The distal airway is more important in the regulation of airflow resistance than is the proximal airway, and volatile anesthetics have a greater inhibitory effect on distal airway muscle tone. The authors investigated the different reactivities of airway smooth muscles to volatile anesthetics by measuring porcine tracheal or bronchial (third to fifth generation) smooth muscle tension and intracellular concentration of free Ca2+ ([Ca2+]i) and by measuring inward Ca2+ currents (ICa) through voltage-dependent Ca2+ channels (VDCs).
Intracellular concentration of free Ca2+ was monitored by the 500-nm light emission ratio of Ca2+ indicator fura-2. Isometric tension was measured simultaneously. Whole-cell patch clamp recording techniques were used to investigate the effects of volatile anesthetics on ICa in dispersed smooth muscle cells. Isoflurane (0-1.5 minimum alveolar concentration) or sevoflurane (0-1.5 minimum alveolar concentration) was introduced into a bath solution.
The volatile anesthetics tested had greater inhibitory effects on carbachol-induced bronchial smooth muscle contraction than on tracheal smooth muscle contraction. These inhibitory effects by the anesthetics on muscle tension were parallel to the inhibitory effects on [Ca2+]i. Although tracheal smooth muscle cells had only L-type VDCs, some bronchial smooth muscle cells (approximately 30%) included T-type VDC. Each of the two anesthetics significantly inhibited the activities of both types of VDCs in a dose-dependent manner; however, the anesthetics had greater inhibitory effects on T-type VDC activity in bronchial smooth muscle.
The existence of the T-type VDC in bronchial smooth muscle and the high sensitivity of this channel to volatile anesthetics seem to be, at least in part, responsible for the different reactivities to the anesthetics in tracheal and bronchial smooth muscles.
VOLATILE anesthetics at clinically relevant concentrations have a potent and direct inhibitory effect on airway smooth muscle. 1–3The direct effects of these anesthetics on airway smooth muscle are thought to be ultimately caused by a decrease in intracellular concentration of free Ca2+([Ca2+]i), 2,3a primary regulator of smooth muscle tone. 4This decrease is, in part, a result of a blockade of Ca2+influx through L-type voltage-dependent Ca2+channels (VDCs). 5In studies performed in vivo , 6,7variations in airway resistance in the larger, more proximal airway, but not the more distal airway, 8have been evaluated. In studies performed in vitro , 9the direct effects of anesthetics on the trachea, but only as distal as the segmental bronchus, have been evaluated.
The direct effects of volatile anesthetics on distal airway smooth muscle may be more important clinically. The lung region that is important in regulation of airflow resistance is between the third- and seventh-generation bronchi, 10,11and a series of studies have shown that there were significant physiological and pharmacologic differences between tracheal and bronchial smooth muscles. 12,13Mazzeo et al. 14,15demonstrated, by measuring muscle tension, that volatile anesthetics had a more inhibitory effect on distal airway muscle tone than on proximal airway muscle tone. On the other hand, Croxton et al. 16showed that peripheral airway smooth muscle was more resistant to dihydropyridine-sensitive (L-type) VDC antagonists than was tracheal smooth muscle, indicating that L-type VDCs are the predominant mechanism for Ca2+entry in tracheal smooth muscle. Recently, Janssen 17found T-type VDCs as well as L-type VDCs in canine bronchial smooth muscle cells by using the whole-cell patch clamp technique. We therefore speculated that the difference in distributions of T- and L-type VDCs is related to the difference in reactivities to volatile anesthetics 14,15and to dihydropyridine-sensitive VDC antagonists in proximal and distal airway smooth muscles. 16,18
This study was conducted to test this hypothesis by simultaneously measuring porcine tracheal or bronchial (third to fifth generation) smooth muscle tension and [Ca2+]iusing the fluorescence technique 2,4and by measuring inward Ca2+currents through VDCs (ICa) using patch clamp techniques. 5,19We also investigated the inhibitory effects of the volatile anesthetics isoflurane and sevoflurane on these muscle tones with changes in [Ca2+]iand on these channels’ activities.
