacylglycerol. The elevation in IP3levels after acetylcholine stimulation and its inhibition by U73122 are therefore consistent with activation of PLC. Agonist-induced IP3is metabolized via  specific phosphatases. Metabolism of IP3may be regulated by other mechanisms such as protein kinase C, 26 which has been thought to inhibit agonist-induced IP3either by inhibition of PLC or activation of phosphatases, or by protein kinase A 26 acting in a similar fashion. After elevation of IP3, release of Ca2+occurs via  IP3-gated receptor channels of the SR. Previous studies have shown that the IP3receptor channel displays a bell-shaped dependence on the level of [Ca2+]iitself, 27,28 such that at a fixed concentration of IP3, Ca2+conductance is low when [Ca2+]iis also low, but conductance increases with increasing [Ca2+]ito a point. Accordingly, during unstimulated conditions, there is only a low, background level of Ca2+release through IP3receptor channels.
Exposure to halothane resulted in marked elevation of IP3concentrations in ASM cells. In this regard, it is of significance that inhibition of PLC by U73122, which should theoretically blunt any halothane effects on PLC per se  , resulted in an extremely small effect on the halothane-induced elevation of IP3concentrations. These data suggest that, in addition to an effect on PLC itself, halothane may also influence the activity of other regulatory mechanisms such as phosphatases, thus altering the time course of IP3formation and degradation. For example, previous studies have demonstrated that halothane can inhibit the effects of PKC in smooth muscle. Accordingly, PKC-modulated degradation of IP3may be delayed in the presence of halothane, resulting in continued elevation of IP3concentrations even when PLC is inhibited by U73122. Whether halothane affects these specific intracellular regulatory mechanisms remains to be determined.
The increase in IP3concentrations induced by halothane may have a complex effect on [Ca2+]iregulation in the cell. On the one hand, elevated IP3concentrations may induce SR Ca2+release, thus partially accounting for the transient [Ca2+]iresponse observed in single ASM cells. On the other hand, increased IP3concentrations will lead to faster inactivation of the IP3receptor channel, especially if [Ca2+]iis also somewhat elevated, as with concurrent acetylcholine stimulation (see review by Taylor 28 on the interaction between [Ca2+]iand IP3receptor function). Such inactivation would inhibit subsequent SR Ca2+release, resulting in ASM relaxation.
The halothane-induced elevation in IP3concentrations observed in the current study sharply contrasts with the findings of Yamakage et al.  7 In that study using canine ASM, halothane was found to decrease IP3concentrations in the presence of muscarinic stimulation with carbachol. The reasons underlying this discrepancy are not entirely clear but may be related either to species differences in the sensitivity of the IP3regulatory mechanisms to anesthetics, including PLC versus  phosphatase activities, or to anesthetic concentrations (approximately 0.45 mm in the current study vs.  0.75–1.15 mm in the study by Yamakage et al.  7). Furthermore, in the study by Yamakage et al.  , the time course of examining IP3concentrations was also more extended compared with the current study. It is entirely possible that with prolonged exposure to halothane, IP3concentrations are indeed reduced. However, the focus of the current study was the immediate time period after acetylcholine exposure, which was consistent with time course of acetylcholine-induced [Ca2+]ioscillations in our previous study. 14 Indeed, other studies have shown that halothane induces formation of IP3in neuroblastoma cells 29 and erythrocytes. 30 Furthermore, our comparisons have been performed both in the presence and absence of muscarinic stimulation.
Effect of Halothane on Intracellular Ca2+Concentration
The transient [Ca2+]iresponse of both intact and β-escin–permeabilized ASM cells to halothane is consistent with previous evidence from other tissues such as pituitary cells, 8 cardiac muscle, 6,31 vascular smooth muscle, 32,33 and our recent study on ASM. 14 Because all of the experiments in this study were performed under conditions where both Ca2+influx and efflux across the plasma membrane were blocked, the results clearly indicate that the elevation in [Ca2+]iis caused by SR Ca2+release. The current study demonstrates that the decrease in SR Ca2+content is mediated by increased Ca2+leak through both IP3receptor and RyR channels.
