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Anesth Analg 2002;94:84-88
© 2002 International Anesthesia Research Society

ANESTHETIC PHARMACOLOGY

The Repolarizing Effects of Volatile Anesthetics on Porcine Tracheal and Bronchial Smooth Muscle Cells

Michiaki Yamakage, MD PhD, Xiangdong Chen, MD, Akira Kimura, MD, Sohshi Iwasaki, MD, and Akiyoshi Namiki, MD PhD

Department of Anesthesiology, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan

Address correspondence and reprint requests to Michiaki Yamakage, MD, PhD, Department of Anesthesiology, Sapporo Medical University School of Medicine, South 1, West 16, Chuo-ku, Sapporo, Hokkaido 060-8543, Japan. Address e-mail to yamakage{at}sapmed.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
This study was conducted to determine the effects of volatile anesthetics (potent bronchodilators) on membrane potentials in porcine tracheal and bronchial smooth muscle cells. We used a current-clamp technique to examine the effects of the volatile anesthetics isoflurane (1.5 minimum alveolar anesthetic concentration [MAC]) and sevoflurane (1.5 MAC) on membrane potentials of porcine tracheal and bronchial (third- to fifth-generation) smooth muscle cells depolarized by a muscarinic agonist, carbachol (1 µM). The effects of volatile anesthetics on muscarinic receptor binding affinity were also investigated by using a radiolabeled receptor assay technique. The volatile anesthetics isoflurane and sevoflurane induced significant repolarization of the depolarized cell membranes in the trachea (from -19.8 to -23.6 mV and to -24.8 mV, respectively) and bronchus (from -24.7 to -29.3 mV and -30.4 mV, respectively) without affecting carbachol binding affinity to the muscarinic receptor. The repolarizing effect was abolished by a Ca2+-activated Cl- channel blocker, niflumic acid. These results indicate that volatile anesthetic-induced repolarization of airway smooth muscle cell membranes might be caused by a change in Ca2+-activated Cl- channel activity and that the different repolarized effects of the volatile anesthetics could in part contribute to the different effects of volatile anesthetics on tracheal and bronchial smooth muscle contractions.

IMPLICATIONS: By use of a current-clamp technique, the volatile anesthetics isoflurane and sevoflurane repolarized porcine airway smooth muscle cell membranes depolarized by a muscarinic agonist. This effect might be caused mainly by change in Ca2+-activated Cl- channel activity, not in K+ channel activity.


   Introduction
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 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Alteration in the electrical activity of smooth muscle cell membranes plays an important role in regulation of contractile and relaxant properties of the cells in various physiological conditions (1,2). Contractile agonists act on smooth muscle in part through membrane depolarization, leading to activation of voltage-dependent Ca2+ channels (VDCCs) and contraction (3). The changes in membrane conductance, underlying agonist-evoked depolarization in airway smooth muscle cells, include activation of Ca2+-activated Cl- (ClCa) channel conductance and suppression of K+ channel conductance (4). Volatile anesthetics are potent bronchodilators (5), and one of the main mechanisms of the direct relaxant effect of these anesthetics is a decrease in intracellular concentration of free Ca2+ (6) by inhibition of Ca2+ influx through VDCCs (7). The relaxant effects of volatile anesthetics are significantly smaller in tracheal smooth muscle than in bronchial smooth muscle (8,9). The distal airway is important in the regulation of airflow resistance (10). Hyperpolarization of cell membranes by volatile anesthetics has also been suggested to be one of the mechanisms of the anesthetic action in other types of cells (11,12). However, there is little information on the direct effects of volatile anesthetics on membrane potential in airway smooth muscle. Possible different effects on membrane potential in tracheal and bronchial smooth muscles could also explain the regional variations in the inhibitory actions of anesthetics on airway smooth muscle tone.

We therefore conducted the present study to clarify the roles of membrane potentials in the different inhibitory effects of volatile anesthetics on proximal and distal airway smooth muscle tones by measuring a muscarinic agonist-depolarized membrane potential with a current-clamp technique. Because there is a possibility that volatile anesthetics interfere with the muscarinic agonist-receptor binding affinity, resulting in repolarization of cell membranes, we also investigated the effects of volatile anesthetics on muscarinic receptor binding affinity by using a radiolabeled receptor assay technique.


