Secondary Logo

Journal Logo

Interactions of Volatile Anesthetics with Cholinergic, Tachykinin, and Leukotriene Mechanisms in Isolated Guinea Pig Bronchial Smooth Muscle

Wiklund, C. U., MD, PhD*,; Lindsten, U., MD*,; Lim, S., MD†,; Lindahl, S. G.E., MD, PhD*

doi: 10.1097/00000539-200212000-00032

We studied relaxation of airway smooth muscle by sevoflurane, desflurane, and halothane in isolated guinea pig bronchi. Ring preparations were mounted in tissue baths filled with physiological salt solution and continuously aerated with 5% CO2 in oxygen. Electrical field stimulation induced contractions sensitive to tetrodotoxin, indicating nerve-mediated responses. These consisted of an atropine-sensitive cholinergic phase and a nonadrenergic noncholinergic (NANC) phase sensitive to SR48968, a neurokinin-2 receptor antagonist. Anesthetics were added to the gas aerating the tissue baths. Sevoflurane and desflurane at 1.0 minimum alveolar anesthetic concentration and halothane at 1.0–2.0 minimum alveolar anesthetic concentrations inhibited both cholinergic and NANC contractions to electrical field stimulation. None of the anesthetics affected responses to exogenously applied neurokinin A, a likely mediator of NANC contractions, suggesting prejunctional inhibition of NANC neurotransmission. The anesthetics did not affect the initiation of contractile responses to leukotriene C4 (LTC4), a mediator of asthmatic bronchoconstriction. However, sevoflurane and desflurane both relaxed bronchi in a steady-state contraction achieved by LTC4. Surprisingly, halothane did not relax LTC4 contractions. Concerning LTC4-elicited bronchoconstriction, sevoflurane and desflurane were more potent airway smooth muscle relaxants in vitro.

*Department of Anesthesiology and Intensive Care Medicine, Karolinska Hospital and Institute, Stockholm, Sweden; and †Division of Pediatric Cardiology, University of Michigan, Ann Arbor

Supported, in part, by funds from the Karolinska Institute, the Swedish Society of Medicine, and the Medical Research Council K2001–73X-10401–09A.

July 24, 2002.

Address correspondence and reprint requests to Claes U. Wiklund, MD, PhD, Department of Anesthesiology and Intensive Care, Karolinska Hospital, S-171 76 Stockholm, Sweden. Address e-mail to

Asthma is an increasingly common disease. The prevalence of asthma in an adult Medicaid population in Kentucky was reported to be 4.6%(1). More frequent prevalences have been found in children. A 20-yr survey of school children in Patras, Greece, demonstrated that the prevalence of asthma increased from 1.5% to 6.0% and lifetime prevalence in the same group from 8% to 9.6%(2). A significant number of fatal cases occur each year (3). Volatile anesthetics are relaxants of airway smooth muscle. Their mechanism of action is well studied, especially their direct action on smooth muscle cells and on Ca-dependent intracellular functions (4,5). However, there are fewer studies on nonadrenergic noncholinergic (NANC) nerve activity and on asthmatic mediators such as leukotrienes (6,7).

In clinical practice, halothane has been successfully used in patients with status asthmaticus (8), but there are negative effects, such as halothane-induced hepatitis and catecholamine-sensitizing effects on myocardial cells, that limit its use (9,10). However, in a small number of severely bronchoconstricted patients, volatile anesthetics must be used in the intensive care unit when other therapies prove insufficient (11).

Two newer inhaled anesthetics, sevoflurane and desflurane, relax airway smooth muscle. Their effects on neuroeffector transmission and on contractile responses to asthmatic mediators in airway smooth muscle have not previously been extensively studied (6).

In this study, we were especially interested in actions of these volatile anesthetics on postganglionic cholinergic and NANC neurotransmission and on contractile responses to leukotriene C4 (LTC4), an important mediator of asthmatic airway constriction.

