K+ and Ca2+-activated Cl- (ClCa) channel currents have been shown to contribute to the alteration of membrane electrical activity in airway smooth muscle. This study was conducted to investigate the effects of volatile anesthetics, which are potent bronchodilators, on the activities of these channels in porcine tracheal and bronchial smooth muscles.
Whole-cell patch clamp recording techniques were used to investigate the effects of superfused isoflurane (0-1.5 minimum alveolar concentration) or sevoflurane (0-1.5 minimum alveolar concentration) on K+ and ClCa channel currents in dispersed smooth muscle cells.
Isoflurane and sevoflurane inhibited whole-cell K+ currents to a greater degree in tracheal versus bronchial smooth muscle cells. More than 60% of the total K+ currents in tracheal smooth muscle appeared to be mediated through delayed rectifier K+ channels compared with less than 40% in bronchial smooth muscle. The inhibitory effects of the anesthetics were greater on the delayed rectifier K+ channels than on the remaining K+ channels. Cl- currents through ClCa channels were significantly inhibited by the anesthetics. The inhibitory potencies of the anesthetics on the ClCa channels were not different in tracheal and bronchial smooth muscle cells.
Volatile anesthetics isoflurane and sevoflurane significantly inhibited Cl- currents through ClCa channels, and the inhibitory effect is consistent with the relaxant effect of volatile anesthetics in airway smooth muscle. Different distributions and different anesthetic sensitivities of K+ channel subtypes could play a role in the different inhibitory effects of the anesthetics on tracheal and bronchial smooth muscle contractions.
VOLATILE anesthetics are potent bronchodilators, and the bronchodilation is a result of a direct effect on the airway smooth muscle as well as an indirect effect on the reflex neural pathway. 1–3One proposed mechanism for the direct relaxant effect of these anesthetics is a decrease in intracellular concentration of free Ca2+([Ca2+]i), 4,5produced mainly by an inhibition of Ca2+influx through voltage-dependent Ca2+channels (VDCCs). 6Most previous studies focused on the direct effects of anesthetics on the larger, more proximal airway; however, the distal airway, especially between the third- and seventh-generation bronchi, is important in the regulation of airflow resistance. 7,8Brown et al. 9and Mazzeo et al. 10,11, using high-resolution computed tomography and by measuring muscle tension, respectively, recently showed that volatile anesthetics have a greater inhibitory effect on distal airway muscle tone than on proximal airway muscle tone. In a more recent study, 12we demonstrated, by the use of patch clamp techniques, that T-type VDCCs existed in bronchial smooth muscle. The high sensitivity of this channel to volatile anesthetics may be partly responsible for the different reactivities to anesthetics in tracheal and bronchial smooth muscles. However, the mechanisms of the direct inhibitory action of volatile anesthetics on tracheal and bronchial smooth muscle contraction remain to be fully elucidated.
It is well established that membrane depolarization is an important contributor to activation of VDCCs, leading to Ca2+influx into cytosol and subsequent smooth muscle contraction. 13Contractile agonists such as acetylcholine, histamine, and serotonin depolarize the membranes of airway smooth muscle cells, 14,15whereas a relaxant agent such as isoproterenol hyperpolarizes the membrane. 16These observations strongly suggest that alteration in the electrical activity of smooth muscle cell membranes plays an important role in regulation of the contractile and relaxant properties of the cells in various physiologic conditions. The mechanisms of cell membrane depolarization of airway smooth muscle have been demonstrated to be caused by activation of Ca2+-activated Cl−(ClCa) channels and inhibition of K+channels. 17–20Because the Cl−equilibrium potential in smooth muscle is thought to range from −40 to −20 mV, 21opening of Cl−channels can lead to depolarization in resting cells. 17,18The ClCachannel, 18,22the large-conductance Ca2+-activated K+(KCa) channel, 19,23and delayed rectifier K+(KDR) channel 19,23have carefully been characterized at the whole-cell or single-channel current levels in airway smooth muscle. Hyperpolarization of cell membranes by volatile anesthetics has been suggested to be one of the mechanisms for the anesthetic action in several cell types. 24–26However, little is known about the effects of volatile anesthetics on K+–Cl−channel activities in airway smooth muscle. Possible different effects of volatile anesthetics on K+or Cl−channel activities in tracheal and bronchial smooth muscles could also explain the regional variations in the inhibitory actions of anesthetics on airway smooth muscle tone.