   Methods
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 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
This study was approved by the Animal Care and Use Committee of our institution. By use of previously described methods (9), adult pigs of either sex (weighing 30–45 kg) were sedated, anesthetized, and then killed by exsanguination. The lungs and cervical trachea were removed and placed in ice-cold Krebs-Ringer solution aerated with 95% oxygen and 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.

For a radioligand-binding receptor study, crude membranes were prepared with a previously described method (13). The tracheal and bronchial smooth muscle tissues were minced in 0.25 M ice-cold sucrose buffer with Tris-HCl (pH 7.4) and were homogenized twice. The homogenate was centrifuged at 1,500g for 10 min at 4°C, and the supernatant was filtered through 120-µm mesh and then centrifuged at 100,000g for 30 min at 4°C. Protein concentrations in the membranes were determined by the method of Lowry et al. (14).

For current-clamp measurements, the tracheal and bronchial smooth muscle tissues were minced and digested for 20 min at 37°C in Ca2+-free Tyrode’s solution to which 0.08% (wt/vol) collagenase had been added (9). Cells were then dispersed by trituration, filtered through mesh, and centrifuged. The pellet was resuspended in a modified Kraftbrühe solution (15) and stored at 4°C for up to 5 h before use.

In a preliminary study, the effect of a single dose of one of two volatile anesthetics, isoflurane (1.5 [2.7% at the vaporizer] minimum alveolar anesthetic concentration [MAC]) (16) and sevoflurane (1.5 [4.2%] MAC) (17), on muscarinic receptor binding affinity was confirmed by using (-)-[3H]quinuclidinyl benzilate ([3H]QNB, 40.2 Ci/mM) (18). Nonspecific binding was determined in the presence of 10-6 M atropine. Labeled and unlabeled drugs were added as 20-µL aliquots to give a final assay volume of 540 µL. After incubation with or without either anesthetic at 37°C for 30 min, the plate was placed on ice, and 50-µL aliquots of buffer were removed for determination of the free (equilibrium) concentration of [3H]QNB. The remaining buffer was pipetted out, and the wells were washed twice for 5 min. Punches were removed, placed in a vial, and counted. The density of receptors (Bmax) and the dissociation constant for the ligand were determined with linear regression and Scatchard transformation (n = 5 each). In another experiment, agonist (carbachol) binding was measured with or without either anesthetic in competition studies, in which 150 pM [3H]QNB and different concentrations of agonist were used. Equilibrium binding data were fitted by nonlinear regression analysis to a two-receptor population model (n = 5 each).

All experiments were performed at 37°C. Micropipettes were pulled from soda lime tubing (GC-1.5; Narishige, Tokyo, Japan) by use of a horizontal puller (model P-97; Sutter Instruments, Novato, CA). These had resistances of 3–5 M{Omega} when filled with solution. An aliquot 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 G{Omega}) with slight suction, the patch membrane was disrupted by strong negative pressure.

Membrane potential was recorded in the tight-seal whole-cell configuration (19) by using a CEZ-2400 patch clamp amplifier (Nihon Kohden, Tokyo, Japan). To measure the membrane potential, the following bath and pipette solutions were used (in mM): NaCl 135, KCl 5.4, MgCl2 1.0, CaCl2 1.8, glucose 10, and HEPES 10 (pH 7.4 with Tris) for the bath solution, and KCl 130, MgCl2 2.0, EGTA 5.0, CaCl2 1.0, Na2ATP 5.0, and HEPES 10 (pH 7.2 with Tris) for the pipette solution. Experimental protocols were performed in control solutions for more than 5 min to obtain a stable baseline. Cells were pretreated with 1 µM carbachol, a potent muscarinic agonist, and then exposed to bath solution equilibrated with one of the two volatile anesthetics: isoflurane (1.5 [2.7% at the vaporizer] MAC) (16) or sevoflurane (1.5 [4.2%] MAC) (17). Charybdotoxin (CHTX; 100 nM), a specific Ca2+-activated K+ channel blocker (20); 4-aminopyridine (4-AP; 5 mM), a specific delayed rectifier K+ channel blocker (20); and niflumic acid (10 µM), a specific ClCa channel blocker (21), were used to investigate the roles of these channels in the effects of volatile anesthetics on membrane potentials.

Anesthetic concentrations were measured according to a previously described method (22). Briefly, the vaporizers for isoflurane and sevoflurane were calibrated with an infrared anesthetic gas monitor (5250 RGM; Datex-Ohmeda, Madison, WI). Concentrations of the anesthetics 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 reported previously (22).

