Low-temperature Modification of the Inhibitory Effects of Volatile Anesthetics on Airway Smooth Muscle Contraction in Dogs

The protocol for this study was approved by the Sapporo Medical University Ethical Committee on Animal Research. Adult mongrel dogs weighing 10–15 kg were anesthetized with 10 mg/kg intramuscular ketamine and killed by exsanguination. The tracheas were excised quickly, and the epithelium, cartilage, and connective tissue were stripped from the smooth muscle.

Tissue preparation was performed according to the previously described method. 8Briefly, tracheal smooth muscle strips (1 mm wide and 8 mm long) were pretreated with 5 μm acetoxymethyl ester of fura-2, an indicator of Ca²⁺, in a physiologic salt solution containing 0.02% (vol/vol) cremophor EL for 6 or 7 h at room temperature (22–24°C). The 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. The temperature of the bath solution was monitored continuously using a thermometer (CTM-205; Terumo, 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 (R^340/380) was calculated and used as an indicator of [Ca²⁺]ⁱ. 8,9Contractions were induced by high-K⁺(72.7 mm) and carbachol (1 μm), a potent muscarinic receptor agonist. After the contractions reached a steady state, the temperature of the organ bath was changed from 37°C to 34 or 31°C. In another experiment, the tissues were exposed to a bath solution equilibrated using one of two volatile anesthetics: isoflurane (1.0, 2.0, or 3.0% at the vaporizer) or sevoflurane (1.0, 2.0, or 3.0%), with simultaneous changes in the temperature of the bath solution (34°C or 31°C).

Because there is a possibility that changes in temperature per se  can change the affinity of fura-2 for Ca²⁺, 10we confirmed the effect of temperature on R^340/380of the tracheal smooth muscle strips by the use of the skinned fiber technique. 11The muscle strips (0.1–0.2 mm wide) were superfused for 20 min using a relaxing solution containing 50 μg/ml saponin and 5 μm acetoxymethyl ester of fura-2. The relaxing solution was made with the following composition using the algorithm of Fabiato and Fabiato:12Mg-ATP: 7.5 mm; EGTA: 4.0 mm; imidazol: 20 mm; dithiothreitol: 1.0 mm; free Mg²⁺: 1.0 mm; free Ca²⁺: 1 nm; creatine phosphate: 10 mm; and creatine phosphokinase: 0.1 mg/ml. After incubation with 0.3 μm free Ca²⁺, the temperature of the organ bath was changed within the range between 37 and 31°C. R^340/380, an indicator of [Ca²⁺]ⁱ, did not change within the temperature range tested with or without 1 μm carbachol (n = 4, data not shown).

We used conventional whole cell patch clamp techniques to observe inward Ca²⁺currents (I^Ca) through VDCCs. 13Tracheal smooth muscle tissue was minced and digested for 20 min at 37°C in Ca²⁺-free Tyrode’s solution to which 0.08% (wt/vol) collagenase was added. 14Cells were dispersed by trituration, filtered through nylon mesh, and centrifuged. The pellet was resuspended in a modified Kraftbrühe solution 15and stored at 4°C for up to 5 h before use. The modified Kraftbrühe solution contained the following: KCl: 85 mm; K²HPO⁴: 30 mm; MgSO⁴: 5.0 mm; Na²ATP: 5.0 mm; pyruvic acid: 5.0 mm; creatine: 5.0 mm; taurine: 20 mm; β-hydroxybutyrate: 5.0 mm; and 0.1% (wt/vol) fatty acid–free bovine serum albumin, with pH adjusted to 7.25 with 0.5 m tris[hydroxymethyl]aminomethane.

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). Resistances were 3–5 MΩ if filled with solution. The pipette solution contained the following: CsCl: 130 mm; MgCl²: 4.0 mm; EGTA: 10 mm; Na²ATP: 5.0 mm; and HEPES: 10 mm, with pH adjusted to 7.2 with tris[hydroxymethyl]aminomethane. The bath solution contained the following: tetraethylammonium chloride: 130 mm; MgCl²: 1.0 mm; CaCl²: 10 mm; glucose: 10 mm; and HEPES: 10 mm, with pH adjusted to 7.4 with tris[hydroxymethyl]aminomethane. An aliquot (approximately 0.5 ml) of the cell suspension was placed in a chamber on the stage of an inverted microscope. A micromanipulator was used to position the patch pipette against the membrane of a tracheal smooth muscle cell. After obtaining a high-resistance seal (> 5 GΩ), 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.

