Volatile anesthetics relax airway smooth muscle (ASM) by altering intracellular Ca2+ concentration ([Ca2+]i). The authors hypothesized that relaxation is produced by decreasing sarcoplasmic reticulum Ca2+ content via increased Ca2+ "leak" through both inositol trisphosphate (IP3) and ryanodine receptor channels.
Enzymatically dissociated porcine ASM cells were exposed to acetylcholine in the presence or absence of 2 minimum alveolar concentration (MAC) halothane, and IP3 levels were measured using radioimmunoreceptor assay. Other cells were loaded with the Ca2+ indicator fluo-3 and imaged using real-time confocal microscopy.
Halothane increased IP3 concentrations in the presence and absence of acetylcholine. Inhibition of phospholipase C blunted the IP3 response to halothane. Exposure to 2 MAC halothane induced a transient [Ca2+]i response, suggesting depletion of sarcoplasmic reticulum Ca2+. Exposure to 20 microM Xestospongin D, a cell-permeant IP3 receptor antagonist, resulted in a 45+/-13% decrease in the [Ca2+]i response to halothane compared with halothane exposure alone. In permeabilized cells, Xestospongin D or 0.5 mg/ml heparin decreased the [Ca2+]i response to halothane by 65+/-13% and 68+/-22%, respectively, compared with halothane alone. In both intact and permeabilized cells, 20 microM ryanodine blunted the [Ca2+]i response to halothane by 32+/-13% and 39+/-21%, respectively, compared with halothane alone. Simultaneous exposure to Xestospongin D and ryanodine completely inhibited the [Ca2+]i response to halothane.
The authors conclude that halothane reduces sarcoplasmic reticulum Ca2+ content in ASM cells via increased Ca2+ leak through both IP3 receptor and ryanodine receptor channels. Effects on IP3 receptor channels are both direct and indirect via elevation of IP3 levels.
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. 1–3These effects of halothane are partly attributable to an inhibition of Ca2+influx. 3–5Other studies in vascular smooth muscle 3and cardiac tissue 6have indicated that halothane depletes Ca2+stores (content) of the sarcoplasmic reticulum (SR) by increasing Ca2+“leakage.” In a recent study, Yamakage et al. 7demonstrated 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. 8,9Whether 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. 10More 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. 11–13In 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. 14Our 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
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.
Inositol Triphosphate Measurements
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
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. 11,12,15Briefly, 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 (Kd) 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 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. 19Using 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. 21In the solutions used to examine the effects of halothane, the concentrations for 2 MAC halothane were 0.47 ± 0.03 mm.
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. 14,15During 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.
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. 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.
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. 11,15In 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.
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.
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.
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.
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. 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.
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).
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.
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).
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 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 1–3through 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
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, 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.
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, 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.