In a recently published study, we demonstrated that clinically relevant concentrations of halothane affect acetylcholine-induced [Ca2+]ioscillations in ASM cells. 14 We had previously established that acetylcholine-induced [Ca2+]ioscillations represent repetitive SR Ca2+release and reuptake, where initiation of oscillations is dependent on Ca2+release through IP3receptor channels, but sustenance of oscillations occurs through Ca2+-induced Ca2+release mechanisms via  RyR channels. 11,12,15 The amplitude of [Ca2+]ioscillations thus represented SR Ca2+content, and the frequency represented Ca2+-induced Ca2+release sensitivity. We found that halothane decreases both the amplitude and frequency of the oscillations and therefore decreases SR Ca2+content and reduces the sensitivity for Ca2+-induced Ca2+release. However, the mechanisms underlying halothane effects on the SR were not examined.
The current study demonstrates a direct effect of halothane on Ca2+release through IP3receptors in ASM. This is supported by the interactions between PLC inhibition via  U73122 and IP3receptor channel blockage with XeD. Clearly, part of the [Ca2+]iresponse to halothane does occur because of elevated IP3concentrations alone, as indicated by the decreased [Ca2+]iresponse in the presence of U73122. However, the fact that XeD produces further decrement in the [Ca2+]iresponse indicates a direct effect on the IP3receptor channels. Halothane-induced SR Ca2+release through IP3receptor channels has been previously demonstrated in pituitary cells. 8 On the other hand, other studies in different cell systems have found that halothane inhibits the [Ca2+]iresponse to agonists known to work predominantly via  the IP3mechanism. 34–36 These differing results may be related to a number of factors, including the type of IP3receptor channel involved and the relative sensitivities of the channel to IP3itself versus  halothane (given the fact that IP3concentrations are also differentially affected).
Another major finding in the current study was the effect of halothane on SR Ca2+release through RyR channels, resulting in decreased SR Ca2+content. These data are also consistent with our previous study on halothane effects on acetylcholine-induced [Ca2+]ioscillations, 14 which involve repetitive release through these channels. 11,15 Our data are also consistent with studies in cardiac muscle, 6,9,31 skeletal muscle, 37 and vascular smooth muscle 33,34 demonstrating that volatile anesthetics increase SR Ca2+leak through RyR channels. Therefore, it is likely that halothane-induced Ca2+release through RyR channels also contributes to ASM relaxation via  decreased SR Ca2+content.
In addition to a direct effect on the RyR channel itself, halothane may have effects on upstream [Ca2+]iregulatory mechanisms. In ASM cells, acetylcholine stimulation also leads to production of cADPR, which has been shown to be a major second messenger system in a number of cell types (see review by Lee 38). In a recent study, 13 we demonstrated that cADPR indirectly releases SR Ca2+through RyR channels in ASM cells. Further studies are required to determine the effects of halothane on acetylcholine-induced elevation of cADPR concentrations in ASM cells. These effects would only further contribute to decreased [Ca2+]i.
In summary, halothane affects [Ca2+]iregulation in porcine ASM cells by decreasing SR Ca2+content, mediated through increased Ca2+leak through both IP3and RyR channels. These effects likely contribute to anesthetic-induced decrease in the ASM response to receptor stimulation.
The authors thank Thomas Keller, B.S. (Research Technician, Department of Anesthesiology, Mayo Clinic and Foundation, Rochester, MN), for technical assistance in the studies.