Back to Top | Article Outline


The study was approved by the local ethics committee for animal studies at the Karolinska Institute. Guinea pigs of either sex (300–600 g) were killed by CO2 narcosis and exsanguination. The lungs were excised and put in ice-cold physiological salt solution (PSS, composition in millimoles per liter: Na 149, K 2.9, Ca 1.8, Mg 0.5, Cl 144, HCO3 23.8, H2PO4 0.4, and glucose 5.5) and aerated continuously with 5% CO2 in oxygen. Rings of proximal bronchi, three cartilages wide, two preparations from each lung, were dissected free from connective tissue and mounted in 25-mL water-jacketed tissue baths made of glass (Section of Engineering, Mayo Clinic, Rochester, MN). The temperature was increased from 4°C to 37°C during 60 min, after which electrical field stimulation (EFS) was initiated. An initial muscle tone of 5 mN was applied, which was found optimal for tension recording (12). The rings were allowed to equilibrate for 2–3 h before experiments were started. Motor activity was recorded with Grass force-displacement transducers (FT03) (Grass Instruments, Quincy, MA), and tracings were made on a Grass Polygraph (Grass Instruments).

Isolated ring preparations of guinea-pig bronchi were stimulated supramaximally (0.3-ms pulse duration, monophasic pulses, 15 V, 10 Hz, 100 pulses; i.e., 10-s stimulations at 10-min intervals) by EFS, applied via a direct current amplifier (Section of Engineering, Mayo Clinic), triggered by a stimulator via two parallel platinum electrodes (10 × 50 mm), 8 mm apart. They responded with biphasic contractions consisting of a rapid atropine-sensitive phase followed by a slow phase resistant to atropine and propranolol (Fig. 1). The slow phase was abolished by SR48968, a selective antagonist of neurokinin-2 (NK2) receptors, suggesting responses to cholinergic and NANC nerve activity.

Figure 1

Figure 1

Pulse duration-response curves were obtained to determine optimal stimulation variables and to identify nerve-mediated stimulation (Fig. 2). EFS was applied (15 V, 10 Hz, trains of 100 pulses; i.e., 10-s stimulation duration at 10-min intervals), and pulse duration was increased gradually from 0.01–5.0 ms. This process was repeated in the presence of tetrodotoxin (3 × 10−7 mmol/L), a blocker of fast neuronal sodium channels (13). At the 0.3-ms pulse duration, responses were maximal, with negligible responses remaining in the presence of tetrodotoxin. Thus, responses to EFS with 0.3-ms pulse duration and 10-s stimulation duration were regarded as nerve mediated and were used throughout the study (Fig. 3).

Figure 2

Figure 2

Figure 3

Figure 3

Two bronchial ring preparations were used for neurokinin A (NKA) and two for LTC4. NKA or LTC4 (10−9–10−7 mol/L) were added cumulatively to the tissue baths in log-increments, allowing 8–10 min for stabilization of the contractile response of each concentration. The stabilized contractile response to NKA or LTC4 (10−7 mol/L) after the first cumulative application was regarded as the maximal response and set to 100%. After the first application, preparations were washed thoroughly and allowed to equilibrate for 30 min before repeated cumulative dose-response applications were performed. After a 15-min equilibration, a volatile anesthetic was added to two baths that received NKA or LTC4. The other two test rings served as controls for the effect of time. In some preparations, the basal tone was slightly lower after the first cumulative dose-response application, resulting in that some cases had time-adjusted control contractions slightly larger than 100% (Fig. 4). The maximal contractile response to LTC4 was slightly attenuated by repeated application resulting in time-adjusted control contractions smaller than 100% (Fig. 5).

Figure 4

Figure 4

Figure 5

Figure 5

In two ring preparations, single doses of LTC4 (10−8 mol/L) were applied. 10−8 mol/L was close to the 50% effective dose (ED50) of LTC4 and produced a contractile response in the range 100%–200% of contractile response to nerve stimulation. Ten minutes was allowed for stabilization of contractile response to LTC4. This stabilized response was used as a control contractile response and set at 100%.

A volatile anesthetic was added to one of the tissue baths, whereas the other served as control for the effect of time. Volatile anesthetics relaxed the bronchial rings with a slow onset such that a stabilized effect resulted after 20 min.

Desflurane, sevoflurane, and halothane were added, via vaporizers, to the gas aerating the PSS. Concentrations of anesthetics were measured by gas chromatography, with a Varian gas chromatograph 7400, as described previously (13). Effects were studied at 1.0 minimum alveolar anesthetic concentration (MAC). Halothane was also studied at 2.0 MAC. With our experimental setup, larger concentrations than 1.0 MAC in the PSS were not achievable for desflurane and sevoflurane using maximal concentration from vaporizers with maximal aerating intensity in tissue chambers.