Therefore, the objective of the current study was to further clarify the effect of volatile anesthetics on K+and Cl−channel activities in both proximal and distal airway smooth muscle using whole-cell patch clamp methodology.
Materials and Methods
Isolation of Single Smooth Muscle Cells from Pig Tracheae and Bronchi
The experimental protocol used in this study was approved by the Sapporo Medical University Animal Care and Use Committee (Sapporo, Japan). Using previously described methods, 12adult pigs of both sexes (Sus scrofa , weighing 30–45 kg) were sedated with ketamine (25 mg/kg administered intramuscularly), anesthetized with pentobarbital sodium (7–8 mg/kg administered intravenously), and then killed by exsanguination. The lungs and cervical trachea were removed and placed in ice-cold Krebs-Ringer bicarbonated solution aerated with 95% O2and 5% CO2. The tracheae were excised, and the epithelium, cartilage, and connective tissue were stripped from the smooth muscle. Intrapulmonary bronchi of third to fifth generations were dissected from the surrounding parenchymal tissue, and cartilage and connective tissue were stripped from the smooth muscle. The epithelial layer was removed by gently rolling the tissue across moistened filter paper.
The tracheal and bronchial smooth muscle tissues were minced and digested for 20 min at 37°C in Ca2+-free Tyrode solution, to which 0.08% (wt/vol) collagenase had been added. 12Cells were then dispersed by trituration, filtered through nylon mesh, and centrifuged. The pellet was resuspended in a modified Kraftbrühe solution 27and stored at 4°C for up to 5 h before use. The modified Kraftbrühe solution contained 85 mm KCl, 30 mm K2HPO4, 5.0 mm MgSO4, 5.0 mm Na2ATP, 5.0 mm pyruvic acid, 5.0 mm creatine, 20 mm taurine, 5.0 mm β-hydroxybutyrate, and 0.1% (wt/vol) fatty acid-free bovine serum albumin (pH 7.25 with Tris).
Measurements of K+and Cl−Channel Activities
All experiments were performed at 37°C. Micropipettes were pulled from soda lime tubings (GC-1.5; Narishige, Tokyo, Japan) using a brown-flaming horizontal puller (model P-97; Sutter Instrument, Novato, CA). These had resistances of 3–5 MΩ when filled with solution. An aliquot (approximately 0.5 ml) of the cell suspension was placed in a perfusion chamber on the stage of an inverted microscope (IX-70; Olympus, Tokyo, Japan). A micromanipulator was used to position the patch pipette against the membrane of a tracheal or bronchial smooth muscle cell. After obtaining a high-resistance seal (5–20 GΩ) with slight suction, the patch membrane was disrupted by strong negative pressure.