Materials and Methods
The protocol for this study was approved by the Sapporo Medical University Ethical Committee on Animal Research. Adult pigs of either sex (Sus scrofa , weighing 30–45 kg) were sedated with ketamine (25 mg/kg intramuscularly) and anesthetized with pentobarbital sodium (7–8 mg/kg intravenously). The animals were 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. 20
Simultaneous Measurement of Muscle Tension and [Ca2+]i
Tracheal (1 mm wide and 8 mm long) and bronchial (1 mm wide and 5 mm long) smooth muscle strips were loaded with 5 μm acetoxymethyl ester of fura-2, an indicator of Ca2+, in a physiological salt solution containing 0.02% (vol/vol) cremophor EL for 6 or 7 h at room temperature (22–24°C). The physiological salt solution contained 136.9 mm NaCl, 5.4 mm KCl, 1.5 mm CaCl2, 1.0 mm MgCl2, 23.9 mm NaHCO3, 5.5 mm glucose, and 0.01 mm EDTA. This solution was saturated with a gas mixture of 95% O2–5% CO2at 37°C (pH ∼7.4). Each fura-2–loaded muscle strip was held in a temperature-controlled (37°C) organ bath, and one end of the muscle strip was connected to a strain gauge transducer (LVS-20GA; Kyowa, Tokyo, Japan). Experiments were performed using a fluorescence spectrometer (CAF-100; Japan Spectroscopic, Tokyo, Japan). Excitation light was passed through a rotating filter wheel (48 Hz) that contained 340- and 380-nm filters. The light emitted from the muscle strip at 500 nm was measured using a photomultiplier. The ratio of the fluorescence resulting from excitation at 340 nm to that at 380 nm (R340/380) was calculated and used as an indicator of [Ca2+]i. 2,4
Physiological salt solution aerated with 95% O2–5% CO2was used for the control bath solution, and the airway smooth muscle strips were allowed to equilibrate for 30 min after being mounted in the bath. To establish an optimal length, the resting tension was adjusted to 2 g for tracheal and 1 g for bronchial smooth muscle strips. These values were selected as the optimal values for maximal active force generation determined in preliminary experiments using repeated carbachol contractions and various baseline tensions. 2,14–16Both tissues were contracted with submaximal effect (∼ED95) concentrations of carbachol (1 μm), a stable potent muscarinic receptor agonist. After the contractions had reached a steady state, the tissues were exposed to a bath solution equilibrated with one of two volatile anesthetics: isoflurane (0.5 [0.9% at the vaporizer], 1.0 [1.8%], or 1.5 [2.7%] minimum alveolar concentration [MAC] in the pig 21) or sevoflurane (0.5 [1.4%], 1.0 [2.8%], 1.5 [4.2%] MAC in the pig 22). Similar to this experiment, the tissue strips were exposed to 1 μm nifedipine, a dihydropyridine-sensitive VDC antagonist, during carbachol-induced contraction.
Measurement of Voltage-dependent Ca2+Channel Activity
We used conventional whole-cell patch clamp techniques 19to observe inward Ca2+currents (ICa) through VDCs. 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 was added. 5Cells were dispersed by trituration, filtered through nylon mesh, and centrifuged. The pellet was resuspended in a modified Kraftbrühe solution 23and 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 adjusted to 7.25 with tris-[hydroxymethyl]aminomethane [Tris]).
The experiments were performed at 37°C. Micropipettes were pulled from soda lime “hematocrit” tubing (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. Recording solutions were chosen to inhibit Na+–K+currents and enhance Ca2+currents. The pipette solution contained 130 mm CsCl, 4.0 mm MgCl2, 10 mm EGTA, 5.0 mm Na2ATP, and 10 mm HEPES (pH adjusted to 7.2 with Tris). The bath solution contained 130 mm tetraethylammonium chloride, 1.0 mm MgCl2, 10 mm CaCl2, 10 mm glucose, and 10 mm HEPES (pH adjusted to 7.4 with Tris). 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 (3–20 MΩ) with slight suction, the patch membrane was disrupted by strong negative pressure. Membrane currents were monitored using a CEZ-2400 patch clamp amplifier (Nihon Kohden, Tokyo, Japan), and the amplifier output 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.