References
Jones KA, Housmans PR, Warner DO, Lorenz RR, Rehder K: Halothane alters cytosolic calcium transient in tracheal smooth muscle. Am J Physiol 1993; 265: L80–6Jones, KA Housmans, PR Warner, DO Lorenz, RR Rehder, K
Jones KA, Lorenz RR, Morimoto N, Sieck GC, Warner DO: Halothane reduces force and intracellular Ca2+in airway smooth muscle independently of cyclic nucleotides. Am J Physiol 1995; 268: L166–72Jones, KA Lorenz, RR Morimoto, N Sieck, GC Warner, DO
Yamakage M, Kohro S, Kawamata T, Namiki A: Inhibitory effects of four inhaled anesthetics on canine tracheal smooth muscle contraction and intracellular Ca2+concentration. Anesth Analg 1993; 77: 67–72Yamakage, M Kohro, S Kawamata, T Namiki, A
Warner DO, Jones KA, Lorenz RR: The effects of halothane pretreatment on manganese influx induced by muscarinic stimulation of airway smooth muscle. Anesth Analg 1997; 84: 1366–71Warner, DO Jones, KA Lorenz, RR
Yamakage M, Hirshman CA, Croxton TL: Volatile anesthetics inhibit voltage-dependent Ca2+channels in porcine tracheal smooth muscle cells. Am J Physiol 1995; 268: L187–91Yamakage, M Hirshman, CA Croxton, TL
Connelly TJ, Coronado R: Activation of the Ca2+release channel of cardiac sarcoplasmic reticulum by volatile anesthetics. A nesthesiology 1994; 81: 459–69Connelly, TJ Coronado, R
Yamakage M, Kohro S, Matsuzaki T, Tsuchida H, Namiki A: Role of intracellular Ca2+stores in the inhibitory effect of halothane on airway smooth muscle contraction. A nesthesiology 1998; 89: 165–73Yamakage, M Kohro, S Matsuzaki, T Tsuchida, H Namiki, A
Hossain MD, Evers AS: Volatile anesthetic-induced efflux of calcium from IP3-gated stores in clonal (GH3) pituitary cells. A nesthesiology 1994; 80: 1379–89Hossain, MD Evers, AS
Lynch Cr, Frazer MJ: Anesthetic alteration of ryanodine binding by cardiac calcium release channels. Biochim Biophys Acta 1994;1194:109–17
Coburn RF, Baron CB: Coupling mechanisms in airway smooth muscle. Am J Physiol 1990; 258: L119–33Coburn, RF Baron, CB
Kannan MS, Prakash YS, Brenner T, Mickelson JR, Sieck GC: Role of ryanodine receptor channels in Ca2+oscillations of porcine tracheal smooth muscle. Am J Physiol 1997; 272: L659–64Kannan, MS Prakash, YS Brenner, T Mickelson, JR Sieck, GC
Sieck GC, Kannan MS, Prakash YS: Heterogeneity in dynamic regulation of intracellular calcium in airway smooth muscle cells. Can J Physiol Pharmacol 1997; 75: 878–88Sieck, GC Kannan, MS Prakash, YS
Prakash YS, Kannan MS, Walseth TF, Sieck GC: Role of cyclic ADP ribose in the regulation of [Ca2+]iin porcine tracheal smooth muscle. Am J Physiol 1998; 274: C1653–60Prakash, YS Kannan, MS Walseth, TF Sieck, GC
Pabelick CM, Prakash YS, Kannan MS, Jones KA, Warner DO, Sieck GC: Effect of halothane on intracellular calcium oscillations in porcine tracheal smooth muscle cells. Am J Physiol 1999; 276: L81–9Pabelick, CM Prakash, YS Kannan, MS Jones, KA Warner, DO Sieck, GC
Prakash YS, Kannan MS, Sieck GC: Regulation of intracellular calcium oscillations in porcine tracheal smooth muscle cells. Am J Physiol 1997; 272: C966–75Prakash, YS Kannan, MS Sieck, GC
Challiss RA, Batty IH, Nahorski SR: Mass measurements of inositol(1,4,5)trisphosphate in rat cerebral cortex slices using a radioreceptor assay: Effects of neurotransmitters and depolarization. Biochem Biophys Res Commun 1988; 157: 684–91Challiss, RA Batty, IH Nahorski, SR
Bredt DS, Mourey RJ, Snyder SH: A simple, sensitive, and specific radioreceptor assay for inositol 1,4,5-trisphosphate in biological tissues. Biochem Biophys Res Commun 1989; 159: 976–82Bredt, DS Mourey, RJ Snyder, SH
Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265–75Lowry, OH Rosenbrough, NJ Farr, AL Randall, RJ
Takahashi A, Camacho P, Lechleiter JD, Herman B: Measurement of intracellular calcium. Physiol Rev 1999; 79: 1089–1125Takahashi, A Camacho, P Lechleiter, JD Herman, B
Prakash YS, van der Heijden HF, Gallant EM, Sieck GC: Effect of beta-adrenoceptor activation on [Ca2+]iregulation in murine skeletal myotubes. Am J Physiol 1999; 276: C1038–45Prakash, YS van der Heijden, HF Gallant, EM Sieck, GC
Van Dyke RA, Wood CL: Binding of radioactivity from 14 C-labeled halothane in isolated perfused rat livers. A nesthesiology 1973; 38: 328–32Van Dyke, RA Wood, CL
Fabiato A: Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Meth Enzymol 1988; 157: 378–416Fabiato, A
Godt RE, Lindley BD: Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog. J Gen Physiol 1982; 80: 279–97Godt, RE Lindley, BD
Gafni J, Munsch JA, Lam TH, Catlin MC, Costa LG, Molinski TF, Pessah IN: Xestospongins: Potent membrane permeable blockers of the inositol 1,4,5-trisphosphate receptor. Neuron 1997; 19: 723–33Gafni, J Munsch, JA Lam, TH Catlin, MC Costa, LG Molinski, TF Pessah, IN
Yang CM, Ong R, Chen YC, Hsieh JT, Tsao HL, Tsai CT: Effect of phorbol ester on phosphoinositide hydrolysis and calcium mobilization induced by endothelin-1 in cultured canine tracheal smooth muscle cells. Cell Calcium 1995; 17: 129–40Yang, CM Ong, R Chen, YC Hsieh, JT Tsao, HL Tsai, CT
Ding KH, Husain S, Akhtar RA, Isales CM, Abdel-Latif AA: Inhibition of muscarinic-stimulated polyphosphoinositide hydrolysis and Ca2+mobilization in cat iris sphincter smooth muscle cells by cAMP-elevating agents. Cell Signal 1997; 9: 411–21Ding, KH Husain, S Akhtar, RA Isales, CM Abdel-Latif, AA
Bezprozvanny I, Watras J, Ehrlich BE: Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature 1991; 351: 751–4Bezprozvanny, I Watras, J Ehrlich, BE
Taylor CW: Inositol trisphosphate receptors: Ca2+-modulated intracellular Ca2+channels. Biochim Biophys Acta 1998; 1436: 19–33Taylor, CW
Smart D, Smith G, Lambert DG: Halothane and isoflurane enhance basal and carbachol-stimulated inositol (1,4,5) triphosphate formation in SH-SY5Y human neuroblastoma cells. Biochem Pharmacol 1994; 47: 939–45Smart, D Smith, G Lambert, DG
Rooney TA, Hager R, Stubbs CD, Thomas AP: Halothane regulates G-protein-dependent phospholipase C activity in turkey erythrocyte membranes. J Biol Chem 1993; 268: 15550–6Rooney, TA Hager, R Stubbs, CD Thomas, AP
Wheeler DM, Katz A, Rice RT, Hansford RG: Volatile anesthetic effects on sarcoplasmic reticulum Ca content and sarcolemmal Ca flux in isolated rat cardiac cell suspensions. A nesthesiology 1994; 80: 372–82Wheeler, DM Katz, A Rice, RT Hansford, RG
Fehr DM, Larach DR, Zangari KA, Schuler HG: Halothane constricts bovine pulmonary arteris by release of intracellular calcium. J Pharmacol Exp Ther 1996; 277: 706–13Fehr, DM Larach, DR Zangari, KA Schuler, HG
Su JY, Zhang CC: Intracellular mechanisms of halothane’s effect on isolated aortic strips of the rabbit. A nesthesiology 1989; 71: 409–17Su, JY Zhang, CC
Sill JC, Uhl C, Eskuri S, Van Dyke R, Tarara JG: Halothane inhibits agonist-induced inositol phosphate and Ca2+signaling in A7r5 cultured vascular smooth muscle cells. Mol Pharmacol 1991; 40: 1006–13Sill, JC Uhl, C Eskuri, S Van Dyke, R Tarara, JG
Loeb A: Alteration of calcium mobilization in endothelial cells by volatile anesthetics. Biochem Pharmacol 1993; 45: 1137–42Loeb, A
Kohro S, Yamakage M: Direct inhibitory mechanisms of halothane on human platelet aggregation. A nesthesiology 1996; 85: 96–106Kohro, S Yamakage, M