The following drugs and chemicals were used: carbachol, EGTA, CHTX, 4-AP, niflumic acid (Sigma Chemical, St. Louis, MO), type I collagenase (Gibco Laboratories, Grand Island, NY), protease (Calbiochem, La Jolla, CA), [3H]QNB (New England Nuclear, Boston, MA), sevoflurane (Maruishi, Osaka, Japan), and isoflurane (Ohio Medical, Madison, WI).

Data are expressed as means ± SD. Changes in measured variables in this study were compared by using the paired or unpaired two-tailed Student’s t-test. In all comparisons, P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
The membranes of cells in tracheal smooth muscle had significantly higher Bmax than did those of cells in bronchial smooth muscle (Table 1). Treatment of the airway smooth muscle cell membranes with a single dose (1.5 MAC) of each volatile anesthetic tested had no effect on muscarinic Bmax. Forty-three percent of the receptors displayed high-affinity binding (KH; 1.67 µM) in tracheal smooth muscle, whereas 65% of the receptors bound carbachol with KH (3.42 µM) in bronchial smooth muscle. The remaining receptors showed low binding affinity in both tracheal (low-affinity binding [KL] = 247 µM) and bronchial smooth muscles (KL = 163 µM). Equilibration of the membranes with isoflurane (1.5 MAC) or sevoflurane (1.5 MAC) did not significantly change either the distribution or the binding affinity of high-binding and low-binding receptors in tracheal and bronchial smooth muscle.


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  		Table 1. Effects of the Volatile Anesthetics Isoflurane and Sevoflurane on the Characteristics of Muscarinic Receptor Binding in Membranes of Tracheal and Bronchial Smooth Muscle Cells
 
Figure 1 shows some representative results of current-clamp experiments in tracheal smooth muscle cells. The resting membrane potential of tracheal smooth muscle cells (-45.6 ± 2.9 mV, n = 34) was significantly higher than that of bronchial smooth muscle cells (-51.9 ± 4.7 mV, n = 39) (Table 2). Application of 1 µM carbachol significantly depolarized the cell membranes by approximately 26 mV, and isoflurane significantly hyperpolarized the carbachol-induced membrane depolarization (Fig. 1A and Table 2). Sevoflurane showed a similar repolarizing effect (raw data not shown). We further investigated the effects of K+ channel blockers (CHTX for Ca2+-activated K+ channel blocker and 4-AP for delayed rectifier K+ channel blocker) and a ClCa channel blocker, niflumic acid, on the repolarizing actions of volatile anesthetics in tracheal and bronchial smooth muscle cells. As shown in Figure 1B, applications of 100 nM CHTX and 5 mM 4-AP had little effect on carbachol-induced depolarization of a tracheal smooth muscle cell. Under these conditions, sevoflurane (1.5 MAC) still had a significant hyperpolarizing effect on the membrane potential. Isoflurane also showed a similar repolarizing effect (raw data not shown). In contrast, application of 10 µM niflumic acid resulted in a significant hyperpolarization of the carbachol-induced membrane depolarization. After pretreatment of 10 µM niflumic acid, 1.5 MAC sevoflurane did not have any significant effect on the membrane potential (Fig. 1C). Similar results were obtained when isoflurane was used.



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  		Figure 1. Representative data of the effects of the volatile anesthetics tested on membrane potentials in tracheal smooth muscle cells. A, Effect of 1.5 minimum alveolar anesthetic concentration (MAC) isoflurane on carbachol (1 µM)-induced depolarization of the membrane. Isoflurane significantly hyperpolarized the membrane potential. B, Effect of 1.5 MAC sevoflurane on carbachol (1 µM)-induced depolarization of the membrane pretreated with 100 nM charybdotoxin (CHTX), a Ca2+-activated K+ channel blocker, and 5 mM 4-aminopyridine (4-AP), a delayed rectifier K+ channel blocker. Sevoflurane significantly repolarized the membrane potential, whereas CHTX and 4-AP had little effect on the membrane potential. C, Effect of 1.5 MAC sevoflurane on carbachol (1 µM)-induced depolarization of the membrane pretreated with 10 µM niflumic acid, a Ca2+-activated Cl- channel blocker. Sevoflurane had little effect on the membrane potential, whereas niflumic acid significantly hyperpolarized the membrane.