Inward Ca2+currents were elicited by 100-ms depolarizing pulses (−60 to +40 mV) from a holding potential of −80 or −40 mV. A holding potential of −40 mV was used to elicit ICathrough L-type VDCs. 17,24ICathrough T-type VDCs was obtained by digital subtraction of ICaobtained at a holding potential of −40 mV from total ICaelicited from a holding potential of −80 mV in the same cell. 24We also confirmed the presence of two types of ICas in these cells by pharmacologic identification. The Ca2+channel blockers with a dihydropyridine structure in these cells are relatively selective for the ICathrough L-type VDCs. 17,18
Voltage-pulse protocols were performed in control solutions for more than 5 min to obtain a stable baseline. Cells were then exposed to bath solution equilibrated with one of two volatile anesthetics: isoflurane (0.5 [0.9% at the vaporizer], 1.0 [1.8%], or 1.5 [2.7%] MAC) or sevoflurane (0.5 [1.4%], 1.0 [2.8%], or 1.5 [4.2%] MAC). The temperature-controlled perfusion chamber (MT-1; Narishige) consisted of a glass coverslip bottom, with needles placed for rapid solution changes. 25The chamber volume was approximately 1 ml, and complete solution changes in the chamber could be obtained within 1 min using a peristaltic pump (CTP-3; Iuchi, Tokyo, Japan) attached to the input and output ports. After 6-min exposure, the perfusate was switched again to the control solution.
Inactivation curves were determined, using a double-pulse protocol that consisted of a 3-s prepulse to a potential in the range of −80 to +10 mV, followed by a 100-ms depolarization to +10 mV. To observe the inactivation curve of Ca2+currents through T-type VDCs, we used another double-pulse protocol that consisted of a 3-s prepulse to a potential in the range of −120 to −10 mV, followed by a 100-ms depolarization to −10 mV. 17The peak change in the current was expressed as a fraction of that obtained using the −80- or −20-mV prepulse, and this quantity was least-squares fitted to a Bolzman expression to estimate the potential of half-maximal inactivation (V1/2) and the slope factor (k). 26Similar to the ICaexperiment, the effects of the volatile anesthetics on the inactivation curves were also shown.
Measurement of Anesthetic Concentrations in the Gas Phase and in the Bath Solution
Anesthetic concentrations were measured according to the previously described method. 25Briefly, the vaporizers for isoflurane and sevoflurane were calibrated using an infrared anesthetic gas monitor (5250 RGM; Datex-Ohmeda, Madison, WI). Concentrations of the anesthetic agents in bath solution samples were analyzed using a gas chromatograph (GC-17A; Shimadzu, Kyoto, Japan). The 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 concentrations 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. There were no significant differences between the concentrations of these anesthetics in the perfusion chamber for patch clamp recording and those in the bath solution of a spectrometer (n = 4, data not shown).
The following drugs and chemicals were used:β-hydroxybutyrate, cremophor EL, fatty acid–free bovine serum albumin, Na2ATP, pyruvic acid, creatine, taurine, nifedipine, EGTA (Sigma Chemical, St. Louis, MO), acetoxymethyl ester of fura-2, (Dojindo, Kumamoto, Japan), EDTA (Katayama, Osaka, Japan), 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. For the measurement of [Ca2+]iand muscle tension, carbachol-induced sustained changes in [Ca2+]i(indicated by R340/380) and muscle tension were used as references (100%). Changes in measured parameters with exposure to each anesthetic were compared at each point (concentrations or applied potential) using the paired two-tailed t test. One-way analysis of variance for repeated measurements and the Fisher exact test were used to determine the concentration-dependent effects. In all comparisons, P < 0.05 was considered to be significant.
Effects of Volatile Anesthetics on Tension and [Ca2+]iin Tracheal and Bronchial Smooth Muscle Strips
As has been reported previously, 2,27R340/380, an indicator of [Ca2+]i, was rapidly increased by 1 μm carbachol with a concomitant contraction in a tracheal smooth muscle strip (fig. 1A). During carbachol-induced contraction, 1.5 MAC isoflurane significantly decreased both the muscle tension and [Ca2+]i. In a bronchial smooth muscle strip (fig. 1B), carbachol similarly increased R340/380with a concomitant contraction; however, the maximum tension (1.1 ± 0.3 g) was significantly lower than that (4.0 ± 0.7 g) of tracheal smooth muscle strips. Isoflurane (1.5 MAC) similarly and significantly decreased both muscle tension and [Ca2+]iin a bronchial smooth muscle strip; however, the inhibitory effects of isoflurane on them seemed to be greater than those in a tracheal smooth muscle strip. Sevoflurane had similar inhibitory effects on muscle tension and [Ca2+]iin both tracheal and bronchial smooth muscle tissues (raw data not shown). The relations between anesthetic potencies (MAC) and percentage of responses of muscle tension and [Ca2+]iare shown in figure 2. In both tracheal and bronchial smooth muscle tissues, the volatile anesthetics tested significantly decreased muscle tension and [Ca2+]iin a dose-dependent manner, and there were no significant differences between these anesthetics in the inhibitory potencies on both muscle tension and [Ca2+]i. The inhibitory effects by these anesthetics were, however, significantly greater in bronchial smooth muscles than in tracheal smooth muscles at any MAC tested.