Membrane currents were recorded in the tight-seal whole-cell configuration 28using a CEZ-2400 patch clamp amplifier (Nihon Kohden, Tokyo, Japan). To measure outward whole-cell K+currents (IK), recording solutions to enhance IKwere used. The bath solution contained 135 mm NaCl, 5.4 mm KCl, 1.0 mm MgCl2, 1.8 mm CaCl2, 10 mm glucose, and 10 mm HEPES (pH 7.4 with Tris). Niflumic acid (10 μm) was also added to minimize the Cl−currents (ICl) through ClCachannels (ICl(Ca)). 29The pipette solution contained 70 mm KCl, 60 mm K+-glutamate, 1.0 mm MgCl2, 2.5 mm EGTA, 5.0 mm K2ATP. 1.0 mm CaCl2, and 10 mm HEPES (pH 7.2 with Tris). To measure the whole-cell ICl(Ca), recording solutions to inhibit IKwere used. The bath solution contained 130 mm tetraethylammonium chloride, 1.0 mm MgCl2, 5.0 mm CaCl2, 10 mm glucose, and 10 mm HEPES (pH 7.4 with Tris). The pipette solution contained 130 mm CsCl, 4.0 mm MgCl2, 2.5 mm EGTA, 5.0 mm Na2ATP, 1.8 mm CaCl2, and 10 mm HEPES (pH 7.2 with Tris); the computer-calculated [Ca2+]iwas approximately 10−6m. The calculated reversal potential for Cl−(ECl) was approximately 0 mV. In another experiment, both EGTA and CaCl2were removed from the pipette solution to show the characteristics of ICl(Ca). The amplifier output of the membrane currents was low-pass filtered at 2,000 Hz. Leak currents, estimated by appropriate scaling of currents during 20-mV hyperpolarizing pulses, were subtracted from each of these records. Whole-cell IKwas elicited at 5-s intervals by 250-ms depolarizing pulses (−40 to +60 mV) from a holding potential of −70 mV. Whole-cell IClwas activated by 500-ms depolarizations in 10-mV steps to a series of potentials (−40 to +40 mV) from a holding potential of −70 mV and measured as the slow deactivating tail currents on 500-ms repolarization to the holding potential.
Experimental protocols were performed in control solutions for more than 5 min to obtain a stable baseline. Cells were exposed to a bath solution equilibrated with one of the two volatile anesthetics: isoflurane (0.5 [0.9% at the vaporizer], 1.0 [1.8%], or 1.5 [2.7%] minimum alveolar concentration [MAC]30) or sevoflurane [0.5 (1.4%), 1.0 (2.8%), or 1.5 (4.2%) MAC 31]. Charybdotoxin (100 nm), a specific KCachannel blocker, 324-aminopyridine (5 mm), a KDRchannel blocker, 32and niflumic acid (10 μm), a specific ClCachannel blocker, 29were used to identify the characteristics of membrane currents measured and to investigate the roles of these channels in the effects of the volatile anesthetics on the membrane currents. The temperature-controlled perfusion chamber (MT-1; Narishige) consisted of a glass coverslip bottom, with needles placed for rapid solution changes. 33After 5-min exposure, the perfusate was switched again to the control solution.
Measurement of Anesthetic Concentrations in the Gas Phase and in the Bath Solution
Anesthetic concentrations were measured according to the previously described method. 33Briefly, the vaporizers for isoflurane and sevoflurane were calibrated with an infrared anesthetic gas monitor (5250 RGM; Datex-Ohmeda, Madison, WI). Concentrations of the anesthetic agents in bath solution samples were analyzed with a gas chromatograph (GC-17A; Shimadzu, Kyoto, Japan). The mean concentrations of isoflurane and sevoflurane in the solutions were similar to those that had been reported. 33The mean concentrations of isoflurane in the solution at 37°C (0.9, 1.8, and 2.7% in the gas phase) were 0.25, 0.55, and 0.78 mm, respectively, whereas the mean concentration of sevoflurane in the solution (1.4, 2.8, and 4.2% in the gas phase) were 0.24, 0.56 and 0.82 mm, respectively. Each concentration of the anesthetic had a close linear correlation with each concentration of the agent in the gas phase.
The following drugs and chemicals were used: fatty acid-free bovine serum albumin, Na2ATP, pyruvic acid, creatine, taurine, β-hydroxybutyrate, EGTA, nifedipine, charybdotoxin, 4-amonopyridine, niflumic acid (Sigma Chemical, St. Louis, MO), type-I collagenase (Gibco Laboratories, Grand Island, NY), protease (Calbiochem, La Jolla, CA), sevoflurane (Maruishi, Osaka, Japan), and isoflurane (Ohio Medical, Madison, WI). Nifedipine was dissolved in ethanol (0.01% final concentration).