Acetylcholine chloride, atropine sulfate, propranolol hydrochloride, and tetrodotoxin were purchased from Sigma (St Louis, MO). NKA was from Scientific Marketing Associates (Barnet, Herts, United Kingdom), and LTC4 was purchased from Cascade Biochem Ltd (Reading, Berkshire, United Kingdom). SR48968 was a kind gift from Sanofi Recherche (Montpellier, Cédex, France). Sevoflurane and desflurane were kind gifts from Abbott (Abbott Park, IL) and Pharmacia & Upjohn (Peapack, NJ), respectively. Halothane was purchased from Zeneca AB (Gothenburg, Sweden). The study was supported by Abbott and Pharmacia & UpJohn.

Contractile responses in bronchial rings exposed to volatile anesthetics were compared with responses in rings not exposed to volatile anesthetics. In rings exposed to volatile anesthetics, control contractile responses to NKA and LTC4 were adjusted for the effect of time as described before (13). Responses to EFS were adjusted to the effect of time accordingly. Experimental data were expressed as mean ± sd. Statistical analysis was performed with SPSS (Chicago, IL) for windows. Statistical significance was tested with repeated-measures analysis of variance or Student’s t-test for paired or unpaired variables. P < 0.05 was regarded as statistically significant.

Back to Top | Article Outline


Pretreatment with atropine (10−6 mol/L) and propranolol (10−6 mol/L) did not significantly affect the action by volatile anesthetics on the slow phase of contractile responses to nerve stimulation. Neither did pretreatment with SR48968 (10−6 mol/L) significantly affect the action by volatile anesthetics on the rapid phase of contractile responses to nerve stimulation. Therefore, experiments on responses to EFS were performed in the absence of atropine and propranolol or SR48968. Concentrations of anesthetics were normalized to guinea pig MAC values of approximately 6% for desflurane, 1% for halothane, and 2% for sevoflurane (13). Equianesthetic concentrations were used, and only one concentration of volatile anesthetic was given to each muscle preparation.

Desflurane, halothane, and sevoflurane inhibited contractile responses to EFS in a reversible manner (Figs. 1 and 3). All three anesthetics inhibited both phases of contractile responses to EFS. The rapid atropine-sensitive contraction was more efficiently inhibited by halothane than by sevoflurane or desflurane (Fig. 3A). The NANC phase of contractile responses to EFS was equally inhibited by all three anesthetics (Fig. 3B).

Cumulative dose-response curves for NKA (10−9–10−7 mol/L) showed an ED50 of 3.8 ± 1.6 × 10−8 mol/L. A second group of cumulative dose-response curves were performed during exposure for sevoflurane or desflurane at 1.0 MAC or halothane at 1.0–2.0 MAC (Fig. 4; data for sevoflurane and halothane not shown). None of the volatile anesthetics had any effect on ED50 or efficacy, i.e., force development.

Cumulative dose-response curves to LTC4 (10−9–10−7 mol/L) revealed an ED50 of 6.6 ± 9.9 × 10−9 mol/L. Subsequent cumulative dose-response curves were performed after incubation with sevoflurane or desflurane at 1.0 MAC or halothane at 1.0–2.0 MAC (Fig. 5; data for halothane and desflurane not shown). The volatile anesthetics did not significantly affect ED50 or efficacy, i.e., force development.

In bronchial rings precontracted submaximally with LTC4 (10−8 mol/L), sevoflurane and desflurane at 1.0 MAC induced a slow relaxation. Halothane (1.0–2.0 MAC) did not significantly affect the contractile response to LTC4 (Fig. 6).

Figure 6

Figure 6

Back to Top | Article Outline


The principal findings in this study were that halothane, sevoflurane, and desflurane inhibited contractile responses to cholinergic and NANC neurotransmission. Sevoflurane and desflurane relaxed airway smooth muscle during steady-state contraction by LTC4, whereas halothane did not.

Some volatile anesthetics, for example, isoflurane and desflurane, irritate airways in awake patients. During anesthesia, sevoflurane decreases airway resistance more efficiently than halothane and isoflurane (14). Desflurane increases airway resistance after tracheal intubation in smokers (15). The lack of influence by sympathetic and parasympathetic nerves in isolated smooth muscle may explain why desflurane relaxes bronchial smooth muscle in vitro and increases airway resistance in vivo.