Figure 3shows the effects of 1 μm nifedipine on carbachol-induced muscle contraction and increase in [Ca2+]iin tracheal and bronchial smooth muscle strips. Nifedipine significantly decreased the muscle tension and [Ca2+]iin both tissues (fig. 3A). However, the inhibitory effects on muscle tension and [Ca2+]iwere significantly greater in tracheal smooth muscle tissue (muscle tension by 66 ± 9% and [Ca2+]iby 97 ± 4%, respectively) than in bronchial smooth muscle tissue (muscle tension by 40 ± 6% and [Ca2+]iby 78 ± 8%, respectively) (P < 0.01; n = 7;fig. 3B).
Characteristics of Voltage-dependent Ca2+Channels in Tracheal and Bronchial Smooth Muscle Cells
The ICaobserved in porcine tracheal smooth muscle cells during step depolarizations from −80 mV peaked at approximately 10 ms and was inactivated with a time constant of approximately 50–100 ms (fig. 4A). During baseline conditions, threshold activation of ICaoccurred at −20 mV, and maximum peak current amplitude was obtained at +10 mV (fig. 4B). The maximum peak ICawas −439 ± 97 pA (range, −249 to −754 pA). As shown in figure 4A(a superimposed trace), voltage steps from a holding potential of −40 mV to +10 mV elicited similar currents. All tracheal smooth muscle cells tested (n = 56) showed these characteristics. The addition of 1 μm nifedipine virtually eliminated the ICaof tracheal smooth muscle cells by 96% (n = 7 each).
The commonly encountered ICas from bronchial smooth muscle cells were similar to the ICain tracheal smooth muscle cells as shown in figure 4. In this case, the ICawas maximally activated within 5–10 ms after depolarization and inactivated with a time constant of approximately 50–100 ms. The maximum peak ICawas −372 ± 63 pA (range, −196 to −679 pA). In 29% of bronchial smooth muscle cells (24 of 82), however, ICas were evoked at much more negative potentials and had a more complicated time course as illustrated in figure 5. In this case, membrane depolarizations from a holding potential of −80 mV elicited a rapidly inactivating, low-threshold current at a negative potential (−40 to −20 mV), which was maximally activated at 0 mV (fig. 5A). In contrast, as shown in figure 5B, voltage steps from a holding potential of −40 mV in the same cells elicited only a sustained ICathat resembled the long-lasting current elicited at a holding potential of −80 mV. This sustained ICawas activated at −20 mV with peak activation at +10 mV. The differences between the activation thresholds and the kinetics of inactivation between the two current types suggested the presence of two Ca2+channel types in some of the porcine bronchial smooth muscle cells. The current–voltage relations of the ICas shown in figures 5A and 5Bare illustrated in figure 5C. ICas elicited from the holding potential of −80 mV were larger than those elicited from −40 mV. Subtraction of the latter ICa(holding potential of −40 mV) from the former ICa(holding potential of −80 mV) provided the current–voltage relation of the second Ca2+current, corresponding to the ICathrough transient (T-type) VDCs. 17,24
We confirmed the presence of two types of Ca2+channel currents in bronchial smooth muscle cells by pharmacologic identification. Figure 6illustrates the effect of 1 μm nifedipine, an L-type VDC antagonist, on ICa. The L-type ICawas recorded during depolarizing pulses from a holding potential of −40 mV to +10 mV (fig. 6A), whereas the transient ICawas elicited by depolarizing from −80 mV to −20 mV (fig. 6B). Recordings in figures 6A and 6Bshow that nifedipine decreased the peak amplitude of the L-type ICaby 92 ± 7%, whereas in the same cells it suppressed the T-type ICaby only 14 ± 6% (n = 4).