Data are expressed as mean ± SD. Amplitudes of whole-cell currents (IKor ICl) were measured at the end of depolarizations. Changes in the current amplitudes with exposure to each anesthetic and the measured parameters between trachea and bronchus were compared using the paired and unpaired two-tailed t test, respectively. One-way analysis of variance for repeated measurements and Fisher exact test were used to determine the concentration-dependent effects. In all comparisons, P < 0.05 was considered significant.
Electric Properties of Outward K+Currents in Tracheal and Bronchial Smooth Muscle Cells and the Effects of Volatile Anesthetics on These Currents
Figure 1Ashows a macroscopic outward IKobtained from a freshly dispersed porcine tracheal smooth muscle cell dialyzed with a pipette solution containing a [Ca2+]iof approximately 500 nm, the concentration used to enhance the IKthrough Ca2+-activated K+channels (IK(Ca)). ICl(Ca)was blocked by 10 μm niflumic acid. The IKwas activated progressively by 250-ms depolarizing pulses from a holding potential of −70 mV to consecutively more-positive membrane potentials. Stepwise depolarization from a holding potential of −70 mV to more than −30 mV elicited an outward IKwith a mean amplitude of 1,760 ± 262 pA at +60 mV (n = 57). The kinetics of the outward K+current in this study was similar to that previously reported for IKcurrents in airway smooth muscle cells. 19,34Isoflurane (1.5 MAC) caused an approximately 30% reduction in IKwithout an apparent effect on the time course of the current (fig. 1B), an effect that was reversible when the anesthetic was washed out. Sevoflurane showed a similar inhibitory effect (raw data not shown). The same experimental protocol was also performed in bronchial smooth muscle cells. A similar outward IKwith a mean amplitude of 1,376 ± 180 pA at +60 mV (n = 68) was observed, and both volatile anesthetics tested (1.5 MAC) significantly inhibited IKby approximately 22%. Figure 1Csummarizes the relation between amplitude of IKat +60 mV, expressed as a percentage of the control, and the potencies (minimum alveolar concentration) of the volatile anesthetics tested. Both the volatile anesthetics isoflurane and sevoflurane significantly and dose-dependently inhibited IK; however, the IKof tracheal smooth muscle cells was more sensitive to volatile anesthetics than was that of bronchial smooth muscle cells.
We further investigated the whole-cell IKusing pharmacologic techniques. Figure 2Ashows the result of a typical experiment in a tracheal smooth muscle cell. The addition of 5 mm 4-aminopyridine, a selective KDRchannel blocker, significantly decreased IK, and a supplementary application of 100 nm charybdotoxin, a selective KCachannel blocker, further decreased the IKto almost a zero current level. The current–voltage relation for this experiment is shown in figure 2B. Both K+channel blockers significantly suppressed the IKamplitude over the entire voltage range studied without shifting the voltage dependency of the current–voltage relation. The same experimental protocol was performed in bronchial smooth muscle cells, and figure 2Csummarizes the inhibitory effects of these K+channel blockers on the IKamplitude at +60 mV in tracheal and bronchial smooth muscle cells. Most of the whole IKin both tracheal and bronchial smooth muscle cells was occupied by these charybdotoxin- and 4-aminopyridine–sensitive K+channels. The 4-aminopyridine–sensitive IKcomponent was more than 60% in tracheal smooth muscle cells, whereas it was less than 40% in bronchial smooth muscle cells.