Blanck TJ, Peterson CV, Baroody B, Tegazzin V, Lou J: Halothane, enflurane, and isoflurane stimulate calcium leakage from rabbit sarcoplasmic reticulum. A nesthesiology 1992; 76: 813–21Blanck, TJ Peterson, CV Baroody, B Tegazzin, V Lou, J
Lee HC: Mechanisms of calcium signaling by cyclic ADP ribose and NAADP. Physiol Rev 1997; 77: 1133–64Lee, HC
Fig. 1. Effect of halothane on inositol triphosphate (IP3) concentrations in porcine airway smooth muscle cells. IP3concentrations were measured using radioimmunoreceptor assay. Compared with unstimulated control, IP3concentrations were significantly (P  < 0.05) elevated by exposure to acetylcholine, 2 minimum alveolar concentration (MAC) halothane, and a combination thereof. The IP3response to simultaneous application of acetylcholine and halothane was significantly greater than that induced by either agent alone. Inhibition of phospholipase C by U73122 significantly blunted the IP3response to both acetylcholine and halothane, but the effect on halothane was considerably smaller. *Significant (P  < 0.05) difference from unstimulated control. †Significant difference from acetylcholine exposure alone. §Significant difference from halothane exposure alone.
Fig. 1. Effect of halothane on inositol triphosphate (IP3) concentrations in porcine airway smooth muscle cells. IP3concentrations were measured using radioimmunoreceptor assay. Compared with unstimulated control, IP3concentrations were significantly (P 
	< 0.05) elevated by exposure to acetylcholine, 2 minimum alveolar concentration (MAC) halothane, and a combination thereof. The IP3response to simultaneous application of acetylcholine and halothane was significantly greater than that induced by either agent alone. Inhibition of phospholipase C by U73122 significantly blunted the IP3response to both acetylcholine and halothane, but the effect on halothane was considerably smaller. *Significant (P 
	< 0.05) difference from unstimulated control. †Significant difference from acetylcholine exposure alone. §Significant difference from halothane exposure alone.
Fig. 1. Effect of halothane on inositol triphosphate (IP3) concentrations in porcine airway smooth muscle cells. IP3concentrations were measured using radioimmunoreceptor assay. Compared with unstimulated control, IP3concentrations were significantly (P  < 0.05) elevated by exposure to acetylcholine, 2 minimum alveolar concentration (MAC) halothane, and a combination thereof. The IP3response to simultaneous application of acetylcholine and halothane was significantly greater than that induced by either agent alone. Inhibition of phospholipase C by U73122 significantly blunted the IP3response to both acetylcholine and halothane, but the effect on halothane was considerably smaller. *Significant (P  < 0.05) difference from unstimulated control. †Significant difference from acetylcholine exposure alone. §Significant difference from halothane exposure alone.
×
Fig. 2. Representative examples of the intracellular Ca2+concentration ([Ca2+]i) response of intact (A  ) and β-escin–permeabilized (B  ) airway smooth muscle cells to 2 minimum alveolar concentration (MAC) halothane. In intact cells, Ca2+influx and efflux across the plasma membrane were nonspecifically blocked using zero extracellular Ca2+and lanthanum chloride. During these conditions, exposure to halothane induced a transient [Ca2+]iresponse that was reproducible after an intervening washout. A similar effect was elicited with cells in which plasma membrane effects were eliminated by permeabilization. Amplitude bar for (A  ) only.