Effects of Volatile Anesthetics on T- and L-type Ca2+Channel Currents
Figure 7shows the effects of 1.5 MAC sevoflurane on whole-cell ICain tracheal and bronchial smooth muscle cells. To elicit the ICathrough L-type VDCs in tracheal and bronchial smooth muscle cells, stepwise depolarizations (−30 to +40 mV) from a holding potential of −40 mV were used. 17,24Sevoflurane significantly and similarly inhibited both ICas in tracheal (fig. 7A) and bronchial (fig. 7B) smooth muscle cells without changes in the time course of the currents. The lower figures show the relations between peak ICaand command potential before and after exposure to 1.5 MAC sevoflurane. Sevoflurane significantly inhibited ICathroughout the voltage range studied. There was no apparent shift in the voltage dependence of induced ICa. As shown in figure 7C, a stepwise depolarization of a bronchial smooth muscle cell from a holding potential of −80 mV resulted in the activation of mixed ICacarried through both L- and T-type VDCs. Sevoflurane decreased ICain a bronchial smooth muscle cell without changes in the time course of the currents. Although the anesthetic significantly inhibited ICathroughout the voltage range studied, there was a +10 mV shift of the peak ICaversus command potential curve toward more positive potentials (fig. 7C). Isoflurane showed similar inhibitory effects on the ICas in both tracheal and bronchial smooth muscle cells (data not shown).
We determined the anesthetic potency dependence of the inhibition of peak ICaby each of these volatile anesthetics. The inhibitions of peak ICathrough T-type VDCs in bronchial smooth muscle cells were obtained by digital subtraction of the currents obtained at a holding potential of −40 mV from total currents elicited from a holding potential of −80 mV in the same cells. Figures 8A and 8Bshow the relations between the percent control peak ICaand the anesthetic potencies (MAC). Each of the two volatile anesthetics significantly inhibited peak ICain a dose-dependent manner. Based on the anesthetic potencies, sevoflurane required somewhat greater concentrations to achieve the same inhibitory effect as that of isoflurane. There was no significant difference between tracheal and bronchial smooth muscles in the inhibitory effects on the ICathrough L-type VDCs; however, both anesthetics induced greater inhibitory effects on ICathrough T-type VDCs in bronchial smooth muscles than they did through L-type ICas in either tissue at each MAC tested (P < 0.05; n = 7).
The effects of the volatile anesthetics isoflurane and sevoflurane at equi-effective inhibitory potencies (1.0 and 1.5 MAC, respectively) on the inactivation curves of ICas are summarized in figure 9and table 1. The inactivation curve for T-type Ca2+current in bronchial smooth muscle cells was obtained by using nifedipine (1 μm) to block the Ca2+current through L-type VDCs before examining the voltage dependence of inactivation (fig. 9C). Each of these anesthetics shifted the inactivation curve to more negative potentials in either type of airway smooth muscle tissues or in either type of VDCs. The induced changes in V1/2were indistinguishable between tracheal and bronchial smooth muscle tissues or between T-type and L-type VDCs. The slope factor, k, was not changed by exposure to each anesthetic.
Effects of Volatile Anesthetics and Nifedipine on Muscle Tension and [Ca2+]iin Tracheal and Bronchial Smooth Muscles
One of the major findings of our study is that both isoflurane and sevoflurane had greater inhibitory effects on agonist-induced bronchial smooth muscle contraction than on tracheal smooth muscle contraction (figs. 1 and 2). This result is consistent with that by Mazzeo et al. 14,15These inhibitory effects by the anesthetics on muscle tension were parallel to the inhibitory effects on [Ca2+]i. [Ca2+]i, which plays a central role in the regulation of airway smooth muscle tone, 4is regulated by the influx of Ca2+through membrane-associated Ca2+channels (VDCs and voltage-independent receptor-operated Ca2+channels) and by the release of Ca2+from intracellular Ca2+stores. 28,29Entry of extracellular Ca2+through VDCs is necessary for maintenance of the contraction of airway smooth muscle. 2,4,28–30Our results therefore indicated that volatile anesthetics had different effects on the Ca2+influx through VDCs in tracheal and bronchial smooth muscles.
