Effects of Halothane on Sarcoplasmic Reticulum Calcium Release Channels in Porcine Airway Smooth Muscle Cells

After obtaining porcine tracheae from a local abattoir, the smooth muscle layer was dissected, and ASM cells were dissociated using previously described techniques. 11,12,14,15Briefly, 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.

Measurements of IP³in ASM samples were performed using a radioreceptor assay. 16,17ASM 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). 16,17The measurement technique is similar to that used by other investigators. 7A Lowry protein assay was used to measure protein concentrations for normalization of IP³concentrations. 18 

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. 11,12,15Briefly, 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, 11,12,15a 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.

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 (K^d) of the fluorescent dye differs in vitro  versus  in vivo  (see Takahashi et al.  19for a review). Therefore, as in previous studies, 15,20we 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:

to calculate [Ca²⁺]ⁱlevels from fluorescence values (F), where F^minis the fluorescence at minimal [Ca²⁺]ⁱconcentrations (0 nm in this study) and F^maxis the fluorescence at saturating concentrations, determined using a buffer and ionophore technique similar to the one described above. 19Using the F^min, F^max, and gray level values from our measurements in ASM cells as previously described, we calculated the apparent K^dfor 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 

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. 21In the solutions used to examine the effects of halothane, the concentrations for 2 MAC halothane were 0.47 ± 0.03 mm.

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. 14,15During 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. 11,14This 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, 22with 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.

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. 24In 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. 11,14In 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.

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. 11,15In 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.

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.

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.

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.

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. 14The 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.

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. 14To 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.

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).

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).

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.

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).

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).

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.

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.

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. 14Furthermore, 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 K^dof 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 1–3through voltage-gated L-type Ca²⁺channels. 3,5However, 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 

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, 26which has been thought to inhibit agonist-induced IP³either by inhibition of PLC or activation of phosphatases, or by protein kinase A 26acting 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, 27,28such 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 28on 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.  7In 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. 14Indeed, other studies have shown that halothane induces formation of IP³in neuroblastoma cells 29and erythrocytes. 30Furthermore, our comparisons have been performed both in the presence and absence of muscarinic stimulation.

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, 8cardiac muscle, 6,31vascular smooth muscle, 32,33and our recent study on ASM. 14Because 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. 14We 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. 11,12,15The 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. 8On 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. 34–36These 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, 14which involve repetitive release through these channels. 11,15Our data are also consistent with studies in cardiac muscle, 6,9,31skeletal muscle, 37and vascular smooth muscle 33,34demonstrating 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, 13we 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.