The effects of volatile anesthetics tested on each charybdotoxin- and 4-aminopyridine–sensitive current were examined in tracheal and bronchial smooth muscle cells. After application of 5 mm 4-aminopyridine, the remaining current could be considered as the IK(Ca). As shown in figure 3A(top), 1.5 MAC isoflurane significantly decreased the IK(Ca)in a tracheal smooth muscle cell. Similar effects were observed with sevoflurane (raw data not shown), and the same protocol was performed in bronchial smooth muscle cells. Both isoflurane and sevoflurane significantly inhibited IK(Ca), and the inhibitory effects of the volatile anesthetics tested were indistinguishable (fig. 3A, bottom). Although the IKin the case of application of 100 nm charybdotoxin was considered as the IKmainly through the KDRchannel (IK(DR)), it is possible that the IK(Ca)still existed during the condition of high [Ca2+]i(500 nm). Therefore, in another experiment, we confirmed the characteristics of the IKusing a pipette solution in which [Ca2+]iwas strongly buffered with 10 mm EGTA to minimize the IK(Ca). An application of 100 nm charybdotoxin had no effect on the current (n = 4, data not shown), and 5 mm 4-aminopyridine substantially inhibited the current at +60 mV by more than 90% (n = 4, data not shown). The effects of isoflurane and sevoflurane on the Ca2+-independent IK(DR)were examined in both tracheal and bronchial smooth muscle cells. Both volatile anesthetics significantly inhibited IK(DR), and the inhibitory effects were indistinguishable (fig. 3B). Figure 4summarizes the relation between the percentage control of IK(Ca)(fig. 4A) and IK(DR)(fig. 4B) at +60 mV and the anesthetic potencies of the anesthetics tested in tracheal and bronchial smooth muscle cells. Both isoflurane and sevoflurane significantly and dose-dependently inhibited both the IK(Ca)and IK(DR), and the inhibitory effects of the anesthetics tested on each current were indistinguishable. However, the K(DR)channel was more sensitive than was the K(Ca)channel to the volatile anesthetics.
Electric Properties of Whole-cell Ca2+-activated Cl−Currents in Tracheal and Bronchial Smooth Muscle Cells and the Effects of Volatile Anesthetics on These Currents
As shown in figure 5A, 500-ms depolarizing pulses from a holding potential of −70 mV to a series of potentials elicited an initial inward Ca2+current, which was followed by a sustained current when 130 mm CsCl and no EGTA–Ca2+in the pipette solution and 130 mm tetraethylammonium chloride in the bath solution were used. An inwardly directed tail current was observed after return to the holding potential. The kinetics of this tail current was much slower than those of the inward Ca2+current. A specific ClCachannel blocker, niflumic acid (10 μm), substantially blocked the sustained outward current and inward tail current by more than 80% (fig. 5B). Nifedipine (1 μm), a potent L-type VDCC blocker, dramatically inhibited both the Ca2+current and the tail current (fig. 5B). The patterns of the tail current were similar to those reported for ClCain rabbit coronary artery myocytes 35and rabbit esophageal smooth muscle. 36 Figure 5Cshows the current–voltage relation of the IClthrough ClCachannels (ICl(Ca)) measured at the end of the clamp steps.
To investigate the effects of volatile anesthetics on the ClCachannel activity, a pipette solution containing 2.5 mm EGTA and 1.8 mm CaCl2was used to fix the [Ca2+]ito approximately 1 μm because it is possible that the inhibitory action of volatile anesthetics on VDCCs 6,12have some effect on the ClCachannel activity. During this condition, a similar current was evoked by pulses from a holding potential of −70 mV for 500-ms depolarization in both tracheal and bronchial smooth muscle cells. When the current was evoked by a depolarization pulse from a holding potential of −70 to +20 mV, 1 μm nifedipine completely blocked the initial inward Ca2+current but had little effect on the sustained outward ICl(Ca)(n = 3, data not shown).