Inward Ca²⁺currents were elicited by 100-ms depolarizing pulses (−50 to 40 mV) from a holding potential of −80 mV. Leak currents were subtracted, and membrane capacitance and series resistance were compensated. After a stable baseline of peak I^Cawas obtained at 37°C, the temperature of the bath solution was changed to 31°C. In another experiment, cells were exposed to a bath solution equilibrated using one of two volatile anesthetics, isoflurane (1.0, 2.0, or 3.0%) or sevoflurane (1.0, 2.0, or 3.0%), with simultaneous change in the temperature of the bath solution from 37 to 31°C. Inactivation curves also were determined, using a double-pulse protocol that consisted of a 3-s prepulse to a potential in the range between −80 and 10 mV, followed by a 100-ms depolarization to 10 mV. The peak change in the current was expressed as a fraction of that obtained using the −80-mV prepulse, and this quantity was least-squares fitted to a Boltzman expression 16to estimate the potential of half-maximal inactivation and the slope factor.

Bath temperature during the patch clamp recording was controlled using a circular thermofoil heater 115 mm in diameter (MT-1; Narishige), with a 35-mm central opening. Temperature sensing was performed by means of a small insulated thermocouple situated no more than 5 mm from that cell from which recordings were being made. The overall stability of this system was ±0.1°C from the set point.

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 (1.0, 2.0, and 3.0% in the gas phase) were 0.26, 0.52, and 0.82 mm, respectively, whereas the mean concentrations of sevoflurane in the solution (1.0, 2.0, and 3.0% in the gas phase) were 0.16, 0.35, and 0.54 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, or between the concentrations of different temperatures of the bath solution in the range between 31 and 37°C (n = 4, data not shown).

The following drugs and chemicals were used:β-hydroxybutyrate, cremophor EL (Sigma Chemical, St. Louis, MO), acetoxymethyl ester of fura-2 (Dojindo Laboratories, Kumamoto, Japan), type I collagenase (Gibco Laboratories, Grand Island, NY), isoflurane (Ohio Medical, Madison, WI), and sevoflurane (Maruishi Pharmacoceutical, Osaka, Japan).

Data are expressed as the mean ± SD. For the measurement of [Ca²⁺]ⁱand muscle tension, high-K⁺–induced sustained changes in [Ca²⁺]ⁱ(indicated by R^340/380) and muscle tension at 37°C 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.

At 37°C, the ratio R^340/380, an indicator of [Ca²⁺]ⁱ, was increased rapidly by high K⁺(72.7 mm), with a concomitant contraction (fig. 1) . [Ca²⁺]ⁱand muscle tension reached their respective peaks within 10–50 s. With carbachol, [Ca²⁺]ⁱalso increased rapidly to a concentration similar to that induced by high K⁺. The amount of muscle contraction with carbachol, however, was more than twice that induced by high K⁺, indicating that carbachol induces a greater contraction than does high K⁺at a given [Ca²⁺]ⁱ. Exposure to low temperature (34°C) significantly decreased high-K⁺–induced muscle contraction and increase in [Ca²⁺]ⁱ. Low temperature also decreased the carbachol-induced increase in [Ca²⁺]ⁱ; however, it enhanced carbachol-induced muscle contraction. The effects of 3% isoflurane with a decrease in temperature (31°C) are shown in figure 2. Isoflurane significantly and similarly inhibited high-K⁺–induced muscle contraction, with an increase in [Ca²⁺]ⁱ, and carbachol-induced muscle contraction, with an increase in [Ca²⁺]ⁱ. The relations between anesthetic concentrations and percentage of response of muscle tension or [Ca²⁺]ⁱare shown in figure 3. Linear relations were observed between anesthetic concentrations and percentage of response of muscle tension and between anesthetic concentrations and percentage of response of [Ca²⁺]ⁱ. High-K⁺–induced muscle contraction and increase in [Ca²⁺]ⁱwere significantly decreased by low-temperature exposure in a gradient-dependent manner at all concentrations of isoflurane (fig. 3A, n= 7). Similar effects of low-temperature exposure also were seen with sevoflurane (by 10–18% at 31°C, n = 6; data not shown). Exposure to low temperature, however, significantly prevented the inhibitory effect of sevoflurane on carbachol-induced muscle contraction, and the increase in [Ca²⁺]ⁱcaused by carbachol was inhibited significantly by exposure to low-temperature exposure at all concentrations of sevoflurane (n = 7;fig. 3B). Similar effects of exposure to low temperature were seen with isoflurane (by 11–18% at 31°C, n = 6; data not shown).