Excitatory NANC nerves may induce bronchoconstriction (NK2 receptors), mucus secretion, vasodilation, and vascular hyperpermeability (NK1 receptors) (7). In this study, contractile responses to EFS were attenuated by volatile anesthetics. Possible explanations include inhibition of transmitter release, reduced receptor activation, or interaction with intracellular contractile processes. We found sensitivity to NKA to be unaltered by anesthetics. In previous studies, volatile anesthetics inhibited contractile responses to EFS (13) and excitatory junction potentials (16) without alteration of acetylcholine sensitivity. Halothane inhibited acetylcholine release from preganglionic nerve terminals (17). These investigations support our suggestion of reduced transmitter release. Possible cellular sites of action are blocks of interaction between transmitter containing vesicles and synaptic membranes, stabilization of the axonal membrane, or depressed intracellular Ca2+ mobilization (13).

Neurogenic inflammation can be modulated via altered release of sensory neuropeptides (7). In the current study, the lack of effect by volatile anesthetics on sensitivity to NKA is compatible with the suggestion that they do not affect NK2 receptor activation or intracellular processes responsible for the initiation of NK2 receptor-mediated contraction.

Leukotrienes are produced in increased amounts in airways of asthmatics. They produce bronchoconstriction, recruit inflammatory cells, and enhance excitability of airway sensory nerves (18). Anesthetics did not alter sensitivity to LTC4. Our results are consistent with the suggestion that volatile anesthetics did not influence activation of leukotriene receptors or intracellular processes responsible for the initiation of contractile responses to LTC4. Tudoric et al. (6) reported inhibitory effects by halothane and isoflurane on contractile responses to allergen and leukotriene D4 in vitro. Passive sensitization enhanced LTC4 release and sensitivity in human isolated bronchi (19). We used tissue from nonsensitized animals, whereas Tudoric et al. (6) used sensitized animals, which may explain the different results. This would be of interest in a discussion of drug choice in allergy-mediated bronchoconstriction and in nonallergic bronchial hyperreactivity.

The lack of effect by anesthetics on initiation of contractile response to LTC4 and relaxation of steady-state contraction induced by LTC4 may seem contradictory. Initiation and maintenance of contractile response to muscarinic stimulation may be linked to different intracellular mechanisms. Rapidly cycling phosphorylated cross bridges are responsible for muscle shortening and slowly cycling dephosphorylated cross bridges for maintenance of steady-state tone (20). Separate intracellular processes for initiation and maintenance of leukotriene-mediated contractions are likely. We suggest that sevoflurane and desflurane act on intracellular mechanisms responsible for the maintenance of contractions induced by LTC4, most likely on slowly cycling intracellular cross bridges. Halothane is structurally different from both sevoflurane and desflurane, suggesting different affinity for single or multiple sites of membrane bound proteins (21,22) such as ion channels, which might explain the different effects on contractile response to LTC4. Co-administration of sevoflurane or desflurane and halothane might reduce the relaxing effect on LTC4-induced bronchoconstriction.

In summary, halothane, sevoflurane, and desflurane attenuated airway smooth muscle tone via inhibition of cholinergic and NANC neurotransmission, most likely via both pre- and postjunctional mechanisms. Sevoflurane and desflurane both reduced LTC4-induced bronchoconstriction, whereas halothane did not. The relaxing effects by sevoflurane and desflurane on contraction induced by the asthmatic mediator LTC4 suggest a beneficial role in the treatment of asthmatic patients.