Fig. 2. Representative examples of the intracellular Ca2+concentration ([Ca2+]i) response of intact (A 
	) and β-escin–permeabilized (B 
	) airway smooth muscle cells to 2 minimum alveolar concentration (MAC) halothane. In intact cells, Ca2+influx and efflux across the plasma membrane were nonspecifically blocked using zero extracellular Ca2+and lanthanum chloride. During these conditions, exposure to halothane induced a transient [Ca2+]iresponse that was reproducible after an intervening washout. A similar effect was elicited with cells in which plasma membrane effects were eliminated by permeabilization. Amplitude bar for (A 
	) only.
Fig. 2. Representative examples of the intracellular Ca2+concentration ([Ca2+]i) response of intact (A  ) and β-escin–permeabilized (B  ) airway smooth muscle cells to 2 minimum alveolar concentration (MAC) halothane. In intact cells, Ca2+influx and efflux across the plasma membrane were nonspecifically blocked using zero extracellular Ca2+and lanthanum chloride. During these conditions, exposure to halothane induced a transient [Ca2+]iresponse that was reproducible after an intervening washout. A similar effect was elicited with cells in which plasma membrane effects were eliminated by permeabilization. Amplitude bar for (A  ) only.
×
Fig. 3. Effect of halothane on inositol triphosphate (IP3) receptor channels in intact (A  ) and β-escin–permeabilized (B  ) airway smooth muscle (ASM) cells. During conditions of blocked Ca2+influx and efflux, halothane induced a transient intracellular Ca2+concentration ([Ca2+]i) response even when IP3receptor channels were blocked by a membrane permeant inhibitor, Xestospongin D (XeD). These data indicate that halothane increases sarcoplasmic reticulum Ca2+release also through ryanodine receptor (RyR) channels. The decrease in the [Ca2+]iresponse to halothane during these conditions is consistent with figure 3, indicating the IP3receptor component. Amplitude bar for (A  ) only. MAC = minimum alveolar concentration.
Fig. 3. Effect of halothane on inositol triphosphate (IP3) receptor channels in intact (A 
	) and β-escin–permeabilized (B 
	) airway smooth muscle (ASM) cells. During conditions of blocked Ca2+influx and efflux, halothane induced a transient intracellular Ca2+concentration ([Ca2+]i) response even when IP3receptor channels were blocked by a membrane permeant inhibitor, Xestospongin D (XeD). These data indicate that halothane increases sarcoplasmic reticulum Ca2+release also through ryanodine receptor (RyR) channels. The decrease in the [Ca2+]iresponse to halothane during these conditions is consistent with figure 3, indicating the IP3receptor component. Amplitude bar for (A 
	) only. MAC = minimum alveolar concentration.
Fig. 3. Effect of halothane on inositol triphosphate (IP3) receptor channels in intact (A  ) and β-escin–permeabilized (B  ) airway smooth muscle (ASM) cells. During conditions of blocked Ca2+influx and efflux, halothane induced a transient intracellular Ca2+concentration ([Ca2+]i) response even when IP3receptor channels were blocked by a membrane permeant inhibitor, Xestospongin D (XeD). These data indicate that halothane increases sarcoplasmic reticulum Ca2+release also through ryanodine receptor (RyR) channels. The decrease in the [Ca2+]iresponse to halothane during these conditions is consistent with figure 3, indicating the IP3receptor component. Amplitude bar for (A  ) only. MAC = minimum alveolar concentration.
×
Fig. 4. Effect of halothane on ryanodine receptor channels in intact (A  ) and β-escin–permeabilized (B  ) airway smooth muscle cells. During conditions of blocked Ca2+influx and efflux, halothane induced a transient intracellular Ca2+concentration ([Ca2+]i) response even when ryanodine receptor channels were blocked by high concentrations of ryanodine. These data indicate that halothane increases sarcoplasmic reticulum Ca2+release through inositol triphosphate receptor channels. The decrease in the [Ca2+]iresponse to halothane during these conditions suggests a ryanodine receptor component. Amplitude bar for (A  ) only. MAC = minimum alveolar concentration.