 

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  		Table 2. Effects of the Volatile Anesthetics Isoflurane and Sevoflurane on Membrane Potentials of Tracheal and Bronchial Smooth Muscle Cells
 
As summarized in Table 2, the same protocol was performed in bronchial smooth muscle cells, and similar results were obtained in these cells. Although the degrees of effects of volatile anesthetics and K+/Cl- channel blockers per se were indistinguishable between tracheal and bronchial smooth muscle cells, the absolute values of membrane potentials in tracheal smooth muscle cells were significantly higher than those in bronchial smooth muscle cells in all conditions.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
In the current-clamp experiments, resting membrane potential was measured after full access into the cells had been achieved. The mean values of resting potential (approximately -45 and -52 mV in tracheal and bronchial smooth muscle cells, respectively) were similar to the results previously reported with current-clamp techniques (4) but somewhat higher than those with tissue strips (23,24). The differences between membrane potentials in these studies might be associated with the exchange of monovalent ions (e.g., Cl-) between the patch pipette and cytosol. Application of 1 µM carbachol depolarized the cells by approximately 26–27 mV in both tissues, and the volatile anesthetics tested significantly hyperpolarized the carbachol-induced depolarization by approximately 4–5 mV in both tissues (Fig. 1 and Table 2) without affecting [3H]QNB and muscarinic agonist- (carbachol-) binding affinity (Table 1). It has been reported that the volatile anesthetics halothane and isoflurane have no effect on muscarinic receptor binding affinity in the rat brain (25). Although the carbachol binding was fitted by a two-receptor population model (KH and KL), and the distribution (Bmax) and binding affinity of KH and KL were different in tracheal and bronchial smooth muscles (Table 1), these data were consistent with those of another study (26) in which there were different distributions of muscarinic receptor subtypes (M1–M4) in distal and proximal airways. Accordingly, the inhibitory effect of volatile anesthetics on airway smooth muscle tone could be, at least in part, caused by the hyperpolarizing effect of the anesthetics. The volatile anesthetic-induced repolarization observed in this study was abolished by a specific ClCa channel blocker, niflumic acid, but not by K+ channel blockers, CHTX and 4-AP. It is therefore thought that the anesthetic-induced repolarization of airway smooth muscle cells is caused mainly by change in ClCa channel activity, not in K+ channel activity.

A comparison of the hyperpolarizing effects of the volatile anesthetics in tracheal and bronchial smooth muscle cells showed that there were no significant differences between the effects on these two types of cells. Therefore, the hyperpolarizing effect of the anesthetic per se does not seem to be responsible for the different inhibitory effects of volatile anesthetics on the muscle tone in tracheal and bronchial smooth muscles. However, when exposed to the anesthetic in pretreatment with carbachol, the absolute membrane potential in bronchial smooth muscle cells (-29.3 mV) was significantly lower than that in tracheal smooth muscle cells (-24.7 mV) (Table 1). Under this condition, the VDCC activities in tracheal and bronchial smooth muscle cells should be different because some of the L-type VDCCs were inactivated at -30 mV, whereas T-type VDCCs, which exist only in bronchial smooth muscle cells (9), can open more easily than L-type VDCCs at this potential. The T-type VDCCs are more sensitive to volatile anesthetics in bronchial smooth muscle cells (9). Accordingly, the different membrane potentials per se of tracheal and bronchial smooth muscle cells could contribute to the different effects of volatile anesthetics on tracheal and bronchial smooth muscle contractions (9).

In conclusion, volatile anesthetic-induced repolarization of airway smooth muscle cell membranes depolarized by a muscarinic agonist might be caused mainly by change in ClCa channel activity, not in K+ channel activity. The different hyperpolarized effects per se of tracheal and bronchial smooth muscle cells could in part contribute to the different effects of volatile anesthetics on tracheal and bronchial smooth muscle contractions.


   Acknowledgments
 
This study was supported by a grant-in-aid (12671489, 2000) for research from the Ministry of Education, Science and Culture, Tokyo, Japan, and an incentive grant (III-27, 2000) for research from the Uehara Memorial Foundation, Tokyo, Japan.


   References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Accepted for publication September 14, 2001.





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Lippincott, Williams & Wilkins Anesthesia & Analgesia® is published for the International Anesthesia Research Society® by Lippincott Williams & Wilkins and Stanford University Libraries' HighWire Press®. Copyright 2002 by the International Anesthesia Research Society. Online ISSN: 1526-7598   Print ISSN: 0003-2999 HighWire Press