Voltage–pulse protocols were performed in control solutions for more than 5 min to obtain a stable baseline value. Data from cells that showed unstable I^Caamplitudes, less than 100 pA of peak I^Ca, or a more than 10% reduction in amplitude during the control recording period were discarded. Technically satisfactory data were obtained from 84 cells studied at the holding potential of −80 mV. In all cases, recordings were performed at two different temperatures (37 and 31°C). At any given temperature, there was sufficient consistency in peak current amplitude and time course to justify the pooling of records according to temperature. At 37°C, I^Caseen in dispersed canine tracheal smooth muscle cells during step depolarization from −80 mV peaked at approximately 10 ms and was inactivated with a time constant in the range between 70 and 100 ms (fig. 4A). During baseline conditions, threshold activation of I^Caoccurred at approximately −20 mV, and maximum peak current amplitude was obtained at approximately 10 mV (fig. 4B). The maximum peak I^Cawas −439 ± 48 pA (range, −260–−789 pA). The inactivation parameters obtained in 28 cells during control conditions were a potential of half-maximal inactivation of 14.6 ± 3.2 mV and a slope factor of 7.4 ± 1.2 mV. I^Cacurrents with a similar time course were observed in the inactivation experiments.

As shown in a representative trace for depolarization from −80 to 10 mV (fig. 4A), the amplitude of I^Cawas clearly sensitive to temperature, and exposure to low temperature (31°C) inhibited the magnitude of I^Ca. Figure 4Bshows the relation between peak I^Caand applied potential before and after exposure to low temperature (31°C). Low-temperature exposure significantly inhibited I^Caat step potentials in the range between −10 and 40 mV and decreased peak I^Caat 10 mV by approximately 18% (n = 7). There was no apparent shift in the voltage-dependence of I^Ca. We determined the dose-dependence of the inhibition of peak I^Caby each volatile anesthetic tested. Figure 5shows the relations between the percentage of control peak I^Caat 10 mV and the concentration of each anesthetic, isoflurane (fig. 5A) and sevoflurane (fig. 5B), in the gas phase. Both volatile anesthetics significantly inhibited peak I^Cain a dose-dependent manner (n = 7). Exposure to low tempera- ture significantly inhibited the I^Caat all concentrations of these anesthetics (n = 7).

The effects of the volatile anesthetics isoflurane and sevoflurane at equieffective inhibitory concentrations (2.0 and 3.0%, respectively) on the inactivation curve of I^Cacurrents are summarized in figure 6and table 1. Each of these anesthetics shifted the inactivation curve to a more negative potential (n = 7). Exposure to low temperature also significantly shifted the inactivation curve to a more negative potential (n = 7). The induced changes in the potential of half-maximal inactivation brought about both by these anesthetics and by the low temperature were statistically significant in each case. The slope factor was not changed by exposure to either the anesthetics or low temperature.

One of the major findings of our study is that in canine tracheal smooth muscle in vitro , exposure of the muscle to low temperature significantly decreased high-K⁺–induced muscle contraction and increase in [Ca²⁺]ⁱ, but it enhanced carbachol-induced muscle contraction with a decrease in [Ca²⁺]ⁱ(fig. 1). Our results are in general agreement with those of studies using various species in which exposure to low temperature has been associated with an enhancement of the agonist-induced contraction of airway smooth muscle. 1–3,17–19The results of these studies suggested that the effect of direct exposure to low temperature on airway smooth muscle per se  has some role in low-temperature–induced airway hyperresponsiveness in a clinical situation. Ishii and Shimo 20and Freed and Stream, 21however, showed in in vivo  studies that exposure of the airway to low temperature decreased the response to contractile agonists in guinea pigs and in dogs, respectively. These apparent discrepancies may result from differences in agonist types, concentrations, in vivo  or in vitro  studies, species, or the temperature gradients used in these studies.

Intracellular Ca²⁺is the primary regulator of smooth muscle contraction. 7,22[Ca²⁺]ⁱis regulated by influx of Ca²⁺through cell membrane–associated Ca²⁺channels and by release of Ca²⁺from intracellular Ca²⁺stores, especially the sarcoplasmic reticulum. 7Ishii and Shimo 3reported that increased responsiveness of the rat airway smooth muscle to acetylcholine with lowered temperature might involve the enhancement of increase in [Ca²⁺]ⁱ, resulting from the acceleration of Ca²⁺release from the sarcoplasmic reticulum, inhibition of Ca²⁺extrusion from the cell, or the inhibition of Ca²⁺reuptake by the sarcoplasmic reticulum. Release of Ca²⁺from the sarcoplasmic reticulum is, however, important in initiation, not maintenance, of the muscle contraction, 7–9and we found that the low temperature could enhance the agonist-induced muscle contractility with a decrease in [Ca²⁺]ⁱ(fig. 1). Therefore, the change in [Ca²⁺]ⁱcannot be attributed to the low-temperature–induced contraction of airway smooth muscle shown in this study. Low temperature could increase Ca²⁺sensitivity in a broad sense. Activation of muscarinic receptors also activates protein kinase C concomitant with an increase in [Ca²⁺]ⁱ. Many investigators, 9,23,24including us, 8have shown that protein kinase C activation by muscarinic agonist sensitizes contractile elements to Ca²⁺or activates a Ca²⁺-independent mechanism in airway smooth muscles, because carbachol induces a much greater contraction than does high K⁺at a given [Ca²⁺]ⁱ(fig. 1). Huang et al.  25reported that a potent exogenous protein kinase C activator, phorbol-12,13-diacetate, increased airway smooth muscle tone more at a lower temperature. Therefore, protein kinase C is thought to play a role in regulation of airway smooth muscle contractility at different temperatures. The role of protein kinase C activity in airway smooth muscle tone, however, is controversial. 26,27Investigations of this possibility, such as measurement of muscle tension in β-escin–permeabilized skinned fiber, 28must be conducted in the future.