There are two possible reasons for these different effects on Ca2+influx by volatile anesthetics. The anesthetics could have different inhibitory effects on the same type of VDCs, or the anesthetics could have different inhibitory effects on different subtypes of VDCs in tracheal and bronchial smooth muscles. Comparison of nifedipine-induced relaxation responses from tracheal and bronchial smooth muscle preparations contracted by carbachol (fig. 3) suggested that the two tissues responded differently to the dihydropyridine-sensitive (L-type) Ca2+channel antagonist. This result is similar to the finding of Croxton et al. , 16and it is consistent with the clinical observations that a dihydropyridine-sensitive Ca2+channel antagonist was ineffective in reversing bronchospasm in individuals with asthma. 31,32We also measured changes in [Ca2+]iwith muscle tension and found that the inhibitory effects on muscle tension by nifedipine were parallel to the inhibitory effects on [Ca2+]i. In tracheal smooth muscle, 1 μm nifedipine decreased [Ca2+]iby 97%, whereas the L-type VDC antagonist decreased it by only 78% in bronchial smooth muscle.
The results obtained by using the fluorescence technique in the current study indicate that a dihydropyridine-insensitive and volatile anesthetic–sensitive pathway of Ca2+influx may exist in porcine distal airway smooth muscle. To investigate this mechanism, the ICaactivities through VDCs in smooth muscle cells obtained from porcine tracheae and bronchi were evaluated using the whole-cell patch clamp techniques.
Electric Properties of Inward Ca2+Currents in Tracheal and Bronchial Smooth Muscle
As has previously been reported in porcine 5,33and canine 25,34tracheal smooth muscle cells, we measured depolarization-induced ICain freshly dispersed porcine tracheal smooth muscle cells under ionic conditions designed to inhibit K+/Na+currents and to enhance Ca2+currents. These ICas showed a threshold and peak activation at −20 mV and +10 mV, respectively (fig. 4). The inactivation parameters V1/2and k were −17.6 and 7.6 mV, respectively (table 1). Based on their time and voltage dependences and their sensitivity to blockade by nifedipine (fig. 4), these currents are presumed to reflect the activity of L-type VDCs. 35
The commonly encountered ICas from bronchial smooth muscle cells (approximately 70%) were similar to those seen in tracheal smooth muscle cells. Even when the cells were depolarized from a holding potential of either −80 or −40 mV, we observed similar ICa, indicating that these bronchial smooth muscle cells have only L-type VDCs. 17,24The rest of the bronchial smooth muscle cells (approximately 30%), however, showed different characteristics of ICaduring stepwise depolarizations from a holding potential of −80 mV (fig. 5A). As shown in figures 5A and 5C, the ICas were evoked at much more negative potentials and showed a rapidly inactivating current at a negative potential (−40 to −20 mV). Voltage steps from a holding potential of −40 mV in the same cells elicited only sustained ICas that resembled the L-type ICa(fig. 5B). Subtraction of the latter ICa(holding potential of −40 mV) from the former ICa(holding potential of −80 mV) provided the I-V relation of the second Ca2+current (fig. 5C). This second current was first noted at −50 or −40 mV with maximum activation at −10 mV. Inactivation parameters V1/2and k were −58.7 and 6.1 mV, respectively (table 1), and these currents were insensitive to nifedipine (fig. 6B). These characteristics are consistent with T-type VDCs. 17,24
The results from the current experiments showed that two types (L- and T-types) of VDCs coexist in some porcine bronchial smooth muscle cells; however, no evidence of the existence of a second VDC in tracheal smooth muscle was obtained. It seems that the presence of T-type VDCs in approximately 30% of bronchial smooth muscle cells cannot completely explain the very different responses to nifedipine in tracheal and bronchial smooth muscles. T-type VDC has been suggested to play a prominent role in the initiation of action potentials rather than in [Ca2+]ihomeostasis in other tissues because of their transient opening time and their small conductance. 36However, T-type VDCs induced a window current at potentials ranging from −50 to −10 mV. Depolarization of the cell membrane at potentials in this range would lead to a persistent Ca2+influx through these T-type VDCs, which in turn could contribute to excitation–contraction coupling as well as refilling of the internal Ca2+stores. 37,38Furthermore, T-type ICas are not suppressed during agonist stimulation, 17as is the case for L-type ICas. 39,40The small size of the T-type ICadoes not necessarily lessen the possible importance of their contribution to excitation. Accordingly, it is possible that the different responses to nifedipine between these channels are partly caused by the different electric properties of VDCs in tracheal and bronchial smooth muscles. In addition, as suggested by Croxton et al. , 16another pathway of dihydropyridine-insensitive receptor-operated Ca2+channelss 41may also play a role in the different responses to nifedipine between tracheal and bronchial smooth muscles, although no evidence suggests that the Ca2+influx through receptor-operated Ca2+channels are different in these airway smooth muscle tissues.