During these conditions, isoflurane (1.5 MAC) significantly reduced the nifedipine-insensitive ICl(Ca)in a tracheal smooth muscle cell during a depolarization pulse to +40 mV (fig. 6A). Sevoflurane induced a similar inhibitory effect on the current (raw data not shown). Figure 6Bshows the current–voltage relation of the ICl(Ca)with or without each volatile anesthetic tested in tracheal smooth muscle cells. Both volatile anesthetics, isoflurane and sevoflurane, significantly suppressed the ICl(Ca)amplitude for the entire voltage range studied. Similar experiments were performed in bronchial smooth muscle cells, and figure 6Csummarizes the relation between the percentage control of ICl(Ca)at +40 mV and the anesthetic potencies of isoflurane and sevoflurane in tracheal and bronchial smooth muscle cells. Both the volatile anesthetics tested significantly and dose-dependently inhibited the ICl(Ca), and the inhibitory effects of the anesthetics tested were indistinguishable both between tracheal and bronchial smooth muscle cells and between isoflurane and sevoflurane.
Effects of Volatile Anesthetics on Whole-cell K+Currents in Tracheal and Bronchial Smooth Muscle Cells
In this experiment, whole-cell IKwas recorded in freshly dispersed porcine tracheal and bronchial smooth muscle cells with a pipette solution containing 2.5 mm EGTA and 1.0 mm CaCl2(calculated [Ca2+]i= approximately 500 nm), and the kinetics of the IKwas similar to that previously reported in airway smooth muscles (fig. 1). 19,34The importance of K+conductance for the maintenance of normal electrical behavior in smooth muscle has long been known. 37,38K+channels appear to play important roles in setting resting membrane potential and in limiting electrical responses to excitatory stimuli. 39,40With the K+equilibrium potential at −80 to approximately −90 mV, the opening of K+channels causes membrane hyperpolarization, leading to airway smooth muscle relaxation. Volatile anesthetics are potent bronchodilators; therefore, the hyperpolarization of the cell membrane by anesthetic-induced activation of K+channels has been suggested to be one of the main mechanisms for the relaxant effects. The current study demonstrated, disappointingly, that the volatile anesthetics tested induced inhibition of K+channels in both tracheal and bronchial smooth muscle cells. Although the inhibition of K+channels can depolarize the cell membrane, leading to muscle contraction, it is speculated that a muscarinic receptor agonist, carbachol, already and substantially inhibited the whole K+channel currents in our experimental conditions. 17–20
Although it has been reported that there are many kinds of K+channel subtype in other tissues, only KCaand KDRhave carefully been identified in airway smooth muscle cells using whole-cell or single-channel patch clamp techniques. 19,23Adenosine triphosphate–activated K+channel has also been shown to exist in airway smooth muscle 41; however, simultaneous application of KCaand KDRchannel blockers substantially diminished the whole K+currents in this study, and adenosine triphosphate–activated K+channels can open when the intracellular concentration of adenosine triphosphate is less than 3 mm 41(5 mm in the pipette solution in this study). Furthermore, it has been suggested that the opening of the adenosine triphosphate–activated K+channel is not involved in halothane's relaxant effect on airway smooth muscle. 42
Because the resting membrane potential is more negative in the bronchus (−70 mV) than in the trachea (−60 mV) 43and because both isoflurane and sevoflurane had a greater inhibitory effect on whole-cell IKin tracheal than in bronchial smooth muscle cells (fig. 1), we further examined the effects of the anesthetics on each IK(Ca)or IK(DR)using a pharmacologic technique. The concentrations of K+channel blockers used in this study (5 mm for 4-aminopyridine and 100 nm for charybdotoxin) were roughly equivalent to the drug concentrations required to maximally inhibit the IKthrough KDRand KCachannels, respectively. 44,45In whole-cell studies of dissociated airway smooth muscle cells, a prominent Ca2+-sensitive outward current has been observed by several investigators, 19,40,46but the magnitude of the current varies depending on the [Ca2+]iused. The open-state probability of KCachannels was found to be low in resting conditions ([Ca2+]i= approximately 100 nm) 19,40; therefore, rather high [Ca2+]i(500 nm) was used in the current study, the concentration being similar to the [Ca2+]iduring the condition of agonist-stimulated smooth muscle. 47During this condition, the IK(Ca)component in bronchial smooth muscle cells was greater than 60%, whereas it was less than 40% in tracheal smooth muscle cells (fig. 2). This result is consistent with the finding reported by Snetkov et al. , 46that the voltage-dependent outward K+current was mainly caused by activation of KCachannels in human bronchial smooth muscles during a physiologic condition. The different distributions of the two components of IKin tracheal and bronchial smooth muscle are still not clear. However, it could explain the greater inhibitory effects of the volatile anesthetics on whole IKin tracheal than in bronchial smooth muscle cells, because the KDRchannel component was greater in tracheal than bronchial smooth muscle cells and the inhibitory effects of volatile anesthetics tested were greater on the KDRthan on KCachannel currents (figs. 2 and 4).