Back to Top | Article Outline


1. Piecoro LT, Potoski M, Talbert JC, Doherty DE. Asthma prevalence, cost and adherence with expert guidelines on the utilization of health care services and costs in a state Medicaid population. Health Serv Res 2001; 36: 357–71.
2. Anthracopoulos M, Karatza A, Liolios E, et al. Prevalence of asthma among schoolchildren in Patras, Greece: three surveys over 20 years. Thorax 2001; 56: 569–71.
3. Levy BD, Kitch B, Fanta CH. Medical and ventilatory management of status asthmaticus. Intensive Care Med 1998; 24: 105–17.
4. Yamakage M, Hirshman CA, Croxton TL. Volatile anesthetics inhibit voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells. Am J Physiol 1995; 268: L187–91.
5. Kai T, Bremerich DH, Jones KA, Warner DO. Drug-specific effects of volatile anesthetics on Ca2+ sensitization in airway smooth muscle. Anesth Analg 1998; 87: 425–9.
6. Tudoric N, Coon RL, Kampine JP, Bosnjak ZJ. Effects of halothane and isoflurane on antigen- and leukotriene-D4-induced constriction of guinea pig trachea. Acta Anaesthesiol Scand 1995; 39: 1111–6.
7. Joos GF, Germonpré PR, Pauwels RA. Role of tachykinins in asthma. Allergy 2000; 55: 321–37.
8. Saulnier FF, Durocher AV, Deturck RA, et al. Respiratory and hemodynamic effects of halothane in status asthmaticus. Intensive Care Med 1990; 16: 104–7.
9. Gut J, Christen U, Huwyler J. Mechanisms of halothane toxicity: novel insights. Pharmacol Ther 1993; 58: 133–55.
10. Turner LA, Vodanovic S, Bosnjak ZJ. Interaction of anesthetics and catecholamines on conduction in the canine His-Purkinje system. Adv Pharmacol 1994; 31: 167–84.
11. Parnass SM, Feld JM, Chamberlin WH, Segil LJ. Status asthmaticus treated with isoflurane and enflurane. Anesth Analg 1987; 66: 193–5.
12. Kageyama N, Ichinose M, Igarashi A, et al. Repeated allergen exposure enhances excitatory nonadrenergic noncholinergic nerve-mediated bronchoconstriction in sensitized guinea-pigs. Eur Respir J 1996; 9: 1439–44.
13. Wiklund CU, Lim S, Lindsten U, Lindahl SGE. Relaxation by sevoflurane, desflurane and halothane in the isolated guinea-pig trachea via inhibition of cholinergic neurotransmission. Br J Anaesth 1999; 83: 422–9.
14. Rooke GA, Choi J-H, Bishop MJ. The effect of isoflurane, halothane, sevoflurane, and thiopental/nitrous oxide on respiratory system resistance after tracheal intubation. Anesthesiology 1997; 86: 1294–9.
15. Goff MJ, Arain SR, Ficke DJ, et al. Absence of bronchodilation during desflurane anesthesia: a comparison to sevoflurane and thiopental. Anesthesiology 2000; 93: 404–8.
16. Korenaga S, Takeda K, Ito Y. Differential effects of halothane on airway nerves and muscle. Anesthesiology 1984; 60: 309–18.
17. Bosnjak ZJ, Dujic Z, Roerig DL, Kampine JP. Effects of halothane on acetylcholine release and sympathetic ganglionic transmission. Anesthesiology 1988; 69: 500–6.
18. Barnes NC, Smith LJ. Biochemistry and physiology of the leukotrienes. Clin Rev Allergy Immunol 1999; 17: 27–42.
19. Schmidt D, Rabe KF. The role of leukotrienes in the regulation of tone and responsiveness in isolated human airways. Am J Respir Crit Care Med 2000; 161: S62–7.
20. Torphy TJ, Hay DWP. Biochemical regulation of airway smooth-muscle tone: an overview. In: Agrawal DK, Townley RG, eds. Airway smooth muscle: modulation of receptors and response. Boca Raton, FL: CRC Press, 1990: 39–68.
21. Banks MI, Pearce RA. Dual actions of volatile anesthetics on GABAA IPSCs: dissociation of blocking and prolonging effects. Anesthesiology 1999; 90: 120–34.
22. Raines DE, Zachariah VT. Isoflurane increases the apparent agonist affinity of the nicotinic acetylcholine receptor. Anesthesiology 1999; 90: 135–46.
Back to Top | Article Outline

Attention Authors!

Submit Your Papers Online

You can now have your paper processed and reviewed faster by sending it to us through our new, web-based Rapid Review System. Submitting your manuscript online will mean that the time and expense of sending papers through the mail can be eliminated. Moreover, because our reviewers will also be working online, the entire review process will be significantly faster. You can submit manuscripts electronically via There are links to this site from the Anesthesia & Analgesia website (, and the IARS website ( To find out more about Rapid Review, go to and click on “About Rapid Review.”

© 2002 International Anesthesia Research Society