Fig. 4. Effect of halothane on ryanodine receptor channels in intact (A 
	) and β-escin–permeabilized (B 
	) airway smooth muscle cells. During conditions of blocked Ca2+influx and efflux, halothane induced a transient intracellular Ca2+concentration ([Ca2+]i) response even when ryanodine receptor channels were blocked by high concentrations of ryanodine. These data indicate that halothane increases sarcoplasmic reticulum Ca2+release through inositol triphosphate receptor channels. The decrease in the [Ca2+]iresponse to halothane during these conditions suggests a ryanodine receptor component. Amplitude bar for (A 
	) only. MAC = minimum alveolar concentration.
Fig. 4. Effect of halothane on ryanodine receptor channels in intact (A  ) and β-escin–permeabilized (B  ) airway smooth muscle cells. During conditions of blocked Ca2+influx and efflux, halothane induced a transient intracellular Ca2+concentration ([Ca2+]i) response even when ryanodine receptor channels were blocked by high concentrations of ryanodine. These data indicate that halothane increases sarcoplasmic reticulum Ca2+release through inositol triphosphate receptor channels. The decrease in the [Ca2+]iresponse to halothane during these conditions suggests a ryanodine receptor component. Amplitude bar for (A  ) only. MAC = minimum alveolar concentration.
×
Fig. 5. Effect of simultaneous inhibition of both inositol triphosphate (IP3) and ryanodine receptor (RyR) channels on the intracellular Ca2+concentration ([Ca2+]i) response of intact airway smooth muscle (ASM) cells to halothane. In the presence of blocking concentrations of both Xestospongin D and ryanodine, halothane did not elevate [Ca2+]i, indicating that the effect of halothane on the sarcoplasmic reticulum is mediated entirely via  the two receptor channels. MAC = minimum alveolar concentration.
Fig. 5. Effect of simultaneous inhibition of both inositol triphosphate (IP3) and ryanodine receptor (RyR) channels on the intracellular Ca2+concentration ([Ca2+]i) response of intact airway smooth muscle (ASM) cells to halothane. In the presence of blocking concentrations of both Xestospongin D and ryanodine, halothane did not elevate [Ca2+]i, indicating that the effect of halothane on the sarcoplasmic reticulum is mediated entirely via 
	the two receptor channels. MAC = minimum alveolar concentration.
Fig. 5. Effect of simultaneous inhibition of both inositol triphosphate (IP3) and ryanodine receptor (RyR) channels on the intracellular Ca2+concentration ([Ca2+]i) response of intact airway smooth muscle (ASM) cells to halothane. In the presence of blocking concentrations of both Xestospongin D and ryanodine, halothane did not elevate [Ca2+]i, indicating that the effect of halothane on the sarcoplasmic reticulum is mediated entirely via  the two receptor channels. MAC = minimum alveolar concentration.
×
Fig. 6. Summary of the effects of halothane on inositol triphosphate versus  ryanodine receptor channels. Statistical comparisons were made to the intracellular Ca2+concentration ([Ca2+]i) response to the first exposure to halothane alone. Accordingly, the control for these interactions was a second exposure to halothane alone (first bar). § Significant (P  < 0.05) difference from first halothane exposure. ‡ Significant difference from exposure to Xestospongin D or ryanodine exposure alone.
Fig. 6. Summary of the effects of halothane on inositol triphosphate versus 
	ryanodine receptor channels. Statistical comparisons were made to the intracellular Ca2+concentration ([Ca2+]i) response to the first exposure to halothane alone. Accordingly, the control for these interactions was a second exposure to halothane alone (first bar). § Significant (P 
	< 0.05) difference from first halothane exposure. ‡ Significant difference from exposure to Xestospongin D or ryanodine exposure alone.
Fig. 6. Summary of the effects of halothane on inositol triphosphate versus  ryanodine receptor channels. Statistical comparisons were made to the intracellular Ca2+concentration ([Ca2+]i) response to the first exposure to halothane alone. Accordingly, the control for these interactions was a second exposure to halothane alone (first bar). § Significant (P  < 0.05) difference from first halothane exposure. ‡ Significant difference from exposure to Xestospongin D or ryanodine exposure alone.
×