Effects of Volatile Anesthetics on Whole-cell Cl−Currents in Tracheal and Bronchial Smooth Muscle Cells
Our previous experiments have extensively characterized the inward Ca2+currents through VDCCs in porcine airway smooth muscle. 6,12,33All of these studies were performed using a pipette solution containing 10 mm EGTA and 0 mm Ca2+, and no tail currents were seen on the repolarization after application of depolarizing pulses. When EGTA was eliminated from the pipette solution, we observed a more complicated trace, as illustrated in figure 5. When currents were evoked by depolarization pulses from a holding potential of −70 to +20 mV, nifedipine (1 μm), an L-type VDCC blocker, blocked the initial inward, sustained outward, and inwardly directed tail currents. On the other hand, niflumic acid (10 μm), a specific ClCachannel blocker, significantly decreased the sustained outward and inwardly directed tail currents but had no effects on the initial inward currents. Because the initial inward currents are thought to be Ca2+currents, the sustained currents are consistent with ICl(Ca)described at the whole-cell current level in rabbit ventricular myocytes 48and rabbit smooth muscle cells. 35,36The ICl(Ca)observed in this study is thought to contribute to the regulation of membrane potential in airway smooth muscle. It has been shown that changes in ClCachannel activity was involved in the acetylcholine-induced depolarization in cells from human 17and guinea-pig 18airway smooth muscles. The responses of the ClCachannels are considered to be mediated by agonist-induced increase in [Ca2+]i. 17,18Because the Cl−equilibrium potential in smooth muscle is thought to range from −40 to −20 mV, 21opening of Cl−channels leads to depolarization in resting cells, 17,18but to hyperpolarization in cells that have already been depolarized to potentials more positive than the equilibrium potential.
With regard to the physiologic role of ClCachannels in airway smooth muscle, we examined the effects of the volatile anesthetics isoflurane and sevoflurane on the currents. The volatile anesthetics had similar inhibitory effects on ClCachannel activity in tracheal and bronchial smooth muscle cells. The inhibitory effects of the volatile anesthetics on the Cl−channels would hyperpolarize or repolarize the cell membrane and lead to relaxation of airway smooth muscle. However, the similar inhibitory effects of volatile anesthetics on ClCachannel activities in tracheal and bronchial smooth muscle cells indicate that the effects of volatile anesthetics on ClCachannels could not contribute to regional differences in the inhibitory effects of volatile anesthetics on tracheal and bronchial smooth muscle contractions.
In conclusion, the volatile anesthetics isoflurane and sevoflurane significantly inhibited Cl−currents through ClCachannels, and the inhibitory effect is consistent with the relaxant effect of volatile anesthetics in airway smooth muscle. Different distributions and different anesthetic sensitivities of K+channel subtypes could play a role in the different inhibitory effects of the anesthetics on tracheal and bronchial smooth muscle contractions.
The authors thank Noritsugu Tohse, M.D., Ph.D. (Professor and Chairman, Department of Physiology Section I, Sapporo Medical University School of Medicine, Sapporo, Japan) for his valuable review and comments on the manuscript, and the first author thanks Xianyi Liu, M.D. (Professor and Chairman, Department of Anesthesiology, The Renmin Hospital of Wuhan University School of Medicine, P. R. of China) for support.