Effects of Volatile Anesthetics on T- and L-type Voltage-dependent Ca2+Channel Activities
Since we confirmed that two types of VDCs coexist in some porcine bronchial smooth muscle cells, we evaluated the inhibitory effects of the volatile anesthetics isoflurane and sevoflurane on L-type ICain tracheal and bronchial smooth muscle cells and on T-type ICain bronchial smooth muscle cells separately. Unlike the classical Ca2+channel antagonist nifedipine, which has a much greater inhibitory effect on L-type than on T-type ICas (fig. 6), both isoflurane and sevoflurane had a greater inhibitory effect on T-type ICathan on L-type ICa(figs. 7 and 8). These results are consistent with the results of experiments in which muscle tension and [Ca2+]iwere measured (figs. 1 and 2). A series of investigations has been conducted to examine the possible actions of volatile anesthetics on different types of VDCs, and it has been shown that the activities of both L- and T-type VDCs in cardiac Purkinje cells appeared to be approximately equally suppressed by halothane, isoflurane, and enflurane. 24In clonal (GH3) pituitary cells, however, it has been found that there were different sensitivities to the reduction by halothane between T- and L-type VDCs activities. 42Recent molecular studies have revealed structural heterogeneity between VDCs of different tissues, 43suggesting that these apparent discrepancies may result from the differences of cell types and species or the experimental conditions used. Taking our current findings into account, there seems to be some information concerning the actions of volatile anesthetics at the level of membrane-associated channels, although it remains to be determined whether the action of the anesthetics on Ca2+channels is a direct effect on the channel proteins or whether it is a secondary consequence of, for example, alterations in membrane lipids. 44
Because of the different properties, such as resting membrane potential, of the distal and proximal airway smooth muscles, 45,46the different responses of volatile anesthetics on distal and proximal airway smooth muscles is also likely, in part, to be caused by the different effects of the anesthetics on the common L-type VDCs, which are identified extensively in airway smooth muscle. The absolute magnitude of the resting membrane potential is greater in the bronchus (−70 mV) than in the trachea (−60 mV), 46and this difference is thought to be caused by reduced Na+permeability, not to an increase in K+permeability. 45However, we have obtained direct evidence that the inhibitory effects of volatile anesthetics on L-type ICas in bronchial and tracheal smooth muscles are not different (fig. 8). Therefore, the substantial inhibitory effects of the anesthetics on T-type VDC activity could, at least in part, be caused by the fact that bronchial smooth muscle is more sensitive to volatile anesthetics than is tracheal smooth muscle.
To further examine the inhibitory actions of these volatile anesthetics on VDCs of tracheal (L-type) and bronchial (T- and L-type) smooth muscle cells, we studied the effects of these anesthetics on steady state, voltage-dependent inactivation of ICas. During prolonged depolarization, a fraction of the VDCs enters an unavailable or “inactivated” state. The degree of steady state inactivation depends on the prepulse potential (fig. 9). Each of the two volatile anesthetics tested significantly shifted the inactivation curves to more negative potentials without changing the sigmoid shapes of the curves. A qualitatively similar shift induced by some dihydropyridine-sensitive Ca2+channel antagonists in porcine tracheal smooth muscle cells has been interpreted as evidence of drug-induced stabilization of the inactivated state. 47There were no significant differences in the shift of the inactivation curve either between tracheal and bronchial smooth muscles L-type VDCs or between T- and L-type VDCs of bronchial smooth muscle.
In conclusion, the volatile anesthetics isoflurane and sevoflurane at clinically relevant potencies had greater inhibitory effects on carbachol-induced bronchial smooth muscle contraction than on tracheal smooth muscle contraction. These inhibitory effects on muscle tension induced by the anesthetics were parallel to the inhibitory effects on [Ca2+]i, indicating that the anesthetics have different effects on the Ca2+influx through VDCs. Although tracheal smooth muscle cells have only L-type VDCs, we have found some bronchial smooth muscle cells (∼30%) that have T-type as well as L-type VDCs. Each of the two volatile anesthetics significantly inhibited the activities of both types of VDC in a dose-dependent manner; however, the anesthetics had greater inhibitory effects on T-type VDC activity in bronchial smooth muscle. The existence of the T-type VDC in bronchial smooth muscle and the high sensitivity of this channel to volatile anesthetics seem to be, at least in part, responsible for the different reactivities to the anesthetics in tracheal and bronchial smooth muscles.