High-K⁺solution can depolarize the cell membrane of smooth muscle 29and open VDCCs 30, resulting in a Ca²⁺influx from the extracellular fluid and muscle contraction. Sustained contraction of airway smooth muscle requires the continued entry of extracellular Ca²⁺, 31and blockade of VDCCs suppresses the sustained increase in [Ca²⁺]ⁱin carbachol-stimulated tracheal smooth muscle. 8,9We therefore hypothesized from our results obtained by using the fluorescence technique that the exposure to low temperature could have inhibited the VDCC activity, and we found, by using the whole cell patch clamp technique, that the exposure to low temperature (31°C) inhibited I^Cathrough VDCCs of freshly dispersed canine tracheal smooth muscle cells without an apparent change in the voltage-dependence of I^Ca(fig. 4). These results are consistent with those of previous studies using a variety of species. 32–35Similar effects on the temperature-dependence of channel conductance and gating kinetics have been reported 34,36,37and have been attributed to changes in the fluidity of membrane liquids that, in turn, influence the degree and rate of channel opening. The current findings are compatible with the hypothesis that structural changes within the membrane, which occur during thermotropic phase transition, are capable of altering the function of the intramembranous portion of voltage-sensitive calcium channels and leaving unaffected those portions of the channel that do not lie within the membrane per se . 34The volatile anesthetics tested in this study inhibited the activation of I^Cain a dose-dependent manner (figs. 4 and 5). These anesthetics also shifted the inactivation curve of I^Cato a more negative potential (fig. 6). These effects of volatile anesthetics on VDCCs in airway smooth muscle seem to be additive with the effects of low temperature on VDCCs and are consistent with the results of the effects of these anesthetics on [Ca²⁺]ⁱ.

There is a possibility that changes in temperature per se  could have changed the affinity of fura-2 for Ca²⁺. 10We therefore confirmed the effect of temperature on R^340/380, an indicator of [Ca²⁺]ⁱ, in tracheal smooth muscle strips by the use of the skinned fiber technique, 11and we found that the R^340/380did not change within the temperature range tested (31–37°C). There is another report suggesting that the affinity of a Ca²⁺chelate compound such as EGTA could be changed by pH. 38Grynkiewicz et al. , 39however, reported that the intensity of excitation of fura-2 by 500-nm light was not changed by pH excursion. We therefore believe that the measurement of [Ca²⁺]ⁱby the use of fura-2 in this study was reliable.

Another uncertainty is the variation in solubilities of volatile anesthetics at different temperatures. 40We confirmed that the concentrations of the volatile anesthetics in the bath solution were indistinguishable at the different temperatures tested in this study. That is why the replacement of the bath solution in this study was accomplished by changing the inflow perfusate to one that had been bubbled vigorously with each anesthetic at 37°C, and the temperature of the solution was changed during passage through the tube to the bath. We still do not know whether the solubilities of volatile anesthetics in oil or fat (cell membrane) vary at different temperatures. There is a possibility that the change in the solubility of volatile anesthetics in cell membranes at different temperatures plays a role in the low-temperature–induced suppression of VDCC activity.

Exposure to low temperature significantly decreased both high-K⁺–induced muscle contraction and increase in [Ca²⁺]ⁱ, but it enhanced carbachol-induced muscle contraction with a decrease in [Ca²⁺]ⁱ. Volatile anesthetics showed significant inhibition of both high-K⁺–induced and carbachol-induced airway smooth muscle contraction, with a concomitant decrease in [Ca²⁺]ⁱ. Exposure to low temperature significantly prevented the inhibitory effect of volatile anesthetics on carbachol-induced muscle contraction; the increase in [Ca²⁺]ⁱcaused by carbachol was inhibited significantly by the low temperature at all concentrations of the anesthetics. The inhibition of VDCC activity both by exposure to low temperature and by volatile anesthetics can be attributed, at least in part, to the decrease in [Ca²⁺]ⁱ. Low temperature could sensitize contractile elements to Ca²⁺or activate a Ca²⁺-independent mechanism if the airway smooth muscle is activated by a muscarinic agonist. 8,9